Investigation of pre-existing reactivity to biotherapeutics can uncover potential immunogenic epitopes and predict immunogenicity risk

Abstract

Despite recent advances in the development of tools to predict immunogenicity risk of biotherapeutic molecules, the ability of a protein to elicit the formation of anti-drug antibodies (ADA) remains one of the most common causes for termination of clinical development programs. In this study, we use ADA assays to detect and measure pre-existing reactivity or the ability of a molecule to produce an ADA-like response in serum from treatment-naïve, healthy donors. We report herein that the magnitude of pre-existing reactivity evaluated pre-clinically and expressed as the 90th percentile of Tier 2 inhibition correlates with the subsequent rate of ADA emergence in the clinic. Furthermore, a multi-domain biotherapeutic (IgG-scFv bispecific antibody) showed the highest pre-existing reactivity and incidence of treatment-emergent ADA (TE-ADA) (57% and 93%, respectively). Using the components of the multidomain molecule in the Tier 2 step of the ADA assay, we were able to identify the scFv as the target of the serum pre-existing reactivity. Most importantly, the domain specificity of pre-existing ADA was the same as that of the TE-ADA from patients treated with the molecule. Based on these data, we propose the evaluation of the magnitude and of the domain specificity of pre-existing reactivity as a powerful tool to understand the immunogenic potential of novel biotherapeutics.

Bioanalysis in the Age of New Drug Modalities

“…Large differences in signal readout were observed between naïve individuals during the screening phase of method development for IgG-scFv-based compounds, and trying multiple assay formats and buffers yielded little improvement until a higher MRD (minimum required dilution) and target interference exclusion by immune capture beads were utilized. Others have reported the same phenomenon (51). For those BsAbs with the two functional binding sites distributed in tandem on the CDR (complementarity determining region) of each arm, the two arms of a given ADA molecule would bind to the same BsAb molecule, thus failing to form a bridge complex by simultaneous binding to two drug molecules.…”

Safety and Immunogenicity of LY3415244, a Bispecific Antibody Against TIM-3 and PD-L1, in Patients With Advanced Solid Tumors

“…Pre-existing ADA reactivity was assessed as previously described (20). Briefly, 56 to 60 normal human serum samples (purchased commercially from Bioreclamation IVT) were tested (Supplemental Methods) to obtain the screening ADA signal (Tier 1).…”

“…Briefly, 56 to 60 normal human serum samples (purchased commercially from Bioreclamation IVT) were tested (Supplemental Methods) to obtain the screening ADA signal (Tier 1). Epitope specificity studies were conducted as described above and as previously described (20).…”

“…It has been shown that the incidence of clinical TE-ADA in dosed subjects correlates with detection of pre-existing ADA in the healthy donors, and that the epitope specificity of pre-existing ADA can predict the epitope preference of the clinical ADA (20). Based on this, we investigated whether an early assessment of pre-existing ADA reactivity would have informed the high immunogenicity risk of LY3415244.…”

Toward Drug-Like Multispecific Antibodies by Design

“…It is also possible to determine the domain specificity of ADAs by evaluating antibody binding to individual domains of a therapeutic, a strategy that has been successfully adapted for use in determining ADA specificity in bispecific antibodies [ 245 ]. Interestingly, similar assays have been used to show that pre-existing ADAs for an IgG-scFv corresponded to the same domain as emergent ADAs for the same therapeutic, suggesting that the presence of pre-existing ADAs can be used to predict and even identify regions of immunogenic liability in multispecific antibodies [ 246 ]. Despite the utility of these assays, they require human testing, and are limited in their ability to quantify ADA interactions or precisely determine ADA epitopes [ 247 ].…”