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1.
Cancer Discov ; 11(1): 158-175, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32847940

RESUMO

Agonistic antibodies targeting CD137 have been clinically unsuccessful due to systemic toxicity. Because conferring tumor selectivity through tumor-associated antigen limits its clinical use to cancers that highly express such antigens, 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 antitumor 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. SIGNIFICANCE: Reported CD137 agonists suffer from either systemic toxicity or limited efficacy against antigen-specific cancers. STA551, an antibody designed to agonize CD137 only in the presence of extracellular ATP, inhibited tumor growth in a broad variety of cancer models without any systemic toxicity or dependence on antigen expression.See related commentary by Keenan and Fong, p. 20.This article is highlighted in the In This Issue feature, p. 1.


Assuntos
Trifosfato de Adenosina , Neoplasias , Animais , Anticorpos Monoclonais/farmacologia , Antígenos de Neoplasias , Imunoterapia , Camundongos , Neoplasias/tratamento farmacológico , Microambiente Tumoral , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral
2.
Nat Med ; 18(10): 1570-4, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23023498

RESUMO

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.


Assuntos
Anticorpos Biespecíficos , Fator IXa/imunologia , Fator VIII/fisiologia , Fator X/imunologia , Hemofilia A/terapia , Hemostasia , Animais , Anticorpos Biespecíficos/imunologia , Anticorpos Biespecíficos/farmacologia , Anticorpos Biespecíficos/uso terapêutico , Hemofilia A/imunologia , Macaca fascicularis
3.
Appl Environ Microbiol ; 72(2): 1487-95, 2006 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-16461703

RESUMO

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 CaCl2. 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.


Assuntos
Aldeído Oxirredutases/metabolismo , Ácido Ascórbico/metabolismo , Rhodobacteraceae/enzimologia , Sorbose/análogos & derivados , Aldeído Oxirredutases/química , Aldeído Oxirredutases/genética , Sequência de Aminoácidos , Sequência de Bases , DNA Bacteriano/genética , Expressão Gênica , Genes Bacterianos , Heme/química , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Modelos Biológicos , Dados de Sequência Molecular , Peso Molecular , Mutagênese Insercional , Cofator PQQ/química , Subunidades Proteicas , Rhodobacteraceae/genética , Sorbose/metabolismo
4.
Biosci Biotechnol Biochem ; 69(3): 659-62, 2005 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-15785002

RESUMO

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.


Assuntos
Ácido Ascórbico/biossíntese , Gluconobacter oxydans/metabolismo , Hexoses/metabolismo , Sorbitol/metabolismo , Sorbose/análogos & derivados , Sorbose/metabolismo
5.
Biochim Biophys Acta ; 1647(1-2): 278-88, 2003 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-12686146

RESUMO

Gluconobacter strains effectively produce L-sorbose from D-sorbitol because of strong activity of the D-sorbitol dehydrogenase (SLDH). L-sorbose is one of the important intermediates in the industrial vitamin C production process. Two kinds of membrane-bound SLDHs, which consist of three subunits, were reportedly found in Gluconobacter strains [Agric. Biol. Chem. 46 (1982) 135,FEMS Microbiol. Lett. 125 (1995) 45]. We purified a one-subunit-type SLDH (80 kDa) from the membrane fraction of Gluconobacter suboxydans IFO 3255 solubilized with Triton X-100 in the presence of D-sorbitol, but the cofactor could not be identified from the purified enzyme. The SLDH was active on mannitol, glycerol and other sugar alcohols as well as on D-sorbitol to produce respective keto-aldoses. Then, the SLDH gene (sldA) was cloned and sequenced. It encodes the polypeptide of 740 residues, which contains a signal sequence of 24 residues. SLDH had 35-37% identity to those of membrane-bound quinoprotein glucose dehydrogenases (GDHs) from Escherichia 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 the polypeptide consisting of 126 very hydrophobic residues that is similar to the one-sixth N-terminal region of the GDH. Development of the SLDH activity in E. coli required co-expression of the sldA and sldB genes and the presence of PQQ. The sldA gene disruptant showed undetectable oxidation activities on D-sorbitol in growing culture, and resting-cell reaction (pH 4.5 and 7); in addition, they showed undetectable activities on D-mannitol and glycerol. The disruption of the sldB gene by a gene cassette with a downward promoter to express the sldA gene resulted in formation of a larger size of the SLDH protein and in undetectable oxidation of the polyols. In conclusion, the SLDH of the strain 3255 functions as the main polyol dehydrogenase in vivo. The sldB polypeptide possibly has a chaperone-like function to process the SLDH polypeptide into a mature and active form.


