2E8 binds to the high affinity I-domain in a metal ion-dependent manner: A second generation monoclonal antibody selectively targeting activated LFA-1

Roberto Carreño, Wells S. Brown, Dan Li, Jessica A. Hernandez, Yang Wang, Tae Kon Kim, John W. Craft, Krishna V. Komanduri, Laszlo G. Radvanyi, Patrick Hwu, Jeffrey J. Molldrem, Glen B. Legge, Bradley W. McIntyre, Qing Ma

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Abstract

The activation of leukocyte function-associated antigen-1 (LFA-1) plays a critical role in regulating immune responses. The metal ion-dependent adhesion site on the I-domain of LFA-1 αL subunit is the key recognition site for ligand binding. Upon activation, conformation changes in the I-domain can lead LFA-1 from the low affinity state to the high affinity (HA) state. Using the purified HAI-domain locked by disulfide bonds for immunization, we developed an mAb, 2E8, that specifically binds to cells expressing the HA LFA-1. The surface plasmon resonance analysis has shown that 2E8 only binds to the HA I-domain and that the dissociation constant (KD) for HA I-domain is 197 nM. The binding of 2E8 to the HA I-domain is metal ion-dependent, and the affinity decreased as Mn2+ was replaced sequentially by Mg 2+ and Ca2+. Surface plasmon resonance analysis demonstrates that 2E8 inhibits the interaction of HA I-domain and ICAM-1. Furthermore, we found that 2E8 can detect activated LFA-1 on both JY and Jurkat cells using flow cytometry and parallel plate adhesion assay. In addition, 2E8 inhibits JY cell adhesion to human umbilical vein endothelial cells and homotypic aggregation. 2E8 treatment reduces the proliferation of both human CD4+ and CD8+ T cells upon OKT3 stimulation without the impairment of their cytolytic function. Taken together, these data demonstrate that 2E8 is specific for the high affinity form of LFA-1 and that 2E8 inhibits LFA-1/ICAM-1 interactions. As a novel activation-specific monoclonal antibody, 2E8 is a potentially useful reagent for blocking high affinity LFA-1 and modulating T cell activation in research and therapeutics.

Original languageEnglish (US)
Pages (from-to)32860-32868
Number of pages9
JournalJournal of Biological Chemistry
Volume285
Issue number43
DOIs
StatePublished - Oct 22 2010

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ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Carreño, R., Brown, W. S., Li, D., Hernandez, J. A., Wang, Y., Kim, T. K., Craft, J. W., Komanduri, K. V., Radvanyi, L. G., Hwu, P., Molldrem, J. J., Legge, G. B., McIntyre, B. W., & Ma, Q. (2010). 2E8 binds to the high affinity I-domain in a metal ion-dependent manner: A second generation monoclonal antibody selectively targeting activated LFA-1. Journal of Biological Chemistry, 285(43), 32860-32868. https://doi.org/10.1074/jbc.M110.111591