WO2022023292A9

WO2022023292A9 - Corona virus spike protein-targeting antibodies and use thereof

Info

Publication number: WO2022023292A9
Application number: PCT/EP2021/070904
Authority: WO
Inventors: Hans-Martin JÄCK; Klaus Überla; Thomas Winkler
Original assignee: Friedrich-Alexander-Universität Erlangen-Nürnberg
Priority date: 2020-07-31
Filing date: 2021-07-26
Publication date: 2022-05-05
Also published as: WO2022023292A2; WO2022023292A3

Abstract

Antibodies that bind to the spike (S) protein of the new coronavirus CoV-2 are provided. Some of them bind to the N-terminal domain (NTD), the receptor-binding domain (RBD), the S2 domain, the S1 domain or the trimeric spike ectodomain. Some antibodies inhibit the binding of the CoV-2 spike protein to the human angiotensin-converting enzyme 2 (hACE- 2). Preferably the antibodies neutralize and prevent CoV-2 infection of cells and animals. Also provided are hybridoma cell lines that produce, and most preferably, secrete into cell culture media the antibodies of the invention. Also provided are eukaryotic expression vectors that encode fully human CoV-2 neutralizing antibodies. The antibodies of the invention are useful for diagnosing and treating disorders associated with CoV-2 infections and for preventing symptoms of a CoV-2 infection as well as CoV-2 infection as such.

Description

Corona Virus Spike Protein-targeting antibodies and use thereof

INTRODUCTION

COVID-19 is a new lung disease that is caused by SARS-CoV-2, a new variant of the human Coronavirus family. The disease was first described in December 2019 in Wuhan, China, and the complete DNA sequence was available to the scientific community at the beginning of January 2020. As of today, over 107 million confirmed COVID-19 cases, and 2.3 million deaths had been reported worldwide. Therefore, we are in desperate need of a protective vaccination and an effective treatment to oppose this disease.

Coronaviruses are host cell membrane-enveloped particles with a ca. 30kb-long RNA genome associated with the nucleoprotein (N). The crown-shaped appearance (lat. corona) of the virus is determined by the trimeric spike (S) glycoprotein in the virus envelope (Li , Ann. Rev. Virol. 2020; 3:237). The S protein also mediates the initial binding of coronaviruses to the corresponding host cell receptor via its receptor-binding domain (RBD), thereby determining the infectivity and cell tropism of the virus. CoV-2, for example, infects only cells expressing membrane-anchored angiotensin-converting enzyme 2 (ACE2, Hoffmann et al., Cell 2020; 181 :271). Therefore, the S protein is the Achilles heel of the CoV- 2 virus and the target of almost all active vaccines and passive approaches with potent neutralizing antibodies.

The first two RNA-based vaccines from Biontech/Pfizer (Sahin et al., Nature 2020; 586: 59) and Moderna (Jackson et al. , NEJM 2020; 383:1920) and an adenovirus-based vector vaccine from AstraZeneca (Folegatti et al., Lancet 2020; 396:467) were approved in the EU on December 21, 2020, January 6, 2021 , and January 29, 2021 , respectively. However, it is still unclear whether the currently approved and tested vaccines generate long-lived plasma cells (at least over 2-3 years) and faster-responding memory B cells, both of which are necessary for the maintenance of long-lasting neutralizing antibodies in the blood. However, memory B cells (Hartley et al, Sci. Immunol. 2020, online 10.1126/sciimmunol.abf8891; Gaebler et al, Nature 2021 , online s41586-021-03207-w ) and antibody-secreting plasmablasts could be detected in recovered COVID-19 subjects with relatively low serum antibody levels after 6 months (Rodda, preprint 2020, https://doi.org/10.21203/rs.3. rs- 57112/v16). However, whether this immune memory remains stable over years remains to be shown in longitudinal studies. Recently published data also suggest that antibodies generated, for example, by the Novavax vaccine (News release 28.01.2021 - https://ir.novavax.com/, Wibmer et al., www.biorxiv.org 2021.01.18.427166v) or by natural infection recognize the South African virus variant less well or not at all. This is also true for some antibodies already used or scheduled for therapy. For example, Eli Lilly's LY-CoV555 (approved in the U.S.) was completely inactive against the South African variant, and Regn 10933, one of the two antibodies in an approved antibody cocktail from Regeneron, was 58-fold less effective. The second antibody in the Regeneron cocktail retained its neutralizing ability, as did the complete cocktail (Wang et al, https://www.biorxiv.Org/content/10.1101/2021.01.25.428137v2).

As experienced with the first vaccines from Biontec/Pfizer and Moderna, there is also a shortage of vaccine doses due to limited manufacturing capabilities and logistic problems. Also, low-income counties did not reserve enough vaccine doses in advance. Further, about 20% of U.S. citizens, and very likely E.U. citizens, are either reluctant to get vaccinated or, due to medical conditions, will not respond adequately to vacation or cannot be vaccinated at all. Consequently, it could take years to protect the entire world population by active vaccination.

Another problem is that once the elderly and the risk groups (about 20% of the German population) are vaccinated and the number of deaths and hospitalizations has decreased, there will be immense economic, social, political, and also legal pressure to end the Corona measures. Consequently, there could be a considerable increase in new infections, especially among younger people. Because of the high numbers, intensive care units will fill up with younger people and unvaccinated at-risk groups, and many will die. Besides, ongoing new infections will increase the risk of the emergence of more virulent virus mutants that are resistant to current vaccination protocols or some of the already approved antibodies due to the high specificity of these interventions.

