key: cord-321369-xzu2faol
authors: Andreano, Emanuele; Nicastri, Emanuele; Paciello, Ida; Pileri, Piero; Manganaro, Noemi; Piccini, Giulia; Manenti, Alessandro; Pantano, Elisa; Kabanova, Anna; Troisi, Marco; Vacca, Fabiola; Cardamone, Dario; De Santi, Concetta; Benincasa, Linda; Agrati, Chiara; Capobianchi, Maria Rosaria; Castilletti, Concetta; Emiliozzi, Arianna; Fabbiani, Massimiliano; Montagnani, Francesca; Depau, Lorenzo; Brunetti, Jlenia; Bracci, Luisa; Montomoli, Emanuele; Sala, Claudia; Ippolito, Giuseppe; Rappuoli, Rino
title: Extremely potent human monoclonal antibodies from convalescent Covid-19 patients
date: 2020-10-07
journal: bioRxiv
DOI: 10.1101/2020.10.07.328302
sha: 
doc_id: 321369
cord_uid: xzu2faol

Human monoclonal antibodies are safe, preventive and therapeutic tools, that can be rapidly developed to help restore the massive health and economic disruption caused by the Covid-19 pandemic. By single cell sorting 4277 SARS-CoV-2 spike protein specific memory B cells from 14 Covid-19 survivors, 453 neutralizing antibodies were identified and 220 of them were expressed as IgG. Up to 65,9% of monoclonals neutralized the wild type virus at a concentration of >500 ng/mL, 23,6% neutralized the virus in the range of 100 - 500 ng/mL and 9,1% had a neutralization potency in the range of 10 - 100 ng/mL. Only 1,4% neutralized the authentic virus with a potency of 1-10 ng/mL. We found that the most potent neutralizing antibodies are extremely rare and recognize the RBD, followed in potency by antibodies that recognize the S1 domain, the S-protein trimeric structure and the S2 subunit. The three most potent monoclonal antibodies identified were able to neutralize the wild type and D614G mutant viruses with less than 10 ng/mL and are good candidates for the development of prophylactic and therapeutic tools against SARS-CoV-2. One Sentence Summary Extremely potent neutralizing human monoclonal antibodies isolated from Covid-19 convalescent patients for prophylactic and therapeutic interventions.

The impact of the SARS-CoV-2 pandemic, with more than 35 million cases, over 1 million deaths, 5 trillion impact on the gross domestic product (GDP) and 45 million people filing unemployment in the United States alone, is unprecedented (Aratani, 2020) .

In the absence of drugs or vaccines, non-pharmaceutical interventions such as social distancing and quarantine have been the only way to contain the spread of the virus.

These interventions showed to be efficient when properly implemented but not all countries were able to do so showing the limits of these strategies. The urgency to develop vaccines and therapies is extremely high. The effort to develop vaccines is unprecedented and fortunately in October 2020 we already have several vaccines in advanced Phase III efficacy trials and many others in earlier phase of development. In spite of the big effort, it is predictable this wave of infection will continue to spread globally and it is likely to be followed by additional waves in the next few years until herd immunity, acquired by vaccination or by natural infection, will constrain the circulation of the virus. It is therefore imperative to develop in parallel both vaccines and therapeutic tools to face the next waves of SARS-CoV-2 infections as soon as possible. Among the many therapeutic options available, human monoclonal antibodies (mAbs) are the ones that can be developed in the shortest period of time. In fact, the extensive clinical experience with the safety of more than 50 commercial mAbs approved to treat cancer, inflammatory and autoimmune, disorders provides high confidence on their safety. These advantages, combined with the urgency of the SARS-CoV-2 pandemic, support and justify an accelerated regulatory pathway. In addition, the long industrial experience in developing and manufacturing mAbs decreases the risks usually associated with the technical development of investigational products. Finally, the incredible technical progress in the field allows to shorten the conventional timelines and go from discovery to proof of concept trials in 5-6 months (Kelley, 2020) . Indeed, in the case of Ebola, mAbs were the first therapeutic intervention recommended by the World Health Organization (WHO) and they were developed faster than vaccines or other drugs (Kupferschmidt, 2019) .

