Many experimental cancer vaccines are exploring toll-like receptor agonists (TLRas) such as CpG, a DNA motif that agonizes toll-like receptor 9 (TLR9), to trigger immune responses that are potent and molecularly-specific. The ability to tune the immune response is especially important in the immunosuppressive microenvironments of tumors. Because TLR9 is located intracellularly, CpG must be internalized by immune cells for functionality. Polyplexes can be selfassembled through electrostatics using DNA (anionic) condensed by a positively charged carrier. These structures improve cell delivery and have been widely explored for gene therapy. In contrast, here we use cationic poly (β-amino esters) (PBAEs) to assemble polyplexes from CpG as an adjuvant to target and improve immune stimulation in cells and mouse models. Polyplexes were formed over a range of PBAE:CpG ratios, resulting in a library of complexes with increasingly positive charge and stronger binding as PBAE:CpG ratio increased. Although higher PBAE:CpG ratios exhibited improved CpG uptake, lower ratios of PBAE:CpG—which condensed CpG more weakly, activated DCs and tumorspecific T cells more effectively. In a mouse melanoma model, polyplexes with lower binding affinities improved survival more effectively compared with higher binding affinities. These data demonstrate that altering the polyplex interaction strength impacts accessibility of CpG to TLRs in immune cells. Thus, physiochemical properties, particularly the interplay between charge, uptake, and affinity, play a key role in determining the nature and efficacy of the immune response generated. This insight identifies new design considerations that must be balanced for engineering effective immunotherapies and vaccines.