Development of an enzymatic assay for the detection of neutralizing antibodies against therapeutic angiotensin-converting enzyme 2 (ACE2)

Therapeutic proteins have the potential to elicit immune responses in animals and humans (Mire-Sluis et al., 2004; Yu et al., 2006; Shankar et al., 2008). Contributors to the response could include product related factors such as chemical modifications, impurities that co-purify with product, contaminants, formulation, aggregates, and clinical factors such as dose concentration, dosing frequency, route of drug administration, rate of administration, patient underlying disease, concomitant medication, and genetic status among others (Patten and Schellekens, 2003). Further, an immune response triggered by a therapeutic enzyme may neutralize the endogenous counterpart resulting in a decrease or depletion of the therapeutic and endogenous enzymes imposing safety concerns for patients. Therefore, monitoring of anti-drug antibody (ADA) and neutralizing antibody (NAb) responses to both the recombinant therapeutic enzyme and endogenous enzyme is important during early development and subsequent clinical studies. Testing considerations for NAb detection against therapeutic enzymes have been published mostly for lysosomal storage diseases (Wang et al., 2008). NAb cross-reactivity to the endogenous counterpart has also been characterized (Sominanda et al., 2010). Here, we describe an enzymatic NAb assay which detects neutralizing antibodies to both recombinant and endogenous angiotensin-converting enzyme 2 (ACE2). NAb assay sensitivity was optimized by selecting the assay incubation time as 20 min with an enzyme concentration of 0.5 μg/mL. Four anti-ACE2 antibodies out of a commercial panel of 18 were found to have neutralizing capabilities based upon their ability to abrogate ACE2 enzymatic activity. We demonstrated assay specificity by small peptide inhibitors specific for ACE or ACE2. DX600, an ACE2 specific inhibitor did not cross-react with ACE. Conversely, captopril, an inhibitor of ACE did not inhibit ACE2. The assay specificity for ACE2 neutralizing antibodies was further demonstrated by the lack of reactivity of two species control antibodies and 14 anti-ACE2 antibodies. Moreover, we demonstrated assay specificity to human endogenous ACE2 from human epithelial cells. Three human cell lines (Calu-3, Caco-2, Huh-7) were evaluated for the cell surface expression of ACE2 by flow cytometry and Western blot. Subsequently, whole cell lysates, cell culture supernatant, and live cells were evaluated in the assay. Results demonstrated that Calu-3 had elevated levels of ACE2 compared to Caco-2 or Huh-7. Calu-3 also demonstrated elevated ACE2 enzymatic activity in all three sources and could be inhibited by the ACE2 specific inhibitor DX600 as well as the neutralizing antibodies for the recombinant ACE2. Thus, we describe here a method to detect NAb against a therapeutic enzyme and assess NAb cross-reactivity to the native endogenous enzyme. The approach of method development described here could be applied for the assessment of NAb responses to other enzymatic therapeutics.