SummaryCD25 is expressed at high levels on regulatory T (Treg) cells and was initially proposed as a target for cancer immunotherapy. However, anti-CD25 antibodies have displayed limited activity against established tumors. We demonstrated that CD25 expression is largely restricted to tumor-infiltrating Treg cells in mice and humans. While existing anti-CD25 antibodies were observed to deplete Treg cells in the periphery, upregulation of the inhibitory Fc gamma receptor (FcγR) IIb at the tumor site prevented intra-tumoral Treg cell depletion, which may underlie the lack of anti-tumor activity previously observed in pre-clinical models. Use of an anti-CD25 antibody with enhanced binding to activating FcγRs led to effective depletion of tumor-infiltrating Treg cells, increased effector to Treg cell ratios, and improved control of established tumors. Combination with anti-programmed cell death protein-1 antibodies promoted complete tumor rejection, demonstrating the relevance of CD25 as a therapeutic target and promising substrate for future combination approaches in immune-oncology.

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Fusion‐tag affinity chromatography is a key technique in recombinant protein purification. Current methods for protein recovery from mammalian cells are hampered by the need for feed stream clarification. We have developed a method for direct capture using immobilized metal affinity chromatography (IMAC) of hexahistidine (His6) tagged proteins from unclarified mammalian cell feed streams. The process employs radial flow chromatography with 300–500 μm diameter agarose resin beads that allow free passage of cells but capture His‐tagged proteins from the feed stream; circumventing expensive and cumbersome centrifugation and/or filtration steps. The method is exemplified by Chinese Hamster Ovary (CHO) cell expression and subsequent recovery of recombinant His‐tagged carcinoembryonic antigen (CEA); a heavily glycosylated and clinically relevant protein. Despite operating at a high NaCl concentration necessary for IMAC binding, cells remained over 96% viable after passage through the column with host cell proteases and DNA detected at ∼8 U/mL and 2 ng/μL in column flow‐through, respectively. Recovery of His‐tagged CEA from unclarified feed yielded 71% product recovery. This work provides a basis for direct primary capture of fully glycosylated recombinant proteins from unclarified mammalian cell feed streams. Biotechnol. Bioeng. 2016;113: 130–140. © 2015 Wiley Periodicals, Inc.