RESUMO
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.
RESUMO
Bispecific antibodies (BisAbs) are biotherapeutics that amalgamate the specificities of two distinct antibodies into one molecule, however, their engineering requires genetic modification and remains time-consuming. Therefore, we used AJICAP second-generation technology, which drives the production of site-specific conjugation without genetic modification requirements, to generate BisAbs. Using haloketone chemistry as an alternative to maleimide chemistry, we successfully produced site-specific antibody conjugates. Pharmacokinetic studies revealed that the haloketone-based antibody conjugate was stable in the rat plasma. The resultant BisAbs were rigorously evaluated, and surface plasmon resonance measurements and flow cytometry analyses confirmed that the antigen binding remained intact. Additionally, the affinity for the neonatal Fc receptor (FcRn) was retained after conjugation. Further cytotoxicity evaluation emphasized the pronounced activity of the generated BisAbs. This novel approach introduces a fully chemical, site-specific strategy capable of producing BisAbs, heralding a new era in the field of biotherapeutics.
RESUMO
PURPOSE: Sinusoidal obstruction syndrome (SOS)/hepatic veno-occlusive disease (VOD) is a fatal complication after hematopoietic stem cell transplantation. We previously reported the usefulness of an ultrasonographical (US) scoring system, the Hokkaido US-based scoring system consisting of ten parameters (HokUS-10): (1) hepatomegaly in the left lobe and (2) right lobe, (3) dilatation of the main portal vein (PV), (4) hepatofugal flow in the main PV, (5) decreased velocity of the PV, (6) dilatation of the para-umbilical vein (PUV), (7) appearance of blood flow signal in the PUV, (8) gallbladder (GB) wall thickening, (9) ascites, and (10) increased resistive index of the hepatic artery, for the diagnosis of SOS/VOD. However, the reliability of this system among operators remains elusive. Therefore, we prospectively evaluated the reliability of HokUS-10. METHODS: Twenty-four healthy volunteers and 40 patients with liver dysfunction were enrolled. Inter- and intra-operator reliabilities were analyzed using three sonographers. RESULTS: The median concordance rate of HokUS-10 among three sonographers and intra-operator in 24 volunteers was 92% (95% CI: 73-98%) and 98% (95% CI: 92-100%), respectively. In all 64 cases, in terms of the reliability between two sonographers for three representative US parameters (amount of ascites, GB wall thickening, and appearance of PUV blood flow signal), the median concordance rate was more than 98% (95% CI: 86-106%). CONCLUSION: The inter- and intra-reliabilities of HokUS-10 were excellent. Thus, US might be a reliable tool for SOS/VOD diagnosis.