益科思特2020年5月在美国冷泉港发表了新冠病毒治疗抗体文章-与世界分享我们的努力和成果

益科思特(Excyte)公司在美国冷泉港运营的bioRxiv发表新冠病毒治疗抗体文章，与世界分享我们的努力和成果。医药无国界，行医治，行拯救是我们的初衷与梦想

https://www.biorxiv.org/content/10.1101/2020.05.19.104281v1

bioRxiv preprint doi: https://doi.org/10.1101/2020.05.19.104281. this version posted May 19, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license. Isolation of and Characterization of Neutralizing Antibodies to Covid-19 from a Large Human Naïve scFv Phage Display Library Andy Q. Yuan1,2,*, Likun Zhao2 , Lili Bai2 , Qingwu Meng1,2 , Zhenguo Wen3,4 ,Yanhu Li2 , Daqing Guo2 , Shanshan Zhen2 , Xiaojun Chen2，Ji Yang2，Xiaoying Xue2 1. Excyte LLC, MD USA, 15601 Crabbs Branch Way, W123, Rockville MD 20855, USA 2. Yikesite (Beijing) Biopharma Development LLC, Suite B-306, 5 Kaituo Rd，Haidian District, Beijing, 100085, China 3. Beijing Institute of Petrochemical Technology, 19 Qingyuan N Rd, Daxing District, Beijing, 102617, China 4. Bonuo Biotech LLC. Suite. A-0141, 1 Qianping Road, Daxing District, Beijing, 102604, China *. Correspondence:Andy Yuan, PhD., Excyte LLC, 15601 Crabbs Branch Way, W123, Rockville MD 20855 USA Email: andyyuan@iexcyte.com Abstract SARS-CoV-2 (Covid-19) has caused currently ongoing global plague and imposed great challenges tohealth managing systems all over the world, with millions of infections and hundreds of thousands of deaths.In addition to racing to develop vaccines, neutralizing antibodies (nAbs) to this virus have been extensivelysought and are expected to provide another prevention and therapy tool against this frantic pandemic. Tooffer fast isolation and shortened early development, a large human naïve phage display antibody library,was built and used to screen specific nAbs to the receptor-binding domain, RBD, the key for Covid-19 virusentry through a human receptor, ACE2. The obtained RBD-specific antibodies were characterized byepitope mapping, FACS and neutralization assay. Some of the antibodies demonstrated spike-neutralizingproperty and ACE2-competitiveness. Our work proved that RBD-specific neutralizing binders from human naïve antibody phage display library are promising candidates to for further Covid-19 therapeutics development.Keywords: Covid-19, phage display, neutralizing antibody, screening, epitope Introduction Around December of 2019, pneumonia patients afflicted by an unknown type of coronavirus, which laternamed as 2019-nCoV, SARS-CoV-2 or Covid-19 by WHO, had emerged in Wuhan China. In the past 5 months, the highly contagious virus has since then quickly spread to almost every nation in the world,infecting more than 5 million people and resulting in more than 320,000 deaths1 . To this date, this pandemic marked the third highly pathogenic coronavirus human infection in the 21st century, after the outbreak of the deadlier Middle East Respiratory Syndrome coronavirus (MERS-CoV) and the Severe AcuteRespiratory Syndrome coronavirus (SARS-CoV-1). The whole world was caught off guard to this challenge.To flatten the incident curve, social measures such as quarantine, lockdown and distancing were widelyapplied, leading to a sudden pause of most economic activities. Among all the measures mobilized tocombat the plague, like to many others, scientific approach is the decisive and ultimate answer.In past months biological studies have found strong conservatives and resemblances between SARS-CoV- 1 and Covid-19 in genomic sequences, mode of infection and to certain extent, clinical pathology2,3,4 . Likedealing many infectious diseases, the first effective step whenever possible is always preventing theinitiation of an infection by isolation or prophylaxis measures such as antibody or vaccine. Vaccinespotentiating host immune responses and eliciting protective antibody production will quickly mobilize bioRxiv preprint doi: https://doi.org/10.1101/2020.05.19.104281. this version posted May 19, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license. humoral and cellular immunity defenses against subsequent viral invasion. When lack of vaccine, thepassive immunity conferred by specific monoclonal antibodies has well been recognized in the treatmentof many viral diseases, for example, FDA approved anti-RSV drug Synagis5,6 . Based on the limitedscientific data, treating Covid-19 patients with convalescent plasma from recovered donors appears safe, clinically effective and reduces mortality7 . Significant efforts have been made to develop therapeutic antiviral antibodies against SARS-CoV8 and MERS9. High viral biological similarities and promising animalefficacy of these antibodies highlight the potential to obtain Covid-19 specific neutralizing antibodies(nAbs) for its therapy and prophylaxis. The entry of Covid-19 to human cells has been recently reported that Covid-19 virus binds to the humanangiotensin-converting enzyme 2 (ACE2) through it spike protein, or S-protein, which is almost identicalin the infection of SARS-CoV-1 or MERS4,10 . The portion in S-protein, called receptor binding domain(RBD), is the exact motif that interacts with ACE211 . Details of the molecular interaction between RBD and ACE2 have been analyzed to atom level12, facilitating the design of