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: 8013862
• 负责人: ULRICH RODECK
• 金额: $ 16.9万
• 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 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8013862
• 关键词: Address; Adverse effects; Animal Model; Antibodies; Antibody Affinity; Antigen Targeting; Antigens; Binding; Biological Testing; Cell Proliferation; Cell Survival; Cells; Cetuximab; Chimeric Proteins; Clinical; Clinical Management; Clinical Trials; Complex; Development; Diagnostic; Dimerization; Disease; Dose-Limiting; EGFR inhibition; 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（由申请方提供）：近年来，识别肿瘤相关抗原的单克隆抗体（mAb）已越来越多地整合到肿瘤的临床管理中，预计这一趋势将持续下去。基于抗体的治疗的突出靶点是ErbB家族成员，包括表皮生长因子受体（EGFR/ErbB 1）本身和ErbB 2。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，向肿瘤患者施用这些药剂与剂量限制性不良副作用相关，这是由于抑制正常组织，特别是皮肤和胃肠道中的EGFR信号传导。因此，非常需要开发基于抗体的治疗剂，其特异性识别肿瘤环境中而不是正常组织上的靶抗原。在这里，我们提出了一种新的方法来实现这一目标。其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。预期抗原片段与mAb CDR的结合封闭融合蛋白的抗原识别位点，从而降低正常组织反应性。为了恢复肿瘤部位处的高亲和力抗原-抗体相互作用，将蛋白水解切割位点工程化到阻断部分与抗体片段之间的接头中。该位点被设计为在稳态条件下对在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（包括外周血）中几乎没有活性或没有活性。在肿瘤部位切割后，由于质量作用原理，预测“未掩蔽的”抗体分配至肿瘤相关抗原。拟议的工作将测试基于EGFR拮抗性mAb C225和425的抗体衍生物的生物学特性，所述抗体衍生物识别人EGFR（EGFRdIII）的胞外结构域上的不同表位。在初步工作中，制备了双（Ab-Ag）2复合物，由此将C225和425 scFv共价连接至EGFRdIII序列，所述EGFRdIII序列被工程化以促进彼此的抗原识别位点的二聚化和相互封闭（交叉掩蔽）。在MMP 9处理后，这些复合物显示解离，随后与纯化的EGFR和表达EGFR的肿瘤细胞的结合显著增强。这些有希望的结果形成了IgG样形式的交叉掩蔽Ab的重新格式化和体外测试（特异性目的1）以及通过这些构建体在体内测定小鼠中的肿瘤归巢和生长抑制人肿瘤异种移植物（特异性目的2）的基础。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中进一步测试，即小鼠中的人类肿瘤异种移植。 公共卫生关系：近年来，单克隆抗体（mAb）已越来越多地用于某些形式的癌症和选择其他疾病的临床管理。目前用于癌症治疗的大多数mAb识别肿瘤相关抗原，这些抗原也存在于正常组织上。这种情况导致限制这些药剂在患者中的应用和功效的不良副作用。该提案描述了一种新的概念，以工程抗体衍生物，克服这些限制。为此，我们将重点关注目前在临床使用或临床试验中的两种EGFR拮抗性单克隆抗体，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临床使用或开发的广泛的mAb。

项目成果

Meditope-Fab interaction: threading the hole.

Mediope-Fab 相互作用：穿入孔。

• DOI: 10.1107/s2053230x17016272
• 发表时间: 2017
• 期刊: Acta crystallographica. Section F, Structural biology communications
• 作者: Bzymek,KrzysztofP;Ma,Yuelong;Avery,KendraN;Horne,DavidA;Williams,JohnC
• 通讯作者: Williams,JohnC

Engineering a high-affinity peptide binding site into the anti-CEA mAb M5A.

将高亲和力肽结合位点设计到抗 CEA mAb M5A 中。

• DOI: 10.1093/protein/gzx016
• 发表时间: 2017
• 期刊: Protein engineering, design & selection : PEDS
• 作者: Zer,Cindy;Avery,KendraN;Meyer,Kassondra;Goodstein,Leah;Bzymek,KrzysztofP;Singh,Gagandeep;Williams,JohnC
• 通讯作者: Williams,JohnC

Improving theranostics in pancreatic cancer.

• DOI: 10.1002/jso.24625
• 发表时间: 2017-07
• 期刊: Journal of surgical oncology
• 影响因子: 2.5
• 作者: King J;Bouvet M;Singh G;Williams J
• 通讯作者: Williams J