Development of a cell-penetrating beta-catenin antagonist peptide as a therapeutic candidate for Wnt-driven breast cancer

摘要 在>20%的所有癌症中发现Wnt/β-连环蛋白信号的失调过表达和过度活化， 50%的乳腺癌，特别是三阴性乳腺癌（TNBC）。然而，尽管广泛的努力， Wnt/β-catenin信号通路，没有靶向Wnt通路的药物被批准，主要是由于不能 将β-连环蛋白的致癌功能与稳态功能分开。BCL 9是一种转录辅因子， 调节致癌β-连环蛋白介导的基因反式激活的激活剂。其过表达增强Wnt/β- 连环蛋白信号传导，导致细胞增殖、迁移和体外侵袭，并诱导肿瘤发生 和体内进展。小鼠肠上皮细胞中Bcl 9/Bcl 91基因切除减少肿瘤生长 无表型异常。因此，β-连环蛋白与BCL 9相互作用的特异性拮抗作用是 假设证明是一种安全有效的癌症治疗策略。 Sapience Therapeutics试图开发一种β-连环蛋白拮抗剂肽，旨在与β-连环蛋白相互作用。 我们的方法将氨基酸（AA）取代引入天然HD2结合结构域序列，以增强HD2结合结构域的功能。 靶向亲和力，并且包括用于细胞内进入的短细胞穿透结构域（CPD）。此外，为了克服 Sapience的肽包括D-对映体AA（D-AA）， 显著增加稳定性和生物利用度，同时降低潜在的免疫原性。 通过广泛的构效关系（SAR）研究，我们筛选了一个肽库， 破坏β-连环蛋白与BCL 9的致癌关联，并选择ST 316作为主要候选物。ST 316显示器 在依赖于Wnt的肿瘤细胞中有效和选择性抑制Wnt信号传导和诱导细胞毒性 信号传导（但不是Wnt非依赖性肿瘤）。此外，ST 316表现出稳定性、上级生物利用度和 溶解度和体内效力。在该SBIR的I期中，ST 316表现出纳摩尔结合亲和力， catenin和低微摩尔（2.7µM）EC 50值抑制β-catenin体外转录活性。安全 在50 mg/kg剂量下无组织病理学发现的研究表明，治疗窗至少为10倍。 在这一概念证明之后，该II期申请提出了严格的IND使能毒理学和phar- 支持ST 316临床开发的宏观学研究。具体目标#1将决定首次在人类 (FIH)通过确定大鼠中的无不良作用水平（NOAEL）和最高非严重毒性剂量确定ST 316剂量 （HNSTD）的28天重复给药GLP毒性研究中的小型猪。在具体目标#2中，我们提出了一系列 确定ST 316在小鼠患者来源的异种移植物（PDX）中的药理学活性剂量（PAD）的实验 模型，并确定与PAD相关的药效学（PD）生物标志物，可用于临床 以评估ST 316活性。然后，PAD将用于具体目标#3，以评价ST 316对 肿瘤免疫微环境（TME），即巨噬细胞极化、肿瘤浸润淋巴细胞（TIL） 肿瘤中的表达以及与PD-1抑制剂组合的影响。

ABSTRACT Dysregulated overexpression and hyperactivation of Wnt/-catenin signaling is found in >20% of all cancers and in 50% of breast cancer, particularly triple negative breast cancer (TNBC). Yet despite extensive efforts to inhibit Wnt/-catenin signaling, no drugs that target the Wnt pathway have been approved, mainly due to the inability of inhibitors drugs to separate oncogenic from homeostatic functions of -catenin. BCL9 is a transcriptional co- activator that regulates oncogenic β-catenin-mediated gene transactivation. Its overexpression enhances Wnt/β- catenin signaling, resulting in cell proliferation, migration and invasiveness in vitro, and induces tumor initiation and progression in vivo. Genetic ablation of Bcl9/Bcl9l in mouse intestinal epithelium reduces tumor growth without phenotypic abnormalities. Therefore, specific antagonism of the interaction of β-catenin with BCL9 is hypothesized to prove a safe and efficacious strategy for cancer therapy. Sapience Therapeutics sought to develop a -catenin antagonist peptide, designed to interact with -catenin. Our approach introduces amino acid (AA) substitutions to the native HD2 binding domain sequence to enhance target affinity and includes a short cell penetrating domain (CPD) for intracellular entry. Additionally, to overcome challenges encountered by traditional peptides, Sapience’s peptides include D-enantiomer AAs (D-AAs), which dramatically increase stability and bioavailability, while reducing potential immunogenicity. Through extensive structure-activity relationship (SAR) studies, we screened a library of peptides designed to disrupt β-catenin’s oncogenic association with BCL9 and selected ST316 as the lead candidate. ST316 displays potent and selective inhibition of Wnt signaling and induction of cytotoxicity in tumor cells dependent on Wnt signaling (but not Wnt-independent tumors). Further, ST316 demonstrates stability, superior bioavailability and solubility and in vivo potency. In Phase I of this SBIR, ST316 demonstrated nanomolar binding affinity to β- catenin and a low micromolar (2.7µM) EC50 value for inhibition of β-catenin transcriptional activity in vitro. Safety studies with no histopathological findings at 50mg/kg suggested a therapeutic window of at least 10x. Following this proof of concept, this Phase II application proposes rigorous IND-enabling toxicology and phar- macology studies to support clinical development of ST316. Specific Aim #1 will determine the first-in-human (FIH) ST316 dose by identifying the no adverse effect level (NOAEL) in rats and highest non-severely toxic dose (HNSTD) in minipigs in 28-day repeat dose GLP toxicity studies. In Specific Aim #2, we propose a series of experiments to determine the ST316 pharmacologic active dose (PAD) in mouse patient-derived xenograft (PDX) models, and identify pharmacodynamic (PD) biomarkers associated with the PAD, which can be used clinically to assess ST316 activity. The PAD will then be used in Specific Aim #3 to evaluate the impact of ST316 on the tumor immune microenvironment (TME), namely macrophage polarization, tumor infiltrating lymphocyte (TIL) expression in tumors and the impact of combination with a PD-1 inhibitor.