A novel immunoadjuvant, its preparation method and application

Abstract

The application discloses a novel immunoadjuvant, a preparation method and application thereof, and the molecular formula of the novel immunoadjuvant is as follows: The adjuvant can produce a stronger immunization effect, the adjuvant can significantly reduce the antigen dosage, and the adjuvant is convenient to prepare and easy to preserve.

Description

Technical Field

[0001] This invention relates to a novel immune adjuvant, its preparation method, and its application, belonging to the field of bioengineering technology. Background Technology

[0002] An adjuvant is a substance added to a vaccine to stimulate and enhance the strength and duration of the immune response. The traditional development of new vaccine adjuvants has been described as one of the slowest processes in medical history. For over 70 years since it was first licensed in the 1920s, aluminum salts (alum) remained the sole adjuvant in many vaccine products, such as those for hepatitis B, diphtheria, tetanus, and pertussis or human papillomavirus. However, in the late 1990s, the oil-in-water emulsion adjuvant MF59 was first used in Europe as an adjuvant for a trivalent inactivated vaccine against seasonal influenza, and was licensed for use in adults over 65 years of age. Over the past 20 years, four other adjuvants have been incorporated into other products: AS01 (for the shingles vaccine Shingrix and the malaria vaccine Mosquirix), AS04 (for the hepatitis B vaccine Fendrix and the human papilloma vaccine Cervarix), AS03 (for the pandemic influenza vaccines Pandemrix and Arepanrix), and cytosine guanosine monophosphate (CpG) 1018 (for the hepatitis B vaccine Heplisav-B). Although many other adjuvants have shown high efficacy in preclinical models during this period, most have not yet been approved for human use, often due to safety or tolerability issues. Furthermore, despite the widespread use of existing adjuvants (including alum, MF59, and the AS0 adjuvant system), the molecular mechanisms by which they actually function in humans are not fully understood.

[0003] Due to the existence of various infectious diseases, including COVID-19, in recent years, attention to vaccine adjuvants has grown rapidly. Vaccine manufacturers and public health agencies such as the WHO have set multiple goals to enhance current vaccines and develop new ones, and new candidate vaccines targeting infectious diseases, allergies and autoimmune diseases, as well as cancer and fertility treatments have emerged in the past few years. In many cases, because the vaccine antigens themselves have low immunogenicity, these vaccines require safe and effective immune adjuvants. Summary of the Invention

[0004] The purpose of this invention is to provide a novel immune adjuvant, its preparation method, and its application.

[0005] To achieve the above and other related objectives, the present invention provides a novel immune adjuvant with the following molecular formula:

[0006]

[0007] To achieve the above and other related objectives, the present invention provides a method for preparing a novel immune adjuvant, comprising the following steps:

[0008] Step 1: Swell 2-CTC resin with dichloromethane, then add Fmoc-Cys(Trt)-OH and DIPEA, react for 0.8-1.2 hours, then add methanol to end-cap, then filter to remove dichloromethane, wash with dimethylformamide to obtain Fmoc-Cys(Trt)-CTC resin;

[0009] Step 2: Add 15-25% of the volume of piperidine / dimethylformamide solution to Fmoc-Cys(Trt)-CTC resin, then purge with nitrogen for 20-40 minutes, and dry to obtain H2N-Cys(Trt)-CTC resin.

[0010] Step 3: Wash the H2N-Cys(Trt)-CTC resin obtained in Step 2 with dimethylformamide;

[0011] Step 4: Add Fmoc-Acp-OH amino acid, DIPEA and HBTU to dimethylformamide for reaction to obtain Fmoc-Acp-Cys(Trt)-CTC resin;

[0012] Step 6: Wash the Fmoc-Acp-Cys(Trt)-CTC resin obtained in Step 4 with dimethylformamide;

[0013] Step 7: Add 15-25% of the volume of piperidine / dimethylformamide solution to Fmoc-Acp-Cys(Trt)-CTC resin, then purge with nitrogen for 20-40 minutes, and dry to obtain H2N-Acp-Cys(Trt)-CTC resin.

