WO2021110120A1

WO2021110120A1 - Anti-tumor vaccine molecule, preparation method therefor and use thereof

Info

Publication number: WO2021110120A1
Application number: PCT/CN2020/133798
Authority: WO
Inventors: 郭军; 杨光富; 杜晶晶; 王昌伟
Original assignee: 华中师范大学
Priority date: 2019-12-04
Filing date: 2020-12-04
Publication date: 2021-06-10
Also published as: CN112516300A

Abstract

An anti-tumor vaccine molecule, a preparation method therefore and use thereof. The anti-tumor vaccine molecule has a structure as shown in formula (I). In the formula (I), A is an adjuvant, B is an antigen, m of As are respectively covalently connected to a protein by means of a covalent connecting arm, and n of Bs are respectively covalently connected to the protein by means of a covalent connecting arm. The anti-tumor vaccine is a novel anti-tumor molecule, has good immune performance, can produce an IgG antibody with a high titer, has relatively strong cellular immunity, has good thermal stability, and is easy to store and transport. A_m-Protein-B_n Formula (I)

Description

An anti-tumor vaccine molecule and its preparation method and application

Cross-references to related applications

This application claims the rights and interests of the Chinese patent application 201911228567.3 filed on December 4, 2019, the content of which is incorporated herein by reference.

Technical field

The invention relates to the field of anti-tumor vaccines, in particular to an anti-tumor vaccine molecule and its preparation method and application.

Background technique

Extensive research in cell biology and biochemistry has shown that tumor-associated antigens (TACAs, such as MUC1 glycoprotein) are ideal targets for immunotherapy and are vigorously pursued in the development of immunotherapy anti-cancer strategies.

So far, immunotherapy has proven to be one of the most promising cancer treatments, offering many possibilities.

However, due to the commonly used tumor-associated antigens as self-antigens, they have poor antigenicity and poor immune regulation tolerance, resulting in failure to elicit an effective immune response. Therefore, improving the immunogenicity of tumor-associated antigens has become an urgent problem to be solved.

A large number of studies have tried to obtain better immunogenic tumor-associated antigens, and linkers that link the antigens to different immunostimulatory components: a fully synthesized two-component anti-cancer vaccine with built-in adjuvant, containing Th cells Three-component anti-cancer vaccines with epitopes and built-in adjuvants or multi-component anti-cancer vaccines with Th, Tc cell epitopes and built-in adjuvants. Commonly used adjuvants include Toll-like receptor 2 lipopeptide ligands (Pam ₃ CSK ₄ ), monophosphoryl lipid A (MPLA), CpG, NKT cell agonist (αGalCer), etc. Animal experiments show that with the increase in the number of vaccine components, the immune response of mice to tumor-associated antigens may gradually increase, but the difficulty of synthesis further increases.

Furthermore, some studies have disclosed that the vaccine is equipped with optimized uncovalently linked MUC1 or nicotine antigen and αGalCer, which significantly improves the immunogenicity of the antigen.

In addition, semi-synthetic anti-cancer vaccines usually combine tumor-associated antigens with different carrier proteins, and then mix them with adjuvants to form a vaccine. The carrier proteins include bovine serum albumin (BSA), CRM197 (non-toxic diphtheria toxin mutant), and Cold toxoid (TTOX) and keyhole limpet hemocyanin (KLH). Given that the carrier protein has multiple Tc and Th epitopes, it can enhance the presentation of antigens, and thus can easily improve the immune response of the vaccine.

However, in order to develop more effective anti-tumor immunotherapy strategies, current challenges still exist.

In addition, Toll-like receptor (TLR) is an intracellular pattern recognition receptor that can recognize highly conserved components of a variety of pathogens and induce the host's innate and adaptive immune response. Second, it has the potential to regulate the activation of antigen-presenting cells and enhance the secretion of costimulatory molecules and many cytokines.

Summary of the invention

The purpose of the present invention is to overcome the problem of poor immunogenicity of tumor-associated antigens in the prior art.

In order to achieve the above objective, the first aspect of the present invention provides an anti-tumor vaccine molecule having a structure represented by formula (I). In said formula (I), A is an adjuvant, B is an antigen, and m The A is covalently connected to the protein through a covalent linking arm, and the n Bs are covalently connected to the protein through a covalent linking arm, and the number of amino acid molecules in the protein is greater than or equal to 100; In formula (I), m is an integer greater than or equal to 1, and n is an integer greater than or equal to 1;

A _m -protein-B _n formula (I).

The second aspect of the present invention provides a method for preparing the anti-tumor vaccine molecule having the structure represented by formula (I) as described in the first aspect, the method comprising:

Perform a first coupling between the antigen and the protein, and perform a second coupling between the obtained first intermediate and the adjuvant; or

The adjuvant is coupled to the protein for the third time, and the obtained second intermediate is coupled to the antigen for the fourth time.

The third aspect of the present invention provides the application of the anti-tumor vaccine molecule having the structure represented by formula (I) as described in the aforementioned first aspect in an anti-tumor vaccine.

There are obvious differences in the structure, preparation and properties of the peptides and proteins used in vaccines: (1) The number of amino acids in the peptides is small (<100, generally used peptides <50 amino acids), and the molecular weight is small; proteins are composed of amino acids. Large numbers (>100), large molecular weight, large volume; (2) Polypeptides are generally synthesized chemically, with few modification sites, and are easy to modify during chemical synthesis or after synthesis. They can be purified by HPLC; proteins are generally synthesized through biosynthesis. There are many modifiable sites, which are mainly modified in the aqueous phase after synthesis, and cannot be purified by HPLC; (3) The peptide is small in size, easy to diffuse, and not easy to be enriched in lymphoid tissues. Generally, it contains less than 3 Th and Tc epitopes. The immunogenicity is weak; the protein is large in size, not easy to spread, and is easy to be enriched in lymphoid tissues. It contains many Th and Tc epitopes, and the immunogenicity is strong.

The design of the built-in adjuvant protein conjugate strategy provided by the present invention is an effective strategy for designing high-efficiency immunotherapy anticancer vaccines. Compared with the traditional vaccine strategy, the anti-tumor vaccine of the present invention is a new anti-tumor molecule, has good immune performance, can produce higher titer IgG antibodies and stronger cellular immunity, has good thermal stability, is easy to store and transport.

Description of the drawings

Fig. 1 is an anti-tumor vaccine molecule according to a preferred embodiment of the present invention. Fig. 1A shows the anti-tumor vaccine molecule according to a preferred embodiment of the present invention. In Fig. 1A, the immune agonist is an adjuvant, LK1 and LK2 both represent covalently linked arms; Figure 1B shows an anti-tumor vaccine molecule according to another more preferred embodiment of the present invention. In Figure 1B, the TLR agonist is an adjuvant, and both LK1 and LK2 represent Covalently connected arms.

Figure 2 shows the cytokine IFN-γ and IL-6 test for the serum taken 2h after the first immunization as described in Example 9.

Figure 3 shows the test results of the IgG antibody titer against MUC1 of the three immunity described in Example 10.

Figure 4 shows the comparison of the IgG antibody titer test results against MUC1 for the first, second, and third immunity described in Example 10.

Figure 5 shows the comparison of IgM antibody titer test results against MUC1 for the first, second, and third immunity described in Example 10.

Fig. 6 shows the comparison of the test results of the IgG subtype antibody titers against MUC1 of the three immunity described in Example 10.

Fig. 7 shows the comparison of the IgG antibody titer test results of the three immunity against BSA described in Example 11.

Fig. 8 shows the MTT method described in Example 12 to measure the survival rate of MCF-7 cells.

Figure 9 shows the measurement of the binding of vaccine-induced antiserum to MCF-7 cells by flow cytometry as described in Example 13.

Figure 10 shows the lysis rate of MCF-7 cells described in Example 15.

Detailed ways

The endpoints and any values of the ranges disclosed herein are not limited to the precise range or value, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, between the end values of each range, between the end values of each range and individual point values, and between individual point values can be combined with each other to obtain one or more new numerical ranges. These values The scope should be considered as specifically disclosed herein.

As mentioned above, the first aspect of the present invention provides an anti-tumor vaccine molecule having a structure represented by formula (I). In said formula (I), A is an adjuvant, B is an antigen, and m The A is covalently connected to the protein through at least one covalent linking arm, and the n Bs are covalently connected to the protein through at least one covalent linking arm. The number of amino acid molecules in the protein is greater than or equal to 100; In the formula (I), m is an integer greater than or equal to 1, and n is an integer greater than or equal to 1;

A _m -protein-B _n formula (I).

