AJICAP Second Generation: Improved Chemical Site-Specific Conjugation Technology for Antibody–Drug Conjugate Production

Abstract

The site-directed chemical conjugation of antibodies remains an area of great interest and active efforts within the antibody–drug conjugate (ADC) community. We previously reported a unique site modification using a class of immunoglobulin-G (IgG) Fc-affinity reagents to establish a versatile, streamlined, and site-selective conjugation of native antibodies to enhance the therapeutic index of the resultant ADCs. This methodology, termed “AJICAP”, successfully modified Lys248 of native antibodies to produce site-specific ADC with a wider therapeutic index than the Food and Drug Administration-approved ADC, Kadcyla. However, the long reaction sequences, including the reduction–oxidation (redox) treatment, increased the aggregation level. In this manuscript, we aimed to present an updated Fc-affinity-mediated site-specific conjugation technology named “AJICAP second generation” without redox treatment utilizing a “one-pot” antibody modification reaction. The stability of Fc affinity reagents was improved owing to structural optimization, enabling the production of various ADCs without aggregation. In addition to Lys248 conjugation, Lys288 conjugated ADCs with homogeneous drug-to-antibody ratio of 2 were produced using different Fc affinity peptide reagent possessing a proper spacer linkage. These two conjugation technologies were used to produce over 20 ADCs from several combinations of antibodies and drug linkers. The in vivo profile of Lys248 and Lys288 conjugated ADCs was also compared. Furthermore, nontraditional ADC production, such as antibody–protein conjugates and antibody–oligonucleotide conjugates, were achieved. These results strongly indicate that this Fc affinity conjugation approach is a promising strategy for manufacturing site-specific antibody conjugates without antibody engineering.

Scale-Up Synthesis of Site-Specific Antibody–Drug Conjugates Using AJICAP Second-Generation Technology

“…A previous in vivo efficacy and pharmacokinetic study revealed that the biological profile of Lys248 ADC was like that of Lys288 ADC. 11 However, in the present stability study, the intermediates exhibited a dissimilar behavior. Lys248-related compounds were found to be more stable than Lys288-related compounds.…”

“…MC-VC-MMAE (CAS#646502-53-6) was purchased from NJ Biopharmaceuticals, LLC (USA). Peptide reagents (1a and 1b) were prepared according to a published method . All other chemical reagents were purchased from Sigma-Aldrich (USA).…”

“…With this affinity-based strategy, two different AJICAP peptide reagents were successfully created, which allowed access to two distinct conjugation sites (Lys248 and Lys288) after fine-tuning of the linker length and reactive groups. 11 Over 20 ADCs were produced using these two conjugation technologies, employing various combinations of antibodies and drug linkers. Early biological studies, which included in vivo efficacy and toxicology evaluations, indicated that site-specific AJICAP-ADCs (including both Lys248-and Lys288-based ADCs) exhibited an enhanced therapeutic window when compared with traditional stochastic ADCs.…”

“…The limited scope of Fc-affinity technologies for site-specific ADC generation prompted us to develop a versatile and facile direct antibody modification technology that circumvents antibody engineering. 11 Optimization of the reaction sequence led to the present improved technology, termed "AJICAP second generation". The technology provides a promising chemical platform, which is potentially applicable to the production of a wide variety of site-specific ADCs (Figure 1).…”

A Manufacturing Strategy Utilizing a Continuous-Mode Reactor toward Homogeneous PEGylated Bioconjugate Production

“…34 To establish a robust and reliable PEGylation using the continuous mode, some challenges still remain. For example, process development including understanding critical process parameters, PEG reagent screening to replace NHS ester having risk of overreactions, 35 detailed conjugation site analysis by peptide mapping, and normal operating range identification was not complete in the present study. Further investigations to establish the ideal continuousmode-mediated PEGylation are currently underway.…”