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 self-assembled 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 tumor-specific 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.

中文翻译：

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多聚体相互作用强度是癌症免疫疗法中效能的驱动力

许多实验性癌症疫苗正在探索诸如CpG的收费型受体激动剂（TLRas），CpG是一种激动收费型受体9（TLR9）的DNA基序，以触发有效且分子特异性的免疫反应。调节免疫应答的能力在肿瘤的免疫抑制微环境中尤其重要。由于TLR9位于细胞内，因此CpG必须被免疫细胞内在化才能发挥功能。可以使用带正电的载体缩合的DNA（阴离子）通过静电作用自组装多聚体。这些结构改善了细胞的传递，并已广泛用于基因治疗。相反，这里我们使用阳离子聚（β-氨基酯）（PBAEs）从CpG组装多聚体作为佐剂，以靶向和改善细胞和小鼠模型中的免疫刺激。在一定范围的PBAE：CpG比率范围内形成了复合物，从而形成了一个带正电荷的复合物库，并且随着PBAE：CpG比率的增加，结合力更强。尽管较高的PBAE：CpG比率显示出改善的CpG吸收，但是较低的PBAE：CpG比率（较弱地浓缩CpG），更有效地激活了DC和肿瘤特异性T细胞。在小鼠黑色素瘤模型中，与较高的结合亲和力相比，具有较低的结合亲和力的多链体可更有效地提高生存率。这些数据表明改变多态相互作用强度会影响免疫细胞中CpG对TLR的可及性。因此，理化性质，特别是电荷，摄取和亲和力之间的相互作用，在确定所产生的免疫应答的性质和功效中起关键作用。