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

描述（由申请人提供）：耐药乳腺癌细胞是复发性疾病的主要原因。癌细胞存活和耐药性的一种机制是GRP 78/BiP的动员，其是未折叠蛋白反应和ER应激的主调节剂。由于其作用，GRP 78/BiP被认为是乳腺癌化疗反应性的新预测因子。我们假设靶向破坏乳腺癌细胞中的GRP 78/BiP将增加批准的化疗药物的疗效。为了验证这一假设，我们构建了一个融合蛋白，包括人表皮生长因子（EGF）和SLiP，一种新的细菌毒素SubAB的催化亚基，其独特的和唯一的底物是GRP 78/BiP。我们的初步结果表明，在EGFR阳性细胞中，EGF-SLiP在皮摩尔浓度下切割GRP 78/BiP并抑制细胞生长。对于该项目至关重要的是，阿霉素抗性癌细胞比亲本细胞对EGF-SLiP表现出更大的敏感性。由于EGFR过表达也与乳腺癌细胞的耐药性增加有关，我们预计通过EGFR靶向SLiP将特别有益。我们还期望EGF-SLiP和化疗药物之间基于机制的互补性，以及针对SLiP的预先存在的免疫力的缺乏，将为EGF-SLiP提供相对于先前不成功的基于EGF的细胞毒素的明显优势。为了回应对原始提案的批评，并考虑到新的数据，我们重新设计了提案，并专注于原位乳腺癌模型的动物研究。在该项目中将探索关于EGF-SLiP用于治疗乳腺癌的潜在效用的关键问题：1）肿瘤渗透和GRP 78/BiP切割，2）全身毒性和免疫原性，以及3）与多柔比星组合用于治疗多柔比星抗性肿瘤的功效。为了回应重新提交的批评，我们更详细地解释了BiP的主调节作用，它允许EGF-SLiP与各种损伤协同作用，并添加了额外的数据。该项目第一阶段的成功完成将提供关于体内关键EGF-SLiP特征的关键信息。我们预计，I期的结果将足以合理决定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靶向药物的并行比较、正式毒理学、GMP生产以及向第一阶段临床试验的过渡。公共卫生相关性：我们建议测试使用一种新的细胞毒素EGF-SLiP在乳腺癌化疗过程中靶向耐药细胞的可行性。细胞毒素的毒性部分SLiP是最近表征的细菌蛋白酶，其在哺乳动物细胞中仅切割一种蛋白质GRP 78/BiP，其控制细胞对化疗药物的防御，并且是乳腺癌对化疗的抗性的新预测因子。EGF-SLiP将细胞毒素靶向表达EGF受体的乳腺癌细胞，这一特征也与癌症进展和耐药性有关。使用乳腺癌的原位小鼠模型，我们将确定EGF-SLiP在切割肿瘤中的GRP 78/BiP中的功效，评估全身毒性和免疫原性，并确定其作为治疗多柔比星抗性肿瘤的组合方案的一部分的功效。我们期望该数据确立EGF-SLiP临床开发的可行性。

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.