Antibody to CD137 Activated by Extracellular Adenosine Triphosphate Is Tumor Selective and Broadly Effective In Vivo without Systemic Immune Activation.

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.

Improvement of the VEGF binding ability of DNA aptamers through in silico maturation and multimerization strategy.

Aptamers are mainly selected by in vitro selection using random nucleic acid libraries. These aptamers have often shown insufficient affinity for biomedical applications. We improved DNA aptamer binding affinity for vascular endothelial growth factor (VEGF) through in silico maturation (ISM) and aptamer multimerization. ISM is one of a number of evolutionary approaches and aptamer multimerization is one of several semi-rational strategies to improve function. We first reselected VEGF-binding aptamers using a partially randomized DNA library and identified two aptamers with higher binding ability than that of a known aptamer. We conducted ISM using the re-selected aptamers to optimize the key loop sequences created by a three-way junction structure. After five ISM rounds, we identified aptamer 2G19 [dissociation constant (Kd), 52 nM] as a local optimum of the defined search space. We characterized the aptamer and found that a specific stem-loop structure was involved in aptamer VEGF recognition. To further improve its affinity for VEGF, we multimerized 2G19 or its stem-loop structure. The designed SL5-trivalent aptamer (Kd, 0.37 nM) with three binding motifs significantly increased binding affinity, representing a 500-fold improvement from systematic evolution of ligands by exponential enrichment-selected aptamers.

Development of a novel biosensing system based on the structural change of a polymerized guanine-quadruplex DNA nanostructure.

By inserting an adenosine aptamer into an aptamer that forms a G-quadruplex, we developed an adaptor molecule, named the Gq-switch, which links an electrode with flavin adenine dinucleotide-dependent glucose dehydrogenase (FADGDH) that is capable of transferring electron to a electrode directly. First, we selected an FADGDH-binding aptamer and identified that its sequence is composed of two blocks of consecutive six guanine bases and it forms a polymerized G-quadruplex structure. Then, we inserted a sequence of an adenosine aptamer between the two blocks of consecutive guanine bases, and we found it also bound to adenosine. Then we named it as Gq-switch. In the absence of adenosine, the Gq-switch-FADGDH complex forms a 30-nm high bulb-shaped structure that changes in the presence of adenosine to give an 8-nm high wire-shaped structure. This structural change brings the FADGDH sufficiently close to the electrode for electron transfer to occur, and the adenosine can be detected from the current produced by the FADGDH. Adenosine was successfully detected with a concentration dependency using the Gq-switch-FADGDH complex immobilized Au electrode by measuring response current to the addition of glucose.

Selection of DNA aptamer against prostate specific antigen using a genetic algorithm and application to sensing.

In order to construct an aptasensor, aptamers that show high affinity for target molecules are required. While the systematic evolution of ligands by exponential enrichment (SELEX) is an efficient method for selecting aptamers, it sometimes fails to obtain aptamers with high affinity and so additional improvements are required. We applied a genetic algorithm (GA) to post-SELEX screening as an in silico maturation of aptamers. First, we pre-selected DNA aptamers against prostate specific antigen (PSA) through three rounds of SELEX. To improve the PSA-binding ability of the aptamers, we carried out post-SELEX screening using GA with the pre-selected oligonucleotide sequences. For screening using GA, we replicated the oligonucleotide sequences obtained through SELEX, crossed over and mutated in silico resulting in 20 sequences. Those oligonucleotide sequences were synthesized and assayed in vitro. Then, the oligonucleotides were ranked according to PSA-binding ability and the top sequences were selected for the next cycle of GA operation. After GA operations, we identified the aptamer showing a 48-fold higher PSA-binding ability than candidates obtained by SELEX. The dissociation constant (K(D)) of the obtained aptamer was estimated to be several tens of nM. We demonstrated sensing of PSA using the obtained aptamer and succeeded in sensing PSA concentrations between 40 and 100 nM. This is the first report of a DNA aptamer against PSA and its application to PSA sensing.