Development of drug conjugates of R-spondin peptibodies for the treatment of colorectal cancer

项目总结 结直肠癌仍然是世界上癌症相关死亡的主要原因，只有一小部分 患者受益于免疫检查点抑制剂的治疗。抗体-药物结合物(ADC)是 与细胞杀伤药物共价连接的单抗(MAbs) 是抗癌治疗的主要手段。这种方法结合了mAbs的高度特异性 用高效细胞毒药物作为抗原靶点，产生可传递有效载荷的“武装”单抗 (药物)到肿瘤细胞与丰富水平的抗体靶标。这种方法已经成为一种主要的 在过去几年中批准了几个ADC的癌症治疗方法。富含亮氨酸重复序列 含有G蛋白偶联受体4、5和6(LGR4-6)是三种高度相关的受体 在结直肠癌中表达上调。它们与R-Spins(RSPO)结合，RSPO是一组分泌蛋白，与 高亲和力，增强Wnt信号转导。RSPO-LGR信号在肿瘤中的重要作用 形成、发展和抗药性。特别是，LGR5富含于 结肠癌和LGR5阳性细胞推动肿瘤生长和转移。然而，LGR5阳性和 -阴性细胞可以相互转换，靶向LGR5的ADC抑制肿瘤生长，但失败 彻底根除因癌细胞可塑性而产生的肿瘤。值得注意的是，LGR5阴性细胞仍然 LGR4和/或LGR6。我们推断，同时靶向LGR4-6可能会克服 癌细胞的可塑性和耐药性。最近，我们证明了RSPO的一种突变形式 可与LGR4-6高亲和力结合而不增强Wnt信号转导。RSPO的药物结合物 融合到IgG1-Fc结构域的突变体在体内外均能抑制肿瘤细胞的生长。我们有 现在产生了一种基于吡咯洛苯并二氮杂二聚体(PBD)的RSPO2突变体的药物结合物 在体外和体内结肠癌模型中均显示出较强的抗肿瘤作用。在本提案中，我们将 在体内外一系列结肠癌模型中评价药物结合物，并测定其 小鼠的耐受性。这些结果和结论可能第一次验证PBD共轭 RSPO-Fc蛋白作为同时靶向LGR4-6治疗结直肠癌的新方法 治疗和确定进一步开发的候选药物。

PROJECT SUMMARY Colorectal cancer is still a major cause of cancer-related death in the world and only a small subset of patients benefits from therapy by immune checkpoint inhibitors. Antibody–drug conjugates (ADCs) are monoclonal antibodies (mAbs) that are covalently linked to cell-killing drugs and have emerged as a major modality in anti-cancer treatment. This approach combines high specificity of mAbs against their antigen targets with highly potent cytotoxic drugs, resulting in “armed” mAbs that deliver the payload (drug) to tumor cells with enriched levels of the antibody target. The approach has become a major modality of cancer therapeutics with several ADCs approved in the last few years. Leucine-rich repeat containing, G protein-coupled receptor 4, 5 and 6 (LGR4-6) are three related receptors that are highly upregulated in colorectal cancers. They bind R-spondins (RSPOs), a group of secreted proteins with high affinity and potentiate Wnt signaling. Aberrant RSPO-LGR signaling plays critical roles in tumor formation, progression, and drug resistance. In particular, LGR5 is enriched in cancer stem cells of colon cancer and LGR5-positive cells drive tumor growth and metastasis. However, LGR5-positive and -negative cells can interconvert and ADCs targeting LGR5 inhibited tumor growth but failed to completely eradicate tumors due to cancer cell plasticity. Remarkably, LGR5-negative cells still express LGR4 or LGR6 or both. We reasoned that simultaneous targeting LGR4-6 may overcome cancer cell plasticity and drug resistance. Recently, we demonstrated that a mutated form of RSPO can bind to LGR4-6 with high affinity without potentiating Wnt signaling. Drug conjugates of RSPO mutant fused to IgG1-Fc domain was able to inhibit tumor cell growth in vitro and in vivo. We have now generated a pyrrolobenzodiazepine dimer (PBD)-based drug conjugate of an RSPO2 mutant that showed potent anti-tumor effect in colon cancer models in vitro and in vivo. In this proposal we will evaluate the drug conjugate in a series of colon cancer models in vitro and in vivo and determine its tolerability in mice. These results and conclusions may, for the first time, validate PBD-conjugated RSPO-Fc protein as a novel approach for simultaneous targeting of LGR4-6 for colorectal cancer treatment and identify drug candidates for further development.