[0014] Step 8: Wash the H2N-Acp-Cys(Trt)-CTC resin obtained in Step 7 with dimethylformamide;

[0015] Step 9: Add Fmoc-Ser(PO3HBzl)-OH amino acid, DIPEA and HBTU to dimethylformamide to react and obtain Fmoc-Ser(PO3HBzl)-Acp-Cys(Trt)-CTC resin.

[0016] Step 10: Wash the Fmoc-Ser(PO3HBzl)-Acp-Cys(Trt)-CTC resin obtained in step 9 with dimethylformamide;

[0017] Step 11: Add 15-25% by volume of piperidine / dimethylformamide solution to Fmoc-Ser(PO3HBzl)-Acp-Cys(Trt)-CTC resin, then purge with nitrogen for 20-40 minutes, and dry to obtain H2N-Ser(PO3HBzl)-Acp-Cys(Trt)-CTC resin;

[0018] Step 12: Wash the H2N-Ser(PO3HBzl)-Acp-Cys(Trt)-CTC resin obtained in Step 11 with dimethylformamide;

[0019] Step 13: Prepare according to the method in steps 9-12:

[0020] H2N-Ser(PO3HBzl)-Ser(PO3HBzl)-Acp-Cys(Trt)-CTC resin;

[0021] Step 14: Acetic anhydride and DIPEA are added to dimethylformamide to react and obtain Ac-Ser(PO3HBzl)-Ser(PO3HBzl)-Acp-Cys(Trt)-CTC resin;

[0022] Step 15: Wash Ac-Ser(PO3HBzl)-Ser(PO3HBzl)-Acp-Cys(Trt)-CTC resin with methanol;

[0023] Step 16: Mix the cutting fluid with the product from Step 15, cut for 1.5-2.5 hours under stirring, then filter to remove the resin, precipitate the filtrate with ice-cold anhydrous diethyl ether, wash the precipitate with ice-cold anhydrous diethyl ether, and finally place the precipitate in a vacuum drying oven and dry at room temperature to obtain the crude product Ac-Ser(PO3H2)-Ser(PO3H2)-Acp-Cys.

[0024] The preferred technical solution is that the degree of substitution of the 2-CTC resin is 0.8-1.2 mmol / g.

[0025] The preferred technical solution is as follows: In step 4, the molar ratio between Fmoc-Acp-OH amino acid, DIPEA, HBTU and resin is 2.8-3.2:5-7:2.8-2.9:1.

[0026] The preferred technical solution is as follows: In step 9, the molar ratio between Fmoc-Ser(PO3H2Bzl)-OH amino acid, DIPEA, HBTU and resin is 2.8-3.2:5-7:2.8-2.9:1.

[0027] To achieve the above and other related objectives, the present invention provides the following technical solution: the application of a novel immune adjuvant, wherein the novel immune adjuvant is used in vaccine preparation.

[0028] Due to the application of the above technical solution, the advantages of this invention compared with the prior art are:

[0029] 1. The adjuvant of the present invention can produce a stronger immune effect.

[0030] 2. The adjuvant of the present invention can significantly reduce the amount of antigen used.

[0031] 3. The adjuvant of the present invention is easy to prepare and easy to store. Attached Figure Description

[0032] Figure 1 RBD antigen expression plasmid map.

[0033] Figure 2 RBD antigen identification results.

[0034] Figure 3 Schematic diagram of RBD antigen-adjuvant coupling.

[0035] Figure 4 Serum titers of mice in each group were measured. Detailed Implementation

[0036] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification.

[0037] Please see Figure 1-4 It should be understood that the structures, proportions, sizes, etc., illustrated in the accompanying drawings are merely for illustrative purposes to aid those skilled in the art and to facilitate understanding and reading. They are not intended to limit the scope of the invention and therefore have no substantial technical significance. Any modifications to the structure, changes in proportions, or adjustments to size, without affecting the effectiveness and purpose of the invention, should still fall within the scope of the technical content disclosed in this invention. Furthermore, the terms such as "upper," "lower," "left," "right," "middle," and "one" used in this specification are merely for clarity and not intended to limit the scope of the invention. Changes or adjustments to their relative relationships, without substantially altering the technical content, should also be considered within the scope of the invention's implementation.