According to the first preferred embodiment, m in the formula (I) is 1, and n is an integer greater than or equal to 1; for example, m is 1, and n is 1, 2, 3, 4, 5, 6, 7 , 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, etc.

According to a second preferred embodiment, m in the formula (I) is 1, and n is an integer greater than or equal to 2; for example, m is 1, and n is 2, 3, 4, 5, 6, 7, 8. , 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, etc.

According to a third preferred embodiment, m in the formula (I) is an integer greater than or equal to 2, and n is an integer greater than or equal to 1, for example, m is 2, 3, 4, 5, 6, 7, 8. , 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, etc., n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, etc.

According to a fourth preferred embodiment, m in the formula (I) is an integer greater than or equal to 2, and n is an integer greater than or equal to 2; for example, m is 2, 3, 4, 5, 6, 7, 8. , 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, etc., n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 etc.

In the present invention, the adjuvant and the protein can be covalently linked to each other through any covalently linked sites, and the antigen and the protein can also be covalently linked to each other through any covalently linked sites.价连接。 Price connection.

Preferably, the antigen includes at least one of a tumor-associated antigen, a tumor-specific antigen, a pathogen antigen, a biotoxin, and a biomolecular antigen.

The tumor-associated antigens of the present invention refer to antigen molecules present on tumor cells or normal cells, including, for example, embryonic proteins, glycoprotein antigens, squamous cell antigens, etc., and are commonly used in clinical tumor diagnosis. Tumor-associated antigens are not unique to tumor cells. Normal cells can be synthesized in small amounts and are highly expressed when tumor cells proliferate, so they are called "related antigens."

The tumor-specific antigen of the present invention refers to a new antigen that is only expressed on the surface of a certain tumor cell but does not exist on normal cells, so it is also called a unique tumor antigen.

Preferably, the tumor-associated antigen is selected from at least one of a tumor-associated polypeptide antigen, a tumor-associated glycopeptide antigen, and a tumor-associated carbohydrate antigen.

Preferably, the antigen contains at least one polypeptide or glycopeptide selected from MUC1, MUC16, NY-ESO-1, MAGE-A1/3/4, WT1, STAT3, HER2 and GP100.

The MUC1 of the present invention is a glycosylated, high molecular weight (Mr>200×103) mucin 1, which is a transmembrane molecule whose transmembrane sequence is inserted into the cell membrane like a rod, and has a structure consisting of 69 amino acid residues. The composed tail extends into the cytoplasm.

The MUC16 of the present invention, also known as CA125, is a high-molecular-weight glycoprotein expressed on the surface of various types of epithelial cells, and mainly plays the role of protecting and repairing the epithelium.

The NY-ESO-1 of the present invention represents New York esophageal squamous cell carcinoma 1 (NY-ESO-1), which is a cancer-testis antigen (CTA) and is re-expressed in many tumors.

The MAGE-A1/3/4 of the present invention means the detection of tumor-testis antigen (CTA) melanin antigen A (MAGE-A).

The WT1 of the present invention represents the Wilms tumor protein, which is expressed by the human WT1 gene.

The STAT3 of the present invention represents signal transduction and activation transcription factor 3.

The HER2 of the present invention represents human epidermal growth factor receptor-2 (HER2).

The GP100 of the present invention represents a melanin-related antigen.

Preferably, the antigen contains MUC1, and more preferably, the MUC1 antigen is selected from at least one of the following structures:

Wherein, in the structure of the antigen containing MUC1, each of R ¹ , R ² , R ³ , R ⁴ and R ⁵ is connected to an amino acid residue modification group, and each of R ¹ , R ² , R ³ , R ⁴ and R ⁵ are each independently selected from hydrogen and the sugar structure shown below:

Preferably, the tumor-associated carbohydrate antigen is selected from at least one of the following structures:

In the anti-tumor vaccine molecule of the present invention, the protein is preferably selected from bovine serum albumin (BSA), chicken ovalbumin (OVA), keyhole hemocyanin (KLH), tetanus toxoid (TT), Diphtheria toxoid (DT), Haemophilus influenzae D protein, group B meningococcal outer membrane protein complex (OMP), pertussis toxoid, typhoid flagella, pneumolysin (PLY) and non-toxic mutations of diphtheria toxin At least one of the body (CRM197).

Preferably, in the anti-tumor vaccine molecule of the present invention, the adjuvant is a pattern recognition receptor agonist.

Preferably, the pattern recognition receptor agonist is selected from at least one of Toll-like receptor agonists and NKT agonists; more preferably, the Toll-like receptor agonist is selected from TLR7 agonists, TLR8 agonists , At least one of TLR9 agonist, TLR3 agonist, TLR2 agonist and TLR4 agonist.

In a particularly preferred case, the representative structure of a TLR7 agonist is as follows:

among them,

In the above-mentioned representative structure of TLR7 agonist, R _{1 is} selected from any one of the following structures:

In the above-mentioned representative structure of TLR7 agonist, R _{2 is} selected from any one of the following structures:

In other preferred cases, the representative structure of the TLR7 agonist is as follows:

In a particularly preferred case, the representative structure of a TLR8 agonist is as follows:

In a particularly preferred case, the TLR9 agonist is CpG-ODN, and the representative structure is as follows:

Wherein, 5-(P=S)TCCATGACGTTCCTGACGT represents a nucleic acid sequence.

Particularly preferably, the TLR3 agonists are poly(I:C) and poly-ICLC, and the representative structures are as follows:

Poly(I:C) is polyinosic acid, which is polycreatic acid-polycytidysic acid, which is an analog of double-stranded RNA, one chain is Poly(I) and the other chain is Poly(C).

Poly-ICLC is a synthetic double-stranded polyriboinosine-polyribocytidylic acid Poly(I:C) stabilized with polylysine and carboxymethylcellulose (LC).

In a particularly preferred case, the representative structure of a TLR2 agonist is as follows:

In a particularly preferred case, the representative structure of a TLR4 agonist is as follows:

R ₃ represents a hydrogen atom or a phosphate group;

R ₄ , R ₅ , R ₆ , and R ₇ each independently represent a fatty acyl group with a carbon number of 1-20;

R ₈ represents a hydrogen atom or a phosphate group;

R ₉ represents a hydroxyl group, an amino group or a carboxyl group;

a, b, c, and d represent an alkyl group having 1-20 carbon atoms.

R ⁸ represents a hydrogen atom or a phosphate group;

R ⁹ represents a hydroxyl group, an amino group, a carboxyl group, a phosphoric acid group;

R ¹⁰ represents a hydrogen atom or a methyl group, including R/S configuration;

R ¹¹ and R ¹² each independently represent a hydroxyl group, an amino group, or a carboxyl group;

R ¹³ , R ¹⁴ and R ¹⁵ each independently represent a fatty acyl group with a carbon number of 1-20;

X represents oxygen or sulfur or selenium atom or amino group;

Z represents oxygen or amino;

e, f and g represent alkyl groups with 1-20 carbon numbers;

h, i, j, and k each independently represent an integer of 0-6.

In a particularly preferred case, the representative structure of the NKT cell agonist is as follows:

Preferably, in the anti-tumor vaccine molecule of the present invention, the structure of each covalent linking arm is independently selected from the following structures:

