Scaffolds within scaffolds: Generating ion channel blocking antibodies by fusing knottin to peripheral CDR loops

Cysteine-knot miniproteins (knottins) have potential as therapeutic agents to block ion channels involved in cancer, autoimmunity and pain but suffer from manufacturing difficulties, short half-lives and a lack of specificity. IONTAS have invented a novel molecular format wherein a peripheral CDR loop (e.g. VL CDR2) of an antibody has been removed and replaced by a naturally occurring knottin. In this novel format (termed a KnotBodyTM), the knottin enjoys the extended half-life of an antibody molecule and the peripheral CDRs gain additional diversity within a scaffold which is pre-disposed to blockade of ion channels. This example of successful fusion of one structural domain within another was initially achieved by inserting a trypsin binding knottin (EETI-II) flanked by diverse repertoire of short linker sequences into the CDR2 position of naïve antibody light chain sequences. Functional KnotBodies were selected from this library using phage display technology on the basis of retained trypsin binding and the correct folding of both domains were confirmed using X-ray crystallography. To further demonstrate the merits of this novel format, the modular nature of the KnotBody binding surface was exploited to: (i) improve existing knottin binding by introducing additional VHcontacts; (ii) create a bispecific molecule by introducing a VH chain that binds to a different target; (iii) engineer novel binding specificity on the knottin scaffold by loop diversification; (iv) substitute the selected (EETI-II trypsin binding) knottin with ion channel blocking knottins.