CN114306586A - Tumor vaccine based on TF antigen and chitosan oligosaccharide endogenous adjuvant, method and application - Google Patents
Tumor vaccine based on TF antigen and chitosan oligosaccharide endogenous adjuvant, method and application Download PDFInfo
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Abstract
The invention discloses a tumor vaccine prepared by covalently coupling TF antigen and chitosan oligosaccharide endogenous adjuvant with CRM197 carrier protein, wherein the structural formula of the synthetic tumor vaccine is as follows:
the result of an immune experiment shows that the coupled chitosan oligosaccharide adjuvant group (TF-CRM197-COS) can obviously produce IgG antibody with higher titer, and the value is 25600. Compared with a simple physical mixed antigen and adjuvant group, the TF-CRM197-COS group can more effectively stimulate the T cell immunity of mice, and the humoral immunity titer is lower.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and relates to preparation and immunological evaluation of a tumor vaccine based on Thomsen-Friedenreich antigen and a chitosan oligosaccharide adjuvant, in particular to a tumor vaccine based on TF (Thomsen-Friedenreich) antigen and a chitosan oligosaccharide endogenous adjuvant, a method and application thereof.
Background
Tumor vaccines have become an ideal method for treating tumors, and anti-tumor vaccines are designed and synthesized according to Tumor Associated Carbohydrate Antigens (TACAs), which are over-expressed on the surface of tumor cells, and are used for stimulating the immune system of a human body to generate immune response, so that the growth, reproduction and diffusion of tumors are inhibited, and the tumors are finally killed. Thomsen-Friedenreich (TF) antigen is one of the most important tumor targets, and is overexpressed in colon, breast, bladder and stomach cancers. The TF antigen is a hapten, the basic structure of the TF antigen is Gal beta 1,3GalNAc alpha Ser/Thr, and the TF antigen is the I-type core structure of a mucin O-oligosaccharide chain; the precursor of the TF antigen is the Tn antigen (GalNAc. alpha. Ser/Thr). However, native carbohydrate antigens do not directly elicit cellular immunity that can produce high affinity IgG antibodies. In order to solve the defects of the saccharide vaccine, the saccharide antigen is coupled with carrier protein to prepare a whole antigen, and an immune adjuvant is added to prepare the effective vaccine.
In the case of carbohydrate immune adjuvants, researches in recent years show that polysaccharides have the function of immune adjuvants, can enhance the immune effect of vaccines, and can promote organisms to generate specific immunity and non-specific immunity. Therefore, research on the degradation of polysaccharides to prepare functional oligosaccharides for use as vaccine adjuvants has also been carried out. Chitosan Oligosaccharides (COS), namely chitosan oligosaccharides and oligochitosan, are oligosaccharide products with the polymerization degree of 2-20, which are obtained by degrading chitosan through a special biological enzyme technology (or a chemical degradation technology and a microwave degradation technology), have the molecular weight of less than or equal to 3200Da, good water solubility and good biocompatibility, are wide in source, purely natural and easy to absorb, and are more worthy of attention, the chitosan oligosaccharides can effectively activate the cellular immunity and the humoral immunity of organisms, and the chitosan oligosaccharides and the oligochitosan have good application prospects when being developed into vaccine adjuvants. It is reported that Liu and the like use chitosan oligosaccharide as an adjuvant of formalin inactivated vibrio anguillarum vaccine, can obviously promote humoral immunity of organisms, effectively inhibit invasion of pathogenic bacteria, and further define the activity of chitosan oligosaccharide as an adjuvant (Fish & shellfish immunology,2015,47: 855) 860).
Currently, the most common method of use of adjuvants is by direct physical mixing with the antigen. Simple physical mixing, while somewhat immunologically active, is not necessarily the best way to exert its adjuvant activity. The literature reports that the coexistence of antigens and adjuvants in a single delivery system can enhance immunogenicity, such as flagellin, CpG, and LPS, etc. (Bioconjugate Chem,2007,18: 77-83; Vaccine,2009,27: 3013-3021). Studies have shown that T cells are activated only when Antigen Presenting Cells (APC) present certain amounts of antigen and adjuvant, and that only one is presented and not T cells (Nature,2006,440: 808-163812; Vaccine,2008,26: 1626-1637). Studies by Slurter et al have found that chemically coupling an antigen to an adjuvant into a whole APC can present more antigen and thus activate the T cell immune response. (Journal of controlled Release,2010,143: 207-. Therefore, the defect that the physical mixed antigen and the adjuvant cannot present APC cells simultaneously exists in the synthetic vaccine technology, and in order to solve the problem, the TF carbohydrate antigen and the carrier protein are covalently coupled and the chitosan oligosaccharide is introduced as the immunologic adjuvant through covalent coupling, so that the novel tumor vaccine based on the Thomsen-Friedenreich antigen and the endogenous chitosan oligosaccharide adjuvant is designed and synthesized.
Through detection, no patent publication related to the present patent application has been found.
