Generating potent and selective inhibitors of Kv1.3 ion channels by fusing venom derived mini proteins into peripheral CDR loops of antibodies
Pathogenic T cell effector memory (TEM) cells drive many autoimmune disorders and are uniquely dependent on the Kv1.3 channel. A number of venom derived knottin (cysteine-rich mini-protein) inhibitors of Kv1.3 are being developed as potential drug candidates, but can suffer from manufacturing difficulties, short half-lives and a lack of specificity. We have developed a novel molecular format wherein a peripheral CDR loop of an antibody has been replaced by a knottin. In this novel KnotBodyTM format, the knottin benefits from the improved therapeutic functionality of an antibody and the antibody gains additional diversity by the addition of a scaffold which is pre-disposed to blockade of ion channels.
A proof-of-concept fusion protein of one structural domain within another was initially achieved by inserting a trypsin inhibiting knottin (EETI-II) flanked by diverse repertoire of short linker sequences into the CDR2 position of naïve antibody light chain sequences. Functional KnotBodyTM molecules were selected from this library using phage display technology on the basis of retained trypsin binding, with the correct folding of both domains confirmed using X-ray crystallography.
To further demonstrate the benefits of this novel format, the modular nature of the KnotBodyTM binding surface was exploited to: (i) improve existing knottin binding by introducing additional VH contacts; (ii) create a bispecific molecule by introducing a VH chain that binds to a different target; (iii) substitute the proof-of-concept knottin (EETI-II, a trypsin inhibitor) with ShK, a Kv1.3 ion channel blocking toxin; (iv) develop a panel of low-nM Kv1.3 inhibitors with selectivity exceeding 3000-fold over the Kv1.1 channel, a closely related Kv family member.