The SARS-CoV-2 spike glycoprotein (S-protein) has a pivotal role in viral pathogenesis and it is considered the main target to elicit potent neutralizing antibodies and the focus for the development of therapeutic and prophylactic tools against this virus , Tay et al., 2020 . Indeed, SARS-CoV-2 entry into host cells is mediated by the interaction between S-protein and the human angiotensin converting enzyme 2 (ACE2) (Wang et al., 2020b . The S-protein is a trimeric class I viral fusion protein which exists in a metastable prefusion conformation and in a stable postfusion state. Each Sprotein monomer is composed of two distinct regions, the S1 and S2 subunits. Structural rearrangement occurs when the receptor binding domain (RBD) present in the S1 subunit binds to the host cell membrane. This interaction destabilizes the prefusion state of the Sprotein triggering the transition into the postfusion conformation which in turn results in the ingress of the virus particle into the host cell (Wrapp et al., 2020) . Single-cell RNAsequencing analysis revealed that ACE2 expression was ubiquitous in different human organs have suggesting that SARS-CoV-2, through the S-protein, can invade human cells in different major physiological systems including the respiratory, cardiovascular, digestive and urinary systems, thus enhancing the possibility of spreading and infection (Zou et al., 2020) .

During the first few months of this pandemic, several groups have been active in isolating and characterizing human monoclonal antibodies from Covid-19 convalescent patients or from humanized mice and some of them have been progressing quickly to clinical trials for the prevention and cure of SARS-Cov-2 infection (Shi et al., 2020 , Hansen et al., 2020 , Wang et al., 2020a , Pinto et al., 2020 , Zost et al., 2020 , Rogers et al., 2020 , Andreano et al., 2020 . So far, most of the work on human monoclonal antibodies against SARS-CoV-2 started from one or few patients and it allowed to successfully isolate and characterize the first interesting antibodies with moderate neutralization potency. In many cases an hundreds of nanograms to micrograms of antibodies were required to neutralize the virus in vitro therefore grams of antibodies will be needed per single patient. In this scenario intravenous delivery results to be the only possible administration route for their therapeutic and prophylactic use. Only recently very potent human antibodies have been isolated and these can be considered for intramuscular or subcutaneous administration. A striking example is a monoclonal antibody against RSV which delivered intramuscularly to premature babies has shown very promising results in clinical settings (Griffin et al., 2020) .

Furthermore, giving the initial rush in isolating potential antibody candidates for clinical development, the whole picture on different types of neutralizing antibodies generated after infection is not yet clear.

To identify and characterize potent mAbs against SARS-CoV-2, we isolated over 4,200 Sprotein specific-memory B cells (MBCs) derived from 14 Covid-19 convalescent. From the screening of thousands of B cells, three extremely potent monoclonal antibodies were identified and are excellent candidates for further development.

Isolation and characterization of S-protein specific antibodies from SARS-CoV-2 convalescent patients To retrieve mAbs specific for SARS-CoV-2 S-protein, peripheral blood mononuclear cells (PBMCs) from fourteen convalescent patients enrolled in this study were collected and stained with fluorescent labelled S-protein trimer to identify antigen specific memory B cells (MBCs). Fig. 1 summarizes the overall experimental strategy. The gating strategy described in Fig. S1 was used to single cell sort into 384-well plates IgG + and IgA + MBCs binding to the SARS-CoV-2 S-protein trimer in its prefusion conformation. The sorting strategy aimed to specifically identify class-switched MBCs (CD19 + CD27 + IgD -IgM -) to identify only memory B lymphocytes that went through maturation processes. A total of 4,277 S-protein-binding MBCs were successfully retrieved with frequencies ranging from 0,17% to 1,41% (Table 1) . Following the sorting procedure, S-protein + MBCs were incubated over a layer of 3T3-CD40L feeder cells in the presence of IL-2 and IL-21 stimuli for two weeks to allow natural production of immunoglobulins (10). Subsequently, MBC supernatants containing IgG or IgA were tested for their ability to bind either the SARS-CoV-2 S-protein trimer in its prefusion conformation or the S-protein S1 + S2 subunits ( Fig.   2A -B) by enzyme linked immunosorbent assay (ELISA). A panel of 1,731 mAbs specific for the SARS-CoV-2 S-protein were identified showing a broad range of signal intensities (Table 1) .

