key: cord-0920544-qqwfreu4
authors: Ma, Huan; Zeng, Weihong; Meng, Xiangzhi; Huang, Xiaoxue; Yang, Yunru; Zhao, Dan; Zhou, Peigen; Wang, Xiaofang; Zhao, Changcheng; Sun, Yong; Wang, Peihui; Ou, Huichao; Hu, Xiaowen; Xiang, Yan; Jin, Tengchuan
title: Potent in vitro Neutralization of SARS-CoV-2 by Hetero-bivalent Alpaca Nanobodies Targeting the Spike Receptor-Binding Domain
date: 2021-02-03
journal: bioRxiv
DOI: 10.1101/2021.02.02.429311
sha: 73b65e58a8b9653f3c868123794dbdd1b3c240bb
doc_id: 920544
cord_uid: qqwfreu4

Cell entry by SARS-CoV-2 requires the binding between the receptor-binding domain (RBD) of the viral Spike protein and the cellular angiotensin-converting enzyme 2 (ACE2). As such, RBD has become the major target for vaccine development, while RBD-specific antibodies are pursued as therapeutics. Here, we report the development and characterization of SARS-CoV-2 RBD-specific VHH/nanobody (Nb) from immunized alpacas. Seven RBD-specific Nbs with high stability were identified using phage display. They bind to SARS-CoV-2 RBD with affinity KD ranging from 2.6 to 113 nM, and six of them can block RBD-ACE2 interaction. The fusion of the Nbs with IgG1 Fc resulted in homodimers with greatly improved RBD-binding affinities (KD ranging from 72.7 pM to 4.5 nM) and nanomolar RBD-ACE2 blocking abilities. Furthermore, fusion of two Nbs with non-overlapping epitopes resulted in hetero-bivalent Nbs, namely aRBD-2-5 and aRBD-2-7, with significantly higher RBD binding affinities (KD of 59.2 pM and 0.25 nM) and greatly enhanced SARS-CoV-2 neutralizing potency. The 50% neutralization dose (ND50) of aRBD-2-5 and aRBD-2-7 was 1.22 ng/mL (∼0.043 nM) and 3.18 ng/mL (∼0.111 nM), respectively. These high-affinity SARS-CoV-2 blocking Nbs could be further developed into therapeutics as well as diagnosis reagents for COVID-19. Importance To date, SARS-CoV-2 has caused tremendous loss of human life and economic output worldwide. Although a few COVID-19 vaccines have been approved in several countries, the development of effective therapeutics including SARS-CoV-2 targeting antibodies remains critical. Due to their small size (13-15 kDa), highly solubility and stability, Nbs are particularly well suited for pulmonary delivery and more amenable to engineer into multi-valent formats, compared to the conventional antibody. Here, we report a serial of new anti-SARS-CoV-2 Nbs isolated from immunized alpaca and two engineered hetero-bivalent Nbs. These potent neutralizing Nbs showed promise as potential therapeutics against COVID-19.

as therapeutics for treating COVID-19 patients [19] [20] [21] . In addition to the conventional mAbs, a distinct 72 type of antibody fragment derived from camelid immunoglobulins, termed VHH or nanobody (Nb), is an 73 attractive alternative for COVID-19 treatment. Compared to the conventional antibody, VHH is cheaper 74 to produce, has an enhanced tissue penetration, and is more amenable to engineering into multivalent 75 and multi-specific antigen-binding formats [22] . Moreover, Nbs are particularly well suited for 76 pulmonary delivery because of their small size (13-15 kDa), highly solubility and stability [23, 24] .

Cell entry by SARS-CoV-2 requires the interaction between the RBD of the viral Spike protein and 78 the receptor ACE2, which is also the receptor for SARS-CoV-1 [25] [26] [27] [28] [29] . The RBD of SARS-CoV-2 binds 79 to ACE2 with a KD of ~15 nM, which about 10-to 20-fold better than that for SARS-CoV-1 RBD [30] . 80

In this study, we report the development and characterization of seven anti-RBD Nbs isolated from 81 alpcacas immunized with SARS-CoV-2 RBD. Furthermore, two high-affinity hetero-bivalent Nbs were 82 developed by fusing two Nbs with distinct epitopes, resulting in antibodies with strong SARS-CoV-2 83 neutralizing potency.

Results 87

Our aim was to develop potent SARS-CoV-2 neutralizing antibodies with favorable biological 89 characteristics. Towards this goal, we immunized two alpacas 3 times with highly purified recombinant 90 SARS-CoV-2 RBD (Fig. S1) . Total RNA was extracted from 1 x 10 7 PBMCs from the immunized alpacas 91 and used as the template for synthesizing cDNA. The VHH coding regions were amplified from the cDNA 92 and cloned into a phagemid vector, generating a library with about 1.6 x 10 7 independent clones. Phages 93 displaying VHH were prepared from the library with the helper phage and selected with SARS-CoV-2 94 RBD via two rounds of biopanning. Titration of the output phages after each round of panning indicated 95 that the RBD-binding phages were effectively enriched (Fig. 1A) .

