CN107522772B

CN107522772B - Short peptide, application of short peptide as vaccine adjuvant and vaccine using short peptide as vaccine adjuvant

Info

Publication number: CN107522772B
Application number: CN201710667286.2A
Authority: CN
Inventors: 杨志谋; 王忠彦; 王玲; 石芳
Original assignee: Nankai University
Current assignee: Nankai University
Priority date: 2017-08-07
Filing date: 2017-08-07
Publication date: 2020-05-19
Anticipated expiration: 2037-08-07
Also published as: CN107522772A

Abstract

The invention provides a short peptide, application thereof as a vaccine adjuvant and a vaccine using the short peptide as the vaccine adjuvant, wherein the short peptide is introduced with a blocking group with an anti-inflammatory effect and can form hydrogel, and the hydrogel can be simply and physically mixed with an antigen, so that the antigen immune response capability can be effectively enhanced, and the hydrogel can be used for completely inhibiting the growth of a tumor in a preventive tumor vaccine. The sequence of the short peptide is X-G^DF^DF^DY, wherein X is Fbp or Car.

Description

Short peptide, application of short peptide as vaccine adjuvant and vaccine using short peptide as vaccine adjuvant

Technical Field

The invention relates to a short peptide, application thereof as a vaccine adjuvant and a vaccine using the short peptide as the vaccine adjuvant.

Background

The establishment of the inflammatory microenvironment is essential for the development and progression of tumors. Inflammation plays an important role in various stages of tumor development, such as initiation, promotion, malignant change, invasion and metastasis. Recent studies have shown that tumor-associated inflammation can significantly promote tumorigenesis and cancer cell survival by recruiting leukocytes, expressing pro-tumor cytokines and chemokines, inducing angiogenesis. At the same time, these tumor-associated inflammations can also suppress the immune response against tumors by impairing immune surveillance and immune editing. Therefore, inhibition of tumor-associated inflammation is important for activating anti-tumor adaptive immune responses. A recent study of the Sousa topic group showed that: the combined application of aspirin and immune checkpoint therapy (anti-PD-1) can obviously enhance the effect of immunotherapy, because aspirin can inhibit tumor inflammation, so that the tumor microenvironment is changed into the tumor-inhibiting microenvironment, and therefore, the therapeutic efficacy of anti-PD-1 is improved.

Hydrogels formed by short peptides have been developed greatly, and they are applied to three-dimensional culture of cells, drug release, sensing application, cancer cell inhibition and immune regulationThe aspect shows great potential. To form hydrogels, it is often necessary to attach an aromatic capping group to the short peptide. Our previous studies found that the aromatic group-terminated tetrapeptide G^DF^DF^DThe hydrogel formed by Y is an excellent vaccine adjuvant, and the short peptide hydrogel can be used for wrapping antigens with the antigens through a simple physical mixing method, so that the antibody titer is greatly improved. However, these short peptide hydrogels do not have anti-inflammatory effects and are not significant for anti-tumor prevention and treatment.

The aspirin structure contains benzene rings, and if the aspirin structure is used as a blocking group to be introduced into the short peptide, the obtained short peptide can form hydrogel, and the hydrogel may have an anti-inflammatory effect, so that the anti-tumor vaccine adjuvant is obtained. However, the results of studies by the group of professors of the xumenon (Beilstein, j. org. chem.,2013,9,908-917) show that short peptides blocked with aspirin do not form hydrogels and are not useful as vaccine adjuvants for encapsulation of various types of antigens.

Disclosure of Invention

Object of the Invention

An object of the present invention is to provide a short peptide, which introduces a blocking group having an anti-inflammatory effect and can form a hydrogel that not only effectively enhances the ability of an antigen to respond to an immune response after being physically mixed with an antigen simply but also can completely inhibit the growth of a tumor when used in a prophylactic tumor vaccine.

Another purpose of the invention is to provide the application of the short peptide as a vaccine adjuvant.

Another objective of the invention is to provide a vaccine using the above-mentioned short peptide as a vaccine adjuvant.

Summary of The Invention

According to a first aspect of the invention, there is provided a short peptide having the sequence X-G^DF^DF^DY, wherein X is Fbp or Car.

