Synthesis And Activity Evaluation Of Tumor-Associated Tn Antigen Vaccine Conjugated With NKT And TLR4 Agonists

ObjectiveAt present,the main challenges for synthetic vaccines including how to induce the antigen-specific immune responses,especially high titers IgG antibodies and produce immune memory by immune system,overcoming immune tolerance;which are expected to enhance the immunogenicity of antigens.The Tn antigen is one of the most excellent and potential tumor-associated carbohydrate antigens(TACAs),which is expressed in a variety of tumor cells including breast cancer cells.However,the Tn antigen is a T cell-independent antigen,which was found hardly to produce high-affinity IgG antibodies and easily to be cleared in vivo.These factors plague the application of the Tn antigen.To solve this problem,many researches have been done,one of the effective strategies is connectting the Tn antigen to a carrier as well as an adjuvant.In this work,we have rationally designed a three-component vaccine containing the Tn antigen,MPLA(a TLR4 agonist that has been approved by the FDA for use as an adjuvant)and KRN7000(iNKT cell agonist).What’s more,two-component vaccine(MPLA-Tn and KRN7000-Tn),which provide the basis for subsequent immunological activity research and structure-activity relationship exploration.MethodsSynthesis of KRN7000 building block:D-galactose was used as the starting material,which hydroxy group were protected by acetyl group,the O-1 position is selectively protected by p-toluene thiophenol and all acetyl groups are removed.Benzaldehyde aldehyde acetal selectively protected the C-4 and C-6 hydroxyl groups,and benzyl protected the C-2 and C-3 hydroxyl groups.The O-6 position was reduced to the hydroxyl group and replaced by sodium azide to gave the glycosylation donor.Glycosylation donor was glycosylated with phytosphingosine receptor 4 to give a undeprotected KRN7000;azide was reduced to an amino group,which reacted with adipic anhydride to give a 6-position modified KRN7000 building block.Synthesis of Tn building block:D-aminogalactose hydrochloride was used as the starting material;which O-2 position replaced by azide;O-1 position,O-3 position,O-4 position and O-6 position protected by acetyl groups;and the O-1 position,which acetyl group was removed,reacted with trichloroacetonitrile to give a glycosylation donor.The glycosylation donor was glycosylated with a threonine acceptor,the azide at the C-2 position was reacted to form an acetyl group,and the Fmoc was removed to obtain the Tn antigen building block.Synthesis of MPLA building blocks:Compound 24 is the starting material;acetic anhydride replaced the hydroxyl group at position C-3 to obtain a glycosylation donor.The hydroxyl group at the C-3 position of the compound 24 was protected by benzyl bromide,the p-tolylthio group at the C-1 position was substituted by azide ethanol,and the hydroxyl group of 6-position was selectively reduced to obtain a glycosylation acceptor.The glycosylation donor was glycosylated with a glycosylation acceptor to give a disaccharide,which phthaloyl groups and acetyl group were removed,and the self-made fatty acid chain were attached.the 1-position modified MPLA,which was obtained by selectively disconnecting the 4-position and attaching a phosphate group to obtain an MPLA building block.Coupling of MPLA-Tn-KRN7000 three-component saccharide antigen anti-tumor vaccine:KRN7000 building block and Tn building block were esterified to give the Tn-KRN7000 building block.The alkyne group of the Tn-KRN7000 building block and the azide on the MPLA building block were coupled by a classic click reaction to obtain a MPLA-Tn-KRN7000 three-component glycoantigen antitumor vaccine.Coupling of two-component saccharide antigen anti-tumor vaccine:MPLA-Tn and KRN7000-Tn two-component glycoantigen antitumor vaccines were synthesized by the classical coupling reaction.Preliminary activity evaluation:Three compounds were immunized on mice on day 1,day 14,day 21,and day 28,and blood samples were taken on day 21,which titers of IgG antibodies were measured.Results and ConclusionsThe MPLA-Tn-KRN7000,MPLA-Tn and KRN7000-Tn anti-tumor vaccine were successfully synthesized by a series of organic chemistry methods.The structure of the target product compounds were confirmed by 1H NMR、13C NMR、HHCOSY、HSQC and high resolution mass spectrometry,etc.,indicating that the spectrum is consistent with the structure.At the same time,we synthesized the carbohydrate vaccine by the synthetic method with the advantages of clear synthetic route,stable and controllable product quality.Preliminary immunization studies showed that the MPLA-Tn-KRN7000 three-component vaccine induced IgG titers significantly higher than the MPLA-Tn and KRN7000-Tn two-component vaccine.It is proved that the dual adjuvant of MPLA-Tn-KRN7000 three-component vaccine can effectively improve the immunogenicity of Tn saccharide antigen.The adjuvants MPLA and KRN7000 have synergistic effects in the specific immunization of Tn antigen...