In the present study, we conducted ADA analysis in a large set of plasma and ocular samples from minipigs and cynomolgus monkeys treated with different intraocular biotherapeutics. Our analysis revealed a significant association between ADA incidence in plasma and ocular fluids in full agreement with our previous preliminary observations (18). In particular, none of the animals had detectable ocular ADAs in the absence of detectable ADAs in plasma. ADAs appeared in plasma at median 22 days postdose, about 16 days earlier than in AH. Taken together, our data showed that ocular ADAs appear only in animals with preceding systemic ADA response, thus suggesting the systemic origin of ocular ADAs.

Only about a half of the animals with positive test results in plasma had detectable ADAs in ocular fluids. Given that ocular ADAs seem to originate from the systemic circulation, it is likely that this discordance in the test results was due to low levels of ocular ADAs caused by the anatomical and physiological barriers to the diffusion of macromolecules into in the eye (the blood-aqueous and the blood-retinal barriers). Thus, ocular ADA levels may not reach the detection limit, especially when systemic ADA levels are low. This effect was indeed apparent in AH, which has low concentration of proteins due to both the ocular barriers and its high turnover rate (20). Consequently, ADA incidence in AH was the lowest among the three matrices, and ADA-negative results in AH did not fully exclude ADA-positivity in VH. The disruption of the blood-ocular barrier seen in the target indications of IVT biotherapeutics (e.g., due to inflammatory neo-vascularization) can facilitate the diffusion of macromolecules into the eye and potentially result in a better concordance between the presence of systemic and ocular ADAs in patients. However, leaky vessels do not appear to be a prerequisite for the appearance of ocular ADAs, as ocular ADAs were detected in our study in healthy animals with an intact blood-ocular barrier.

The observed kinetics of ADA response to IVT administered drugs appears to be in line with the systemic origin of ocular ADAs. Slow permeation of systemic ADAs through the ocular barriers can explain the observed time lag between the appearance of ADAs in plasma and AH. Although the immune complex assay used for ocular matrices cannot detect the IgM isotype, which appears at early stages of an immune response, this limitation unlikely impacts the results observed in our study because IgM were not detected or detected at very low levels in AH of animals and humans (21,22,23). However, IgM was detected in the eyes of animals and humans with inflammatory conditions (24, 25) and in patients treated with IVT administered drugs (26), indicating that the onset and composition of ocular ADAs may differ in healthy and patient populations.

Plasma and AH samples in our study were taken over a short period of time after a single IVT drug administration, and only one terminal VH sample was available for each animal. Therefore, the duration of ADA responses could not be reliably assessed based on the data in our study; the observed decrease of ADA signal in the late AH samples should be interpreted cautiously in the absence of titration data. Moreover, the kinetics of ocular ADA response after a single dose can differ from that after multiple doses because high drug concentration in the eye expected after chronic administration can lead to an accumulation of ADAs in ocular fluids in form of drug-ADA complexes. Thus, we expect a higher concordance between ADA-positive plasma and ocular samples in studies with chronic drug treatment and longer sampling period.

The data available in the study only allowed an assessment based on signal levels, which provide limited comparability regarding the magnitude of the immune response—a fundamental problem of ADA assays (27). This can be partially compensated by sample titration, which allows a quasi-quantitative measure of the magnitude of the ADA response, yet ocular samples cannot be obtained in large amount required for such approach. Therefore, the comparisons based on ADA signal levels ought to be interpreted as exploratory owing to the lack of titer data, use of two different assays, and some measurements being close to the upper limit of the assay signal range.

Immunogenicity in animals is per se not predictive of the incidence of clinical immunogenicity owing to immunologic incompatibility of preclinical species with human or humanized biotherapeutics (1, 3). However, given the anatomical and physiological similarity between minipig, monkey and human eyes (28, 29), it is likely that ocular ADA response in humans has the same underlying mechanism as in these animals, namely, the formation of systemic ADAs first and their subsequent entry into VH. The possible contribution of ocular ADAs to intraocular inflammation could not be analyzed in this study due to the lack of histopathology data. It is worth noting that other factors, such as drug impurities and protein aggregates, can play a role in intraocular inflammation after IVT biologic drug administration (10).

Our study has provided so far the most comprehensive analysis of ocular ADAs in preclinical species. More studies are certainly needed to further elucidate the mechanisms of ocular antidrug immune response and their clinical relevance. Isotyping and neutralizing assays can be used to characterize ADA response and predict potential clinical sequalae, such as loss of efficacy without apparent adverse reactions caused by neutralizing non-compliment binding antibodies. Moreover, such in-depth analysis may provide another line of evidence for the systemic origin of ocular ADAs by showing that ocular and systemic ADAs have the same characteristics. To our knowledge, no studies with conventional isotyping or neutralizing assays have been reported for ocular matrices likely because the analysis of ocular samples poses a substantial bioanalytical challenge. The novel immune complex assay format can be potentially adapted to selectively detect ADA isotypes (30, 31), enabling detailed analysis of ocular ADA response.

Overall, our data support the use of plasma as a surrogate matrix for the detection of ocular ADA response. Apart from better concordance with the test results in VH, plasma offers other advantages over AH in the clinical setting. These include much better availability, which enables frequent monitoring and extensive bioanalysis, such as re-analysis and titration, and the early onset of ADAs in plasma, which allows timely implementation of mitigation measures before ADAs appear in the eye. Moreover, spared AH samples can be used for exploratory investigations, such as biomarker or target measurements. The key question yet to be answered is however not the detection of ADAs per se but whether we can determine a threshold for the magnitude of the systemic ADA response above which the achieved ocular ADA levels would lead to clinical consequences.