Development of Anti-EGFR-huEndo-P125A: A combined Vasculogenic Mimicry-Angiogenesis inhibitory therapeutic for Triple Negative Breast Cancer

项目总结/摘要 三阴性乳腺癌（TNBC）是侵袭性乳腺癌（BC）亚型，其包括 大约20%的新诊断的BC。尽管表皮生长因子受体（EGFR）在人的前列腺中表达， ~60%的TNBC和65-72%的基底细胞样BC，抗EGFR单抗西妥昔单抗（爱必妥®）几乎没有反应。 作为单药或与化疗联合使用。TNBC的侵袭性转移行为与 具有形成由肿瘤细胞自身排列的血管通道的能力，称为血管生成拟态（VM）， 以及称为“侵入伪足”的细胞突起的形成，其促进细胞迁移、内渗和 转移尽管血管生成在BC生长和转移中起着核心作用，但联合治疗仍是治疗BC的关键。 血管生成和VM的靶向作用在很大程度上还没有经过测试。我们已经合成了一种抗体融合蛋白， aEGFR-huEndo-P125 A（EEPA 125）通过将抗EGFR IgG 1的重链与高度抗EGFR IgG 1的重链连接， 血管生成“有效负载”，huEndoP 125 A，其是人内皮抑制素的P125 -> A125突变体。EEPA 125抑制 血管生成和VM，这在单独使用西妥昔单抗或huEndo-P125 A时均未观察到。的组合 非融合形式的抗EGFR IgG 1和huEndo-P125 A不抑制VM，表明递送二聚体 通过EEPA 125融合的huEndo-P125 A对于VM抑制是必需的。我们成功地制造并提纯了 从稳定的CHO细胞池中分离EEPA 125至>95%纯度。EEPA 125的体内测试显示 MDA-MB-468 TNBC肿瘤生长和转移扩散的抑制。EEPA125 更有效 也明显 静脉内施用嗜肺MDA-MB-231-4175 TNBC细胞后肺转移减少，和 与西妥昔单抗治疗相比，小鼠存活率提高。EEPA 125也表现出ADCC活性， 显著抑制Wnt/β-连环蛋白信号传导、TNBC运动性和迁移以及侵袭伪足形成。 此外，与对照组相比，它减少了TNBC细胞分泌可溶性MMP 2和MT 1-MMP蛋白。 用huEndo-P125 A或西妥昔单抗治疗。我们还描述了EEPA 125的血清消除模式 在免疫受损的小鼠中。我们现在建议进行EEPA 125的体内功效验证试验 使用临床相关的TNBC PDX（患者来源的异种移植物）模型，并研究EEPA 125在 预防用抗血管生成药物治疗后诱导的VM增加和“反弹血管生成” VEGFR抑制剂舒尼替尼。与化疗剂如紫杉醇与EEPA 125的组合将是有利的。 测试.我们将生成稳定的CHO细胞克隆，用于EEPA 125的工艺工程和cGMP生产， 并在免疫功能低下和免疫功能正常的啮齿动物中进行PK/PD和毒理学/安全性研究， 在食蟹猴中。我们还将建立一个表达EGFR的小鼠乳腺癌模型，并研究 EEPA 125与抗PD-L1检查点抑制剂联合使用时的免疫作用 小鼠生成的结果将使风险-获益评估成为可能，为与FDA的IND前会议做准备 并最终提交IND进行I/II期试验。

PROJECT SUMMARY/ABSTRACT Triple-negative breast cancer (TNBC) is an aggressive breast cancer (BC) subtype which comprises approximately 20% of newly diagnosed BCs. Although epidermal growth factor receptor (EGFR) is expressed in ~60% of TNBC and 65-72% of basal-like BCs, the anti-EGFR MAb, Cetuximab (Erbitux®), elicits little response as a single-agent or when combined with chemotherapy. Aggressive metastatic behavior of TNBC correlates with the ability to form vascular channels lined by tumor cells themselves, known as Vasculogenic Mimicry (VM), and the formation of cellular protrusions called ‘invadopodia’ that facilitate cellular migration, intravasation and metastasis. Although angiogenesis plays a central role in BC growth and metastasis, combined therapeutic targeting of angiogenesis and VM has largely been untested. We have synthesized an antibody-fusion protein, aEGFR-huEndo-P125A (EEPA125) by linking the heavy chains of an anti-EGFR IgG1 with a highly anti- angiogenic “payload”, huEndoP125A, which is a P125 --> A125 mutant of human endostatin. EEPA125 inhibited both angiogenesis, and VM, which was not seen with cetuximab nor huEndo-P125A alone. A combination of anti-EGFR IgG1 and huEndo-P125A in non-fused form did not inhibit VM suggesting that the delivery of dimeric huEndo-P125A by EEPA125 fusion is essential for VM inhibition. We successfully produced and purified EEPA125 from a stable pool of CHO cells to >95% purity. Testing of EEPA125 in vivo showed inhibition of MDA-MB-468 TNBC tumor growth and metastatic spread than cetuximab. EEPA125 more effective also markedly reduced lung metastasis following intravenous administration of lung tropic MDA-MB-231-4175 TNBC cells, and improved survival of mice compared to cetuximab treatment. EEPA125 also demonstrated ADCC activity, and it markedly inhibited Wnt/b-catenin signaling, TNBC motility and migration, and invadopodia formation. Furthermore, it reduced the secretion of soluble MMP2 and MT1-MMP proteins by TNBC cells compared to treatment with huEndo-P125A, or cetuximab. We also characterized the serum elimination pattern of EEPA125 in immunocompromised mice. We now propose to carry out confirmatory in vivo efficacy testing of EEPA125 using clinically relevant TNBC PDX (patient derived xenograft) models, and to study EEPA125 efficacy in preventing increased VM and “rebound angiogenesis” induced following treatment with the anti-angiogenic VEGFR inhibitor sunitinib. Combination with chemotherapeutic agents such as paclitaxel with EEPA125 will be tested. We will generate a stable CHO cell clone for process engineering and cGMP manufacture of EEPA125, and perform PK/PD and toxicology/safety studies in immunocompromised and immunocompetent rodents and in cynomolgus monkeys. We will also develop an EGFR expressing murine breast cancer model and investigate the immune effects of EEPA125 when combined with of an anti PD-L1 checkpoint inhibitor in immunocompetent mice. Results generated will enable a risk-benefit assessment in preparation for a pre-IND meeting with the FDA and the eventual filing of an IND for Phase I/II testing.