Antibody Engineering Program

虽然基于抗体的疗法已经成为癌症治疗的主要组成部分，但针对重要靶点如细胞表面受体和离子通道的抗体的产生仍然很困难。这些蛋白质含有通常无法通过经典的小鼠或人IgG基抗体到达的隐藏的功能位点。单结构域抗体已经显示出靶向困难抗原和隐藏表位的有希望的能力。NCI的Mitchell Ho博士已经证明，单结构域抗体能够靶向癌症信号复合物中的隐藏功能位点[Feng et al. PNAS，2013; Gao et al Nature Communications，2015; Li et al. PNAS，2017; Li et al. Hepatology，2019]。Ho实验室构建了大型鲨鱼和骆驼单结构域（“纳米抗体”）文库，并分离了多种抗原的结合物[Feng et al. Antibody Therapeutics，2019]，这表明噬菌体展示的单结构域抗体文库可以成为分离治疗性抗体的有价值来源。AEP正在与合作者使用Ho实验室创建的鲨鱼和骆驼单域噬菌体文库进行两个领域的研究。一种是通过纳米抗体与MHC相关肽复合物的结合来靶向细胞内癌症靶标。另一个是将纳米抗体分离到重要的和新兴的病毒，如拉沙病毒和SARS-CoV-2。在2021财年，AEP发表了两篇文章和其他几篇准备中的手稿。一篇文章发表在Molecular Cancer Therapeutics [Duan and Ho Mol Cancer Ther 2021]。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，靶库限于表面或可溶性抗原，其是癌症蛋白质组的相对小的百分比。人类蛋白质组的大多数蛋白质是细胞内的。来自细胞内靶点的短肽可以由细胞表面的主要组织相容性复合物I类（MHC-I）分子呈递，使其成为癌症免疫治疗的潜在靶点。可以开发抗体来靶向这些肽/MHC复合物，类似于T细胞受体（TCR）对此类复合物的识别。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有助于了解它们的作用机制和免疫治疗靶表位的选择。我们的审查总结和讨论的细胞内抗原的选择，肽/MHC复合物的生产，分离的TCRm抗体的治疗应用，TCRm抗体的局限性，以及可能的方式来推进TCRm抗体为基础的方法在临床上。在发表于Antibody Therapeutics的另一篇文章中[Sun and Ho，AntiB Ther 2020]，我们总结了针对SARS-CoV-2的中和抗体的发展，重点讨论了各种抗体发现策略（动物免疫、噬菌体展示和B细胞克隆），描述了结合表位并比较了中和活性。针对SARS-CoV-2和SARS-CoV刺突蛋白的广泛中和抗体可能有助于治疗COVID-19和未来的感染。基于S309的VIR-7831/7832是唯一处于后期临床开发阶段的抗体，尽管其不直接阻断病毒受体结合，但可中和SARS-CoV-2和SARS-CoV。到目前为止，唯一的交叉中和抗体也是受体结合阻断剂是纳米抗体VHH-72。开发纳米抗体作为治疗COVID-19和其他呼吸系统疾病的吸入药物的可行性是一个值得探索和测试的有吸引力的想法。鸡尾酒策略，如REGN-COV 2，或工程化的多价和多特异性分子，组合两种或更多种抗体可能会提高疗效，并防止由于病毒逃逸突变体引起的耐药性。除了受体结合结构域之外，其他病毒抗原如刺突蛋白的S2亚基和病毒附着位点如宿主细胞上的硫酸乙酰肝素蛋白聚糖也值得研究。

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 FY21, the AEP published two articles and several other manuscripts in preparation. One article was published in Molecular Cancer Therapeutics [Duan and Ho Mol Cancer Ther 2021]. 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 major histocompatibility complex 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 understand their mechanisms of action and selection of target epitopes for immunotherapy. Our review summarized and discuss 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 in the clinic. In the other article published in Antibody Therapeutics [Sun and Ho, Antib Ther 2020], we summarized the development of neutralizing antibodies against SARS-CoV-2, with a focus on discussing various antibody discovery strategies (animal immunization, phage display and B cell cloning), describing binding epitopes and comparing neutralizing activities. Broad-neutralizing antibodies targeting the spike proteins of SARS-CoV-2 and SARS-CoV might be helpful for treating COVID-19 and future infections. VIR-7831/7832 based on S309 is the only antibody in late clinical development, which can neutralize both SARS-CoV-2 and SARS-CoV although it does not directly block virus receptor binding. Thus far, the only cross-neutralizing antibody that is also a receptor binding blocker is nanobody VHH-72. The feasibility of developing nanobodies as inhaled drugs for treating COVID-19 and other respiratory diseases is an attractive idea that is worth exploring and testing. A cocktail strategy such as REGN-COV2, or engineered multivalent and multispecific molecules, combining two or more antibodies might improve the efficacy and protect against resistance due to virus escape mutants. Besides the receptor-binding domain, other viral antigens such as the S2 subunit of the spike protein and the viral attachment sites such as heparan sulfate proteoglycans that are on the host cells are worth investigating.