Trastuzumab and rituximab underwent assessment, as demonstrated in Table . In every instance, sufficient conversion was observed between both AJICAP-M reagents (1 and 2) and these mAbs, facilitating the site-specific installation of azide groups. The conjugation sites for the resultant trastuzumab-Lys248 or 288 azides were identified using peptide mapping analysis (Figure and S5 in the SI). A comparison of the azide adducts (and the corresponding amines reduced during pretreatment for peptide mapping, figure and) and naked trastuzumab underscored the targetspecific modification of the AJICAP-antibody-azides at Lys248 and Lys288. Subsequently, the effectiveness of newly installed azides was assessed using two commercially available payload linkers (DBCO-maytansinoid and DBCO-VC-PAB-MMAE) (Table ). Sitespecific ADCs synthesized from trastuzumab-Lys248-azide, trastuzumab-Lys288-azide, or rituximab-Lys248-azide exhibited drug-to-antibody ratios (DARs) exceeding 1.8. The DAR was determined using two analytical methods: quadrupole time-of-flight mass spectrometry (Q-TOF MS) and hydrophobic interaction chromatography (HIC)-HPLC (SI, Figures S4-S11 and S18-S23). Analysis via size exclusion chromatography (SEC) revealed that the aggregation percentages were maintained post-modification in all reaction scenarios (SI, Figures ). Subsequently, the biological profiles of the synthesized ADCs were evaluated. To elucidate the stability of the novel AJICAP-M linkage against traditional cysteine-haloamide conjugation, ADCs were synthesized using the latter method to serve as a control group. Considering the known risks associated with the thiol-maleimide linkage in commonly employed maleimide-based compounds-principally the potential for retro Michael reactions leading to inadvertent payload release -alternative linkers to maleimide were incorporated in the control ADCs. Haloamides, frequently utilized as substitutes within the maleimide group , were selected for this purpose, thereby directly comparing the stability between the AJICAP-M ADCs and controls. A series of cell-based assays were performed to assess the in vitro cytotoxicity. These assays involved the use of cell lines with varying levels of HER2 antigen expression: NCI-N87 and HCC1954 for high HER2 expression and PC-3 for low HER2 expression, as illustrated in Figure . The results indicated that AJICAP-M and control ADC demonstrated significant antitumor efficacy against cell lines with high HER2 antigen expression. Conversely, negligible activity was observed in cell lines with low HER2 antigen expression, highlighting the specificity of ADCs.