Disclosure of Invention
The invention mainly aims to overcome the defects in the prior art, provides a tumor vaccine, a method and application of the tumor vaccine, wherein TF antigen and chitosan oligosaccharide are used as adjuvants to be covalently coupled with diphtheria toxin mutant CRM197, and immunological evaluation is carried out on the tumor vaccine.
In order to achieve the purpose, the invention adopts the following technical scheme:
a tumor vaccine prepared by covalently coupling a TF antigen and a chitosan oligosaccharide endogenous adjuvant with CRM197 carrier protein, wherein the structural formula of the tumor vaccine is as follows:
furthermore, the vaccine is synthesized by a chemical method to obtain TF-adipic acid succinimide ester, the TF-adipic acid succinimide ester is combined with diphtheria toxin mutant CRM197 carrier protein to obtain a glycoprotein conjugate, and the conjugate is coupled with a thiolated chitosan oligosaccharide vaccine adjuvant through a heterobifunctional connecting arm.
Further, the sulfhydrylation agent in the sulfhydrylation chitosan oligosaccharide vaccine adjuvant is 2-iminosulfane hydrochloride, thioglycolic acid or 3- (2-pyridinedimercapto) propionic acid N-hydroxysuccinimide ester; or the heterobifunctional linking arm is 3-benzoylmaleimide-N-hydroxysuccinimide, 3-maleimidopropionic acid hydroxysuccinimide ester or 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid succinimide ester.
Further, the weight ratio of the thiolated chitosan oligosaccharide vaccine adjuvant to the glycoprotein conjugate is 0.3: 1-0.6: 1.
Further, the vaccine is an anti-tumor vaccine.
A method for preparing the tumor vaccine comprises the following steps:
step one, synthesizing to obtain TF-adipic acid succinimide ester by a chemical method;
combining TF-adipic acid succinimide ester with diphtheria toxin mutant CRM197 carrier protein at a molar ratio of 15: 1-20: 1 to obtain a glycoprotein conjugate;
performing sulfhydrylation derivatization on the chitosan oligosaccharide adjuvant to obtain a sulfhydrylation chitosan oligosaccharide vaccine adjuvant;
step four, reacting the heterotype bifunctional connecting arm with the conjugate prepared in the step two in a molar ratio of 8: 1-10: 1 to obtain an intermediate;
reacting the thiolated chitosan oligosaccharide vaccine adjuvant obtained in the step three with the intermediate obtained in the step four, wherein the weight ratio of the thiolated chitosan oligosaccharide vaccine adjuvant to the intermediate is 0.3: 1-0.6: 1, and obtaining a target conjugate;
and step six, performing ultrafiltration and centrifugation on the conjugate obtained in the step five, and performing freeze-drying operation to obtain the target synthetic tumor vaccine.
Further, the specific steps are as follows:
step one, galactosamine hydrochloride is used as an initial raw material, 2-site azido, 1-site glycosylation and 4, 6-site benzaldehyde dimethyl acetal protection of a sugar ring are carried out to obtain a glycosyl acceptor, disaccharide glycosylation reaction is carried out on the glycosyl acceptor and a glucosinolate glycosyl donor to obtain TF disaccharide, then an azido reduction, deprotection and protection operation are carried out to obtain a TF antigen compound, the total yield of 7 steps of reaction is 12%, and the TF antigen and adipic acid bis-succinimide ester are reacted according to the molar ratio of 12: 1-15: 1 to obtain TF-adipic acid succinimide ester;
step two, combining TF-adipic acid succinimide ester with diphtheria toxin mutant CRM197 carrier protein, dissolving the combined protein and the carrier protein in a PBS buffer solution, wherein the molar ratio of the TF-adipic acid succinimide ester to the diphtheria toxin mutant CRM197 carrier protein is 15: 1-20: 1, and performing ultrafiltration, centrifugation and purification to obtain a glycoprotein conjugate;
performing sulfhydrylation derivatization on the chitosan oligosaccharide adjuvant to obtain a sulfhydrylation chitosan oligosaccharide vaccine adjuvant;
step four, dissolving the heterobifunctional connecting arm and the conjugate prepared in the step two in a PBS buffer solution, reacting at a molar ratio of 8: 1-10: 1, and performing ultrafiltration centrifugal purification to obtain an intermediate;
reacting the thiolated chitosan oligosaccharide vaccine adjuvant obtained in the step three with the intermediate obtained in the step four, wherein the weight ratio of the thiolated chitosan oligosaccharide vaccine adjuvant to the intermediate is 0.3: 1-0.6: 1, and obtaining a target conjugate;
step six, performing ultrafiltration and centrifugation on the conjugate obtained in the step five, and performing freeze-drying operation to obtain a target synthetic tumor vaccine;
wherein the PBS buffer is 0.01M and the pH is 8.0.
The tumor vaccine is applied to the preparation or the application as a tumor medicament.
