Immunotherapy allows increased specificity and efficacy of cancer treatment. However, toxicity can be a problem when molecular targets are present on non-diseased cells. Demibodies are novel antibody derivatives that are designed to form bispecific heterodimers that recognise unusual pairs of cell-surface antigens expressed in the context of disease. A demibody pair comprises two chains, each containing a soluble single chain variable fragment (scFv) for antigen binding, an action domain for detection or cell-killing upon heterodimerisation, and a leucine zipper for heterodimerisation. Heterodimerisation occurs through complementary zippers only after each demibody is confined to the target cell surface through antigen binding to confer increased selectivity over current immunotherapies.
Previous model demibody constructs contained mCherry and mOrange fluorescent action domains for Förster Resonance Energy Transfer (FRET) detection on leukemia cells. Expression in SHuffle E. coli cells resulted in moderate yields of soluble and fluorescent demibodies. Although live-cell FRET experiments showed some indications of FRET, the experiments were complicated by quenching and non-specific binding to the cell-surface that may be attributed to partially misfolded demibodies.
Thus, variant demibody constructs were expressed and secreted from mammalian Expi293 cells. In these constructs, a new superfolder GFP (sfGFP) was used in place of mOrange to pair with the mCherry constructs both to optimise the detection of FRET, and for compatability with oxidising environments in the secretory pathway. This approach led to improved yield and purity of demibodies. In particular, preparations of sfGFP-containing constructs predominantly contained monodisperse demibodies. Preparations of mCherry-containing constructs contained two additional lower molecular weight species that co-purified with the target protein forming multimers in solution. Mass spectrometric analysis showed that these species contained partial mCherry sequence. Therefore, mCherry may not be suitable for demibody constructs. Alternative fluorescent proteins that can FRET pair with sfGFP, and alternative action domain constructs are being investigated.