Receptor-Targeted Fusion Protein With Cleavage-Activated Cell Killing
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Solution Overview
Problem
Current treatments for human cytomegalovirus (HCMV) infections, such as immunotoxins, suffer from limited selectivity and insufficient internalization leading to inadequate cell killing, and are associated with significant adverse side effects and drug resistance.
Innovation Solution
A fusion protein comprising a first peptide that binds to a receptor, such as US28, and a second peptide with an optimized cleavage site, like ArgX1X2Arg, which upon binding and internalization, releases a toxin like Exotoxin A domains II and III to kill infected cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If existing immunotoxins are used to target CMV-infected cells, then some cell killing activity is achieved, but cell-targeting selectivity is poor and internalization is insufficient
Solution Approach 1:
The immunotoxin is divided into three functional segments: a targeting peptide (first peptide) that binds to CMV-specific receptors, a cleavage site peptide (second peptide) containing furin recognition sequences, and a toxin moiety. This segmentation allows each component to perform its specific function optimally - targeting, conditional activation, and cell killing - thereby improving both selectivity and internalization efficiency.
Solution Approach 2:
The cleavage site peptide acts as an intermediary between the targeting peptide and the toxin. It remains intact during circulation and targeting, then gets cleaved by furin proteases inside the target cell, mediating the transition from a stable circulating complex to an active intracellular toxin, thus improving both selectivity and internalization.
2Reliability
If current drugs are used for HCMV treatment, then infection is treated, but adverse side effects and drug resistance occur
Solution Approach 1:
The toxin is extracted and delivered only after specific intracellular cleavage events occur within CMV-infected cells. This ensures the toxic effect is taken out (activated) only where needed - in infected cells - rather than systemically, thereby maintaining treatment effectiveness while minimizing adverse side effects and reducing selection pressure for drug resistance.
Solution Approach 2:
The immunotoxin undergoes parameter changes upon internalization - the cleavage site peptide is cleaved by furin proteases, changing the molecular weight and conformation of the complex, and activating the toxin. This parameter change (from inactive precursor to active toxin) occurs only inside target cells, providing selective effectiveness while sparing healthy cells from adverse effects.
3Reliability
If a fusion protein with cleavage site is designed, then selectivity is improved, but manufacturing complexity increases
Solution Approach 1:
The cleavage site peptide contains specific local quality features - furin recognition sequences (e.g., RQRF, RRQR motifs) - that are localized at the junction between the targeting peptide and toxin. This local quality enhancement ensures selective cleavage only in cells expressing furin proteases (infected cells), improving selectivity without requiring complex manufacturing processes.
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
Enhances targeting and killing of HCMV-infected cells, including latently infected cells, with improved selectivity and reduced side effects by utilizing a cleavage site motif that increases potency towards virus-encoded receptors.
Implementation Method 1
a first peptide which binds to at least one receptor expressed on a cell
Implementation Method 2
a second peptide which comprises a cleavage site and a toxin, wherein the cleavage site has an amino acid sequence ArgX1X2Arg
Data Source
AI summary
A fusion protein is provided which comprises a first and a second peptide. The first peptide enables the fusion protein to bind to a receptor expressed on a cell, and the second peptide having a cleavage site that enables the fusion protein to kill said cell. The fusion protein is thus useful for the prevention or treatment of an infection caused by a pathogen. Nucleic acids encoding the fusion protein and methods of making and using the fusion protein are also provided.