Purpose: We are interested in identifying mechanisms of resistance to the current generation of antibody-drug conjugates (ADC) and developing ADCs that can overcome this resistance.Experimental Design: Pinatuzumab vedotin (anti-CD22-vc-MMAE) and polatuzumab vedotin are ADCs that contain the microtubule inhibitor monomethyl auristatin E (MMAE) attached to the antibody by the proteasecleavable linker maleimidocaproyl-valine-citrulline-p-aminobenzoyloxycarbonyl (MC-vc-PAB). Early clinical trial data suggest that these ADCs have promising efficacy for the treatment of nonHodgkin lymphoma (NHL); however, some patients do not respond or become resistant to the ADCs. Anthracyclines are very effective in NHL, but ADCs containing the anthracycline doxorubicin were not clinically efficacious probably due to the low drug potency and inadequate linker technology. The anthracycline analogue PNU-159682 is thousands of times more cytotoxic than doxorubicin, so we used it to develop a new class of ADCs. We used the same MC-vc-PAB linker and antibody in pinatuzumab vedotin but replaced the MMAE with a derivative of PNU-159682 to make anti-CD22-NMS249 and tested it for in vivo efficacy in xenograft tumors resistant to MMAE-based ADCs.Results: We derived cell lines from in vivo xenograft tumors that were made resistant to anti-CD22-vc-MMAE and anti-CD79b-vc-MMAE. We identified P-gp (ABCB1/MDR1) as the major driver of resistance to the vc-MMAE-based conjugates. Anti-CD22-NMS249 was at least as effective as anti-CD22-vc-MMAE in xenograft models of the parental cell lines and maintained its efficacy in the resistant cell lines.Conclusions: These studies provide proof of concept for an anthracycline-based ADC that could be used to treat B-cell malignancies that are resistant to vc-MMAE conjugates.
[3H]P1075 binding to membrane preparations of rabbit skeletal muscle were observed in the presence of nucleotide triphosphates or diphosphates but not AMP, cAMP, adenosine, tripolyphosphate, or pyrophosphate. Nonhydrolyzable or poorly hydrolyzable ATP analogs inhibited MgATP-supported binding. The EC50 value for MgATP-supported binding (0.4 mM) was decreased approximately 10-fold in the presence of an ATP-regenerating system, and significant metabolism by membrane nucleotidases was confirmed by high performance liquid chromatographic analysis. [3H]P1075 bound to skeletal muscle with a Kd value of 37 +/- 3 nM and a Bmax value of 280 +/- 14 fmol/mg of protein. [3H]P1075 binding to subcellular fractions was highest in membranes enriched in T tubules. Specific binding was reversible, trypsin-sensitive, maximal at pH 8, and stereoselective for the (3S,4R)-enantiomer of cromakalim. Potassium channel openers exhibited a rank order of potency of P1075 > pinacidil > levcromakalim = BMS-180448 > nicorandil > diazoxide = BRL 38226. Fluorescein analogs (ethyleosin, phloxine B, and rose bengal) were relatively potent inhibitors of binding (Ki = 200-300 nM). The potassium channel openers cromakalim and BMS-180448 were competitive inhibitors of [3H]P1075 binding. In contrast, rose bengal and the ATP-regulated potassium channel antagonist glyburide increased the rate of [3H]P1075 dissociation in a manner consistent with noncompetitive interaction.
1 This study compares the activity of BMS-180560 (2-butyl-4-chloro-1-[[1-[2-(2H-tetrazol-5-yl)phenyl]-1H-indol-4-yl]methyl]-lH-imidazole-5-carboxylic acid), an insurmountable angiotensin II (All) receptor antagonist, with that of losartan and EXP3174 in functional and biochemical models of AII-receptor activation.2 BMS-180560 selectively inhibited ['25I]-Sar'Ile8AII (['25I]SI-AII) binding to rat aortic smooth muscle (RASM) cell and rat adrenal cortical AT, receptors (Ki = 7.6 ± 1.2 and 18.4 ± 3.9 nM respectively) compared to adrenal cortical AT2 receptors (Ki = 37.6 ± 1.3 JiM). The K, values of BMS-180560 and EXP3174, but not losartan, varied as a function of the BSA concentration used in the assays, indicating that the diacid drugs bound to albumin.3 BMS-180560 (3-300 nM) increased the KD of SI-AII for RASM cell AT, receptors. Only at high concentrations of BMS-180560 (300nM) were Bmax values decreased.4 BMS-1 80560 inhibited AII-stimulated contraction of rabbit aorta with a calculated KB = 0.068 ± 0.048 nM and decreased maximal AlI-stimulated contraction at 1 nM BMS-180560 by 75%. In the presence of 0.1% BSA, a higher KB value (5.2 ± 0.92 nM) was obtained. Losartan behaved as a competitive antagonist with a KB = 2.6 ± 0.13 nM. Contraction stimulated by endothelin-1, noradrenaline, KCl, or the TXA2 receptor agonist U-46619 were unaffected by BMS-180560 (1 nM).5 All stimulated the acidification rates of RASM cells as measured by a Cytosensor microphysiometer with an EC50 of 18 nM. Losartan (30 nM) shifted the All concentration-effect curves in a competitive manner whereas BMS-180560 (0.01 and 0.1 nM) decreased the maximum responses by 60 and 75% respectively. Inhibition by losartan and BMS-180560 could be reversed following washout although recovery took longer for BMS-180560. (17-55%). BMS-180560 (3 and 10 nM) increased the EC50 for All and decreased the maximum response by 30 and 80% respectively. The inhibition by EXP3174 and BMS-180560 could be reversed by inclusion of losartan (200 nM) indicating that the inhibition was not irreversible. 7 In conclusion, BMS-180560 is a potent, specific, predominantly competitive, reversible All receptor antagonist, which displays insurmountable receptor antagonism. At concentrations of BMS-180560 which have no effect on receptor number, BMS-180560 produced insurmountable antagonism of AII-stimulated second messenger formation, extracellular acidification, and smooth muscle contraction.
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