Generation of Neutralizing Monoclonal Antibodies Against SARS-CoV-2 for Prevention and Therapy in Patients with COVID-19

生成针对 SARS-CoV-2 的中和单克隆抗体，用于预防和治疗 COVID-19 患者

• 批准号: 10927958
• 负责人: Patrizia Farci
• 金额: $ 4.8万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10927958
• 关键词: 2019-nCoV; ACE2; Acute; Adenovirus Vector; Affinity; African American population; Animal Model; Animals; Antibodies; Antibody titer measurement; Autoantibodies; Binding; Biological Assay; Blood donor; Blood specimen; Body Weight decreased; COVID-19; COVID-19 complications; COVID-19 pandemic; COVID-19 patient; COVID-19 vaccine; Cardiac Surgery procedures; Caucasians; Chiroptera; Clinical; Collaborations; Complication; Cryoelectron Microscopy; Data; Developing Countries; Development; Disease; Dissociation; Distal; Dose; Economics; Effectiveness; Emergency department visit; Ensure; Epitopes; Equilibrium; Evolution; Exhibits; Female; Future; Generations; Genetic Variation; Goals; Hamsters; Heparin; Hispanic Populations; Hospitalization; Human; Immune; Immune Evasion; Immunoassay; Individual; Libraries; Life; Lung; Mesocricetus auratus; Modeling; Monoclonal Antibodies; Mutation; Nasal turbinate bone structure; Normal Range; Nose; Orthopedic Surgery; PF4 Gene; Pathogenicity; Patients; Phage Display; Platelet Activation; Platelet Count measurement; Preclinical Testing; Predisposition; Prevention; Proteins; RNA; RNA Viruses; Resolution; Respiratory Disease; SARS coronavirus; SARS-CoV-2 B.1.351; SARS-CoV-2 B.1.617.2; SARS-CoV-2 BA.1; SARS-CoV-2 antibody; SARS-CoV-2 infection; SARS-CoV-2 spike protein; SARS-CoV-2 variant; Sarbecovirus; Serum; Severity of illness; Side; Syndrome; Technology; Therapeutic; Therapeutic Intervention; Therapeutic antibodies; Thrombocytopenia; Thrombosis; Vaccination; Vaccines; Variant; Viral; Virus; combinatorial; experience; fighting; global health; heparin-induced thrombocytopenia; in vivo; in vivo evaluation; insight; male; nanomolar; neutralizing antibody; neutralizing monoclonal antibodies; novel coronavirus; pandemic disease; peripheral blood; prophylactic; receptor; receptor binding; reconstruction; severe COVID-19; social; therapeutic vaccine; variants of concern

1. Potent monoclonal antibodies neutralize Omicron sublineages and other SARS-CoV-2 variants A. Generation and characterization of anti-SARS-CoV-2 mAbs using phage-display libraries from COVID-19 convalescent patients. Peripheral blood samples were collected from 12 convalescent COVID-19 patients with high neutralizing serum antibody titers against SARS-CoV-2, and total RNA was extracted and used for the construction of four phage-display Fab libraries, derived from either a single donor or multiple donors combined. Three sequential cycles of panning were carried out to enrich for specific clones using a stabilized trimeric spike protein (S-2P) derived from the original SARS-CoV-2 strain, Wuhan-Hu-1. We obtained 18 Fabs that exhibited high-affinity binding to the viral spike with equilibrium dissociation constants (KD) in the picomolar or low nanomolar range. B. Potent neutralization of diverse SARS-CoV-2 variants of concern. The neutralizing activity of our 18 mAbs was evaluated using a pseudotype virus neutralization assay. Eleven showed potent neutralizing activity against the original SARS-CoV-2 strain (WA-1) and the Alpha variant, while 7 also neutralized the Delta variant in the picomolar range. In contrast, only 4 of the 11 mAbs were effective against the Beta variant. Of note, one of them, NA8, neutralized Beta, Omicron BA.1 and BA.2 sublineages with a picomolar potency, as well as BA.2.12.1 and BA.4 sublineages at nanomolar concentration. Since clinically approved mAbs are generally used in combination to ensure coverage against multiple variants, we compared side-by-side the neutralization potency of the combination of NA8 and NE12, used at equimolar concentrations (1:1), with that of 6 clinically approved antibodies used in paired combinations. Against the most recent variants, BA.2.12.1 and BA.4, the NA8/NE12 combination retained neutralizing activity at low nanomolar concentrations as did the REGN-10933/REGN-10987 combination, while COV2-2130/COV2-2196 was the most potent combination and LY-CoV555/LY-CoV016 was ineffective. These results suggest that NA8 and NE12, when used in combination, are one of the most broad and potent antibody pairs with picomolar or low nanomolar neutralizing activity against the major SARS-CoV-2 variants of concern. Based on these results, NA8 and NE12 were selected for further characterization and preclinical testing. C. Structural analysis of antibodies NE12 and NA8. To gain structural insight into the interactions of antibodies NE12 and NA8 with the SARS-CoV-2 spike glycoprotein, in collaboration with Peter Kwong and Yaroslav Tsybovsky, we obtained cryo-EM reconstructions of Fab NE12 and Fab