Reporter Cell Line Engineering for Therapeutic Antibody Evaluation in Animal Health

Summary

We have developed complex custom reporter cell lines designed to support IND-enabling studies and validate lead therapeutic antibodies against the ligands of two different companion animals’ target classes: a dual-chain receptor and a GPCR. Using custom vector design, a hyperactive transposase, optimized selection and sorting strategies, and assay development expertise, we generated clonal reporter cell lines with high signal induction and reproducible IC₅₀ measurements. The assays demonstrated sub-nanomolar and double-digit nanomolar IC₅₀ values for antibodies targeting the dual-chain receptor and GPCR ligands, respectively. Reporter cell lines, validated assays, and protocols were successfully transferred to the client to support their IND submission.

Problem

Sensitive and robust clonal reporter cell lines are often required by regulatory entities to support IND submissions and be used for different purposes from determining mechanism of action, potency assessment or lot release. Companion animal reporter cell lines are often not commercially available and have scarce supporting literature available. Moreover, reporter cell line development for these receptor classes can be challenging, particularly for GPCRs, due to complex signalling profiles, difficulties in achieving stable receptor expression, maintaining cell fitness, and generating assays with a high dynamic range.

Approach

The DNA-binding domain of key binding transcription factors were identified and crossed with publicly available information to ensure suitable responsive elements were used during vector design. Multiple proteins were overexpressed, by using a two-vector strategy, to reconstitute the animal activation pathway. High-copy number and stable integration was achieved by using a licensed hyperactive transposase technology allowing the successful integration of all sequences in-between the ITRs in one single transfection round. Using tagged ligands, sorting of high-expressing clones was achieved. Clones were screened using different ligand-activation protocols to find the best-performing cell lines. Top selected clones were used for functional assay development and determine lead antibodies IC₅₀.

Outcome

Two reporter cell lines for two complex targets reached validated, IND-ready clones in under 3.5 months from vector design. Lead antibodies showed full blocking, with IC₅₀ near 0.29 nM (dual-chain) and 6.9 nM (GPCR), and the assays were transferred to the client.

Key Results