A versatile structure-based therapeutic platform for development of VHH-based antitoxin and antiviral agents

摘要 在以前的NIH赞助的研究中，我们成功地测试了整合结构和功能的假设。 促进了将机制信息引入基于异多聚体VHH的中和剂（VNA）设计中 开发具有更高功效和多功能性的抗毒素。在本更新建议中，我们将应用这些 研究结果来测试假设，我们的设计VNA平台，这是快速响应新的威胁， 允许开发高度实用的下一代抗毒素和抗病毒产品， 治疗中毒或病毒感染的效力，并对广泛的天然病原体有效 变体。我们的研究将集中在两个病原体，这是目前的主要威胁，可以受益于下一个- 新一代治疗药物：肉毒杆菌神经毒素（BoNT）和SARS-CoV-2。我们提出了两个具体目标， 将在五年的研究中同时进行。在目标1中，我们将开发一个小规模的 抗毒素VNA，其针对三种流行BoNT血清型（A、B和E）的所有亚型提供保护。BONT是 CDC第1层选择代理。然而，针对BoNT的少数可用的抗毒素治疗主要来自于 大型动物多克隆抗血清，如马肉毒杆菌抗毒素HBAT，其遭受多种 制造和存储的挑战。我们的目标是测试该平台生产高度实用的VNA的能力 作为下一代BoNT抗毒素产品，可能以RNA纳米颗粒的形式提供， 目前HBAT抗毒素产品的效力和天然变体特异性，并对 潜在的新威胁。在目标2中，我们将开发一种单一的VNA抗病毒剂， SARS-CoV-1和SARS-CoV-2的变体。SARS-CoV-2是持续的COVID-19大流行的病毒原因。 快速开发治疗的一个有希望的策略是开发SARS-CoV-2中和抗体， 特别是靶向刺突蛋白的抗体，用于预防性或被动免疫治疗。然而，小说 导致感染和传播增强的SARS-CoV-2变异体已经出现， 危险的变种会进化。直接关注的是部分逃脱的变体 目前基于Ab的治疗和接种疫苗或既往感染过COVID-19的患者的中和作用， 在这些变异流行率高的某些地区，疫苗效力降低。我们提出一种mRNA- 一旦施用，以极高的病毒中和作用增强VNA的表达， 力量VNA将含有多个共价连接的VHH，其结合至刺突蛋白的保守表位。 这种方法将测试该平台开发一种产品的能力，这种产品可以最大限度地降低免疫逃逸的风险 通过进化和选择SARS-CoV-2和SARS-CoV-1的临床毒株。如果成功，这 技术平台可以在为各种各样的疾病创造实用疗法方面具有广泛的应用， 新出现的和潜在的大流行性病毒感染、生物恐怖威胁因子和其他传染病。

ABSTRACT In previous NIH sponsored research we successfully tested the hypothesis that integrating structural and mechanistic information into heteromultimeric VHH-based neutralizing agent (VNA) design facilitated development of antitoxins with even greater efficacy and versatility. In this renewal proposal, we will apply these findings to test the hypothesis that our designer VNA platform, which is rapidly responsive to new threats, will permit development of highly practical, next-generation antitoxin and antiviral products that possess excellent potencies in treating intoxications or viral infections and are effective against a broad range of natural pathogen variants. Our research will focus on two pathogens that are major current threats which could benefit from next- generation therapeutics: botulinum neurotoxin (BoNT) and SARS-CoV-2. We propose two Specific Aims which will be underway simultaneously throughout the five years of research. In Aim 1, we will develop a small pool of antitoxin VNAs that protect against all subtypes of the three prevalent BoNT serotypes (A, B and E). BoNTs are CDC Tier 1 select agents. However, the few available antitoxin treatments against BoNTs primarily derive from large animal polyclonal antisera, such as the equine botulism antitoxin HBAT, which suffer from multiple manufacturing and storage challenges. Our goal is to test the platform’s ability to produce highly practical VNAs as a next-generation BoNT antitoxin product, likely delivered as RNA nanoparticles, which improves on the potencies and natural variant specificities of the current HBAT antitoxin product and is rapidly responsive to potential new BoNT threats. In Aim 2 we will develop a single VNA antiviral agent that protects against known variants of SARS-CoV-1 and SARS-CoV-2. SARS-CoV-2 is the viral cause of the ongoing COVID-19 pandemic. A promising strategy for rapid development of a therapy is development of SARS-CoV-2 neutralizing antibodies, especially antibodies targeting the spike protein, for prophylactic or passive immunotherapies. However, novel variants of SARS-CoV-2, which cause enhanced infection and transmission, have emerged, and more dangerous variants are expected to evolve. Of immediate concern are variants that partially escape neutralization by current Ab-based therapies and in vaccinated or previously-infected COVID-19 patients, leading to reduced vaccine efficacy in certain areas with a high prevalence of these variants. We propose an mRNA- based antiviral product that, once administered, elicits expression of a VNA with extremely high virus neutralizing potency. The VNA will contain multiple covalently linked VHHs binding to conserved epitopes of the spike protein. This approach will test the platform’s ability to develop a product that minimizes the risks of immune escape through evolution and selection of clinical strains of SARS-CoV-2 and SARS-CoV-1. If successful, this technology platform could have broad applications in creating practical therapeutics for a wide variety of emerging and potential pandemic viral infections, bioterror threat agents, and other infectious diseases.