NA8 bound to a stabilized spike trimer (S-6P) at nominal resolutions of 3.1 and 2.9 , respectively. The epitope of NE12 overlaps the ACE2 receptor footprint, indicating that NE12 directly blocks the spike-receptor interaction. Similar to Fab NE12, Fab NA8 shows extensive contacts with the receptor-binding ridge, but this antibody binds to a unique epitope along the outer side of the RBD distal from the tip. There is a moderate overlap between the NA8 epitope and the ACE2-binding interface of the RBD. These structural data further confirm that NE12 and NA8 provide a pair of potent complementary mAbs for potential clinical use. D. Antibodies NE12 and NA8 protect hamsters from SARS-CoV-2 infection. In collaboration with Ulla Buchholz, we tested the in vivo prophylactic efficacy of mAbs NE12 and NA8 in the golden Syrian hamster model, which closely mimics the severity of the disease in humans. Treatment with NA8 significantly protected hamsters from weight loss induced by two viral variants, WA-1 and Beta, while NE12 was highly effective against the WA-1 strain. Accordingly, the viral titers were significantly reduced in both the lungs and the nasal turbinates. Overall, these data demonstrate that mAb NA8 exerted strong prophylactic protection in vivo from two antigenically distinct SARS-CoV-2 variants, and NE12 exerted strong prophylactic protection against the original WA-1 strain in a highly susceptible animal model, despite the relatively low antibody doses used. In summary, these results show that broad and potent human antibodies can overcome the continuous immune escape of evolving SARS-CoV-2 variants. E. Development of a universal neutralizing mAb against all SARS-CoV2 variants and other sarbecoviruses The identification of potent, broadly neutralizing antibodies against SARS-CoV-2 variants of concern that resist neutralization by most mAbs, such as the B.1.351/Beta and the Omicron subvariants, is critical for creating an arsenal of therapeutic antibodies with high potency against both present and future variants of concern that will continue to emerge because of the sustained worldwide spread of this virus, leading to escape from current prophylactic and therapeutic interventions. Capitalizing on the data so far obtained, now we are focusing on the development of universal mAbs that are able to neutralize not only SARS-CoV-2 but also SARS-CoV-1 and other sarbecoviruses from other animal species such as bats. These antibodies are extremely important to prepare for potential new coronavirus-related pandemics caused by spill over from animals to humans. 2. Identification of Circulating Anti-PF4 Antibodies Associated with Disease Severity in Patients with COVID-19 The pathogenic mechanisms that trigger the most severe complications of COVID-19, which are often fatal, are still largely unknown. Severe COVID-19 is frequently associated with thrombosis and thrombocytopenia, which are reminiscent of the clinical presentation of two life-threatening syndromes: heparin-induced thrombocytopenia (HIT), which develops in patients treated with high doses of heparin after cardiac or orthopedic surgery, and vaccine-induced thrombosis with thrombocytopenia (VITT), which is a rare complication of adenovirus-vectored COVID-19 vaccines. The pathogenic mechanism of both the HIT and VITT syndromes involves the elicitation of autoantibodies that target partially cryptic epitopes in platelet factor 4 (PF4 or CXCL4), which are fully revealed upon binding to heparin or other polyanionic molecules. Using a clinically validated immunoassay, we evaluated the presence of antibodies to PF4 in 100 hospitalized patients with COVID-19 with moderate or severe disease, 25 acute COVID-19 cases visiting the Emergency Department and 65 convalescent individuals. Anti-PF4 antibodies were detected in 95 of 100 (95.0%) hospitalized COVID-19 patients irrespective of prior heparin treatment, while patients hospitalized for severe acute respiratory disease unrelated to COVID-19 had markedly lower levels. Higher antibody levels were detected in males than in females, and in African Americans and Hispanics than in White Caucasians (Liu Q, et al. 2022). Anti-PF4 antibody levels were correlated with the maximum disease severity score and with significant reductions in circulating platelet counts during hospitalization. In convalescent individuals from COVID-19, the antibody levels returned to near-normal values. Sera from COVID-19 patients induced higher levels of platelet activation than sera from healthy blood donors, but the results were not correlated with the levels of anti-PF4 antibodies. These results demonstrate that the vast majority of patients with severe COVID-19 develop anti-PF4 antibodies, which may play a role in the clinical complications of COVID-19, and have implications for the therapeutic management of the most severe cases.

