Ramos Kevin E, Okba Nisreen M A, Tan Jessica, Bandawane Pooja, Meade Philip S, Loganathan Madhumathi, Francis Benjamin, Shulenin Sergey, Holtsberg Frederick W, Aman M Javad, McMahon Meagan, Krammer Florian, Lai Jonathan R
Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York, USA.
Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
mBio. 2024 Jul 17;15(7):e0108524. doi: 10.1128/mbio.01085-24. Epub 2024 Jun 20.
Monoclonal antibodies (mAbs) are an attractive therapeutic platform for the prevention and treatment of influenza virus infection. There are two major glycoproteins on the influenza virion surface: hemagglutinin (HA), which is responsible for viral attachment and entry, and neuraminidase (NA), which mediates viral egress by enzymatically cleaving sialic acid to release budding particles from the host cell surface. Broadly neutralizing antibodies (bNAbs) that target the conserved HA central stalk region, such as CR9114, can inhibit both viral entry and egress. More recently, broadly binding mAbs that engage and inhibit the NA active site, such as 1G01, have been described to prevent viral egress. Here, we engineered bispecific antibodies (bsAbs) that combine the variable domains of CR9114 and 1G01 into a single molecule and evaluated if simultaneous targeting of two different glycoproteins improved antiviral properties and . Several CR9114/1G01 bsAbs were generated with various configurations of the two sets of the variable domains ("bsAb formats"). We found that combinations employing the addition of a single-chain variable fragment in the hinge region of an IgG scaffold had the best properties in terms of expression, stability, and binding. Further characterization of selected bsAbs showed potent neutralizing and egress-inhibiting activity. One such bsAb ("hSC_CR9114_1G01") provided higher levels of prophylactic protection from mortality and morbidity upon challenge with H1N1 than either of the parental mAbs at low dosing (1 mg/kg). These results highlight the potential use of bsAbs that simultaneously target HA and NA as new influenza immunotherapeutics.
Infection by the influenza virus remains a global health burden. The approaches utilized here to augment the activity of broadly protective influenza virus antibodies may lead to a new class of immunotherapies with enhanced activity.
单克隆抗体（mAb）是预防和治疗流感病毒感染的一个有吸引力的治疗平台。流感病毒粒子表面有两种主要糖蛋白：血凝素（HA），负责病毒的附着和进入；神经氨酸酶（NA），通过酶促裂解唾液酸介导病毒释放，从而使芽生颗粒从宿主细胞表面释放。靶向保守的HA中央茎区域的广泛中和抗体（bNAb），如CR9114，可以抑制病毒的进入和释放。最近，已描述了结合并抑制NA活性位点的广泛结合单克隆抗体，如1G01，可预防病毒释放。在此，我们构建了双特异性抗体（bsAb），将CR9114和1G01的可变域组合成一个单一分子，并评估同时靶向两种不同糖蛋白是否能改善抗病毒特性。通过两组可变域的各种配置（“bsAb形式”）产生了几种CR9114/1G01双特异性抗体。我们发现，在IgG支架的铰链区添加单链可变片段的组合在表达、稳定性和结合方面具有最佳特性。对所选双特异性抗体的进一步表征显示出强大的中和和释放抑制活性。一种这样的双特异性抗体（“hSC_CR9114_1G01”）在低剂量（1mg/kg）下比任何一种亲本单克隆抗体都能提供更高水平的预防保护，防止H1N1攻击后的死亡和发病。这些结果突出了同时靶向HA和NA的双特异性抗体作为新型流感免疫疗法的潜在用途。
流感病毒感染仍然是全球健康负担。这里采用的增强广泛保护性流感病毒抗体活性的方法可能会导致一类具有增强活性的新型免疫疗法。
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