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Acute B-Cell Inhibition by Soluble Antigen Arrays is ValencyDependent and Predicts Immunomodulation in Splenocytes J. Daniel Griffin, Martin A Leon, Jean R Salash, Michael Shao, Brittany L Hartwell, Chad J Pickens, Joshua O Sestak, and Cory Berkland Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.9b00328 • Publication Date (Web): 17 Apr 2019 Downloaded from http://pubs.acs.org on April 23, 2019
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Biomacromolecules
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Acute B-Cell Inhibition by Soluble Antigen Arrays
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is Valency-Dependent and Predicts
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Immunomodulation in Splenocytes
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J. Daniel Griffin1, Martin A. Leon2, Jean R. Salash3, Michael Shao4, Brittany L. Hartwell1, Chad J.
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Pickens3, Joshua O. Sestak3, and Cory Berkland1,3,4
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1Bioengineering
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2Department
of Chemistry, University of Kansas, Lawrence, KS
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3Department
of Pharmaceutical Chemistry, University of Kansas, Lawrence, KS
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4Department
of Chemical and Petroleum Engineering, University of Kansas, Lawrence, KS
Graduate Program, University of Kansas, Lawrence, KS
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*Corresponding author:
[email protected]; 2030 Becker Drive, Room 320E, Lawrence, KS
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66047
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ABSTRACT
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Antigen valency plays a fundamental role in directing the nature of an immune response to
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be stimulatory or tolerogenic. Soluble Antigen Arrays (SAgAs) are an antigen-specific
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immunotherapy that combats autoimmunity through the multivalent display of autoantigen. While
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mechanistic studies have shown SAgAs to induce T and B-cell anergy, the effect of SAgA valency
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has never been experimentally tested. Here, SAgAs of discrete antigen valencies were synthesized
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by click chemistry and evaluated for acute B-cell signaling inhibition as well as downstream
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immunomodulatory effects in splenocytes. Initial studies using the Raji B-cell line demonstrated
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SAgA valency dictated the extent of calcium flux. Lower valency constructs elicited the largest
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reductions in B-cell activation. In splenocytes from mice with experimental autoimmune
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encephalomyelitis, the same valency-dependent effects were evident in the downregulation of the
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costimulatory marker CD86. The reduction of calcium flux observed in Raji B-cells correlated
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strongly with downregulation in splenocyte CD86 expression after 72 hours. Here, a thorough
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analysis of SAgA antigenic valency illustrates that low, but not monovalent, presentation of
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autoantigen was ideal for eliciting the most potent immunomodulatory effects.
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Keywords: Soluble Antigen Array, Antigen-Specific Immunotherapy, Valency, Calcium Flux,
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Anergy, Click Chemistry
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Biomacromolecules
INTRODUCTION
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Antigen presentation, especially with regards to valency (the number of antigens presented)
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can be a powerful therapeutic tool for either stimulating or suppressing immunity. More
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importantly, tuning ligand density on a macromolecule or colloid can vastly amplify or diminish
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immune signaling down either pathway 1-5. Targeting antigen-specific cell surface receptors may
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also provide a targeted approach to directing immune responses. Moving from monovalent antigen
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toward a multivalent antigen display can increase therapeutic potency by virtue of increasing
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receptor engagement through avidity
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immunity matured in the late 1970s with seminal work by Howard Dintzis which illustrated starkly
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different immunological outcomes. By tuning properties including molecular size and ligand
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valency, Dintzis observed that large (>100 kD) polymers grafted with high ligand density (>20
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ligands/polymer, or 1 ligand per 5 kD) were immunogenic while smaller constructs (