The beneficial effects obtained by the invention are as follows:
1. the coupled chitosan oligosaccharide adjuvant group (TF-CRM197-COS) of the invention, namely the vaccine of the invention, can obviously produce IgG antibody with higher titer, and the value is 25600. Compared with a simple physical mixed antigen and adjuvant group, the TF-CRM197-COS group can more effectively stimulate the cellular immunity of mice and reduce the humoral immunity.
2. The coupled chitosan oligosaccharide adjuvant group (TF-CRM197-COS) of the invention, namely the vaccine of the invention, generates lower humoral immunity level, and the IgM antibody titer value is 228. In comparison, the titer of humoral immune IgM antibody produced by the physical antigen and adjuvant combination group (TF-CRM197/COS) was higher, with a value of 800.
3. The chitosan oligosaccharide adjuvant can enhance immune response, has good adjuvant effect, and can be used as an adjuvant of an attenuated vaccine, a protein vaccine, a nucleic acid vaccine or a polypeptide vaccine. The chitosan oligosaccharide can be produced in large scale, and the preparation method is mature and stable. The invention lays a foundation for the research of the action mechanism of the chitosan oligosaccharide adjuvant and provides a reference for finding the oligosaccharide with better adjuvant effect.
Drawings
FIG. 1 is a nuclear magnetic hydrogen spectrum of compound 5 in deuterated chloroform according to the invention;
FIG. 2 is a nuclear magnetic hydrogen spectrum of compound 7 in deuterated chloroform according to the invention;
FIG. 3 is a nuclear magnetic hydrogen spectrum of compound 9 in deuterated chloroform according to the invention;
FIG. 4 is a nuclear magnetic hydrogen spectrum of compound 11 in deuterated chloroform according to the invention;
FIG. 5 is a MALDI-TOF-MS mass spectrum of Compound 12 of the present invention;
FIG. 6 is a MALDI-TOF-MS mass spectrum of Compound 13 of the present invention;
FIG. 7 is a graph showing IgG antibody titer levels induced by vaccines of the present invention;
FIG. 8 is a graph showing IgM antibody titer levels induced by the vaccine of the present invention.
Detailed Description
The present invention will be further described in detail with reference to examples for better understanding, but the scope of the present invention is not limited to the examples.
The raw materials used in the invention are all conventional commercial products if not specified, the method used in the invention is all conventional in the field if not specified, and the mass of each substance used in the invention is all conventional use mass.
A tumor vaccine prepared by covalently coupling a TF antigen and a chitosan oligosaccharide endogenous adjuvant with CRM197 carrier protein, wherein the structural formula of the tumor vaccine is as follows:
preferably, the vaccine is synthesized by a chemical method to obtain TF-adipic acid succinimide ester, the TF-adipic acid succinimide ester is combined with diphtheria toxin mutant CRM197 carrier protein to obtain a glycoprotein conjugate, and the conjugate is coupled with a thiolated chitosan oligosaccharide vaccine adjuvant through a heterobifunctional connecting arm.
Preferably, the sulfhydrylation agent in the sulfhydrylation chitosan oligosaccharide vaccine adjuvant is 2-iminosulfane hydrochloride, thioglycolic acid or 3- (2-pyridinedimercapto) propionic acid N-hydroxysuccinimide ester; or the heterobifunctional linking arm is 3-benzoylmaleimide-N-hydroxysuccinimide, 3-maleimidopropionic acid hydroxysuccinimide ester or 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid succinimide ester.
Preferably, the weight ratio of the thiolated chitosan oligosaccharide vaccine adjuvant to the glycoprotein conjugate is 0.3: 1-0.6: 1.
Preferably, the vaccine is an anti-tumor vaccine.
A method for preparing the tumor vaccine comprises the following steps:
step one, synthesizing to obtain TF-adipic acid succinimide ester by a chemical method;
combining TF-adipic acid succinimide ester with diphtheria toxin mutant CRM197 carrier protein at a molar ratio of 15: 1-20: 1 to obtain a glycoprotein conjugate;
performing sulfhydrylation derivatization on the chitosan oligosaccharide adjuvant to obtain a sulfhydrylation chitosan oligosaccharide vaccine adjuvant;
step four, reacting the heterotype bifunctional connecting arm with the conjugate prepared in the step two in a molar ratio of 8: 1-10: 1 to obtain an intermediate;
reacting the thiolated chitosan oligosaccharide vaccine adjuvant obtained in the step three with the intermediate obtained in the step four, wherein the weight ratio of the thiolated chitosan oligosaccharide vaccine adjuvant to the intermediate is 0.3: 1-0.6: 1, and obtaining a target conjugate;
and step six, performing ultrafiltration and centrifugation on the conjugate obtained in the step five, and performing freeze-drying operation to obtain the target synthetic tumor vaccine.
