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

抗 EGFR-huEndo-P125A 的开发：针对三阴性乳腺癌的血管生成拟态-血管生成联合抑制疗法

• 批准号: 10481695
• 负责人: Rathin C Das
• 金额: $ 40万
• 依托单位: SYNERGYS BIOTHERAPEUTICS, INC.
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-20 至 2023-03-27
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10481695
• 关键词: Antibodies; Behavior; Blood Vessels; Breast Cancer Cell; Breast Cancer Model; Cetuximab; Chimeric Proteins; Chinese Hamster Ovary Cell; Clone Cells; Cyclic GMP; Development; Endostatins; Engineering; Epidermal Growth Factor Receptor; Erbitux; Growth; Human; IgG1; Immune; Immune checkpoint inhibitor; Immunocompetent; Immunocompromised Host; KDR gene; Link; Lung; MDA MB 231; MDA-MB-468; MMP14 gene; MMP2 gene; Macaca fascicularis; Metastatic Neoplasm to the Lung; Mus; Neoplasm Metastasis; Newly Diagnosed; Paclitaxel; Pattern; Phase; Play; Preparation; Process; Proteins; Risk-Benefit Assessment; Rodent; Role; Serum; Signal Transduction; Testing; Therapeutic; Toxicology; Tumor Cell Line; aggressive breast cancer; angiogenesis; anti-PD-L1; beta catenin; breast cancer diagnosis; cancer subtypes; cell motility; chemotherapeutic agent; chemotherapy; clinically relevant; dimer; efficacy testing; improved; in vivo; inhibitor; intravenous administration; malignant breast neoplasm; meetings; migration; mimicry; mutant; patient derived xenograft model; pharmacokinetics and pharmacodynamics; prevent; response; safety study; therapeutic target; triple-negative invasive breast carcinoma; tumor growth

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.

项目摘要/摘要 三阴性乳腺癌(TNBC)是侵袭性乳腺癌(BC)亚型，包括 约20%的新诊断的BCS。尽管表皮生长因子受体(EGFR)在 ~60%的TNBC和65%-72%的基底样BCS，抗EGFR单抗，西妥昔单抗(Erbitux®)，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其相关 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)， 以及细胞突起的形成，这种突起被称为“内向突起”，它促进了细胞的迁移、血管内和 转移。尽管血管生成在结直肠癌的生长和转移中起着核心作用，但联合治疗 血管生成和VM的靶向性在很大程度上还没有得到测试。我们已经合成了一种抗体融合蛋白， AEGFR-huEndo-P125A(EEPA125)通过将抗EGFR IgG1的重链与高度抗EGFR的 血管生成“有效载荷”，huEndoP125A，它是人内皮抑素的P125--&GT；A125突变体。EEPA125被抑制 血管生成和Vm，这不是西妥昔单抗或huEndo-P125A单独观察到的。一种组合 非融合形式的抗EGFR IgG1和huEndo-P125A不抑制VM，提示二聚体的递送 通过EEPA125融合的huEndo-P125A对VM的抑制是必不可少的。我们成功地生产和提纯了 从稳定的CHO细胞池中提取EEPA125以获得95%的纯度。EEPA125体内检测显示 西妥昔单抗对MDA-MB-468肿瘤生长和转移的抑制作用优于西妥昔单抗。EEPA125 更有效 也很明显 静脉注射亲肺MDA-MB-231-4175TNBC细胞可减少肺转移； 与西妥昔单抗治疗相比，提高了小鼠的存活率。EEPA125也表现出ADCC活性，它 显著抑制Wnt/b-catenin信号转导，抑制TNBC的运动和迁移，抑制内翻足的形成。 此外，它还减少了TNBC细胞分泌的可溶性MMP2和MT1-MMP2蛋白。 使用huendo-P125A或西妥昔单抗治疗。我们还表征了EEPA125的血清清除模式 在免疫功能低下的小鼠身上。我们现在建议进行EEPA125的体内确证疗效测试 使用临床相关的TNBC PDX(患者来源的异种移植)模型，并研究EEPA125在 预防抗血管生成药物治疗后Vm增加和“反跳血管生成” 血管内皮生长因子受体抑制剂孙尼替尼。与紫杉醇等化疗药物联合EEPA125将是 测试过。我们将为EEPA125的工艺工程和cGMP制造产生稳定的CHO细胞克隆， 并对免疫受损和免疫活性的啮齿动物进行PK/PD和毒理学/安全性研究 在食蟹猴身上。我们还将建立一种表达EGFR的小鼠乳腺癌模型，并研究 EEPA125与抗PD-L1检查点抑制剂联用的免疫活性研究 老鼠。生成的结果将使风险-效益评估成为可能，为与FDA举行IND前会议做准备 以及最终提交IND以进行第一阶段/第二阶段测试。