Characterization of glucose-regulated protein 170 as a molecular chaperone and its use in cancer immunotherapy

The 170 kDa glucose regulated protein 170 (grp170) is an endoplasmic reticulum resident chaperone protein and a member of heat shock protein (hsp). Mouse grp170 was cloned into the Baculovirus expression system and recombinant protein was purified from insect cells. Grp170 function was assessed by determining its ability to bind to and prevent aggregation of heat denatured Luciferase. Grp170 maintains denatured Luciferase in a soluble, folding competent state and grp170-bound Luciferase is able to partially recover function in the presence of rabbit reticulocyte lysate after heat denaturation. Domain deletion mutants of grp170 were designed using the structure of DnaK and hsp110 as guides. Testing chaperoning activity showed that grp170 has two functional domains, the classical β-sandwich peptide binding domain and the C-terminal α-helical region.; Hsp was studied as a tumor rejection antigen. Hsp and antigen presenting cell (APC) interaction is the first step of immune system activation. Fluorescein isothiocyanate (FITC)-conjugated grp170 binds to the surface of RAW264.7 macrophages and other APCs in a saturable manner. Excess unlabeled grp170 competes with cell surface bound FITC-grp170. Scavenger receptors and Toll-like receptors are suggested to be a candidate for a grp170 receptor on the APC. Functional domain deletion mutants of the grp170 bind to RAW264.7 cell surface with varying affinities. In each case surface binding is inhibited by the full-length grp170.; Grp170 was also found to bind to and chaperone the melanoma antigen gp100 following heat stress in vitro. The grp170/gp100 chaperone complex elicited a gp100 specific T-cell immune response as determined by interferon gamma (IFN-γ) secretion from extracted lymphocytes. Grp170 domain deletion mutants, which have chaperoning activity, were created and complexed with gp100 by heat shock. Grp170 mutant-gP100 complexes also activated gp100 specific T-cells. Vaccination with the grp170/gp100 or grp170 mutants/gp100 complexes delayed the growth of implanted B16 melanoma tumor in C57BL/6 mice.; The chaperoning activity of grp170 and its domain deletion mutants can be used for cancer immunotherapy since grp170/tumor antigen complex binds to APC and activate immune system.