blockade agents, including antibody to intervene the entry process. In the meantime, in a race to win this unprecedented battle, every secondmatters in the generation of lead antibody molecules. Instead of immunizing animals and screeningsubsequent hybridoma, we took advantages of an in-house very large naïve human antibody phage displaylibrary built in our lab. Here we summarized the construction of the library, the isolation andcharacterization of RBD nAbs, providing strong basis for further development of potential antibodyarsenals for COVID-19.Results Human antibody phage display library Human antibody phage display library has become the dominant route13, 14 to quickly obtain antibody leadsand develop antibody-based biotherapeutics to an almost given target. A large human naïve antibody phagedisplay library (Named as EHL Library) was built in-house based upon conventional phage displaytechnique, in a similar protocol reported earlier15 . To ensure the library to be a high-value research resource,we adopted multiple measures to achieve extensive diversity and super-size during the whole process.PBMCs of 37 donors from a variety of ethnic groups and nationalities across the world, including Latinos,African American, Caucasian, Chinese, Russians, Hindus, Vietnamese, Philippines, Native American,Japanese and Jewish etc. were collected to enhance the diversity range of antibody variable genes to beused in the scFv assembly. 7 subfamilies of variable heavy (VH) genes, 7 subfamilies of variable kappa genes ( Vκ ) and 11 subfamilies of variable lambda genes ( Vλ) were separately amplified and recovered.Two types of scFv libraries, VH-Vκ and VH- Vλ, were separately assembled and cloned into phagemidpADL-10b, resulting two types of scFv libraries, each containing more than 5×1010 colony forming unit(cfu) in size, after total 100 repeats of electroporation of ligated phagemid constructs. Random sequencingof 100 colonies of each library revealed over 80% of correct scFv-coding and full usage of all antibodysubfamily genes (data not shown). The utility and quality of the EHL Library were validated by successfulscreening of specific hits over a dozen of selected human targets, mostly tumor-associated antigens, withaverage KD of two digits nM (data not shown). Validation of Covid-19 spike-expressing cell lineA surrogate tool is essential to research on any very deadly contagious pathogens such as Covid-19, whosespike protein is of great needs not only as purified form but also as membrane one. A cell membrane protruding spike may not behave the same as it presents in its viral particle, the availability of such cell linecan serve most needs to some extent such as antibody binding examination and competition assays. Amammalian cell line ID8 was generated by transfection of 293FT with a pseudo lenti vector that containsgene encoding the full-length Covid-19 spike protein, a transmembrane motif (TM) and a 3xFLAG tag at C-terminal (Fig. 1, A) and selected under Blasticidin S hydrochloride. To verify the spike expression,structure and function, ID8 cells was stained by anti-FLAG-FITC (Fig. 1, B) or human ACE2-mFc,followed by anti-mouse-FITC (Fig. 1, C). and analyzed by FACS. Results showed that both stains exhibitedsignificant right shift of ID8 population (Fig.1, B and C, red peaks), validated the ID8’s ability to bindACE2, indicating the spike is in trimeric membrane status and mimics the function of Covid-19 spike16 .Biopanning naïve phage antibody library yielded multiple RBD hitsAiming to obtain antibodies that directly target to RBD of Covid-19 spike so that they have limited bindingsites, and great chance of interference to the interaction between spike and ACE2, we used RBD fusion(RBD-mFc) instead of full-length or subunit of Covid-19 spike protein as bait in the biopanning from EHL library in a solid phase approach17. 3 rounds of biopanning yielded vast enrichment as output titer increasedover 100 times (data not show) over that of 1st round. Upon monophage ELISA screening on the 3rd -roundoutput, more than 95% of clones were positive. After sequencing the scFv genes of positive hits, we foundvery focused clone specificity (2 uniqueness among 90 available scFv sequences, data not shown). Toharvest diversified binders, we turned to 2nd round output for ELISA screening (Fig2, A). Among 160positive hits we identified 42 unique clones, with many have minor differences in amino acids. Throughmulti-alignment of the scFv primary sequences, a phylogenetic tree was generated (Fig 2, B) to measurehomology gap between them.FLAG Spike ACE2-mFc Fig.1 Validation of Covid-19spike expression cell line A mammalian cell line ID8 wasgenerated by transfecting 293FTwith a pseudo virus that containsfull-length Covid-19 S protein, atransmem-brane motif (TM) and a Cterminal 3xFLAG tag. A. The cartoonillustration of the ID8 cells andmembrane anchoring of Covid-19spike (green), C-terminal FLAG(yellow) and binding of ACE2-mFc(blue) to S trimer. B. FACS plot ofintracellular staining of FLAG withanti-FLAG-FITC. C. FACS plot of ID8cell membrane sequential staining ofACE2-mFc, anti-mouse FITC.AB C was not certified by peer review) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/2020.05.19.104281. this version posted May 19, 2020. The copyright holder for this preprint (which By checking the amino acid sequences, we saw majority of the hit clones (37/42) belong to lambda scFvand only five belong to kappa scFv (data not shown), though equal amount of kappa and lambda scFvlibrary aliquots were mixed before first panning. Among the lambda scFv hits, most of them (30/37) havealmost same complementarity-determining region (CDR) compositions in both VH and Vλ, with sporadicdifferences in the framework region (FR), implying fast enrichment of antibody recognizing certain epitope. Membrane spike bound antibodies are specific for Covid-19Phage antibody hits tend to be false positive by ELISA detection, due to the stickiness of phage proteins18 and epitope/conformation differences between purified/truncated protein and membrane/full-lengthparental molecule19 or lose the reactivity because the once exposed epitopes in protein fragment becomeinaccessible in native protein. This is very true because in current case, the panning bait is a dimeric andfragmented RBD (RBD-mFc), while Covid-19 native spike is a trimer20 . To verify the authenticity of thephage antibody binders and check their ability to recognize cell membrane trimeric Covid-19 Spike, wefirst generated soluble antibodies to facilitate further characterization of their properties. Since many clonesare highly similar in CDR residues and only differ by a few FR residues, to reduce the work we transferred22 scFv genes (one of every two close members from above alignment) from phagemid to an in-housemammalian expression vector pFP and expressed them as scFv-huFc (human Fc) fusion. Upon protein-Apurification and aggregates removal to make sure monomer achieved over 95%, the antibodies were testedby FACS on above-generated ID8 cells to screen positive membrane covid-19 spike binders, which are thepotential neutralizing antibody candidates.8 out of the 22 expressed scFv-huFc antibodies demonstrated significant ID8 cell-binding capabilities withvaried shifts by FACS (Fig.3). We tried to rank the binders roughly by the positive percentages, which is 0 0.5 1 1.5 2 2.5 1 2 3 4 5 6 7 8 9 10 11 12 ELISA of Covid-19 RBD2nd round panning outputFig.2 Monophage ELISA screening Covid-19 RBD phage antibodies EHL library was panned with Covid-19 for 2 rounds. Single colonies form 2nd -round output was picked to preparemonoclonal phage antibody solutions, examined by ELISA with RBD, mouse Fc and 293F cells. Only RBD positivehits were resorted to DNA sequencing of scFv genes. A. Chart of absorbance (A450) on RBD plate ELISA. B. Aminoacid sequence multi-alignment of unique scFv clones positive for RBD binding.A B was not certified by peer review) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/2020.05.19.104281. this version posted May 19, 2020. The copyright holder for this preprint (whichthe reflection of apparent affinity instead of intrinsic affinity. Clone 1B1 seems to be the strongest spike binder among them. 1B11 and 5C2 are comparable intermediate ones and the rest five are close as mildones. Reviewing their amino acid sequences (data not shown), we noticed that 1B11 share almost the sameCDR compositions are the rest five hits except 5C2 and 1B1but differs in several FR residues, however1B11 exhibited significant higher P2 than its cognate members, indicating affinity beneficial effect of thosedifferent FR residues in 1B11. Future kinetics measurement should reveal the quantitative affinity gap. It is now known that the amino acid sequence homologies between MERS-CoV, SARS-CoV-1 and SARSCoV-2 are very high, especially the latter two, approximates to 75% for the spike proteins and are 73.7%for RBDs3 . More conserved in structure than primary sequence, the RBDs in both viruses bind the sameACE2 in different affinities21. Genetically it has been revealed that in RBD, some area is conserved, andsome are hypervariable22 . To investigate whether the 8 ID8 cell-binding positive antibodies from above have any cross reaction to closely related spike proteins of SARS and MERS coronavirus, they were furtherexamined by ELISA across SARS spike protein (SARS-S), MERS Spike protein (MERS-S) and Covid-19spike protein (Covid-19-S). As it showed in Fig.4, All 8 hits are exclusively specific for Covid-19 spike and none of them has any cross reactivity to the other two spikes, indicating structurally conserved RBDsare not quite immunogenic conservative among these coronaviruses. Fig.3 FACS examination of RBD-binding antibodies to Covid-19 membrane spike on ID8 cells. Soluble scFv-huFcof 22 hits that were RBD ELISA-positive were incubated with equal amount of ID8 cells, followed by goat anti-humanFc (PE conjugated). Stained cells were analyzed to draw dot-plot by FACS. P2 Gating was set based on the backgroundof secondary antibody staining and considered positive. Only clones having positive percentages were shown.Individual clone was labeled in each plot.1A12 1H2 1C10 5C2 1A5 1G6 1B1 1B11 was not certified by peer review) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/2020.05.19.104281. this