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。有效的单克隆抗体中和Omicron Sublineages和其他SARS-COV-2变体 A.使用COVID-19康复患者的噬菌体播放文库对抗SARS-COV-2 MAB的产生和表征。 从12例疗养院Covid-19患者中收集外周血样本，具有高中和SARS-COV-2的高中和血清抗体滴度，并提取总RNA并用于构建四个噬菌体 - 播放Fab文库，这些含量是从一个单个供体或多个捐赠者组合的。使用稳定的三聚体尖峰蛋白（S-2P）进行了三个顺序循环以富集特定的克隆，该克隆源自原始的SARS-COV-2菌株Wuhan-hu-1。我们获得了18个Fab，在皮摩尔或低纳莫尔范围内具有平衡分离常数（KD）与病毒尖峰表现出高亲和力结合。 B.各种关注的SARS-COV-2变体的有效中和。 使用伪型病毒中和测定法评估了我们18个单击的中和活性。十一显示了针对原始SARS-COV-2菌株（WA-1）和α变体的有效中和活性，而7还中和picomolor范围内的三角变体。相比之下，11个mAb中只有4个有效地针对Beta变体。值得注意的是，其中一个是Na8，中和β，Omicron BA.1和BA.2 Sublineages具有皮摩尔效力，以及BA.2.12.1和BA.4纳莫尔浓度下的Sublineages。由于通常将经临床认可的mAB组合使用以确保对多种变体的覆盖范围，因此我们并排比较了在等摩尔浓度（1：1）的Na8和Ne12组合的中和效力，并与6种临床认可的抗体中的6种使用。对于最近的变体BA.2.12.1和BA.4，NA8/NE12组合与REGN-10933/REGN-10987组合一样，保持中和活性，而COV2-2130/COV2-2196是最有效的组合和Ly-Cov55555/ly-cov016均为cov2-2130/cov2-2196。这些结果表明，NA8和NE12在组合组合时是最宽，有效的抗体对之一，具有皮摩尔或低纳摩尔中和活性，与主要关注的主要SARS-COV-2变体。基于这些结果，选择了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。 为了与抗体Ne12和Na8与SARS-COV-2尖峰糖蛋白的相互作用获得结构性见解，与Peter Kwong和Yaroslav Tsybovsky合作，我们获得了Fab Ne12和Fab Na8的冷冻em重建，并在NOMINDAL（s-6pike Resimal）中获得了稳定的Spike Spike spime spike and Nimallimals and Nimals and Nimally and。 NE12的表位与ACE2受体足迹重叠，表明NE12直接阻止了尖峰受体相互作用。与Fab Ne12相似，Fab Na8与受体结合脊显示了广泛的接触，但是该抗体与尖端远端RBD外侧的独特表位结合。 RBD的Na8表位与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为潜在的临床使用提供了一对有效的互补mAB。 D.抗体NE12和NA8保护仓鼠免受SARS-COV-2感染。 与Ulla Buchholz合作，我们在金叙利亚仓鼠模型中测试了MABS NE12和NA8的体内预防性疗效，该模型密切模仿了人类疾病的严重性。用NA8处理两个病毒变体WA-1和β诱导的重量减肥，而NE12对WA-1菌株非常有效。因此，在肺和鼻涡轮上，病毒滴度均显着降低。总体而言，这些数据表明，MAB Na8在两个抗原不同的SARS-COV-2变体中在体内施加了强烈的预防性保护，尽管使用了相对较低的抗体剂量，但在高度易感动物模型中，在高度易感动物模型中对原始WA-1菌株施加了强烈的预防性保护。总而言之，这些结果表明，广泛而有效的人类抗体可以克服不断发展的SARS-COV-2变体的连续免疫逃逸。 E.针对所有SARS-COV2变体和其他SARBECOVIRASE的通用中和MAB的开发 鉴定出对SARS-COV-2变异的有效，广泛中和抗体的抗体，这些变体的抗体抗体，大多数mAb（例如B.1.351/beta和Omicron subvariants）等大多数mAb中和抗体，对于创造具有高度抗体的逃生而导致这种逃生的逃生是至关重要的当前的预防性和治疗性干预措施。利用迄今为止获得的数据，现在我们着重于通用mAB的发展，这些mAB不仅能够中和SARS-COV-2，还可以从其他动物物种（例如蝙蝠）中获得SARS-COV-1和其他SARBECOVIRES。这些抗体对于准备潜在的冠状病毒相关的新大流星非常重要。 2。鉴定Covid-19患者中与疾病严重程度相关的循环抗PF4抗体 触发最严重的COVID-19并发症的致病机制通常是致命的，但基本上还不清楚。严重的Covid-19通常与血栓形成和血小板减少症有关，这让人联想到两种威胁生命的综合征的临床表现：肝素诱发的血小板细胞减少症（HIT），在患者中以高剂量的心脏或矫形外科手术和疫苗的疫苗促进（VITT）在患者中发育，并在患者中进行了疫苗，并引起了疫苗的影响，并引起了疫苗的影响。腺病毒载体的罕见并发症是COVID-19-19疫苗。 HIT和VITT综合征的致病机制涉及启发在血小板因子4（PF4或CXCL4）中部分隐性表位的自身抗体，这些自身抗体在与肝素或其他多丙二醇分子结合时充分揭示。使用经过临床验证的免疫测定法，我们评估了100例住院的COVID-19患者患有中度或重度疾病的患者中对PF4的存在，25急性共同Covid-19患者访问了急诊科和65个康复患者。在100例（95.0％）住院的Covid-19患者中，有95例（95.0％）的患者在95例中检测到抗PF4抗体，而与先前的肝素治疗无关，而因与Covid-19无关的严重急性呼吸道疾病而住院的患者的水平明显降低。在男性中检测到的抗体水平高于女性，在非洲裔美国人和西班牙裔中，抗体水平高于白人高加索人（Liu Q等，2022）。抗PF4抗体水平与最大疾病严重程度评分相关，并在住院期间显着降低了循环血小板计数。在COVID-19的疗养个体中，抗体水平恢复为近正常值。来自COVID-19患者的血清比健康献血者的血清诱导的血小板激活水平更高，但结果与抗PF4抗体的水平无关。这些结果表明，绝大多数患有严重COVID-19的患者会产生抗PF4抗体，这可能在Covid-19的临床并发症中起作用，并且对最严重病例的治疗管理具有影响。