Monoclonal antibodies are widely used to target disease-related antigens. However, because conventional antibody binds to the antigen but cannot eliminate the antigen from plasma, and rather increases the plasma antigen concentration by reducing the clearance of the antigen, some clinically important antigens are still difficult to target with monoclonal antibodies because of the huge dosages required. While conventional antibody can only bind to the antigen, some natural endocytic receptors not only bind to the ligands but also continuously eliminate them from plasma by pH-dependent dissociation of the ligands within the acidic endosome and subsequent receptor recycling to the cell surface. Here, we demonstrate that an engineered antibody, named sweeping antibody, having both pH-dependent antigen binding (to mimic the receptor-ligand interaction) and increased binding to cell surface neonatal Fc receptor (FcRn) at neutral pH (to mimic the cell-bound form of the receptor), selectively eliminated the antigen from plasma. With this novel antigen-sweeping activity, antibody without in vitro neutralizing activity exerted in vivo efficacy by directly eliminating the antigen from plasma. Moreover, conversion of conventional antibody with in vitro neutralizing activity into sweeping antibody further potentiated the in vivo efficacy. Depending on the binding affinity to FcRn at neutral pH, sweeping antibody reduced antigen concentration 50- to 1000-fold compared to conventional antibody. Thereby, sweeping antibody antagonized excess amounts of antigen in plasma against which conventional antibody was completely ineffective, and could afford marked reduction of dosage to a level that conventional antibody can never achieve. Thus, the novel mode of action of sweeping antibody provides potential advantages over conventional antibody and may allow access to the target antigens which were previously undruggable by conventional antibody.

Agonistic antibodies targeting CD137 have been clinically unsuccessful due to systemic toxicity. Since conferring tumor selectivity through tumor-associated antigen limits its clinical use to cancers that highly express such antigen, we exploited extracellular adenosine triphosphate (exATP), which is a hallmark of the tumor microenvironment and highly elevated in solid tumors, as a broadly tumor selective switch. We generated a novel anti-CD137 switch antibody, STA551, which exerts agonistic activity only in the presence of exATP. STA551 demonstrated potent and broad anti-tumor efficacy against all mouse and human tumors tested and a wide therapeutic window without systemic immune activation in mice. STA551 was well tolerated even at 150 mg/kg/week in cynomolgus monkeys. These results provide a strong rationale for the clinical testing of STA551 against a broad variety of cancers regardless of antigen expression, and for the further application of this novel platform to other targets in cancer therapy.

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In the course of screening for an inhibitor of ER stress-induced XBP1 activation, we isolated a new member of the triene-ansamycin group compound, trierixin, from a culture broth of Streptomyces sp. AC 654. Trierixin was purified by column chromatography on silica gel and by HPLC. The molecular formula of trierixin is C 37 H 52 N 2 O 8 S. Trierixin inhibited thapsigargin-induced XBP1-luciferase activation in HeLa/XBP1-luc cells and endogenous XBP1 splicing in HeLa cells with an IC 50 of 14 ng/ml and 19 ng/ml, respectively. Moreover, in the process of isolating trierixin, we isolated structurally related mycotrienin II and trienomycin A as inhibitors of ER stress-induced XBP1 activation from a culture broth of a trierixin-producing strain. This study provides the first observation that triene-ansamycins have a novel inhibitory effect against XBP1 activation.

The pH-dependent antigen binding antibody, termed a recycling antibody, has recently been reported as an attractive type of second-generation engineered therapeutic antibody. A recycling antibody can dissociate antigen in the acidic endosome, and thus bind to its antigen multiple times. As a consequence, a recycling antibody can neutralize large amounts of antigen in plasma. Because this approach relies on histidine residues to achieve pH-dependent antigen binding, which could limit the epitopes that can be targeted and affect the rate of antigen dissociation in the endosome, we explored an alternative approach for generating recycling antibodies. Since calcium ion concentration is known to be lower in endosome than in plasma, we hypothesized that an antibody with antigen-binding properties that are calcium-dependent could be used as recycling antibody. Here, we report a novel anti-interleukin-6 receptor (IL-6R) antibody, identified from a phage library that binds to IL-6R only in the presence of a calcium ion. Thermal dynamics and a crystal structure study revealed that the calcium ion binds to the heavy chain CDR3 region (HCDR3), which changes and possibly stabilizes the structure of HCDR3 to make it bind to antigen calcium dependently (PDB 5AZE). In vitro and in vivo studies confirmed that this calcium-dependent antigen-binding antibody can dissociate its antigen in the endosome and accelerate antigen clearance from plasma, making it a novel approach for generating recycling antibody.