Improved immunogenicity of a self tumor antigen by covalent linkage to CD40 ligand

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Abstract
The interaction between the CD40 ligand (CD40L) and CD40 on antigen‐presenting cells (APCs) is critical in promoting humoral and cellular immune responses. Agonistic anti‐CD40 monoclonal antibody and soluble CD40L can act as powerful adjuvants to promote vaccination, but usually require repeated high‐dose treatment. In this study, we demonstrate that the adjuvant effect of CD40L can be greatly improved by directly linking the antigen to CD40L. We constructed a fusion protein (Id‐CD40L) consisting of the extracellular domain of CD40L and the idiotype (Id) protein, a weakly immunogenic tumor‐specific antigen derived from the murine 38C13 B‐cell lymphoma. The soluble Id‐CD40L fusion protein retained CD40 binding activity and stimulated CD80 and CD86 upregulation and interleukin (IL)‐12 production by macrophages. Immunization of mice with Id‐CD40L without adjuvants resulted in high titers of anti‐Id Abs dominated by the IgG1 isotype and protected the mice from subsequent lethal tumor challenge. In a dose‐response study, we demonstrated that Id‐CD40L elicited anti‐Id antibody (Ab) responses in all immunized animals, even at a dose as low as 0.5 μg. Immunization with free Id and an IgG‐CD40L fusion protein, which was identical in structure to Id‐CD40L but lost the Id determinant, resulted in significant lower anti‐Id responses, indicating that physical linkage between the tumor antigen and CD40L was required for the optimal immune response. These results demonstrate that fusing CD40L to a candidate antigen can greatly improve the adjuvant activity of CD40L. This approach may be useful in developing vaccines for a variety of malignant and infectious diseases.