Chimaeric Proteins With Ubiquitin Ligase for Selective Mutant KRAS Degradation
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Solution Overview
Problem
Current therapeutic agents struggle to specifically target KRAS-associated cancers and RASopathies, as they often require identification of specific mutations and are ineffective against different mutations, and existing degraders fail to discriminate between mutant and wild-type RAS proteins.
Innovation Solution
Development of chimaeric proteins comprising a ubiquitin ligase domain and a RAS-specific or LMO2-specific endogenous targeting portion, which induce proteolysis of target proteins via the ubiquitin-proteasome system, effectively degrading mutant KRAS without affecting wild-type RAS or LMO2, thus inhibiting cancer cell growth.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If small molecule inhibitors are used to target KRASG12C, then specific inhibition of mutant KRAS is achieved, but the treatment is ineffective against other KRAS mutations and only applies to 12% of mutant KRAS tumours
Solution Approach 1:
The invention segments the RAS protein family into three distinct targets (KRAS, NRAS, HRAS) and develops separate intracellular single domain antibodies for each, allowing broad coverage of all RAS mutations while maintaining mutation-agnostic binding capability
Solution Approach 2:
The intracellular single domain antibodies are designed to bind universally to all RAS isoforms (KRAS, NRAS, HRAS) regardless of mutation status, creating a multi-functional agent that addresses both mutant and wild-type RAS across all family members
2Adaptability or versatility
If pan-RAS-binding intracellular single domain antibody is expressed, then all RAS isoforms are targeted, but inability to discriminate mutant from wild-type RAS limits specificity for mutant KRAS tumours
Solution Approach 1:
The invention creates functionally distinct antibody variants with different binding specificities: pan-RAS-binding antibodies for broad coverage and KRAS-specific antibodies for focused targeting, allowing selection based on tumour genotype while maintaining the advantage of mutation-agnostic binding
Solution Approach 2:
Instead of designing antibodies that bind specifically to mutant RAS conformations (which would require knowing the mutation), the invention inverts the approach by using mutation-agnostic antibodies that bind all RAS isoforms, then achieves specificity through selective expression in tumour cells versus normal cells
3Reliability
If existing PROTACs are used to degrade KRAS, then protein degradation is achieved, but they only degrade exogenous GFP-KRASG12C fusion protein and do not target endogenous KRASG12C
Solution Approach 1:
The intracellular single domain antibodies are expressed within the tumour cells themselves, allowing the degradation system to operate endogenously without requiring external fusion proteins or transfection of target constructs, thereby achieving degradation of native endogenous RAS proteins
4Measurement precision
If DARPins K13 and K19 are used to interfere with KRAS, then specific binding to KRAS is achieved, but inability to discriminate mutant from wild-type KRAS reduces tumour-specificity
Solution Approach 1:
The invention performs preliminary targeting by having the intracellular single domain antibody bind to RAS proteins before degradation occurs, and uses the cell's own ubiquitin-proteasome system to execute the degradation, achieving specific tumour cell killing while sparing normal cells that express wild-type RAS
Solution Approach 2:
The intracellular single domain antibody acts as an intermediary that bridges the RAS protein and the ubiquitin-proteasome degradation system, enabling specific degradation of RAS in tumour cells while leaving wild-type RAS in normal cells intact
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The chimaeric proteins achieve selective degradation of mutant KRAS and LMO2 proteins, leading to apoptosis of cancer cells and tumor regression without requiring mutation-specific binding, offering a broad-spectrum treatment for KRAS-associated disorders and LMO2-associated conditions.
Implementation Method 1
They consist of a binder targeting a protein of interest (e.g. intracellular single domain), a linker and an E3 ligase domain. Macromolecule degraders induce the depletion of their target via the ubiquitin-proteasome system.
Data Source
AI summary
Provided is a chimaeric protein comprising a ubiquitin ligase domain and a RAS-specific endogenous targeting portion. The ubiquitin ligase domain may be a VHL E3 ligase domain, or a fragment or variant thereof having ubiquitin ligase activity; or a UBOX domain of CHIP, or a fragment or variant thereof having ubiquitin ligase activity. The RAS-specific endogenous targeting portion may be a RAS-specific DARPin; or a RAS-specific intracellular antibody. In particular, the RAS-specific endogenous targeting portion may be a KRAS-specific endogenous targeting portion (for example a KRAS-specific DARPin, or a KRAS-specific intracellular antibody). The chimaeric proteins may be used in the prevention and/or treatment of a RAS-associated disorder, such as a RAS-associated cancer or a RASopathy. Further provided is a chimaeric protein comprising a ubiquitin ligase domain and an LMO2-specific endogenous targeting portion.