version posted May 19, 2020. The copyright holder for this preprint (whichThe Covid-19 RBD specific antibodies bind to different epitopes Having obtained 8 Covid-19 spike specific human antibodies, we wondered how their binding epitopeslocating in its RBD and, more importantly, what is the potential impact on the binding of RBD to ACE2.Biolayer Interferometry (BLI) based instrument Blitz offers a simple straight forward evaluation method ofantibody-receptor interaction at protein level23 . Antibodies, RBD or ACE2 were loaded to appropriatesensors sequentially, typically that the previous step has been well equilibrated. The ascending or flatresponse curves recorded corresponding component incubated with sensors implied yes/no interactionamong proteins between adjacent layers (Fig.5). Some observations are clear to see from the Blitz results:All hit clones bind to RBD by Blitz (Fig.5, A,B,C); 1B1 can concurrently bind to RBD with the other 6antibodies (such as 1B11 etc.）except 5C2 (Fig.5 B,D); 1B1 mutually competes with 5C2 (Fig.5, C). Consistent with the competition between 1B1 and 5C2, when RBD is bound by 1B1, it can no longer bebound by 5C2, and vice versa. (Fig.5, D). 01234 1A12 1G6 1A5 1B11 1B1 1H2 1C10 5C2 Specificity of Covid-19 Ab hitsSARS-S MERS-S Covid-19-S Fig.4 Cross-reaction examination of Covid-19RBD hits by ELISA Binding of 8 COVid-19 RBD-hits was examinedby ELISA to the spike proteins (named as SARSS, MERS-S and Covid-19-S) for SARS-COV-1,MERS-COV and SARS-COV-2 (Covid-19) andmeasured by absorbance (A450). All hitsdemonstrated strong positive to Covid-19-S(yellow bars) and none cross-reacted to the othertwo spikes (green and blue bars).A B C D Fig. 5 BLITZ epitope mapping of Covid-19 RBD-hits antibodies (A). Rotational mutual interaction examination of the six hits (1B11, 1A5, 1G6, 1C10,1A12 and 1H2) was captured by protein A sensors, followed by RBD binding and oneof the six antibodies plus ACE2. RBD was bound well by individual antibodies but notconcurrent binding. (B). 1B1 concurrently bound to RBD with any one of the six otherantibodies ( 1B11, 1A5, 1G6, 1C10, 1A12 and 1H2). (C). 5C2 concurrently bound toRBD with any one of the six other antibodies ( 1B11, 1A5, 1G6, 1C10, A12 and 1H2). (D). 1B1 and 5C2 mutually competed in binding to RBD was not certified by peer review) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/2020.05.19.104281. this version posted May 19, 2020. The copyright holder for this preprint (whichCombining the amino acid sequence information and this BLI study, we drew a putative Venn epitope mapof the 8 RBD-antibodies and ACE2 on Covid-19 RBD (Fig.6). For the convenience of description, wecategorized the epitope as group Ⅰ (1B1 antibody), group Ⅱ (5C2 antibody) and group Ⅲ (antibody 1B11,1A5, 1A12, 1G6, 1H2 and 1C10). Spike+ cells are bound simultaneously by RBD-antibodies and ACE2In silicon examined by Blitz done above on the obtained RBD-antibodies implied their potential variedinterferences to the ACE2 interaction with Covid-19 Spike. Since their epitopes/affinities are different, westarted to investigate whether competition exists, or how much is it, between these antibodies and ACE2 inbinding to ID8 cells. A preliminary experiment was done to check if there was dual binding when bothACE2 and individual antibodies were added to ID8 cells. Equal concentration of ACE2-mFc fusion (mouseFc, MW 110KD) and any one of the 8 RBD-specific scFv-huFc (human Fc, MW 100KD) were mixed andincubated with ID8 cells before appropriate 2nd fluorescent reagents were added for FACS analysis. Dotplots of PE and FITC channels were depicted (Fig.7) for every sample.Dot-plots of all groups demonstrated strong ACE2 binding (FITC channel), which is consistent with earlierstudy that ID8 expresses functional Covid-19 Spike. On one side, the fact that ACE2+ population in allgroups are greater than 95% (Fig.7) implied limited blockade of RBD-antibodies to prevent Covid-19 spikefrom binding to ACE2 when both are present simultaneously at around equal concentration. On the otherside, all antibodies demonstrated positive binding to the ID8 cells as well, at varied extent from~11% (1H2)Fig. 6 Venn map of Covid-19 hits and ACE2 on RBDEpitope locations on RBD by the isolated 8 hit antibodies were deduced from theabove Blitz results and largely categorized into 3 groups (Ⅰ, Ⅱ and Ⅲ). 1B1 and 5C2have large overlapped sites in RBD and may block the most part of RBD-ACE2interaction interface. The group Ⅲ hits (1B11, 1A5, 1G6, 1C10, 1A12 and 1H2)binds to an independent site from 1B1 or 5C2, however may block a minor side ofACE2-RBD interaction interface. Fig.7 Dual binding examination of Covid-19 RBD-hit antibodies All 8 RBD hits separately were incubated together and ACE2-mFc at around equal concentrations before adding toID8 cells. Both antibody binding and ACE2 binding were detected by corresponding specific secondary antibodies(different fluorescent conjugation). Upper row from left: FITC-conjugated secondary control, 1A5, 1H2, 1C10, 5C2.Lower row from left: ACE2-mFc (alone, positive control), 1A12, 1G6, 1B11 and 1B1was not certified by peer review) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/2020.05.19.104281. this