Purpose: Gene therapy is an emerging therapeutic platform delivering a healthy gene to a patient to restore normal cell function. This complex drug modality comprised of a nucleotide sequence packaged in a vector to deliver a functional transgene protein requires the generation of critical reagents to each component to support the development of specific assays across various platforms. Generation of de novo antibody reagents to the vector faced challenges due to the reported internalization of the vector upon immunization as well as its unique and complex structure. De novo reagent antibody production to the human transgene protein also had obstacles to circumvent attributable to the high homology in animal hosts requiring breaking of tolerance to elicit an antibody response. In this case study, we report the strategies to produce a neutralizing antibody (nAb) positive control (PC) to the vector and an antidrug antibody (ADA) PC to the transgene protein which entailed de novo immunizations, phage display, and assessment of commercial reagents.
Methods: Anti-vector nAb PC: (1) De novo mAbs: A repetitive immunization multiple sites (RIMMS) protocol was employed to reintroduce the antigen every three days to the mice to circumvent internalization and induce a high antibody titer. Mouse antisera, fusion clones and purified antibodies were characterized by two vendors to test specificity (antibody titer by ELISA, nAb activity in cell based ligand binding assay (LBA)) which required detailed logistics to transport samples between two different vendor sites. (2) Supernatant of a commercial mAb was protein L purified prior to evaluation.
Anti-transgene protein ADA PC: A multi-tiered approach was taken to prepare a reagent antibody to the transgene protein. (1) For de novo antibody production, mice and rabbits were immunized with KLH conjugated transgene protein. Antibody titers from bleeds were obtained by ELISA and counter screened against an irrelevant protein to ensure specificity. (2) Phage display was explored as alternative methodology, and in vitro panning of the transgene against a diverse naïve scFv phage library with three rounds of enrichment was performed to identify specific binders. Unique clones were converted from scFv to mouse IgG1 framework. (3) A commercial mAb was purchased and protein G purified to assess as a reagent.
Results: Anti-vector nAb PC: (1): De novo mAbs: The RIMMS immunization protocol elicited high antibody titers in the mice which successfully generated 10 reagent antibodies in a 5 month period. The IC50 values of the de novo mAbs were determined to be 14.4-112.7 ng/mL in the nAb LBA with a KD range to the vector of 53-438 pM. (2) The purified commercial antibody reported IC50 and KD results comparable to the mAbs.
Anti-transgene protein ADA PC: (1) De novo antibody generation in mice and rabbits had limited success. One clone with weak binding to the transgene protein was derived from the mouse immunizations, and the antibody response in rabbits was low to minimal despite double dosing with the antigen. (2) Phage display delivered seven unique scFv clones. Characterization of the mouse IgG1 constructs by LBA demonstrated specificity to the transgene protein with a broad span of affinities at173 pM- 5.6 nM. (3) The purified commercial antibody had a KD value of 280 pM and a similar performance in the LBA as the phage derived antibodies.
Conclusion: Commercial antibodies were suitable reagents in their respective assays; however, supply issues precluded their use for the lifetime of the LBA’s. Internal reagent development was successful, but two approaches were required to produce positive controls against the vector and transgene protein, de novo mAbs and phage display, respectively.
Glenn Miller– Andover, Massachusetts
Lee Walus– Andover, Massachusetts
Renee Ramsey– Andover, Massachusetts
Rosemary Lawrence-Henderson– Andover, Massachusetts
Boris Gorovits– Senior Director PDM-NBE, Pfizer Inc., Andover, Massachusetts
Alison Joyce– Pfizer Inc., Andover, Massachusetts