De Novo-Designed Minibinders Expand the Synthetic Biology Sensing Repertoire for Engineered Cell Therapies

Core Concepts

De novo-designed minibinders can be readily adapted as antigen sensors across diverse synthetic receptor platforms, including proteolytic receptors and chimeric antigen receptors, enabling expanded sensing capabilities for engineered cell therapies.

Abstract

This study demonstrates that de novo-designed protein minibinders can function as versatile antigen sensors when integrated with various synthetic receptor systems. The key findings are:

The minibinders LCB1 and LCB3, which target the SARS-CoV-2 Spike protein, were successfully coupled to two different types of synthetic proteolytic receptors - synNotch and SNIPR. Both minibinders enabled specific activation of these receptors in the presence of Spike-expressing cells.

The LCB1 minibinder, when coupled to the SNIPR receptor, was able to detect live SARS-CoV-2 virus, an improvement over the previously reported SARSNotch system which could only detect cell-expressed Spike protein.

The LCB1 and LCB3 minibinders were also integrated into a chimeric antigen receptor (CAR) system. The minibinder-CARs were able to specifically activate primary human CD8+ T cells and induce cytotoxicity against Spike-expressing target cells, though with lower efficacy compared to a benchmark CD19-CAR.

Optimization of the linker sequence connecting the minibinder to the synthetic receptor was explored, with a short flexible linker found to be optimal for both proteolytic and CAR receptor function.

Overall, these results demonstrate the versatility of de novo-designed minibinders as antigen sensors that can be readily adapted across diverse synthetic receptor platforms, expanding the sensing capabilities available for engineering therapeutic cell lines.

Stats

The expression level of the different minibinder-coupled synthetic receptors on Jurkat cells ranged from a median fluorescence of ~2,000 to ~6,000 counts.
The fraction of the Jurkat cell population that was activated (expressing BFP) in response to Spike-expressing target cells ranged from ~45% to ~96% depending on the specific receptor-minibinder combination.
The maximal cytotoxicity of the minibinder-CARs against Spike-expressing K562 cells was ~40-45% lysis at a 2:1 effector:target ratio.

Quotes

"Minibinders represent a novel class of antigen sensors that have the potential to dramatically expand the sensing repertoire of cell engineering tools."
"Our findings suggest that minibinders can be easily deployed across synthetic receptors as antigen sensors."
"Computational approaches for creating de novo-designed binders against practically any protein... position minibinders as attractive new antigen sensors."

Key Insights Distilled From

De novo-designed minibinders expand the synthetic biology sensing repertoire

by Weinberg,Z. ... at www.biorxiv.org 01-15-2024

https://www.biorxiv.org/content/10.1101/2024.01.12.575267v1

Deeper Inquiries

How could the affinity and binding kinetics of minibinders be further optimized to improve their performance as antigen sensors in synthetic receptor systems?

Minibinders' affinity and binding kinetics can be optimized through several strategies. One approach is to utilize computational design methods to fine-tune the binding interface between the minibinder and its target antigen. By iteratively designing and testing different binding interfaces, researchers can select variants with the optimal affinity and kinetics for the desired application. Additionally, incorporating structural information about the target antigen can guide the design of minibinders with specific binding properties.
Another strategy is to explore different linker sequences between the minibinder and the receptor to modulate the binding characteristics. Short, flexible linkers have been shown to be effective in maintaining the functionality of minibinders in synthetic receptor systems. By systematically testing different linker lengths and compositions, researchers can identify the optimal linker design that enhances the performance of minibinders as antigen sensors.
Furthermore, leveraging advanced computational tools, such as deep learning-assisted methods, can expedite the design process and enable the generation of minibinders with enhanced affinity and kinetics. These tools can analyze large datasets of binding interactions to predict and optimize the binding properties of minibinders, leading to improved performance in synthetic receptor systems.

What are the potential limitations or drawbacks of using minibinders compared to traditional antibody-derived antigen sensors, and how could these be addressed?

While minibinders offer several advantages, such as faster production timelines and unique binding properties, they also have limitations compared to traditional antibody-derived antigen sensors. One potential drawback is the size of minibinders, which may limit their versatility in certain cellular engineering applications where payload size is constrained. Additionally, the generalizability of minibinders across different synthetic receptors and target antigens may vary, leading to suboptimal performance in some systems.
To address these limitations, researchers can explore different minibinder designs, such as smaller variants or engineered minibinders with optimized properties for specific applications. By tailoring the minibinder structure and composition, it may be possible to overcome size constraints and enhance the adaptability of minibinders to diverse synthetic receptor systems. Additionally, conducting thorough characterization and validation studies across a range of receptors and antigens can help identify the most effective minibinder variants for specific applications.
Furthermore, ongoing advancements in computational design tools and high-throughput screening methods can facilitate the rapid optimization of minibinders for improved performance. By integrating experimental data with computational modeling, researchers can iteratively refine minibinder designs to address the limitations and drawbacks associated with their use in synthetic receptor systems.

Given the versatility of minibinders demonstrated in this study, what other types of synthetic receptor systems or cellular engineering applications could they be explored for in the future?

The versatility of minibinders opens up a wide range of possibilities for their application in various synthetic receptor systems and cellular engineering contexts. One potential area of exploration is the development of minibinder-coupled biosensors for detecting specific biomolecules or pathogens in complex biological samples. By integrating minibinders with sensor platforms, researchers can create highly sensitive and selective detection systems for diagnostic or research purposes.
Minibinders could also be utilized in the design of novel cell-based therapies, such as engineered immune cells with customized antigen recognition capabilities. By incorporating minibinders into chimeric antigen receptors (CARs) or synthetic Notch receptors, researchers can engineer immune cells to target specific antigens with high precision, potentially enhancing the efficacy of immunotherapies for cancer or infectious diseases.
Moreover, minibinders could be integrated into synthetic signaling pathways to modulate cellular behavior in response to external stimuli. By coupling minibinders with transcriptional regulators or signaling domains, researchers can create synthetic circuits that enable precise control over cellular functions, such as proliferation, differentiation, or apoptosis. This approach could have applications in regenerative medicine, tissue engineering, and biotechnology.
Overall, the versatility of minibinders offers a wealth of opportunities for innovation in synthetic biology, cell engineering, and biomedical research. Continued exploration and development of minibinder-based technologies hold great promise for advancing our understanding of cellular processes and developing novel therapeutic interventions.