However, as shown by the data presented in this work, detailed understanding of the catabolites in the tumors (which is not routinely performed), together with their DNA binding properties, are required to explain the dramatic efficacy differences exhibited by the cyclopropyl-containing and cyclobutyl-containing ADCs

However, as shown by the data presented in this work, detailed understanding of the catabolites in the tumors (which is not routinely performed), together with their DNA binding properties, are required to explain the dramatic efficacy differences exhibited by the cyclopropyl-containing and cyclobutyl-containing ADCs. good examples to demonstrate that the chemical nature and concentration of intratumor catabolites depend on the linker type used for drug conjugation, and the potency of the released drug moiety ultimately determines the ADC in vivo efficacy. Introduction Antibody drug conjugates (ADC) have a complex structure that combines an antibody with a small-molecule drug (often cytotoxin) through a chemical linker (Senter and Sievers, 2012; Chari et al., 2014; Polakis, 2016). Figure 1A shows a simplified diagram of such an ADC with a drug-to-antibody ratio (DAR) of 2 that can undergo deconjugation to generate DAR1 and DAR0 species as well as a cytotoxic drug. Great efforts have been made to characterize the heterogeneous and dynamic mixtures of these ADC species in circulation (Xu et al., 2011; Alley and Anderson, 2013), each of which could have its own pharmacokinetic and biologic activity profile. However, the best ADC species in circulation to use for exposure-response correlation (both safety and efficacy) is not currently known (Kamath and Iyer, 2015; Khot et al., 2015; Singh et al., 2015; Wang et al., 2016). An important question is to determine key parameters involving in ADC in vivo efficacy. Open in a separate window Fig. 1. (A) Deconjugation and catabolism of THIOMAB ADC. (B) Catabolism of disulfide-linked ADC. (C) Catabolite formation of methyl- and cyclobutyl-containing PBD-dimer conjugates in tissues. (D) Catabolite formation of cyclopropyl-containing PBD-dimer conjugate in tissues. The ADC linker determines the mechanism and rate of payload release, both of which affect exposure of normal and tumor tissues to BAY-545 a drug payload; thus, the ADC linker is a critical part of an ADC. Recently, Pillow et al. (2016) discovered a self-immolating disulfide linker (found, 585.2708, and calculated, 585.2711, C33H36N4O6, and by major fragments at 504.2144, 492.2144, 411.1570, 327.1724, 259.1096, and 246.1139. Cyclopropyl thiol was identified by molecular ion at found, 733.2901, and calculated, 733.2902, C38H44N4O9S, and BAY-545 by major fragments at 715.2814, 585.2716, 536.2040, 504.2140, and 492.2140. An affinity capture approach using protein-A magnetic beads was used to enrich the cyclopropyl-disulfide-PBD-dimer and cyclobutyl-disulfide-PBD-dimer ADCs from the mouse-tissue homogenate in the phosphate-buffered saline solution buffer, pH 7.4. The bound ADCs were subject to on-bead proteolysis with trypsin following standard protein denaturation, reduction, and alkylation processing steps. Briefly, quantification of the total antibody concentration was achieved by using LC-MS/MS measurement of its surrogate peptide(s) produced by proteolytic digestion. A surrogate peptide TTPPVLDSDGSFFLYSK, generated from the human unique Fc region to allow the differentiation of cyclopropyl-disulfide-PBD-dimer and cyclobutyl-disulfide-PBD-dimer ADCs from the endogenous matrix components, was quantified by a MRM transition of 938.0/836.7. In addition, several other peptides characteristic with the human Cetrorelix Acetate Fc region were monitored for the conformation and troubleshooting purposes as described previously (Xu et al., 2014). The DAR was determined as described previously (Xu et al., 2011). Briefly, an appropriate volume of mouse plasma after intravenous administration of ADCs was incubated at room temperature with the biotinylated CD22 target antigen, which was coupled to the streptavidin paramagnetic beads (Invitrogen/Thermo Fisher Scientific). The bead-captured ADC analytes were washed and deglycosylated at 37C overnight. The resulting samples in 30% acetonitrile in water containing 1% formic acid were injected onto a Triple TOF 5600 mass spectrometer (AB Sciex) coupled with HPLC using a reversed-phase HPLC column. The compounds were eluted by a gradient of BAY-545 mobile phase A (water with 0.1% formic acid) and mobile phase B (acetonitrile with 0.1% formic acid) at a flow rate of 5 = 8). Mean (S.E.M.) tumor volumes are plotted BAY-545 over BAY-545 time (days postdose). The control anti-Napi2b conjugates did not show efficacy in a separate experiment. (B) Tolerability.