pH-switchable shark-derived single domain antibodies for biomedical applications

几十年来，抗体在诊断和治疗领域的各种应用中一直是不可替代的分子。同源抗原的高度特异性与Fc介导的免疫效应功能相结合，使抗体成为医学应用的吉祥工具。除了具有经典的重链和轻链组成的抗体外，鲨鱼的获得性免疫谱系还包括一个仅有重链的同型抗体，称为IgNAR，其中抗原结合仅由一个称为vNAR的小而高度稳定的结构域介导。近年来，vNAR片段已经发展成为有希望的靶向结合支架，可以为医学和生物技术应用量身定制。这些结构域具有高亲和力和特异性、小尺寸、高物理化学稳定性和低生产成本等有益特性。在拟议的项目中，我们着手于产生和利用pH响应型vNAR结构域用于生物治疗。这种pH可调的vNAR结构域对于在酸性肿瘤微环境中特异性靶向抗原可能很有吸引力。因此，在酸性pH下对肿瘤相关抗原具有较高亲和力的变体，但在生理条件下亲和力显著较低的变体，可以减少传统抗体疗法中经常观察到的脱靶效应。此外，还将开发pH依赖和抗独特型vNAR变体，旨在提高现有治疗性抗体的靶向疗效。这些掩蔽性VNAR在生理pH下对抗体具有高亲和力，但在弱酸性条件下亲和力较低。这种作用会阻止健康组织中的治疗性抗体，而在到达酸性肿瘤微环境时会被揭开。

For decades, antibodies have been irreplaceable molecules for various applications encompassing the field of diagnostics as well as therapy. The high specificity for a cognate antigen combined with Fc-mediated immune effector functions renders antibodies auspicious tools for medical applications. In addition to antibodies with the classical composition of heavy and light chains, the adaptive immune repertoire of sharks also includes a heavy-chain only isotype named IgNAR, where antigen binding is mediated exclusively by a small and highly stable domain referred to as vNAR. In recent years, vNAR fragments have evolved as promising, target-binding scaffolds that can be tailored for applications in medicine and biotechnology. These domains comprise beneficial properties such as high affinity and specificity, small size, high physicochemical stability and a low cost of production. In the proposed project we set out to generate and utilize pH-responsive vNAR domains for biotherapeutic applications. Such pH-switchable vNAR domains might be attractive for the specific targeting of antigens in the acidic tumor microenvironment. As such, variants comprising higher affinity for a tumor-associated antigen at acidic pH but significantly lower affinity under physiological conditions could reduce off-target effects which are often observed for conventional antibody therapeutics. In addition, pH-dependent and anti-idiotypic vNAR variants will be developed aimed at increasing the on-target efficacy of already available therapeutic antibodies. These masking vNARs comprise high affinity for the antibody at physiological pH, but low affinity under slightly acidic conditions. This effect would block the therapeutic antibody in healthy tissue, while being unmasked upon reaching the acidic tumor microenvironment.