# A nucleotide‐independent, pan‐RAS‐targeted DARPin elicits anti‐tumor activity in a multimodal manner

## TL;DR

A new protein called DARPin 784_F5 can block cancer-causing RAS proteins in both active and inactive states, potentially leading to tumor regression.

## Contribution

A nucleotide-independent DARPin that targets all RAS isoforms and inhibits their function in both GDP and GTP states.

## Key findings

- The DARPin 784_F5 binds to RAS with low nanomolar affinity regardless of nucleotide state.
- It inhibits RAS activation, downstream signaling, and nanoclustering, reducing cancer cell growth.
- Expression of 784_F5 led to tumor regression in a colorectal xenograft model.

## Abstract

The KRAS oncoprotein is a frequent tumor driver in lung, pancreatic, and colorectal cancers and has proven to be a challenging pharmaceutical target. The first KRAS‐targeted therapeutics are now being tested in clinical trials but the consequences of preferentially targeting the GDP or GTP state of KRAS and the relevance of RAS nanoclustering have remained unclear. Here we report a Designed Ankyrin Repeat Protein (DARPin) that recognizes the RAS switch I/II region with low nm affinity, independently of the nucleotide bound (GDP‐ or GTP state). This DARPin, termed ‘784_F5’, occupies the effector recognition lobe, resulting in interference with SOS‐mediated activation, RAS downstream effector interactions, and KRAS nanoclustering. Consequently, this anti‐RAS DARPin potently blocks downstream signaling, leading to a strong reduction in proliferation and anchorage‐independent growth in RAS‐dependent cell lines. We showed that the expression of ‘784_F5’, the pan‐RAS, nucleotide‐independent DARPin can lead to tumor regression in a colorectal xenograft model which may hold promise for further investigation and development.

We report a Designed Ankyrin Repeat Protein that binds and inhibits RAS proteins, which serve as central cell signaling hubs and are essential for the progression of many cancers. Its unique feature is that it does not discriminate between different RAS isoforms or mutations and is capable of binding to RAS in both its active (GTP‐bound) and inactive (GDP‐bound) states.