Biophysical characterization of 53-6.7 antibody binding to CD8 immunoreceptor
Author(s)
Srinivasan, Shreyaa
Advisor(s)
McShan, Andrew
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Abstract
Expressing on CD8+ T cells, the CD8 co-receptor binds the membrane-proximal domain of the MHC-I molecule away from the membrane-distal domains that present the peptide for the TCR binding. CD8 contributes to antigen recognition and T cell signaling. While CD8 is widely recognized as a co-stimulatory molecule for conventional CD8+ αβ T cells, recent reports highlight its multifaceted role in both adaptive and innate immune responses. Recognizing CD8’s increasing role in immune response underscores the significance of modulating its function, which delves into finding new strategies that can influence CD8 to augment or repress T-cell signaling. Monoclonal antibodies have been deeply explored as crucial immunomodulators, particularly with T-cell receptors. While there have been few observations about the impact of these antibodies on T-cell signaling and immune regulation via CD8, the biophysical interactions between antibodies and CD8 have not been extensively studied, prompting the investigation into this important area. To address this knowledge gap, my thesis research aimed to conduct phylogenetic analysis, generate recombinant CD8 protein, and perform biophysical characterization of therapeutically relevant CD8/monoclonal antibody (mAB) complexes. Recombinant expression constructs for CD8α and CD8β ectodomains and ectodomain-hinges were designed and expressed in E. coli cells. Proteins were expressed in insoluble inclusion bodies after induction, which required solubilization and in vitro refolding to prepare a properly conformed CD8 homodimer and heterodimeric proteins. Successful refolding of CD8αα was confirmed via circular dichroism spectroscopy. Surface plasmon resonance revealed high-affinity interactions between CD8αα and antibodies 53-6.7 and YTS 169.4, but not antibody OKT8. Refolding attempts for CD8αβ and CD8αβ linker constructs failed, indicating different structural dynamics between CD8αβ and CD8αα, and potential requirement of using eukaryotic expressions. I also wrote and submitted a Review Article (to Frontiers in Immunology – Chapters 1 – 3) that outlines the unique structure and function of different CD8 domains (ectodomain, hinge, transmembrane and cytoplasmic tail), in addition to various ways of harnessing CD8 as an immunomodulator through engineering CD8 mutations and utilizing monoclonal antibodies binding to CD8 for venturing potential medical applications. Overall, these studies serve as a useful resource, guiding both fundamental research in CD8-related immunology and translational efforts towards targeted immunotherapy.
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Date
2024-04-30
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