Targeting CD33+ Acute Myeloid Leukemia with GLK-33, a Lintuzumab-Auristatin Conjugate with a Wide Therapeutic Window.

CD33 (Siglec-3) is a cell surface receptor expressed in approximately 90% of acute myeloid leukemia (AML) blasts, making it an attractive target for therapy of AML. Although previous CD33-targeting antibody-drug conjugates (ADC) like gemtuzumab ozogamicin (GO, Mylotarg) have shown efficacy in AML treatment, they have suffered from toxicity and narrow therapeutic window. This study aimed to develop a novelADCwith improved tolerability and a wider therapeutic window. GLK-33 consists of the anti-CD33 antibody lintuzumab and eight mavg-MMAU auristatin linkerpayloads per antibody. The experimental methods included testing in cell cultures, patient-derived samples, mouse xenograft models, and rat toxicology studies. GLK-33 exhibited remarkable efficacy in reducing cell viability within CD33-positive leukemia cell lines and primary AML samples. Notably, GLK-33 demonstrated antitumor activity at single dose as low as 300 mg/kg in mice, while maintaining tolerability at single dose of 20 to 30 mg/kg in rats. In contrast with both GO and lintuzumab vedotin, GLK-33 exhibited a wide therapeutic window and activity against multidrug-resistant cells. The development of GLK-33 addresses the limitations of previous ADCs, offering a wider therapeutic window, improved tolerability, and activity against drug-resistant leukemia cells. These findings encourage further exploration of GLK-33 in AML through clinical trials.

Simultaneous Measurement of Tricarboxylic Acid Cycle Intermediates in Different Biological Matrices Using Liquid Chromatography-Tandem Mass Spectrometry; Quantitation and Comparison of TCA Cycle Intermediates in Human Serum, Plasma, Kasumi-1 Cell and Murine Liver Tissue.

The tricarboxylic acid (TCA) cycle is a central part of carbon and energy metabolism, also connecting to glycolysis, amino acid, and lipid metabolism. The quantitation of the TCA cycle intermediate within one method is lucrative due to the interest in central carbon metabolism profiling in cells and tissues. In addition, TCA cycle intermediates in serum have been discovered to correspond as biomarkers to various underlying pathological conditions. In this work, an Liquid Chromatography-Mass Spectrometry/Mass Spectrometry-based quantification method is developed and validated, which takes advantage of fast, specific, sensitive, and cost-efficient precipitation extraction. Chromatographic separation is achieved while using Atlantis dC18 2.1 mm × 100 mm, particle size 3-µm of Waters column with a gradient elution mobile phase while using formic acid in water (0.1% v/v) and acetonitrile. Linearity was clearly seen over a calibration range of: 6.25 to 6400 ng/mL (r2 > 0.980) for malic acid; 11.72 to 12,000 ng/mL (r2 > 0.980) for cis-aconitic acid and L-aspartic acid; 29.30 to 30,000 ng/mL (r2 > 0.980) for isocitric acid, l-serine, and l-glutamic acid; 122.07 to 125,000 ng/mL (r2 > 0.980) for citric acid, glycine, oxo-glutaric acid, l-alanine, and l-glutamine; 527.34 to 540,000 ng/mL (r2 > 0.980) for l-lactic acid; 976.56 to 1,000,000 ng/mL (r2 > 0.980) for d-glucose; 23.44 to 24,000 ng/mL (r2 > 0.980) for fumaric acid and succinic acid; and, 244.14 to 250,000 ng/mL (r2 > 0.980) for pyruvic acid. Validation was carried out, as per European Medicines Agency (EMA) "guidelines on bioanalytical method validation", for linearity, precision, accuracy, limit of detection (LOD), limit of quantification (LLOQ), recovery, matrix effect, and stability. The recoveries from serum and tissue were 79-119% and 77-223%, respectively. Using this method, we measured TCA intermediates in serum, plasma (NIST 1950 SRM), and in mouse liver samples. The concentration found in NIST SRM 1950 (n = 6) of glycine (246.4 µmol/L), l-alanine (302.4 µmol/L), and serine (92.9 µmol/L).