Purpose: Cystine knot peptides, also know as knottins, are an emerging class of biotherapeutic molecules that can be engineered to bind to a diverse range of targets. Much like therapeutic antibodies, there is a need to develop critical reagents for pharmacokinetic (PK) and immunogenicity assays to support preclinical development, though this new class of molecules can present different challenges and considerations for reagent discovery. To this end, monoclonal and polyclonal reagent antibodies against a cystine-knot fusion protein (CKFP) were generated and used for PK and ADA assay development.
Methods: Using robust antibody discovery methods, monoclonal antibodies were generated through the rapid immunization of mice with the target knottin peptide conjugated to BSA carrier protein, followed by fusion and high-throughput ELISA screening to identify anti-CKFP specific monoclonal hybridomas. Polyclonal antibodies were generated by the rapid immunization of rabbits with either knottin peptide-BSA conjugate or full-length CKFP, followed by multi-step affinity chromatography of antisera to yield anti-CKFP specific purified antibody. PK and ADA assays for rat and monkey samples were developed, which each assay step optimized to maximize signal-to-noise and working range while minimizing %CV and relative error.
Results: A panel of 50 anti-CKFP monoclonal antibodies was generated from two cohorts of CKFP immunized mice. The antibodies were characterized with both ELISA and label-free biolayer interferometry (ForteBio Octet) to identify complementary pairs that could be used in sandwich ELISA for CKFP quantification. Lead antibodies were purified and used to develop two PK ELISA assays for the measurement of CKFP in rat and monkey serum samples, one for quantitation of total knottin peptide and one for quantitation of total fusion protein. The final optimized anti-knottin PK assay gave sensitivity down to 15.6 ng/mL CKFP, while the anti-fusion protein PK assay gave sensitivity down to 0.469 mg/mL CKFP.
Two purified rabbit polyclonal antibodies were generated, one from rabbits immunized with knottin peptide-BSA conjugate and one from rabbits immunized with full length CKFP. The two antibodies were used a positive controls in the method development of a Bridging ADA ELISA for rat and monkey serum samples. The anti-knottin peptide antibody gave higher sensitivity in the Bridging ELISA (below 100 ng/mL) compared to the anti-fusion protein antibody (below 200 ng/mL).
Conclusion: Monoclonal and polyclonal reagent antibodies against a cystine-knot fusion protein (CKFP) have been generated and used to develop PK and ADA assays suitable for a cystine knot fusion protein.
Joshua Lowitz
– Antibody Solutions, SunnyvaleCatherine Vo
– Antibody SolutionsGlen Lin
– Antibody SolutionsRick Chang
– Antibody SolutionsShannon Jones-Iatauro
– Antibody SolutionsRonald Gamatero
– Antibody Solutions
John Kenney
– President, Antibody Solutions, Sunnyvale, California
John Kenney
– President, Antibody Solutions, Sunnyvale, California