version posted May 19, 2020. The copyright holder for this preprint (whichbioRxiv preprint doi: https://doi.org/10.1101/2020.05.19.104281. this version posted May 19, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license. to ~94% (1B1). Again, 1B1 championed the ranking, followed by 1B11 and 5C2. By the percentage of dualpositive population, we can easily rank the antibodies in order of 1B1>1B11>5C2>1G6 etc. for theircompetitiveness against ACE2. Surprisingly the rest four antibodies (1H2, 1C10, 1A5 and 1A12), who share the same CDR compositions (but not exact amino acids in FR) as 1B11 and 1G6 (thus the similar/sameepitope), showed significant less binding on cells, implying weaker affinities caused by the different FRresidues. Overall the data here proved that there is varied extent of competitiveness between these RBDantibodies and ACE2 in binding to Covid-19 spike. RBD nAbs preventively block interaction between Covid-19 spike and ACE2 Next we explored another scenario where antibodies are much abundant than ACE2 (this is possible sinceantibody can be administered beforehand in large amount as prophylaxis measure). In this case we wantedto find out if any of these RBD-hits can significantly reduce the binding of spike to ACE2, i.e., neutralizethe virus (here again we used Covid-19 spike-expressing ID8 as surrogate). We first titrated theconcentration of ACE2-mFc on fixed amount of ID8 cells. ACE2 is abundantly and widely expressed inhuman epithelial cells, the total (the soluble and membrane) expression level is yet to know，although ACE2 in the serum level has been reported24 . We found that when ACE2-mFc was around 0.02 µg/ml ,~100% ID8 cells were positive by FACS (Fig.8, green histograms), indicating very strong interactionbetween Covid-19 spike and ACE2. For the neutralization assay, we set up a serial dilution (2 times down)of every candidate antibody from the 10 µg/ml to ~0.31 µg/ml (a point where there was minimal impact on ACE2-mFc binding to spike+ cells in a preparatory experiment) , added them individually to same fixedamount of ID8 cells and incubated enough time to saturate the cells (spike). Next, the saturationconcentration of ACE2-mFc was loaded to compete out antibody. Finally, only signals from FITC channel was collected, which were the MFI (mean fluorescence intensity) numbers of ACE2-mFc binding to ID8cells. Histograms of part of the diluted sample concentrations (shown in Fig.8) clearly demonstrated thatsome the RBD-hits, such as 5C2, can objectively block Covid-19 spike from binding to ACE2 at variousconcentrations. When there is competition between any RBD hit antibody and ACE2 in binding to Covid-19 spike, anincreased antibody concentration would lead to reduced ACE2 attachment, quantitatively represented byMFI. Indeed, after grouping the MFIs as shown in Fig.9, at 10 µg/ml (a clinically achievable serumconcentration) we observed among 8 of the antibodies, at least 3 hit antibodies (1G6, 1C10 and 5C2)demonstrated significant ACE2 attachment reduction, decreasing the MFI of ACE2-mFc by as much as60%, in comparing to negative antibody control. The rest 5 hits caused little to moderate inhibition at thisconcentration. As antibody concentration lowered down (less antibody binding), MFI increased, moreACE2-mFc occupied the ID8 cells. Overall a dose-dependent manner is observed for the antibody inhibition.Among them, 5C2 performed the best in neutralizing Covid-19 spike protein in binding to ACE2-mFcacross all dilutions to the lowest point, with around 50% reduction of MFI. Surprisingly, contrary to theprevious ranks where 1B1 seemed to be best in binding to RBD and probably have large portion ofoverlapped epitopes with 5C2, it demonstrated much mild inhibition of spike in binding to ACE2 in thisassay. This finding highlighted the importance of epitope location in order to be an efficient blocker besidesdecent affinity.Discussions In the report we described the construction of a naïve human antibody phage display library and the usageof it in isolation meaningful weapons against Covid-19. We have preliminarily characterized the epitopes,specificity and neutralization potentials of the obtained human antibodies to Covid-19 spike. Our studyoffered promising human nAb candidates for further development of antibody-based weapons against the SARS-COV-2.0 5000 10000 15000 20000 1B1 1B11 5C2 1A12 1A5 1G6 1C10 1H2 Iso-Ctrl MFI of ACE2 competed by RBD-specific Antibodies 10 5 2.5 1.25 0.625 0.31 Fig.8 Competition FACS evaluation of neutralizing potentials of RBD-hit antibodies Serial diluted RBD antibodies were preincubated separately with ID8 cells before adding of ACE2-mFc, whosebinding was analyzed by APC-conjugated secondary antibody. The FACS plot of ACE2-mFc with or withoutantibody is shown. Negative control (red), no antibody ACE2-mFc control (green), and only histograms of threeantibody concentrations 10 µg/ml plots (pink), 2.5 µg/ml plots (yellow) and 0.31 µg/ml plots (aqua) were shown. Upper row from left: 1B1, 1A12, 1B11, 1A5. Lower row from left: 1G6, 1C10, 1H2 and 5C2 .Fig.9. MFI change of ACE2 binding to ID8 cells in the presence of serial diluted RBD-antibodies. 