Preferably, the specific steps are as follows:
step one, galactosamine hydrochloride is used as an initial raw material, 2-site azido, 1-site glycosylation and 4, 6-site benzaldehyde dimethyl acetal protection of a sugar ring are carried out to obtain a glycosyl acceptor, disaccharide glycosylation reaction is carried out on the glycosyl acceptor and a glucosinolate glycosyl donor to obtain TF disaccharide, then an azido reduction, deprotection and protection operation are carried out to obtain a TF antigen compound, the total yield of 7 steps of reaction is 12%, and the TF antigen and adipic acid bis-succinimide ester are reacted according to the molar ratio of 12: 1-15: 1 to obtain TF-adipic acid succinimide ester;
step two, combining TF-adipic acid succinimide ester with diphtheria toxin mutant CRM197 carrier protein, dissolving the combined protein and the carrier protein in a PBS buffer solution, wherein the molar ratio of the TF-adipic acid succinimide ester to the diphtheria toxin mutant CRM197 carrier protein is 15: 1-20: 1, and performing ultrafiltration, centrifugation and purification to obtain a glycoprotein conjugate;
performing sulfhydrylation derivatization on the chitosan oligosaccharide adjuvant to obtain a sulfhydrylation chitosan oligosaccharide vaccine adjuvant;
step four, dissolving the heterobifunctional connecting arm and the conjugate prepared in the step two in a PBS buffer solution, reacting at a molar ratio of 8: 1-10: 1, and performing ultrafiltration centrifugal purification to obtain an intermediate;
reacting the thiolated chitosan oligosaccharide vaccine adjuvant obtained in the step three with the intermediate obtained in the step four, wherein the weight ratio of the thiolated chitosan oligosaccharide vaccine adjuvant to the intermediate is 0.3: 1-0.6: 1, and obtaining a target conjugate;
step six, performing ultrafiltration and centrifugation on the conjugate obtained in the step five, and performing freeze-drying operation to obtain a target synthetic tumor vaccine;
wherein the PBS buffer is 0.01M and the pH is 8.0.
The tumor vaccine is applied to the preparation or the application as a tumor medicament.
Specifically, the preparation and detection are as follows:
example 1: chemical synthesis method of Thomsen-Friedenreich antigen active ester
1. Synthesis of Compound 5
Taking galactose amino hydrochloride 3(10g, 46.4mmol) to a 500mL round-bottom flask, adding 100mL methanol, and adding imidazole-1-sulfonyl azide hydrochloride (11.7g, 55.7mmol), K respectively at room temperature2CO3(14.7g,106.7mmol) and stirred vigorously at room temperature for 12 h. By TLC (EA: MeOH: H)2O: AcOH ═ 6: 2: 1: 0.5) monitor the completion of the reaction of the starting materials. The resulting mixture was concentrated under reduced pressure and purified by column chromatography on silica gel eluting with (EA: MeOH 4: 1) to give product 4(7.2g, 35.1mmol, 76%) as a white solid.
Taking compound 4(0.8g multiplied by 9, 3.90mmol) and putting in a 50mL round-bottom flask, respectively adding 7.5mL 3-chloro-1-propanol, under the protection of argon, cooling the reaction system to 0 ℃, starting dropwise adding acetyl chloride (459.1mg, 5.9mmol), returning to room temperature after the dropwise adding is completed and transferring to 70 ℃ under the protection of argon and stirring for 8 h. By TLC (EA: MeOH: H)2O: AcOH ═ 10: 2: 1: 0.5) monitor the completion of the reaction of the starting materials. The resulting mixture was concentrated under reduced pressure, purified by silica gel column chromatography, and eluted with (EA: PE ═ 2: 1) to give product 5(3.48g, 12.33mmol, 35%) as a white solid. The nuclear magnetic detection result of the compound 5 is shown in fig. 1, and the specific nuclear magnetic attribution result is as follows:1HNMR(400MHz,CDCl3)δ5.01(d,J＝3.2HZ,1H),4.17-4.16(m,1H),4.06-4.03(m,1H),3.99-3.97(m,2H),3.96-3.89(m,1H),3.87-3.85(m,1H),3.70-3.68(m,2H),3.67-3.58(m,1H),3.54(dd,J＝3.2HZ,J＝10.4HZ,1H),3.39(s,1H),2.17-1.99(m,2H).
2. synthesis of Compound 7
Taking compound 5(3.48g, 12.3mmol), adding 30mL of acetonitrile into a 100mL round-bottom flask respectively, cooling the reaction system to 0 ℃ under the protection of argon, starting dropwise adding benzaldehyde dimethyl acetal (3.76g, 5.85mmol), adding p-toluenesulfonic acid (1.06g, 6.18mmol) completely, and then returning to room temperature under the protection of argon and stirring for 12 h. By TLC (EA: MeOH: H)2O: AcOH ═ 10: 2: 1: 0.5) monitor additional benzaldehyde dimethyl acetal (1.88g, 12.35mmol) and p-toluenesulfonic acid (531.83mg, 3.09mmol) until the starting material is reacted completely. With Et3The reaction solution was adjusted to pH 7.0 with N, concentrated under reduced pressure, purified by silica gel column chromatography, and eluted with (PE: EA ═ 5: 1) to give product 6 as a white solid.
