1. Preclinical Development of an anti-5T4 Antibody-Drug Conjugate: Pharmacokinetics in Mice, Rats, and NHP and Tumor/Tissue Distribution in Mice
Wei Song, Leslie Lorello, Judy Lucas, Nicole Duriga, Quazi Shakey, Puja Sapra, Yanhua Zhang, Brian Rago, James McNally, JoAnn Wentland, Eugene Kadar, Mauricio Leal, Xiaogang Han, Franklin Spriggs Bioconjug Chem . 2015 Nov 18;26(11):2223-32. doi: 10.1021/acs.bioconjchem.5b00205.
The pharmacokinetics of an antibody (huA1)-drug (auristatin microtubule disrupting MMAF) conjugate, targeting 5T4-expressing cells, were characterized during the discovery and development phases in female nu/nu mice and cynomolgus monkeys after a single dose and in S-D rats and cynomolgus monkeys from multidose toxicity studies. Plasma/serum samples were analyzed using an ELISA-based method for antibody and conjugate (ADC) as well as for the released payload using an LC-MS/MS method. In addition, the distribution of the Ab, ADC, and released payload (cys-mcMMAF) was determined in a number of tissues (tumor, lung, liver, kidney, and heart) in two tumor mouse models (H1975 and MDA-MB-361-DYT2 models) using similar LBA and LC-MS/MS methods. Tissue distribution studies revealed preferential tumor distribution of cys-mcMMAF and its relative specificity to the 5T4 target containing tissue (tumor). Single dose studies suggests lower CL values at the higher doses in mice, although a linear relationship was seen in cynomolgus monkeys at doses from 0.3 to 10 mg/kg with no evidence of TMDD. Evaluation of DAR (drug-antibody ratio) in cynomolgus monkeys (at 3 mg/kg) indicated that at least half of the payload was still on the ADC 1 to 2 weeks after IV dosing. After multiple doses, the huA1 and conjugate data in rats and monkeys indicate that exposure (AUC) increases with increasing dose in a linear fashion. Systemic exposure (as assessed by Cmax and AUC) of the released payload increased with increasing dose, although exposure was very low and its pharmacokinetics appeared to be formation rate limited. The incidence of ADA was generally low in rats and monkeys. We will discuss cross species comparison, relationships between the Ab, ADC, and released payload exposure after multiple dosing, and insights into the distribution of this ADC with a focus on experimental design as a way to address or bypass apparent obstacles and its integration into predictive models.
2. Inhibition of Megakaryocyte Differentiation by Antibody-Drug Conjugates (ADCs) is Mediated by Macropinocytosis: Implications for ADC-induced Thrombocytopenia
Sara Gulesserian, Zhilan Zeng, Sher Karki, Karen Morrison, Lisa Do, Josh Snyder, Fernando Doñate, Sathish Kumar Ganesan, Veronica Robles, Hui Zhao, Jimmy Ou, David R Stover, Hector Aviña Mol Cancer Ther . 2017 Sep;16(9):1877-1886. doi: 10.1158/1535-7163.MCT-16-0710.
Thrombocytopenia is a common adverse event in cancer patients treated with antibody-drug conjugates (ADC), including AGS-16C3F, an ADC targeting ENPP3 (ectonucleotide pyrophosphatase/phosphodiesterase-3) and trastuzumab emtansine (T-DM1). This study aims to elucidate the mechanism of action of ADC-induced thrombocytopenia. ENPP3 expression in platelets and megakaryocytes (MK) was investigated and shown to be negative. The direct effect of AGS-16C3F on platelets was evaluated using platelet rich plasma following the expression of platelet activation markers. Effects of AGS-16C3F, T-DM1, and control ADCs on maturing megakaryocytes were evaluated in anin vitrosystem in which human hematopoietic stem cells (HSC) were differentiated into MKs. AGS-16C3F, like T-DM1, did not affect platelets directly, but inhibited MK differentiation by the activity of Cys-mcMMAF, its active metabolite. FcγRIIA did not appear to play an important role in ADC cytotoxicity to differentiating MKs. AGS-16C3F, cytotoxic to MKs, did not bind to FcγRIIA on MKs. Blocking the interaction of T-DM1 with FcγRIIA did not prevent the inhibition of MK differentiation and IgG1-mcMMAF was not as cytotoxic to MKs despite binding to FcγRIIA. Several lines of evidence suggest that internalization of AGS-16C3F into MKs is mediated by macropinocytosis. Macropinocytosis activity of differentiating HSCs correlated with cell sensitivity to AGS-16C3F. AGS-16C3F was colocalized with a macropinocytosis marker, dextran-Texas Red in differentiating MKs. Ethyl isopropyl amiloride (EIPA), a macropinocytosis inhibitor, blocked internalization of dextran-Texas Red and AGS-16C3F. These data support the notion that inhibition of MK differentiation via macropinocytosis-mediated internalization plays a role in ADC-induced thrombocytopenia.Mol Cancer Ther; 16(9); 1877-86. ©2017 AACRSee related article by Zhao et al., p. 1866.
3. Overcoming challenges associated with the bioanalysis of cysteine-conjugated metabolites in the presence of antibody-drug conjugates
Sharon Boram, Christopher A Evans, Jonathan Kehler, Kristen E Pannullo, Hermes Licea-Perez, Andrew P Mayer Bioanalysis . 2021 Sep 23. doi: 10.4155/bio-2021-0171.
Aim:Investigations have shown that for the antibody-drug conjugate (ADC) belantamab mafodotin, concentrations of the cysteine-conjugated metabolite, Cys-mcMMAF, were overestimated in the presence of the ADC during sample processing when utilizing a historical SPE method.Results:A new assay was developed utilizing an acidic protein precipitation to remove the ADC early in the extraction process, thus eliminating the risk of overestimating Cys-mcMMAF in the presence of belantamab mafodotin.In vitroexperiments demonstrated a linear relationship between the concentration of belantamab mafodotin and the release of Cys-mcMMAF. Extensive stability assessments were performed to cover storage of study samples.Conclusion:This work emphasized the critical importance of understanding the performance of a bioanalytical method for free toxic payload in the presence of the ADC.
