Browsing by Subject "Activity"

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Determining the relationship between TCR affinity and T cell response
(2018-08-16) Stevens, Christopher A.; Maynard, Jennifer Ann, 1974-; Georgiou, George; Alper, Hal; Ehrlich, Lauren; Jiang, Ning; Upton, Jason
The T-cell response is a critical component of controlling many cancers and viral diseases and recently developed T-cell based therapies have become very popular and effective treatment methods. Adoptive cell therapy involves the isolation of a patient’s or donor’s T-cells, expansion or modification ex vivo, and then administration into the patient. The recent FDA approval of an adoptive cell therapy involving an engineering antigen receptor has solidified this technique from a passing fad to a legitimate and effective technique for treating cancer. T-cells are able to recognize a vast range of target antigens through the use of a T-cell receptor (TCR) which targets small peptide fragments presented on the surface of diseased cells in the form of major histocompatibility complexes (pMHC). Further improvement of these therapies and methods requires a deeper understanding of how these TCR-pMHC interactions relate to functionality. Cytomegalovirus is a persistent viral infection that can reactivate periodically during periods of immunodepression. Viral control is mediated by a strong T-cell response and it can cause life-threatening complications in immunosuppressed individuals.Additionally, the T-cell response after chronic CMV reactivation becomes focused to a few individual clonotypes bearing ‘public’ TCRs that have shared sequences between individuals and are often high affinity. This makes CMV an excellent model for studying TCR-pMHC interactions. Several attempts to improve functionality of natural TCRs by engineering for higher affinity have resulted loss of specificity or reduced activation. Methods for direct selection of TCRs with improved activity are currently being developed, however they are very limited, whereas there are a variety of methods for affinity maturation of TCRs. A firm understanding of how TCR-pMHC binding affinity relates to activation would greatly benefit all T cell therapies. This work attempts to provide a better understanding of the relationship between TCR-pMHC affinity and T-cell activity by characterizing TCRs engineered in multiple platforms to obtain a range of affinities while maintaining antigen specificity. Additionally, we observe the relationship between affinity and activity of in vivo derived TCRs against CMV to help aid adoptive cell therapies, as well as the development of a vaccine to elicit potent T-cell responses.
Enzyme immobilization on gold surfaces : effects of surface chemistry and attachment strategies on binding and activity
(2023-04-20) Correira, Joshua Manuel; Webb, Lauren J.; Samanta, Devleena; Baiz, Carlos R; Gordon, Vernita; Shear, Jason B
Functional enzymes are the basis for many biotechnological systems, including biosensors, bio-fuel cells, and heterogeneous biocatalysts. In these systems, enzymes are often immobilized on a solid support or surface to capture their catalytic activity. Immobilization has the advantage of improving enzyme stability and reusability but often results in a significant loss of catalytic activity (1-2 orders of magnitude) when compared to the native enzyme. This inactivation is due to direct interactions between the enzyme and the solid support. Here, we developed methods to study enzyme immobilization and resulting inactivation. The aim of this work was to identify optimal surface chemistries and attachment strategies that promote high binding efficiencies while minimizing activity losses. Subsequently, we studied this using three enzymes (acetylcholinesterase (AChE), β-galactosidase (β-gal), horseradish peroxidase (HRP)) immobilized on gold surfaces by direct adsorption, covalent attachment, and DNA-directed attachment. First, AChE was directly adsorbed onto a variety of gold surfaces modified with self-assembled monolayers (SAMs) terminated with -COO⁻, -NH₃⁺, -OH, and -CH₃ functional groups at varying mole % to study the effect of surface hydrophobicity and charge on binding and activity. We found that binding was directly proportional to surface hydrophobicity (r = 0.75) and activity was inversely proportional to surface hydrophobicity (r = -0.62). The highest binding observed was ~40% of a monolayer on the most hydrophobic surfaces and the lowest binding observed was ~10% of a monolayer on the most hydrophilic surfaces. Conversely, on the most hydrophobic surfaces AChE retained <10% of its native activity, and on the most hydrophilic surfaces AChE retained ~40% of its native activity. This illuminated an inherent problem with direct adsorption: high binding and high activity are mutually exclusive. Due to these findings, we next immobilized β-gal and HRP on DNA-functionalized gold surfaces using DNA-DNA interactions, to avoid direct interactions between the enzyme and surface. We found that β-gal retained 62% of its native activity following immobilization, a significant improvement over previous direct adsorption strategies.