Human monoclonal antibodies produced by biopharmaceutical techniques are an excellent solution to prevent this scenario. Human antibodies are precise magic bullets without the expectation of any severe side effects. They attack the coronavirus immediately after infection, bridging a hole that vaccines cannot fill: they can be used as an emergency drug to immediately treat Corona-infected people at risk for a more severe disease progression and slow down the spread of the viruses. By far the best target for antibodies is the S protein on the surface of CoV-2, which is necessary for virus entry into the target cell. Several human monoclonal CoV-2-neutralizing antibodies isolated either from convalescent COVID-19 patients, from synthetic human antibody phage libraries, or from CoV-2-immunized transgenic mice with human antibody genes have been described (review in Shi et al., Stem Cell Res. 2020, j.scr.2020.102125). Clinicaltrials.gov currently lists studies with 12 single antibodies and 6 cocktails with two antibodies, all targeting the spike protein of CoV-2. Three of these approaches have reached phase 2/3 clinical trials (Glaxo and Celltrion with single antibodies and AstraZeneca with a cocktail of two antibodies), and last December two [from Regeneron (Weinreich et al. NEJM 2020, 384:238) and Eli Lilly (Chen et al., NEJM 2021 ; 384:229)] received emergency approval from the U.S. Food and Drug after only 10 months in development. The latest news from both Regeneron and Eli Lilly is very encouraging. For example, Regeneron's combination of two antibodies completely prevented symptomatic infections in a group of 186 people exposed to the virus through a familial constellation. In contrast, 8 of 223 people in the placebo group showed symptomatic infection (Amber Tong, Endpoint Views, January 21, 2021). And recent data from Eli Lilly show "that two antibodies are better than one." For example, the combination of two antibodies reduced to-do cases and hospitalizations by 70% in coronavirus patients. In a group of 1,000 patients, there were only 11 such events in the treatment group and 36 in the placebo group (Gottlieb et al, JAMA 2021, jama.2021.0202).

Recently published data, however, suggest that antibodies generated, for example, by the Novavax vaccine (News release 28.01.2021 - https://ir.novavax.com/, Wibmer et al., www.biorxiv.org 2021.01.18.427166v) or by natural infection recognize the South African virus variant less well or not at all. This is also true for some antibodies already used or scheduled for therapy. For example, Eli Lilly's LY-CoV555 (approved in the U.S.) was completely inactive against the South African variant, and Regn10933, one of the two antibodies in an approved antibody cocktail from Regeneron, was 58-fold less effective. The second antibody in the Regeneron cocktail retained its neutralizing ability, as did the complete cocktail (Wang et al, htps://www.biorxiv.org/content/10.1101/2021.01.25.428137v2).

Hence, a small set of approved neutralizing antibodies from only a few companies might not secure the local population and will not be available in sufficient quantities to meet demand during an outbreak triggered by vaccine- and antibody-resistant virus mutants. Therefore, a comprehensive arsenal of clinically tested therapeutic antibodies, preferentially as a cocktail of at least two non-overlapping from local production pipelines, will prevent a more severe disease progression in COVID-19 patients in an early stage of their disease and slow down virus dissemination.

Consequently, there is a need in the art for an antibody useful in the treatment and prevention of COVID-19 infections.

SUMMARY OF THE INVENTION

The isolation and characterization of new, highly potent antibodies that bind against the spike protein of CoV-2, or in particular to the membrane-bound CoV-2 spike protein are disclosed. Some of these antibodies prevent virus infection in vitro and in vivo.

Such antibodies can neutralize CoV-2 by interfering with virus entry into the host cells. For example, antibodies can neutralize by directly blocking the binding of the Spike protein’s receptor-binding domain (RBD) to its host entry receptor angiotensin-converting enzyme 2 (ACE-2). Alternatively, neutralizing antibodies could affect virus infection from interacting with sites other than the RBD. Furthermore, antibodies against CoV-2 that alone do not neutralize CoV-2 could enhance the activity of neutralizing antibodies. All these activities of anti-CoV-2 spike antibodies are valuable drugs to either protect from infection or treat CoV- 2-infected people or to detect CoV-2.

The new antibodies are preferably highly potent human monoclonal antibodies that bind to spike protein of CoV-2, specifically preventing virus infection.

The present inventors, i.e., the CoronoVirus Erlangen (CoVER) team, in a research program identified CoV-2 spike protein-binding antibodies by the conventional hybridoma technology from the spleens of a CoV-2 spike-vaccinated mice producing only antibodies with whole human variable (V) regions (Fig. 1). This so-called TRIANNI line was established by replacing all mouse VH, VK and VL gene segments with the corresponding human gene regions (Fig. 1 , patent U.S. 2013/0219535 A1). This innovative platform allows the access of the entire human antibody repertoire in a single organism,

The first fusion from a TRIANNI mouse immunized once with CoV2 spike-encoding plasmid DA and boosted twice with purified trimeric spike protein identified in a flow cytometric assay 21 hybridoma lines secreting CoVER (CVR, equivalent nomenclature: TRES) antibodies against the membrane-bound CoV-2 spike protein (Fig. 2 and 3), but surprisingly to none of the other five human Coronaviruses (data not shown). Nine of the CoVER hybridoma antibodies (CVR) that bound to CoV-2 spike protein expressed on the surface of HEK cells (Fig. 2 and 3) neutralized CoV-2 in cell culture (Fig. 4). The other antibodies bound to different regions of CoV-2 spike protein and can be used for diagnostic assays. Three (CVR6, CVR224 and CVR328) of the 11 neutralizing CVR antibodies were selected and used for in vivo experiments with mice transgenic for human ACE2. As shown in Figure 5, all three efficiently protected mice from COVID-19 (Fig. 5).

