Purpose: An immunogenicity assay for an IgG4 therapeutic was developed to detect and monitor patient immune responses in support of a Phase II safety and efficacy study. The therapeutic is a humanized monoclonal IgG4 antibody, without a modified Fc hinge region (unlocked IgG4), therefore IgG4 arm exchange is expected to occur in vivo between the therapeutic antibody and endogenous circulating antibody, and in vitro between antibodies found in normal human serum used for assay controls and labeled drug used as assay reagents (Aalberese et al, Immunology, 2002). The hybrid antibodies formed during these in vitro exchange events may alter assay signal which could impact interpretation and reporting of results of patient immune responses to the therapeutic. The arm exchange presents challenges in assay development and consideration should be given to several factors to minimize the reporting of false negative results (Partridge et al, Bioanalysis, 2017).
Methods: The effects of hybrid IgG4 antibody formation in the immunogenicity assay were evaluated by measuring the change in assay signal after combining the labeled drug mixture (LDM) over incubation periods ranging from 18 hours to 1 minute prior to performing the assay. To determine the effect of the presence of serum on hybrid antibody formation, two treatments were conducted: Set 1 consisted of the assay low positive control (ADA spiked into serum) or negative control (normal pooled human serum) added to the LDM in assay buffer at each sequential incubation period, and Set 2 in which the same controls were added to all LDM incubations immediately prior to running the assay.
Results: The results showed significant differences between the two treatments. In Set 1 a correlation was observed between increases in signal in all 3 controls when adding to LDM at timepoint 0. In Set 2 there was no apparent correlation between signal change when controls were added at each incubation. The relative signal increase seen in Set 1 NC compared to LPC could have the effect of increasing false negative results due to disproportionate increase in assay cutpoint with longer incubation times. LDM incubated simultaneously with the samples resulted in a lesser signal increase which may be due to endogenous IgG4 present in the serum which produces slightly more half labeled antibodies and fewer hybrid LDM antibodies.
Conclusion: Several factors in the assay were critical to generating consistent and accurate results from the assay. (1) Preparation of labeled bridging reagents must occur within a consistent time window prior to conducting assay. (2) Samples and controls must be incubated simultaneously with labeled bridging reagents, and (3) the % matrix in all assay samples and controls must be consistent. When these factors are controlled, the effects of hybrid IgG4 antibody formation is mitigated in the bridging assay, allowing for consistent and accurate assessments of patient immunogenic responses to unlocked IgG4 therapeutics.
Christian Braithwaite– Scientist II, Biogen, Cambridge