Monoclonal Antibody-based Prodrugs - Novel Tools for Cancer Therapy

描述(申请人提供)：近年来，识别肿瘤相关抗原的单抗越来越多地被整合到肿瘤的临床治疗中，这一趋势预计将继续下去。以抗体为基础的治疗的主要靶点是ErbB家族成员，包括表皮生长因子受体(EGFR/ErbB1)本身和ErbB2。目前临床使用的EGFR特异性抗体通常抑制EGFR依赖的信号转导，并诱导抗体依赖的细胞介导的细胞毒作用。然而，给肿瘤患者使用这些药物会产生剂量限制的副作用，因为EGFR信号在正常组织中受到抑制，特别是皮肤和胃肠道。因此，非常需要开发基于抗体的治疗方法，在肿瘤环境中特异性识别其靶抗原，但在正常组织中不识别。在这里，我们提出了一种新的方法来实现这一目标。它的基础是构建抗体衍生品，其中抗原识别位点被抗原片段可逆地掩盖。抗原片段与单抗CDRs的结合有望封闭融合蛋白的抗原识别部位，从而降低正常组织的反应性。为了在肿瘤部位恢复高亲和力的抗原-抗体相互作用，在封闭部分和抗体片段之间的连接物中设计了一个蛋白水解性切割位点。该部位对肿瘤组织中高活性的蛋白水解酶(即基质金属蛋白酶，MMPs)很敏感，而在包括外周血在内的正常组织中，在动态平衡条件下很少或没有活性。在肿瘤部位发生裂解后，根据群体作用原理，未被掩蔽的抗体被预测将分配给肿瘤相关抗原。这项拟议的工作将测试基于EGFR拮抗性mAbC225和425的抗体衍生物的生物学特性，这些mAbC225和425识别人EGFR(EGFRdIII)细胞外域的不同表位。在初步工作中，C225和425单链抗体被共价连接到EGFRdIII序列，以鼓励彼此的抗原识别位点的二聚化和相互封闭(交叉掩蔽)。经MMP9处理后，这些复合体被解离，随后与纯化的EGFR和表达EGFR的肿瘤细胞的结合显着增强。这些有希望的结果构成了重组和体外测试类免疫球蛋白抗体(特异性目标1)的基础，以及通过这种结构在体内确定肿瘤归巢和抑制人肿瘤生长的异种移植(特异性目标2)的基础。在未来的工作中，这些试剂作为诊断试剂(成像)的用途将在适当的动物模型中进一步测试，即人类肿瘤在小鼠体内的异种移植。 与公共卫生相关：近年来，单抗越来越多地被用于某些形式的癌症和某些其他疾病的临床治疗。目前用于癌症治疗的大多数单抗识别肿瘤相关抗原，这些抗原也存在于正常组织中。这种情况导致了不良副作用，限制了这些药物在患者中的应用和疗效。这项建议描述了一种新的概念来设计抗体衍生物，以克服这些限制。为此，我们将重点研究目前正在临床使用或临床试验中的两种EGFR拮抗型单抗，即C225和425的分子修饰。如果成功，这里要测试的概念可能适用于目前正在临床使用或开发的多种单抗。

DESCRIPTION (provided by applicant): In recent years monoclonal antibodies (mAbs) recognizing tumor-associated antigens have increasingly been integrated into the clinical management of neoplasia and this trend is expected to continue. Prominent targets for antibody-based therapies are ErbB family members, including the epidermal growth factor receptor (EGFR/ErbB1) itself and ErbB2. EGFR-specific antibodies currently in clinical use typically inhibit EGFR-dependent signal transduction and induce antibody-dependent cell-mediated cytotoxicity. Administration of these agents to tumor patients is, however, associated with dose-limiting adverse side effects due to inhibition of EGFR signaling in normal tissues, specifically skin and the gastrointestinal tract. It is, therefore, highly desirable to develop antibody-based therapeutics that specifically recognize their target antigen in the tumor environment but not on normal tissues. Here we propose a novel approach to achieve this goal. It is based on the construction of antibody derivatives in which antigen recognition sites are reversibly masked by antigen fragments. Association of the antigen fragment with the mAb CDRs is expected to occlude the antigen recognition site of the fusion protein and, thus, reduce normal tissue reactivity. To restore high affinity antigen- antibody interaction at tumor sites, a proteolytic cleavage site is engineered into the linker between the blocking moiety and the antibody fragment. This site is designed to be susceptible to proteases with high activity in tumor tissues (i.e. matrix metalloproteases (MMPs)) but little or no activity in normal tissues including peripheral blood under homeostatic conditions. Upon cleavage at tumor sites, the 'unmasked' antibody is predicted to partition to the tumor-associated antigen due to mass action principles. The proposed work will test biological properties of antibody derivatives based on the EGFR antagonistic mAbs C225 and 425 that recognize distinct epitopes on the extracellular domain of the human EGFR (EGFRdIII). In preliminary work dual (Ab-Ag)2 complexes were made whereby C225 and 425 scFvs were covalently linked to EGFRdIII sequences engineered to encourage dimerization and reciprocal occlusion (crossmasking) of the antigen recognition sites of each other. Upon MMP9 treatment these complexes were shown to dissociate followed by markedly enhanced binding to purified EGFR and to tumor cells expressing EGFR. These promising results form the basis for the reformatting and in vitro testing of crossmasked Abs in an IgG-like format (Specific Aim 1) and the determination of tumor homing and growth inhibition human tumor xenotransplants in mice by such constructs in vivo (Specific Aim 2). In future work, the utility of these reagents as diagnostic agents (imaging) will be further tested in appropriate animal models, i.e. human tumor xenotransplants in mice. PUBLIC HEALTH RELEVANCE: In recent years monoclonal antibodies (mAbs) have been used increasingly in the clinical management of certain forms of cancer and select other diseases. Most mAbs currently used in cancer therapy recognize tumor-associated antigens, which are also present on normal tissues. This circumstance leads to adverse side effects that limit the application and efficacy of these agents in patients. This proposal describes a novel concept to engineer antibody derivatives that overcome these limitations. To this end, we will focus on molecular modification of two EGFR antagonistic monoclonal antibodies currently in clinical use or clinical trials, i.e. C225 and 425, respectively. If successful, the concept to be tested here may be applicable to a wide range of mAbs currently in clinical use or development.