Fully synthetic Tn-based three-component cancer vaccine using covalently linked TLR4 ligand MPLA and iNKT cell agonist KRN-7000 as built-in adjuvant effectively protects mice from tumor development

We present a new strategy for self-adjuvanting vaccine development that has different types of covalently-linked immunostimulants as the carrier molecule.Using Tn antigen as the model,a three-component vaccine(MPLA-Tn-KRN7000)containing the TLR4 ligand MPLA and the iNKT cell agonist KRN7000 was designed and synthesized.This expands fully synthetic self-adjuvanting vaccine studies that use a single carrier to one with two different types of carriers.The corresponding two-component conjugate vaccines Tn-MPLA,Tn-KRN7000 and Tn-CRM197 were also synthesized,as controls.The immunological evaluation found that MPLA-Tn-KRN7000 elicits robust Tn-specific and T cell-dependent immunity.The antibodies specifically recognized,bound to and exhibited complement-dependent cytotoxicity against Tn-positive cancer cells.In addition,MPLA-Tn-KRN7000 increased the survival rate and survival time of tumor-challenged mice,and surviving mice reject further tumor attacks without any additional treatment.Compared to the glycoprotein vaccine Tn-CRM197,the two-component conjugate vaccines,Tn-MPLA and Tn-KRN7000,and the physical mixture of Tn-MPLA and Tn-KRN7000,MPLA-Tn-KRN7000 showed the most effect at combating tumor cells both in vitro and in vivo.The comparison of immunological studies in wild-type and TLR4 knockout mice,along with the test of binding affinity to CD1d protein suggests that the covalently linked MPLA-KRN7000 immunostimulant induces a synergistic activation of TLR4 and iNKT cell that improves the immunogenicity of Tn.This work demonstrates that MPLA-Tn-KRN7000 has the potential to be a vaccine candidate and provides a new direction for fully synthetic vaccine design.