Three of 11 neutralizing antibodies (CVR6, CVR224 and CVR567 - identical with TRES- 6.15, TRES-224.2 and TRES-567.4 respectively) efficiently blocked the binding of membrane-bound CoV-2 Spike-2 to angiotensin-converting enzyme 2 (ACE-2, hACE-2), the host receptor of CoV-2, with a half maximal inhibitory concentration (EC50) of < 5nM, e.g., between 0.1 and 1 ,2 nM (Fig. 6), recognized an epitope in the receptor-binding domain, RBD, by Elisa and immunblots (Fig. 7) with an affinity of KD< 1 nM (Fig. 8) and substantially neutralized wildtype CoV-2 in vitro with IC50 values < 1 nM, e.g., between 0.01 and 0.11 nM (Fig. 4B).

Sequence analysis confirmed the clonal relationship of the three cluster 1 antibodies CVR6, CVR224 and CVR567 (see SEQ ID NOs.: 1-6). All three carry an identical L chain with 5 somatic mutations in the antigen bindings loops (complementary determining regions, CDR) and one in the framework (FR). They utilize the same VDJ H chain exon with four somatic mutations in the framework. CVR224 contains two additional mutations in the CDR1 and CDR2 and an unusual change of a Tyr at position 115 (position 95 without leader sequence, which ends at position 19 of SEQ ID NOs 1 , 3 and 5) that is conserved in 98% of all human VH regions.

The CoV-2 binding (Fig. 9A) and virus-neutralizing activity (Fig. 9B) could be confirmed with all three cluster 1 antibodies carrying the human VH and VL regions from the CVR hybridomas and the constant human gammal and human kappa region, respectively.

The other six CoV-2 neutralizing antibodies are secreted from a group (cluster 2) of clonally related CVR hybridomas (mature peptide sequences SEQ ID NOs.: 37-48). Sequence analysis revealed identical and productive VDJ and VL joining sequences for the H and L chains. The six H chains differ in the number and positions of amino acid changes in their H and L chains (Fig. 11 B). All six CVR cluster 2 antibodies bound to the spike protein (Fig. 8B) and neutralized CoV-2 (Fig. 4) with affinities and IC50 values, respectively, in the subnanomolar range. Moreover, and most importantly, CVR328 (the only one sofar used in this experiment) prevented virus replication and death in CoV-2 infected mice expressing the human ACE-2 entry receptor (Fig. 6). The six cluster 2 CVR antibodies did not recognize RBD in Elisa assays (Fig. 7B), did not interfere with ACE-2 binding to the spike’s RBD (Fig. 6A), and interacted with a not yet identified epitope in the so-called N-terminal domain (NTD) of the spike (Fig. 7A). Further, competition experiments clearly showed that none of the six cluster 2 antibodies prevented the binding of the cluster 1 antibodies CVR6 or CVR224 to the S protein (Fig. 10). Therefore, cluster 2 CVR antibodies bind to an S protein epitope distinct from the RBD epitope of the cluster 2 antibodies CVR6 and CVR224.

The invention provides antibodies that bind to the CoV-2 spike protein, preferably to the membrane-bound CoV-2 spike protein. The sequence of the entire SARS-CoV-2 genome (gene bank accession NC_045512) and the Spike protein are disclosed in Wu et al., Nature 579 (7798), 2020, 265-269. The receptor-binding domain (RBD) of the CoV-2 Spike protein is disclosed in Hoffmann et al. Cell 181 (2), 2020, 271-280 and in Walls et al., Cell 181 (2), 2020, 281-292.

Preferably, in a first aspect of the invention, the antibodies inhibit binding of RBD of the CoV- 2 Spike protein to angiotensin-converting enzyme 2 (ACE-2, hACE-2), more preferably the antibodies inhibit binding of CoV-2 to angiotensin-converting enzyme 2 (ACE-2, hACE-2), most preferably, the antibodies neutralize and prevent CoV-2 infection of cells. These antibodies may be cluster 1 antibodies (see Figure 3). Such antibodies are also useful for diagnosis and allow for specific labelling of the RBD subdomain of the CoV-2 Spike protein. Examples of the antibodies of the first aspect of the invention are the antibodies characterized by SEQ ID NOs: 1-6.

The invention - in a second aspect - also provides antibodies that neutralize and prevent CoV-2 infection of cells albeit they do not bind to RBD of the CoV-2 Spike protein. These antibodies may be cluster 2 antibodies as described herein below - see Figure 3. Such antibodies preferably bind to the NTD of the CoV-2 Spike protein. Such antibodies are also useful for diagnosis and allow for specific labelling of the NTD subdomain of the CoV- 2 Spike protein. Examples of the antibodies of the second aspect of the invention are the antibodies characterized by SEQ ID NOs: 37-48.

The invention - in a third aspect - also provides antibodies that may not neutralize nor prevent CoV-2 infection of cells and bind to S1 of the CoV-2 Spike protein. These antibodies may be cluster 3 antibodies as described herein below - see Figure 3. Such antibodies are useful for diagnosis and allow for specific labelling of the S1 subdomain of the CoV-2 Spike protein. Examples of the antibodies of the third aspect of the invention are the antibodies characterized by SEQ ID NOs: 49-60.

The invention - in a fourth aspect - also provides antibodies that may not neutralize nor prevent CoV-2 infection of cells and bind to various subdomains of the CoV-2 Spike protein. Some of these antibodies are exemplified in Figure 3. Such antibodies are useful for diagnosis and allow for specific labelling of respective CoV-2 Spike protein subdomains. Examples of the antibodies of the fourth aspect of the invention are the antibodies characterized by SEQ ID NOs: 61-102; see also Tables 1 and 2.

