Hemophilia A is a bleeding disorder resulting from coagulation factor VIII (FVIII) deficiency. Exogenously provided FVIII effectively reduces bleeding complications in patients with severe hemophilia A. In approximately 30% of such patients, however, the 'foreignness' of the FVIII molecule causes them to develop inhibitory antibodies against FVIII (inhibitors), precluding FVIII treatment in this set of patients. Moreover, the poor pharmacokinetics of FVIII, attributed to low subcutaneous bioavailability and a short half-life of 0.5 d, necessitates frequent intravenous injections. To overcome these drawbacks, we generated a humanized bispecific antibody to factor IXa (FIXa) and factor X (FX), termed hBS23, that places these two factors into spatially appropriate positions and mimics the cofactor function of FVIII. hBS23 exerted coagulation activity in FVIII-deficient plasma, even in the presence of inhibitors, and showed in vivo hemostatic activity in a nonhuman primate model of acquired hemophilia A. Notably, hBS23 had high subcutaneous bioavailability and a 2-week half-life and would not be expected to elicit the development of FVIII-specific inhibitory antibodies, as its molecular structure, and hence antigenicity, differs from that of FVIII. A long-acting, subcutaneously injectable agent that is unaffected by the presence of inhibitors could markedly reduce the burden of care for the treatment of hemophilia A.

Engaging inhibitory FcγRIIb by Fc region has been recently reported to be an attractive approach for improving the efficacy of antibody therapeutics. However, the previously reported S267E/L328F variant with enhanced binding affinity to FcγRIIb, also enhances binding affinity to FcγRIIaR131 allotype to a similar degree because FcγRIIb and FcγRIIaR131 are structurally similar. In this study, we applied comprehensive mutagenesis and structure-guided design based on the crystal structure of the Fc/FcγRIIb complex to identify a novel Fc variant with selectively enhanced FcγRIIb binding over both FcγRIIaR131 and FcγRIIaH131. This novel variant has more than 200-fold stronger binding affinity to FcγRIIb than wild-type IgG1, while binding affinity to FcγRIIaR131 and FcγRIIaH131 is comparable with or lower than wild-type IgG1. This selectivity was achieved by conformational change of the CH2 domain by mutating Pro to Asp at position 238. Fc variant with increased binding to both FcγRIIb and FcγRIIa induced platelet aggregation and activation in an immune complex form in vitro while our novel variant did not. When applied to agonistic anti-CD137 IgG1 antibody, our variant greatly enhanced the agonistic activity. Thus, the selective enhancement of FcγRIIb binding achieved by our Fc variant provides a novel tool for improving the efficacy of antibody therapeutics.

Acetic acid bacteria, especially Gluconobacter species, have been known to catalyze the extensive oxidation of sugar alcohols (polyols) such as D-mannitol, glycerol, D-sorbitol, and so on. Gluconobacter species also oxidize sugars and sugar acids and uniquely accumulate two different keto-D-gluconates, 2-keto-D-gluconate and 5-keto-D-gluconate, in the culture medium by the oxidation of D-gluconate. However, there are still many controversies regarding their enzyme systems, especially on D-sorbitol and also D-gluconate oxidations. Recently, pyrroloquinoline quinone-dependent quinoprotein D-arabitol dehydrogenase and D-sorbitol dehydrogenase have been purified from G. suboxydans, both of which have similar and broad substrate specificity towards several different polyols. In this study, both quinoproteins were shown to be identical based on their immunocross-reactivity and also on gene disruption and were suggested to be the same as the previously isolated glycerol dehydrogenase (EC 1.1.99.22). Thus, glycerol dehydrogenase is the major polyol dehydrogenase involved in the oxidation of almost all sugar alcohols in Gluconobacter sp. In addition, the so-called quinoprotein glycerol dehydrogenase was also uniquely shown to oxidize D-gluconate, which was completely different from flavoprotein D-gluconate dehydrogenase (EC 1.1.99.3), which is involved in the production of 2-keto-Dgluconate. The gene disruption experiment and the reconstitution system of the purified enzyme in this study clearly showed that the production of 5-keto-D-gluconate in G. suboxydans is solely dependent on the quinoprotein glycerol dehydrogenase.Acetic acid bacteria are obligate aerobes well known as vinegar producers and also known to be able to oxidize various sugars and sugar alcohols such as D-glucose, glycerol, D-sorbitol, and so on, in addition to ethanol. Such oxidation reactions are called oxidative fermentation, since they involve incomplete oxidations of such alcohols or sugars accompanied by an accumulation of the corresponding oxidation products in large amounts in the culture medium. Of the two genera of acetic acid bacteria, Gluconobacter species extensively catalyze the oxidation of sugars and sugar alcohols except for ethanol, while Acetobacter species have a high ability to oxidize ethanol to acetic acid. These oxidation reactions of sugars or sugar alcohols seem to be carried out by membrane-bound dehydrogenases linked to the respiratory chain located in the cytoplasmic membrane of the organism (14). Of these oxidative fermentations of acetic acid bacteria, vinegar production from ethanol and 2-keto-D-gluconate (2KGA) production from glucose have each been shown to be carried out by sequential membranebound alcohol and aldehyde dehydrogenases and by glucose and gluconate dehydrogenases, respectively (14).There is still controversy about the mechanism of L-sorbose and 5-keto-D-gluconate (5KGA) production in Gluconobacter species. Three different membrane-bound enzymes have been proposed to be involved in L-sorbose production fr...

