Antibody Engineering Program

虽然基于抗体的治疗方法已经成为癌症治疗的主要组成部分，但产生针对细胞表面受体和离子通道等重要靶点的抗体仍然很困难。这些蛋白质含有隐藏的功能位点，这些位点通常是传统的小鼠或人类基于igg的抗体无法到达的。单域抗体已显示出针对困难抗原和隐藏表位的有希望的能力。美国国家癌症研究所的Mitchell Ho博士已经证明，单域抗体能够靶向癌症信号复合物中隐藏的功能位点[Feng等人]。PNAS, 2013;Gao et al . Nature Communications, 2015；Li等人。PNAS, 2017;Li等人。肝脏病学,2019]。Ho实验室已经构建了大型鲨鱼和骆驼单域（“纳米体”）文库，并分离了多种抗原的结合物[Feng等人]。Antibody Therapeutics, 2019]，表明噬菌体显示的单域抗体文库可以成为分离治疗性抗体的有价值来源。AEP正在进行两个领域的研究，合作者使用何氏实验室创建的鲨鱼和骆驼单域噬菌体文库。一种是通过纳米体与MHC相关肽复合物的结合来靶向细胞内癌症靶点。另一种方法是将纳米体与拉沙病毒和SARS-CoV-2等重要和新出现的病毒分离开来。在FY22， AEP发表了三篇综述文章[Duan和Ho。Molecular Cancer Therapeutics 2021；段和何。Antibody Therapeutics 2022；耶拉罕和何。Antibody Therapeutics 2022]和一篇方法学论文[Duan et al.]现行议定书[2022]。基于抗体的免疫疗法在各种癌症类型中显示出临床有效性。然而，靶标库仅限于表面或可溶性抗原，这是癌症蛋白质组中相对较小的百分比。人类蛋白质组的大多数蛋白质都是细胞内的。来自细胞内靶点的短肽可以通过MHC I类（MHC-I）分子在细胞表面呈现，使其成为癌症免疫治疗的潜在靶点。可以开发针对这些肽/MHC复合物的抗体，类似于t细胞受体（TCR）对这些复合物的识别。这些抗体被称为t细胞受体模拟物（TCRm）或tcr样抗体。正在进行的临床前和临床研究将帮助我们了解它们的作用机制和免疫治疗目标表位的选择。我们发表在《Molecular Cancer Therapeutics》（AACR）上的综述文章[Duan and Ho 2021]总结并讨论了细胞内抗原的选择、肽/MHC复合物的产生、用于治疗的TCRm抗体的分离、TCRm抗体的局限性，以及将基于TCRm抗体的方法推进到临床的可能途径。单结构域抗体，包括鲨鱼免疫球蛋白新抗原受体的抗原结合可变结构域和重链的骆驼可变区域，是最小的抗原识别结构域（15 kDa），与常规抗体相比具有独特的特征。它们能够结合经典抗体难以获得的表位，也可用于治疗或诊断，或作为多结构域结构体、抗体-药物偶联物、免疫毒素或嵌合抗原受体治疗的模块构建块。我们的方法论文包含了使用细菌和哺乳动物细胞表达系统纯化和验证与SARS-CoV-2刺突蛋白的S2亚基结合的两种单域抗体（一种鲨鱼和一种骆驼）的详细程序[Duan等人]。现行议定书。2022]。为生产高产量、高质量、高纯度的单域抗体提供了全面参考。在2022财年，AEP还作为共同作者发表了两篇研究论文[Hong et al.]。PNAS 2022;潘等人。分子癌症治疗[j]。

While antibody-based therapeutics have emerged as a major component in cancer treatment, the generation of antibodies to important targets such as cell surface receptors and ion channels remains difficult. These proteins contain buried functional sites that are often unreachable by classical mouse or human IgG-based antibodies. Single domain antibodies have shown a promising ability to target difficult antigens and hidden epitopes. Dr. Mitchell Ho at the NCI has demonstrated that single domain antibodies are capable of targeting buried functional sites in cancer signaling complexes [Feng et al. PNAS, 2013; Gao et al Nature Communications, 2015; Li et al. PNAS, 2017; Li et al. Hepatology, 2019]. The Ho lab has constructed large shark and camel single-domain ('nanobody') libraries and isolated binders to a wide range of antigens [Feng et al. Antibody Therapeutics, 2019], indicating that the phage-displayed single domain antibody libraries can be a valuable source to isolate therapeutic antibodies. Two areas of research are being pursued at the AEP with collaborators using the shark and camel single domain phage libraries created by the Ho lab. One is focused on targeting intracellular cancer targets via binding of nanobodies to the MHC associated peptide complexes. The other is to isolate nanobodies to important and emerging viruses such as Lassa virus and SARS-CoV-2. In FY22, the AEP published three review articles [Duan and Ho. Molecular Cancer Therapeutics 2021; Duan and Ho. Antibody Therapeutics 2022; Yerabham and Ho. Antibody Therapeutics 2022] and one methodology paper [Duan et al. Current Protocols 2022]. Antibody-based immunotherapies show clinical effectiveness in various cancer types. However, the target repertoire is limited to surface or soluble antigens, which are a relatively small percentage of the cancer proteome. Most proteins of the human proteome are intracellular. Short peptides from intracellular targets can be presented by MHC class I (MHC-I) molecules on cell surface, making them potential targets for cancer immunotherapy. Antibodies can be developed to target these peptide/MHC complexes, similar to the recognition of such complexes by the T-cell receptor (TCR). These antibodies are referred to as T-cell receptor mimic (TCRm) or TCR-like antibodies. Ongoing preclinical and clinical studies will help us understand their mechanisms of action and selection of target epitopes for immunotherapy. Our review article published in Molecular Cancer Therapeutics (AACR) [Duan and Ho 2021] summarized and discussed the selection of intracellular antigens, production of the peptide/MHC complexes, isolation of TCRm antibodies for therapeutic applications, limitations of TCRm antibodies, and possible ways to advance TCRm antibody-based approaches into the clinic. Single-domain antibodies, including the antigen-binding variable domains of the shark immunoglobulin new antigen receptor and the camelid variable region of the heavy chain, are the smallest antigen recognition domains (15 kDa) and have unique characteristics compared to conventional antibodies. They are capable of binding epitopes that are hard to access for classical antibodies and can also be used for therapeutics or diagnostics or as modular building blocks for multi-domain constructs, antibody-drug conjugates, immunotoxins, or chimeric antigen receptor therapy. Our method paper contains detailed procedures for the purification and validation of two single-domain antibodies (one shark and one camel), which bind to the S2 subunit of the SARS-CoV-2 spike protein, using both bacterial and mammalian cell expression systems [Duan et al. Current Protocols. 2022]. It provides a comprehensive reference for the production of single-domain antibodies with high yield, good quality, and purity. In FY2022, the AEP also published two research papers as co-authors [Hong et al. PNAS 2022; Pan et al. Molecular Cancer Therapeutics 2022].