Collecting glycosyl donor compound 2(1.84g, 4.06mmol) and glycosyl acceptor 6(1g, 2.70mmol), and drying in 100mL container
In a flask with a mouth, 10mL of anhydrous dichloromethane is added under the protection of argon, the reaction system is stirred for 30 minutes under the protection of argon at room temperature, the reaction system is cooled to-15 ℃, N-iodosuccinimide (2.58g, 14.93mmol) and trifluoromethanesulfonic acid (373.35mg, 2.49mmol) are added, and after complete dropwise addition, the reaction system is returned to room temperature and stirred for 3 hours under the protection of argon. The reaction was completed by TLC (PE: EA ═ 1: 1). Et was added dropwise3The reaction mixture was adjusted to pH 7.0 with N, filtered under reduced pressure, concentrated and purified by silica gel column chromatography eluting with (PE: EA ═ 3: 1) to give product 7(2.87g, 4.10mmol, 82%) as a white solid. The nuclear magnetic detection result of the compound 7 is shown in fig. 2, and the specific nuclear magnetic attribution result is as follows:1HNMR(400MHz,CDCl3)δ7.54-7.52(m,2H),7.37-7.36(m,3H),5.55(s,1H),5.41-5.40(m,1H),5.29(t,J＝10HZ,1H),5.04-5.01(m,2H),4.78(d,J＝7.6HZ,1H),4.39(s,1H),4.28-4.19(m,2H),3.97-3.91(m,2H),3.85-3.81(m,1H),3.61-3.70(m,4H),2.16-2.05(m,12H),1.98(s,3H).
3. synthesis of Compound 9
Taking compound 7(2.87g, 4.10mmol), adding 15mL tetrahydrofuran into a 100mL single-neck screw bottle to completely dissolve the compound, adding 10mL acetic acid, 5mL acetic anhydride, adding 21g zinc powder into the system, and stirring for 12h at room temperature under a sealed condition. Detection by TLC (PE: EA ═ 1: 1), after completion of the reaction, the zinc powder was filtered through celite under reduced pressure, concentrated, and purified by silica gel column chromatography, eluting with (PE: EA ═ 3: 1) to give product 9 as a white solid.
Taking the compound 8(2.1g, 2.93mmol), adding 15mL of anhydrous acetonitrile into a 50mL single-mouth eggplant-shaped bottle under the protection of argon, cooling the reaction system to 0 ℃, starting to add tetrabutylammonium fluoride (5.56mL, 5.86mmol) and azidotrimethylsilane (675.7mg, 5.86mmol), completely dropwise adding, heating to 60 ℃, and stirring for 8 hours under the protection of argon. Detection was by TLC (PE: EA ═ 1: 1) and the reaction was complete. Concentrated under reduced pressure and purified by silica gel column chromatography eluting with (PE: EA ═ 3: 1) to give product 9(1.68g, 2.32mmol, 79%) as a white solid. The nuclear magnetic assay results of compound 9 are shown in fig. 3, and the specific nuclear magnetic assignment results are as follows:1HNMR(400MHz,CDCl3)δ7.52-7.51(m,2H),7.36-7.26(m,3H),5.73(d,J＝8.8HZ,1H),5.53(s,1H),5.36-5.35(m,1H),5.27(s,1H),5.19-5.15(m,1H),5.00-4.94(m,2H),4.74(d,J＝8.0HZ,1H),4.63(s,1H),4.29-4.22(m,2H),4.15-4.02(m,4H),3.96-3.90(m,2H),3.89-3.79(m,1H),3.62(s,1H),3.52-3.50(m,3H),3.42-3.35(m,3H),2.12-2.11(m,3H),2.05-2.00(m,6H),1.96-1.92(m,6H).
4. synthesis of compound 11Thomsen-Friedenreich antigen active ester
Taking compound 9(1.68g, 2.32mmol), adding 15mL of anhydrous methanol into a 50mL single-neck eggplant-shaped bottle under the protection of argon, cooling the reaction system to 0 ℃, starting to add sodium methoxide, adjusting the pH of the reaction system to 9.0, and stirring for 2 hours. TLC (EA: MeOH: H)2O: AcOH ═ 10: 1: 1: 0.5) monitoring, after the raw materials completely react, adding 1M hydrochloric acid for regulationWhen the reaction solution is neutral, the reaction solution is concentrated under reduced pressure to obtain a crude product which is directly used in the next step. Dissolving the crude compound in the previous step in 60% acetic acid water solution, stirring for 3H at 60 deg.C in oil bath, TLC (EA: MeOH: H)2O: AcOH ═ 6: 2: 1: 0.5) monitoring the reaction of the raw materials, concentrating under reduced pressure and pumping to obtain a crude compound which is directly used in the next step.
