Antigenic peptide delivery to antigen-presenting cells using a CD40-coiled coil affinity-based platform

Delivery of antigenic peptides to antigen presenting cells (APCs) such as dendritic cells (DCs) using monoclonal antibodies (mAbs) is an attractive approach to evoke antigen-specific T cell activation and improve drug efficacy. Peptide linkage to mAbs has previously been achieved through genetic fusion, chemical conjugation, nano-engineered platforms and high affinity peptides. In this study, we have developed a flexible antibody-peptide linking technology using oppositely charged coiled coil domains to non-covalently link peptides to mAbs. The technology comprises (1) an anti-CD40 mAb connected with negatively charged E domains and (2) an immunogenic OVA peptide (SIINFEKL) from ovalbumin used as a model antigenic peptide fused with positively charged K domains. Combining these constructs leads to the formation of complexes that can be targeted to CD40 expressed on cells. Proof of concept antibody constructs connected with E domains generated from transient expressions exhibited good manufacturability, binding, and stability attributes comparable to a control mAb. Also, optimal repeat lengths for coiled-coil oligomerization domains were identified in these studies. Binding kinetics studies showed that connecting E domains to mAbs do not impede Fc gamma and neonatal receptor interactions. Additionally, formation of stable complexes capable of binding CD40 expressing cells was demonstrated in vitro. In vivo functionality evaluations showed that treatment of human CD40 transgenic mice with complexes elicited expansion of OVA peptide-specific CD8+ T cells and potent antitumor effects superior to peptide monotherapies. Overall, these findings demonstrate that the technology has great potential for application as an in vivo tool for antigenic peptide delivery.

利用单克隆抗体（monoclonal antibodies, mAbs）将抗原肽递送至树突状细胞（dendritic cells, DCs）等抗原呈递细胞（antigen presenting cells, APCs），是诱导抗原特异性T细胞活化、提升药物疗效的极具吸引力的策略。此前，肽与单克隆抗体的连接可通过基因融合、化学偶联、纳米工程平台以及高亲和力肽等方式实现。本研究开发了一种灵活的抗体-肽连接技术，借助带相反电荷的卷曲螺旋结构域实现肽与单克隆抗体的非共价连接。该技术包含两大组件：（1）与带负电的E结构域相连的抗CD40单克隆抗体；（2）以卵清蛋白来源的免疫原性OVA肽（SIINFEKL）作为模式抗原肽，与带正电的K结构域融合。将上述两种构建体结合即可形成复合物，可靶向结合细胞表面表达的CD40。经瞬时表达制备的带E结构域的概念验证抗体构建体，展现出与对照单克隆抗体相当的可制造性、结合特性与稳定性。本研究同时确定了适用于卷曲螺旋寡聚化结构域的最优重复长度。结合动力学研究表明，将E结构域连接至单克隆抗体不会阻碍其与Fcγ受体及新生儿Fc受体的相互作用。此外，体外实验证实，所形成的稳定复合物可结合表达CD40的细胞。体内功能评价结果显示，对人CD40转基因小鼠施用该复合物，可诱导OVA肽特异性CD8+ T细胞扩增，且其强效抗肿瘤效果优于肽单药治疗。综上，本研究结果表明，该技术作为抗原肽体内递送工具具备巨大的应用潜力。