Development of retargeted adenovirus to tumor-associated antigen sialyl Lewis X

Abstract

[한글]

Sialyl Lewis X (sLewX)는 종양관련항원으로 암세포가 내피세포에 부착될때 리간드로 작용하여 암세포의 전이를 돕고, sLewX 발현이 증가될수록 위암, 대장암, 폐암, 난소암과 같은 다양한 암의 예후가 나쁘다는 보고가 있다. 따라서 우리는 sLewX에 대한 in vitro random phage display법을 이용하여 20여개의 phage를 분리하여 sLewX 특이적 결합능과 펩타이드들의 염기서열을 확인하였고, sLewX가 많이 발현되는 암세포에 선택적으로 감염되는 아데노바이러스벡터를 개발하였다.

특정조직 및 세포로 표적화된 아데노바이러스를 개발하기 위해서는 바이러스 본래의 세포수용체와의 결합능을 없애고, 새로운 세포수용체와의 결합능을 첨가해주어야 한다. 우리는 CAR 결합능이 배제된 아데노바이러스인 YKL-1/420A를 개발하였고, 그 결과 비교군의

아데노바이러스에 비해 유전자 전달효율이 급격히 감소하는 것을 확인하였다. 여기에 phage display법으로 분리한 펩타이드 중 3개를 선택하여 바이러스의 fiber C 말단에 결합시켜 YKL-1-420A/7mer, YKL-1-420A/12mer, YKL-1-420A/p30을 만들었다. YKL-1-420A/p30은 YKL-1-420A에 비해 10-100배 가량 증가된 세포살상력을 보였으며, xenograft 종양모델에 처치한 결과 YKL-1-420A에 비해 급격하게 종양크기가 감소하였다. 따라서 아데노바이러스의 재표적화는 유전자치료에 매우 유용하게 이용될 수 있으며 특히 sLewX에 특이적으로 결합하는 아데노바이러스인 YKL-1-420A/p30은 세포특이적이며 CAR에 비의존적인 유전자전달을 가능하게 하였다.

[영문]

Sialyl Lewis X (sLewX) is a tumor-associated carbohydrate antigen. Several studies indicate that sLewX serves as a ligand in the adhesion of cancer cells to endothelial cells, resulting in hematogenous metastasis of human cancer cells.

Increased expression of sLewX has been reported to correlate with poor prognoses in various cancers including gastric, colorectal, lung and ovarian cancers. In this perspective, we adopted an in vitro random phage display library panning on sLewX to develop adenoviral vectors capable of the selective infection of the cancer cells with respect to the normal cells. More than 20 individually isolated phage were tested for the ability to bind specifically to sLewX and the sequences of these novel peptides were identified. The development of tissue cell selective targeting adenovirus requires the generation of adenovirus vectors which lack native receptor binding and additionally contain domains which redirect the vector to tissue/cell specific receptors. Towards this goal, we have generated CAR binding-ablated adenovirus YKL-1/420A using the backbone of an E1B55kD deleted oncolytic adenoviral vector YKL-1. Ykl-1/420A adenovirus exerted dramatic reduction of transduction efficiency compared to control adenovirus. Among the peptides isolated by in vitro random phage display, 3 peptides were chosen and fused to the C-terminus of the fiber protein of YKL-1-420A, generating three sLewX-targeted adenoviruses of YKL-1-420A/7mer, YKL-1-420A/12mer, and YKL-1-420A/p30.

YKL-1-420A/p30 exerted about 10-100 times greater cytolytic ability than control adenovirus YKL-1-420A. Furthermore, treatment with YKL-1-420A/p30 significantly suppressed tumor growth in xenograft tumor model when compared with control

adenovirus YKL-1-420. Taken together, these studies demonstrate that strategy to alter adenovirus tropism may allow greatly improved utilities of adenovirus for gene therapy applications. In particular, sLewX-specific targeted adenovirus YKL-1-420A/p30 allowed CAR-independent gene delivery as well as cell-specific gene delivery, and showed significant promise as a targeting replicative adenovirus vector in cancer gene therapy.