Tolerability Studies of Trastuzumab-6 and Kadcyla? Sprague-Dawley rats (two females per group), six weeks old at the beginning of the experimental phase, obtained from Charles River Laboratories, Wilmington, MA, USA, were treated by intravenous (bolus) injection using a microflex infusion set introduced into a tail vein (2 mL/kg at 1 mL/min) with trastuzumab-6 or Kadcyla? (at 20, 35, 50, or 60 mg/kg). next-generation ADCs with enhanced therapeutic index. Keywords: antibodyCdrug conjugates (ADCs), therapeutic index, spacer technology, carbamoyl sulfamide, chemoenzymatic, glycan-remodeling, copper-free click chemistry 1. Introduction It took more than 50 years since the pioneering studies by Math et al. on targeted tumor treatment with methotrexate for the first antibodyCdrug conjugate (ADC), Adcetris?, to reach market approval in 2011. Since then, three more ADCs were approved or re-approved, i.e., Kadcyla? (in 2013), Besponsa? (2017), and Mylotarg? (2017). Mechanistically, ADCs exert their activity by (a) selective binding of antibody to tumor; (b) internalization; and (c) lysosomal degradation and liberation of toxic payload, leading to cytotoxic cell death [1]. In recent years, however, evidence has emerged that conjugation technology is also a critical component in determining quality attributes of an ADC [2,3]. For example, although the majority (~65%) of clinical ADCs are based on random payload attachment [4], a Lobucavir clear trend toward Lobucavir site-specifically conjugated ADCs based on engineered cysteine can be noted (currently >10 in the clinic), generally showing an increased therapeutic index [5,6]. A range of other site-specific conjugation technologies has been explored in the past decade [1,4,7,8,9], but to date only genetic encoding [10] of a nonnatural amino acid (= 0 and tumor size was monitored over time (= 5); (B) In vivo efficacy in Karpas-299 xenograft (CDX) of ADCs derived from brentuximab-1 conjugated with either HydraSpace? BCN-MMAE construct 5a or PEG-only variant 5b. A single dose of either ADC (1 mg/kg) was administered on = 0 and tumor size was monitored over time (= 8). Open in a separate window Figure 4 (A) Structures of branched HydraSpace? BCN-payload constructs 6 and 7; (B) In vivo efficacy in T226 patient-derived xenograft (PDX) of GlycoConnect? ADC derived from trastuzumab-1 conjugated with HydraSpace? BCN-maytansine construct 6 versus Kadcyla?. A single dose of Lobucavir ADC (9 mg/kg) was administered on = 0 and tumor size was monitored over time (= 5). Number of complete responders (CR) is indicated in the graph. (C) In vivo efficacy in Karpas-299 cell-derived xenograft (CDX) of GlycoConnect? ADC derived from brentuximab-1 conjugated with HydraSpace? BCN-MMAE construct 7 versus Adcetris?. A single dose of either ADC (1 mg/kg) was administered on = 0 and tumor size was monitored over time (= 7). Number of complete responders (CR) is indicated in the graph. 3.3. Xenograft Studies with Karpas-299 Model Female CB.17 SCID mice (eight to 12 weeks old at the beginning of the experimental phase, obtained from Charles River Laboratories, Wilmington, MA, USA) were injected with 1 107 Karpas-299 tumor cells in a 50% Matrigel subcutaneous in the flank (Karpas-299 cell xenograft model). When the tumor volume was in the range of 100C150 mm3, groups of eight mice were injected i.v. with a single dose of either vehicle (control), brentuximab-5a or brentuximab-5b (all at 1 mg/kg) and tumors were measured twice weekly as depicted in Figure 3B. In another study, groups of seven mice were injected i.v. with a single dose of either vehicle, Adcetris? or brentuximab-7 (all at 1 cxadr mg/kg) and tumors were measured twice weekly as depicted in Figure 4C. 3.4. Tolerability Studies of Trastuzumab-6 and Kadcyla? Sprague-Dawley rats (two females per group), six weeks old at the beginning Lobucavir of the experimental phase, obtained from Charles River Laboratories, Wilmington, MA, USA, were treated by intravenous (bolus) injection using a microflex infusion set introduced into a tail vein (2.