-CO-, -O-CO-, -NH-CO-, -NH(C=NH)-, -SO ₂ -, -O-SO ₂ -, -NH-, -NH-CO-CH ₂ -, -CH ₂ -, -C ₂ H ₄ -, -C ₃ H ₆ -, -C ₄ H ₈ -, -C ₅ H ₁₀ -, -C ₆ H ₁₂ -, -C ₇ H ₁₄ -, -C ₈ H ₁₆ -, -C ₉ H ₁₈ -, -C ₁₀ H ₂₀ -, -CH(CH ₃ )-, -C[(CH ₃ ) ₂ ]-, -CH ₂ -CH(CH ₃ )-, -CH (CH ₃ )-CH ₂ -, -CH(CH ₃ )-C ₂ H ₄ -, -CH ₂ -CH(CH ₃ )-CH ₂ -, -C ₂ H ₄ -CH(CH ₃ )-,- CH ₂ -C[(CH ₃ ) ₂ ]-, -C[(CH ₃ ) ₂ ]-CH ₂ -, -CH(CH ₃ )-CH(CH ₃ )-, -C[(C ₂ H ₅ ) (CH ₃ ))-, -CH(C ₃ H ₇ )-, -(CH ₂ -CH ₂ -O) _p -CH ₂ -CH ₂ -, -CO-CH ₂ -, -CO-C ₂ H ₄ -, -CO-C ₃ H ₆ -, -CO-C ₄ H ₈ -, -CO-C ₅ H ₁₀ -, -CO-C ₆ H ₁₂ -, -CO-C ₇ H ₁₄ -, -CO- C ₈ H ₁₆ -, -CO-C ₉ H ₁₈ -, -CO-C ₁₀ H ₂₀ -, -CO-CH(CH ₃ )-, -CO-C[(CH ₃ ) ₂ ]-, -CO- CH ₂ -CH(CH ₃ )-, -CO-CH(CH ₃ )-CH ₂ -, -CO-CH(CH ₃ )-C ₂ H ₄ -, -CO-CH ₂ -CH(CH ₃ )- CH ₂ -, -CO-C ₂ H ₄ -CH(CH ₃ )-, -CO-CH ₂ -C[(CH ₃ ) ₂ ]-, -CO-C[(CH ₃ ) ₂ ]-CH _2- , -CO-CH(CH ₃ )-CH(CH ₃ )-, -CO-C[(C ₂ H ₅ )(CH ₃ )]-, -CO-CH(C ₃ H ₇ )- or -CO- (CH ₂ -CH ₂ -O) _p -CH ₂ -CH ₂ -;

Wherein, in the structure of the covalent link arm,

Each x is independently selected from an integer of 1-60;

Each Y is independently selected from at least one of -NH-, -O-, -S- and -S-S-;

Each p is independently selected from an integer of 1-60.

The present invention does not specifically limit the method for preparing the aforementioned anti-tumor vaccine molecule. Those skilled in the art can combine the structural characteristics of the anti-tumor vaccine molecule in the art and the conventional synthetic methods in the field to determine a suitable method to prepare the anti-tumor vaccine molecule. And, the example part of the present invention exemplarily lists the specific preparation methods of some anti-tumor vaccine molecules, and those skilled in the art can also combine the exemplified preparation methods of the present invention to determine the specific preparation of all anti-tumor vaccine molecules of the present invention. However, those skilled in the art should not understand that this is a limitation of the present invention.

Similarly, the adjuvants, antigens and proteins in the anti-tumor vaccine molecules of the present invention can be synthesized by using existing methods, or can be obtained commercially, and the present invention is not limited to this.

Preferably, as mentioned above, the second aspect of the present invention provides a method for preparing the anti-tumor vaccine molecule according to the first aspect of the present invention, the method comprising:

It should be noted that the aforementioned "first", "second", "third", "fourth", etc. are only used for distinction, which means that the above definitions are not the same process, but do not indicate the order of sequence. The skilled person should not understand that this is a limitation of the present invention.

As mentioned above, there are at least two methods for preparing the anti-tumor vaccine molecules in the present invention. The first one is: the first coupling of the antigen and the protein, and the second coupling of the obtained first intermediate and the adjuvant. The second method is to perform a third coupling between the adjuvant and the protein, and perform a fourth coupling between the obtained second intermediate and the antigen.

The present invention has no particular restrictions on the specific conditions of each coupling, and those skilled in the art can determine suitable conditions in combination with the context of the present invention and the routine in the art.

As mentioned above, the third aspect of the present invention provides the application of the anti-tumor vaccine molecule described in the first aspect of the present invention in an anti-tumor vaccine.

Several preferred specific embodiments of the present invention are provided below.

In a preferred embodiment of the present invention, the present invention provides an anti-tumor vaccine molecule with the structure shown in Figure 1A. In the anti-tumor vaccine molecule, the antigen molecule and the adjuvant molecule are shared with the same protein (that is, the carrier protein). After valency connection, an embedded adjuvant three-in-one protein conjugate is obtained, which can produce high-titer IgG antibody immune response when used as an anti-tumor vaccine molecule. As shown in Fig. 1A, in the structural formula of the anti-tumor vaccine molecule, both LK1 and LK2 represent covalent link arms. In this preferred embodiment, the adjuvant, antigen, protein, and covalent linking arm are all as described above.

In particular, the inventors of the present invention found that when TLR7 agonists are used as adjuvants, they can significantly better enhance immune stimulating activity and reduce side effects. Therefore, in another more preferred embodiment of the present invention, a TLR7 agonist is used as an adjuvant, and BSA is used as a protein, and the three components of the tumor-associated glycopeptide antigen MUC1 are covalently combined to form an adjuvant-protein-antigen vaccine molecule, The specific structure of the vaccine molecule is shown in Figure 1B, where LK1 and LK2 both represent covalent link arms. In this preferred embodiment, the antigen, protein, and covalent linking arm are all as described above. The anti-tumor vaccine molecule not only produces significant IgG antibodies, but also induces a relatively high level of IgG2a, resulting in the antibody type biased towards Th1 type cellular immunity.

The present invention provides a three-in-one protein-binding vaccine strategy with a novel structure, that is, covalently binding an adjuvant to a carrier protein bound to an antigen. This solution is first applied to the field of anti-cancer (ie anti-tumor) vaccines.

In the preferred embodiment, the present invention also has the following specific advantages:

(1) Due to the cluster arrangement of the bound TLR7 agonist on the carrier protein-bound antigen conjugate, it will greatly change the pharmacokinetic properties, promote the transport of the conjugate to the lymph nodes, enhance immunostimulatory activity and reduce side effects.

(2) Activate antigen presenting cells (APCs) and activate T cells.

(3) Produce high-affinity IgG antibodies against the antigen and higher IgG2a antibodies.

(4) The antibodies produced can recognize cancer cells and can initiate the lysis of the recognized cancer cells by activating, for example, the complement-dependent cytotoxicity (CDC) of rabbit serum.

(5) Stimulate cytotoxic T lymphocyte killing effect (CTL) and mediate stronger T cell immunity.

The three-in-one protein conjugate (that is, the anti-tumor vaccine molecule) provided by the present invention is an effective strategy for designing high-efficiency immunotherapy anti-cancer vaccines.

Hereinafter, the present invention will be described in detail through examples. In the following examples, the test methods used, unless otherwise specified, are all conventional methods. The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

Example 1: Preparation of TLR7 agonist

Synthesis of compound 2: 2-chloroadenine (5.4g, 31.8mmol) and sodium (5.0g, 217mmol) were mixed in ethylene glycol monomethyl ether (235mL, 3.1mol), and the mixture was stirred and refluxed at 140°C for 10h . Then add 25 mL of water, add 1 mol/L hydrochloric acid to pH=7, concentrate, wash with water and filter to obtain compound 2, and proceed to the next step without purification.

Synthesis of compound 3: Compound 2 (0.7g, 3.34mmol), potassium carbonate (3.2g, 23.44mmol), methyl 4-bromomethylbenzoate (1.5g, 6.68mmol) were added to 10mL dry N,N-di In Methylformamide (DMF), stir and reflux at 60°C for 8h. Then, 5% by mass citric acid aqueous solution was added until no bubbles were generated, extracted with chloroform, washed with brine, dried with MgSO ₄ , filtered, and concentrated. The reaction mixture was purified by column chromatography to obtain a white solid 3. ¹ H NMR (400MHz, DMSO-d ₆ ) δ (ppm) 8.10 (s, 1H), 7.95 (d, J = 8.0 Hz, 2H), 7.43 (s, 1H), 7.42 (d, J = 8.1 Hz, 2H, 2 × benzene ring), 7.30 (s, 2H, 2 × benzene ring), 5.38 (s, 2H, NH ₂ ), 4.32 (t, J = 4.7 Hz, 2H, 2H, CH ₂ -O), 3.85 (s, 3H, CH ₃ -O), 3.61 (t, J = 4.7 Hz, 2H, 2H, CH ₂ -O), 3.28 (s, 3H, CH ₃ -O). ¹³ C NMR (101MHz, DMSO- d ₆ )δ (ppm) 166.40 (C=O), 161.82 (C ₂ ), 157.23 (C ₆ ), 151.66 (C ₄ ), 142.93 (C ₈ ), 140.03, 130.02, 129.38, 128.23 (benzene), 115.59 (C ₅ ), 70.74(O- C H ₂ -), 65.82(- C H ₂ -O), 58.55(-O C H ₃ ), 52.64(-O C H ₃ ), 46.18( C H ₂ -C ₆ H ₄ COOH). HRMS calculated value C ₁₇ H ₂₀ N ₅ O ₄ ⁺ [M+H] ⁺ : 358.1510. Measured value: 358.1513.