[0038] Example 1: A novel immune adjuvant, its preparation method, and its application

[0039] A novel immune adjuvant, with the following molecular formula:

[0040]

[0041] Preparation method: The Fmoc solid-phase peptide synthesis method was used.

[0042] Synthesis of R1O1(Ac-Ser(PO3H2)-Ser(PO3H2)-Acp-Cys).

[0043] Step 1: Take 1g of CTC resin (degree of substitution approximately 1.0 mmol / g), swell it with 5ml of DCM for 5 minutes. After full swelling, add 0.4mmol of Fmoc-Cys(Trt)-OH and 0.8mmol of DIPEA, react for 1 hour, add 1ml of methanol to cap the resin for half an hour, filter off the DCM, and wash the resin 3 times with DMF. At this point, the resin is Fmoc-Cys(Trt)-CTC resin (degree of substitution approximately 0.4 mmol / g).

[0044] Step 2: Add 3 times the resin volume of 20% Pip / DMF solution, purge with nitrogen for 30 minutes, and then dry under vacuum. This yields H2N-Cys(Trt)-CTC resin (with Fmoc groups removed).

[0045] Step 3: Wash the resin from the previous step 5 times with DMF at a volume of 2:1 (washing the resin removes residual solvent and prepares it for the next reaction).

[0046] Step 4: Take 1.2 mmol of Fmoc-Acp-OH amino acid, 2.4 mmol of DIPEA, and 1.14 mmol of HBTU. React with an appropriate amount of DMF solvent for 30 minutes to obtain Fmoc-Acp-Cys(Trt)-CTC resin. The molar ratio of amino acid:DIPEA:HBTU:resin is 3:6:2.85:1.

[0047] Step 5: Wash the resin from the previous step three times with DMF at a volume of 2 (washing the resin removes residual solvent and prepares it for the next reaction).

[0048] Step 6: Add 3 times the resin volume of 20% Pip / DMF solution, purge with nitrogen for 30 minutes, and then dry under vacuum. This yields H2N-Acp-Cys(Trt)-CTC resin (with Fmoc groups removed).

[0049] Step 7: Wash the resin from the previous step 5 times with DMF at a volume of 2 times the resin volume (washing the resin removes residual solvent and prepares it for the next reaction).

[0050] Step 8: Take 1.2 mmol of Fmoc-Ser(PO3HBzl)-OH amino acid, 2.4 mmol of DIPEA, and 1.14 mmol of HBTU. React with an appropriate amount of DMF solvent for 30 minutes to obtain Fmoc-Ser(PO3HBzl)-Acp-Cys(Trt)-CTC resin. Amino acid:DIPEA:HBTU:resin = 3:6:2.85:1 (molar ratio).

[0051] Step 9: Wash the resin from the previous step three times with DMF at a volume of 2 (washing the resin removes residual solvent and prepares it for the next reaction).

[0052] Step 10: Add 3 times the volume of resin of 20% Pip / DMF solution, purge with nitrogen for 30 minutes, and then dry under vacuum. This yields H2N-Ser(PO3HBzl)-Acp-Cys(Trt)-CTC resin (with Fmoc groups removed).

[0053] Step 11: Wash the resin from the previous step 5 times with DMF at a volume of 2 times the resin volume (washing the resin removes residual solvent and prepares it for the next reaction).

[0054] Step 12: Repeat steps 8, 9, 10, and 11 to obtain H2N-Ser(PO3HBzl)-Ser(PO3HBzl)-Acp-Cys(Trt)-CTC resin.

[0055] Step 13: Take 2.4 mmol of acetic anhydride and 4.8 mmol of DIPEA. React with an appropriate amount of DMF solvent for 30 minutes. Ac-Ser(PO3HBzl)-Ser(PO3HBzl)-Acp-Cys(Trt)-CTC resin is obtained. Acetic anhydride:DIPEA:resin = 6:12:1 (molar ratio).

[0056] Step 14: Wash the resin three times with methanol and then dry the resin (in preparation for cutting).