1. 有效的单克隆抗体可中和 Omicron 亚系和其他 SARS-CoV-2 变体 A. 使用来自 COVID-19 恢复期患者的噬菌体展示文库生成抗 SARS-CoV-2 mAb 并对其进行表征。 从 12 名具有高中和血清抗体滴度的 SARS-CoV-2 恢复期 COVID-19 患者中采集外周血样本，提取总 RNA 并用于构建四个噬菌体展示 Fab 文库，这些文库来自单个供体或多个捐助者联合起来。使用源自原始 SARS-CoV-2 毒株 Wuu-Hu-1 的稳定三聚刺突蛋白 (S-2P) 进行了三个连续的淘选循环，以富集特定克隆。我们获得了 18 个 Fab，它们与病毒刺突表现出高亲和力结合，平衡解离常数 (KD) 在皮摩尔或低纳摩尔范围内。 B. 有效中和各种令人关注的 SARS-CoV-2 变体。 使用假型病毒中和测定评估了我们的 18 种 mAb 的中和活性。 11 种对原始 SARS-CoV-2 毒株 (WA-1) 和 Alpha 变体表现出有效的中和活性，而 7 种还可以在皮摩尔范围内中和 Delta 变体。相比之下，11 种单克隆抗体中只有 4 种对 Beta 变体有效。值得注意的是，其中之一 NA8，以皮摩尔浓度中和了 Beta、Omicron BA.1 和 BA.2 亚系，以及纳摩尔浓度下的 BA.2.12.1 和 BA.4 亚系。由于临床批准的单克隆抗体通常联合使用以确保针对多种变异的覆盖，因此我们并排比较了等摩尔浓度 (1:1) 下使用的 NA8 和 NE12 组合与 6 种临床批准的单克隆抗体的中和效力。配对组合中使用的抗体。针对最新的变体 BA.2.12.1 和 BA.4，NA8/NE12 组合在低纳摩尔浓度下保留了中和活性，REGN-10933/REGN-10987 组合也是如此，而 COV2-2130/COV2-2196 是最有效的组合而 LY-CoV555/LY-CoV016 无效。这些结果表明，NA8 和 NE12 联合使用时，是最广泛、最有效的抗体对之一，对主要 SARS-CoV-2 变体具有皮摩尔或低纳摩尔的中和活性。根据这些结果，选择 NA8 和 NE12 进行进一步的表征和临床前测试。 C.抗体NE12和NA8的结构分析。 为了深入了解抗体 NE12 和 NA8 与 SARS-CoV-2 刺突糖蛋白的相互作用，我们与 Peter Kwong 和 Yaroslav Tsybovsky 合作，获得了与稳定刺突三聚体结合的 Fab NE12 和 Fab NA8 的冷冻电镜重建图（ S-6P），标称分辨率分别为 3.1 和 2.9。 NE12 的表位与 ACE2 受体足迹重叠，表明 NE12 直接阻断刺突受体相互作用。与 Fab NE12 类似，Fab NA8 显示与受体结合脊的广泛接触，但该抗体与沿远离尖端的 RBD 外侧的独特表位结合。 NA8 表位和 RBD 的 ACE2 结合界面之间存在适度的重叠。这些结构数据进一步证实 NE12 和 NA8 为潜在的临床应用提供了一对有效的互补单克隆抗体。 D. 抗体 NE12 和 NA8 可保护仓鼠免受 SARS-CoV-2 感染。 我们与 Ulla Buchholz 合作，在金色叙利亚仓鼠模型中测试了 mAb NE12 和 NA8 的体内预防功效，该模型非常模拟人类疾病的严重程度。 