An antibody-drug conjugate with intracellular drug release properties showing specific cytotoxicity against CD7-positive cells.

Refractory T cell acute leukaemias that no longer respond to treatment would benefit from new modalities that target T cell-specific surface proteins. T cell associated surface proteins (the surfaceome) offer possible therapy targets to reduce tumour burden but also target the leukaemia-initiating cells from which tumours recur. Recent studies of the T cell leukaemia surfaceome confirmed that CD7 is highly expressed in overt disease. We have used an anti-CD7 antibody drug conjugate (ADC) to show that the binding of antibody to surface CD7 protein results in rapid internalization of the antigen together with the ADC. As a consequence, cell killing was observed via induction of apoptosis and was dependent on cell surface CD7. The in vitro cytotoxic activity (EC50) of the anti-CD7 ADC on T cell acute leukaemia (T-ALL) cells Jurkat and KOPT-K1 was found to be in the range of 5-8 ng/mL. In a pre-clinical xenograft model of human tumour growth expressing CD7 antigen, growth was curtailed by a single dose of ADC. The data indicate that CD7 targeting ADCs may be developed into an important second stage therapy for T cell acute leukaemia, for refractory CD7-positive leukaemias and for subsets of acute myeloid leukaemia (AML) expressing CD7.

Precursor-directed biosynthesis of nonribosomal lipopeptides with modified glutamate residues.

Precursor-directed biosynthesis of calcium dependent antibiotics (CDAs) with modified 3-trifluoromethyl and 3-ethyl glutamate residues was achieved by feeding synthetic glutamate analogues to a mutant strain of Streptomyces coelicolor impaired in the biosynthesis of the natural precursor (2S,3R)-3-methyl glutamic acid.

Biosynthesis of the (2S,3R)-3-methyl glutamate residue of nonribosomal lipopeptides.

The calcium-dependent antibiotics (CDAs) and daptomycin are therapeutically relevant nonribosomal lipopeptide antibiotics that contain penultimate C-terminal 3-methyl glutamate (3-MeGlu) residues. Comparison with synthetic standards showed that (2S,3R)-configured 3-MeGlu is present in both CDA and daptomycin. Deletion of a putative methyltransferase gene glmT from the cda biosynthetic gene cluster abolished the incorporation of 3-MeGlu and resulted in the production of Glu-containing CDA exclusively. However, the 3-MeGlu chemotype could be re-established through feeding synthetic 3-methyl-2-oxoglutarate and (2S,3R)-3-MeGlu, but not (2S,3S)-3-MeGlu. This indicates that methylation occurs before peptide assembly, and that the module 10 A-domain of the CDA peptide synthetase is specific for the (2S,3R)-stereoisomer. Further mechanistic analyses suggest that GlmT catalyzes the SAM-dependent methylation of alpha-ketoglutarate to give (3R)-methyl-2-oxoglutarate, which is transaminated to (2S,3R)-3-MeGlu. These insights will facilitate future efforts to engineer lipopeptides with modified glutamate residues, which may have improved bioactivity and/or reduced toxicity.