Detection of Memory B Activity Against a Therapeutic Protein in Treatment-Naïve Subjects

Bridging immunoassays commonly used to detect and characterize immunogenicity during biologic development do not provide direct information on the presence or development of a memory anti-drug antibody (ADA) response. In this study, a B cell ELISPOT assay method was used to evaluate pre-existing ADA for anti-TNFR1 domain antibody, GSK1995057, an experimental biologic in treatment naive subjects. This assay utilized a 7-day activation of PBMCs by a combination of GSK1995057 (antigen) and polyclonal stimulator followed by GSK1995057-specific ELISPOT for the enumeration of memory B cells that have differentiated into antibody secreting cells (ASC) in vitro. We demonstrated that GSK1995057-specific ASC were detectable in treatment-naïve subjects with pre-existing ADA; the frequency of drug-specific ASC was low and ranged from 1 to 10 spot forming units (SFU) per million cells. Interestingly, the frequency of drug-specific ASC correlated with the ADA level measured using an in vitro ADA assay. We further confirmed that the ASC originated from CD27⁺ memory B cells, not from CD27^--naïve B cells. Our data demonstrated the utility of the B cell ELISPOT method in therapeutic protein immunogenicity evaluation, providing a novel way to confirm and characterize the cell population producing pre-existing ADA. This novel application of a B cell ELISPOT assay informs and characterizes immune memory activity regarding incidence and magnitude associated with a pre-existing ADA response.