"short peptide" is a term commonly used in the art and refers to a short chain peptide consisting of 3-9 amino acid residues.

Specifically, the structural formula of the short peptide when X is Fbp is as follows:

when X is Car, the structural formula of the short peptide is as follows:

the short peptides can be synthesized by the well-known FMOC-solid phase synthesis method.

According to a second aspect of the invention, there is also provided the use of the short peptides as vaccine adjuvants.

The short peptide can be selected from various physical forms when being used as a vaccine adjuvant, and the capability of the short peptide for enhancing immune response is not substantially influenced, for example, the short peptide can be uniformly mixed with water and then mixed with an antigen to obtain a vaccine, or the short peptide can also be used in the form of short peptide hydrogel because the short peptide has good gelling property, specifically, the water mixture of the short peptide is heated and cooled to form the short peptide hydrogel, and then the short peptide hydrogel is mixed with the antigen and is used as the vaccine after standing. The vaccine may be a protein vaccine. Preferably, the vaccine is an anti-tumor vaccine. As a general knowledge in the art, the aqueous mixture of the short peptide should have good biocompatibility, and therefore, it is required to mix the short peptide with an aqueous solution having good biocompatibility to obtain the aqueous mixture of the short peptide. Typically, the aqueous solution with good biocompatibility may be a physiological saline or PBS solution. The preparation method of the vaccine is simple and practical and has controllable components. The heating and cooling method for forming the hydrogel is a known method, and specifically comprises the following steps: heating the water mixture of the short peptide by using a heating device such as a hair drier or an oil bath pan until the short peptide is completely dissolved (generally heating to more than 90 ℃), and cooling to form gel (generally cooling to 20-40 ℃). Whether the gel is formed or not is judged by a method of inverting the bottle, the hydrogel is remained at the bottom of the bottle, and the hydrogel is liquid if the hydrogel is flowing.

In a third aspect of the invention, there is also provided a vaccine comprising the above-described short peptide as a vaccine adjuvant. The short peptides can be conveniently physically mixed with an antigen to obtain a vaccine. Preferably, the short peptide is mixed with water and then heated and cooled to form the short peptide hydrogel, and then the short peptide hydrogel is mixed with antigen and is used as a vaccine after standing. The vaccine may be a protein vaccine. Preferably, the vaccine is an anti-tumor vaccine (e.g., a prophylactic tumor vaccine or a therapeutic tumor vaccine). The amount of the short peptide can be determined by a person skilled in the art through simple experiments, and when the short peptide is generally used in a protein vaccine, the mass ratio of the short peptide to the antigen is 5:1-30:1, and more preferably 5:1-20: 1.

The inventor finds that the short peptide can be used as a vaccine adjuvant to enhance the immunogenicity of antigens so that a main body generates strong antigen-specific cellular immunity and humoral immunity response, the polypeptide hydrogel has an additional anti-inflammatory effect by using Fbp and Car as end capping groups, and the anti-tumor effect is surprisingly improved in the technical field of the existing immunotherapy, and particularly the anti-tumor effect can completely inhibit the generation of tumors in a preventive tumor vaccine (compared with the vaccine adjuvant discovered earlier by the inventor, namely the tetrapeptide G with the end capped by an aromatic group^DF^DF^DY, superior in anti-tumor vaccine potency). The short peptide is easy to prepare and has single and controllable component.

The invention increases the types of immunologic adjuvants, and provides valuable information for developing immunologic adjuvants and anti-tumor vaccines which can be applied to tumors.

Drawings

FIG. 1: the antibody titer of the protein vaccines OVA, Alum-OVA, vac-1, vac-2 and vac-3 for triggering immune response;

FIG. 2: the preventive immune effect of PBS, OVA, vac-1, vac-2 and vac-3 against B16-OVA tumors;

FIG. 3: therapeutic immune effects of PBS, OVA, vac-1, vac-2, vac-3 against EG7-OVA tumors.

Detailed Description

The invention is further described below with reference to examples, which are intended to be illustrative only and are not intended to be limiting.

