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.

Chemical site-specific conjugation technology utilizing immunoglobulin-G (IgG) Fc-affinity reagents is a versatile and promising tool for producing next-generation antibody–drug conjugates (ADCs). Our research group recently reported a novel Fc-affinity peptide-mediated conjugation method, termed AJICAP second-generation. This technology, based on thioester chemistry, produces site-specific ADCs with low aggregation. Herein, we report further investigations into the AJICAP second-generation technology. By varying the parameters of the peptide conjugation step, it was found that this reaction is feasible under a wide range of reaction conditions. All synthetic intermediates of the AJICAP-ADCs were sufficiently stable, indicating that each synthetic step is a possible holding point in ADC manufacturing. The Lys248- and Lys288-conjugated ADCs were prepared on a gram-scale using two different Fc-affinity peptide reagents, employing a scale-down manufacturing approach involving tangential flow filtration. The overall product yield was >80%, and ultimately, 13.2 g of trastuzumab-Lys248-MMAE and 1.26 g of trastuzumab-Lys288-MMAE were obtained with target drug to antibody ratios (DARs). Moreover, ADCs were synthesized at various scales, and it was verified that the DAR and aggregation rates could be replicated consistently across different scales. The results strongly indicate that the AJICAP second-generation process is a robust and practical approach for the manufacture of ADCs.

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.