Phage display is a well-established procedure to isolate binders against a wide variety of antigens that can be performed on purified antigens, but also on intact cells. As selection steps are performed in vitro, it is possible to focus the outcome of the selection on relevant epitopes by performing some additional steps, such as depletion or competitive elutions. However in practice, the efficiency of these steps is often limited and can lead to inconsistent results. We have designed a new selection method named masked selection, based on the blockade of unwanted epitopes to favor the targeting of relevant ones. We demonstrate the efficiency and flexibility of this method by selecting single-domain antibodies against a specific portion of a fusion protein, by selecting binders against several members of the seven transmembrane receptor family using transfected HEK cells, or by selecting binders against unknown breast cancer markers not expressed on normal samples. The relevance of this approach for antibody-based therapies was further validated by the identification of four of these markers, Epithelial cell adhesion molecule, Transferrin receptor 1, Metastasis cell adhesion molecule, and Sushi containing domain 2, using immunoprecipitation and mass spectrometry. This new phage display strategy can be applied to any type of antibody fragments or alternative scaffolds, and is especially suited for the rapid discovery and identification of cell surface markers. Molecular & Cellular Proteomics
Organic cation transporter (OCT) 3 (SLC22A3) is a widely expressed drug transporter, handling notably metformin and platinum derivatives, as well as endogenous compounds like monoamine neurotransmitters. OCT3 has been shown to be inhibited by a few marketed tyrosine kinase inhibitors (TKIs). The present study was designed to determine whether additional TKIs may interact with OCT3. For this purpose, the effects of 25 TKIs toward OCT3 activity were analyzed using OCT3overexpressing HEK293 cells. 13/25 TKIs, each used at 10 µM, were found to behave as moderate or strong inhibitors of OCT3 activity, that is, they decreased OCT3-mediated uptake of the fluorescent dye 4-(4-(dimethylamino)styryl)-Nmethylpyridinium iodide by at least 50% or 80%, respectively. This OCT3 inhibition was correlated to some molecular descriptors of TKIs, such as the percentage of H atoms and that of cationic forms at pH = 7.4. It was concentration-dependent, notably for brigatinib, ceritinib, and crizotinib, which exhibited low half maximal inhibitory concentration (IC 50 ) values in the 28-106 nM range. Clinical concentrations of these three marketed TKIs, as well as those of pacritinib, were next predicted to inhibit in vivo OCT3 activity according to regulatory criteria. Cellular TKI accumulation experiments as well as trans-stimulation assays, however, demonstrated that OCT3 does not transport brigatinib, ceritinib, crizotinib, and pacritinib, thus discarding any implication of OCT3 in the pharmacokinetics of these TKIs. Taken together, these data suggest that some TKIs may act as potent inhibitors of OCT3 activity, which may have consequences in terms of drug-drug interactions and toxicity.
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