In the following examples, the presence or absence of hydrogel formation was examined by inverting the vial as is commonly used in the art.

The sources of the formulations referred to in the following examples are as follows:

medium, RMPI 1640, purchased from seimer fisher Scientific, sterile;

fetal bovine serum, purchased from seimer feishel Scientific, sterile;

the 2-Cl-Trt resin is purchased from Tianjin Nankai and science and technology Limited and has the activity of 1.2 mmol/mL;

n, N-diisopropylethylamine (DIEPA below) was purchased from Sigma Aldrich (Sigma-Aldrich) at 99% purity;

benzotriazole-N, N, N ', N' -tetramethyluronium hexafluorophosphate (hereinafter referred to as HBTU) available from Gill Biochemical (Shanghai) Co., Ltd., purity 98%;

trifluoroacetic acid (hereinafter indicated as TFA), purchased from Sigma Aldrich (Sigma-Aldrich), 99% pure;

triisopropylsilane (hereinafter referred to as TIS), purchased from Sigma Aldrich (Sigma-Aldrich) with a purity of 99%;

d-configuration amino acid is purchased from Gill Biochemical (Shanghai) Co., Ltd, and has purity of 98%;

endotoxin-free egg white protein (hereinafter referred to as OVA protein) was purchased from InvivoGen and has a purity of 99%;

aluminum hydroxide adjuvant (hereinafter referred to as aluminum adjuvant) was purchased from the zemer feishel scientific (thermo fisher scientific) with a purity of 99%;

naphthylacetic acid was purchased from Sigma Aldrich (Sigma-Aldrich) with a purity of 99%;

fluoroibuprofen, carprofen, was purchased from Aladdin Chemicals, Inc. (Aladdin) with a purity of 99%.

Cancer cells B16-OVA, purchased from Van. hainanensis, and cultured as follows:

1) heating the water bath to 37 deg.C in advance, preheating culture medium and serum in the water bath, and simultaneously turning on ultra-clean bench ultraviolet lamp to irradiate for half an hour;

2) the frozen cancer cells B16-OVA were taken out of the liquid nitrogen tank, immediately placed in a water bath at 37 ℃ to thaw the cells, and then immediately transferred to a clean bench to perform the following operations: carefully transferring the cell-containing solution into a centrifuge tube containing a culture medium by using a pipette, centrifuging for 5 minutes, removing a supernatant, resuspending the cell-containing solution by using a culture medium containing 10% fetal calf serum, transferring the cell-containing solution into a culture dish containing a culture medium containing 10% fetal calf serum, and then putting the culture dish into a 37 ℃ incubator for culture;

3) observing the cell state on the next day, and carrying out the following experiment after the cell state is good and first generation;

4) collecting cell culture solution, sucking into centrifuge tube, centrifuging at 1000rpm for 4min, removing solution, adding fresh culture medium, blowing with gun, and counting with cell counting plate to 5 × 10⁷Each per milliliter.

Cancer cells EG7-OVA, purchased from vanda, shanghai song, and cultured as follows:

2) the frozen cancer cells EG7-OVA were removed from the liquid nitrogen tank, quickly placed in a 37 ℃ water bath to thaw the cells, and then quickly transferred to a clean bench for the following operations: carefully transferring the solution containing the cells into a centrifuge tube containing a culture medium by using a pipette, centrifuging for 5 minutes, removing a supernatant, resuspending the cell-containing solution by using the culture medium containing 10% fetal calf serum and 0.4mg/mL G418, transferring the cell-containing solution into a culture dish containing 10% fetal calf serum and 0.4mg/mL G418, and then putting the culture dish into a 37 ℃ incubator for culture;

The remaining reagents were all commercially available analytical grade reagents.

The OVA protein solution is prepared by dissolving OVA protein in PBS (pH 7.4) to obtain 5mg/mL OVA protein solution.

