Background To induce a prominent anti-tumor T-cell response, a viral or tumor derived antigen epitope imbedded in a longer synthetic peptide (SLP) can be used, which also requires internalization and processing by antigen presenting cells (APCs) to enable T cell priming. Herein we present the design and evaluation of a CD40 targeting tetravalent bispecific antibody, binding peptides through affinity as an antibody-drug conjugate. APC activation as well as in vitro and in vivo T-cell proliferation studies demonstrate retained agonistic activity as well as improved T cell proliferation/expansion in vitro and in vivo, compared to non-linked peptide/antibody mixes.

Methods T-cell priming was evaluated with B3Z assay or a cytomegalovirus (CMV) model and displayed superior uptake to non-bound peptide in the co-stimulatory independent B3Z assay. In addition, intracellular peptide release in APCs was analysed using a unique quenching strategy displaying peptide release after around 4-6 hour post antigen.

Results Peptide stability in vitro, when bound to the antibody, was analysed by mass spectrometry and displayed prolonged peptide stability in serum, increasing the peptide half-life by 15 times in vitro (

Conclusions Data support that the novel delivery system can improve antigen targeting to dendritic cells, but can also provide a prolonged peptide half-life as well as a peptide delivery to APCs. Combined this improves the efficiency of both antigen delivery and CD40 agonistic functionality.