RESUMEN
A glucose-tolerant strain of the cyanobacterium Synechocystis sp. PCC 6803, generally referred to as wild type, produces a hemolysin-like protein (HLP) located on the cell surface. To analyze the function of HLP, we constructed a mutant in which the hlp gene was disrupted. The growth rate of the mutant was reduced when the cells were stressed by treatment with CuSO(4), CdCl(2), ZnCl(2), ampicillin, kanamycin, or sorbitol in liquid medium, suggesting that HLP may increase cellular resistance to the inhibitory effects of these compounds. Uptake assays with (109)Cd(2+) using the silicone-oil layer centrifugation technique revealed that both wild type and mutant cells were labeled with (109)Cd(2+) within 1 min. Although the total radioactivity was much higher in the wild-type cells, (109)Cd(2+) incorporation was clearly much higher in the mutant cells after adsorbed (109)Cd(2+) was removed from the cell surface by washing with EDTA. These findings suggest that HLP functions as a barrier against the adsorption of toxic compounds.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas Hemolisinas/metabolismo , Synechocystis/metabolismo , Proteínas Bacterianas/genética , Cadmio/metabolismo , Tamaño de la Célula , Desecación , Proteínas Hemolisinas/genética , Calor , Mutagénesis Insercional , Estrés Fisiológico , Synechocystis/genéticaRESUMEN
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.
Asunto(s)
Adenosina Trifosfato , Neoplasias , Animales , Anticuerpos Monoclonales/farmacología , Antígenos de Neoplasias , Inmunoterapia , Ratones , Neoplasias/tratamiento farmacológico , Microambiente Tumoral , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis TumoralRESUMEN
Dysregulation of the complement system is linked to the pathogenesis of a variety of hematological disorders. Eculizumab, an anti-complement C5 monoclonal antibody, is the current standard of care for paroxysmal nocturnal hemoglobinuria (PNH) and atypical hemolytic uremic syndrome (aHUS). However, because of high levels of C5 in plasma, eculizumab has to be administered biweekly by intravenous infusion. By applying recycling technology through pH-dependent binding to C5, we generated a novel humanized antibody against C5, SKY59, which has long-lasting neutralization of C5. In cynomolgus monkeys, SKY59 suppressed C5 function and complement activity for a significantly longer duration compared to a conventional antibody. Furthermore, epitope mapping by X-ray crystal structure analysis showed that a histidine cluster located on C5 is crucial for the pH-dependent interaction with SKY59. This indicates that the recycling effect of SKY59 is driven by a novel mechanism of interaction with its antigen and is distinct from other known pH-dependent antibodies. Finally, SKY59 showed neutralizing effect on C5 variant p.Arg885His, while eculizumab does not inhibit complement activity in patients carrying this mutation. Collectively, these results suggest that SKY59 is a promising new anti-C5 agent for patients with PNH and other complement-mediated disorders.
Asunto(s)
Anticuerpos Neutralizantes/inmunología , Complemento C5/antagonistas & inhibidores , Complemento C5/inmunología , Animales , Anticuerpos Neutralizantes/administración & dosificación , Anticuerpos Neutralizantes/química , Complemento C5/química , Cristalografía por Rayos X , Hemoglobinuria Paroxística/tratamiento farmacológico , Humanos , Macaca fascicularis , Unión Proteica , Conformación ProteicaRESUMEN
The hemolysin-like protein (HLP) Sll1951, characterized by the GGXGXDXUX nonapeptide motif implicated in Ca(2+) binding, was purified from the glucose-tolerant strain (GT) of Synechocystis sp. strain PCC 6803. HLP was eluted at 560 kDa after gel filtration chromatography. Atomic absorption spectroscopy indicated that the protein bound calcium. The bound Ca(2+) was not chelated with EGTA; however, it was released after being heated at 100 degrees C for 1 min, and it rebound to the Ca(2+)-depleted protein at room temperature. The apparent HLP molecular mass increased to 1,000 kDa and reverted to 560 kDa during the release and rebinding of Ca(2+), respectively. The monomers of the respective forms appeared at 90 and 200 kDa after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. HLP showed no apparent hemolytic activity against sheep erythrocytes; however, a slight hemolytic activity was detected during the conformational change caused by the rebinding of Ca(2+). Immunoelectron microscopy using polyclonal antibodies against the 200-kDa monomer revealed that HLP is located in the cell surface layer. The localization and Ca(2+)-induced reversible conformational change suggest that HLP is a member of the repeat in toxin (RTX) protein family despite its latent and low toxicity. In some other cyanobacteria, RTX proteins are reported to be necessary for cell motility. However, the GT was immotile. Moreover, the motile wild-type strain did not express any HLP, suggesting that HLP is one of the factors involved in the elimination of motility in the GT. We concluded that the involvement of RTX protein in cyanobacterial cell motility is not a general feature.