(T1330-02-08) Production and Characterization of Critical Biotin and DIG Conjugated RESCA-Anti-CD8 VHH Reagents to Enable the Non-Clinical and Clinical Anti-Drug Antibody Assay Development

Purpose: The anti-CD8 ImmunoPET (iPET) agent was developed as a clinical imaging tool to quantify cytotoxic T-cells to guide clinical development in cancer immunotherapy and autoimmune diseases. The imaging agent was derived from a humanized single-domain antibody (VHH) conjugated with a complexing agent (RESCA), which became the imaging reagent by chelation of radioisotope ¹⁸F. To support non-clinical and clinical anti-drug antibody (ADA) assay development, it was necessary to produce biotin and digoxigenin (DIG) conjugated RESCA-anti-CD8 VHH critical reagents. Unlike traditional monoclonal antibodies that contain ~90 potential conjugation sites, the parental anti-CD8 VHH molecule only contains 4 conjugation sites.  Even fewer conjugation sites on RESCA-anti-CD8 VHH are available for biotin or DIG conjugation because some of these are occupied by RESCA.   In addition, the dual payload (biotin/DIG and RESCA) coupled with the low molecular weight of this molecule introduce additional complexities to standard Liquid Chromatography / Mass Spectrometry (LC-MS) and Size Exclusion Chromatography-Multi Angle Light Scattering (SEC-MALS) characterization methods. These factors pose unique challenges to generating and characterizing Biotin and DIG conjugated RESCA-anti-CD8 reagents and require fit-for-purpose reagent generation and characterization approaches.

Methods: RESCA-anti-CD8 VHH was conjugated to biotin (Sulfo-NHS-LC-biotin) and digoxigenin (DIG) (3-amino-3 deoxydigoxigenin hemisuccinamide, succinimidyl ester) based on an amine chemistry reaction at different challenge ratios. To ensure the quality of the conjugated reagents, the reagents were characterized by several biophysical characterization methods including 2-hydroxyazobenzene-4'-carboxylic acid (HABA) test, LC-MS, SEC-MALS, and Surface Plasmon Resonance (SPR).

Results: Small-scale preparations of biotin and DIG conjugated RESCA-anti-CD8 VHH were initially made at different challenge ratios (CRs). Based on the LC/MS characterization data, a 30:1 challenge ratio for biotin RESCA-anti-CD8 VHH and 10:1 for DIG RESCA-anti-CD8 VHH were selected. As shown in Table 1, the incorporation ratios for the selected biotin and DIG conjugated reagents are 1.9 and 2.0 per VHH, and the amounts of unconjugated RESCA-anti-CD8-VHH are 5.6% and 5.5%, respectively. However, when large-scale (10 mg) preparations of conjugated reagents were produced, it was found that the incorporation ratio for both biotin and DIG RESCA-anti-CD8-VHH dropped to less than one label per molecule and the percentage of unconjugated RESCA-anti-CD8 VHH increased to around 40%. The low incorporation ratio and increased percentage of unconjugated RESCA-anti-CD8-VHH in these large-scale conjugates made them unacceptable for assay development. To understand and resolve this issue, we examined different parameters associated with the conjugation process. The loading volume used during the pre-conjugation buffer exchange was found to be the key parameter that affected the quality of the labeled reagents; this minimizes interference from the source material storage buffer. Based on the HABA test results and LC-MS data (Table 2), the optimal loading volume was found to be 1.5 mL or below. The optimal loading volume was applied to produce multiple subsequent lots of biotin and DIG conjugates at large scales, and all lots showed consistent quality with incorporation ratio ranging from 2.1-2.2 and the amounts of unconjugated RESCA-anti-CD8-VHH less than 3.5%. Additional reagent characterization by SEC-MALS and SPR showed that the conjugates contained low aggregation levels ( < 4%) and retained high binding activity after conjugation (ranging from 94-101%).

Conclusion: A fit-for-purpose reagent production strategy in conjunction with several biophysical characterization methods was applied to produce biotin and DIG RESCA-anti-CD8 VHH with optimized conjugation conditions and satisfied quality. The conjugated RESCA anti-CD8 reagents were successfully incorporated into the ADA assays to support the non-clinical and clinical studies.

Table 1. Characterization of biotin and DIG conjugated RESCA-anti-CD8 with different challenge ratio by LC/MS

Table 2. Impact of loading volume (mL) on the quality of biotin and DIG conjugated RESCA-anti-CD8 VHH determined by HABA test and LC-MS