Humanization of lead ancestral L-asparaginase candidates identified through ancestral sequence reconstruction Open Access

Abstract

Acute lymphoblastic leukemia (ALL) is the most prevalent form of pediatric cancer. Current chemotherapy treatments utilize a bacterial L-Asparaginase (L-ASNase) derived from either Escherichia coli or Erwinia chrysanthemi. Without the L-ASNase treatment, the patient’s chances of remission decline. ~30% of patients have an immunological response to the bacterial L-ASNases, so research for another less immunogenic asparaginase is warranted. Previous research established the effectiveness of the Cavia porcellus (Guinea Pig) L-ASNase but found issue with gathering ample Guinea Pig serum. Our research utilized in silico mapping to find the most recent common ancestor between guinea pigs and Homo sapiens (humans) and, using Ancestral Sequence Reconstruction, generated ancestral L-ASNases to determine which were closest in similarity to human L-ASNases while retaining comparable clinical-grade activity seen in existing bacterial L-ASNases. Recombinant L-ASNases were generated through the retaining of active sites of the ancestral L-ASNases, with the ancestral C-termini replaced with the human C-terminus. The recombinant enzymes were cloned into mammalian expression plasmids and transfected into two types of mammalian cells: Human Embryonic Kidney (HEK) Expi293F and Baby Hamster Kidney (BHK) cells. Early asparaginase assays measuring the production of aspartate and ammonia (asparaginase byproducts) demonstrated the success of the recombinant enzymes, but subsequent attempts to generate large amounts of the proteins were unsuccessful. Multiple attempts at generating stable cell lines for each L-ASNase of interest were also unsuccessful. The current stable cell line of BHK cells transfected with the Ancestral 104 recombinant (An104) plasmid shows promise in the generation of a stable cell line that produces the An104 L-ASNase, warranting the scale-up of the stable line to create a kinetic profile for the enzyme. Research into other ancestral recombinant L-ASNases that are closer in genetic similarity to humans is also warranted to determine the ideal candidate which retains clinical-grade asparaginase activity while increasing the similarity to the human asparaginase to reduce immunogenicity. 

Table of Contents

Table of Contents

Introduction. 1

Background. 1

Hypothesis. 3

Aims of Study. 3

Methods and Materials. 4

Results and Discussion. 11

Experimental Successes. 11

Experimental Failures and Troubleshooting. 15

Conclusion and Future Directions 19

Reflection. 20

Figures. 22

References 25

 

 

About this Honors Thesis

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School	Emory College
Department	Biology
Degree	B.S.
Submission	Honors Thesis
Language	English
Research Field	Biology, Cell Biology, Molecular
Keyword	L-asparaginase ALL Ancestral Sequence Reconstruction
Committee Chair / Thesis Advisor	Christopher Doering, Emory University
Committee Members	Kate O'Toole, Emory University Sunil Raikar, Emory University

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