IL-2 Muteins with Superagonist Activity via Interface Mutations
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Solution Overview
Problem
Existing IL-2 superagonists face challenges with stability, solubility, and developability, which affect their expression levels, aggregation propensity, and thermal stability, ultimately limiting their biological activity and therapeutic efficacy.
Innovation Solution
Identification of discrete sets of mutations in the IL-2 sequence, specifically in the vicinity of the IL-2 receptor beta chain binding interface, which increase the cytokine's binding capacity to the beta subunit, leading to enhanced superagonist activity. These mutations include changes at positions 81, 83, 84, and 87, and the introduction of the I92L substitution, resulting in a favourable developability profile.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If individual mutations or discrete sets of mutations are introduced to modify functional properties of cytokines, then binding capacity to receptor subunits is improved, but stability and solubility of the molecules deteriorate
Solution Approach 1:
The patent combines multiple mutations (including K35E, R81D, R83Q, S87E, and I92L) into a single integrated mutein construct. This merging approach allows the cumulative effect of individual mutations to enhance binding capacity while the specific combination is optimized to maintain molecular stability and solubility, resolving the contradiction between improved binding and maintained stability.
Solution Approach 2:
The patent systematically varies amino acid residues at specific positions (81, 83, 84, 87, and 92) to optimize the balance between binding capacity and stability. By changing parameters such as charge distribution (introducing negative charges at positions 81, 83, and 87) and hydrophobicity (I92L substitution), the muteins achieve enhanced beta-chain binding while maintaining favorable developability profiles including stability and solubility.
2Reliability
If individual mutations or discrete sets of mutations are introduced to modify functional properties of cytokines, then binding capacity to receptor subunits is improved, but solubility of the molecules deteriorates
Solution Approach 1:
The patent modifies charge distribution parameters by introducing negative charges at positions 81, 83, and 87 through mutations such as K35E, R81D, and R83Q. These parameter changes enhance electrostatic interactions with the beta chain while the specific combination of mutations is optimized to prevent excessive charge accumulation that could compromise solubility, thus maintaining ease of manufacture.
Solution Approach 2:
The patent applies mutations locally at the binding interface (positions 81-87) and at position 92, concentrating functional improvements where they are most needed for beta-chain interaction. This localized modification approach minimizes disruptions to the overall molecular structure and solubility properties, allowing enhanced binding capacity without sacrificing solubility and ease of manufacture.
3Stability of the object's composition
If extensive in silico optimisation and receptor affinity selection are used to redesign stable agonists, then stability is improved, but deviation from primary sequence of natural cytokines increases leading to potential immunogenicity
Solution Approach 1:
The patent applies mutations primarily at the binding interface (positions 81-87) and at position 92, preserving the overall native IL-2 sequence structure. This localized modification strategy maintains sequence similarity to natural cytokines, reducing the risk of immunogenicity while achieving enhanced stability through targeted changes at critical positions that affect both stability and binding properties.
Solution Approach 2:
The patent makes controlled parameter changes at specific positions rather than extensive sequence redesign. By limiting modifications to seven specific positions and using conservative substitution strategies (such as I92L and charged residue changes at 81, 83, and 87), the muteins achieve improved stability while maintaining sufficient sequence homology to natural IL-2 to minimize immunogenicity concerns.
Data Source
AI summary
The present invention relates to the Biotechnology branch and is based on the identification of sets of IL-2 mutations in the vicinity of the interface with the receptor beta chain, through the selection of variants from filamentous phage libraries by affinity to the extracellular domain of the beta chain. Recombinant proteins derived from these variants show a very favourable developability profile, in terms of high expression levels, low tendency to aggregate, and high thermal stability. In addition, compared to the original unmutated IL-2 and to other described superagonist muteins, they have a higher capacity to stimulate immune effector populations carrying the dimeric IL-2 receptor and a higher anti-tumour activity in vivo.