[0057] Step 15: Cutting: Cutting solution with 6 times the volume of resin (volume ratio: trifluoroacetic acid: anisole sulfide: 1,2-ethanedithiol: phenol: water = 87.5%: 5%: 2.5%: 2.5%: 2.5%), shake on a shaker for 2 hours, filter off the resin, precipitate the filtrate with ice-cold anhydrous ether, and wash the precipitate 3 times with ice-cold anhydrous ether. Finally, place the precipitate in a vacuum drying autoclave and dry at room temperature for 24 hours to obtain crude product Ac-Ser(PO3H2)-Ser(PO3H2)-Acp-Cys;

[0058] Ac-Ser(PO3H2)-Ser(PO3H2)-Acp-Cys was purified by HPLC with a purity of over 95%.

[0059] Purification steps: First, analyze the crude peptide using an analytical column (C18 analytical column) with a fast gradient. The mobile phase was: Aqueous phase A: 0.1% TFA / water; Organic phase B: 0.1% TFA / acetonitrile; Gradient: 5%-70% for 20 minutes. The main peak of the crude peptide was observed around 7 minutes.

[0060] Peptide separation was performed using a C18 column, and the target peak was collected. Mass spectrometry analysis revealed that the molecular weight of the collected target peak matched that of Ac-Ser(PO3H2)-Ser(PO3H2)-Acp-Cys, and the HPLC purity was found to be above 95%.

[0061] The collected target peaks were first frozen solid with liquid nitrogen, and then vacuum dried into powder using a freeze dryer.

[0062] One mg of the lyophilized powdered peptide was dissolved in water and analyzed by HPLC and MS. The results showed that the HPLC purity was above 95%, and the molecular weight remained unchanged by MS.

[0063] Pip is piperidine; TFA is trifluoroacetic acid; DIPEA is a base used to adjust the pH during condensation reactions; HBTU is a condensing agent; Wang resin is a type of resin.

[0064] In step 8, the part in parentheses represents the side chain protecting group of the corresponding amino acid. In step 10, all of these are removed at once; this step is usually referred to as cleavage.

[0065] I. Preparation of RBD antigen for COVID-19 vaccine

[0066] The coronavirus Spike gene was purchased from Nanjing Genscript Biotech Co., Ltd. (Catalog No.: C0425FA280-6). Primers were designed and synthesized as follows:

[0067] RBD-F:GCTCTGGGTTTCCAGGTTCCACCGGTAGGGTGCAGCCAACCGAGTCTATC

[0068] RBD-R:CACTGTGCTGGATATCTGCAGAATTCATTAGTGATGATGGTGGTGGTGATGATGGAAGTTCACGCACTTGTTCTTCACC

[0069] Using the Spike gene as a template and RBD-F / RBD-R as primers, the RBD-HIS gene fragment was amplified under the following conditions: 94℃, 5 min; (94℃, 30 s; 58℃, 30 s; 72℃, 1 min) x 30; 72℃, 5 min. The amplification system consisted of: 10X buffer (containing Mg2+), 5 μL; 2.5 mM dNTP, 2 μL; 10 μM primer1, 1 μL; 10 μM primer2, 1 μL; template, 2 μL; Taq, 0.5 μL; ddH2O, 38.5 μL. Using the HSA gene as a template and HSA-F1 / HSA-R2 as primers, the HSA fragment was amplified under the following conditions: 94℃, 5 min; (94℃, 30 s; 55℃, 30 s; 72℃, 1 min) x 30; 72℃, 5 min. Amplification system: 10X buffer (containing Mg2+), 5 μL; 2.5 mM dNTP, 2 μL; 10 μM primer 1, 1 μL; 10 μM primer 2, 1 μL; template, 1 μL; Taq, 0.5 μL; ddH2O, 38.5 μL. The PCDNA3.1 plasmid vector was digested with HindIII / EcoRI, and the large fragment was recovered using a gel extraction kit (purchased from Tiangen Biotech, catalog number: DP209). The operating procedure was as follows: RBD-HIS PCR product was digested with HindIII / EcoRI. The digestion system was: Buffer 2X: 2 μL, EcoRI: 0.5 μL, HindIII: 0.5 μL, template PCR product: 2 μL, water: 15 μL. The reaction was carried out at 37°C for 30 minutes, followed by gel extraction using a gel extraction kit (purchased from Tiangen Biotech, catalog number: DP209). The operating procedure was as follows. The digested and recovered vector and fragment were ligated using T4 DNase for 30 minutes (ThermoFisher, catalog number: 46300018). The ligation product was transformed into FAST1 competent E. coli cells and incubated overnight at 37°C. Clones were picked the next day for sequencing identification. For clones with correct sequencing, recombinant plasmids were extracted from the top 10 E. coli cells using an endotoxin-free plasmid extraction kit and named: pCDNA3.1-RBD HIS.