Dissolving the crude compound in the last step into 20mL of methanol, adding a proper amount of Pd/C, and stirring for 2h at room temperature under the condition of hydrogen. By (EA: MeOH: H)2O: AcOH ═ 4: 2: 1: 0.5) detecting, and finishing the reaction. Celite was filtered, concentrated under reduced pressure, and purified by gel column chromatography eluting with MeOH to give product 10 as a white solid.
Weighing TF disaccharide Gal beta 1-3GalNAc alpha ProNH2(10mg, 22.7. mu. mol), 2.4mL of N, N-dimethylformamide was added to a 10mL single-neck eggplant-shaped bottle, the reaction system was cooled to 0 ℃ and bis-succinimidyl adipate (105mg, 340.6. mu. mol) was added to the bottle, and PBS buffer (0.1M, 0.6mL, pH 8.0) was added thereto, wherein the volume ratio of N, N-dimethylformamide to PBS was 4: 1, stirring the mixture at room temperature for 24 hours under the protection of argon. By (EA: MeOH: H)2O: AcOH ═ 2: 2: 1: 0.5) detection of the developing agent, and after the reaction is finished. The solvent was evaporated under reduced pressure, the crude compound was washed 10 times with ethyl acetate, and the purified activated TF sugar chain white solid 11(12mg, 18.47umol, 81.36%) was evaporated under reduced pressure. The nuclear magnetic assay results of compound 11 are shown in fig. 4, and the specific nuclear magnetic assignment results are as follows:1HNMR(400MHz,CDCl3)δ4.85(d,J＝3.6HZ,1H),4.47(d,J＝7.4HZ,1H),4.34-4.31(m,1H),4.23(d,J＝2.4HZ,1H),4.05-3.96(m,2H),3.91(d,J＝3.2HZ,1H),3.80-3.74(m,5H),3.71(s,3H),3.67-3.61(m,3H),3.54-3.45(m,2H),3.39-3.24(m,2H),2.43-2.39(m,2H),2.28-2.24(m,2H),2.03(s,3H),1.84-1.81(m,2H),1.61-1.59(m,4H).
example 2: ligation of TF active esters to Carrier proteins
TF-adipimidate 11 and protein CRM197 or BSA were mixed at room temperature according to the sugar chain: protein 20:1 (molar mass ratio) was mixed in PBS buffer (0.1M, 2.5mL, pH 8.0) and stirred at 100rpm at 30 ℃ for 72 hours. Thereafter, the solution was diluted to 10mL with distilled water, purified by ultrafiltration using an ultrafiltration centrifuge (30KD, Amicon Ultra, Millipore), centrifuged at 3000rpm/min at 4 ℃ for 30 min/time for several times to remove excess sugar chains, and unreacted sugar chains were recovered. And (3) collecting glycoprotein remained in the membrane after ultrafiltration and centrifugation, concentrating the glycoprotein into ultrapure water of which the volume is less than or equal to 1mL, uniformly mixing, and freeze-drying the solution to obtain white fluffy powder, namely the target glycoprotein conjugate 12. The average molecular weight was determined by MALDI-TOF-MS (as shown in FIG. 5).
Example 3: preparation of chitosan oligosaccharide adjuvant
1. Thiolated derivatization of chitooligosaccharide COS
COS and 2-iminosulfane hydrochloride (IT) were weighed in the required amounts, respectively, and IT was weighed in a molar ratio of IT: COS 5:1, dissolved in 20mM PBS (pH 7.4), mixed, and reacted at room temperature for 3 hours.
TF-CRM197 saccharide antigen maleinized derivatization
According to the required amount, 5mg of TF-CRM197 sugar antigen is sucked, 3-benzoylmaleimide-N-hydroxysuccinimide (MBS) is weighed and dissolved in ACN to prepare 35mg/mL solution for later use, MBS is weighed and diluted by PBS buffer (0.1M, 2.5mL, pH 8.0) according to the mol ratio of MBS to TF-CRM197 being 50:1, TF-CRM197 is added for mixing, and the reaction is carried out for 12 hours at 4 ℃. Thereafter, the reaction solution was transferred to an ultrafiltration membrane tube having a molecular weight cut-off of 3000, and centrifuged at 6000rpm for 30min to remove unreacted substances.
Chemical coupling of TF-CRM197 with COS
Respectively taking the above-derived compounds, mixing according to the mass ratio of 1:1, and reacting at 4 ℃ overnight. Centrifuging at 4 deg.C and 6000rpm/min, centrifuging for 20 min/time for several times in batches, removing unreacted sugar ester, washing glycoprotein in membrane with pure water for several times, and freeze drying to obtain target product 13 of glycoprotein coupled chitosan oligosaccharide. And subjected to MALDI-TOF-MS to determine the average molecular weight (as shown in FIG. 6).
Example 4: vaccine adjuvant activity assay for Chitosan oligosaccharide vaccine adjuvant
The chitooligosaccharide conjugate of example 3 was tested for immunological activity, and the antibody titer was determined by subcutaneous injection into immunized mice.
The specific method comprises the following steps:
experimental animals: balb/c mice, 6-8 weeks old, 6/group, female.