Identification of S-protein specific mAbs able to neutralize SARS-CoV-2

The 1,731 supernatants containing S-protein specific mAbs, were screened in vitro for their ability to block the binding of the S-protein to Vero E6 cell receptors. and for their ability to neutralize authentic SARS-CoV-2 virus by in vitro microneutralization assay. In the neutralization of binding (NoB) assay, 339 of the 1,731 tested (19,6%) S-protein specific mAbs were able to neutralize the antigen/receptor binding showing a broad array of neutralization potency ranging from 50% to over 100% (Table 1 and Fig. 2C ).

As for the authentic virus neutralization assay, supernatants containing naturally produced IgG or IgA were tested for their ability to protect the layer of Vero E6 cells from the cytopathic effect triggered by SARS-CoV-2 infection (Fig. S2) . To increase the throughput of our approach, supernatants were tested at a single point dilution and to increase the sensibility of our first screening a viral titer of 25TCID 50 was used. For this first screening mAbs were classified as neutralizing, partially neutralizing and not-neutralizing mAbs based on their inability to protect Vero E6 cells from infection, or to their ability to partially or completely prevent the cytopathic effect. Out of 1,731 mAbs tested in this study, a panel of 453 (26,2%) mAbs neutralized the live virus and prevented infection of Vero E6 cells (Table 1 ). The percentage of partially neutralizing mAbs and neutralizing mAbs (nAbs) identified in each donor was extremely variable ranging from 2,6 -29,7% and 2,8 -26,4% respectively ( Fig. 3A and Table S1 ). The majority of nAbs were able to specifically recognize the S-protein S1 domain (57,5%; N=244) while 7,3% (N=53) of nAbs were specific for the S2 domain and 35,2% (N=156) did not recognize single domains but only the S-protein in its trimeric conformation ( Fig. 3B ; Table S2 ). From the panel of 453 nAbs, we recovered the heavy and light chain amplicons of 220 nAbs which were expressed as full length IgG1 using the transcriptionally active PCR (TAP) approach to characterize their neutralization potency. The vast majority of nAbs identified (65,9%; N=145) had a low neutralizing potency and required more than 500 ng/ml to achieve an IC 100 . A smaller fraction of the antibodies had an intermediate neutralizing potency (23,6%; N=52) requiring between 100 and 500 ng/ml to achieve the IC 100 , while 9,1% (N=20) required between 10 and 100 ng/ml. Finally, only 1,4% (N=3) of the expressed nAbs were classified as extremely potently nAbs, showing an IC 100 lower than 10 ng/mL ( Fig. 3C -D; Table S3 ).

Based on the first round of screening, 14 nAbs were selected for further characterization.

All nAbs were able to bind the SARS-CoV-2 S-protein in its trimeric conformation (Fig. 4A ).

The mAbs named J08, I14, F05, G12, C14, B07, I21, J13 and D14 were also able to specifically bind the S1 domain (Fig. 4B ). The nAbs named H20, I15, F10 and F20 were not able to bind single S1 or S2 domains but only the S-protein in its trimeric state, while the nAb L19 bound only the S2 subunit ( Fig. 4B -C) . Among the group of S1 specific nAbs only J08, I14, F05, G12, C14 and B07 were able to bind the S1 receptor binding domain (RBD) and to strongly inhibit the interaction between the S-protein and Vero E6 receptors Table 2 ). On the other hand I21, J13 and D14, despite showing S1 binding specificity, did not show any binding to the RBD and NoB activity ( Fig S3 and Table 2). Based on this description four different groups of nAbs against SARS-CoV-2 were identified. The first group (Group I) is composed by S1-RBD specific nAbs (J08, I14, F05, G12 and C14) and showed extremely high neutralization potency against both the WT and D614G live viruses ranging from 3,91 to 157,49 ng/mL ( Fig. 4D -F; Table 2 ). The second group (Group II) is composed by S1 nAbs that did not bind the RBD (B07, I21, J13 and D14). These antibodies also showed good neutralization potency ranging from 49,61 to 500 ng/mL (Fig. 4D -F; Table 2 ) but inferior to S1-RBD directed nAbs. Antibodies belonging to Group I and II showed picomolar affinity to the S-protein with a KD ranging from 0.78 to 6.30 E -10 M (Fig. S4 ). The third group (Group III) is composed by antibodies able to bind the S-protein only in its whole trimeric conformation (H20, I15, F10 and F20).