[0070] >RBD HIS AA

[0071] RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFHHHHHHHH*。

[0072] >RBD HIS NT

[0073] AGGGTGCAGCCAACCGAGTCTATCGTGCGCTTTCCTAATATCACAAACCTGTGCCCATTTGGCGAGGTGTTCAACGCAACCCGCTTCGCCAGCGTGTACGCCTGGAATAGGAAGCGGATCAGCAACTGCGTGGCCGACTATAGCGTGCTGTACAACTCCGCCTCTTTCAGCACCTTTAAGTGCTATGGCGTGTCCCCCACAAAGCTGAATGACCTGTGCTTTACCAACGTCTACGCCGATTCTTTCGTGATCAGGGGCGACGAGGTGCGCCAGATCGCCCCCGGCCAGACAGGCAAGATCGCAGACTACAATTATAAGCTGCCAGACGATTTCACCGGCTGCGTGATCGCCTGGAACAGCAACAATCTGGATTCCAAAGTGGGCGGCAACTACAATTATCTGTACCGGCTGTTTAGAAAGAGCAATCTGAAGCCCTTCGAGAGGGACATCTCTACAGAAATCTACCAGGCCGGCAGCACCCCTTGCAATGGCGTGGAGGGCTTTAACTGTTATTTCCCACTCCAGTCCTACGGCTTCCAGCCCACAAACGGCGTGGGCTATCAGCCTTACCGCGTGGTGGTGCTGAGCTTTGAGCTGCTGCACGCCCCAGCAACAGTGTGCGGCCCCAAGAAGTCCACCAATCTGGTGAAGAACAAGTGCGTGAACTTCCATCATCACCACCACCATCATCAC。

[0074] II. Antigen-Adjuvant Coupling

[0075] The antigen and adjuvant were conjugated using the Sulfo-SMCC cross-linking kit (ThermoFisher catalog number: 22122). A schematic diagram illustrating the conjugation principle is shown below. Figure 3 As shown.

[0076] 1. Add an appropriate amount of cross-linking agent (Sulfo-SMCC) to the RBD HIS antigen protein solution. The molar ratio of RBD HIS antigen to cross-linking agent should be controlled within the range of 1:5-1:100.

[0077] 2. Incubate the reaction mixture at room temperature for 30 minutes.

[0078] 3. Use a desalting column equilibrated with coupling buffer to remove excess crosslinking agent.

[0079] 4. Combine R101 and the desalted RBD His antigen in a molar ratio of 1:1 to 10:1.

[0080] 5. Incubate the reaction mixture at room temperature for 30 minutes or at 4°C for 2 hours.

[0081] III. Mouse Immunization

[0082] Male Balb / C healthy mice aged approximately 6–8 weeks were selected and immunized at multiple sites on their backs. The immunization groups were as follows; the immunization schedule was: initial immunization dose of 10 μg antigen / mouse, and aluminum adjuvant (B / C group) dose of 100 μL / mouse. A second immunization with the same dose was administered 15 days later; 7 days later, blood was collected from the tail and serum titers were measured using an indirect ELISA method.

