新冠病毒

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7. Guttler等以刺突蛋白S1区作为靶标蛋白，筛选得到60个纳米抗体，其中有43个能有效阻止病毒感染。They use VHH72, a single-domain Camelid antibodies developed against the RBD by Wrapp et al. [20]. The virus was nearly entirely inhibited by pre-incubation with 500 nM VHH-72 (7 mg/L), but lower concentrations had no impact.
Re5D06 was another nanobody that all epitope-binders blocked the ACE2-RBD interaction (e.g., Re5D06, Re6H06, or Re9B09).
Re9F06 prevents docking to ACE2. The structure reveals a remarkable affinity between this nanobody and the RBD.
Four tyrosines (Y449, Y453, Y489, Y505) and three phenylalanines (F456, F486, F490) are particularly notable on the RBD side, implying that the nanobody covers a “sticky” portion of the RBD. The long CDR3 loop considerably enhances the shape complementarity with the RBD surface. CDR3 has three highly exposed tyrosines that are required for RBD binding. This indicates that the loop conformation has been strongly stabilized. Re6B06 and Re6H06 are both hyperthermostable, and Re6H06 is one of the most potent RBD binders (KD ≤ 1 pM) and neutralizers (at 50 pM). They designed an approach that increased avidity and discovered that homo-trimerization is a better way to gain avidity. The NC1 trimerization domains of human collagens XV and XVIII were identified as possibilities. They evaluated different designs and discovered that VHH-spacer-collagen XVIII NC1 fusions had the best neutralizing potency. The trimerization also reduced the nanobody Re9F06’s minimal neutralizing concentration from 50 nM to 167 pM. The best VHHs for the trimerized format and thermostability were Re6B06-spacer-ColXVIII. The Alpha/UK B.1.1.7 variant with an N501Y mutation did not influence the binding of Re5D06 or any other nanobody that was examined. The Re5D06-RBD interaction was reduced by beta/South African or gamma/Brazilian mutations. They’re looking for nanobodies with a high affinity for all SARS-CoV-2 variations and showed that Re9F06 isn’t affected by the escape mutations. They fused two structures (Re9F06-R28) and demonstrated this tandem bound mutated RBD variant remarkably well. Re9F06-Re6H06 and the collagen XVIII trimer of the Re9F06 neutralization effect were more efficient (17 pM).

四个酪氨酸(Y449, Y453, Y489, Y505)和三个苯丙氨酸(F456, F486, F490)在RBD侧表现得尤为明显，表明纳米体覆盖了RBD的“粘性”部分。长CDR3环路大大增强了形状与RBD表面的互补。CDR3有三个高度暴露的酪氨酸，它们是RBD结合所必需的。这表明环构象已被强稳定。Re6B06和Re6H06都是超耐热的，Re6H06是最有效的RBD粘合剂(KD≤1 pM)和中和剂(50 pM)之一。他们设计了一种增加贪婪的方法，并发现同质三聚化是获得贪婪的更好方法。人类胶原XV和XVIII的NC1三聚域被确定为可能。他们评估了不同的设计，发现vhh -间隔-胶原蛋白XVIII NC1融合具有最好的中和效力。三聚化还将纳米体Re9F06的最小中和浓度从50 nM降低到167 pM。三聚体的最佳VHHs为Re6B06-spacer-ColXVIII。带有N501Y突变的Alpha/UK B.1.1.7变异不影响Re5D06或任何其他被检测的纳米体的结合。beta/南非或gamma/巴西突变降低了Re5D06-RBD的相互作用。他们正在寻找对所有SARS-CoV-2变异具有高亲和力的纳米体，并表明Re9F06不受逃避突变的影响。他们融合了两个结构(Re9F06-R28)，并证明这种串联结合突变的RBD变体非常好。Re9F06- re6h06和胶原XVIII三聚体对Re9F06的中和作用更有效(17 pM)。
Re9F06, collagen XVIII-trimerized, tolerates RBD escape mutations better than the others and appears to be an ideal therapeutic candidate. Because of their exceptional potency, great mutation tolerance, and hyperthermostability, Re6H06 are excellent candidates [19].
研究表明，将纳米体融合到fc片段能提高其抗病毒活性，胶原XVIII的三聚化结构域看起来是一个合适的替代融合伙伴。Re9F06，胶原xviii -三聚体，对RBD逃脱突变的耐受性优于其他蛋白，似乎是理想的治疗候选者。
Re6H06由于其特殊的效力，巨大的突变耐受性和很好的热稳定性，是极好的候选治疗抗体。

