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. 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 homogenous drug-to-antibody ratio of 2 were produced using this technology after a proper modification of the spacer linkage of Fc-affinity peptide reagents. 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, non-traditional ADC applications, such as antibody-protein conjugates and antibody-oligonucleotide conjugates, were performed. These results strongly indicate that this Fc affinity conjugation approach is a promising strategy for manufacturing site-specific antibody conjugates.

Bispecific Antibodies Produced via Chemical Site-Specific Conjugation Technology: AJICAP Second Generation

“…In previous studies, our research group successfully enhanced the therapeutic index of site-specific ADCs 11 . The stability of these ADCs was confirmed by rat PK studies, and the newly formed thiol-maleimide bond, specifically linked to the Lys248 position of the antibody Fc region, was stable.…”

“…However, these published methods are mainly used for research purposes and are therefore not scalable or robust. Therefore, we applied our preoperative technology, utilizing Fc-affinity peptide reagents, to generate a novel format for BisAb production 11 . This technology, termed "AJICAP second generation," allows for a non-engineering preparation of site-specific antibodydrug conjugates (ADCs) (Figure 1).…”

“…This unique strategy produced two site-specific ADCs with two different conjugation sites (Lys248 and Lys288). In vivo biological studies have shown that these site-specific ADCs have a wider therapeutic window than conventional ADCs produced using the stochastic conjugation method 11 . Furthermore, process development toward gram-scale ADC preparation was completed, indicating that this chemical conjugation technology is a practical approach for manufacturing sitespecific antibody conjugates 12 .…”

Chemical technology principles for selective bioconjugation of proteins and antibodies

“…330 For example, a peptide-equipped NHS ester was used to synthesize IgG-MMAE conjugates (119c). [331][332][333][334] Alternatively, the electrophilicity of bioconjugation reagents can be reduced to take advantage of kinetic preferences. In this perspective, pentafluorophenyl ester (120a) was shown to render improved selectivity.…”

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

“…The limited scope of Fc-affinity technologies for sitespecific ADC generation prompted us to develop a versatile and facile direct antibody modification technology that circumvents antibody engineering. 7 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).…”