Synthesis of compound 4: Compound 3 (0.8 g, 2.2 mmol) was added to chloroform (10 mL), elemental bromine (227 μL, 4.4 mmol) was added, and the mixture was stirred at room temperature for 8 h. Then saturated Na ₂ S ₂ O _{3 was added to} remove excess bromine, extracted with chloroform, the organic phase was washed with brine, dried with MgSO ₄ , filtered, and concentrated. The reaction mixture was purified by column chromatography to obtain 4 as a white solid. ¹ H NMR (400MHz, DMSO-d ₆ ) δ (ppm): 7.96 (d, J = 8.1 Hz, 2H, 2 × benzene ring), 7.50 (s, 2H, NH ₂ ), 7.36 (d, J = 8.1 Hz, 2H, 2 × benzene ring), 5.36 (s, 2H, CH ₂ -C ₆ H ₄ CO ₂ CH ₃ ), 4.33 (t, J = 4.7 Hz, 2H, CH ₂ -O), 3.85 (s, 3H,CH ₃ -O),3.61(t,J=4.7Hz,2H,CH ₂ -O), 3.29(s,3H,CH ₃ -O). ¹³ C NMR(101MHz,DMSO-d ₆ )δ( ppm): 166.34 (C=O), 161.86 (C ₂ ), 156.20 (C ₆ ), 152.92 (C ₄ ), 141.78, 130.13, 129.51, 127.82 (benzene), 124.29 (C ₈ ), 115.87 (C ₅ ) , 70.68(O- C H ₂ -), 66.02(- C H ₂ -O), 58.56(-O C H ₃ ), 52.66(-O C H ₃ ), 46.59( C H ₂ -C ₆ H ₄ COOH ). HRMS calculated value C ₁₇ H ₁₉ BrN ₅ O ₄ ⁺ [M+H] ⁺ : 436.0615, measured value: 436.0619.

Synthesis of compound 5 (defined as TLR7a): Compound 4 (0.5g) was added to 20ml of 6M NaOH:MeOH=4:1 (v/v), and the mixture was stirred and refluxed at 100°C for 4h. Then add 1mol/L hydrochloric acid to adjust to pH=7, concentrate and wash with water, dissolve the solid with methanol, and purify the reaction mixture by column chromatography to obtain compound 5. ¹ H NMR (400MHz, DMSO-d ₆ ) δ (ppm): 10.09 (s, 1H, CO ₂ H), 7.90 (d, J = 7.9 Hz, 2H, 2 × benzene ring), 7.37 (d, J = 7.9Hz, 2H, 2×benzene ring), 6.53 (s, 2H, NH ₂ ), 4.92 (s, 2H, CH ₂ -C ₆ H ₄ CO ₂ H), 4.24 (t, J = 4.7 Hz, 2H, CH ₂ -O),3.56(t,J=4.8Hz,2H,CH ₂ -O), 3.25(s,3H,CH ₃ -O). ¹³ C NMR(101MHz,DMSO-d ₆ )δ(ppm) :167.58(C=O), 160.35(C ₂ ), 152.71(C ₆ ), 149.59(C ₄ ), 148.29(C ₈ ), 142.48, 130.41, 130.07, 127.90 (benzene), 98.86(C ₅ ), 70.67 (O- C H ₂ -), 65.79(- C H ₂ -O), 58.52(-O C H ₃ ), 42.64( C H ₂ -C ₆ H ₄ COOH). HRMS calculated value C ₁₆ H ₁₉ N ₅ O ₅ ⁺ [M+H] ⁺ : 360.1302. Found: 360.1304.

Example 2: Preparation of coupling of TLR7 agonist and carrier protein BSA

Synthesis of compound 14: Under the protection of argon, dissolve compound 5 (0.03mmol) and EDCI (0.09mmol) in DMF (1mL), and finally add NHS (N-hydroxysuccinimide) (0.09mmol), 25 Stir for 3h at °C; then spin-dry the resulting mixture with an oil pump to obtain compound 13.

BSA (0.3μmol, purchased from Wuhan Chucheng Zhengmao Technology Engineering Co., Ltd., brand CC1050003) was dissolved in PBS (2mL), compound 13 (0.006mmol) was added to DMF to dissolve, the two solutions were mixed and placed on a shaker at 25°C Reaction for 48h. The obtained compound 14 (defined as TLR7a-BSA) was purified by centrifugal filtration using an ultrafiltration tube (Millipore UFC910096 15M, 10KD) and lyophilized. Using MALDI-TOF-MS test, the average number of TLR7a and BSA covalently linked is 6-7 after analysis.

Example 3: Preparation of antigen MUC1

Compound 8 was synthesized by manual solid phase, and the operation steps are as follows:

(1) Resin activation of Rink Amide AM (purchased from Gill Biochemical (Shanghai) Co., Ltd.)

Weigh 306 mg (0.2 mmol) of Rink Amide AM resin into a solid phase synthesis reaction tube, and use dry dichloromethane (DCM) (4 mL) to swell for 30 min under the protection of nitrogen. Washing: use dry DCM (3×3mL) and dry (

Spherical molecular sieve dried) N,N-dimethylformamide (DMF) (3×3mL) was washed alternately and repeatedly.

(2) Deprotection of Fmoc group

Under nitrogen agitation, add a 20% piperidine/DMF solution (3 mL, 3×5 min) to the reaction tube, and wash: use DCM (3×3 mL) and DMF (3×3 mL) to alternately rinse the resin repeatedly.

(3) Kaiser-test

Kaiser-test judges whether the amino group is deprotected by observing the color development of the resin. The main components of the developer are ninhydrin, phenol and pyridine.

Dip several resin particles into a clean test tube with a clean medicine spoon, add two drops each of the color developer ninhydrin, phenol and pyridine, and then use a hot air gun at 120°C for 1 minute to observe the color of the resin particles. If it turns blue, it means that the amino group is exposed and the Fmoc protecting group is removed; if there is no obvious color change, it means that the Fmoc protecting group is not removed, that is, the amino group is not exposed.

(4) Coupling of amino acids

In a sample bottle (10 mL), weigh the amino acid Fmoc-AA-OH (3.0 equivalents), and add PyBOP (3.0 equivalents) to dry DMF solvent (2-3 mL) to completely dissolve the amino acids. Then add DIPEA (6.0 equivalent) and mix well, add it to the cleaned and swollen resin, and react at room temperature for about 2 hours under the protection of nitrogen.

(5) Kaiser-test: Add a color developer, heat with a hot air gun, and observe that there is no obvious color change, indicating that the reaction is complete.

(6) Repeat the steps of (1), (2), (3), (4), (5) until the amino acid connection is completed.

(7) Removal of acetyl group

Under nitrogen agitation, add hydrazine hydrate: DMF: methanol=1:1:1 (v/v/v, 3mL, 2×15min), washing: use DCM (3×3mL) and DMF (3×3mL) alternately and repeatedly wash Resin.

(8) Cut the peptide chain from the resin.

After the peptide chain is synthesized, _{completely deprotect it with TFA/TIPS/H 2} O (95:2.5:2.5, v/v/v) for two hours, spin dry with an oil pump, and precipitate with ether to obtain the crude product compound 8 as a white crude product solid .

(9) Analysis, identification and purification of crude products.

