Masked IL12 Protein via Bioorthogonal Trigger
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Solution Overview
Problem
Existing methods for masking IL12 proteins to minimize side effects and achieve targeted activation in vivo are inefficient, relying on overexpression of proteases, non-target expression, and genetic engineering of the IL12 protein, which can lead to off-target toxicity and require complex constructs.
Innovation Solution
A masked IL12 protein is developed with specific lysine residues modified to include a dienophile or tetrazine Trigger, allowing for controlled activation in vivo by reacting with a diene or dienophile, independent of the target site's microenvironment, and without the need for genetic engineering.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If protease-cleavable linkers are used to mask IL12 protein, then the IL12 protein can be activated in vivo, but off-target toxicity occurs due to non-target expression and unintended linker cleavage
Solution Approach 1:
The patent removes the protease-cleavable linker component from the masking system and replaces it with a chemically stable mask that is removed by non-enzymatic chemical reduction. This extraction of the protease dependency eliminates off-target cleavage events while maintaining targeted activation capability through controlled chemical reduction at the target site
Solution Approach 2:
The patent changes the activation mechanism from enzymatic cleavage to chemical reduction by changing the oxidation state of a disulfide bond. This parameter change from proteolytic processing to redox chemistry provides more controlled and predictable activation kinetics while reducing off-target effects, as the chemical reduction can be more precisely controlled than protease activity
2Reliability
If genetically engineered IL12 protein constructs are used to include masking elements, then masking functionality is achieved, but the complexity of protein production increases
Solution Approach 1:
The patent segments the masking functionality into a separate chemically attachable mask component rather than incorporating it into the IL12 protein sequence itself. This allows the mask to be applied post-production to wild-type IL12 protein, avoiding the need for genetically engineered fusion proteins and simplifying both protein expression and mask attachment processes
Solution Approach 2:
The patent introduces a disulfide bond as an intermediary linkage between the IL12 protein and the masking element. This reversible chemical bond serves as a mediator that allows the mask to be attached to wild-type protein through simple chemical conjugation and subsequently removed by chemical reduction, eliminating the need for complex genetic engineering while maintaining stable masking during circulation
3Reliability
If the IL12 receptor is used to mask IL12 protein, then masking is achieved, but co-expression is required which increases production labor and anti-drug antibody response
Solution Approach 1:
The patent extracts the masking functionality from the IL12 receptor system and replaces it with a chemically attached mask that does not require receptor co-expression. This eliminates the need for complex dual-expression systems while maintaining the ability to mask IL12 activity during circulation and enable targeted activation at the disease site
Solution Approach 2:
The patent creates a simplified copy of the masking function that mimics the desired outcome of receptor-mediated masking without requiring the actual receptor protein. The chemically attached mask replicates the masking effect through steric hindrance and chemical modification of the IL12 surface, providing equivalent functionality with much simpler production requirements
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 approach significantly reduces off-target effects, allows for precise timing of IL12 activation, and improves the ratio of unmasked to masked IL12 activity, while maintaining rapid unmasking kinetics and avoiding the use of IL12 receptor components.
Implementation Method 1
at least one lysine residue of the IL12 protein has a structure according to Formula (1), in which the wiggly lines indicate bonds to other amino acid residues of the IL12 protein; the Trigger is a dienophile or a tetrazine; the dienophile or the tetrazine is linked to the ε-amine of the lysine residue of Formula (1) via a bond CA
Data Source
AI summary
Disclosed herein are masked IL12 proteins that can be activated both in vitro and in vivo. Surprisingly, a small, bioorthogonal group is sufficient to deactivate or mask the IL12 protein, which can thereafter be activated by contacting the masked IL12 protein with the bioorthogonal reactive partner of the Trigger joining the IL12 protein to the mask (either a dienophile or a diene). The invention also relates to methods for using and preparing said masked IL12 proteins.