Garg Gunjal, Gibbs Jesse, Belt Brian, Powell Matthew A, Mutch David G, Goedegebuure Peter, Collins Lynne, Piwnica-Worms David, Hawkins William G, Spitzer Dirk
Department of Surgery, Washington University School of Medicine, St, Louis, MO 63110, USA.
BMC Cancer. 2014 Jan 21;14:35. doi: 10.1186/1471-2407-14-35.
The targeted delivery of cancer therapeutics represents an ongoing challenge in the field of drug development. TRAIL is a promising cancer drug but its activity profile could benefit from a cancer-selective delivery mechanism, which would reduce potential side effects and increase treatment efficiencies. We recently developed the novel TRAIL-based drug platform TR3, a genetically fused trimer with the capacity for further molecular modifications such as the addition of tumor-directed targeting moieties. MUC16 (CA125) is a well characterized biomarker in several human malignancies including ovarian, pancreatic and breast cancer. Mesothelin is known to interact with MUC16 with high affinity. In order to deliver TR3 selectively to MUC16-expressing cancers, we investigated the possibility of targeted TR3 delivery employing the high affinity mesothelin/MUC16 ligand/receptor interaction.
Using genetic engineering, we designed the novel cancer drug Meso-TR3, a fusion protein between native mesothelin and TR3. The recombinant proteins were produced with mammalian HEK293T cells. Meso-TR3 was characterized for binding selectivity and killing efficacy against MUC16-positive cancer cells and controls that lack MUC16 expression. Drug efficacy experiments were performed in vitro and in vivo employing an intraperitoneal xenograft mouse model of ovarian cancer.
Similar to soluble mesothelin itself, the strong MUC16 binding property was retained in the Meso-TR3 fusion protein. The high affinity ligand/receptor interaction was associated with a selective accumulation of the cancer drug on MUC16-expressing cancer targets and directly correlated with increased killing activity in vitro and in a xenograft mouse model of ovarian cancer. The relevance of the mesothelin/MUC16 interaction for attaching Meso-TR3 to the cancer cells was verified by competitive blocking experiments using soluble mesothelin. Mechanistic studies using soluble DR5-Fc and caspase blocking assays confirmed engagement of the extrinsic death receptor pathway. Compared to non-targeted TR3, Meso-TR3 displayed a much reduced killing potency on cells that lack MUC16.
Soluble Meso-TR3 targets the cancer biomarker MUC16 in vitro and in vivo. Following attachment to the tumor via surface bound MUC16, Meso-TR3 acquires full activation with superior killing profiles compared to non-targeted TR3, while its bioactivity is substantially reduced on cells that lack the tumor marker. This prodrug phenomenon represents a highly desirable property because it has the potential to enhance cancer killing with fewer side-effects than non-targeted TRAIL-based therapeutics. Thus, further exploration of this novel fusion protein is warranted as a possible therapeutic for patients with MUC16-positive malignancies.
癌症治疗药物的靶向递送是药物开发领域中一项持续存在的挑战。肿瘤坏死因子相关凋亡诱导配体（TRAIL）是一种很有前景的抗癌药物，但其活性特征可通过癌症选择性递送机制得到改善，这将减少潜在的副作用并提高治疗效率。我们最近开发了基于TRAIL的新型药物平台TR3，这是一种基因融合三聚体，具有进一步进行分子修饰的能力，例如添加肿瘤靶向部分。粘蛋白16（MUC16，癌抗原125）是包括卵巢癌、胰腺癌和乳腺癌在内的几种人类恶性肿瘤中特征明确的生物标志物。间皮素已知与MUC16具有高亲和力相互作用。为了将TR3选择性递送至表达MUC16的癌症，我们研究了利用间皮素/MUC16高亲和力配体/受体相互作用进行TR3靶向递送的可能性。
利用基因工程，我们设计了新型抗癌药物间皮素-TR3（Meso-TR3），它是天然间皮素与TR3之间的融合蛋白。重组蛋白由哺乳动物HEK293T细胞产生。对Meso-TR3针对MUC16阳性癌细胞和缺乏MUC16表达的对照细胞的结合选择性和杀伤效力进行了表征。采用卵巢癌腹腔异种移植小鼠模型在体外和体内进行了药物疗效实验。
与可溶性间皮素本身相似，Meso-TR3融合蛋白保留了强大的MUC16结合特性。高亲和力配体/受体相互作用与抗癌药物在表达MUC16的癌症靶点上的选择性积累相关，并与体外和卵巢癌异种移植小鼠模型中增强的杀伤活性直接相关。利用可溶性间皮素的竞争性阻断实验证实了间皮素/MUC16相互作用对于将Meso-TR3附着于癌细胞的相关性。使用可溶性死亡受体5-免疫球蛋白（DR5-Fc）和半胱天冬酶阻断实验的机制研究证实了外源性死亡受体途径的参与。与非靶向TR3相比，Meso-TR3对缺乏MUC16的细胞显示出显著降低的杀伤效力。
可溶性Meso-TR3在体外和体内靶向癌症生物标志物MUC16。通过表面结合的MUC16附着于肿瘤后，与非靶向TR3相比，Meso-TR3获得完全激活并具有卓越的杀伤特性，而其在缺乏肿瘤标志物的细胞上的生物活性显著降低。这种前药现象代表了一种非常理想的特性，因为它有可能在比非靶向TRAIL疗法副作用更少的情况下增强癌症杀伤作用。因此，有必要进一步探索这种新型融合蛋白作为MUC16阳性恶性肿瘤患者可能的治疗方法。
