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。这些蛋白含有隐藏的功能位点，这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。在2020财年，AEP发表了两篇文章，并合作编写了其他几篇手稿。一篇是评论文章，分析了由Ho实验室[English等人]开发的鲨鱼库。Antibody Therapeutics 2020；PMID: 32118195)。另一篇是一篇方法文章，描述了rabbit single domain antibody for cancer target B7-H3 （CD276）的分离[Feng et al.]。Antibody Therapeutics 2020；PMID: 32166218)。鲨鱼免疫球蛋白新抗原受体（IgNAR）的抗原结合可变结构域（VNAR）大约在5亿年前进化而来，它是动物王国中最小的抗体片段之一，大小为12-15 kDa。在这篇综述文章中，AEP评估了鲨鱼VNAR单域序列的现有知识，以及鲨鱼VNAR作为研究工具和潜在治疗方法的持续发展，特别强调了最近对120万条鲨鱼VNAR序列的下一代测序分析，以及从6条成年护士鲨（Ginglymostoma cirratum）中构建的大型噬菌体展示鲨鱼VNAR文库[English等]。Antibody Therapeutics 2020；PMID: 32118195)。大型噬菌体展示的VNAR单域文库涵盖了所有四种已知的VNAR类型（类型I-IV）和许多以前未知的类型。正在进行的临床前开发将有助于确定鲨鱼VNAR单结构域作为治疗癌症和其他人类疾病的潜在新候选药物家族的效用。兔抗体可以识别多种表位，包括小鼠和人类免疫原性较差的表位。AEP已经建立了一种分离兔VH单域抗体的方法，用于潜在的癌症治疗[Feng等]。Antibody Therapeutics 2020；PMID: 32166218)。用重组人B7-H3 （CD276）蛋白免疫家兔，构建了具有109个多样性的噬菌体展示兔VH单域文库，并从噬菌体筛选中分离出A1和B1两种结合物。两种兔VH单结构域均与B7-H3阳性的肿瘤细胞系表现出抗原依赖性结合，而与B7-H3敲除的肿瘤细胞系不表现出抗原依赖性结合。我们的研究表明，蛋白质免疫后噬菌体展示筛选可用于兔单域抗体的分离。AEP报道的两种单域抗体可能在癌症免疫治疗中有潜在的应用。我们也在支持针对SARS-CoV-2的中和纳米体的开发。

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 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. In FY20, the AEP published two articles and collaborated in several other manuscripts in preparation. One is a review article that analyzes the shark library developed by the Ho lab [English et al. Antibody Therapeutics 2020; PMID: 32118195]. The other is a method article that describes the isolation of rabbit single domain antibodies for the cancer target B7-H3 (CD276) [Feng et al. Antibody Therapeutics 2020; PMID: 32166218]. The antigen binding variable domain (VNAR) of the shark immunoglobulin new antigen receptor (IgNAR) evolved approximately 500 million years ago and it is one of the smallest antibody fragments in the animal kingdom with sizes of 12-15 kDa. In the review article, the AEP evaluates the current knowledge of the shark VNAR single domain sequences and ongoing development of shark VNARs as research tools as well as potential therapeutics, in particular highlighting the recent next-generation sequencing analysis of 1.2 million shark VNAR sequences and construction of a large phage displayed shark VNAR library from six adult nurse sharks (Ginglymostoma cirratum) [English et al. Antibody Therapeutics 2020; PMID: 32118195]. The large phage-displayed VNAR single domain library covers all the four known VNAR types (Types I-IV) and many previously unknown types. Ongoing preclinical development will help define the utility of shark VNAR single domains as a potentially new family of drug candidates for treating cancer and other human diseases. Rabbit antibodies can recognize diverse epitopes, including those that are poorly immunogenic in mice and humans. The AEP has established a method to isolate rabbit VH single domain antibodies for potential cancer therapy [Feng et al. Antibody Therapeutics 2020; PMID: 32166218]. We immunized rabbits with recombinant human B7-H3 (CD276) protein, made a phage-displayed rabbit VH single domain library with a diversity of 109, and isolated two binders (A1 and B1) from phage panning. Both rabbit VH single domains exhibited antigen-dependent binding to B7-H3-positive tumor cell lines but not B7-H3 knockout tumor cell lines. Our study shows that protein immunization followed by phage display screening can be used to isolate rabbit single domain antibodies. The two single domain antibodies reported by the AEP may have potential applications in cancer immunotherapy. We are also supporting the development of neutralizing nanobodies against SARS-CoV-2.