Peptide solid-phase synthesis was carried out with 306mg of Rinke Amide AM resin. After the peptide chain was completely synthesized, it was directly _{deprotected with TFA/TIPS/H 2} O (95:2.5:2.5, v/v/v). The reaction was carried out for 2h, and the oil pump was rotated. After drying and precipitation with ether, compound 8 was obtained. It was identified by HPLC, HRMS (EI) and NMR. HPLC chromatographic conditions: 5-90% D solution (CH ₃ CN) in E solution (H ₂ O+0.1% TFA), after 30 min, λ = 220 nm, retention time is 6.699 min. Compound 8 HRMS (EI data, calculated value C ₅₉ H ₉₈ N ₁₈ O ₂₃ , calculated value [M+H+Na] ²⁺ , calculated value m/z=725.3509. Observed value: 725.3215. ¹ H NMR (600MHz, D ₂ O)δ(ppm): 4.87(d,1H,H ₁ ), 4.64-4.63(m, 2H, D _α , R _α ), 4.59-4.57(m, 2H, A _α ), 4.54-4.43( m,6H,P _α ,S _α ,T _α ),4.29-4.27(m,1H,V _α ),4.11-4.09(m,4H,T _β ),4.05-4.02(m,1H,H ₃ ), 3.99-3.88 (m, 5H, G _α , H ₅ ), 3.86-3.63 (m, 12H, S _β , P _δ , H ₂ , H ₄ , H ₆ ), 3.50-3.23 (m, 2H, R _δ ) , 2.96–2.79(m,2H,D _β ), 2.34–2.29(m,3H,P _β1 ), 2.16–2.08(m,1H,V _β ), 2.05–2.01(m,6H,P _β2 ,Ac- NH),2.00-1.87(m,6H,P _γ ),1.73-1.71(m,4H,R _β ,R _γ ),1.42-1.33(m,6H,A _β ),1.27-1.23(m,6H, T _γ ), 0.99-0.97 (m, 6H, V _γ ). ¹³ C NMR (151MHz, D ₂ O) δ (ppm): 174.79,174.08,173.73,173.70,173.48,173.45,173.16,173.05,172.65,171.47 ,171.07,170.95,170.74,167.22,163.05 (15×C=O), 156.65 (R _ζ ), 98.52 (C ₁ ), 75.36 (C ₅ ), 71.32 (C ₄ ), 68.45 (C ₃ ), 67.96, 67.94(T _β ),66.96(C ₆ ),61.26(S _β ),60.97,60.65,60.27(P _α ),59.88(V _α ),59.56,58.87(T _α ),57.04(S _α ),55.15( C ₂ ), 51.13 (R _α ), 50.22 (D _α ), 49.59, 47.83, 47.74 (P _δ ), 47.68, 47.58 (A _α ), 41.99 (R _δ ), 40.40, 40.26 (G _α ), 36.30( D _β ),30.09(V _β ), 29.34 (R _β ), 29.22, 29.17, 27.28 (P _β ), 24.64 (R _γ ), 24.56, 24.50, 24.08 (P _γ ), 22.25 (Ac-NH), 18.68, 18.32 (T _γ ), 17.37 (V _γ ), 15.43, 15.09 (A _β ).

Example 4: Preparation of antigen MUC1 squaraine monoamide

Compound 10 was synthesized by artificial solid phase, and the operation steps are as follows:

(1) Deprotection of Fmoc group of resin 7

(2) Kaiser-test

(3) Coupling of amino acids

In a sample bottle (10 mL), weigh the amino acid Fmoc-Gly-OH (3.0 equivalents), and add PyBOP (3.0 equivalents) to dry DMF solvent (2-3 mL) to completely dissolve the amino acids. Then add DIPEA (6.0 equivalent) and mix well, add it to the cleaned and swollen resin, and react at room temperature for about 2 hours under the protection of nitrogen.

(4) Kaiser-test: add a color developer, heat with a hot air gun, and observe that there is no obvious color change, indicating that the reaction is complete.

(5) Repeat steps (1) and (2).

(6) Removal of acetyl group

(7) Diethyl squarate (6.0 equivalents) and DIPEA (6.0 equivalents) were added to dry DMF solvent (2-3 mL), mixed uniformly, and reacted at room temperature for about 2 hours under the protection of nitrogen.

(8) Cut the peptide chain from the resin.

After the peptide chain is synthesized, _{completely deprotect it with TFA/TIPS/H 2} O (95:2.5:2.5, v/v/v) for two hours, spin dry by oil pump, and precipitate with ether to obtain the crude product compound 10 as a white crude product solid .

(9) Analysis, identification and purification of crude products.

Peptide solid-phase synthesis was performed with 306 mg of Rinke Amide AM resin. After the peptide chain was completely synthesized, it was directly _{deprotected with TFA/TIPS/H 2} O (95:2.5:2.5, v/v/v) and reacted for two hours. The oil pump was spin-dried, and ether was precipitated to obtain compound 10. It is identified by HPLC, HRMS and NMR. HPLC chromatographic conditions: 5-90% D solution (CH ₃ CN) in E solution (H ₂ O+0.1% TFA), after 30 min, λ = 220 nm, retention time is 7.152 min. HRMS (EI) data of compound 10, calculated value C ₆₇ H ₁₀₅ N ₁₉ O ₂₇ , [M+2H] ²⁺ calculated value m/z=805.3803. Observed value: 805.3833. [M+H+Na] ²⁺ Calculated value m/z=816.3713. Observed value: 816.3729. ¹ H NMR (600MHz, CD ₄ O) δ (ppm): 4.81 (d, 1H, H ₁ ), 4.77-4.71 (m, 2H, D _α , R _α ), 4.63-4.56 (m, 2H, A _α ), 4.48--4.45(m,2H,-CH ₂ -), 4.43--4.34(m,6H,P _α ,S _α ,T _α ),4.23-4.21(m,2H,V _α ),4.17(m, 1H, T _β ), 4.12-4.08 (m, 1H, H ₃ ), 3.99-3.83 (m, 7H, G _α , H ₅ ), 3.81-3.60 (m, 12H, S _β , P _δ , H ₂ , H ₄ ,H ₆ ), 3.26–3.17(m,2H,R _δ ), 2.95–2.83(m,3H,D _β ), 2.27–2.20(m,3H,P _β1 ), 2.13–2.11(m,1H ,V _β ),2.07-1.99(m,6H,P _β2 ,Ac-NH),1.99-1.86(m,6H,P _γ ),1.70-1.67(m,4H,R _β ,R _γ ),1.47- 1.43(m,3H,CH ₃ -),1.42–1.33(m,6H,A _β ),1.26–1.18(m,6H,T _γ ),1.00–0.98(m,6H,V _γ ). ¹³ C NMR (151MHz, CD ₄ O)δ(ppm): 188.92,183.79,177.49,174.25,173.78,173.56,173.10,173.04,173.00,172.65,172.60,172.23,172.09,172.05,171.96,170.84,170.67,170.62,170.30( 18C=O), 160.71, 160.46 (C=C), 157.11 (R _ζ ), 98.56 (C ₁ ), 74.25 (C ₅ ), 71.62 (C ₄ ), 69.56 (C ₃ ), 69.50, 68.86 (T _β ), 68.70 (C ₆ ), 66.69 (-CH ₂ -), 61.48 (S _β ), 61.35, 60.72, 59.83 (P _α ), 56.91 (V _α ), 56.33, 56.19 (T _α ), 56.05 (S _α ), 55.91 (C ₂ ), 55.76 (R _α ), 55.31 (D _α ), 50.57, 50.57, 50.04 (P _δ ), 49.83, 48.15 (A _α ), 46.03 (R _δ ), 42.00, 41.84, 40.62 (G _α ), 34.71 (D _β ), 30.00 (V _β ), 29.11 (R _β ), 29.03, 28.96, 27.99 (P _β ), 24.76 (R _γ ), 24.70, 24.52, 24.42 (P _γ ), 22.05 (Ac-NH), 18.36, 18.17 (T _γ ), 15.99, 15.86 (V _γ ), 15.74, 15.30 (A _β ).

Example 5: Preparation of coupling of antigen MUC1 squaraine monoamide and carrier protein BSA

Synthesis of compound 12: Compound 10 (0.03 mmol) and BSA (0.06 μmol) were dissolved in 5 mL of 0.07M Na ₂ B ₄ O ₇ /0.035M KHCO ₃ buffer with pH=9.5. The reaction was carried out on a shaker at 25°C for 48 hours, and the obtained compound 12 (defined as BSA-MUC1) was purified by centrifugal filtration using an ultrafiltration tube (Millipore UFC910096 15M, 10KD) and lyophilized. Tested by MALDI-TOF-MS, the average number of covalent linkages between MUC1 and BSA is 9-11.

Example 6: Preparation of BSA-MUC1 conjugate and TLR7 agonist coupling, and the anti-tumor vaccine molecule is defined as TLR7a-BSA-MUC1.

Synthesis of compound 15: Dissolve BSA-MUC1 (compound 12) (0.12 μmol) in PBS (1 mL), add compound 13 (0.006 mmol) dissolved in DMF, and react on a shaker at 25° C. for 48 hours to obtain compound 15 ( TLR7a-BSA-MUC1) was purified by centrifugal filtration with an ultrafiltration tube (Millipore UFC910096 15M, 10KD) and lyophilized. Tested by MALDI-TOF-MS, the average number of covalent linkages between TLR7a and BSA-MUC1 is calculated to be 6 to 7.