[0083] Example 2: A novel immune adjuvant, its preparation method, and its application

[0084] A novel immune adjuvant, with the following molecular formula:

[0085]

[0086] A method for preparing a novel immune adjuvant includes the following steps:

[0087] Step 1: 2-CTC resin was swollen with dichloromethane, then Fmoc-Cys(Trt)-OH and DIPEA were added and reacted for 0.8 hours. Then methanol was added to end the reaction, followed by filtration to remove dichloromethane and washing with dimethylformamide to obtain Fmoc-Cys(Trt)-CTC resin.

[0088] Step 2: Add 15% by volume of piperidine / dimethylformamide solution to Fmoc-Cys(Trt)-CTC resin, then purge with nitrogen for 20 minutes, and dry to obtain H2N-Cys(Trt)-CTC resin.

[0089] Step 3: Wash the H2N-Cys(Trt)-CTC resin obtained in Step 2 with dimethylformamide;

[0090] Step 4: Add Fmoc-Acp-OH amino acid, DIPEA and HBTU to dimethylformamide for reaction to obtain Fmoc-Acp-Cys(Trt)-CTC resin;

[0091] Step 6: Wash the Fmoc-Acp-Cys(Trt)-CTC resin obtained in Step 4 with dimethylformamide;

[0092] Step 7: Add 15% of the volume of piperidine / dimethylformamide solution to Fmoc-Acp-Cys(Trt)-CTC resin, then purge with nitrogen for 20 minutes, and dry to obtain H2N-Acp-Cys(Trt)-CTC resin.

[0093] Step 8: Wash the H2N-Acp-Cys(Trt)-CTC resin obtained in Step 7 with dimethylformamide;

[0094] Step 9: Add Fmoc-Ser(PO3HBzl)-OH amino acid, DIPEA and HBTU to dimethylformamide to react and obtain Fmoc-Ser(PO3HBzl)-Acp-Cys(Trt)-CTC resin.

[0095] Step 10: Wash the Fmoc-Ser(PO3HBzl)-Acp-Cys(Trt)-CTC resin obtained in step 9 with dimethylformamide;

[0096] Step 11: Add 15% of the volume of piperidine / dimethylformamide solution to Fmoc-Ser(PO3HBzl)-Acp-Cys(Trt)-CTC resin, then purge with nitrogen for 20 minutes, and dry to obtain H2N-Ser(PO3HBzl)-Acp-Cys(Trt)-CTC resin.

[0097] Step 12: Wash the H2N-Ser(PO3HBzl)-Acp-Cys(Trt)-CTC resin obtained in Step 11 with dimethylformamide;

[0098] Step 13: Prepare according to the method in steps 9-12:

[0099] H2N-Ser(PO3H2Bzl)-Ser(PO3HBzl)-Acp-Cys(Trt)-CTC resin;

[0100] Step 14: Acetic anhydride and DIPEA are added to dimethylformamide to react and obtain Ac-Ser(PO3HBzl)-Ser(PO3HBzl)-Acp-Cys(Trt)-CTC resin;

[0101] Step 15: Wash Ac-Ser(PO3HBzl)-Ser(PO3HBzl)-Acp-Cys(Trt)-CTC resin with methanol;

[0102] Step 16: Mix the cutting fluid with the product from Step 15, cut for 1.5 hours under stirring, then filter to remove the resin, precipitate the filtrate with ice-cold anhydrous diethyl ether, wash the precipitate with ice-cold anhydrous diethyl ether, and finally place the precipitate in a vacuum drying oven and dry at room temperature to obtain the crude product Ac-Ser(PO3H2)-Ser(PO3H2)-Acp-Cys.

[0103] The preferred technical solution is that the degree of substitution of the 2-CTC resin is 0.8 mmol / g.

[0104] The preferred technical solution is as follows: In step 4, the molar ratio between Fmoc-Acp-OH amino acid, DIPEA, HBTU and resin is 2.8:5:2.8:1.

[0105] The preferred technical solution is as follows: In step 9, the molar ratio between Fmoc-Ser(PO3HBzl)-OH amino acid, DIPEA, HBTU and resin is 2.8:5:2.8:1.

[0106] The application of the novel immune adjuvant in vaccine preparation.

[0107] The above description is merely a preferred embodiment for explaining the present invention and is not intended to limit the present invention in any way. Therefore, any modifications or changes made to the present invention under the same inventive spirit should still be included within the scope of protection intended by the present invention.

