RESUMO
Greater control is desirable in the stochastic conjugation technology used to synthesize antibody-drug conjugates (ADC). We have shown recently that a fluorescent dye can be stably conjugated to a mAb using a bifunctional platinum(II) linker. Here, we describe the general applicability of this novel linker technology for the preparation of stable and efficacious ADCs. The ethylenediamine platinum(II) moiety, herein called Lx, was coordinated to Desferal (DFO) or auristatin F (AF) to provide storable "semifinal" products, which were directly conjugated to unmodified mAbs. Conjugation resulted in ADCs with unimpaired mAb-binding characteristics, DAR in the range of 2.5 to 2.7 and approximately 85% payload bound to the Fc region, presumably to histidine residues. To evaluate the in vivo stability of Lx and its effect on pharmacokinetics and tumor targeting of an ADC, Lx-DFO was conjugated to the HER2 mAb trastuzumab, followed by radiolabeling with 89Zr. Trastuzumab-Lx-DFO-89Zr was stable in vivo and exhibited pharmacokinetic and tumor-targeting properties similar to parental trastuzumab. In a xenograft mouse model of gastric cancer (NCI-N87) or an ado-trastuzumab emtansine-resistant breast cancer (JIMT-1), a single dose of trastuzumab-Lx-AF outperformed its maleimide benchmark trastuzumab-Mal-AF and FDA-approved ado-trastuzumab emtansine. Overall, our findings show the potential of the Lx technology as a robust conjugation platform for the preparation of anticancer ADCs. Cancer Res; 77(2); 257-67. ©2016 AACR.
Assuntos
Anticorpos Monoclonais/farmacologia , Antineoplásicos/farmacologia , Imunoconjugados/farmacologia , Trastuzumab/farmacologia , Aminobenzoatos , Animais , Antineoplásicos/química , Linhagem Celular Tumoral , Desferroxamina , Desenho de Fármacos , Humanos , Imunoconjugados/química , Camundongos , Oligopeptídeos , Compostos de Platina , Traçadores Radioativos , Neoplasias Gástricas/patologia , Trastuzumab/química , Ensaios Antitumorais Modelo de Xenoenxerto , ZircônioRESUMO
The potential of platinum(II) as a bifunctional linker in the coordination of small molecules, such as imaging agents or (cytotoxic) drugs, to monoclonal antibodies (mAbs) was investigated with a 4-nitrobenzo-2-oxa-1,3-diazole (NBD) fluorophore and trastuzumab (Herceptin™) as a model antibody. The effect of ligand and reaction conditions on conjugation efficiency was explored for [Pt(en)(L-NBD)Cl](NO3 ) (en=ethylenediamine), with L=N-heteroaromatic, N-alkyl amine, or thioether. Conjugation proceeded most efficiently at pHâ 8.0 in the presence of NaClO4 or Na2 SO4 in tricine or HEPES buffer. Reaction of N-coordinated complexes (20â equiv) with trastuzumab at 37 °C for 2â h, followed by removal of weakly bound complexes with excess thiourea, afforded conjugates with an NBD/mAb ratio of 1.5-2.9 that were stable in phosphate-buffered saline at room temperature for at least 48â h. In contrast, thioether-coordinated complexes afforded unstable conjugates. Finally, surface plasmon resonance analysis showed no loss in binding affinity of trastuzumab after conjugation.
Assuntos
Anticorpos Monoclonais/química , Corantes Fluorescentes/química , Imunoconjugados/química , Compostos Organoplatínicos/química , Oxidiazóis/química , Trastuzumab/química , Modelos Moleculares , Estrutura Molecular , Compostos Organoplatínicos/síntese químicaAssuntos
Aminofenóis/síntese química , Aminofenóis/farmacologia , Antibacterianos/síntese química , Antibacterianos/farmacologia , Compostos Policíclicos/síntese química , Compostos Policíclicos/farmacologia , Aminofenóis/química , Antibacterianos/química , Cristalografia por Raios X , Enterococcus faecalis/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Conformação Molecular , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Compostos Policíclicos/química , Estereoisomerismo , Streptococcus pneumoniae/efeitos dos fármacosRESUMO
An efficient total synthesis of integric acid is described starting from the Wieland-Miescher ketone. Key steps involve a one-step orthogonal deprotection/protection strategy of a thioacetal/aldehyde and the selective oxidative cleavage of a prenyl group in the presence of two other unsaturated moieties. The synthesis of both C4' diastereoisomers of integric acid delivered unambiguous evidence for (S)-stereochemistry at the C4' position.