Targeting GRP78/BIP As An Adjunct Therapy For EGFR-positive Breast Cancer

描述(申请人提供)：耐药乳腺癌细胞是复发疾病的主要原因。癌细胞存活和耐药的一个机制是GRP78/Bip的动员，GRP78/Bip是未折叠蛋白反应和内质网应激的主要调节因子。由于GRP78/Bip的作用，GRP78/Bip被认为是预测乳腺癌化疗反应的新指标。我们假设，靶向破坏乳腺癌细胞中的GRP78/Bip将增加经批准的化疗药物的疗效。为了验证这一假设，我们构建了一个包含人表皮生长因子(EGF)和SLIP的融合蛋白，SLIP是一种新的细菌毒素SubAB的催化亚基，其唯一的底物是GRP78/Bip。我们的初步结果表明，在EGFR阳性的细胞中，EGF-SLIP在皮摩尔浓度下裂解GRP78/Bip并抑制细胞生长。对该项目至关重要的是，耐多柔比星的癌细胞比亲本细胞对EGF-SLIP表现出更高的敏感性。由于EGFR的过度表达也与乳腺癌细胞的耐药性增加有关，我们预计通过EGFR的靶向滑动将特别有益。我们还预计，EGF-SLIP与化疗药物之间基于机制的互补性，以及对SLIP缺乏预先存在的免疫力，将使EGF-SLIP具有相对于先前未成功的EGF-Slip细胞毒素的独特优势。为了回应对原始建议的批评，并考虑到新的数据，我们重新设计了建议，并专门专注于原位乳腺癌模型的动物研究。本项目将探讨EGF-SLIP用于乳腺癌治疗的关键问题：1)肿瘤穿透性和GRP78/Bip裂解；2)全身毒性和免疫原性；3)联合阿霉素治疗阿霉素耐药肿瘤的疗效。为了回应对重新提交的批评，我们更详细地解释了BIP的主调节角色，它允许EGF-SLIP与各种侮辱协同作用，并增加额外的数据。该项目第一阶段的成功完成将提供体内关键的EGF滑移特征的关键信息。我们预计，第一阶段的结果将足以合理地决定EGF-SLIP临床开发的可行性。该项目的第二阶段将包括与现有EGFR导向药物的并列比较、正式毒理学、GMP生产，以及过渡到第一阶段临床试验。公共卫生相关性：我们建议测试在乳腺癌化疗过程中使用一种新的细胞毒素EGF-SLIP靶向耐药细胞的可行性。细胞毒素的有毒部分SLIP是一种最近表征的细菌蛋白水解酶，它只分解哺乳动物细胞中的一种蛋白质GRP78/Bip，它控制细胞对化疗药物的防御，是乳腺癌化疗耐药的新预测因子。EGF-SLIP靶向表达EGF受体的乳腺癌细胞的细胞毒素，这一特征也与癌症进展和耐药性有关。利用小鼠原位乳腺癌模型，我们将建立EGF-SLIP在肿瘤中裂解GRP78/Bip的有效性，评估其系统毒性和免疫原性，并将其作为联合方案治疗阿霉素耐药肿瘤的一部分。我们期望这一数据为EGF-SLIP的临床开发提供可行性。

DESCRIPTION (provided by applicant): Drug-resistant breast cancer cells are the main cause of recurrent disease. One mechanism of cancer cell survival and drug resistance is mobilization of GRP78/BiP, master-regulator of unfolded protein response and ER stress. Because of its role, GRP78/BiP is considered as a novel predictor of responsiveness to chemotherapy in breast cancer. We hypothesize that targeted destruction of GRP78/BiP in breast cancer cells would increase the efficacy of approved chemotherapeutic drugs. To test this hypothesis, we constructed a fusion protein comprising human epidermal growth factor (EGF) and SLiP, a catalytic subunit of a novel bacterial toxin SubAB, whose unique and the only substrate is GRP78/BiP. Our preliminary results indicate that in EGFR-positive cells, EGF-SLiP cleaves GRP78/BiP and inhibits cell growth at picomolar concentrations. Critically for this project, doxorubicin-resistant cancer cells display greater sensitivity to EGF-SLiP than parental cells. Since EGFR overexpression is also associated with increased drug resistance of breast cancer cells, we expect that targeting SLiP via EGFR would be especially beneficial. We also expect that the mechanism-based complementarity between EGF-SliP and chemotherapeutic drugs, as well as the absence of pre-existing immunity against SLiP, will provide EGF-SLiP with distinct advantages relative to previous unsuccessful EGF-based cytotoxins. In response to critique of the original proposal and in view of the new data, we redesigned the proposal and focused exclusively on animal studies in an orthotopic breast cancer model. The key questions regarding potential utility of EGF-SLiP for treatment of breast cancer will be explored in this project: 1) tumor penetration and GRP78/BiP cleavage, 2) systemic toxicity and immunogenicity, and 3) efficacy of combination with doxorubicin for treatment of doxorubicin- resistant tumors. In response to the critique of the resubmission we explain in more details the master regulator role of BiP which allows EGF-SLiP to act synergistically with various insults and add additional data. Successful completion of Phase I of this project will provide critical information on key EGF- SLiP characteristics in vivo. We expect that the results of Phase I will be sufficient for rational decision on feasibility of clinical development of EGF-SLiP. Phase II of this project will include side-by-side comparison with existing EGFR-directed drugs, formal toxicology, GMP- production, and transition to Phase I clinical trials. PUBLIC HEALTH RELEVANCE: We propose to test the feasibility of using a new cytotoxin, EGF-SLiP for targeting drug-resistant cells in the course of chemotherapy of breast cancer. The toxic part of the cytotoxin, SLiP, is a recently characterized bacterial protease that cleaves only one protein in mammalian cells, GRP78/BiP, which controls cellular defenses against chemotherapeutic drugs and is a novel predictor of resistance to chemotherapy of breast cancer. EGF-SLiP targets cytotoxin to breast cancer cells that express EGF receptors, a feature that is also implicated in cancer progression and drug resistance. Using orthotopic mouse model of breast cancer, we will establish efficacy of EGF-SLiP in cleaving GRP78/BiP in tumors, assess systemic toxicity and immunogenicity, and establish its efficacy as part of a combination regimen in treating doxorubicin-resistant tumor. We expect that this data establish feasibility of clinical development for EGF-SLiP.