Example 7: Preparation of Coating Antigen Biotin-MUC1

Compound 11 was synthesized by manual solid-phase synthesis, and the operation steps were as follows:

(1) Deprotection of Fmoc group of resin 9

(2) Kaiser-test

Dip several resin particles into a clean test tube with a clean medicine spoon, add two drops each of the color developer ninhydrin, phenol and pyridine, and then use a 150°C hot air gun to heat for 2 minutes to observe the color of the resin particles. If it turns blue, it means that the amino group is exposed and the Fmoc protecting group is removed; if there is no obvious color change, it means that the Fmoc protecting group is not removed, that is, the amino group is not exposed.

(3) Biotin coupling

In a sample bottle (10 mL), weigh the amino acid biotin (3.0 equivalents), HATU (2.0 equivalents) and HOAt (2.0 equivalents) into dry DMF solvent (2-3 mL) to completely dissolve the amino acids. Then add DIPEA (6.0 equivalent) and mix well, add it to the cleaned and swollen resin, and react at room temperature for about 2 hours under the protection of nitrogen.

(5) Removal of acetyl group

(6) Cut the peptide chain from the resin.

After the peptide chain is synthesized, _{completely deprotect it with TFA/TIPS/H 2} O (95:2.5:2.5, v/v/v) for two hours, spin dry by oil pump, and precipitate with ether to obtain the crude product compound 11 as a white crude product solid .

(7) Analysis, identification and purification of crude products.

Peptide solid-phase synthesis was carried out with 306mg of Rinke Amide AM resin. After the peptide chain was completely synthesized, it was directly _{deprotected with TFA/TIPS/H 2} O (95:2.5:2.5, v/v/v). The reaction was carried out for 2h, and the oil pump was rotated. After drying and precipitation with ether, compound 11 was obtained. Identification by HPLC, HRMS and NMR, HPLC chromatographic conditions: 5-60% D solution (CH ₃ CN) in E solution (H ₂ O + 0.1% TFA), after 20 min, λ = 220nm, retention time is 8.498 min. HRMS (EI) data of biotin-MUC1 glycopeptide 11: calculated value C ₇₁ H ₁₁₅ N ₂₁ O ₂₆ S, [M+H+Na] ²⁺ calculated value m/z=866.9004. Observed value: 866.8993. [M +2Na] ²⁺ calculated value m/z=877.8914, measured value: 877.8909. ¹ H NMR (600MHz, CD ₄ O) δ (ppm): 4.80 (d, 1H, H ₁ ), 4.77-4.74 (m, 2H ,H ₁₃ ,H ₁₄ ), 4.62-4.55(m,2H,D _α ,R _α ),4.51-4.41(m,6H,P _α ,S _α ,T _α ), 4.37-4.25(m,5H,V _α ,T _β ),4.21–4.19(m,1H,H ₃ ), 4.00–3.87(m,7H,G _α ,H ₅ ), 3.87–3.65(m,12H,S _β ,P _δ ,H ₂ , H ₄ ,H ₆ ), 3.65–3.60(m,1H,H ₁₃ ), 3.24–3.18(m,2H,R _δ ), 2.95–2.91(m,2H,D _β ), 2.87–2.69(m,2H ,H ₁₂ ), 2.32–2.29(m,2H,H ₇ ), 2.26–2.18(m,3H,P _β1 ), 2.12–2.10(m,V _β ), 2.05–1.87(m,12H,P _β2 , Ac-NH, P _γ ), 1.87-1.84 (m, 1H, H _10' ), 1.75-1.73 (m, 2H, H ₈ ), 1.70-1.67 (m, 4H, R _β , R _γ ), 1.48- 1.46(m,2H,H ₉ ),1.33--1.30(m,1H,H _10' ),1.25-1.20(m,6H,T _γ ),1.01-0.98(m,6H,V _γ ). ¹³ C NMR (151MHz, CD ₄ O)δ(ppm): 176.71,174.78,174.32,174.27,174.22,173.94,173.87,173.82,173.48,173.32,173.19,172.47,172.13,172.11,171.92,171.86,171.54,165.97 (18C= O), 158.34 (R _ζ ), 99.80 (C ₁ ), 75.48 (C ₅ ), 72.84 (C ₄ ), 70.08 (C ₃ ), 69.91, 67.88 (T _β ), 63.04 (C ₆ ), 62.56 (C ₁₃ ),61.94(S _β ),61.49,61 .19,61.05(P _α ),60.23(C ₁₄ ),58.14(V _α ),57.41,57.28(T _α ),57.13(S _α ),56.99(C ₁₁ ),56.76(C ₂ ),56.55(R _α ), 54.40 (D _α ), 51.80, 51.27, 51.05 (P _δ ), 49.77, 49.37 (A _α ), 43.61 (R _δ ), 43.46, 43.06, 41.85 (G _α ), 40.85 (C ₁₂ ), 36.20 (C ₇ ), 35.95 (D _β ), 31.17 (V _β ), 30.34, 30.26, 30.19 (P _β ), 29.43 (C ₉ ), 29.23 (C ₁₀ ), 26.35 (C ₈ ), 25.99 (R _γ ) , 25.94, 25.75, 25.65 (P _γ ), 23.28 (Ac-NH), 19.88, 19.59 (T _γ ), 18.73 (V _γ ), 16.58, 16.54 (A _β ).

Example 8: Immunization of mice

Purchase 35 female BALB/c mice aged 6 to 8 weeks (purchased from the Animal Experiment Center of Huazhong Agricultural University). The specific immunization time course is the first day, the 15th day and the 29th day, respectively, and the injection method is intraperitoneal injection. The blood collection method is tail-end blood collection. Blood collection time: Take blank blood before immunization, take blood 2 hours after the first immunization, and take blood 14 days after each immunization injection, of which the last time (day 42) is the orbital blood collection . After the blood was taken out, it was centrifuged in a centrifuge and the serum was taken out and stored at -80°C.

Table 1: The composition of each group of vaccines

Example 9: Determination of cytokines in vivo

(1) Antigen coating: add 100 μL of the diluted capture antibody (diluted 200 times with the coating solution) to each well, seal the 96-well plate with plastic wrap and incubate overnight in a refrigerator at 4°C after adding the solution.

(2) Washing: Take the 96-well plate out of the refrigerator and return to room temperature (25°C, the same below), shake off the coating liquid, and gently absorb the remaining liquid with absorbent paper. Add 200 μL of PBST solution to each well, shake off the washing solution, and blot dry with absorbent paper. Repeat 4 times.

(3) Filling protein: add 200 μL of 1% BSA in PBS solution (w/v) to each well, seal it with plastic wrap and place it at room temperature, shake for 1 hour, and wash as above.

(4) Add standard/serum:

a) Add standard diluted in concentration gradient according to the kit instructions.

b) Dilute the serum 20 times with the diluent, add 100 μL to each well, and set up two duplicate wells. Seal it with plastic wrap and shake it at room temperature for 2h. Same as above for washing.

(5) Add detection antibody: add 100 μL of diluted detection antibody to each well (dilute 200 times with diluent). After adding the liquid, seal it with plastic wrap and place it at room temperature and shake for 1 hour. washing.

(6) Add Avidin-HRP: add 100 μL of diluted Avidin-HRP to each well (diluted 1000 times with the diluent). After adding the liquid, seal it with plastic wrap and place it at room temperature and shake for 30 minutes.

(7) Wash each well by adding 200 μL of PBST solution and letting it stand for 30 minutes, then shake off the washing solution and blot it dry with absorbent paper. Repeat 5 times.

(8) Substrate color reaction: After mixing the color developer A and color reagent B in equal volumes, add 100 μL per well to a 96-well plate. Place the 96-well plate at room temperature and shake for 30 minutes in the dark. Finally, 100μL stop solution should be added to each well. Put the 96-well plate into the microplate reader immediately and measure the absorbance at 450nm. Draw a standard curve with the absorbance value of the standard, and substitute the absorbance value of the serum into the standard curve to obtain the cytokine content in the serum.

Figure 2 shows that the serum taken 2h after the first immunization was used for the determination of IFN-γ and IL-6. Each bar represents the average content of 5 mice. The serum of each mouse was repeated three times independently, and the value was taken for three times. The mean value, the error bar indicates the standard error of the mean (SEM). The significant difference is relative to the PBS group: **P<0.01; ****P<0.0001; ns, the significant difference is not obvious. Comparison between vaccines: **P<0.01; ****P<0.0001; ns, the significant difference is not obvious.

Example 10: Indirect non-competitive ELISA to determine the content of antibodies in the blood

(1) Coating: Coating antigen biotin-MUC1 and avidin (avidin) are mixed at a molar ratio of 4:1, diluted with pH=9.5 coating solution, antigen (MUC1) concentration is 0.1μg/mL, per well 100μL, placed in a refrigerator at 4°C overnight.