8 Covid-19 spike positive antibody binders (1B1, 1B11, 5C2, 1A12, 1A5, 1G6, 1C10 and 1H2)and one isotype negative control antibody (iso-ctrl in the figure) were mixed with ID8 cells at serial diluted concentrations (10 µg/ml down to 0.31 µg/ml, 2 times dilution) before adding ofhuman ACE2-mFc, which has strong interaction with ID8. MFIs (shown in vertical axis) ofindividual antibody/concentrations cell group (horizonal axis) were complied.was not certified by peer review) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/2020.05.19.104281. this version posted May 19, 2020. The copyright holder for this preprint (whichbioRxiv preprint doi: https://doi.org/10.1101/2020.05.19.104281. this version posted May 19, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license. Antibody Phage display technology has contributed antibody drugs such as FDA-approved blockbuster(Humira) and many clinical stage testing candidates25 . Generally, immune libraries are preferred for thegeneration of antibodies against infectious disease related targets due to the biased antibody repertoires asa result of exposure and in vivo evolution of immune response to the infection26 . However, the targetspecific libraries must be constructed each time to get enriched antibodies against antigens from differentinfections. A human PBMC-derived naïve phage naïve display antibody library is a ready-to-go tool, beingparticularly valuable resources to general targets and shortened R&D process when developing antibodybased clinical therapeutics. Diversity and library size are the two most critical parameters in generating auseful library for any given target27. In the study we first reported how such a library was built and became a key resource to the R&D of antibody-based diagnostics and biotherapeutics .With that advantage we quickly launched a campaign to screen hits, targeting Covid-19 spike (S) protein.Since the outbreak of SARS, many reports have published to elucidate how the spike of corona virus is responsible for interacting with host cell receptors, mainly human ACE2 to initiate infection28,29 .Structurally, the receptor binding domain (RBD) locating from AA 321- 535 in S protein, is the exactportion that interacts with ACE212,22 . Aiming to have better chance in obtain potent nAbs that can efficientlyblock RBD-ACE2 interaction, we used engineering expressed RBD as bait in solid phase biopanning.Dozens of unique primary positive phage antibodies to RBD were selected by monophage ELISA andproved to be very specific for Covid-19, with no cross binding to the other two closely related corona virusmembers, SARS-CoV-1 and MERS-CoV. Through BLI analysis on Blitz these antibodies span at least twoindependent groups of epitopes, each occupying part if not the complete ACE2 binding site in RBD.Additionally, their viral neutralizing potency was evaluated by competition FACS. 3 of the 8 hitsdemonstrated mild to significant competition in a concentration dependent mode to reduce spike bindingto ACE2, suggesting their preventative and therapeutic potentials in combating the Covid-19 pandemic. One of the hits championed the competition assay, exhibited promising development value. Preliminarymicroneutralization study on Covid-19 pseudo virus has proved some of the RBD-antibodies in this reportblocked viral infection (raw data).Since outbroke last December to this date the Covid-19 viral destruction is still ongoing with no foreseeableend. Without any validated medicines clinically available currently, diagnostic, preventive and therapeuticremedies are desperately needed should the virus last long and come back soon. NAbs are historicallyeffective in fighting against viral pandemics as they effectively inhibit virus’s entry by preventing viralattachment or membrane fusion30,31 and very likely to play a critical role to fight Covid-1932 . As late asfrom the outbreaking of SARS, MERS, Ebola to Zika, to Covid-19 convalescent plasma7, 33,34 which maycontain polyclonal nAbs collected from recovered patients have been tested to treat seriously ill people andhave shown certain reduction protection in some cases. Scientific studies of the nAbs to SARS, MERS onanimal models proved their protection effect. Currently researchers all over the world are racing to isolatednAbs from Covid-19 immunized animals (llama)35 , available SAR-Cov-1 neutralizing antibody36 and/orhuman B cells of infected patients37 to develop potential therapeutics. Classical antibody neutralization is strictly defined as the reduction in viral infectivity by the binding ofantibodies to the surface of viral particles (virions), thereby blocking a step in the viral replication cyclethat precedes virally encoded transcription or synthesis38,39. In that regard nAbs are the best correlate ofprotection from viral infection after vaccination. Nabs offers the direct function of abolition of a pathogen’sinfectivity upon binding, with no participation of any other components of the innate or adaptive immunesystem. So, neutralization is probably the most robust and powerful function that antibodies exert againstviruses. In a broadly sense