(W1030-09-52) Generation of Rabbit Anti-Peptide Monoclonal Antibodies for Microdystrophin and Dystrophin Quantitation

Purpose: Duchenne muscular dystrophy is an X-linked developmental disorder caused by mutations in the dystrophin (Dys) gene leading to progressive muscular weakness and premature death due to respiratory and/or cardiac failure. RGX-202 is a recombinant adeno-associated virus of serotype 8 (AAV8) with an optimized human microdystrophin (mDys) transgene under the control of a promoter designed to increase expression in muscle (Spc5-12). Quantitation of mDys levels in skeletal muscles from subjects dosed with RGX-202 is an important factor to understand the treatment effect of the gene therapy.  To develop a specific and sensitive LC-MS assay for the measurement of Dys and mDys protein levels, it is necessary to generate peptide antibodies, with high specificity and affinity, which can be used to extract specific Dys- and mDys-peptides from the complex trypsin-digested muscle tissue matrix.

Methods: A novel approach has been utilized to raise rabbit anti-peptide monoclonal antibodies instead of the traditional mouse hybridoma method. A mDys-specific peptide and two peptides with identical sequence between Dys and mDys proteins were selected, synthesized, and conjugated with keyhole limpet hemocyanin (KLH) for immunization (4 rabbits/peptide), followed by PBMC isolation and antigen-positive B cell enrichment and expansion. Supernatant of clones were screened and characterized for their binding specificities to the corresponding peptides using titer ELISA, MSD, and LC-MS methods.

Results: All rabbits have demonstrated immune response to the peptide antigens. One rabbit from each group with titer of 64,000 or higher and minimal cross-reactivity to other peptide antigens was selected for mAb cloning. Two mAb clones (one against mDys specific peptide and one against peptide with identical sequence) were finally selected and sequenced. Specificities of the mAbs to the peptide targets have been demonstrated in the confirmatory assay format using MSD. The two peptides with corresponding chosen rabbit mAbs were used to develop an LC-MS method for the quantitation of Dys and mDys based on bioanalytical method validation guidance recommendations.  Using the mAbs to capture peptides in trypsin-digested muscle tissue followed by quantitative LC-MS, the calibration curve ranges for mDys and total Dys were both 12.5-2500 fmol/mg (r² = 0.992 and 0.993 respectively). The overall recovery of both peptides (digest & immunocapture efficiency) was more than 75% with CV < 9.2% (n = 3). The precision and accuracy were within 20% for both analytes.

Conclusion: The rabbit mAb approach has successfully generated antibodies against Dys and mDys peptides for the development of an LC-MS method. The final produced mAbs have demonstrated specificity, high affinity to targets and suitability for the LC-MS transgene protein assay supporting phase 1/2 RGX-202 clinical study.