Many naturally occurring peptides have poor proteolytic stability, which limits their therapeutic applications. Cyclotides are plant-derived cyclic peptides that resist proteolysis due to their highly constrained structure, comprising a head-to-tail cyclic backbone and three disulfide bonds that form a cystine-knotted core. This structure makes them useful as scaffolds onto which peptide sequences (epitopes) can be grafted. In this study, VHH7, an alpaca-derived nanobody that targets murine class II MHC molecules, was used for the targeted delivery of cyclotides to antigen-presenting cells (APCs). The cyclotides MCoTI-I, and MCoTI-I with a HA-tag (YPYDVPDYA) grafted into loop 6 (MCoTI-HA), were tested for immunogenic properties. To produce the requisite VHH7-peptide conjugates, a site-specific sortase A-catalyzed reaction in combination with a copper-free strain-promoted cycloaddition reaction was used. MCoTI-I alone did not display any obvious antibody response, thus showing the capacity of cyclotides as immunologically silent scaffolds. By contrast, MCoTI-I conjugated to VHH7 elicited antibodies against cyclic or linear MCoTI-I, thus suggesting a simple and robust approach for targeting cyclotides to APCs, and potentially to other cell types. A similar antibody response was observed when MCoTI-HA was conjugated to VHH7, but there was no reactivity toward a linear HA-tag itself, suggesting differences in conformational constraint between cyclotide-presented and linear epitopes. Studies of commercially available HA antibodies applied to MCoTI-HA confirmed that the conformation of peptide immunogens affects their reactivity. Thus, the production of antibodies that recognize constrained epitopes may benefit from engraftment onto scaffolds such as cyclotides. More broadly, this study validates that a prototypic cyclotide, a member of a peptide family that has proven to be useful as drug design scaffolds in many other studies, can efficiently reach a specific target in vivo.

中文翻译：

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通过结合到纳米抗体上有针对性地递送环氧化物。

许多天然存在的肽的蛋白水解稳定性差，这限制了它们的治疗应用。环肽是植物来源的环肽，由于其高度受限的结构而可抵抗蛋白水解，包括头尾环的主链和三个二硫键，形成胱氨酸打结的核心。这种结构使它们可用作可移植肽序列（表位）的支架。在这项研究中，靶向羊II类MHC分子的羊驼来源的纳米抗体VHH7用于将环氧化物靶向递送至抗原呈递细胞（APC）。测试了接枝到环6（MCoTI-HA）中的具有HA标签的环化物MCoTI-1和MCoTI-1（YPYDVPDYA）的免疫原性。为了产生必需的VHH7-肽缀合物，将位点特异性分选酶A催化的反应与无铜应变促进的环加成反应结合使用。单独的MCoTI-1没有显示任何明显的抗体应答，因此显示了环肽作为免疫沉默支架的能力。相比之下，缀合至VHH7的MCoTI-1引发了针对环状或线性MCoTI-1的抗体，从而提出了一种简单而稳健的方法将环氧化物靶向APC，并可能靶向其他细胞类型。当MCoTI-HA与VHH7偶联时，观察到相似的抗体反应，但对线性HA-tag本身没有反应性，表明在环肽和线性表位之间的构象限制不同。应用于MCoTI-HA的市售HA抗体的研究证实，肽免疫原的构象会影响其反应性。因此，识别受约束的表位的抗体的产生可受益于植入到支架如环氧化物上。从更广泛的意义上说，这项研究验证了原型环肽是一种肽家族的成员，该肽家族已在许多其他研究中被证明可以用作药物设计支架，可以有效地达到特定的靶标。体内。