Also provided by this invention are hybridoma cell lines that produce, and most preferably, secrete into cell culture media the antibodies of the invention. In addition, expression vectors are constructed that allow the production of completely human antibodies that bind to the CoV-2 spike protein (Fig. 9) and neutralize the virus. The antibodies of the invention are useful for detecting CoV-2 spike protein in diagnostic assays and some of them (i.e., cluster 1 and cluster 2 CVR antibodies, for treating various disorders associated with CoV-2 infections and for preventing several symptoms of a CoV-2 infection as well as CoV-2 infection as such.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 depicts the Immunization scheme of human antibody mice (TRIANNI mice) with CoV-2 spike protein. TRIANNI mice were generated by replacing mouse VH, VK and Vlambda gene segments with all human VH, Vkappa and Vlambda gene segments. The mice were immunized either by electroporation with a vector encoding the SARS-CoV-2 spike protein (pCG1_CoV_2019S) and purified soluble trimeric spike in MPLA adjuvants (Fusions 1 and 2) or with self-amplifying SARS-CoV-2 spike protein-encoding RNA (nCoV saRNA, Fusion 3). Spleens were fused by the PEG method with the hybridoma line Sp2/0. MPLA, monophosphoryl Lipid A from Salmonella Minnesota R595 - TLR4-based adjuvant; PEG, polyethylene glycol.

Figure 2 depicts the flow cytometric identification of CoV2 spike binding hybridoma antibodies.

(A) HEK-293T cells were co-transfected with the PEI method with a GFP reporter plasmid and a pCG1-based expression vector for the spike protein of SARS- CoV-2 (CoV-2: position 21580 - 25400 from accession no. NC_045512). 2 days after transfection, cells were stained for 30 min on ice with hybridoma CVR antibodies. (B) Bound antibodies were detected in the gated GFP-positive fraction of transfected HEK293T cells by flow cytometry with a fluorochrome-conjugated goat anti-mouse pan-IgG antibodies. Fl, fluorescence intensity.

Figure 3 summarizes the activities of the CVR antibodies of fusion 1.

Flow binding assays are described in Fig. 2, affinity assay by Elisa assays in Fig. 8, ACE2 blocking assays in Fig. 6, and virus neutralization assays in Fig. 4. CVR, CoronaVirus Erlangen; y, yes; n, no signals; +. Signal detected; Muc, CoV2 isolate from a COVID-19 patient in Munich; ER-1, the first CoV2 isolate from a COVID19 patient in Erlangen.

Figure 4 depicts in vitro CoV2 neutralization assays with CVR hybridoma antibodies.

Vero E6 cells were incubated with wild type SARS-CoV-2 Erlangen-1 with increasing concentrations of the respective (A) CVR hybridoma antibodies or (B) purified CVR hybridoma antibodies. SARS-CoV-2 infection was quantitated after 20 to 24 hours by staining with purified IgG from a convalescent COVID-19 patient and a fluorescence-labeled anti-human IgG using an ELISPOT reader. One representative experiment of at least two experiments performed in triplicates with SEM and the mean IC50 of all experiments is shown. IC50s were calculated with inhibitor vs. variable slope (four parameters) fitting curve with GraphPad Prism 7.02. Figure 5 depicts the therapeutic and prophylactic In vivo efficacy studies with CVR lead antibodies.

(A) Experimental set-up to determine the prophylactic and therapeutic efficacy of CVR lead antibodies. K18-hACE2 transgenic mice express human ACE2, the receptor used by severe acute respiratory syndrome coronavirus (SARS-CoV) to gain cellular entry. The human keratin 18 promoter directs expression to epithelia, including airway epithelia, where infections typically begin (https://www.jax.org/strain/034860 ). (B) Prophylactic efficacy study - viral titer in BAL. Female hACE-2 mice treated with either with 5.25 mg/kg CVR224 (blue), CVR6 (red) or a monoclonal isotype control (grey) by i.v. injection five days before virus inoculation. Intranasal infection was performed under isoflurane anesthesia with 300 FFU of SARS-CoV-2 in 50 pl. Mice were euthanized on day four after virus inoculation, and bronchoalveolar lavage (BAL) was collected. 20 - 100 pl BAL were used for the infection of 2 x 10⁴ Vero cells for 3 hours. The supernatant was removed, and cells were incubated for another 27 hours. SARS-CoV-2-infected cells were visualized using spike protein-specific antibody staining. Mean values and corresponding standard deviations (n = 7 - 8 animals per group) are shown. Statistical evaluation of the data was performed by Kruskal-Wallis test (one-way analysis of variance (ANOVA) and Dunn’s Pairwise Multiple Comparison Procedures as post hoc test. (C) Therapeutic efficacy study - Quantification of SARS-CoV2- RNA loads in BALs. Female hACE-2 mice (n = 6) were infected intranasally under isoflurane anesthesia with 300 FFU of SARS-CoV-2 in 50 pl total volume. 24 hours after virus inoculation, mice received 5.25 mg/kg of CVR6 (red) CVR328 (blue) or isotype control II (grey) by intravenous injection. Cohort 1 was euthanized on day 4, and lungs were homogenized in 2 ml PBS after collection. Viral RNA was isolated from 140 pl of homogenates using QIAamp Viral RNA Mini Kit (Qiagen). RT-qPCR reactions were performed with 5 pl of isolated RNA as a template using TaqMan® Fast Virus 1-Step Master Mix (Thermo Fisher). Synthetic SARS-CoV-2-RNA (Twist Bioscience) was used as a quantitative standard to obtain viral copy numbers. Data points shown represent the viral copy number of each animal with the mean of each group. Calculated reduction is shown in comparison to the isotype control. Statistical evaluation of the data was performed by Kruskal-Wallis test (one-way ANOVA) and Dunn’s Pairwise Multiple Comparison Procedures as post hoc test. (D) Therapeutic studies - Survival of SARS-CoV2-infected mice. Female hACE-2 mice were infected intranasally under isoflurane anesthesia with 300 FFU of SARS- CoV-2 in 50 pl total volume. 24 hours after virus inoculation, they received either 5.25 mg/kg of CVR6 (red) CVR328 (blue) or isotype control II (grey) by intravenous injection. The percentages of surviving animals (n = 8) according to humane endpoints are shown. Overlapping lines are partially offset for better readability. (D) Therapeutic studies - Quantification of SARS-CoV2 titer in BAL. Female hACE-2 mice (n = 6) were infected intranasally under isoflurane anesthesia with 300 FFU of SARS-CoV-2 in 50 l total volume. 24 hours after virus inoculation, the mice were i.v. injected either with 5.25 mg/kg I.V6.183 antibody (red) or 5.25 mg/kg CVR328.5 antibody (blue) or 5.25 mg/kg idiotype control II 480 (grey). According to humane endpoints, cohort one was euthanized at day 4 and cohort 2 at day 10 after virus inoculation. Bronchoalveolar lavages (BAL; around 1.4 ml) were collected, and 20 - 100 pl of BAL were used for infection of 2 x 10⁴ Vero cells for 3 hours. After replacing supernatant with fresh medium, cells were incubated for further 27 hours. SARS- CoV-2 infected cells were visualized using SARS-CoV-2 S-protein specific immunochemistry staining. Mean values and corresponding standard deviations (n=6, measured in duplicates) are shown. Statistical evaluation of the data was performed by Kruskal-Wallis test (one-way ANOVA) and Dunn’s Pairwise Multiple Comparison Procedures as post hoc test.