NA8 治疗可显着保护仓鼠免受两种病毒变种 WA-1 和 Beta 引起的体重减轻，而 NE12 对 WA-1 病毒株非常有效。因此，肺部和鼻甲骨中的病毒滴度显着降低。总体而言，这些数据表明，mAb NA8 对两种抗原不同的 SARS-CoV-2 变体在体内发挥了强大的预防性保护作用，而 NE12 在高度易感的动物模型中对原始 WA-1 毒株发挥了强大的预防性保护作用，尽管抗体相对较低使用的剂量。总之，这些结果表明，广泛而有效的人类抗体可以克服不断进化的 SARS-CoV-2 变体的持续免疫逃逸。 E. 开发针对所有 SARS-CoV2 变种和其他 sarbecovirus 的通用中和 mAb 鉴定针对所关注的 SARS-CoV-2 变体的有效、广泛中和抗体，这些抗体可抵抗大多数 mAb 的中和作用，例如 B.1.351/Beta 和 Omicron 亚变体，对于创建高效能的治疗性抗体库至关重要由于该病毒在全球范围内持续传播，当前和未来令人担忧的变种将继续出现，从而导致人们逃避当前的预防和治疗干预措施。利用迄今为止获得的数据，我们现在专注于开发通用单克隆抗体，这些单克隆抗体不仅能够中和 SARS-CoV-2，还能够中和 SARS-CoV-1 和来自其他动物物种（例如蝙蝠）的其他 sarbeco 病毒。这些抗体对于应对因从动物传播到人类而引起的潜在的新型冠状病毒相关大流行极其重要。 2. 识别与 COVID-19 患者疾病严重程度相关的循环抗 PF4 抗体 引发 COVID-19 最严重并发症（通常是致命的）的致病机制仍然很大程度上未知。严重的 COVID-19 经常与血栓形成和血小板减少症相关，这让人想起两种危及生命的综合征的临床表现：肝素诱导的血小板减少症 (HIT)，发生于心脏或骨科手术后接受高剂量肝素治疗的患者，疫苗诱导的血栓形成伴血小板减少症 (VITT)，这是腺病毒载体的 COVID-19 疫苗的罕见并发症。 HIT 和 VITT 综合征的致病机制均涉及引发针对血小板因子 4（PF4 或 CXCL4）中部分隐性表位的自身抗体，这些表位在与肝素或其他聚阴离子分子结合后得到充分揭示。我们使用经过临床验证的免疫测定法，评估了 100 名患有中度或重度疾病的 COVID-19 住院患者、25 名急诊科就诊的急性 COVID-19 病例和 65 名康复者中是否存在 PF4 抗体。无论之前是否接受过肝素治疗，100 名住院的 COVID-19 患者中，有 95 名 (95.0%) 检测到了抗 PF4 抗体，而因与 COVID-19 无关的严重急性呼吸道疾病住院的患者的抗体水平明显较低。男性中检测到的抗体水平高于女性，非裔美国人和西班牙裔中检测到的抗体水平高于白人（Liu Q 等人，2022）。抗 PF4 抗体水平与最大疾病严重程度评分以及住院期间循环血小板计数的显着减少相关。在 COVID-19 康复者中，抗体水平恢复到接近正常值。来自 COVID-19 患者的血清比来自健康献血者的血清诱导更高水平的血小板活化，但结果与抗 PF4 抗体的水平无关。这些结果表明，绝大多数重症COVID-19患者会产生抗PF4抗体，这可能在COVID-19的临床并发症中发挥作用，并对最严重病例的治疗管理具有影响。