Preparation of example 1

preparation of vaccine vac-1 with short peptide hydrogel loaded with OVA protein at pH 7.4 and room temperature of 20 DEG C

(1) Synthesis of D-configuration short peptide Fbp-G by FMOC-solid phase synthesis method^DF^DF^DY, the structural formula is as follows:

the method comprises the following specific steps:

1) weighing 0.5mmol of 2-Cl-Trt resin, adding 10mL of anhydrous dichloromethane (hereinafter represented by DCM) into a solid phase synthesizer, and placing on a shaker to shake for 5min to fully swell the 2-Cl-Trt resin;

2) removing DCM from the solid phase synthesizer containing 2-Cl-Trt resin by washing the ear with an ear bulb;

3) dissolving 0.75mmol of Fmoc-protected amino acid in 10mL of anhydrous DCM, adding 0.75mmol of DIEPA, transferring to the solid phase synthesizer, supplementing 0.75mmol of DIEPA, and reacting at room temperature for 1 h;

4) and (3) sealing: removing reaction liquid in a solid phase synthesizer by using an aurilave, washing with 10mL of anhydrous DCM for 1min each time for 5 times, adding 20mL of prepared solution with the volume ratio of anhydrous DCM to DIEPA to methanol being 17: 1: 2, and reacting at room temperature for 10 min;

5) removing reaction liquid in the solid phase synthesizer by using an aurilave, washing by using anhydrous DCM for 5 times, washing by using N, N-dimethylformamide (hereinafter referred to as DMF) for 10mL each time for 1min, washing for 5 times, adding 10mL of DMF containing 20% by volume of piperidine for reaction for 25min, reacting by using 10mL of DMF containing 20% by volume of piperidine for 5min, washing by using DMF for 1min, washing for 5 times, and carrying out next reaction by using 10mL of DMF for 10mL each time for 1 min;

6) adding 1mmol of second Fmoc-protected amino acid, 1.5mmol of HBTU, 2mmol of DIEPA and 10ml of DMF, adding the prepared solution into the solid phase synthesizer, and reacting for 2 h;

7) repeating the steps 5) and 6) to sequentially add the needed amino acid or the end capping group (fluorobenzene ibuprofen); then washing the mixture for 5 times by using DMF (dimethyl formamide), washing the mixture for 5 times by using dichloromethane, and carrying out the next reaction;

8) as 95% TFA, 2.5% TIS, 2.5% H₂Adding 10mL of solution consisting of O in volume percent into the solid phase synthesizer, reacting for half an hour (or preparing a TFA solution with the volume percent concentration of 1% by volume by using a TFA/DCM volume ratio of 1: 99), adding 3mL of the TFA solution into the solid phase synthesizer for ten times, wherein the reaction time is 1min each time), cutting the product from the 2-cl-Trt resin, concentrating in vacuum, removing the solvent to obtain a crude product, and then separating and purifying by using HPLC.

Its structural characterization data are as follows:

¹H NMR(400MHz,DMSO)δ9.22(s,1H),8.32–7.90(m,4H),7.25(ddd,J＝79.3,44.2,8.3Hz,19H),6.66(d,J＝6.8Hz,2H),4.61–4.33(m,3H),3.82–3.54(m,3H),3.05–2.63(m,6H),1.33(d,J＝6.3Hz,3H).

(2) 1mg of D-configuration short peptide Fbp-G is taken^DF^DF^DAnd placing the Y into a 1.5 ml glass bottle, adding 400 microliters of PBS (pH 7.4), adjusting the pH value to 7.4 by using a sodium carbonate solution, heating to boil to completely dissolve the compound, and cooling to room temperature to obtain the short peptide hydrogel.

(3) 20 microliters of an OVA protein solution of 5mg/mL was added to the hydrogel prepared in (2), and the volume was adjusted to 500 microliters with a PBS solution (pH 7.4), followed by physical mixing, standing for 3 minutes, and gelling was performed, thereby obtaining a protein vaccine vac-1 (final short peptide concentration was 2mg/mL, and OVA protein concentration was 0.2 mg/mL).

Preparation of example 2

Short peptide hydrogel Car-G at pH 7.4 and room temperature of 20 deg.C^DF^DF^DPreparation of vaccine vac-2 of Y-loaded OVA protein

(1) Synthesis of D-configuration oligopeptide Car-G by FMOC-solid phase synthesis method^DF^DF^DY, the structural formula is as follows:

the specific procedure was as described in preparative example 1, with the end capping group being carprofen.