Antibodies belonging to this group showed lower affinity to the S-protein (KD 7.57 E -8 M -6.40 E -9 M) compared to Group I and II nAbs and medium neutralization potencies ranging from 155,02 to 492,16 ng/mL ( Table 2 ).

This work describes a systematic screening of memory B cells from convalescent people to identify extremely potent human monoclonal antibodies against the spike protein of the SARS-CoV-2 virus, to be used for prevention and therapy of Covid-19. We found that approximately 10% of the total B cells against the spike protein produce neutralizing antibodies and these can be divided into 4 different groups recognizing the S1-RBD, S1domain, S2-domain and the S-protein trimer. We found that the most potent neutralizing antibodies are extremely rare and that they recognize the RBD, followed in potency by the antibodies recognizing the S1 domain, the trimeric structure and the S2 subunit. The L19 antibody against the S2 subunit, which had the lowest neutralizing potency, is representative of several other S2 specific antibodies identified in the preliminary screening. From these data we conclude that in convalescent patients most of the observed neutralization titers are mediated by the antibodies with medium-high neutralizing potency. Indeed, the extremely potent antibodies and the antibodies against the S2 subunit are unlikely to contribute to the overall neutralizing titers because they are too rare and too poor neutralizers respectively to be able to make the difference. The observed antibody repertoire of convalescent patients may be a consequence of the loss of Bcl-6-expressing follicular helper T cells and the loss of germinal centers in Covid-19 patients which may limit and constrain the B cell affinity maturation (Kaneko et al., 2020) . It is therefore important to perform similar studies following vaccination as it is likely that the repertoire of neutralizing antibodies induced by vaccination may be different from the one described here.

Out of 453 neutralizing antibodies that were tested and characterized three showed extremely high neutralization potency against both the initial SARS-CoV-2 strain isolated in Wuhan and the D614G variant currently spread worldwide. During the last few months several groups reported the isolation, structure and passive protection in animal models of neutralizing antibodies against SARS-CoV-2. Most of these studies, with few exceptions report antibodies which require from 20 to several hundreds more ng/mL to neutralize 50% of the virus in vitro. These antibodies are potentially good for therapy. However, they will require a high dosage which will result in elevated cost of goods, low capacity to numbers large quantities of doses and intravenous infusion.

The extremely potent candidates described in our study will allow to use small quantities of antibodies to reach the prophylactic and therapeutic dosage and as consequence decrease the cost of goods and implement sustainable development and manufacturability. This solution may increase the number of doses produced annually and therefore increase antibodies availability in high income countries as well as low-and middle-income countries (LMICs). Our work combined with institutions such as the ELISA assay with S1 and S2 subunits of SARS-CoV-2 S-protein

The presence of S1-and S2-binding antibodies in culture supernatants of monoclonal S- 