After each round of panning, thirty-one individual phages were randomly picked and their RBD-97 binding activity evaluated with phage ELISA. Nineteen and thirty phages were found to be positive for 98 RBD binding after the first and second round of panning, respectively (Fig. 1B) . Sequencing of the 99 positive phage clones after two rounds of panning revealed seven unique Nbs (Fig. 1C) , which were 100 named as aRBD-2, aRBD-3, aRBD-5, aRBD-7, aRBD-41, aRBD-42 and aRBD-54. All seven phages 101 can bind to the S1 domain of SARS-CoV-2 in ELISA, and one (aRBD-41) can also bind to SARS-CoV-102 1 RBD (Fig. S2 ).

Binding characteristics of the identified nanobodies

The identified Nbs were expressed with a mammalian expression vector in 293F cells. To configure the 106 Nb into IgG-like molecule, we fused the C-terminus of the identified Nbs to a TEV protease cleavage 107 site and a human IgG1 Fc in a mammalian expression vector. The homo-bivalent Nb-TEV-Fc fusions 108 were purified from the culture supernatant using protein A (Fig. S3A) . All of the Nb-TEV-Fc fusions 109 showed more than 100 mg/L yield after three days of expression (data not shown). To prepare Nb 110 monomers without the Fc, the fusion proteins were digested with the TEV enzyme (6His tagged) and 111 passed through protein G and Ni NTA column. Highly purified Nbs were obtained from the flow-through 112 (Fig. S3B) . The conformational stability of the seven Nbs were tested using circular dichroism, and the 113 results showed that they were highly stable in solution, with the melting temperature exceeding 70°C 114 ( Fig. S4) .

The SARS-CoV-2 RBD-binding abilities of the seven Nbs were first verified using size-exclusion 116 chromatography (SEC). All seven Nbs formed stable complexes with RBD in solution ( Fig. 2A-I) .

Furthermore, most Nb-Fc fusions demonstrated strong binding to both RBD and the entire ectodomain 118 (S1+S2) of SARS-CoV-2 spike in ELISA, with EC50 of low nM. Compared to the human ACE2-Fc 119 recombinant protein, they bind to the RBD with a higher affinity (Fig. 2J) , while all but aRBD-42 bind 120 to the entire ectodomain of spike protein with a higher affinity (Fig. 2K) . In addition, we also tested the 121 binding ability between the 7 Nbs and a RBD variant that contains N501Y point mutation derived from 122 a recent new SARS-CoV-2 lineage that was rapidly spreading in UK [31] . As expected, N501Y variant 123 showed an enhanced binding activity with ACE2-Fc than original RBD. Interestingly, all of the 7 Nbs 124 exhibited similar binding activity to the variant and original RBD (Fig. S5) .

The binding affinity of the Nbs to RBD were also measured using Surface Plasmon Resonance (SPR).

Six Nbs showed a high binding affinity, with KD values of 2.60, 3.33, 16.3, 3.31, 21.9 and 5.49 nM for 127 aRBD-2, aRBD-3, aRBD-5, aRBD-7, aRBD-41 and aRBD-54, respectively ( Fig. 3A-E and G) .

Consistent with ELISA, aRBD-42 had a relatively weak binding affinity with a KD of 113 nM (Fig. 3F) .

The affinities of Nb-Fc fusions were also measured by SPR. Probably due to dimerization, they showed an enhanced binding capability, with KD values ranging from 4.49 nM to 72.7 pM (Fig. S6) .

Nbs block RBD-ACE2 interaction 133 SARS-CoV-2 infection is initiated by the interaction of RBD and ACE2. To assess the ability of the Nbs 134 in blocking RBD-ACE2 interaction, we performed competitive ELISA. Except for aRBD-42, which has 135 the lowest RBD-binding affinity, all other Nbs (Fig. 4A) and their Fc fusions (Fig. 4B) 

High affinity hetero-bivalent antibodies constructed depend on epitope grouping 142 To find out whether the Nbs bind to overlapping epitopes, the ability of the Nbs to compete with each 143 other for ACE2 binding was studied with ELISA. The Nbs were serially diluted (ranging from 2.5 to 144 10240 nM) and used to compete with 5 nM of a Nb-TEV-Fc fusion to bind SARS-CoV-2 RBD coated 145 on plates ( Fig. 5A-F) . The competition was summarized in Fig. 5G . Based on these grouping and an 146 additional SEC results ( Fig. 6A and B) , we engineered two hetero-bivalent Nbs, namely aRBD-2-5 and 147 aRBD-2-7, by connecting aRBD-2 head-to-tail with aRBD-5 and aRBD-7 through a (GGGGS)3 flexible 148 linker, respectively. They were also expressed in 293F cells and purified as above (Fig. 6C) . SEC 149 indicated aRBD-2-5 and aRBD-2-7 were monomeric in solution ( Fig. 6D and E) , and circular dichroism 150 spectrum analysis showed they were also highly stable in solution (Fig. S4h, i) .