Its structural characterization data are as follows:

¹H NMR(400MHz,DMSO)δ11.32(s,1H),9.19(s,1H),8.56–8.30(m,2H),8.20–7.96(m,3H),7.86(d,J＝8.3Hz,1H),7.51–7.30(m,3H),7.26–7.01(m,11H),6.89(t,J＝6.3Hz,2H),6.65(d,J＝8.3Hz,2H),4.62–4.35(m,3H),3.83–3.41(m,4H),2.98(dd,J＝9.7,4.0Hz,1H),2.78–2.56(m,3H),2.33(t,J＝11.5Hz,1H),1.35(d,J＝5.8Hz,3H).

(2) 1mg of Car-G was taken^DF^DF^DAnd placing the Y into a 1.5 ml glass bottle, adding 400 microliters of PBS (pH 7.4), adjusting the pH value to 7.4 by using a sodium carbonate solution, heating to boil to completely dissolve the compound, and cooling to room temperature to obtain the short peptide hydrogel.

(3) 20 microliters of an OVA protein solution of 5mg/mL was added to the hydrogel prepared in (2), and the volume was adjusted to 500 microliters with a PBS solution (pH 7.4), followed by physical mixing, and standing for 5 minutes to form a gel, thereby obtaining a protein vaccine vac-2 (the final short peptide concentration was 2mg/mL, and the OVA protein concentration was 0.2 mg/mL).

Comparative preparation example 1

20 microliters of 5mg/mL OVA protein solution was taken, and the volume was adjusted to 500 microliters with PBS solution (pH 7.4), to obtain an adjuvant-free protein vaccine OVA (final OVA protein concentration was 0.2 mg/mL).

Comparative preparation example 2

(1) 62.5. mu.l of 40mg/mL aluminum adjuvant was added, and the volume was adjusted to 250. mu.l with a PBS solution (pH 7.4), to obtain an aluminum adjuvant dispersion.

(2) 20. mu.l of OVA protein solution (5 mg/mL) was added to the aluminum adjuvant dispersion solution prepared in (1), and the mixture was dissolved in 500. mu.l of PBS (pH 7.4) and physically mixed to obtain an aluminum adjuvant-containing protein vaccine Al-OVA. (final concentration of aluminum adjuvant 5mg/mL, OVA protein concentration 0.2mg/mL)

Comparative preparation example 3

preparation of short peptide hydrogel Nap-gel at pH 7.4 and room temperature of 20 deg.C

(1) Synthesis of short peptide Nap-G by FMOC-solid phase synthesis method^DF^DF^DY, the structural formula is as follows:

(2) 1mg of Nap-G was taken^DF^DF^DAnd placing the Y into a 1.5 ml glass bottle, adding 400 microliters of PBS (pH 7.4), adjusting the pH value to 7.4 by using a sodium carbonate solution, heating to boil to completely dissolve the compound, and cooling to room temperature to obtain the short peptide hydrogel.

(3) 20 microliters of an OVA protein solution of 5mg/mL was added to the hydrogel prepared in (2), and the volume was adjusted to 500 microliters with a PBS solution (pH 7.4), followed by physical mixing, and standing for 8 minutes to form a gel, thereby obtaining a protein vaccine vac-3 (the final short peptide concentration was 2mg/mL, and the OVA protein concentration was 0.2 mg/mL).

Comparative preparation example 4

Sterile 1 XPBS

Immunization example 1

(1) First immunization of mice

The protein vaccines vac-1, vac-2, OVA, Al-OVA and vac-3 prepared in production example 1, production example 2, comparative production example 1, comparative production example 2 and comparative production example 3 were taken from 6-8 weeks of mice, and the first injection time was taken as 0 day, and the hydrogel was broken into viscous solutions by a vortex machine and then subcutaneously injected into the groin of the mice at a dose of 100 μ l per mouse, respectively.

