We will employ in vitro laboratory directed evolution to engineer blood brain barrier (BBB)-traversing granulocyte colony-stimulating factor (G-CSF) variants that substantially augment cognitive performance improvements observed in Alzheimer's Disease (AD) patients treated with standard G-CSF (Amgen's Filgrastim). In addition to showing promise in treating AD, G-CSF has been trialed in Amyotrophic Lateral Sclerosis (ALS) and stroke patients and reduces motor deficits in Parkinson's Disease (PD) primate models. As such, BBB-traversing G-CSF variants can have a broad impact in treating a range of nervous system disorders, improving thousands of lives and saving millions of dollars in healthcare costs. The anticipated therapeutic efficacy of evolved G-CSF variants is based on the hypothesis that increasing G-CSF's ability to escape lysosomal degradation will improve its ability to cross the BBB and extend its lifetime after entering the brain. Our unique application o a yeast surface display protein engineering platform enables development of such variants. Specifically, we will evolve variants with highly pH- sensitive G-CSF receptor (G-CSFR) binding affinity. This sensitivity promotes G-CSF/G-CSFR complex dissociation within the acidic endosomal environment, sparing G-CSF from being routed to lysosomes for degradation and promoting recycling to the cell exterior. We will use yeast display to evolve G-CSF variants with a range of binding affinities at neutral pH and reduced affinities at endosomal pH (5.5-6.0). Leading variants will be purified and binding properties validated by surface plasmon resonance. Cell culture studies will validate variants'increased ability to cross an endothelial cell model BBB, escape lysosomal degradation in mature neurons, and promote neural stem cell proliferation. Binding and cell culture assay data will elucidate relationships among pH sensitivity, binding affinity, BBB model transcytosis and cell activation potency, guiding our choice of one or two leading G-CSF variants for follow-on AD mouse studies. Our collaborators in the lab of Dr. Juan Sanchez-Ramos, the first group to trial G-CSF in AD, will initiate these experiments immediately after this two-year performance period. We are optimistic that these animal studies will be a preface to realizing our goal of conducting BBB-traversing G-CSF variant human trials within the next five years. Beyond making G-CSF a more clinically-relevant agent for treating AD and other neurodegenerative conditions, the methods employed apply to evolving additional candidate therapeutic neurotrophic factors, particularly granulocyte macrophage colony-stimulating factor (GM-CSF) and erythropoietin (EPO), for increased BBB-traversal and neurotrophic activity duration. As such, this work will be an important initial step toward developing multiple new neurotrophic agents for widespread administration, possibly in combination, as effective therapeutics for treating AD and other nervous system disorders.
Granulocyte colony-stimulating factor (G-CSF) improved patient cognitive performance in an Alzheimer's Disease (AD) clinical trial, has shown promise in treating Amyotrophic Lateral Sclerosis (ALS) and stroke, and reduces motor function deficits in a primate model of Parkinson's Disease (PD). These encouraging outcomes have been observed despite only a small fraction of intravenously injected G-CSF traversing the blood brain barrier (BBB). Increasing passage of G-CSF across the BBB will enhance G-CSF's ability to promote the development of new neurons and protect mature neurons from premature death, thus increasing G-CSF's efficacy in treating AD and other nervous system disorders. In this work, we will use laboratory directed evolution to engineer improved versions of G-CSF that traverse the BBB more efficiently than standard G-CSF and have longer active lifetimes in brain tissue. Our collaboration with the group that conducted the AD trial provides valuable clinical study expertise that will allow us to realize the objective of validating BBB-traversing G-CSF variant therapeutic efficacy in human AD patients within the next five years. Beyond making G-CSF more effective in treating AD and other neurodegenerative conditions, our laboratory directed evolution methods apply to engineering additional candidate therapeutic neurotrophic factors, in particular, granulocyte macrophage colony-stimulating factor (GM-CSF) and erythropoietin (EPO), for increased BBB traversal and extended active lifetimes within the brain. As such, this G-CSF engineering research is a key initial step toward our ultimate goal of widespread administration of BBB- traversing versions of G-CSF, GM-CSF, EPO, and possibly combinations thereof, as effective treatments for AD and other nervous system disorders.
|Heinzelman, Pete; Schoborg, Jennifer A; Jewett, Michael C (2015) pH responsive granulocyte colony-stimulating factor variants with implications for treating Alzheimer's disease and other central nervous system disorders. Protein Eng Des Sel 28:481-9|
|Heinzelman, Pete; Carlson, Sharon J; Cox, George N (2015) Cytokine refacing effect reduces granulocyte macrophage colony-stimulating factor susceptibility to antibody neutralization. Protein Eng Des Sel 28:461-6|