Generation of a multimeric form of CD40L with potent immunostimulatory activity using streptavidin as a chaperon.
Effective aggregation of cell surface immune receptors with their ligands is critical in promoting humoral and cellular immune responses. Simulation of these interactions using soluble multimeric ligands having potent adjuvant effects may prove an effective alternative to agonistic antibodies as immunotherapeutics. Multimeric ligands may effectively engage their receptors, leading to aggregation and effective signal transduction. We exploited the structural characteristics of streptavidin (SA) for the generation of multimeric chimeric proteins. Streptavidin forms stable tetramers and oligomers under physiological conditions, and, as such, chimeric molecules with SA are expected to possess similar features. Two chimeric molecules consisting of the extracellular domains of human and mouse CD40L and a modified form of core streptavidin were generated. These proteins form stable oligomers that could only be dissociated into monomers by heating at 100 degrees C, but not 60 degrees C, under denaturing conditions. The chimeric proteins vigorously stimulated B cells, monocytes, and dendritic cells for the production of cytokines and chemokines and upregulation of immunostimulatory molecules. The use of SA as a chaperon presents a novel approach to generate multimeric immunological molecules with potent activities and their use as potential therapeutics for the treatment of cancer and other immune-based disorders.
Institute for Cellular Therapeutics, University of Louisville, Louisville, KY 40202-1760, USA.
Mehmet O
MO
Kilinc
Lata
L
Mukundan
Esma S
ES
Yolcu
Narendra P
NP
Singh
Jill
J
Suttles
Haval
H
Shirwan
D000818:Animals; D001402:B-Lymphocytes / Q000187:drug effects / Q000276:immunology; D015153:Blotting, Western; D023201:CD40 Ligand / Q000737:chemistry* / Q000276:immunology* / Q000494:pharmacology; D045744:Cell Line, Tumor; D003713:Dendritic Cells / Q000187:drug effects / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D006801:Humans; D051379:Mice; D018832:Molecular Chaperones / Q000737:chemistry* / Q000276:immunology / Q000494:pharmacology; D009000:Monocytes / Q000187:drug effects / Q000276:immunology; D011108:Polymers / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology; D019809:Streptavidin / Q000737:chemistry* / Q000276:immunology / Q000494:pharmacology; D014162:Transfection
D018832:Molecular Chaperones; D011108:Polymers; D023201:CD40 Ligand; D019809:Streptavidin