(2) Second immunization of mice

At the 14 th day time point, the protein vaccines vac-1, vac-2, OVA, Al-OVA and vac-3 prepared in preparation example 1, preparation example 2, comparative preparation example 1, comparative preparation example 2 and comparative preparation example 3 were taken and injected subcutaneously at the groin of mice at a dose of 100 microliters per mouse, respectively, after the hydrogel was broken into viscous solutions by a vortex machine.

(3) Measurement of antibody titer

Mouse sera were taken at day 21 and the corresponding antibody titers were measured by ELISA using a BioTek plate reader. The results are shown in FIG. 1.

(4) Analysis of results

From the results in fig. 1 it is seen that: wherein IgG represents total antibody titer; IgG1 was one of the typing, indicative of a humoral immune response; IgG2a and IgG2b were also one of the typing, indicating cellular immunity levels. Compared with the OVA protein control group vaccine without adjuvant (comparative preparation example 1), the vaccine (vac-1) of the experimental group using the hydrogel adjuvant of the invention can improve the IgG antibody titer by 1476 times, the vaccine (vac-2) of the experimental group using the hydrogel adjuvant of the invention can improve by 929 times, while the vaccine (Al-OVA) of the control group using the aluminum adjuvant can only improve by 125 times, and the vaccine (vac-3) of the control group using the hydrogel adjuvant improves by 446 times; compared with the OVA protein control group vaccine without adjuvant (comparative preparation example 1), the vaccine (vac-1) in the experimental group using the hydrogel adjuvant can improve the IgG1 antibody titer by 2844 times, the vaccine (vac-2) in the experimental group using the hydrogel adjuvant can improve by 2844 times, the vaccine (Al-OVA) in the control group using the aluminum adjuvant can improve by 133 times, and the vaccine (vac-3) in the control group using the hydrogel adjuvant can improve by 867 times; compared with the OVA protein control group vaccine without adjuvant (comparative preparation example 1), the vaccine (vac-1) in the experimental group using the hydrogel adjuvant of the invention can improve the IgG2a antibody titer by 16 times, the vaccine (vac-2) in the experimental group using the hydrogel adjuvant of the invention can improve by 13 times, while the vaccine (Al-OVA) in the control group using the aluminum adjuvant can improve by only 3 times, and the vaccine (vac-3) in the control group using the hydrogel adjuvant can improve by only 9 times; compared with the OVA protein control vaccine without adjuvant (comparative preparation example 1), the vaccine (vac-1) in the experimental group using the hydrogel adjuvant of the present invention can improve the IgG2b antibody titer by 155 times, the vaccine (vac-2) in the experimental group using the hydrogel adjuvant of the present invention can improve by 91 times, while the vaccine (Al-OVA) in the control group using the aluminum adjuvant can improve by only 4 times, and the vaccine (vac-3) in the control group using the hydrogel adjuvant can improve by only 74 times.

Immunization example 2

Preventive immunization experiment against B16-OVA tumors:

(1) first immunization of mice:

the vaccines vac-1, vac-2, OVA, vac-3 and PBS prepared in preparation example 1, preparation example 2, comparative preparation example 1, comparative preparation example 3 and comparative preparation example 4 were taken from 6-8 weeks of C57BL/6J mice, and the first injection time was taken as 0 day, and the hydrogel was broken into viscous solutions by a vortex machine and then subcutaneously injected into the groin of the mice at a dose of 100 microliters per mouse, respectively.

(2) Second immunization of mice

At the 14 th day time point, the vaccines vac-1, vac-2, OVA, vac-3 and PBS prepared in preparation example 1, preparation example 2, comparative preparation example 1, comparative preparation example 3 and comparative preparation example 4 were taken, and subcutaneous injection was performed at the groin of mice at a dose of 100 μ l per mouse after the hydrogel was broken into viscous solution by a vortex machine.

(3) Tumor inoculation

The right dorsal subcutaneous injection of 50. mu.L containing 5X 10 of each C57BL/6J mouse was administered on day 21⁵Sterile PBS of individual B16-OVA cells and recorded as day 0 of tumor inoculation or tumor growth at this time point. As the tumor grew, a macroscopic tumor appeared on the back of the mice, and from day 6 of tumor inoculation, the tumor volume reached 100mm³On the left and right, tumor size was recorded, and tumor volume was measured every 3 days until day 24 of tumor inoculation, to evaluate the tumor suppression effect. The test method is as follows: the length (longest diameter) and width (width perpendicular to the longest diameter direction) of the mouse tumor were measured with a vernier caliper, and the tumor volume was calculated according to the formula [ length × width)/2 ]. The results are shown in FIG. 2, where the abscissa refers to the number of days after tumor inoculation.