antibodies can neutralize viral infectivity in several additional ways40,41. Theymay block viral uptake into cells, prevent uncoating of the genomes in endosomes, cause aggregation ofvirus particles, or lyse the viral membrane through by antibody-dependent cellular cytotoxicity (ADCC)and antibody dependent cellular phagocytosis (ADCP). These protective antibodies are targetingcomponents in other viral cycle steps, as well as utilizing human immune system to clear virus42 . bioRxiv preprint doi: https://doi.org/10.1101/2020.05.19.104281. this version posted May 19, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license. While there is little doubt that clinical Covid-19 nAbs can be developed sooner or later, there is a big hurdle in front of us to overcome, that is, strain coverage and escape. Diversities of collected 86 variant covid-19genomes have been recently unveiled43 . Of note, deletions in spike glycoprotein and mutations located inRBD have been found. More mutations are likely to emerge as the virus continues to transmit across ethicsand global regions. Subsequently monoclonal antibody reacting to specific epitope in RBD is probablyvulnerable to the mutations and glycosylation modifications. Additional studies to dissect the exactrecognizing residues of any candidate neutralizing Covid-19 antibody are necessary before moving tofurther development. Alternatively, as backup it’s rational to develop broad nAbs to more conservativeregions in spike44,45. In fact, nAbs targeting a highly conserved epitope in RBD has been isolated fromconvalescent plasma of SARS patient46 . Combination therapy with multiple non-competing nAbs recognizing different epitopes on the RBD/spike may be another ideal direction to avoid immune escapeand increase coverage47 . In summary, we have reported here the isolation and characterization of nAbs to Covid-19 RBD from ahuman naïve phage display library. Our work provided promising antibody candidates as potentialdiagnostic, prophylaxis and therapeutic reagents for further development. Materials and MethodsConstruction of EHL human naïve phage display library A very large naïve human single chain variable fragment (scFv) phage display library was constructedsimilarly as previously reported15. Briefly, PBMCs (10 million/donor, purchased from BuyPBMCs,Precision for Medicine, https://buypbmcs.com/) collected from healthy donors to extract the total RNA(Cat#74104, RNeasy®, QIAGEN, Valencia, CA). First strand cDNA was generated by usingSuperScript™ III First-Strand Synthesis System (ThermofisherScientific, cat#18080051) fromevery RNA sample (5 µg RNA /rxn) with random hexamer primers or Anchored Oligo(dT)20(ThermofisherScientific, cat#12577011) before pooling. Variable heavy (VH) and light (Vκ and Vλ) geneswere amplified separately from the pooled cDNA samples by two rounds of PCR using HotStarTaq (Qiagen,Cat# 203443) and human antibody sequences primers derived from previous report15 . A pair of SfiI siteswere incorporated into the 5’-end of VH primers and 3’-end of Vκ and Vλ primers to facilitate followed phagemid cloning. Overlapping of VH-Vκ or VH-Vλ scFv genes were assembled separately. AmplifiedscFv PCR fragments were purified with Gel Extraction kit (Qiaquick Gel Extraction Kit, Cat# 28706,QIAGEN), and subjected to SfiI (Fast Digest SfiI, ThermofisherScientific, cat# 1824) digestion.To construct the scFv phagemid library, vector pADL-10b (Antibody Design laboratories, cat#PD0105)was completely digested with SfiI (ThermoFisherScientific, cat#FD1824) followed by dephosphorylationtreatment (CIAP, ThermoFisherScientific, cat#18009027) before purification with PCR purification kit(QIAquick PCR Purification Kit, cat#28104). Digested scFv and pADL-10b was ligated by the ratio of 3:1,using T4 DNA ligase (NEB, Cat#M0202) and incubation overnight at 12°C. Ligation mixture was purifiedand electroporated into TG1 E. coli (Lucigen, Cat# 60502-2) over fifty shocks for each library. CombinedTG1 slurry culture was grown in non-expression (NE) media (2YT containing 1% glucose and 100 µg/ml carbenicillin) at 37°C for one hour before a serial of 10 times dilutions were made to measure theapproximate size of the kappa or lambda scFv library. The rest culture was expanded in NE media, grewto OD600 around 2, and aliquots of culture were frozen as seeds for future production of phage libraries.Propagation of combinatorial phage libraries rescued by M13K07 helper phage (Antibody Designlaboratories, cat#PH010L) was performed as published procedures47. Phage antibody library aliquots werestored at -70°C in a solution of 50% glycerol and named as EHL library. Construction of Covid-19 spike-expressing cell linebioRxiv preprint doi: https://doi.org/10.1101/2020.05.19.104281. this version posted May 19, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license. To express the spike (S) protein of SARS-CoV-2 in mammalian cells, a codon optimized cDNA(GenBank:NC_045512.2) encoding the full-length S protein, a transmembrane motif (TM) and 3xFLAGtag was synthesized and cloned into a pRRL-derived vector (R48) by XbaI/XmaI, yielding pRRL-19SFLAG-BSD. 293FT cells were transfected with lenti-viral