Pan RAS-binding compounds selected from a chemical library by inhibiting interaction between RAS and a reduced affinity intracellular antibody

Abstract

Intracellular antibodies are valuable tools for target validation studies for clinical situations such as cancer. Recently we have shown that antibodies can be used for drug discovery in screening for chemical compounds surrogates by showing that compounds could be developed to the so-called undruggable RAS protein family. This method, called Antibody-derived compound (Abd) technology, employed intracellular antibodies binding to RAS in a competitive surface plasmon resonance chemical library screen. Success with this method requires a high affinity interaction between the antibody and the target. We now show that reduction in the affinity (dematuration) of the anti-active RAS antibody facilitates the screening of a chemical library using an in vitro AlphaScreen method. This identified active RAS specific-binding Abd compounds that inhibit the RAS-antibody interaction. One compound is shown to be a pan-RAS binder to KRAS, HRAS and NRAS-GTP proteins with a Kd of average 37 mM, offering the possibility of a new chemical series that interacts with RAS in the switch region where the intracellular antibody binds. This simple approach shows the druggability of RAS and is generally applicable to antibody-derived chemical library screening by affording flexibility through simple antibody affinity variation. This approach can be applied to find Abd compounds as surrogates of antibody-combining sites for novel drug development in a range of human diseases.

Evolution of nanobodies specific for BCL11A

“…Chemical series identified through competition assays inside cells occupy the effector binding region and interfere with protein–protein interactions and signal transduction. 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 ).…”

Engineered fast-dissociating antibody fragments for multiplexed super-resolution microscopy

“…Most studies aiming to engineer the affinity of antibodies have focused the mutagenesis on the binding interface or CDR loops. Point mutations in the middle of CDRs often lead to the loss of antigen recognition rather than reducing the affinity ( Clark et al., 2006 ; Tanaka et al., 2021 ; Yamashita et al., 2019 ). Due to the high diversity of the CDR sequences, mutation sites for each antibody have to be explored individually when no structure information is available.…”

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

“…We have used a dematuration method to decrease iDAb affinity based on CDR sequences (26) such as it enabled an alpha-screen of RAS G12V -binding compounds and analysis of in vitro-derived RAS-binding Abd compounds (27). On the basis of the LMO2-iDAb LMO2 structural information (23), we introduced additional mutations on the CDRs of iDAb LMO2 dm that would affect the interaction between key amino acids from the iDAb and LMO2 with alanine or glycine substitution while still retaining specific binding (Fig.…”

“…In addition, manipulating the affinity of intracellular antibody fragments, unlike natural partners, is potentially straightforward without structural data, since their CDRs, defined by primary sequence, are major actors of their interaction with their target antigen (34). This process is called intracellular antibody dematuration (27). Furthermore, intracellular antibodies can discriminate family members (isoforms or paralogs), which themselves may have common binding partners [e.g., KRAS (16)].…”

“…In contrast to the cSPR Abd method where a high-affinity antibody is needed (19), the measured in vitro affinity of the iDAbs for their target is not a limitation with our cell-based assay. Highaffinity iDAbs could be dematured to reduce their binding for use in the cell-based assay such as in a RAS compound selection assay (27). Alternatively, an iDAb already with a lower affinity could be directly used in the cell-based assay without prior dematuration step, which makes this a flexible approach.…”