Grouping experiments: (1) blank control (PBS); (2) individual vaccine groups (CRM 197): PBS + CRM 197; (3) TF-CRM197 group; (4) TF-CRM197/COS group; (5) TF-CRM197-COS group; (6) and (4) a chitosan oligosaccharide COS group. Before injection, the mixture was mixed in equal volume, 100. mu.L/mouse, and injected subcutaneously.
Immunization protocol: animals were grouped according to immunization and mice were immunized on days 0,14, and 28, respectively. Tail vein blood is taken at 0, 7, 14, 21, 28, 35 and 42 days after the first immunization, and the antibody titer in the serum is determined by ELISA method.
Preparing reagents for an ELISA method:
1. coating liquid: accurately weigh 8.4g NaHCO3Dissolving in 1L distilled water (DDW), adjusting pH to 9.6 with 1M NaOH solution after the solid is completely dissolved, and storing the prepared coating solution at 4 deg.C.
2. Washing liquid: 0.5ml of LTween-20 was added to 1L of 0.01M PBS solution, and the mixture was mixed well and left at room temperature.
3. Sealing liquid: 20g of BSA was precisely weighed and added to 1L of 0.01M PBS solution, the BSA powder which was not dissolved in the solution was subjected to ultrasonic treatment, and when the solid in the solution was completely dissolved and the solution was pale yellow, the solution was stored in a refrigerator at 4 ℃ for further use.
4. Antibody dilution: 2.5g BSA was precisely weighed and dissolved in 250mL 0.01M PBS solution, after the solid was completely dissolved, 1.25mL TWEEN-20 was added thereto, and after mixing well, it was stored at 4 ℃ for use.
5. Color development liquid: 0.1M citric acid: 19.2g citric acid was added to distilled water (DDW) to 1000mL (A)0.2M disodium hydrogen phosphate: 28.4g of anhydrous disodium hydrogen phosphate was added to 1000mL of distilled water (DDW), (B) 24.3mL of 0.1M citric acid solution (A), 25.7mL of 0.2M phosphate buffer (B), and 50mL of DDW water. Currently used OPD (o-phenylenediamine) is added in 50mg, and 30% H is added2O20.15 mL。
ELISA method for measuring antibody titer in mouse serum
6. Stopping liquid: 2M H2SO4: concentrated sulfuric acid 55.5mL, DDW added to 500 mL.
TF-CRM197 antigen was diluted to 20. mu.g/mL with coating solution, added to 96-well plates, 100. mu.L/well, and coated overnight at 4 ℃.
2. And (3) sealing: spin-drying the coating solution in the well plate, adding 100. mu.L of blocking solution into each well, and incubating at 37 ℃ for 2 h.
3. And (3) drying: and (5) spin-drying the confining liquid, incubating for 1-2 h at 37 ℃, and till all liquid at the bottom of the pore plate is dried.
4. Immunization: diluting mouse serum by 50 times with purchased antibody diluent, sequentially diluting by 102400 times with self-prepared antibody diluent, adding diluted serum antibody into a pore plate, incubating at 37 deg.C for 1.5h at 100 μ L/pore; spin-drying the liquid in the well plate, adding washing liquid, 300 μ L/well, slowly shaking for 40s, repeating this step three times, adding 1: HRP-rabbit anti-mouse IgG/IgM antibody at 2000 dilution, 100. mu.L/well, incubated at 37 ℃ for 1 h; and (3) spin-drying the liquid in the pore plate, adding washing liquor, repeating the plate washing step, adding developing solution under the condition of keeping out of the sun, reacting at room temperature for 20min, and then measuring the absorbance at 450nm by using an enzyme-labeling instrument.
The experimental results are as follows:
the immune antibody titers of each group were examined. The results showed that the PBS group, CRM197 group and physical mixed group only produced lower IgG antibody titers. The covalently coupled chitosan oligosaccharide adjuvant group (TF-CRM197-COS) can obviously produce IgG antibody with high titer, and the titer is 25600. In contrast, the antigen was physically mixed with the chitosan oligosaccharide COS adjuvant and the IgG antibody produced by immunization was 637 (shown in figure 7).
Meanwhile, the titer of the humoral immunity antibodies of the three groups is determined, the titer of the humoral immunity IgM antibody generated by the physical mixed antigen and adjuvant group (TF-CRM197/COS) is higher and is 800, and the titer of the IgM antibody generated by the coupled chitosan oligosaccharide adjuvant group (TF-CRM197-COS) is lower and is 228 (shown in figure 8).
The experiment results show that compared with simple physical mixed antigen and adjuvant, the glycoprotein conjugate antigen containing the chitosan oligosaccharide adjuvant can more effectively stimulate the cellular immunity of mice, the humoral immunity is lower, and the chitosan oligosaccharide immune adjuvant effectively plays a role.
Although the embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that: various substitutions, changes and modifications are possible without departing from the spirit and scope of the invention and the appended claims, and therefore the scope of the invention is not limited to the disclosure of the embodiments and the accompanying drawings.
