ABSTRACT
CD27 is a tumor necrosis factor (TNF) receptor, which stimulates lymphocytes and promotes their differentiation upon activation by TNF ligand CD70. Activation of the CD27 receptor provides a costimulatory signal to promote T cell, B cell, and NK cell activity to facilitate antitumor and anti-infection immunity. Aberrant increased and focused expression of CD70 on many tumor cells renders CD70 an attractive therapeutic target for direct tumor killing. However, despite their use as drug targets to treat cancers, the molecular basis and atomic details of CD27 and CD70 interaction remain elusive. Here we report the crystal structure of human CD27 in complex with human CD70. Analysis of our structure shows that CD70 adopts a classical TNF ligand homotrimeric assembly to engage CD27 receptors in a 3:3 stoichiometry. By combining structural and rational mutagenesis data with reported disease-correlated mutations, we identified the key amino acid residues of CD27 and CD70 that control this interaction. We also report increased potency for plate-bound CD70 constructs compared with solution-phase ligand in a functional activity to stimulate T-cells in vitro. These findings offer new mechanistic insight into this critical costimulatory interaction.
Subject(s)
CD27 Ligand/chemistry , Multiprotein Complexes/chemistry , Tumor Necrosis Factor Receptor Superfamily, Member 7/chemistry , CD27 Ligand/genetics , CD27 Ligand/immunology , Crystallography, X-Ray , Humans , Multiprotein Complexes/genetics , Multiprotein Complexes/immunology , Protein Structure, Quaternary , T-Lymphocytes/immunology , Tumor Necrosis Factor Receptor Superfamily, Member 7/genetics , Tumor Necrosis Factor Receptor Superfamily, Member 7/immunologyABSTRACT
Ultra-scale down (USD) methodology developed by University College London for cell broth clarification with industrial centrifuges was applied to two common cell lines (NS0 and GS-CHO) expressing various therapeutic monoclonal antibodies. A number of centrifuges at various scales were used with shear devices operating either by high speed rotation or flow-through narrow channels. The USD methodology was found effective in accounting for both gravitational and shear effects on clarification performance with three continuous centrifuges at pilot and manufacturing scales. Different shear responses were observed with the two different cell lines and even with the same cell line expressing different products. Separate particle size analysis of the treated broths seems consistent with the shear results. Filterability of the centrifuged solutions was also evaluated to assess the utility of the USD approach for this part of the clarification operation.