Vector digestions were carried out with the respective restriction enzymes AgeI, SalI and Xho as previously described (Tiller et al., 2008, Wardemann and Busse, 2019) . Briefly, 75 ng of IgH, Igλ and Igκ purified PCRII products were ligated by using the Gibson Assembly NEB into 25 ng of respective human Igγ1, Igκ and Igλ expression vectors. The reaction was performed into 5 μL of total volume. Ligation product was 10-fold diluted in nucleasefree water (DEPC) and used as template for transcriptionally active PCR (TAP) reaction which allowed the direct use of linear DNA fragments for in vitro expression. The entire process consists of one PCR amplification step, using primers to attach functional promoter (human CMV) and terminator sequences (SV40) onto the fragment PCRII products. TAP reaction was performed in a total volume of 25 μL using 5 μL of Q5 polymerase (NEB), 5 μL of GC Enhancer (NEB), 5 μL of 5X buffer,10 mM dNTPs, 0,125 µL of forward/reverse primers and 3 μL of ligation product. TAP reaction was performed by using the following cycles: 98°/2', 35 cycles 98°/10'', 61°/20'', 72°/1' and 72°/5' as final extention step. TAP products were purified under the same PCRII conditions, quantified by

Expi293F cell line using manufacturing instructions.

The SARS-CoV-2 virus was propagated in Vero E6 cells cultured in DMEM high Glucose supplemented with 2% FBS, 100 U/mL penicillin, 100 µg/mL streptomycin. Cells were seeded at a density of 1x10 6 cells/mL in T175 flasks and incubated at 37°C, 5% CO 2 for 18-20 hours. The sub-confluent cell monolayer was then washed twice with sterile Dulbecco's phosphate buffered saline (DPBS). Cells were inoculated with 3,5 mL of the virus properly diluted in DMEM 2% FBS at a multiplicity of infection (MOI) of 0.001, and incubated for 1h at 37°C in a humidified environment with 5% CO 2 . At the end of the incubation, 50 mL of DMEM 2% FBS were added to the flasks. The infected cultures were incubated at 37°C, 5% CO 2 and monitored daily until approximately 80-90% of the cells exhibited cytopathic effect (CPE). Culture supernatants were then collected, centrifuged at 4°C at 1,600 rpm for 8 minutes to allow removal of cell debris, aliquoted and stored at -80°C as the harvested viral stock. Viral titers were determined in confluent monolayers of Vero E6 cells seeded in 96-well plates using a 50% tissue culture infectious dose assay (TCID 50 ). Cells were infected with serial 1:10 dilutions (from 10-1 to 10-11) of the virus and incubated at 37°C, in a humidified atmosphere with 5% CO 2 . Plates were monitored daily for the presence of SARS-CoV-2 induced CPE for 4 days using an inverted optical microscope. The virus titer was estimated according to Spearman-Karber formula (Kundi, 1999) and defined as the reciprocal of the highest viral dilution leading to at least 50% CPE in inoculated wells.

The neutralization activity of culture supernatants from monoclonal was evaluated using a CPE-based assay as previously described negative for SARS-CoV-2 in ELISA and neutralization assays). Following expression as full-length IgG1 recombinant antibodies were quantitatively tested for their neutralization potency against both the wild type and D614G strains. The assay was performed as previously described but using a viral titer of 100TCID 50 . Antibodies were prepared at a starting concentration of 20 µg/mL and diluted step 1:2. Technical triplicates were performed for each experiment.

Characterization of SARS-CoV-2 RBD-Antibodies binding by Flow cytometry containing the captured mAb for 180 sec at a flow rate of 80 µl/min. Dissociation was followed for 800 sec, regeneration was achieved with a pulse (60 sec) of Glycine pH 1.5.

Kinetic rates and affinity constant of SPIKE protein binding to each mAb were calculated applying a 1:1 binding as fitting model using the Bia T200 evaluation software 3.1. ng/mL). In all graphs selected antibodies are shown in dark red, pink, gray and light blue based on their ability to recognize the SARS-CoV-2 S1-RBD, S1-domain, S-protein trimer only and S2-domain respectively. Table S1 . Identification of neutralizing antibodies. The Table shows numbers and percentages of neutralizing, partially neutralizing and not-neutralizing antibodies identified for each donor assessed in this study. Table S2 . S-protein binding distribution of neutralizing antibodies. The Table summarizes the binding distribution of neutralizing antibodies against the S1-domain, S2-domain and S-protein trimer. Table S3 . Potency distribution of SARS-CoV-2 S-protein specific nAbs. The Table reports the number of recombinant nAbs expressed per each subject and their distribution based on the neutralization potency. 

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