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域（“纳米抗体”）文库和分离的结合剂与广泛的抗原结合[Feng等人，2013; Gao等人，2015; Li等人，2017; Hong等人，2022; Li等人，2023; Buffington等人2023]，表明噬菌体展示的单结构域抗体文库可以是分离治疗性抗体的有价值的来源。在2023财年，AEP发表了一篇研究文章[Buffington et al. FASEB Journal，2023]、一篇方法论文[Zhang et al. Current Protocols，2023]和一篇合作论文[Fayn et al. Journal Nuclear Medicine 2023]。SARS-CoV-2是COVID-19大流行的病原体。靶向刺突蛋白（特别是SARS-CoV-2的S1亚基或受体结合结构域（RBD））的基于抗体的治疗方法因其在治疗诊断为COVID-19的患者中的临床疗效而受到关注。常规抗体疗法的替代方案是使用鲨鱼新抗原可变受体结构域（VNAR）抗体。VNAR很小（15 kDa），可以深入靶抗原的口袋或凹槽。在2023财年，AEP通过噬菌体淘选从Mitchell Ho博士实验室构建和提供的幼稚护士鲨VNAR噬菌体展示文库中分离出53个与S2亚基结合的VNAR。在这些结合剂中，S2 A9显示出对原始假型SARS-CoV-2病毒的最佳中和活性。包括S2 A9在内的几种结合剂显示出与其他β冠状病毒S2亚基的交叉反应性。此外，在假病毒和活病毒中和试验中，S2 A9显示出对从α到Omicron的所有相关变体（VOC）（包括BA 1、BA 2、BA 4和BA 5）的中和活性。我们的研究结果表明，S2 A9可能是一个有前途的领导分子，用于开发针对SARS-CoV-2和新出现的变体的广泛中和抗体。Ho实验室创建的护士鲨VNAR噬菌体文库提供了一个新的平台，可用于快速分离针对新兴病毒病原体的单域抗体。使鲨鱼VNAR单结构域在临床上发挥作用的一个关键障碍是其在人类中的潜在免疫原性。鲨鱼单结构域抗体的VNAR在进化上与哺乳动物免疫球蛋白的可变区（VH）相距甚远，但我们假设它可能仍然具有参与抗原识别的互补决定区（CDR），至少对于抗原识别的大部分，因此可以通过将这些CDR移植到人VH同源物的框架中来进行人源化。为了验证这一假设，我们显示了VNAR CDR的基础上分析目前可用的VNAR -抗原结构复合物的全球蛋白质数据库存档的三维结构数据，并描述了详细的协议，以人源化VNAR的CDR移植，使用B6（抗假单胞菌外毒素VNAR），最常见的类型（II型）的鲨鱼VNAR s，作为一个例子。我们在Current Protocols（Buffington J，Duan Z，Kwon HJ，Hong J，Li D，Feng M，Xie H，Ho M）中公开了该方法。鉴定护士鲨VNAR单域抗体靶向SARS-CoV-2的刺突S2亚基。FASEB J. 2023 Jun;37（6）：e22973. doi：10.1096/fj.202202099RR.）这样其他科学家也可以测试我们的方法正在进行的努力将进一步优化工程鲨鱼VNARs治疗癌症和其他人类疾病的方案。于2023财政年度，AEP亦与Freddy Escorcia博士合作，于NCI CCR的分子成像分支发表一篇研究论文[Fayne et al. J Nucl Med 2023]。正在进行的AEP合作旨在与伊拉·帕斯坦博士、詹姆斯·古利博士（NCI）、格伦·梅尔利诺博士（NCI）、彼得·邝博士（NIAID）、埃坦·鲁平博士（NCI）和克里斯蒂安·欣里克斯博士（罗格斯大学）开发治疗性抗体和工程免疫蛋白和细胞。

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. 2013; Gao et al 2015; Li et al. 2017; Hong et al. 2022; Li et al. 2023; Buffington et al. 2023], indicating that the phage-displayed single domain antibody libraries can be a valuable source to isolate therapeutic antibodies. In FY23, the AEP published one research article [Buffington et al. FASEB Journal, 2023], one method paper [Zhang et al. Current Protocols, 2023], and one collaborative paper [Fayn et al. Journal Nuclear Medicine 2023]. SARS-CoV-2 is the etiological agent of the COVID-19 pandemic. Antibody-based therapeutics targeting the spike protein, specifically the S1 subunit or the receptor binding domain (RBD) of SARS-CoV-2, have gained attention due to their clinical efficacy in treating patients diagnosed with COVID-19. An alternative to conventional antibody therapeutics is the use of shark new antigen variable receptor domain (VNAR) antibodies. VNAR s are small (15 kDa) and can reach deep into the pockets or grooves of the target antigen. In FY2023, the AEP isolated 53 VNAR s that bind to the S2 subunit by phage panning from a naive nurse shark VNAR phage display library constructed and provided by Dr. Mitchell Ho's laboratory. Among those binders, S2A9 showed the best neutralization activity against the original pseudotyped SARS-CoV-2 virus. Several binders, including S2A9, showed cross-reactivity against S2 subunits from other beta coronaviruses. Furthermore, S2A9 showed neutralization activity against all variants of concern (VOCs) from alpha to omicron (including BA1, BA2, BA4, and BA5) in both pseudovirus and live virus neutralization assays. Our findings suggest that S2A9 could be a promising lead molecule for the development of broadly neutralizing antibodies against SARS-CoV-2 and emerging variants. The nurse shark VNAR phage library created by the Ho lab offers a novel platform that can be used to rapidly isolate single-domain antibodies against emerging viral pathogens. One critical obstacle to getting a shark VNAR single domain to work in the clinic is its potential immunogenicity in humans. The VNAR of shark single domain antibodies is evolutionarily distant from the variable regions (VH) of mammalian immunoglobulins, yet we hypothesize that it may still have complementarity-determining regions (CDRs) that are involved in antigen recognition, at least for the most part of the antigen recognition, therefore making it possible to humanize by grafting these CDRs to the framework of human VH homologs. For testing this hypothesis, we show the VNAR CDR based on an analysis of currently available VNAR -antigen structure complexes in the global Protein Data Bank archive of 3D structure data, and describe the detailed protocol to humanize VNAR by CDR grafting, using B6 (an anti-Pseudomonas exotoxin VNAR), the most common type (Type II) of shark VNAR s, as an example. We published the methods in Current Protocols (Buffington J, Duan Z, Kwon HJ, Hong J, Li D, Feng M, Xie H, Ho M. Identification of nurse shark VNAR single-domain antibodies targeting the spike S2 subunit of SARS-CoV-2. FASEB J. 2023 Jun;37(6):e22973. doi: 10.1096/fj.202202099RR.) so other scientists can also test our methodology. Ongoing efforts will further optimize the protocol for engineering shark VNARs for treating cancer and other human diseases. In FY2023, the AEP also published one research paper as co-authors in collaboration with Dr. Freddy Escorcia in Molecular Imaging Branch at the NCI CCR [Fayne et al. J Nucl Med 2023]. Ongoing AEP collaborations aim to develop therapeutic antibodies and engineer immune protein and cells with Dr. Ira Pastan, Dr. James Gulley (NCI), Dr. Glenn Merlino (NCI), Dr. Peter Kwong (NIAID), Dr. Eytan Ruppin (NCI) and Dr. Christian Hinrichs (Rutgers University).