Assuntos
Proteínas de Bactérias/metabolismo , Gluconobacter/enzimologia , L-Iditol 2-Desidrogenase/metabolismo , Sequência de Aminoácidos , Catálise , Cromatografia Líquida de Alta Pressão , L-Iditol 2-Desidrogenase/genética , L-Iditol 2-Desidrogenase/imunologia , Dados de Sequência Molecular , Peso Molecular , Especificidade por Substrato
6.
Appl Environ Microbiol ; 69(4): 1959-66, 2003 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-12676670

RESUMO

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 immuno-cross-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-D-gluconate. 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.


Assuntos
Oxirredutases do Álcool/metabolismo , Gluconatos/metabolismo , Gluconobacter/enzimologia , Desidrogenase do Álcool de Açúcar/metabolismo , Oxirredutases do Álcool/química , Oxirredutases do Álcool/genética , Sequência de Aminoácidos , Sequência de Bases , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Análise de Sequência de DNA , Sorbitol/metabolismo , Desidrogenase do Álcool de Açúcar/química , Desidrogenase do Álcool de Açúcar/genética , Álcoois Açúcares/metabolismo
7.
Biosci Biotechnol Biochem ; 66(2): 262-70, 2002 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-11999397

RESUMO

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.


Assuntos
Escherichia coli/genética , Gluconobacter/enzimologia , L-Iditol 2-Desidrogenase/genética , Quinolonas/metabolismo , Quinonas/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , DNA Bacteriano , L-Iditol 2-Desidrogenase/metabolismo , Dados de Sequência Molecular , Cofator PQQ , Reação em Cadeia da Polimerase , Homologia de Sequência de Aminoácidos
8.
Biosci Biotechnol Biochem ; 66(11): 2314-22, 2002 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-12506966

RESUMO

The D-sorbitol dehydrogenase gene, sldA, and an upstream gene, sldB, encoding a hydrophobic polypeptide, SldB, of Gluconobacter suboxydans IFO 3255 were disrupted in a check of their biological functions. The bacterial cells with the sldA gene disrupted did not produce L-sorbose by oxidation of D-sorbitol in resting-cell reactions at pHs 4.5 and 7.0, indicating that the dehydrogenase was the main D-sorbitol-oxidizing enzyme in this bacterium. The cells did not produce D-fructose from D-mannitol or dihydroxyacetone from glycerol. The disruption of the sldB gene resulted in undetectable oxidation of D-sorbitol, D-mannitol, or glycerol, although the cells produced the dehydrogenase. The cells with the sldB gene disrupted produced more of what might be signal-unprocessed SldA than the wild-type cells did. SldB may be a chaperone-like component that assists signal processing and folding of the SldA polypeptide to form active D-sorbitol dehydrogenase.


Assuntos
Proteínas de Ciclo Celular/genética , Gluconobacter/enzimologia , L-Iditol 2-Desidrogenase/genética , L-Iditol 2-Desidrogenase/metabolismo , Sequência de Bases , Western Blotting , Membrana Celular/enzimologia , DNA Bacteriano/genética , Escherichia coli/genética , Glucose/metabolismo , Concentração de Íons de Hidrogênio , Metilfenazônio Metossulfato/farmacologia , Dados de Sequência Molecular , Peso Molecular , Plasmídeos/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sorbose/biossíntese , Sorbose/química , Álcoois Açúcares/metabolismo
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