Cetuximab ± chemotherapy enhances dendritic cell-mediated phagocytosis of colon cancer cells and ignites a highly efficient colon cancer antigen-specific cytotoxic T-cell response in vitro.

Cetuximab is a human/mouse chimeric IgG1 monoclonal antibody (mAb) to epidermal growth factor receptor, approved for colorectal carcinoma treatment in combination with chemotherapy. The immune-mediated effects elicited by its human fraction of crystallization moiety might critically contribute to the overall anti-tumor effectiveness of the antibody. We therefore investigated cetuximab ability to promote colon cancer cell opsonization and phagocytosis by human dendritic cells (DCs) that are subsequently engaged in antigen-cross presentation to cytotoxic T-lymphocyte (CTL) precursors. Human colon cancer cell lines were evaluated for susceptibility to DC-mediated phagocytosis before and after treatment with chemotherapy ± cetuximab in vitro. Human DCs loaded with control or drug-treated cetuximab-coated colon cancer cells were used to in vitro generate cytotoxic T cell clones from peripheral blood mononuclear cells of human leucocyte antigen-A(*)02.01(+) donors. T-cell cultures were characterized for immune-phenotype and tumor-antigen specific CTL activity. The results confirmed that treatment of tumor cells with irinotecan + L-folinate + 5-flurouracil (ILF) or with gemcitabine + ILF increased tumor antigen expression. Moreover, malignant cells exposed to chemotherapy and cetuximab were highly susceptible to phagocytosis by human DCs and were able to promote their activation. The consequent DC-mediated cross-priming of antigens derived from mAb-covered/drug-treated cancer cells elicited a robust CTL anti-tumor response. On the basis of our data, we suggest a possible involvement of CTL-dependent immunity in cetuximab anti-cancer effects.

A molecular dynamics study in explicit water of the reduced and oxidized forms of yeast iso-1-cytochrome c--solvation and dynamic properties of the two oxidation states.

Molecular dynamics calculations have been performed over long trajectories with the inclusion of explicit solvent molecules on the reduced and the oxidized states of yeast iso-1-cytochrome c. The resulting structures have been analyzed and compared both in terms of structural properties and dynamical behavior. The structure of the buried water molecules around the heme has been also analyzed for the two oxidation states and compared with the experimental observations on the X-ray and the solution NMR structures. From the overall analysis we learn that, as also observed experimentally through NMR, no significant differences are present between the structures of the two oxidation states beside the arrangement of a few side chains. Also the internal mobility is similar for the two oxidation states, even if interesting differences are observed for some residues, as for Tyr67, a residue present at the heme site. The location and the mobility of the ordered water molecules, observed in solution by NMR, are completely reproduced in the molecular dynamics simulations, which have been able to predict the different displacements of the catalytically relevant water molecule WAT166, similar to those observed in solution for the two oxidation states, at variance with that observed in the starting crystallographic structures. The relevance of these findings with respect to the prediction of structural and dynamical properties is discussed.

PSEUDYANA for NMR structure calculation of paramagnetic metalloproteins using torsion angle molecular dynamics.

The program DYANA, for calculation of solution structures of biomolecules with an algorithm based on simulated annealing by torsion angle dynamics, has been supplemented with a new routine, PSEUDYANA, that enables efficient use of pseudocontact shifts as additional constraints in structure calculations of paramagnetic metalloproteins. PSEUDYANA can determine the location of the metal ion inside the protein frame and allows to define a single tensor of magnetic susceptibility from a family of conformers. As an illustration, a PSEUDYANA structure calculation is provided for a metal-undecapeptide complex, where simulated pseudocontact shifts but no NOE restraints are used as conformational constraints.