Characterization of activating mutations of NOTCH3 in T-cell acute lymphoblastic leukemia and anti-leukemic activity of NOTCH3 inhibitory antibodies.

Notch receptors have been implicated as oncogenic drivers in several cancers, the most notable example being NOTCH1 in T-cell acute lymphoblastic leukemia (T-ALL). To characterize the role of activated NOTCH3 in cancer, we generated an antibody that detects the neo-epitope created upon gamma-secretase cleavage of NOTCH3 to release its intracellular domain (ICD3), and sequenced the negative regulatory region (NRR) and PEST (proline, glutamate, serine, threonine) domain coding regions of NOTCH3 in a panel of cell lines. We also characterize NOTCH3 tumor-associated mutations that result in activation of signaling and report new inhibitory antibodies. We determined the structural basis for receptor inhibition by obtaining the first co-crystal structure of a NOTCH3 antibody with the NRR protein and defined two distinct epitopes for NRR antibodies. The antibodies exhibit potent anti-leukemic activity in cell lines and tumor xenografts harboring NOTCH3 activating mutations. Screening of primary T-ALL samples reveals that 2 of 40 tumors examined show active NOTCH3 signaling. We also identified evidence of NOTCH3 activation in 12 of 24 patient-derived orthotopic xenograft models, 2 of which exhibit activation of NOTCH3 without activation of NOTCH1. Our studies provide additional insights into NOTCH3 activation and offer a path forward for identification of cancers that are likely to respond to therapy with NOTCH3 selective inhibitory antibodies.

pH, salt, and buffer dependent swelling in ionizable copolymer gels: tests of the ideal Donnan equilibrium theory.

We present results of equilibrium swelling studies of the ionizable copolymer gel, methyl methacrylate/N,N-dimethylaminoethyl methacrylate 70/30 mol%, in buffered and unbuffered electrolyte solutions. The experimental conditions were designed to demonstrate the sensitivity of swelling in ionized gels to the electrolyte composition of the external solution. In general, gel swelling as a function of solution ionic strength is shown to be highly nonmonotonic and is particularly sensitive to the valence and concentrations of ions present in solution. A rigorous test of ideal Donnan equilibrium theory shows that the latter is unable to explain all the data in a self-consistent manner. However, a heuristic procedure based on the ideal Donnan theory can predict qualitatively the observed trends. While not quantitative, this heuristic approach provides considerable insight into the mechanisms underlying the swelling behavior under various solution conditions. Possible causes of nonideal behavior are discussed, and some observed specific ion effects are reported and discussed.

Buffer effects on swelling kinetics in polybasic gels.

The swelling kinetics of polybasic gels consisting of copolymers of methyl methacrylate and dimethylaminoethyl methacrylate are studied in solutions at various acidic pH values, with monoacidic derivatives of acetic acid added as buffers. The effects of solution pH, as well as buffer pKa and concentration, on swelling rate are assessed. Gel swelling rate shows a nearly linear dependence on the concentration of nonionized buffer in the solution, as determined by the Henderson-Hasselbach equation. This result is explained in terms of the increased availability of protons that are carried by the nonionized buffer to bare amines on the gel. In fact, the so-called pH sensitivity of these gels, under these conditions, can be attributed mainly to the effect of pH on the nonionized buffer concentration. A practical consequence is that these gels may not reliably mediate pH-sensitive swelling-controlled release in oral applications, since the levels of buffer acids in the stomach (where swelling and release are expected to occur) generally cannot be controlled. However, the gels may be useful as mediators of pH-triggered release when precise rate control is of secondary importance.

Selection of a derivative of the antiviral agent 9-[(1,3-dihydroxy-2-propoxy)-methyl]guanine (DHPG) with improved oral absorption.

Various diesters of 9-[(1,3-dihydroxy-2-propoxy)-methyl]guanine (DHPG) were screened in order to identify a derivative with improved oral absorption. The solubilities and dissolution rates decreased with increasing chain length and branching of the ester group. However, the dipropionate ester showed an anomalously faster dissolution rate. The rates of hydrolysis to DHPG in the presence of intestinal homogenates were found to increase with increasing carbon number for the straight-chain alkyl esters and decreased with branching. The shorter-chain alkyl esters were relatively more stable in intestinal homogenates than in liver homogenates. Therefore they may have a better membrane permeability than DHPG due to their intact ester group. The hydrolysis rates in human blood increased with increasing carbon number for the straight-chain alkyl esters. The dipropionate ester appeared to be the most promising derivative because of its rapid dissolution rate, slower hydrolysis in the intestine, and rapid conversion to DHPG in liver and blood.

A dual-column HPLC method for the simultaneous determination of DHPG (9-[(1,3-dihydroxy-2-propoxy)methyl]guanine) and its mono and diesters in biological samples.

A convenient method for the simultaneous determination of various DHPG species present in biological samples is presented. This method utilizes a cation exchange column (25 cm X 4.6 mm i.d.) coupled in series to a short reversed-phase column (5 cm X 4.6 mm i.d.). The mobile phase consists of methanol:0.005M ammonium phosphate buffer, pH 2.5. There is a large polarity difference between DHPG and its esters due to the non-polar side chain of the ester moiety. The simultaneous determination of the diesters, monoesters, and DHPG in these samples using only the cation exchange or the reversed-phase column is not possible without time-consuming gradient elution. In the reversed-phase mode alone, the esters are highly retained relative to DHPG, whereas the esters are only slightly retained on a cation exchange column and are insensitive to changes in pH and ionic strength of the mobile phase. However, a combination of these two columns provides interesting selectivity for these compounds and offers a unique way of controlling the retention times of these species relative to each other. The retention time of esters can be selectively altered (with respect to DHPG) by changing the composition of methanol in the mobile phase. In contrast, the retention time of DHPG is controlled by changing the buffer strength and pH of the mobile phase.