Current methods for the synthesis of homogeneous antibody–drug conjugates

AbstractDevelopment of efficient and safe cancer therapy is one of the major challenges of the modern medicine. Over the last few years antibody–drug conjugates (ADCs) have become a powerful tool in cancer treatment with two of them, Adcetris® (brentuximab vedotin) and Kadcyla® (ado-trastuzumab emtansine), having recently been approved by the Food and Drug Administration (FDA). Essentially, an ADC is a bioconjugate that comprises a monoclonal antibody that specifically binds tumor surface antigen and a highly potent drug, which is attached to the antibody via either cleavable or stable linker. This approach ensures specificity and efficacy in fighting cancer cells, while healthy tissues remain largely unaffected.Conventional ADCs, that employ cysteine or lysine residues as conjugation sites, are highly heterogeneous. This means that the species contain various populations of the ADCs with different drug-to-antibody ratios (DARs) and different drug load distributions. DAR and drug-load distribution are essential parameters of ADCs as they determine their stability and efficacy. Therefore, various drug-loaded forms of ADCs (usually from zero to eight conjugated molecules per antibody) may have distinct pharmacokinetics (PK) in vivo and may differ in clinical performance. Recently, a significant progress has been made in the field of site-specific conjugation which resulted in a number of strategies for synthesis of the homogeneous ADCs. This review describes newly-developed methods that ensure homogeneity of the ADCs including use of engineered reactive cysteine residues (THIOMAB), unnatural amino acids, aldehyde tags, enzymatic transglutaminase- and glycotransferase-based approaches and novel chemical methods. Furthermore, we briefly discuss the limitation of these methods emphasizing the need for further improvement in the ADC design and development

Specific antibody fragment ligand traps blocking FGF1 activity

Fibroblast growth factor 1 (FGF1) and its receptors (FGFRs) regulate crucial biological processes such as cell proliferation and differentiation. Aberrant activation of FGFRs by their ligands can promote tumor growth and angiogenesis in many tumor types, including lung or breast cancer. The development of FGF1-targeting molecules with potential implications for the therapy of FGF1-driven tumors is recently being considered a promising approach in the treatment of cancer. In this study we have used phage display selection to find scFv antibody fragments selectively binding FGF1 and preventing it from binding to its receptor. Three identified scFv clones were expressed and characterized with regard to their binding to FGF1 and ability to interfere with FGF1-induced signaling cascades activation. In the next step the scFvs were cloned to scFv-Fc format, as dimeric Fc fusions prove beneficial in prospective therapeutic application. As expected, scFvs-Fc exhibited significantly increased affinity towards FGF1. We observed strong antiproliferative activity of the scFvs and scFvs-Fc in the in vitro cell models. Presented antibody fragments serve as novel FGF1 inhibitors and can be further utilized as powerful tools to use in the studies on the selective cancer therapy

Fibroblast growth factor 2 conjugated with monomethyl auristatin E inhibits tumor growth in a mouse model

Worldwide, cancer is the second leading cause of death. Regardless of the continuous progress in medicine, we still do not have a fully effective anti-cancer therapy. Therefore, the search for new targeted anti-cancer drugs is still an unmet need. Here, we present novel protein–drug conjugates that inhibit tumor growth in a mouse model of human breast cancer. We developed conjugates based on fibroblast growth factor (FGF2) with improved biophysical and biological properties for the efficient killing of cancer cells overproducing fibroblast growth factor receptor 1 (FGFR1). We used hydrophilic and biocompatible PEG4 or PEG27 molecules as a spacer between FGF2 and the toxic agent monomethyl auristatin E. All conjugates exhibited a cytotoxic effect on FGFR1-positive cancer cell lines. The conjugate with the highest hydrodynamic size (42 kDa) and cytotoxicity was found to efficiently inhibit tumor growth in a mouse model of human breast cancer