Figure 6 depicts ACE2 inhibition assays with ACR antibodies.

(A) Flow cytometric detection of hACE2-competing antibodies. HEK-293T cells were cotransfected with a GFP reporter plasmid and a pCG1 -based expression vector for the spike protein of CoV-2 as described in Fig. 2. Two days after transfection, cells were incubated with purified CVR antibodies for 30 minutes on ice. 0,7 ug of hACE2-hFc was added. After 30 min on ice, cells were washed, and bound hACE2 was detected in a fluorescence-based flow cytometer with Cy5-labeled goat antibodies against human IgG. Numbers indicate relative Cy5 mean fluorescence intensity (Fl). (B) hACE-2 flow cytometric competition assay to quantitate blocking the activity of CVR antibodies. 2SARS-CoV2 spike -transfected 293T cells (see Fig. 2) were incubated on ice for 10 minutes with 50ul of ACE2-Fc (250ng/ml) produced in HEK293T cells. 50ul of serially diluted Prot G-purified CVR antibodies (250ng/ml-0.9ng/ml) were added, and cells were incubated on ice for an additional 30 minutes. Cells were washed and stained on ice fur 30 minutes with an Alexa647-labelled anti-human IgG-Fc (lgG1) antibody (Biolegend). Alexa567 fluorescence was determined in transfected GFP-positive 293T cells with a FACS Attune next (Thermo Fischer) and analyzed with the software Flow Logic (llnivai Technologies). The EC50 values were determined using Graphpad Prism. (C) Elisa-based ACE2 inhibition assay. Plates were coated with monomeric RBD and incubated with serial dilutions of CVR antibodies and soluble hACE2-hCg1 fusion protein (400ng/ml). Bound hACE2 was quantitated with HRP- coupled antibodies against the hFcy1-Tag of hACE2. Samples were run in triplicates. One representative experiment of two and the mean EC50s of both experiments are shown.

Figure 7 depicts assays to detect binding of CVR antibodies to recombinant CoV-2 spike proteins. (A) Elisa assay to detect CVR antibodies binding to CoV-2 spike protein. 96-well microtiter plates were coated ON at 4°C with 400ng/well recombinant RBD that was purified from the culture medium of transfected 293F cells and 400ng/well NTD (Aero, # S1 D-C52H6). Wells were washed with PBS/0.05% Tween-20 and incubated with purified CVR antibodies (1 pg/ml). Bound CVR antibodies were detected with an HRP-conjugated anti-mouse IgG (1 :4000, Southern Biotech #1030-05) and TMB substrate (BD Bioscience #555214). OD at 450nm was determined in an FLIIO Star Omega Multimode reader (BMG Labtech, Ortenberg, Germany). NTD, N-terminal domain of spike CoV-2; RBD, the receptor-binding domain of spike CoV-2. (B) immunoblot to detect RBD binding antibodies. (B) Western blot analysis to detect the binding of CVR antibodies to denatured and reduced RBD domains of CoV-2. 10ng recombinant CoV-2 RBD with either an huFc(lgGI) or a StrepTag was reduced by beta-mercaptoethanol and separated in a 7% SDS polyacrylamide gel. Proteins were transferred to nylon membranes. The membranes were first incubated without (only the 2^nd antibody) or with 1pg/ml Protein G-purified CVR antibodies in 5% Milk/TBST. Mouse monoclonal Trianni anti-KLH lgG1 antibodies served as a negative control. Bound CVR and anti-KLH antibodies were detected with HRP-conjugated goat sera against mouse lgG2c and mouse lgG1 , respectively, and the chemiluminescence method.

Figure 8 shows ELISA-based affinity measurements with CVR antibodies to RBD and trimeric spike.

(A) KD determination of CVR antibodies binding to RBD. Plates were coated with monomeric recombinant RBD. Serial dilutions of purified CVR hybridoma antibodies were added, and bound CVR antibodies were detected with an HRP-coupled anti-mouse-IgG antibody. KD values were determined using a hyperbolic fitting curve with GraphPad Prism 7.02. One representative experiment out of two and the mean KDS of all experiments are shown. (B) KD determination of cluster 2 antibodies to the trimeric spike protein. KDs were determined as described in (a) using ELISA plates coated with stabilized trimeric spike protein. Shown is one representative experiment out of two and the mean KDS of both experiments.

Figure 9 depicts the binding and virus-neutralization activities of fully recombinant human CVR antibodies.

