Characterization and engineering of Homo sapiens adenosine deaminase isoform 1 for therapeutic applications
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Jennings, Maria Rain
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Abstract
Given the importance of adenosine regulation to every cell in the body, the role of Homo sapiens adenosine deaminase isoform 1 (HsADA1) in normal physiology and pathophysiology cannot be understated. Mutations within the ADA1 gene give rise to dysfunctional HsADA1 expression and adenosine accumulation, which presents clinically as a severe combined immunodeficiency (ADA-SCID) responsible for 15-20 % of all reported SCID cases. The most accessible treatment option for ADA-SCID is enzyme replacement therapy with the ADA1 from Bos taurus (BtADA1). Inversely, HsADA1 expression can also contribute to the pathogenesis of Hairy Cell Leukemia for which the first line treatment is HsADA1 inhibition.
Previously reported yields of HsADA1 from recombinant expression in Escherichia coli were markedly low, so we optimized the expression and purification process through variation of relevant parameters such as codon optimization, culturing conditions, and affinity tag length. Our pure, five milligram per liter yield was conducive to publishing a crystal structure and associated analysis detailing differences between different mammalian ADA1s, presenting a cautionary tale for assumptive inhibitor development. Also, our process enabled kinetic characterization of HsADA1 and generation of clinically relevant variants of the enzyme for further analysis.
During characterization, we serendipitously discovered that HsADA1 lacks catalytic retention in human serum relative to its mammalian homologs in cows and mice. Wildtype HsADA1’s poor catalytic retention in human serum threatens its suitability as a therapeutic enzyme in comparison to the FDA-approved BtADA1. However, BtADA1 is so immunogenic that its administration still incites a reaction from the immunocompromised ADA-SCID population. Patients with other ailments that could benefit from a HsADA1-based intervention such as fibrosis, amyotrophic lateral sclerosis, autoimmune uveitis, or cancer with otherwise functional adaptive immunity may respond poorly to BtADA1 due to its immunogenicity.
Thus, we rationally designed a mutant form of HsADA1 influenced by its serum persistent cow (Bos taurus) and mouse (Mus musculus) homologs for a final form with comparable catalytic retention in human serum but with a reduced immunogenic potential. In our post-hoc phylogenetic analysis of the mutations we induced, we found that HsADA1 is the only ADA1 amongst 34 primate sequences and only one of three vertebrates amongst 490 considered species with a residue variation responsible for a significant reduction in the enzyme’s catalytic retention in human serum. Lastly, we demonstrated our preliminary work with wildtype HsADA1 and our engineered mutant in tumor mouse models and provide suggestions for future work.
For the broader community of scientists and physicians interested in ADA-SCID, we present the first reported in vivo administration of HsADA1 as an alternative to the FDA-approved BtADA1 and propose a mutant thereof for improved catalytic retention in serum. For protein scientists and engineers, we report methods for enhanced recombinant production of complex proteins in E. coli, and a novel approach to engineering therapeutic enzymes for enhanced catalytic retention in serum that is not dependent upon correlation with thermal stability.
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Date
2023-08-04
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Dissertation