High throughput camelid antibody screening as a drug discovery platform

抽象的。单抗用于治疗多种疾病，从癌症到 传染病。然而，开发高亲和力和特异性的抗体仍需时日。 消耗、劳动密集型和不可预测性，尤其是对低免疫原性或毒性靶标。一种新方法 针对各种疾病，特别是针对新出现的疾病的抗体的快速发展是 非常需要。 Abzyme Treateutics LLC正在积极开发无动物抗体发现平台，以加快 诊断和治疗抗体的产生。第一阶段的目标已经圆满完成。 具体地说，我们创建了一个所谓的酵母表面展示骆驼自我多样化的VHH抗体库 或SDALib作为在体外提供多种抗体的单抗生成系统，而不需要 使用体内免疫。该系统的主要特点是能够通过以下方式自生成库 通过抗体编码基因的高突变诱导使抗体多样化。作为原则证明， 我们已经成功地利用该平台分离出一组抗N1神经氨酸酶的单域抗体 (NA)源自H5N1流感病毒。获得的VHH抗体已被证明在 细菌系统为治疗提供了加速放大抗体生产的机会 申请。纯化了抗NA VHH抗体，并对其进行了体外鉴定。作为证明 几种抗NA VHH抗体中的第一种(Flu27-8)，它与H5N1和H5N1的N1 Nas都有反应 H1N1流感病毒，已经人源化，保留了亲本分子的全部抗NA活性。双人- 在细菌系统中以线性融合的形式产生的价抗NA抗体显示出显著的 在体外检测中抗体活性的增加。 第二阶段的后续工作由三个具体目标组成，这些目标将同时进行：(一)推进 一期分离的N1抗NA抗体在体外和体外均可用于临床 活体对抗H5N1和H1N1流感病毒的活性；(Ii)扩大平台的应用范围 开发针对血凝素和其他流感病毒亚型的NAS的抗体，例如H3N2和 H7N9具有不同寻常的人畜共患病潜力，目标是与几种不同的 神经氨酸酶(H5N1、H1N1、H3N2和H7N9)；。(Iii)利用抗体发现平台开发 抗一组肿瘤靶点的VHH抗体及其新型免疫球蛋白样双特异性抗体的制备 人源化抗体。第二阶段的工作是进一步扩大快速技术的应用。 针对治疗靶点的新型抗体的开发和人源化抗流感抗体的发展 在I期分离进入临床研究。

ABSTRACT. Monoclonal antibodies are used for treatment of a wide range of diseases from cancer to infectious diseases. However, development of antibodies with high affinity and specificity is still time- consuming, labor-intensive and unpredictable, especially to low immunogenic or toxic targets. A new approach for rapid development of antibodies against a variety of diseases, especially against emerging diseases is much needed. Abzyme Therapeutics LLC is actively developing animal-free antibody discovery platforms to accelerate generation of diagnostic and therapeutic antibodies. Phase I objectives have been successfully completed. Specifically, we have created a so-called yeast surface display camelid Self-Diversifying VHH Antibody Library or SDALib as a monoclonal antibody generating system providing a diverse array of antibodies in vitro, without using in vivo immunization. The main feature of the system is the ability to self-generate a library with diversified antibodies by means of hypermutation induction in antibody encoding genes. As proof-of-principle, we have successfully used the platform to isolate a set of single-domain antibodies against N1 neuraminidase (NA) derived from influenza H5N1 virus. Obtained VHH antibodies have been shown to express efficiently in a bacterial system providing an opportunity for accelerated scale-up of antibody production for therapeutic application. Anti-NA VHH antibodies have been purified and characterized in in vitro assays. As proof of principle one (Flu27-8) out of several anti-NA VHH antibodies, that is reactive with N1 NAs of both H5N1 and H1N1 influenza viruses, has been humanized preserving the full anti-NA activity of the parental molecule. Bi- valent anti-NA antibodies that have been produced in a bacterial system as linear fusions showed a significant increase in antibody activity in in vitro assays. Subsequent efforts of Phase II consist of three specific aims that will be performed simultaneously: (i) advance the N1 anti-NA antibodies isolated in Phase I toward clinical application by demonstrating both in vitro and in vivo activity against H5N1 and H1N1 influenza viruses; (ii) expand the application of the platform for developing antibodies targeting both hemagglutinin and NAs of other influenza virus subtypes, e.g. H3N2 and H7N9 which have unusual zoonotic potential and aiming for antibodies cross-reacting with several different neuraminidases (H5N1, H1N1, H3N2 and H7N9); (iii) utilize the antibody discovery platform for developing VHH antibodies against a set of cancer targets followed by production of novel IgG-like VHH-based bispecific humanized antibodies. Phase II efforts are to further extend the application of the technology for rapid development of novel antibodies to therapeutic targets and to advance humanized anti-influenza antibodies isolated in phase I into clinical studies.