A cell-based screening method using an intracellular antibody for discovering small molecules targeting the translocation protein LMO2

Abstract

Intracellular antibodies are tools that can be used directly for target validation by interfering with properties like protein-protein interactions. An alternative use of intracellular antibodies in drug discovery is developing small-molecule surrogates using antibody-derived (Abd) technology. We previously used this strategy with an in vitro competitive surface plasmon resonance method that relied on high-affinity antibody fragments to obtain RAS-binding compounds. We now describe a novel implementation of the Abd method with a cell-based intracellular antibody–guided screening method that we have applied to the chromosomal translocation protein LMO2. We have identified a chemical series of anti-LMO2 Abd compounds that bind at the same LMO2 location as the inhibitory anti-LMO2 intracellular antibody combining site. Intracellular antibodies could therefore be used in cell-based screens to identify chemical surrogates of their binding sites and potentially be applied to any challenging proteins, such as transcription factors that have been considered undruggable.

Evolution of nanobodies specific for BCL11A

“…The concept was initially applied in isolating drug leads directed to RAS from two commercial libraries, guided by intracellular antibodies that bind activated RAS isoforms ( 45 , 46 ). Chemical surrogates that bind to LMO2 were also identified using an inhibitory intracellular antibody fragment as a competitor in a compound library screen ( 47 ). Similarly, nanobodies may be suitable for surrogate compound development in cells.…”

Antibody-Based Approaches to Target Pancreatic Tumours

“…Abd uses intracellular antibodies as a guide to select small molecules that would display the same inhibitory mechanism as the intracellular antibody. This strategy has been successfully applied to LMO2 and RAS oncoproteins [ 148 , 149 , 150 , 151 , 152 , 153 ], with notable antiproliferative and cytotoxic effects for the anti-RAS compounds in RAS-mutated cancer cell lines.…”

Intracellular Antibodies for Drug Discovery and as Drugs of the Future

“…The systemic delivery of intracellular antibodies (or similar modalities, such as DARPins [ 31 ] or monobodies [ 32 ]), is a main challenge in this field. When this is achieved, it will transform drug development because selected antibody fragments will be able to be optimised via affinity maturation or dematuration as required [ 33 , 34 ] and/or warheads easily engineered. In this context, macromolecules are macrodrugs [ 35 ] and potentiate a new pharmacology.…”

“…It also implemented the dematured intracellular antibody approach but in a cell-based BRET screen, where the disordered protein was expressed in its normal cellular environment. Thus, this allowed chemical library screening of a disordered protein [ 34 ] that could not be expressed well in recombinant form without co-expression of the iDAb [ 18 ]. This cell-based Abd methodology was exemplified by work on the LMO2 T-cell oncogenic protein, which was discovered to be a chromosomal translocation-activated gene in T-ALL [ 56 , 57 ] (reviewed in [ 58 ]).…”