Claims (8)
1. A tumor vaccine prepared by covalently coupling a TF antigen and a chitosan oligosaccharide endogenous adjuvant with CRM197 carrier protein is characterized in that: the structural formula of the tumor vaccine is as follows:
2. the tumor vaccine of claim 1, wherein: the vaccine is synthesized by a chemical method to obtain TF-adipic acid succinimide ester, the TF-adipic acid succinimide ester is combined with diphtheria toxin mutant CRM197 carrier protein to obtain a glycoprotein conjugate, and the conjugate is coupled with a thiolated chitosan oligosaccharide vaccine adjuvant through a heterobifunctional connecting arm.
3. The tumor vaccine of claim 2, wherein: the sulfhydrylation agent in the sulfhydrylation chitosan oligosaccharide vaccine adjuvant is 2-iminosulfane hydrochloride, thioglycolic acid or 3- (2-pyridinedimercapto) propionic acid N-hydroxysuccinimide ester; or the heterobifunctional linking arm is 3-benzoylmaleimide-N-hydroxysuccinimide, 3-maleimidopropionic acid hydroxysuccinimide ester or 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid succinimide ester.
4. A tumor vaccine according to claims 2 and 3, characterized in that: the weight ratio of the thiolated chitosan oligosaccharide vaccine adjuvant to the glycoprotein conjugate is 0.3: 1-0.6: 1.
5. The tumor vaccine of claim 1, wherein: the vaccine is an anti-tumor vaccine.
6. A method of preparing a tumor vaccine according to any one of claims 1 to 5, wherein: the method comprises the following steps:
step one, synthesizing to obtain TF-adipic acid succinimide ester by a chemical method;
combining TF-adipic acid succinimide ester with diphtheria toxin mutant CRM197 carrier protein at a molar ratio of 15: 1-20: 1 to obtain a glycoprotein conjugate;
performing sulfhydrylation derivatization on the chitosan oligosaccharide adjuvant to obtain a sulfhydrylation chitosan oligosaccharide vaccine adjuvant;
step four, reacting the heterotype bifunctional connecting arm with the conjugate prepared in the step two in a molar ratio of 8: 1-10: 1 to obtain an intermediate;
reacting the thiolated chitosan oligosaccharide vaccine adjuvant obtained in the step three with the intermediate obtained in the step four, wherein the weight ratio of the thiolated chitosan oligosaccharide vaccine adjuvant to the intermediate is 0.3: 1-0.6: 1, and obtaining a target conjugate;
and step six, performing ultrafiltration and centrifugation on the conjugate obtained in the step five, and performing freeze-drying operation to obtain the target synthetic tumor vaccine.
7. The method for preparing a tumor vaccine according to claim 6, wherein: the method comprises the following specific steps:
step one, galactosamine hydrochloride is used as an initial raw material, 2-site azido, 1-site glycosylation and 4, 6-site benzaldehyde dimethyl acetal protection of a sugar ring are carried out to obtain a glycosyl acceptor, disaccharide glycosylation reaction is carried out on the glycosyl acceptor and a glucosinolate glycosyl donor to obtain TF disaccharide, then an azido reduction, deprotection and protection operation are carried out to obtain a TF antigen compound, the total yield of 7 steps of reaction is 12%, and the TF antigen and adipic acid bis-succinimide ester are reacted according to the molar ratio of 12: 1-15: 1 to obtain TF-adipic acid succinimide ester;
step two, combining TF-adipic acid succinimide ester with diphtheria toxin mutant CRM197 carrier protein, dissolving the combined protein and the carrier protein in a PBS buffer solution, wherein the molar ratio of the TF-adipic acid succinimide ester to the diphtheria toxin mutant CRM197 carrier protein is 15: 1-20: 1, and performing ultrafiltration, centrifugation and purification to obtain a glycoprotein conjugate;
performing sulfhydrylation derivatization on the chitosan oligosaccharide adjuvant to obtain a sulfhydrylation chitosan oligosaccharide vaccine adjuvant;
step four, dissolving the heterobifunctional connecting arm and the conjugate prepared in the step two in a PBS buffer solution, reacting at a molar ratio of 8: 1-10: 1, and performing ultrafiltration centrifugal purification to obtain an intermediate;
reacting the thiolated chitosan oligosaccharide vaccine adjuvant obtained in the step three with the intermediate obtained in the step four, wherein the weight ratio of the thiolated chitosan oligosaccharide vaccine adjuvant to the intermediate is 0.3: 1-0.6: 1, and obtaining a target conjugate;
step six, performing ultrafiltration and centrifugation on the conjugate obtained in the step five, and performing freeze-drying operation to obtain a target synthetic tumor vaccine;
wherein the PBS buffer is 0.01M and the pH is 8.0.
8. Use of a tumor vaccine according to any one of claims 1 to 5 in the manufacture of a medicament for treating or preventing a tumor.
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