Site-Specific Conjugation of Monomethyl Auristatin E to Anti-CD30 Antibodies Improves Their Pharmacokinetics and Therapeutic Index in Rodent Models.

Antibody-drug conjugates (ADCs) have demonstrated clinical benefits that have led to the recent FDA approval of KADCYLA and ADCETRIS. Most ADCs that are currently in clinical use or development, including ADCETRIS, are produced by chemical conjugation of a toxin via either lysine or cysteine residues, inevitably leading to heterogeneous products with variable drug-to-antibody ratios (DARs). Here, we describe the in vitro and in vivo characterization of four novel ADCs that are based on the anti-CD30 antibody cAC10, which has the same polypeptide backbone as ADCETRIS, and compare the results with the latter. Bacterial transglutaminase (BTG) was exploited to site-specifically conjugate derivatives of monomethyl auristatin E (all comprising a cleavable linker) to the glutamine at positions 295 and 297 of cAC10, thereby yielding homogeneous ADCs with a DAR of 4. In vitro cell toxicity experiments using two different CD30-positive cell lines (Karpas 299 and Raji-CD30(+)) revealed comparable EC50 values for ADCETRIS (1.8 ± 0.4 and 3.6 ± 0.6 ng/mL, respectively) and the four cAC10-based ADCs (2.0 ± 0.4 to 4.9 ± 1.0 ng/mL). Quantitative time-dependent in vivo biodistribution studies (3-96 h p.i.) in normal and xenografted (Karpas 299 cells) SCID mice were performed with a selected (125)I-radioiodinated cAC10 ADC and compared with that of (125)I-ADCETRIS. The chemo-enzymatically conjugated, radioiodinated ADC showed higher tumor uptake (17.84 ± 2.2% ID/g 24 h p.i.) than (125)I-ADCETRIS (10.5 ± 1.8% ID/g 24 h p.i.). Moreover, (125)I-ADCETRIS exhibited higher nontargeted liver and spleen uptake. In line with these results, the maximum tolerated dose of the BTG-coupled ADC (>60 mg/kg) was significantly higher than that of ADCETRIS (18 mg/kg) in rats. These results suggest that homogeneous ADCs display improved pharmacokinetics and better therapeutic indexes compared to those of chemically modified ADCs with variable DARs.

Thiazolinium and imidazolium chiral ionic liquids derived from natural amino acid derivatives.

Starting from commercially available amino acid derivatives, two novel families of chiral ionic liquids having either a thiazolinium or an imidazolium cation were prepared by simple and straightforward procedures in good overall yields. The properties of these new salts can be finely tuned by careful selection of the anion and the cation.

Translational misreading: a tRNA modification counteracts a +2 ribosomal frameshift.

Errors during gene expression from DNA to proteins via transcription and translation may be deleterious for the functional maintenance of cells. In this paper, extensive genetic studies of the misreading of a GA repeat introduced into the lacZ gene of Escherichia coli indicate that in this bacteria, errors occur predominantly by a +2 translational frameshift, which is controlled by a tRNA modification involving the MnmE and GidA proteins. This ribosomal frameshift results from the coincidence of three events: (1) decreased codon-anticodon affinity at the P-site, which is caused by tRNA hypomodification in mnmE(-) and gidA(-) strains; (2) a repetitive mRNA sequence predisposing to slippage; and (3) increased translational pausing attributable to the presence of a rare codon at the A-site. Based on genetic analysis, we propose that GidA and MnmE act in the same pathway of tRNA modification, the absence of which is responsible for the +2 translational frameshift. The difference in the impact of the mutant gene on cell growth, however, indicates that GidA has at least one other function.