Rationally guided discovery platform for monoclonal antibodies against carbohydrate antigens using virus-like particle conjugate immunization and high throughput selection

使用病毒样颗粒缀合物免疫和高通量选择的合理引导的针对碳水化合物抗原的单克隆抗体的发现平台

• 批准号: 10574738
• 负责人: Daniel Ray Woldring
• 金额: $ 22.76万
• 依托单位: MICHIGAN STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-10 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10574738
• 关键词: Address; Affinity; Antibodies; Antibody Binding Sites; Antigens; Binding; Biological Markers; Breast; Cancer Patient; Carbohydrates; Carrier Proteins; Cell Separation; Cell surface; Cells; Clinical; Complement-Dependent Cytotoxicity; Cytometry; Diagnostic; Directed Molecular Evolution; Disease; Disease Outcome; Engineering; Ensure; Enzymes; Epitope Mapping; Epitopes; Genomics; Glycopeptides; Glycoproteins; Human; Imaging Device; Immune response; Immune system; Immunization; Immunoglobulin Somatic Hypermutation; Individual; Libraries; Light; Malignant Neoplasms; Malignant neoplasm of ovary; Malignant neoplasm of pancreas; Methodology; Methods; Molecular; Monoclonal Antibodies; Mus; Ovarian; Pancreas; Patient-Focused Outcomes; Pattern; Performance; Phylogenetic Analysis; Phylogeny; Polysaccharides; Protein Engineering; Sensitivity and Specificity; Specificity; Technology; Therapeutic; Therapeutic Monoclonal Antibodies; Time; Transgenic Mice; Tumor Cell Line; Tumor-Associated Carbohydrate Antigens; Vaccines; Virus-like particle; Yeasts; biomarker discovery; cancer biomarkers; cancer diagnosis; clinical diagnosis; design; diagnostic tool; diagnostic value; empowerment; glycosylation; guided inquiry; improved; in vivo; interest; lead candidate; lead optimization; malignant breast neoplasm; molecular diagnostics; molecular imaging; neoplastic cell; novel; overexpression; reconstruction; specific biomarkers; tumor; tumorigenesis; tumorigenic; vaccine development

This project will establish a protein engineering platform for evolving monoclonal antibody binding affinity and specificity to solve the notorious challenge of developing clinical mAbs against tumor associated carbohydrate antigens (TACAs). Our central hypothesis is that the merger of Qβ carrier protein-elicited mAb discovery and rationally-guided directed evolution will outpace existing methodologies for discovering powerful antibodies against challenging TACA glycosylated biomarkers. TACAs are unique biomarkers to multiple tumor types, yet they have been underutilized for molecular imaging and diagnostics because of challenges in developing selective, potent binders. Distinct glycosylation patterns of tumor cell surfaces are hallmark features that arise during oncogenesis through changes in expression levels of glyco-processing enzymes. Problematically, these aberrant tumorigenic features are usually undetected by the immune system and rarely identified as non-self. Even when recognized as an antigen, weak binding against monovalent glycans leads to an insufficient immune response. To address this need, we will apply our directed evolution methodology to develop lead candidate mAbs against TACAs selective to cancer with in vivo binding of KD<10nM and specificity >100-fold binding above control cells. This will be accomplished by first generating a diverse panel of TACA-specific antibodies via immunization of transgenic mice with multivalent Qβ vaccines. Dominant antibodies will be isolated and characterized for paratope diversity and the ability to selectively bind the glyco-targets. Next, we use rationally- guided directed evolution to achieve mAb binding affinity and specificity. Multiple TACA-specific mAbs obtained through immunization will undergo high-throughput yeast display directed evolution with site-wise diversification based on structural, stabilizing, and phylogenetic factors to overcome the routinely low affinity of anti-carbohydrate binders. Specificity and affinity will be evaluated against multiple human tumor cell lines. This project will: 1) establish a platform that drastically reduces initial discovery time for translatable molecular imaging and diagnostic tools against carbohydrate antigens; 2) significantly advance understanding of tumorigenic cell glycosylation patterns; and 3) mark a major step towards improving sensitivity and specificity of biomarker-based diagnosis of cancers including ovarian, breast, and pancreatic cancers.

该项目将建立一个蛋白质工程平台，用于不断发展的单克隆抗体结合亲和力和 解决针对肿瘤相关的碳氢化的临床mAb的臭名昭著的挑战的特异性 抗原（炸玉米饼）。我们的中心假设是Qβ载体蛋白质吸收的mAb发现的合并和 理性引导的定向进化将超过现有的发现强大抗体的方法 反对挑战TACA糖基化生物标志物。 塔卡斯（Tacas）是多种肿瘤类型的独特生物标志物 和诊断是由于开发选择性的潜在粘合剂的挑战。不同的糖基化模式 肿瘤细胞表面是通过表达变化在肿瘤发生过程中出现的标志性特征 聚糖加工酶的水平。有问题的是，这些异常的肿瘤特征通常是 未经免疫系统未发现，很少被识别为非自我。即使被公认为抗原， 与单价甘氨酸的弱结合导致免疫激发不足。 为了满足这一需求，我们将采用定向的进化方法来开发主要候选人mabs 反对塔卡斯对癌症的选择性，具有Kd <10nm的体内结合，特异性> 100倍结合 控制细胞。这将通过首先通过生成塔卡特异性抗体的潜水面板来实现 用多价Qβ疫苗对转基因小鼠免疫。主要的抗体将被隔离，并且 特征在于寄生虫多样性和选择性结合糖靶的能力。接下来，我们在理性上使用 指导性进化以实现mAb结合亲和力和特异性。获得的多个TACA特异性mAb 通过免疫将经历高通量的酵母展示的定向进化 基于结构，稳定和系统发育因素的多样化，以克服常规低亲和力 抗碳水化合物粘合剂。特异性和亲和力将针对多个人类肿瘤细胞系进行评估。 该项目将：1）建立一个大幅度减少可翻译分子的初始发现时间的平台 针对碳水化抗原的成像和诊断工具； 2）显着提高了对 肿瘤性细胞糖基化模式； 3）标志着提高灵敏度和特异性的主要步骤 基于卵巢，乳腺癌和胰腺癌等癌症的基于生物标志物的诊断。