Purpose: Pre-existing anti-drug antibodies (pre-ADA) are endogenous antibodies against protein domains that overlap with biotherapeutic epitopes. Consequences of pre-ADA can vary from adverse safety effects, to loss of product efficacy, to no impact at all. It is important to carefully evaluate pre-ADA responses as a potential immunogenicity risk factor, particularly in the case of assessing a new type of biologic treatment for which immunogenicity data is very limited, if it exists at all.
Nanobodies® are a novel class of therapeutic proteins for which there is limited ADA data available. Drug X is a humanized, sequence-optimized checkpoint-inhibitor Nanobody®. To gather more immunogenicity information on this new biologic modality, a larger scale assessment of multiple treatment-naïve serum types including rhesus, normal human, and human cancer serum was initiated to evaluate for, and determine the prevalence of, pre-ADA against Drug X.
Methods: Meso Scale Discovery (MSD) electrochemiluminescence (ECL) technology was used to create a homogenous bridging immunoassay for detection of ADA against Drug X. An anti-Drug X monocolonal antibody was used as the positive control for this assay. For the initial screening assay, samples were diluted into a master mix containing both biotinylated Drug X and sulfo-TAG-labeled Drug X, and incubated at room temperature overnight. The sample/master mix combination was then added to MSD® Streptavidin-coated plates for two hours to allow for the capture of any formed complexes. The complexes were detected by ECL signal measurement on a MESO SCALE DISCOVERY® SECTOR Imager. If a sample tested positive, it would then undergo a confirmatory assay by immunodepletion with 10ug/ml unlabeled Drug X. A signal drop of >50% was reported as positive for immunodepletion.
Results: A minimum of 49 drug-naïve individuals per serum type were analyzed to determine pre-ADA prevalence against Drug X. In total, 90 rhesus, 50 normal human, and 49 cancer human individuals were assayed. 16/90 rhesus, 8/50 normal human, and 7/49 cancer human serum individuals tested positive in the screening assay and underwent confirmation for immunodepletion. Of the samples that initially screened positive, 10/16 rhesus, 6/8 normal human, and 5/7 cancer human individuals were positive for immunodepletion, and thus positive for pre-ADA against Drug X. Those negative for immunodepletion were concluded to be non-specific false positives.
Table 1: Immunogenicity Summary of Pre-ADA Against Drug X (listed as % of total samples analyzed)
A preliminary rhesus PK study was then initiated, and samples were analyzed for pre- and post-treatment ADA. The analysis determined that one animal was positive for pre-ADA, and continued to be ADA positive post-treatment, whereas the remaining animals were all negative for ADA.
Conclusion: An in-depth immunogenicity evaluation confirmed that Drug X, a novel Nanobody® construct, had a significant pre-ADA response in all three of the sample types tested (rhesus, normal human, and cancer human individuals). The pre-ADA response occurs in a similar percentage in each population tested.
A preliminary rhesus PK study concluded that one animal was positive for pre-ADA and continued to be ADA positive post-treatment. However, due to the limited number of animals in the study, more data is needed to determine if true correlation exists between pre-ADA and treatment-emergent ADA.
Minchao Chen– Senior Scientist, Merck Research Laboratories, Palo Alto, California
Derek Wiswell– Palo Alto, California
Shuli Zhang– Principal Scientist, Merck Research Laboratories, Palo Alto, California
Wolfgang Seghezzi– Palo Alto, California