Abstract:
Considering the indispensable role played by proteins in maintaining vital life
processes, it is not surprising that proteins are linked to a broad spectrum of diseases.
Conversely, proteins can be leveraged for effective therapeutic interventions. For
example, secreted growth factors known as cytokines have emerged as promising
candidates for protein-based therapeutics, primarily due to their potent
immunomodulatory properties. An integral element of the innate immune system
consists of neutrophils, the maturation of which is coordinated by a hematopoietic
subprocess known as granulopoiesis. This process requires a complex interplay
among various cytokines and their corresponding receptor molecules. A critical player
in these interactions is the granulocyte-colony stimulating factor receptor (G-CSFR),
which is activated by its native ligand, G-CSF. Unlike most other cytokines, G-CSF
has found clinical use in its native form due to its favorable safety profile and its ability
to increase the number of neutrophils in the blood.
However, G-CSF application is restricted by its stability, production cost and native
activity on G-CSFR. Additionally, there is very limited knowledge regarding non-native
G-CSFR modulators, which could be key not only for understanding G-CSFR related
diseases, but also for the development of innovative therapeutic applications. In order
to unlock the untapped potential of the clinically significant G-CSFR beyond its native
activity, my objective was to employ protein design techniques to craft novel ligands
capable of modulating G-CSFR activity. In addition to customizing receptor activity,
these designs offer enhanced stability and more efficient production compared to their
native counterpart G-CSF.
To this end, I utilized a recently developed hyper-thermostable de novo designed GCSFR
binding module and optimized it with in silico and in vitro high-throughput
methods to obtain a broad spectrum of variants with enhanced binding affinity. I
demonstrate that these enhanced binding modules can be utilized to generate GCSFR
agonists that achieve G-CSF activity in cell-based assays and can also be used
to create ligands capable of modulating G-CSFR activity by tuning receptor geometry.
These ligands featured fine-tuned intracellular signaling, transcriptomic activity, and
primary stem cell differentiation and exhibit in vivo activity in zebrafish and mouse
models. In addition to designing agonists, I show that these enhanced binding modules can be used to generate competitive G-CSFR antagonists with nanomolar inhibitory
activity.
To the best of my knowledge this work is the first demonstration that G-CSFR activity
can be tuned by the design of ligands inducing non-native receptor geometries.
Additionally, this study presents an array of binding modules with diverse affinities to
G-CSFR, along with the newly designed ligands, providing the foundational
components for systematic investigation of G-CSFR activity modulation. These
findings hold significant promise for advancing the development of innovative protein
therapeutics.
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