Targeting metastatic tumors with engineered cellular therapies

总结 转移性脑肿瘤是最常见的颅内肿瘤。晚期乳腺 癌症有很高的倾向转移到脑与人表皮生长因子受体（EGFR） 阳性和三阴性乳腺癌（TNBC;雌激素和孕激素受体和Her 2阴性） 显示脑转移发生率最高的亚型。为了有效治疗多发性高侵袭性脑 转移性乳腺肿瘤（BMBT），迫切需要开发靶向异常信号传导的治疗方法 BMBT的肿瘤微环境（TME）中的肿瘤细胞和免疫细胞中的通路。最近我们 显示鞘内（IT）和颈动脉内（伊卡）注射成人同种异体“现成”间充质 表达同时靶向EGFR和（DR）4/5的双功能蛋白的干细胞（MSC），EvDRL具有 在模拟临床环境的BMBT小鼠模型中的治疗功效。这些发现虽然有希望， 已经提出了关于MSC释放的EvDRL联合诱导的肿瘤细胞杀伤的潜力的基本问题 与同时激活针对BMBT的免疫效应子功能的治疗剂。我们最近 已发表的研究表明，EVDRL的DRL（TRAIL）组分是EVDRL介导的细胞死亡的关键驱动因素 在患者来源的BMBT细胞中。以前的研究表明，除了肿瘤细胞，DRL诱导 骨髓源性抑制细胞（MDSC）和CD 4 + CD 25 + FoxP 3 + T细胞凋亡，同时 增加TME中CD 8 + T细胞的募集。此外，临床和临床前研究使用联合 细胞毒疗法和免疫检查点（ICI）阻断在乳腺转移性癌中显示出增加的疗效。 因此提供了将MSC-EVDRL与免疫调节剂联合收割机治疗BMBT的可能性。在 在本研究中，我们将首先评估MSC-EVDRL诱导TME中肿瘤细胞死亡的有效性和影响。 在由具有不同的细胞毒性的BMBT细胞产生的人源化（hu）NSG乳腺至脑转移肿瘤模型中， 对EVDRL介导的凋亡的应答。接下来，我们将创建双模MSC，释放EVDRL和抗 程序性细胞死亡蛋白（PD）-1纳米抗体（Nb-PD 1），并评估机制联合疗效 在huNSG乳腺至脑转移肿瘤模型中的工程化MSC。我们假设人类MSC-EvDRL 将导致特异性杀死局部和广泛传播的BMBT细胞，Nb-PD 1将靶向募集的T细胞， 的TME。为了便于临床转化，我们将结合可激活的杀伤开关/PET成像剂，疱疹病毒， 单纯病毒胸苷激酶（HSV-TK）进入MSC，并通过PET成像和选择性 HSV-TK激活介导的根除。鉴于工程MSC在非小细胞肺癌中处于I期临床试验， 细胞肺癌患者;目前正在软脑膜中进行使用抗PD-1抗体IT的I/II期试验 转移性患者;我们在原发性脑肿瘤（GBM）患者中的MSC-DRL治疗正在考虑中， FDA;成功执行拟定研究将有助于将我们的策略转化为临床。我们 预计我们的研究结果将对开发基于靶向的新机制做出重大贡献。 BMBT的治疗，从而在挽救许多转移性乳腺癌患者的生命的重大影响。

SUMMARY Metastatic brain tumors are the most commonly observed intracranial tumors. Patients with advanced breast cancer have a high propensity to metastasize to the brain with human epidermal growth factor receptor (EGFR) positive and triple-negative breast cancer (TNBC; estrogen and progesterone receptor and Her2 negative) subtypes showing the highest incidence of brain metastases. To effectively treat multiple highly aggressive brain metastatic breast tumors (BMBT), there is an urgent need to develop therapeutics that target aberrant signaling pathways in tumor cells and the immune cells in the tumor microenvironment (TME) of BMBT. Recently, we have shown that intrathecal (IT) and intracarotid artery (ICA) injection of adult allogeneic “off the shelf” mesenchymal stem cells (MSC) expressing bifunctional protein simultaneously targeting EGFR and (DR)4/5), EvDRL have therapeutic efficacy in mouse models of BMBT that mimic clinical settings. These findings although promising, have raised fundamental questions on the potential of combining MSC released EvDRL induced tumor cell killing with therapeutic agents that simultaneously activate immune effector functions against BMBT. Our recently published studies indicate that DRL (TRAIL) component of EVDRL is the key driver of EVDRL mediated cell death in patient derived BMBT cells. Previous studies have shown that in addition to tumor cells, DRL induces apoptosis in myeloid derived suppressor cells (MDSC) and CD4+ CD25+ FoxP3+ Tregs and simultaneously increases recruitment of CD8+ T cells in the TME. Furthermore, clinical and pre-clinical studies using combined cytotoxic therapy and immune checkpoint (ICI) blockade have shown increased efficacy in breast metastatic tumors thus offering the potential to combine of MSC-EVDRL with immunomodulatory agents to treat BMBT. In this proposal, we will first evaluate the efficacy and influence of MSC-EVDRL induced tumor cell death in the TME in humanized (hu) NSG breast to brain metastatic tumor models generated from BMBT cells that have varying response to EVDRL mediated apoptosis. Next, we will create bimodal MSC releasing EVDRL and anti- programmed cell death protein (PD)-1 nanobodies (Nb-PD1) and evaluate the mechanism combined efficacy of engineered MSC in huNSG breast to brain metastatic tumor models. We hypothesize that human MSC-EvDRL will lead to specific killing of local and widely disseminated BMBT cells and Nb-PD1 will target T cells recruited to the TME. To ease clinical translation, we will incorporate activatable kill switch/PET imaging agent, herpes simplex virus-thymidine kinase (HSV-TK) into MSC and assess their fate by PET imaging and selective eradication mediated by HSV-TK activation. Given that engineered MSC are in phase I clinical trial in non-small cell lung cancer patients; a phase I/II trial using IT of anti-PD-1 antibody is currently ongoing in leptomeningeal metastatic patients; and our MSC-DRL therapy in primary brain tumor (GBM) patients is under consideration by FDA; successful execution of the proposed studies will facilitate translation of our strategy into clinics. We anticipate that our findings will have a major contribution towards developing novel mechanism based targeted therapies for BMBT and thus a major impact in saving the lives of many metastatic breast cancer patients.