Dai Yanshan, Dong Huijin, Gleason Carol, Mora Johanna, Kolaitis Gerry, Balasubramanian Nanda, Surapaneni Sekhar, Kozhich Alexander, Jawa Vibha
Clinical Pharmacology, Pharmacometrics, Disposition & Bioanalysis, Bristol Myers Squibb Company, Princeton, New Jersey, 08543, USA.
AAPS J. 2023 Apr 3;25(3):35. doi: 10.1208/s12248-023-00805-6.
Pre-existing adeno-associated viruses (AAV) neutralizing antibodies (NAb) can prevent AAV vectors from transducing target tissues. The immune responses can include binding/total antibodies (TAb) and neutralizing antibodies (NAb). This study is aimed at comparing total antibody assay (TAb) and cell-based NAb assay against AAV8 to help inform the best assay format for patient exclusion criteria. We developed a chemiluminescence-based enzyme-linked immunosorbent assay to analyze AAV8 TAb in human serum. The specificity of AAV8 TAb was determined using a confirmatory assay. A COS-7-based assay was used to analyze anti-AAV8 NAbs. The TAb screening cut point factor was determined to be 2.65, and the confirmatory cut point (CCP) was 57.1%. The prevalence of AAV8 TAb in 84 normal subjects was 40%, of which 24% were NAb positive and 16% were NAb negative. All NAb-positive subjects were confirmed to be TAb-positive and also passed the CCP-positive criteria. All 16 NAb-negative subjects did not pass the CCP criterion for the positive specificity test. There was a high concordance between AAV8 TAb confirmatory assay and NAb assay. The confirmatory assay improved the specificity of the TAb screening test and confirmed neutralizing activity. We proposed a tiered assay approach, in which an anti-AAV8 screening assay should be followed by a confirmatory assay during pre-enrollment for patient exclusions for AAV8 gene therapy. This approach can be used in lieu of developing a NAb assay and can be also implemented as a companion diagnostic assay for post-marketing seroreactivity assessments due to ease of development and use.
预先存在的腺相关病毒（AAV）中和抗体（NAb）可阻止AAV载体转导靶组织。免疫反应可包括结合/总抗体（TAb）和中和抗体（NAb）。本研究旨在比较针对AAV8的总抗体检测（TAb）和基于细胞的NAb检测，以确定用于患者排除标准的最佳检测形式。我们开发了一种基于化学发光的酶联免疫吸附测定法来分析人血清中的AAV8 TAb。使用确证试验确定AAV8 TAb的特异性。使用基于COS-7的检测法分析抗AAV8 NAb。确定TAb筛查切点因子为2.65，确证切点（CCP）为57.1%。84名正常受试者中AAV8 TAb的患病率为40%，其中24%为NAb阳性，16%为NAb阴性。所有NAb阳性受试者均被确认为TAb阳性，且也通过了CCP阳性标准。所有16名NAb阴性受试者均未通过阳性特异性检测的CCP标准。AAV8 TAb确证试验与NAb试验之间具有高度一致性。确证试验提高了TAb筛查试验的特异性并确认了中和活性。我们提出了一种分层检测方法，即在AAV8基因治疗患者入组前进行抗AAV8筛查试验后应进行确证试验以排除患者。该方法可用于替代开发NAb检测，并且由于易于开发和使用，也可作为上市后血清反应性评估的伴随诊断检测来实施。