(4) Analysis of results

According to the results of FIG. 2, in this experiment against B16-OVA tumors, the unadjuvanted OVA protein group only slightly inhibited tumor growth relative to the PBS group, and the control vaccine vac-3, which used the hydrogel adjuvant without anti-inflammatory effect, significantly inhibited tumor growth, whereas all mice in the experimental group vaccines vac-1 and vac-2, which used the hydrogel adjuvant of the present invention, had no tumor development throughout. In the tumor preventive experiment, the hydrogel adjuvant group vaccine disclosed by the invention has the characteristic of obviously inhibiting tumorigenesis. By applying the strategy, the occurrence of tumors can be effectively eliminated.

Immunization example 3

Therapeutic immunization experiments against EG7-OVA tumors:

(1) tumor vaccination and immunotherapy:

on day 0, each C57BL/6J right dorsal subcutaneous injection of 50 μ L contained 5X 10⁵Sterile PBS of EG7-OVA cells. On day 8, when the tumor volume had grown to about 100mm³At that time, the tumor size was measured from the beginning every 2 days until the 20 th day of tumor inoculation, to evaluate the tumor suppression effect. Two vaccinations were performed on

day

8 and 14, respectively (vaccines vac-1, vac-2, OVA, vac-3 and PBS prepared in preparation example 1, preparation example 2, comparative preparation example 1, comparative preparation example 3 and comparative preparation example 4 were each taken, and subcutaneous injections were performed at the groin of mice at a dose of 100 microliters per mouse, respectively, after the hydrogel was broken into viscous solutions with a vortex machine). Tumor record tumor volume test method is as follows: the length (longest diameter) and width (width perpendicular to the longest diameter direction) of the mouse tumor were measured with a vernier caliper, and the tumor volume was calculated according to the formula [ length × width)/2 ]. The results are shown in FIG. 3, where the abscissa refers to the number of days after tumor inoculation.

(2) Analysis of results

As shown in FIG. 3, in this experiment modeled on EG7-OVA tumors, the unadjuvanted OVA protein group had substantially no tumor-inhibiting effect relative to the PBS group, and the tumor-inhibiting rate was approximately 50% for vaccine vac-3, which is the control group using the hydrogel adjuvant, and approximately 75% for vac-1 and vac-2, which are the experimental groups using the hydrogel adjuvant of the present invention, and in which the tumors of individual mice were completely eliminated. In this immunotherapy experiment, the hydrogel group of the present invention was able to inhibit the growth of tumor more strongly than the control hydrogel group. The method for adjuvant immunotherapy by using the hydrogel of the invention has greater advantages than the method for purely using the immunoadjuvant. The method of the hydrogel adjuvant can simultaneously reduce the inflammation related to the tumor and stimulate CD8⁺T cell immunoreactive doubletHas important effect, and makes the treatment simpler and more efficient.

Claims

1. A short peptide with sequence of X-G^DF^DF^DY, wherein X is Fbp or Car.

2. The use of the short peptide of claim 1 as a vaccine adjuvant, said vaccine being an anti-tumor vaccine.

3. The use of claim 2, wherein the aqueous mixture of short peptides is heated and cooled to form a hydrogel of short peptides, which is then mixed with the antigen and allowed to stand to form a hydrogel for use as a vaccine.

4. The use according to claim 3, wherein the antigen is OVA.

5. A vaccine comprising the short peptide of claim 1 as a vaccine adjuvant, wherein the vaccine is an anti-tumor vaccine.

6. The vaccine of claim 5, wherein the short peptide is mixed with water and then heated to form a hydrogel of the short peptide, and then mixed with the antigen, and the hydrogel formed after standing is used as the vaccine.

7. The vaccine of claim 6, wherein said antigen is OVA.