Luminescence was detected using the EnSpire Plate Reader, and Prism5 for Windows (Graphpad software, Inc., La Jolla, CA, USA) was used for data analysis, including IC50 calculations. 3.8. We studied the differences in the preparation process, in vitro stability, cathepsin B activity and in vitro cytotoxicity of VA-based ADC compared to the ADC of VC. VA had comparable performance to VC, which preliminarily displays its practicability. Additional efficacy and safety studies in a xenograft model indicate this novel ADC exerted potent anti-tumor activity and negligible toxicity. The results of this study show the application potential of VA-based ADC with MMAE as the payload. = 0. After seven days, the AUCs for NAC-12b and NAC-12c were 97.92% and 97.05%, respectively, of the two samples taken immediately at = 0 (Figure 4A). In addition, it could be seen from the HPLC spectrum that the lost fraction was converted to the ring-opened succinimide hydrolysis adduct. Open in a separate window Figure 4 Stability assays of VA- and VC-based conjugates in PBS. Compound in phosphate buffered saline (PBS) (pH 7.4) were incubated at 37 C for seven days. Error bars represent standard deviation from three independent experiments (triplicates). Results are shown as the mean SD. (A) Stability of NAC-12b and NAC-12c; (B) Stability of VA- and VC-based ADCs loaded with four and seven drugs per antibody. Next, we analyzed the in vitro stability of these two ADCs with an average DAR of about 4 and 7. Analogously, the two ADCs having 4 and 7 drugs per antibody, respectively, were diluted with PBS (1 mg/mL, pH 7.4) and incubated at 37 C in a shaking incubator. All of the samples were analyzed by HIC HPLC. As shown in Figure 4B, none of the four ADCs had significant drug off-targets in this test, and VA- and VC-based ADCs that having four payloads decreased stability by 1.58% and 1.34%, respectively, after seven days, whereas those that having seven payloads decreased stability by 2.52% and 3.88%, respectively, under the same test conditions. In short, ADCs with VA- and VC-based linker systems have substantially comparable in vitro stability in TOK-001 (Galeterone) PBS. 2.4. Cathepsin B Reactivity According to previous reports, drug linkers Vegfa containing VA or VC dipeptides are substrates for cathepsin B [28]. Here, we confirmed the cleavable feasibility of VA-based conjugate under the reaction of cathepsin B. The susceptibility TOK-001 (Galeterone) of the dipeptide linker conjugates (NAC-12b and NAC-12c) to enzymatic cleavage was determined by treatment of the acetyl-L-cysteine adduct with cathepsin B from human liver (EC 3.4.22.1, SIGMA-ALDRICH?, St. Louis, MO, USA); NAC-12c was tested in parallel for comparison. The cleavage of dipeptide from both substrates resulted in 1,6-elimination of MMAE, which was identified by liquid chromatography-mass spectrometry (LC-MS; Agilent 1100, Palo Alto, CA, USA). The amount of released MMAE over time is shown in Figure 5. These results demonstrated that the performance of VA-based conjugate is basically the TOK-001 (Galeterone) same as VC, and is likely to be a suitable substrate for cathepsin B. Open in a separate window Figure 5 Cathepsin B reactivity for MMAE release. Typical time course of MMAE cleaved from = 6/group. (A) Comparison of the efficacy of ADC in the middle dose group with the control group; (B) dose-efficacy dependence of the ADC treatment groups; (C) changes in body weight of the mice during the observation period; (D) comparison of the efficacy of ADCs based on VA and VC dipeptides. 2.7. Hematological Analysis To further verify the safety of the ADC (mil40-12b), changes in hematology between vehicle and treatment groups were compared at the end of administration and the observation period. The main test indicators included levels of white blood cells (WBCs), red blood cells (RBCs), platelets (PLTs), neutrophils (Neuts), and lymphocytes (Lymph). In contrast to the vehicle group, all of the above hematological parameters from the three ADC treatment groups were similar on Days 28 and.