made from SARS-CoV-2 S plasmid and othervectors (pLP-1-Gag/Pol, pLP-2-Rev and pLP-VSVG, all in-house made) containing packaging elements. Media was replaced once 12-16 hours after transfection. Pseudo virus stock (named as LV-19S) wascollected from the supernatant and filtered through 0.45 µm membrane. To build stable cell line expressing Covid-19 S, ID8 cells (a gift from Institute of immunotherapy, FujianMedical University) was seeded at 10,000/well in DMEM (Gibco, cat#31600-034) +10% FBS (GeminiBio,cat#100-500 ) in6-well culture plate and incubated overnight. The next morning, 1ml of LV-19S (andvehicle control) was added and incubation continued 24 hours before changing the media with Screeningmedia (DMEM+10% FBS containing 10 µg/ml Blasticidin S hydrochloride (Invitrogen, cat#21001)).Screening media was changed every 3 days until day 7 when non-infected cells all gone. Continue theinfected cell culture, which is the stable cell line, maintained in DMEM+10% FBS containing 8 µg/mlBlasticidin S hydrochloride.Panning of RBD and monophage ELISA Solid phase biopanning of hits from a phage display antibody library was conducted as previously reported49 with minor modifications in library blocking. Briefly Covid-19 spike RBD (Sinobiological, cat#40592- V05H，mouse Fc fusion) protein was coated at 2µg/ml in wells of a 96-well Nunc Maxisorp microplateand placed in a refrigerator overnight. One each of Kappa and lambda EHL Library aliquots were mixedand blocked with 4% milk PBS (MPBS), 10 million 293F cells (to deplete potential binders to residualprotein contaminated in vendor’s RBD) and 10µg/ml mouse IgG (to deplete mouse Fc binders) beforeadding the library stock to the RBD-coating wells. The rest steps are standard protocol49 . Two to three cycles of biopanning were performed before ELISA screening was conducted to select the specific RBDbinding phage antibodies from the output libraries. Monophage ELISA was carried out essentially as described50, 51 with RBD as coating antigen, mouse Fc and 293F cells as negative control.Soluble expression and purification of scFv-Fc Unique scFv clones from DNA sequencing analysis were selected for further subcloning and fusionexpression with human Fc. ScFv genes were cut and paste with SfiI and NcoI sequentially from thephagemid vector into pFP vector (in-house made). Freestyle 293F (ThermoFisherScientific, Cat#R79007)cell was transfected with purified miniprep plasmid DNA (QIAgen minipreps kit, Qiagen, Cat#29107) asdescribed (Lipofectamine™ RNAiMAX Transfection Reagent, ThermoFisher Scientific,Cat#13778500) in 30-ml volume. scFv-Fc product was purified by protein-A affinity Purification column(HiTrap® Protein A High Performance, GE Life science, cat#GE17-0402-01) and size exclusionchromatography (GE SEC, superdex 200, GE Life science, cat#GE17-0612-10) to remove aggregates,whenever necessary. Monomer percentage was analyzed by HPLC-SEC52 . Blitz characterization of hits and epitope mapping The binding sites of obtained antibodies to RBD was examined by Blitz system (ForteBio, PallLifesciences). 10 µg/ml Biotinylated RBD protein was captured by streptavidin biosensor (ForteBio, cat#18-5019), followed by sequential binding of 10 µg/ml RBD antibody A then 10 µg/ml antibody B, in thepresence of 10 µg/ml antibody A, or vice versa. Alternatively, 10 µg/ml antibody A was captured by antihuman Fc biosensor (ForteBio, cat# 18-5060) until saturation, followed by sequential binding of 10 µg/mlRBD, antibody B. Interaction curves were collected and analyzed to determine the relative binding locationsof the antibodies on RBD. FACS examination of hits binding to SpikebioRxiv preprint doi: https://doi.org/10.1101/2020.05.19.104281. this version posted May 19, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license. Spike expressing cell 1D8 was maintained in 1640 media. To examine if spike trimer was expressed, bothFITC conjugated anti-FLAG (CST, cat#8146) and ACE2-mFc (Sinobiologics, cat#10108- H05H) andFITC-anti-mouse-IgG (Biolegend , cat#405305) were incubated with 1-2 million 1D8 cells followingstandard FACS protocol and analyzed by Beckman. To examine if the antibody hits from ELISA screening were still capable of binding to native spike trimer,the individual antibody hit at 5 µg/ml and/or ACE2-mFc was incubated with 1-2 million 1D8 spike+ cellsand detected by anti-human-Fc PE (invitrogen , 12-4998-82) and FITC Goat anti-mouse-IgG (Biolegend ,cat#405305 ) or APC Goat anti-mouse-IgG (Biolegend , cat# 405308 ) following standard FACS protocoland analyzed by Beckman FACS (CytoFLEX, CytExpert2.0).Neutralization assay of RBD-antibodies by FACS To check whether ACE2-Spike interaction can be blocked by the isolated nAbs, individual titrated antibodyfrom 10 ug/ml down to 0.31 ug/ml (2 times down) was incubated with 1-2 spike+ cells for 1 hour at 4°Cbefore adding of 0.02 ug/ml ACE2-mFc followed by FITC conjugated anti-mouse IgG (Biolegend ,cat#405305) and the MFI of FITC and positive percentage were collected.Statement Andy Q. Yuan is the co-founder and CSO of Yikesite Biopharma Development LLC, which wholly-ownsExcyte LLC. 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