VH and VL regions cloned from CVR hybridomas were inserted into the CMV-based pcDNA3.1(+) mammalian expression vector together with the human Cgammal and human Ckappa region, respectively. 293F cells were transiently transfected with combinations of HC and LC expression vectors. Human CVR-lgG1 antibodies were purified by Protein G Sepharose from the supernatant of transfected 293F cells and used for the subsequent functional studies. (A) As described in Fig 2, flow binding assays were performed with CoV-2 spike protein transfected 293T cells and purified human CVR-lgG1. Bound human lgG1 antibodies were detected with AF647-conjugated anti-human-IgG antibodies. Stainings with cell culture medium alone (2nd Ab only) or a purified irrelevant human lgG1 (isotype) were used as negative controls. (B) Test of human CVR-lgG1 antibodies for virus neutralization. The assays were performed as described in Fig. 4.

Figure 10 shows the result of binding competition assays between cluster 1 and cluster 2 antibodies.

SARS-CoV-2-S DNA transfected HEK 293T cells were incubated with 10OpI of recombinant 250ng/ml CVR antibodies with a human Fcy1 region (CVR224h) and serially diluted (ranging from 2.5pg/ml-0.002ng/ml) CVR hybridoma antibodies with a murine Fey (CVR224 as control and CVR328). The cells were incubated for 30 min on ice and washed. Bound antibodies were detected with a mouse lgG2a Alexa647-conjugated antibody directed against human Fey (BioLegend, San Diego, USA #409320). The mean fluorescence intensities of transfected cells were determined with an Attune Nxt (Thermo Fisher Scientific, Waltham, USA) and the Flowlogic software (llnivai Technologies, Mentone, Australia). One representative experiment out of 2, with mean and SEM, is shown. The experiment was performed in duplicates.

Figure 11 A depicts the somatic AA changes in CVR cluster 1 antibodies.

The numbers in blue boxes indicate the number of the amino acid (AA )changes. The isotypes of H and L chains of the hybridoma antibodies are indicated below the CRV antibody. IC50 and % neutralization values are listed in the blue-framed rectangular. CDR, Complementary-determining regions; FR, framework; P, precursors.

Figure 11 B depicts the somatic AA changes in CVR cluster 2 antibodies.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS Antibodies, antigen-binding fragments thereof, and other antigen-binding proteins that bind RBD or other parts of the CoV-2 Spike protein are provided.

Such antigen-binding proteins may selectively inhibit the binding of RBD of the CoV-2 Spike protein to hACE-2.

The antigen-binding protein of the present invention may specifically bind to the complete CoV-2 spike or to subdomains of the CoV-2 Spike protein such as RBD, S1 , NTD (N- terminal domain), S1 , S2 or the ectodomain of the trimeric spike protein with a KD < 0.1 pM, <10 nM, <5 nM, < 1 nM or < 0.1 nM.

In some embodiments, the antigen-binding protein binds explicitly to the complete CoV-2 Spike or to subdomains of the CoV-2 Spike protein such as RBD, S1 or S2 with a KD <100 nM, <10 nM, <5 nM, < 1 nM or < 0.1 nM as determined using a FACS binding assay and analyzed, for example, using methods described in Rathanaswami et al., Biochemical and Biophysical Research Communications 334 (2005) 1004-1013. or using an Elisa assay.

In some embodiments, the antigen-binding protein blocks the binding of hACE-2 to membranes from cells expressing CoV-2 spike protein in a competitive fluorescence-based flow cytometry assays with an EC50 of <100 nM, <10 nM, <1 nM, <0.5 nM or <0.1 nM, with <1 nM or less for preferred antibodies, wherein EC50 is the concentration (i.e. effective concentration) of the antigen-binding protein where the response (or binding) is reduced by half. In some embodiments, the antigen-binding protein blocks the binding of hACE-2 to RBD in an hACE-2- binding Elisa competition assay with EC50 of <100 nM, <50 nM, <20 nM, <10 nM, <1 nM, <0.5 nM or <0.1 nM, with <1 nM or less for preferred antibodies.

In some embodiments, the antigen-binding protein may have an ICso of <100 nM, <50 nM, <10 nM, <1 nM, <0.5 nM, or <0.1 nM in a virus neutralization assay with SARS-CoV-2, with <1 nM or less for preferred antibodies. To determine the potential of antigen-binding proteins, Vero E6 cells were inoculated with wildtype CoV-2 under S3 conditions and incubated overnight. Wildtype CoV-2 infection was quantitated by staining fixed and permeabilized cells with immunoglobulin purified from plasma of a reconvalescent COVID-19 patient and a fluorescence-labeled secondary anti-human IgG antibody.

In another exemplary aspect, the antigen-binding proteins compete for binding to RBD with a reference antibody comprising a heavy chain variable region comprising a sequence selected from the group consisting of SEQ ID NO:1 , 3 and 5 and a light chain variable region comprising a sequence selected from the group consisting of SEQ ID NO:2, 4 and 6. In some embodiments, binding is assessed using, e.g., using a Biacore analysis. In another exemplary aspect, the antigen-binding proteins compete for binding to another part of the CoV-2 Spike protein other than RBD, e.g. NTD with a reference antibody comprising a heavy chain variable region comprising a sequence selected from the group consisting of SEQ ID NO: 37, 39, 41 , 43, 45 and 47 and a light chain variable region comprising a sequence selected from the group consisting of SEQ ID NO: 38, 40, 42, 44, 46 and 48. In some embodiments, binding is assessed using, e.g., using a Biacore analysis.

