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，但针对细胞表面受体和离子通道等重要靶点的抗体的产生仍然困难。这些蛋白质含有埋藏的功能位点，而经典的基于小鼠或人类免疫球蛋白的抗体往往无法达到这些功能位点。单域抗体已显示出针对困难抗原和隐藏表位的前景看好的能力。NCI的Mitchell Ho博士已经证明了单域抗体能够靶向癌症信号复合体中埋藏的功能部位[冯等人]。PNAS，2013；高等人，2015；Li等人。PNAS，2017；Li等人。肝病，2019年]。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剂[冯等人]。2013年；Gao et al 2015年；Li et al.2017；Hong et al.2022；Li等人。2023年；巴辛顿等人。2023]，表明噬菌体展示的单域抗体库可以作为分离治疗性抗体的有价值的来源。在23财年，AEP发表了一篇研究文章[Buffington等人]。参考文献[FASEB Journal，2023]，One方法论文[Zhang et al.当前协议，2023年]，和一篇合作论文[Fayn等人。《2023年核医学杂志》]。SARS-CoV-2是新冠肺炎大流行的病原体。针对S蛋白，特别是SARS-CoV-2的S1亚单位或受体结合结构域的抗体疗法，因其在治疗确诊为新冠肺炎的患者中的临床疗效而受到关注。传统抗体疗法的替代方法是使用SHARK新抗原可变区(VNAR)抗体。VNAR S很小(15 KDa)，可以深入到目标抗原的口袋或凹槽中。在2023财年，AEP通过噬菌体淘洗从由Mitchell Ho博士的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到53个与S2亚基结合的VNAR S。在这些结合剂中，S2A9对原始假型SARS-CoV-2病毒显示出最好的中和活性。包括S2A9在内的几种结合剂显示出与其他贝塔冠状病毒S2亚基的交叉反应。此外，在伪病毒和活病毒中和试验中，S2A9对从α到Omicron的所有变种(VOCs)(包括BA1、BA2、BA4和BA5)都显示出中和活性。我们的发现表明，S2A9可能是一个有希望的先导分子，用于开发针对SARS-CoV-2和新出现的变种的广谱中和抗体。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，可以用于快速分离针对新出现的病毒病原体的单域抗体。鲨鱼VNAR单一结构域在临床上发挥作用的一个关键障碍是它对人类的潜在免疫原性。鲨鱼单域抗体的VNAR在进化上远离哺乳动物免疫球蛋白的可变区(VH)，但我们推测它可能仍然具有参与抗原识别的互补决定区(CDR)，至少在大部分抗原识别中是这样，因此通过将这些CDR嫁接到人VH同源物的框架上使其人源化成为可能。为了验证这一假设，我们基于对全球蛋白质数据库三维结构数据中现有的VNAR-抗原结构复合体的分析，展示了VNAR CDR，并以鲨鱼VNAR最常见的类型(II型)S为例，描述了通过CDR嫁接使VNAR人源化的详细方案。我们在现有的实验方案(Buffington J，Duan Z，Kuan HJ，Hong J，Li D，冯明，谢辉，Ho M.)中发表了这些方法。针对SARS-CoV-2刺突S2亚基的护士鲨VNAR单域抗体的鉴定。FASE B J.2023年6月；37(6)：e22973.DOI：10.1096/fj.202202099RR。)因此，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设计鲨鱼VNAR的方案，用于治疗癌症和其他人类疾病。在2023财年，AEP还发表了一篇研究论文，与NCI CCR分子成像分部的Freddy EScott博士合作发表了一篇研究论文[Fayne等人。J Nucl Med 2023]。AEP正在进行的合作旨在开发治疗性抗体，并与Ira Pastan博士、James Gulley博士(NCI)、Glenn Merlino博士(NCI)、Peter Kuong博士(NIAID)、Eytan Ruppin博士(NCI)和Christian Hinrichs博士(罗格斯大学)共同开发治疗性抗体和设计免疫蛋白质和细胞。

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).