At the 2 2

At the 2 2.5 mg/kg dose, complete regressions (CRs) were achieved for all those mice in the 9c group, but only two CRs were seen in the 1d group, which latter regrew. have different n values and the disulfide-linked ADC 1b. The cell lines Jeg-3 cells (Ag+) and Nam\Luc (Ag?), which are Namalwa cells stably transfected with luciferase, were used. Next, the effect of methylene side chain length (n) on ADC cytotoxicity to cells that homogeneously express target antigen was investigated. The cytotoxicities of 7e and 7c, having n values of 3 and 5, respectively, were most often comparable to each other, and to 1b (Supplementary Table S1). ADCs 6aC6d were tested for their ability to induce bystander killing bystander killing than 6d and 1a. As shown in Physique ?Physique33C, increasing the number of methylene units in the maytansinoid side chain increased the bystander killing of immolative ADCs. Conjugates 7e and 7c, having 3 and 5 methylene groups in the maytansinoid side chain, required approximately 1800 and 700 Jeg-3 Ag+ cells, respectively, to induce 50% Nam/Luc AgC cell killing. Thus, 7c induced 2.5-fold more bystander killing than 7e. Conjugate 7c also induced more bystander killing than 4b and 1b, which required approximately 950 and 2000 Ag+ cells, respectively, to induce 50% cell killing. The comparable cytotoxicities of 7c and 7e could be explained if their metabolites are produced with comparable efficiencies and have comparable potencies once inside cells. However, if the metabolites of 7c are more hydrophobic, as expected, then they could more easily diffuse into other cells to induce more bystander killing. Additional studies will be needed to determine if this is the case; however, because of their lower bystander killing, conjugates with n = 3 were not evaluated further. The metabolites Mouse monoclonal to STAT3 of 8c were identified by incubating Tetracaine the conjugate with the high processing cell line COLO-205 for 24 h followed by cell lysis with organic solvent, using a previously described method.8 As a control, COLO-205 cells were treated the same way but without prior exposure to 8c. Both samples were analyzed by UPLC/MS (Supplementary Physique Tetracaine S1). The 252 nm UPLC trace of cells treated with 8c showed two peaks that were not in the control, a major peak at 12.73 min and a minor peak at 7.72 min having about a fourth of the area of the major peak. The synthesized cytotoxicities of the synthesized Cytotoxicities of studies. Open in a separate window Physique 5 (A) tolerability in mice (8/group) of the immolative ADC 7c vs the anilino ADC 4b and vehicle. * indicates the concentration of ADC based Tetracaine on the antibody component. (B) Pharmacokinetic study of 7c in mice where is the concentration versus time plot of the antibody component and is the concentration versus time plot of the payload component. The clearance of 7c at a dose of 10 mg/kg from circulation in nontumor bearing mice was determined by ELISA as previously described.25 The half-lives of the ADCs antibody and drug components were 15.2 and 10.9 days, respectively, Figure ?Figure55B. The efficacy of maytansinoid ADCs 9c and 1d was compared in mice (8/group) bearing Hs 746T tumor xenografts, where the target c-Met antigen is usually expressed homogeneously, Physique ?Figure66A. The H-Score for these cells,26 a measure of antigen expression, is usually 160. At the 2 2.5 mg/kg dose, complete regressions (CRs) were achieved for all those mice in the 9c group, but only two CRs were seen in the 1d group, which latter regrew. Next anti-huCanAg ADCs (5 mg/kg) were compared in mice (6/group) bearing HT-29 xenografts, where CanAg is only expressed on 20C30% of cancer cells in the tumor, Physique ?Figure66B.27 The greatest antitumor Tetracaine activity was observed with the immolative ADCs 8a (l, l, l) and 8c (l, d, l), resulting in CRs in 6/6 mice, although tumors began to regrow at day 30. The anilino ADC 4c (2/6 CRs) and the disulfide-linked ADC 1c (0/6 CRs) were less efficacious. HT-29 xenograft bearing mice (6/group) were also treated at 2.5 mg/kg; group 8c (2 CRs) had a larger reduction in median tumor size than group 8a (1 CR), Physique ?Figure66C. The efficacy of anti-huFR ADC was compared in mice bearing Tetracaine OV-90 xenografts, which express folate receptor heterogeneously (H-Score = 48), Physique ?Figure66D. The immolative ADC 7c was approximately twice as efficacious as the.