Detection of anti-drug antibodies (ADA) that have a neutralizing capacity is an important aspect of immunogenicity evaluation during development of biotherapeutics, but developing and validating neutralizing antibody (NAb) assays that show direct interference of a biologic function is a challenging and resource-intensive activity. In particular, the need for adequate drug and target tolerance often requires extensive pre-treatment steps that limit assay sensitivity compared with a typical bridging-format assay used to detect binding ADA. Such limitations may complicate data interpretation as a positive ADA followed by a negative NAb result could be due to the presence of non-neutralizing antibodies or could be a false-negative for NAbs due to methodology differences.

To address such issues, we developed a novel assay for Nanobodies® and other antibody-derived therapeutics that solely detects ADA directed against the complementarity-determining regions (CDRs) involved in drug-target interactions. This was achieved by creating a “null variant” of the therapeutic drug, which has mutated CDRs rendering it non-functional for target binding but is otherwise identical to the drug compound. Non-CDR-binding antibodies are pre-complexed with the null variant of the Nanobody leaving only CDR-binding ADA with neutralizing potential (ANP) to be detected in this assay, which is called a NAb Epitope Characterization Assay (NECA). Method qualification results confirmed highly comparable assay characteristics (sensitivity, drug tolerance, selectivity and precision) of both the NECA and a validated ADA assay for the same Nanobody. A panel of purified neutralizing and non-neutralizing antibodies as well as non-clinical and clinical samples were used to further substantiate the fit-for-purpose and advantages of this novel assay format to detect ANP. In the clinical case study, a 20 to 40-fold difference in assay sensitivity existed between the validated ADA assay and NAb assay, which complicated data interpretation. Implementation of the NECA allowed unambiguous comparison of the levels of binding ADA and ANP in study samples which enabled us to delineate the true neutralizing capacity of the responses. Depending on the risk of the therapeutic, this method could be a valuable alternative for NAb testing by enabling earlier detection of ADA with neutralizing potential and ensuring adequate immunogenicity risk assessment.

具有中和能力的抗药物抗体（ADA）的检测是生物疗法开发过程中免疫原性评估的重要方面，但是开发和验证显示出对生物学功能的直接干扰的中和抗体（NAb）检测方法具有挑战性，并且密集活动。特别是，对足够的药物和靶标耐受性的需求通常需要大量的预处理步骤，与用于检测结合ADA的典型桥接形式检测方法相比，其限制了检测方法的敏感性。这样的局限性可能会使数据解释复杂化，因为阳性ADA继之以阴性的NAb结果可能是由于存在非中和性抗体，或者由于方法上的差异可能是对NAb的假阴性。

为了解决这些问题，我们为Nanobodies®和其他抗体衍生的疗法开发了一种新颖的检测方法，该方法仅检测针对与药物-靶标相互作用有关的互补决定区（CDR）的ADA。这是通过创建治疗药物的“无效变体”实现的，该变体具有突变的CDR，使其无法与靶标结合，但与药物化合物相同。将非CDR结合抗体与纳米抗体的无效变体进行预复合，仅留下具有中和潜力（ANP）的CDR结合ADA，以便在此分析中进行检测，这被称为NAb表位表征分析（NECA）。方法鉴定结果证实了高度可比的测定特性（灵敏度，药物耐受性，NECA和同一纳米抗体的经过验证的ADA分析方法的选择性和精密度）。使用一组纯化的中和和非中和抗体以及非临床和临床样品来进一步证实这种新颖的检测形式检测ANP的目的和优势。在临床案例研究中，经过验证的ADA分析和NAb分析之间存在20至40倍的分析灵敏度差异，这使数据解释变得复杂。NECA的实施可以明确比较研究样品中结合ADA和ANP的水平，这使我们能够确定反应的真正中和能力。根据治疗的风险，