(2) Washing: Take the 96-well plate out of the refrigerator and return to room temperature, shake off the coating liquid, and absorb the remaining liquid with absorbent paper. Add 200 μL of PBST solution to each well, shake off the washing solution, and blot dry with absorbent paper. Repeat 3 times.

(3) Blocking: add 100 μL of 1% casein PBS buffer to each well (the concentration of the PBS buffer is a mass percentage, the same below), incubate in a 37°C incubator for 1 hour, and wash the same as above.

(4) Antigen and antibody specific binding: Dilute the serum with 0.1% casein PBS buffer, the dilution factor is 200, 400, 800, 1600, 3200, 64000, 128000, 256000, 512000, 1024000, 2048000, 4096000, 8192000, 16384000. Add 100μL of diluent to each well, set blank and negative controls. Incubate at 37°C for 1h. Same as above for washing.

(5) Add secondary antibody: Dissolve goat anti-mouse IgG-HRP in PBST buffer, dilute to 5000 times with PBST buffer, mix well, add 100 μL of diluent to each well, and incubate at 37°C for 1 hour. The operation of adding secondary antibodies (IgM-HRP, IgG2a-HRP, IgG2b-HRP, IgG1-HRP, IgG3-HRP, IgA-HRP, IgE-HRP) is the same. Same as above for washing.

(6) Color development: add 100μL color development solution to each well (one board needs substrate buffer 9.5mL+0.5mL F solution (2mg/mL TMB/absolute ethanol) + 32uLG solution (35% hydrogen peroxide urea/water solution) ), shake and mix well, store at room temperature in the dark for 5 minutes.

(7) Stop: add 50μL stop solution (2M sulfuric acid) to each well, put it in the microplate reader and mix well and read, and measure the absorbance value of each well at 450nm wavelength.

Figure 3 is an evaluation of the anti-MUC1 titer of the vaccine produced by the serum obtained after the third immunization (IgG antibody titer test result). Each bar represents the average titer of 5 mice, and each value is repeated three times independently. The error bars indicate the standard error of the mean. The significant difference is relative to the PBS group: **P<0.01; ***P<0.001; ns, the significant difference is not obvious. Comparison between vaccines: **P<0.01; ***P<0.001; ns, the significant difference is not obvious.

Figure 4 shows the evaluation of the anti-MUC1 IgG titer produced by the vaccine for the serum obtained after the first, second, and third immunizations. Each bar represents the average titer of 5 mice, and each value is repeated three times independently. The error bars indicate the standard error of the mean.

Figure 5 shows the evaluation of the anti-MUC1 IgM titers produced by the vaccine for the sera obtained after the first, second, and third immunizations. Each bar represents the average titer of 5 mice, and each value is repeated three times independently. The error bars indicate the standard error of the mean.

Figure 6 shows the evaluation of the anti-MUC1 antibody subtypes produced by the vaccine for the serum obtained after the third immunization. Each bar represents the average titer of 5 mice, and each value is repeated three times independently. The error bars indicate the standard error of the mean. Significant difference relative to PBS group: ns, significant difference is not obvious; ****P<0.0001.

Example 11: Indirect non-competitive ELISA to determine the content of antibodies in blood

(1) Coating: Coating antigen BSA, dilute with pH=9.5 coating solution, antigen (BSA) concentration of 1.16μg/mL, 100μL per well, and place in a refrigerator at 4°C overnight.

(3) Blocking: add 100 μL of 1% casein PBS buffer to each well, incubate at 37°C for 1 hour, and wash as above.

(5) Add secondary antibody: Dissolve goat anti-mouse IgG-HRP in PBST buffer, dilute to 4000 times with PBST buffer, mix well, add 100 μL of diluent to each well, and incubate at 37°C for 1 hour. Same as above for washing.

(6) Color development: add 100μL color development solution to each well (one board needs substrate buffer 9.5mL+0.5mL F solution (2mg/mL TMB/absolute ethanol) + 32uL G solution (35% hydrogen peroxide) Urea/water solution)), shake and mix well, and store at room temperature in the dark for 5 minutes.

Figure 7 shows the evaluation of the anti-BSA titer produced by the vaccine for the serum obtained after the third immunization, coated with BSA. Each bar represents the average titer of 5 mice, and each value is repeated three times independently. The error bars indicate the standard error of the mean. Significant difference relative to the PBS group: *P<0.05; **P<0.01; ****P<0.0001. Comparison between vaccines: *P<0.05; ****P<0.0001; ns, the significant difference is not obvious.

Example 12: Determination of MCF-7 cell viability by MTT method

This example aims to explore whether antibodies can mediate complement lysis by activating CDC.

(1) Digest the MCF-7 cells with trypsin, add 10% by volume of FBS/DMEM and pipette evenly, and transfer them to a 96-well cell culture plate (8000 cells per well).

(2) Then wash with PBS 3 times, and add 1:50 diluted mouse serum 1% BSA/PBS (50 μL/well). Continue to place in the incubator for 2h.

(3) Then add rabbit complement (1:50 dilution) 1% BSA/PBS (RC stands for rabbit complement; HIRC stands for high temperature inactivated rabbit complement) (50 μL/well) and incubate for 4 hours.

(4) Prepare 0.5% MTT/PBS solution and add (20μL/well) and incubate at 37°C for 2h.

(5) Finally, aspirate the supernatant, add DMSO (150 μL/well) and mix it by pipetting several times, and measure the absorbance at a wavelength of 490nm. The cell viability determination formula of MCF-7 cells is as follows:

Cell survival rate (%)=(experiment/control)×100%

Figure 8 shows the MTT experiment to evaluate the survival rate of MCF-7 cells for the serum obtained after the third immunization. Each bar represents the average value of 5 mice, and each value is repeated three times independently. The error bars indicate the standard error of the average. Significant difference relative to the PBS group: *P<0.05; ***P<0.001, ****P<0.0001, ns, the significant difference is not obvious. Comparison between vaccines: *P<0.05; ****P<0.0001.

Example 13: Determination of antibody binding to MCF-7 cells by flow cytometry (FACS)

Detect the binding of antibodies induced by candidate vaccines to MCF-7 cells, stain the cells with fluorescent secondary antibodies, and then perform flow cytometry (FACS) analysis.

(1) Digest MCF-7 cells with trypsin, then add culture medium and transfer them to different conical centrifuge tubes (1×10 ⁶ cells/tube), centrifuge at 1500g for 2 minutes, and aspirate the supernatant.

(2) Add washing solution (1% BSA/PBS) (300 μL/tube), centrifuge to make the cells settle at the bottom of the centrifuge tube, aspirate the supernatant, and repeat 3 times. The mouse serum sample (mixed blood of 5 mice) was diluted 50 times with FACS solution (250 μL/tube), and incubated at 0°C for 60 min.

(3) Centrifuge and aspirate the supernatant. Add washing solution (300 μL/tube), centrifuge to make the cells settle at the bottom of the centrifuge tube, aspirate the supernatant, and repeat 3 times. The fluorescently labeled secondary antibody was diluted 50 times with FACS solution (100 μL/tube), and incubated at 0°C for 30 min.

(4) Centrifuge and aspirate the supernatant. Add washing solution (300 μL/tube), centrifuge to make the cells settle at the bottom of the centrifuge tube, aspirate the supernatant, and repeat 3 times. Finally, each tube was dissolved with 250μL FACS solution and mixed well.

(5) Measure the fluorescence intensity by flow cytometry.

Figure 9 shows the flow cytometry (FACS) analysis of the binding of antiserum from immunized mice to MCF-7 cancer cells. Take the PBS group (black) as the control. These images are representative of five independent experiments.

Example 14: Determination of the binding of antibodies to MCF-7 cells by confocal microscopy

MCF-7 cells were stained with mouse serum to determine their potential to recognize MUC1 targets.

(1) First, digest MCF-7 cells with trypsin and add the culture medium, then transfer them to a confocal small dish (1×10 ⁶ cells/dish), and put them in a cell incubator for 12 hours.

(2) Aspirate the medium, wash with 1% BSA/PBS solution 3 times, and then use 1% BSA/PBS solution to dilute the mouse serum 50 times (500 μL/dish) and incubate at 0°C for 1 h.

(3) Aspirate the supernatant, wash 3 times with 1% BSA/PBS solution, dilute the fluorescently labeled secondary antibody with 1% BSA/PBS solution 50 times (500 μL/dish) and incubate at 0°C for 30 min.

