Monoclonal Antibody-based Prodrugs - Novel Tools for Cancer Therapy

基于单克隆抗体的前药 - 癌症治疗的新工具

• 批准号: 7788327
• 负责人: ULRICH RODECK
• 金额: $ 22.28万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-01-18 至 2011-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7788327
• 关键词: Address; Adverse effects; Animal Model; Antibodies; Antibody Affinity; Antigen Targeting; Antigens; Binding; Biological Testing; Cell Survival; Cells; Cetuximab; Chimeric Proteins; Clinical; Clinical Management; Clinical Trials; Complex; Development; Diagnostic; Dimerization; Disease; Dose-Limiting; ERBB2 gene; Engineering; Environment; Epidermal Growth Factor Receptor; Epitopes; Erbitux; Extracellular Domain; Family member; Future; Gastrointestinal tract structure; Goals; Growth; Her2/erbb2/neu Staining Method; Homing; Human; IgG1; Image; Immunoglobulin Fragments; Immunoglobulin G; In Vitro; Label; Ligand Binding; Link; MMP9 gene; Malignant Neoplasms; Masks; Metalloproteases; Methodology; Modification; Molecular; Mono-S; Monoclonal Antibodies; Monoclonal Antibody C225; Multiple Myeloma; Mus; Mutate; Neoplasms; Normal tissue morphology; Nude Mice; Patients; Peptide Hydrolases; Point Mutation; Positron-Emission Tomography; Prodrugs; Property; Reagent; Recombinants; Signal Transduction; Site; Skin; Structure; Surface Plasmon Resonance; Testing; Therapeutic; Toxic effect; Tumor Antigens; Tumor Cell Line; Tumor Tissue; Variant; Work; antibody engineering; antibody-dependent cell cytotoxicity; base; cancer therapy; cell growth; design; extracellular; flexibility; gastrointestinal; in vitro testing; in vivo; inhibiting antibody; neoplastic cell; novel; novel strategies; peripheral blood; polypeptide; public health relevance; receptor; reconstitution; research study; tool; trend; tumor; tumor growth; tumor xenograft

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.

描述（由申请人提供）：近年来，识别肿瘤相关抗原的单克隆抗体（mAb）已越来越多地被整合到肿瘤的临床治疗中，并且预计这种趋势将持续下去。基于抗体的疗法的重要靶点是 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依赖性信号转导并诱导抗体依赖性细胞介导的细胞毒性。然而，对肿瘤患者施用这些药物会因抑制正常组织（特别是皮肤和胃肠道）中的 EGFR 信号传导而产生剂量限制性不良副作用。因此，非常需要开发基于抗体的治疗方法，能够特异性识别肿瘤环境中而非正常组织上的靶抗原。在这里，我们提出了一种实现这一目标的新颖方法。它基于抗体衍生物的构建，其中抗原识别位点被抗原片段可逆地掩蔽。抗原片段与 mAb CDR 的结合预计会封闭融合蛋白的抗原识别位点，从而降低正常组织的反应性。为了恢复肿瘤位点处的高亲和力抗原-抗体相互作用，将蛋白水解切割位点设计到阻断部分和抗体片段之间的连接子中。该位点被设计为对肿瘤组织中具有高活性的蛋白酶（即基质金属蛋白酶（MMP））敏感，但在正常组织（包括稳态条件下的外周血）中具有很少或没有活性。在肿瘤位点裂解后，由于质量作用原理，“未掩蔽”的抗体预计会分配到肿瘤相关抗原。拟议的工作将测试基于 EGFR 拮抗单克隆抗体 C225 和 425 的抗体衍生物的生物学特性，这些抗体衍生物可识别人 EGFR (EGFRdIII) 胞外域上的不同表位。在初步工作中，制备了双 (Ab-Ag)2 复合物，其中 C225 和 425 scFv 与 EGFRdIII 序列共价连接，该序列经工程设计以促进彼此抗原识别位点的二聚化和相互封闭（交叉掩蔽）。经过 MMP9 处理后，这些复合物显示出解离，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以 IgG 样形式重新格式化和体外测试交叉掩蔽抗体的基础（具体目标 1），以及通过体内此类构建体确定小鼠体内肿瘤归巢和生长抑制人肿瘤异种移植物（具体目标 2）。在未来的工作中，这些试剂作为诊断剂（成像）的效用将在适当的动物模型（即小鼠的人类肿瘤异种移植）中进一步测试。 公共卫生相关性：近年来，单克隆抗体 (mAb) 越来越多地用于某些癌症和其他疾病的临床治疗。目前用于癌症治疗的大多数单克隆抗体可识别肿瘤相关抗原，这些抗原也存在于正常组织上。这种情况会导致不良副作用，限制这些药物在患者中的应用和功效。该提案描述了一种新的概念来设计克服这些限制的抗体衍生物。为此，我们将重点关注目前临床使用或临床试验中的两种EGFR拮抗单克隆抗体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临床使用或开发的各种单克隆抗体。