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.

The sldA gene that encodes the D-sorbitol dehydrogenase (SLDH) from Gluconobacter suboxydans IFO 3255 was cloned and sequenced. It encodes a polypeptide of 740 residues, which contains a signal sequence of 24 residues. SLDH had 35-37% identity to the membrane-bound quinoprotein glucose dehydrogenases (GDHs) from E. coli, Gluconobacter oxydans, and Acinetobacter calcoaceticus except the N-terminal hydrophobic region of GDH. Additionally, the sldB gene located just upstream of sldA was found to encode a polypeptide consisting of 126 very hydrophobic residues that is similar in sequence to the one-sixth N-terminal region of the GDH. For the development of the SLDH activity in E. coli, co-expression of the sldA and sldB genes and the presence of pyrrloquinolone quinone as a co-factor were required.

A novel enzyme, L-sorbosone dehydrogenase 1 (SNDH1), which directly converts L-sorbosone to L-ascorbic acid (L-AA), was isolated from Ketogulonicigenium vulgare DSM 4025 and characterized. This enzyme was a homooligomer of 75-kDa subunits containing pyrroloquinoline quinone (PQQ) and heme c as the prosthetic groups. Two isozymes of SNDH, SNDH2 consisting of 75-kDa and 55-kDa subunits and SNDH3 consisting of 55-kDa subunits, were also purified from the bacterium. All of the SNDHs produced L-AA, as well as 2-keto-L-gulonic acid (2KGA), from L-sorbosone, suggesting that tautomerization of L-sorbosone causes the dual conversion by SNDHs. The sndH gene coding for SNDH1 was isolated and analyzed. The N-terminal four-fifths of the SNDH amino acid sequence exhibited 40% identity to the sequence of a soluble quinoprotein glucose dehydrogenase from Acinetobacter calcoaceticus. The C-terminal one-fifth of the sequence exhibited similarity to a c-type cytochrome with a heme-binding motif. A lysate of Escherichia coli cells expressing sndH exhibited SNDH activity in the presence of PQQ and CaCl 2 . Gene disruption analysis of K. vulgare indicated that all of the SNDH proteins are encoded by the sndH gene. The 55-kDa subunit was derived from the 75-kDa subunit, as indicated by cleavage of the C-terminal domain in the bacterial cells. L-Ascorbic acid (L-AA) is an essential nutrient for humans.It is known that L-AA is directly converted from aldonolactones, such as L-gulono-␥-lactone and L-galactono-␥-lactone, by aldonolactone dehydrogenase/oxidases in plants and mammals (except some primates, including humans). The L-gulono-␥-lactone oxidase that catalyzes the conversion of L-gulono-␥-lactone to L-AA was isolated from rat and goat livers and characterized by Nishikimi et al. (19). An L-galactono-␥-lactone oxidase of yeast origin was described by Bleeg (4). In addition, L-sorbosone is also known to be a potential precursor of L-AA in plants. An NADP-dependent L-sorbosone dehydrogenase (SNDH) that converts L-sorbosone to L-AA in spinach leaves has been reported (17), but the enzyme has not been purified or characterized in detail.In current industrial L-AA production processes, as summarized by Hancock and Viola (13), 2-keto-L-gulonic acid (2KGA) is a key intermediate that is chemically converted to L-AA. All of the processes require a large amount of energy and organic solvent, and thus a cheaper and environmentally conscious substitute process, such as enzymatic conversion, is desirable. It has also been reported that L-sorbosone is converted to 2KGA in bacteria. Many microbial L-sorbosone dehydrogenases, including enzymes from Acetobacter liquefaciens (27), Gluconobacter melanogenus UV10 (14), and Gluconobacter oxydans T-100 (23), have been isolated and characterized. Moreover, Ketogulonicigenium vulgare DSM 4025 has been reported to produce aldehyde dehydrogenase (ALDH) (15) and L-sorbose/ L-sorbosone dehydrogenases (SSDHs) (3), which are responsible for sequentially converting L-sorbose to 2KGA via L-sorbosone.The metabolic...

Ketogulonicigenium vulgare DSM 4025, known as a 2-keto-L-gulonic acid producing strain from L-sorbose via L-sorbosone, surprisingly produced L-ascorbic acid from D-sorbitol, L-sorbose, L-gulose, and L-sorbosone as the substrate under a growing or resting condition. As the best result, K. vulgare DSM 4025 produced 1.37 g per liter of L-AA from 5.00 g per liter of L-sorbosone during 4 h incubation time at 30 degrees C under the resting cell condition having 5.70 g per liter of wet cells. The precursor of L-AA formation from D-sorbitol and L-sorbose, except for L-gulose, was thought to be the putative furanose form of L-sorbosone. This is the first time it is reported that bacteria can produce vitamin C via L-sorbosone.

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