Claims

1. A novel immunological adjuvant, characterized in that: Molecular formula is as follows:

2. A process for the preparation of the novel immunoadjuvant as claimed in claim 1, characterized by: The method comprises the following steps: Step 1: 2-CTC resin is swelled with dichloromethane, then Fmoc-Cys(Trt)-OH and DIPEA are added, and the reaction is carried out for 0.8-1.2 hours, then methanol is added for capping, followed by suction filtration to remove dichloromethane, and washing with dimethylformamide to obtain Fmoc-Cys(Trt)-CTC resin; Step 2: 15-25% piperidine / dimethylformamide solution is added to Fmoc-Cys(Trt)-CTC resin, then nitrogen is blown for 20-40 minutes, and H2N-Cys(Trt)-CTC resin is obtained after suction drying; Step 3: H2N-Cys(Trt)-CTC resin obtained in step 2 is washed with dimethylformamide; Step 4: Fmoc-Acp-OH amino acid, DIPEA and HBTU are added to dimethylformamide for reaction to obtain Fmoc-Acp-Cys(Trt)-CTC resin; Step 6: Fmoc-Acp-Cys(Trt)-CTC resin obtained in step 4 is washed with dimethylformamide; Step 7: 15-25% piperidine / dimethylformamide solution is added to Fmoc-Acp-Cys(Trt)-CTC resin, then nitrogen is blown for 20-40 minutes, and H2N-Acp-Cys(Trt)-CTC resin is obtained after suction drying; Step 8: H2N-Acp-Cys(Trt)-CTC resin obtained in step 7 is washed with dimethylformamide; Step 9: Fmoc-Ser(PO3HBzl)-OH amino acid, DIPEA and HBTU are added to dimethylformamide for reaction to obtain Fmoc-Ser(PO3HBzl)-Acp-Cys(Trt)-CTC resin; Step 10: Fmoc-Ser(PO3HBzl)-Acp-Cys(Trt)-CTC resin obtained in step 9 is washed with dimethylformamide; Step 11: 15-25% piperidine / dimethylformamide solution is added to Fmoc-Ser(PO3HBzl)-Acp-Cys(Trt)-CTC resin, then nitrogen is blown for 20-40 minutes, and H2N-Ser(PO3HBzl)-Acp-Cys(Trt)-CTC resin is obtained after suction drying; Step 12: H2N-Ser(PO3HBzl)-Acp-Cys(Trt)-CTC resin obtained in step 11 is washed with dimethylformamide; Step 13: H2N-Ser(PO3HBzl)-Ser(PO3HBzl)-Acp-Cys(Trt)-CTC resin is prepared according to the method of steps 9-12; Step 14: acetic anhydride and DIPEA are added to dimethylformamide for reaction to obtain Ac-Ser(PO3HBzl)-Ser(PO3HBzl)-Acp-Cys(Trt)-CTC resin; ​ Step 15: The Ac-Ser(PO3HBzl)-Ser(PO3HBzl)-Acp-Cys(Trt)-CTC resin is washed with methanol; Step 16: The cleavage solution is mixed with the product of Step 15, stirred for 1.5-2.5 h, then filtered to remove the resin, the filtrate is precipitated with ice-cold anhydrous ether, the precipitate is washed with ice-cold anhydrous ether, and finally the precipitate is placed in a vacuum drying oven and dried at room temperature to obtain the crude product Ac-Ser(PO3H2)-Ser(PO3H2)-Acp-Cys.

3. The method of claim 2, wherein: The degree of substitution of the 2-CTC resin is 0.8-1.2 mmol / g.

4. The method of claim 2, wherein: In Step 4, the molar ratio between Fmoc-Acp-OH amino acid, DIPEA, HBTU and resin is 2.8-3.2:5-7:2.8-2.9:

1.

5. The method of claim 2, wherein: In Step 9, the molar ratio between Fmoc-Ser(PO3HBzl)-OH amino acid, DIPEA, HBTU and resin is 2.8-3.2:5-7:2.8-2.9:1.

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