In another exemplary aspect, the antigen-binding proteins compete for binding to another part of the CoV-2 Spike protein other than RBD, e.g. S1 with a reference antibody comprising a heavy chain variable region comprising a sequence selected from the group consisting of SEQ ID NO: 49, 51 , 53, 55, 57 and 59 and a light chain variable region comprising a sequence selected from the group consisting of SEQ ID NO: 50, 52, 54, 56, 58 and 60. In some embodiments, binding is assessed using, e.g., using a Biacore analysis.

In another exemplary aspect, the antigen-binding proteins compete for binding to another respecitve part of the CoV-2 Spike protein other than RBD, with a reference antibody comprising a heavy chain variable region comprising a sequence selected from the group consisting of SEQ ID NO: 61 , 63, 65, 67, 69, 71 , 73, 75, 77, 79, 81 , 83, 85, 87, 89, 91 , 93, 95, 97, 99 and 101 and a light chain variable region comprising a sequence selected from the group consisting of SEQ ID NO: 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100 or 102. In some embodiments, binding is assessed using, e.g., using a Biacore analysis.

In certain embodiments, the reference antibody comprises (i) a heavy chain defined by a sequence selected from the group consisting of SEQ ID NOs:31 , 32 and 33 and (ii) a light chain defined by a sequence selected from the group consisting of SEQ ID NOs: 34, 35 and 36. In more specific embodiments, the reference antibody comprises a heavy chain and a light chain defined by one of the following pairs of sequences: (i) SEQ ID NO: 31 and SEQ ID NO: 34; (ii) SEQ ID NO: 32 and SEQ ID NO: 35; and (iii) SEQ ID NO: 33 and SEQ ID NO: 36.

In related embodiments, the antigen-binding proteins that compete for binding of ACE-2 to RBD of the CoV-2 Spike protein specifically bind to RBD of the CoV-2 Spike protein with a KD <1 pM, <100 nM, <10 nM, or <5 nM, < 1 nM or < 0.1 nM, e.g., as determined using an Elisa or FACS binding assay and analyzed, for example, using methods described in Rathanaswami et al., Biochemical and Biophysical Research Communications 334 (2005) 1004-1013. In related embodiments, the antigen-binding proteins that compete for binding to RBD of the CoV-2 Spike protein have an EC50 of <100 nM, <10 nM, <1 nM, <0.5 nM or <0.1 nM in an Elisabased hACE-2 binding competition assay with immobilized CoV-2 spike.

In certain embodiments, the antigen-binding proteins that compete for binding to RBD of the CoV-2 Spike protein may be neutralizing antigen-binding proteins, e.g. may have an ICsoof <100 nM, <50 nM, <10 nM, <1 nM, <0.5 nM, or <0.1 nM in a neutralization assay with SARS-CoV-2, with <1 nM or less for preferred antibodies.

In other embodiments, the antigen-binding proteins may compete for binding to a subdomain of the CoV-2 Spike protein, e.g. S1 , NTD or another subdomain different from RBD, and preferably bind to a subdomain of the CoV-2 Spike protein, e.g. S1, NTD or another subdomain different from RBD, with a KD <1 pM, <100 nM, <10 nM, or <5 nM, < 1 nM or < 0.1 nM, e.g., as determined using an Elisa or FACS binding assay and analyzed, for example, using methods described in Rathanaswami et al., Biochemical and Biophysical Research Communications 334 (2005) 1004-1013.

In certain embodiments, the antigen-binding proteins that compete for binding to a subdomain of the CoV-2 Spike protein, e.g. S1, NTD or another subdomain different from RBD, may be neutralizing antigen-binding proteins, e.g. may have an ICsoof <100 nM, <50 nM, <10 nM, <1 nM, <0.5 nM, or <0.1 nM in a neutralization assay with SARS-CoV-2, with <1 nM or less for preferred antibodies.

In another embodiment, antigen-binding proteins do not bind to RBD, but nevertheless, neutralize CoV-2 infection, e.g. have an ICso of <100 nM, <50 nM, <10 nM, <1 nM, <0.5 nM, or <0.1 nM in a neutralization assay with SARS-CoV-2 with <1 nM or less for preferred antibodies. Such antibodies preferably bind to NTD.

In any of the above-mentioned embodiments, the antigen-binding protein that binds to the CoV-2 spike protein, e.g. the membrane-bound CoV-2 spike protein (like all antibodes listed in Fig. 3), or competes for binding to the CoV-2 spike, e.g. RBD of the CoV-2 Spike protein, may be, for example, a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human (e.g., fully human) antibody, a humanized antibody, a chimeric antibody, a multi-specific antibody, or an antigen binding fragment thereof. Further, the antibody fragment of the antigenbinding protein that binds to CoV-2 spike proteins, e.g. the membrane-bound CoV-2 spike protein, or competes for binding to RBD of the CoV-2 Spike protein or neutralizes the infection of CoV-2 in vivo or in vitro can be a Fab fragment, a Fab' fragment, an F(ab')2 fragment, an Fv fragment, a diabody or a single-chain antibody molecule; and maybe, for example, a human monoclonal antibody, e.g., an lgG1 -, lgG2-, lgG3-, or lgG4-, IgM- , lgA1 -, lgA2-type antibody or antibodies that carry mutations in the CH regions that reduce or abolish effector functions, increase chemical stability, and serum half-live. Further, the antigen-binding proteins may be neutralizing antigen-binding proteins, e.g. may have an ICso of <100 nM, <50 nM, <10 nM, <1 nM, <0.5 nM, or <0.1 nM in a neutralization assay with SARS-CoV-2 with <1 nM or less for preferred antibodies.