青花瓷

传统的新冠抗体通常用真核细胞进行表达，不适合大规模生产。纳米抗体可以用原核进行表达，可进行规模化生产。
利用羊驼建立了针对新冠刺突蛋白受体结合域(RBD)的免疫库，分离出45个阻断感染的纳米抗体，其中包括耐95°C的纳米抗体。

最有效的纳米抗体在17-50pm浓度(0.2-0.7μg/L)时中和新冠抗体，结合刺突蛋白的开放和封闭状态，并在x射线和电镜下显示和RBD紧密的相互作用。
好的纳米抗体三聚体在40ng/L的浓度下也能中和病毒。本文中构建了纳米抗体串联，能耐受α、β、Gamma、Epsilon、Iota和Delta/Kappa谱系中出现的K417N/T、E484K、N501Y和L452R免疫逃逸突变。
此外还鉴定了低皮摩尔浓度的纳米抗体能中和β毒株。我们进一步发现，纳米抗体能够促进大肠杆菌胞浆中RBD的自然折叠，而通常情况下RBD的折叠是错误的。
这种“促进折叠”的纳米体可以简化疫苗的生产，以及它们适应病毒逃逸突变株。

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8. Koenig等得到四个针对新冠RBD的中和纳米抗体 (VHHs E, U, V, W) 。将RBD免疫骆驼，The RBD was inoculated to camelids, nanobody libraries were identified by phage display, and it was shown that they are inhibitory. Based on epitope mapping information from X-ray crystallography and SPR as well as detailed data on the conformation of spike-nanobody interactions established by cryo-electron microscopy (cryo-EM) for the determination of biomolecular structures, they have rationally developed multivalent nanobody constructions. They created two types of nanobody fusions: multivalent nanobodies that target the ACE2 binding site on the RBD and biparatopic fusions of two nanobodies that target the non-overlapping binding interfaces that amplify neutralization by activating the spike protein. The nanobodies on the RBD bind to two different epitopes, interfaces “E" and “UVW,” according to X-ray crystallography. The discovery of VHH E, U, and W with two distinct binding mechanisms that early spike activation (maintain the active conformation of the spike) could be a neutralizing mechanism and shows that this form of action is more prevalent than previously assumed. Bi- and trivalent nanobodies with better neutralizing capabilities were created. The replication-deficient vesicular stomatitis virus (VSV) method was used to measure the neutralizing activity. This method was showed that the trivalent VHH EEE prevented infection the most effectively with IC50 of 520 pM) and induced spike fusogenic activity the most significantly. They hypothesized that the nanobody VE attachment to the spike would alter the spike conformation in such a way as to induce an incomplete fusion, thereby deactivating the spike. This activity is called “fusogenic”. Because of their improved neutralizing activity, the multivalent structure-based nanobodies reported here have significant therapeutic potential [21].

青花瓷

9. fu等利用冷冻电镜分析纳米抗体结构，快速有效地得到针对新冠人人源化抗体，亲合力为纳摩尔水平，其中最有效的一株抗体RBD-1-2G (NCATS-BL8125), 能阻断α变异株中RBD的N501Y突变。RBD-1-2G-Fc抗体在体外气道模型实验中，证实能减少WA1和B.1.1.7变异株的病毒载量。

青花瓷

10. Li 等用SARS-CoV RBD-兔IgG-Fc融合蛋白免疫羊驼，建立噬菌体纳米抗体库。经过4轮筛选，筛选得到的4个纳米抗体中，S14和SARS-CoV RBD之间有很强的亲和力活性(KD = 143 pmol/L) 。
并且在假病毒中和活性中，能有效抑制病毒进入细胞，IC50=4.93 ng/mL.
其中和机制显示出剂量-效应关系，阻断可溶性ACE2和SARS-CoV S1蛋白之间的结合。
S14能弱化SARS-CoV RBD和ACE2之间的相互作用。