The RBD binding activities of aRBD-2-5 and aRBD-2-7 were studied with SEC ( Fig. 6D and E) and 

Hetero-bivalent Nbs exhibit potent neutralizing ability against live SARS-CoV-2

To assess the ability of the Nbs in neutralizing SARS-CoV-2, we developed a SARS-CoV-2 micro-159 neutralization assay and assessed representative Nbs in this assay. Nbs that were serially diluted to Interestingly, the hetero-bivalent Nbs exhibited an even higher neutralizing potency than the homo-171 dimeric Nbs. The fitted ND50 for aRBD-2-5 and aRBD-2-7 is 1.22 ng/mL (~0.043 nM) and 3.18 ng/mL 172 (~0.111 nM), respectively (Fig. 7A, B and E) . The Fc fusions of the hetero-bivalent Nbs did not further 173 increase the neutralization potency. The ND50 for aRBD-2-5-Fc and aRBD-2-7-Fc is 11.8 ng/mL (~0.107 nM) and 6.76 ng/mL (~0.0606 nM), respectively (Fig. 7C, D 

Discussion 177 aRBD-41 (Fig. 4A) . However, aRBD-7, which had a similarly high RBD binding affinity of 3.31 nM, 191 only exhibited a weak ACE2-RBD blocking activity (Fig. 4A) . We thus infer that different Nbs may 192 occupy different epitopes on RBD, leading to varying strength of ACE2 binding interference. The 193 epitopes of some Nbs may overlap more closely with that of ACE2. Interestingly, even when the Nbs 194 with a relatively weak ACE2-RBD blocking ability were fused with IgG1 Fc to form homodimers, their 195 blocking ability were increased more than 75-fold ( Fig. 4A and B) . This effect is probably due to the 196 increased apparent RBD-binding affinity by dimerization as well as the additional steric hindrance caused 197 by the increased size. Further investigations are needed to understand the underlying mechanisms.

According to grouping results of the seven Nbs, two hetero-bivalent antibodies were constructed by 199 fusing aRBD-2 to aRBD-5 and aRBD-7 tail-to-head with a flexible linker, which achieved a more than 200 10-fold increase in RBD-binding affinity ( Fig. 6F and G) . Consistent with the increased affinity and 201 steric hindrance, the SARS-CoV-2 neutralization potency of aRBD-2-5 and aRBD-2-7 were greatly 202 enhanced, with ND50 of 1.2 ng/mL (~0.043 nM) and 3.2 ng/mL (~0.111 nM) (Fig. 7) .The neutralization 203 potency of our aRBD-2-5 and aRBD-2-7 appears to be better than other previously reported Nbs, their 

Secondary structure and thermal stabilities of identified Nbs were studied by CD spectra using a

Chirascan Spectrometer (Applied Photophysics). Prior to CD measurements, the sample buffer was 296 changed to phosphate-buffered saline (PBS), and the protein concentration was adjusted to 0.3 mg/ml.

The CD spectra were acquired for each sample from 180 to 260 nm using a 1 mm path length cell. For 298 thermal titration, CD spectra were acquired between 20°C to 95°C with temperature steps of 2.5°C. CD 299 signals at 205 nm were used to characterize the structural changes during thermal titration. Each experiment was repeated twice, and the data were fitted with Prism to obtain the Tm values.

Surface Plasmon Resonance (SPR)

SPR measurements were performed at 25°C using a BIAcore T200 system. SARS-CoV-2 RBD was 304 diluted to a concentration of 15 μg/mL with sodium acetate (pH 4.5) and immobilized on a CM5 chip 305 (GE Healthcare) at a level of ~150 response units (RU). All proteins were exchanged into the running The data are fitted to the model with the drc package in R to obtain the 95% confidence intervals and 327 ND50. It should be noted that all above Nbs were lyophilized at concentration of 2-5 mg/mL and kept at 328 room temperature for one week for transportation. The lyophilized Nbs were re-dissolved in ddH2O 329 before they were used in neutralization assay.

We would like to thanks all the staff who participated in this work for their important contribution. This 

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