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"Generation and Evaluation of ""Next-generation"" Antibody-Toxin-Conjugates for Cancer Therapy"

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New-generation cancer therapy

Traditionally, chemotherapy has been used to treat cancer. Recently, therapeutic antibodies have been approved for the treatment of cancer that promise great selectivity for cancer cell targeting.

Fundamental Research
Health

Antibody drug conjugates (ADC) consist of cellular toxins coupled to therapeutic antibodies. They have demonstrated high efficacy in cancer therapy. However, the chemical conjugation process is random, leading to modification of functional parts of the antibody, and thus, impairing the stability, specificity and overall structure of the antibody. In addition, this approach produces heterogeneous ADCs with variable number of conjugated toxins, and thus, drugs composed of mixtures of ADC molecules. To address the challenges associated with ADC manufacturing, scientists of the EU-funded MABTOX project developed and validated two novel enzymatic conjugation platforms. In particular, they utilised sortase enzymes, a unique class of trans-peptidase enzymes that recognise specific amino acid motifs and can mediate the covalent coupling between proteins. The second conjugation platform employed the split-intein technology, where the two protein domains to be conjugated are attached to the N-terminal and to the C-terminal part of a split-intein respectively. The platforms were tested for the development of already clinically approved ADCs for the treatment of leukaemia, Hodgkin’s lymphoma and solid tumours positive for the glycoprotein NMB (gpNMB). Scientists established and optimised the process for conjugating the MMAF toxin to antibodies against the CD30 and gpNMB surface molecules using sortase enzymes. The process reached up to 80 % conjugation efficiency and produced the next generation ADCs in sufficient quantities. The enzymatically conjugated ADCs were thoroughly characterised by HPLC and mass spectrometry to confirm the addition of the toxin payload to the expected conjugation site, determined by the position of the sortase tag. At the same time, the antibody remained fully intact and displayed the expected molecular weight. In vitro testing of the new ADCs was performed using cell killing assays based on tumour cell lines as models. Results indicated that MMAF-ADCs displayed essentially identical anti-tumour activity against Hodgkin lymphoma and melanoma cells, similar to benchmark ADCs. Importantly, the anti-tumour effect was mediated via the specific binding of the ADCs. Collectively, the activities of the MABTOX project demonstrated that the sortase-enzyme mediated antibody conjugation technology leads to fully functional ADCs, thereby validating the approach for clinical use.

Keywords

Cancer, therapeutic antibodies, ADC, MABTOX, sortase enzyme

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