青花瓷

11. Lu等利用杆状表达系统表达了重组的刺突蛋白和RBD区蛋白，用于筛选纳米抗体。构建的天然纳米抗体库中进行四轮筛选。将筛选得到的纳米抗体插入到表达载体中，在HEK293FT细胞中进行表达。通过中和活性筛选到两个纳米抗体Nb91-hFc和Nb3-hFc。
构建出双价抗体(biNb91-hFc, biNb3-hFc)，双特异性抗体 (Nb91–Nb3-hFc) ，三价抗体(triNb91-hFc, triNb3-hFc) ，和RBD结合活性比单抗要高很多。其中双特异抗体Nb91–Nb3-hFc 的中和活性，IC50=1.54 nM.同时，Nb91-hFc能和S蛋白和RBD蛋白结合，Nb3 –hFc和RBD区结合。这些得到的纳米抗体可以以吸入性方式预防新冠。

青花瓷

从纳米抗体天然库中筛选到4个针对刺突蛋白和RBD蛋白的纳米抗体。 Nb91-hFc和Nb3-hFc能在体外假病毒中和活性中有效抗病毒。多价纳米抗体能进行一步提高中和活性。Nb91-Nb3-hFc双抗展现出很强的RBD结合活性，和SARS-CoV-2假病毒产生中和活性，IC50约为1.54 nM。

青花瓷

12.Ma等人用重组新冠病毒RBD蛋白，免疫羊驼。构建噬菌体纳米抗体库，筛选得到六个纳米抗体(aRBD-2, aRBD-3, aRBD-5, aRBD-7, aRBD-41, aRBD-54) 。构建纳米抗体的真核表达载体，纳米抗体+TEV蛋白酶切割位点+IgG1 Fc，转染HEK 293F细胞，表达和纯化纳米抗体。蛋白经过TBEV切割后，可以得到纳米抗体单体。将这些纳米体用于对N501Y点突变的治疗效果，这是SARS-CoV-2的Alpha、Beta和Gamma变体的一个显著突变，显示N501Y变异不影响纳米抗体和突变体的结合活性。
他们还检测了RBD的点突变对Nbs结合位点的影响，以检查Nbs与RBD的结合位点。选择与ACE2相互作用的RBD特异性突变(Y449A、Q493A、Q498A、V503E、Y505E)。他们报道，在上述突变中，只有Y449A突变显著降低了和纳米抗体的结合，其他4个突变不影响与所选Nbs的结合。尽管这些被选择的突变中没有包含在当前的SARS-CoV-2变体(Alpha、Beta、gamma、Delta)中，但它们可能是SARS-CoV-2中原始RBD中和的强有力的表位。

青花瓷

通过免疫羊驼，从构建的噬菌体纳米抗体库中筛选到7个RBD特异性纳米抗体。亲合力为2.6-113 nM, 其中六个能阻断RBD-ACE2之间的结合作用。将纳米抗体和IgG1 Fc进行融合，RBD结合能力和阻断RBD-ACE2结合的能力提高， (KD 为72.7 pM-4.5 nM) 。将识别无重叠表位的两个纳米抗体构建成双价抗体，aRBD-2-5和aRBD-2-7，RBD结合活性增加 (KD为59.2 pM-0.25 nM) ，SARS-CoV-2中和活性增强。 aRBD-2-5和aRBD-2-7的50%中和剂量分别为1.22 ng/ml (0.043 nM) 和3.18 ng/ml (0.111 nM)。
这些能阻断SARS-CoV-2的纳米抗体可以开发为治疗或者诊断抗体。

青花瓷

新冠病毒包括四个结构蛋白，分别为S蛋白，N蛋白，M蛋白，和E蛋白。
1. S蛋白，刺突糖蛋白（S，Spike Protein）
由1200-1500个氨基酸组成，包含21-35个N-糖基化位点。多个S蛋白以三聚体的形式在冠状病毒表面形成特殊的刺突型花冠结构。
S蛋白与人体细胞表面的血管紧张素转化酶2(ACE2酶)结合，从而进入细胞内部，启动病毒的复制，产生新的病毒。
ACE2酶在人体各器官中具有普遍的表达，其中包括肺部、消化系统、心脏、动脉和肾脏等。 ACE2酶的表达也会随着年龄的增长而增加，在心血管疾病患者中也相对较高。 因此，新冠病毒的易感人群是年老多病者。