NMR-directed evolution of tight-binding nanobodies.
Lead Research Organisation:
University of Manchester
Department Name: Chemistry
Abstract
CONTEXT: Numerous alternatives to antibodies have been developed that circumvent some of the inherent problems of these large disulphide-linked proteins, with the nanobodies produced by camelids perhaps being at the forefront. Libraries of nanobodies can be raised in vivo from Camelids or in vitro, and are subsequently "panned" with recombinant techniques for the specificity of choice. The resulting nanobodies can then be easily produced recombinantly in bacteria or yeast.
CHALLENGE: Frequently, reasonably well-behaved nanobodies can be selected from naïve libraries, with dissociation constants in the range of 0.1-1µM. However, many applications require higher affinity than this, for binding to survive stringent wash conditions, for example, and affinity maturation of the hit nanobody is required. Re-panning of the library will invariably not lead to different nanobodies being produced meaning that re-immunisation of llama or a targeted alteration of the binding site is the best option for increasing binding affinity. Targeted evolution usually requires knowledge of the binding site through crystallographic techniques followed by site-directed mutagenesis. For the majority of protein-protein interactions this is not feasible so we propose to use an NMR-directed strategy for the directed evolution of high affinity nanobodies.
AIMS & OBJECTIVE: To use NMR-directed evolution to reliably produce orders-of-magnitude improvements of binding affinity of nanobodies, for the same cost and time as an additional immunisation or a crystallography-based targeted evolution approach.
This strategy is to be applied (initially) to a variety of small/oligomeric Target molecules namely:
[T1] Heparin oligomers of known sulphation pattern
[T2] phosphopeptides of known phosphorylation state
[T3] Fragments of bacterial lipopolysaccharides
In addition a small protein target (T4), lysozyme will be tested to ascertain applicability of method to small protein targets.
IMPACT: The utility of antibodies as tool compounds for medical and biochemical research and diagnosis is well understood. The ability to produce alternatives quickly and without the requirement for repeated inoculations of animals will vastly reduce the cost of these tool compounds, and allow the direction of techniques that use them to new targets, for example toxic bacterial polysaccharides.
CHALLENGE: Frequently, reasonably well-behaved nanobodies can be selected from naïve libraries, with dissociation constants in the range of 0.1-1µM. However, many applications require higher affinity than this, for binding to survive stringent wash conditions, for example, and affinity maturation of the hit nanobody is required. Re-panning of the library will invariably not lead to different nanobodies being produced meaning that re-immunisation of llama or a targeted alteration of the binding site is the best option for increasing binding affinity. Targeted evolution usually requires knowledge of the binding site through crystallographic techniques followed by site-directed mutagenesis. For the majority of protein-protein interactions this is not feasible so we propose to use an NMR-directed strategy for the directed evolution of high affinity nanobodies.
AIMS & OBJECTIVE: To use NMR-directed evolution to reliably produce orders-of-magnitude improvements of binding affinity of nanobodies, for the same cost and time as an additional immunisation or a crystallography-based targeted evolution approach.
This strategy is to be applied (initially) to a variety of small/oligomeric Target molecules namely:
[T1] Heparin oligomers of known sulphation pattern
[T2] phosphopeptides of known phosphorylation state
[T3] Fragments of bacterial lipopolysaccharides
In addition a small protein target (T4), lysozyme will be tested to ascertain applicability of method to small protein targets.
IMPACT: The utility of antibodies as tool compounds for medical and biochemical research and diagnosis is well understood. The ability to produce alternatives quickly and without the requirement for repeated inoculations of animals will vastly reduce the cost of these tool compounds, and allow the direction of techniques that use them to new targets, for example toxic bacterial polysaccharides.