(4) Wash 3 times with 1% BSA/PBS solution, then wash 2 times with PBS, and finally add 500μL PBS. Observe and take pictures of MCF-7 cells with a confocal microscope (Leica TCS SP8, Wetzlar, Germany) 63 times oil lens .

Example 15: CTL experiment

(1) BALB/c mice (n=5) in each group were injected with candidate vaccine subcutaneously on day 1, day 15 and day 29 respectively, and immunized 3 times. 14 days after the third immunization, the mouse spleen was taken to make a single cell suspension, which was used as an effector cell for CTL detection.

(2) Add freshly isolated splenocytes (1×10 ⁶ cells/well) to RPMI-1640 and incubate with MCF-7 cells (1×10 ⁶ cells/well) for 12 hours. Lactate dehydrogenase (LDH) is then used to detect the cytotoxicity of effector cells to target cells.

(3) 1 hour before the predetermined detection time point, take out the cell culture plate from the cell incubator, add LDH release agent (10% of the original culture fluid volume) to the "sample maximum enzyme activity control well", and add LDH release After mixing, pipetting repeatedly to mix evenly, and then continue to incubate for 1h in the incubator.

(4) After reaching the predetermined time, centrifuge the cell culture plate with a multi-well centrifuge 250g centrifuge for 4 minutes. Then draw 120μL of supernatant from each well to another 96-well microtiter plate, and add LDH detection working solution (60μL/well). The 96-well plate was incubated at room temperature in the dark for 30 minutes, and the absorbance was measured at a wavelength of 490nm. At the same time, MCF-7 cells and splenocytes were incubated separately to determine the spontaneous LDH release value. MCF-7 cells were incubated in RPMI-1640 without FBS lysate, and the maximum release value of LDH was determined. The cell lysis rate is calculated as follows:

Cytotoxicity (%) = (experimental group-spontaneous target cell/absorbance of cell maximum enzyme activity-spontaneous target cell) × 100%

Figure 10 shows the CTL experiment to evaluate the in vitro cytotoxicity of spleen cells to MCF-7 cells after the third immunization. Each bar represents the average of 5 mice, and the error bars indicate the standard error of the average. Comparison between vaccines: *P<0.05; **P<0.01.

It is known from the above vaccine immunity test results that the anti-tumor vaccine molecules provided by the present invention produce significantly high-affinity IgG antibodies against tumor-associated antigens or specific antigens. The antibodies produced can recognize cancer cells and initiate the lysis of the recognized cancer cells by activating the complement-dependent cytotoxicity (CDC) of rabbit serum. These results indicate that the design of the embedded adjuvant protein conjugate strategy provided by the present invention is an effective strategy for designing highly effective immunotherapy anti-cancer vaccines.

The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the scope of the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, including the combination of various technical features in any other suitable manner. These simple modifications and combinations should also be regarded as the disclosed content of the present invention. All belong to the protection scope of the present invention.

Claims

An anti-tumor vaccine molecule having a structure represented by formula (I), characterized in that, in the formula (I), A is an adjuvant, B is an antigen, and m of the A are each passed through at least one covalent The linking arm is covalently connected to the protein, and n of the B are covalently linked to the protein through at least one covalent linking arm, and the number of amino acid molecules in the protein is greater than or equal to 100; the formula (I) Where m is an integer greater than or equal to 1, and n is an integer greater than or equal to 1;

A m -protein-B n formula (I);

Preferably, m in the formula (I) is 1, and n is an integer greater than or equal to 2;

Preferably, m in the formula (I) is an integer greater than or equal to 2, and n is an integer greater than or equal to 2.
The anti-tumor vaccine molecule according to claim 1, wherein the antigen includes at least one of a tumor-associated antigen, a tumor-specific antigen, a pathogen antigen, a biotoxin, and a biomolecular antigen;

Preferably, the tumor-associated antigen is selected from at least one of a tumor-associated polypeptide antigen, a tumor-associated glycopeptide antigen, and a tumor-associated carbohydrate antigen.
The anti-tumor vaccine molecule according to claim 2, wherein the tumor-associated polypeptide antigen or the tumor-associated glycopeptide antigen contains selected from MUC1, MUC16, NY-ESO-1, MAGE-A1/3/4, At least one of WT1, STAT3, HER2, and GP100.
The anti-tumor vaccine molecule according to claim 2, wherein the antigen contains MUC1, preferably, the MUC1 antigen is selected from at least one of the following structures:

Wherein, in the structure of the antigen containing MUC1, each of R 1 , R 2 , R 3 , R 4 and R 5 is independently selected from hydrogen and the sugar structure shown below:
The anti-tumor vaccine molecule according to claim 2, wherein the tumor-associated carbohydrate antigen is selected from at least one of the following structures:
The anti-tumor vaccine molecule according to any one of claims 1-5, wherein the protein is selected from the group consisting of bovine serum albumin, chicken ovalbumin, keyhole hemocyanin, tetanus toxoid, diphtheria toxoid, influenza At least one of haemophilus D protein, group B meningococcal outer membrane protein complex, pertussis toxoid, typhoid flagella, pneumolysin, and non-toxic diphtheria toxin mutant.
The anti-tumor vaccine molecule according to any one of claims 1-6, wherein the adjuvant is a pattern recognition receptor agonist;

Preferably, the pattern recognition receptor agonist is selected from at least one of Toll-like receptor agonists and NKT agonists;

Preferably, the Toll-like receptor agonist is selected from at least one of TLR7 agonist, TLR8 agonist, TLR9 agonist, TLR3 agonist, TLR2 agonist and TLR4 agonist.
The anti-tumor vaccine molecule according to any one of claims 1-5, wherein the structure of each of the covalent link arms is independently selected from the following structures:

-CO-, -O-CO-, -NH-CO-, -NH(C=NH)-, -SO 2 -, -O-SO 2 -, -NH-, -NH-CO-CH 2 -, -CH 2 -, -C 2 H 4 -, -C 3 H 6 -, -C 4 H 8 -, -C 5 H 10 -, -C 6 H 12 -, -C 7 H 14 -, -C 8 H 16 -, -C 9 H 18 -, -C 10 H 20 -, -CH(CH 3 )-, -C[(CH 3 ) 2 ]-, -CH 2 -CH(CH 3 )-, -CH (CH 3 )-CH 2 -, -CH(CH 3 )-C 2 H 4 -, -CH 2 -CH(CH 3 )-CH 2 -, -C 2 H 4 -CH(CH 3 )-,- CH 2 -C[(CH 3 ) 2 ]-, -C[(CH 3 ) 2 ]-CH 2 -, -CH(CH 3 )-CH(CH 3 )-, -C[(C 2 H 5 ) (CH 3 ))-, -CH(C 3 H 7 )-, -(CH 2 -CH 2 -O) p -CH 2 -CH 2 -, -CO-CH 2 -, -CO-C 2 H 4 -, -CO-C 3 H 6 -, -CO-C 4 H 8 -, -CO-C 5 H 10 -, -CO-C 6 H 12 -, -CO-C 7 H 14 -, -CO- C 8 H 16 -, -CO-C 9 H 18 -, -CO-C 10 H 20 -, -CO-CH(CH 3 )-, -CO-C[(CH 3 ) 2 ]-, -CO- CH 2 -CH(CH 3 )-, -CO-CH(CH 3 )-CH 2 -, -CO-CH(CH 3 )-C 2 H 4 -, -CO-CH 2 -CH(CH 3 )- CH 2 -, -CO-C 2 H 4 -CH(CH 3 )-, -CO-CH 2 -C[(CH 3 ) 2 ]-, -CO-C[(CH 3 ) 2 ]-CH 2- , -CO-CH(CH 3 )-CH(CH 3 )-, -CO-C[(C 2 H 5 )(CH 3 )]-, -CO-CH(C 3 H 7 )- and -CO- (CH 2 -CH 2 -O) p -CH 2 -CH 2 -;

Wherein, in the structure of the covalent link arm,

Each x is independently selected from an integer of 1-60;

Each Y is independently selected from at least one of -NH-, -O-, -S- and -S-S-;

Each p is independently selected from an integer of 1-60.
A method for preparing the anti-tumor vaccine molecule of any one of claims 1-8, characterized in that the method comprises:

Perform a first coupling between the antigen and the protein, and perform a second coupling between the obtained first intermediate and the adjuvant; or

The adjuvant is coupled to the protein for the third time, and the obtained second intermediate is coupled to the antigen for the fourth time.
The use of the anti-tumor vaccine molecule of any one of claims 1-8 in an anti-tumor vaccine.