Nanobodies present an appealing class of potential cancer therapeutics. The current study explores the in vivo expression of these molecules through DNA-encoded delivery. We hypothesized that this approach could address the rapid clearance of Nanobodies and, through half-life modulation, increase the produced levels in circulation. We therefore evaluated pharmacokinetics and efficacy of variants of an anti-death receptor 5 Nanobody (NbDR5), either monovalent or multivalent with half-life extension properties, after DNA-based administration. Intramuscular electrotransfer of a monovalent NbDR5-encoding plasmid (pNbDR5) did not result in detectable plasma levels in BALB/c mice. A tetravalent NbDR5-encoding plasmid (pNbDR5₄) provided peak concentrations of 54 ng/mL, which remained above 24 ng/mL during a 12-week follow-up. DNA-based delivery of these Nanobody formats fused to a Nanobody binding to serum albumin (NbSA), pNbDR5-NbSA and pNbDR5₄-NbSA, resulted in significantly higher plasma levels, with peak titers of 5.2 and 7.7 µg/mL, respectively. In an athymic-nude mice COLO 205 colon-cancer model, a quadrupled intramuscular DNA dose led to peak plasma levels of 270 ng/mL for pNbDR5₄ and 38 µg/mL for pNbDR5₄-NbSA. Potent anti-tumor responses were only observed for pNbDR5₄, following either intramuscular or intratumoral delivery. Despite comparable in vitro activity and superior plasma exposure, NbDR5₄-NbSA was less effective than NbDR5₄ in vivo, regardless of whether delivered as DNA or protein. Overall, DNA-based Nanobody delivery resulted in more potent and durable anti-tumor responses than protein-based Nanobody delivery. In conclusion, this study demonstrates pre-clinical proof of concept for DNA-based Nanobodies in oncology and highlights the improved outcome over conventional protein administration.

纳米抗体呈现出一类吸引人的潜在癌症疗法。目前的研究通过 DNA 编码的传递探索了这些分子的体内表达。我们假设这种方法可以解决纳米抗体的快速清除问题，并通过半衰期调节，增加循环中产生的水平。因此，我们在基于 DNA 的给药后评估了抗死亡受体 5 纳米抗体 (NbDR5) 变体的药代动力学和功效，无论是单价的还是具有半衰期延长特性的多价。单价 NbDR5 编码质粒 (pNbDR5) 的肌内电转移在 BALB/c 小鼠中没有导致可检测的血浆水平。四价 NbDR5 编码质粒 (pNbDR5 ₄) 提供了 54 ng/mL 的峰值浓度，在 12 周的随访期间保持在 24 ng/mL 以上。这些基于 DNA 的纳米抗体形式与结合血清白蛋白 (NbSA)、pNbDR5-NbSA 和 pNbDR5 ₄ -NbSA 的纳米抗体融合，导致血浆水平显着升高，峰值滴度分别为 5.2 和 7.7 µg/mL。在无胸腺裸鼠 COLO 205 结肠癌模型中，四倍的肌肉内 DNA 剂量导致 pNbDR5 4 的血浆峰值浓度为 270 ng/mL，pNbDR5 _{4 -NbSA}的血浆浓度峰值为38 µg/mL 。在肌内或肿瘤内递送后，仅观察到 pNbDR5 _{4的有效抗肿瘤反应。}尽管具有相当的体外活性和优越的血浆暴露，NbDR5 ₄-NbSA 在体内不如 NbDR5 ₄有效，无论是作为 DNA 还是蛋白质递送。总体而言，基于 DNA 的纳米抗体递送比基于蛋白质的纳米抗体递送产生更有效和持久的抗肿瘤反应。总之，这项研究证明了基于 DNA 的纳米抗体在肿瘤学中的临床前概念证明，并强调了与传统蛋白质给药相比的改进结果。