In certain embodiments of the first aspect of the invention, the antigen-binding proteins described, e.g., antibodies or fragments thereof, comprise (A) one or more heavy chain complementary determining regions (CDRHs) selected from the group consisting of: (i) a CDRH1 selected from SEQ ID NO:7, 13 and 19; (ii) a CDRH2 selected from SEQ ID NO:8, 14 and 20; (iii) a CDRH3 selected from SEQ ID NO:9, 15 and 21 ; and optionally (iv) a CDRH of (i), (ii) and (iii) that contains one or more amino acid substitutions (e.g., conservative amino acid substitutions), deletions or insertions that collectively total no more than four amino acids; (B) one or more light chain complementary determining regions (CDRLs) selected from the group consisting of: (i) a CDRL1 selected from the group consisting of SEQ ID NQs:10, 16 and 22; (ii) a CDRL2 selected from the group consisting of SEQ ID NOs: 11 , 17 and 23; (iii) a CDRL3 selected from the group consisting of SEQ ID NOs: 12, 18 and 24; and optionally (iv) a CDRL of (i), (ii) and (iii) that contains one or more, e.g., one, two, three, four or more, amino acid substitutions (e.g., conservative amino acid substitutions), deletions or insertions that collectively total no more than four amino acids; or (C) one or more heavy chain CDRHs of (A) and one or more light chain CDRLs of (B). In one embodiment, the total number of amino acid substitutions, deletions, or insertions is no more than two amino acids per CDR. In another embodiment, the amino acid substitutions are conservative substitutions.

In certain embodiments of the second aspect of the invention, the antigen-binding proteins described, e.g., antibodies or fragments thereof, comprise (A) one or more heavy chain complementary determining regions (CDRHs) selected from the group consisting of: (i) a CDRH1 selected from the CDRHIs in SEQ ID NOs:37, 39, 41 , 43, 45 and 47; (ii) a CDRH2 selected from the CDRH2s in SEQ ID NOs: 37, 39, 41 , 43, 45 and 47; (iii) a CDRH3 selected selected from the CDRH3s in SEQ ID NOs: 37, 39, 41 , 43, 45 and 47; and optionally (iv) a CDRH of (i), (ii) and (iii) that contains one or more amino acid substitutions (e.g., conservative amino acid substitutions), deletions or insertions that collectively total no more than four amino acids; (B) one or more light chain complementary determining regions (CDRLs) selected from the group consisting of: (i) a CDRL1 selected from the CDRLIs in SEQ ID NOs:38, 40, 42, 44, 46 and 48; (ii) a CDRL2 selected from the CDRL2s in SEQ ID NOs:38, 40, 42, 44, 46 and 48; (iii) a CDRL3 selected from the CDRL3s in SEQ ID NOs:38, 40, 42, 44, 46 and 48; and optionally (iv) a CDRL of (i), (ii) and (iii) that contains one or more, e.g., one, two, three, four or more, amino acid substitutions (e.g., conservative amino acid substitutions), deletions or insertions that collectively total no more than four amino acids; or (C) one or more heavy chain CDRHs of (A) and one or more light chain CDRLs of (B). In one embodiment, the total number of amino acid substitutions, deletions, or insertions is no more than two amino acids per CDR. In another embodiment, the amino acid substitutions are conservative substitutions.

In certain embodiments of the third aspect of the invention, the antigen-binding proteins described, e.g., antibodies or fragments thereof, comprise (A) one or more heavy chain complementary determining regions (CDRHs) selected from the group consisting of: (i) a CDRH1 selected from the CDRHIs in SEQ ID NOs:49, 51 , 53, 55, 57 and 59; (ii) a CDRH2 selected from the CDRH2s in SEQ ID NOs:49, 51 , 53, 55, 57 and 59; (iii) a CDRH3 selected selected from the CDRH3s in SEQ ID NOs:49, 51 , 53, 55, 57 and 59; and optionally (iv) a CDRH of (i), (ii) and (iii) that contains one or more amino acid substitutions (e.g., conservative amino acid substitutions), deletions or insertions that collectively total no more than four amino acids; (B) one or more light chain complementary determining regions (CDRLs) selected from the group consisting of: (i) a CDRL1 selected from the CDRLIs in SEQ ID NQs:50, 52, 54, 56, 58 and 60; (ii) a CDRL2 selected from the CDRL2s in SEQ ID NOs: 50, 52, 54, 56, 58 and 60; (iii) a CDRL3 selected from the CDRL3s in SEQ ID NOs: 50, 52, 54, 56, 58 and 60; and optionally (iv) a CDRL of (i), (ii) and (iii) that contains one or more, e.g., one, two, three, four or more, amino acid substitutions (e.g., conservative amino acid substitutions), deletions or insertions that collectively total no more than four amino acids; or (C) one or more heavy chain CDRHs of (A) and one or more light chain CDRLs of (B). In one embodiment, the total number of amino acid substitutions, deletions, or insertions is no more than two amino acids per CDR. In another embodiment, the amino acid substitutions are conservative substitutions.

In certain embodiments of the fourth aspect of the invention, the antigen-binding proteins described, e.g., antibodies or fragments thereof, comprise (A) one or more heavy chain complementary determining regions (CDRHs) selected from the group consisting of: (i) a CDRH1 selected from the CDRHIs in SEQ ID NOs:61 , 63, 65, 67, 69, 71 , 73, 75, 77, 79 , 81 , 83, 85, 87, 89, 91 , 93, 95, 97, 99 and 101 ; (ii) a CDRH2 selected from the CDRH2s in SEQ ID NOs:61 , 63, 65, 67, 69, 71 , 73, 75, 77, 79 , 81 , 83, 85, 87, 89, 91 , 93, 95, 97, 99 and 101 ; (iii) a CDRH3 selected selected from the CDRH3s in SEQ ID NOs:61 , 63, 65, 67, 69, 71 , 73, 75, 77, 79 , 81 , 83, 85, 87, 89, 91 , 93, 95, 97, 99 and 101 ; and optionally (iv) a CDRH of (i), (ii) and (iii) that contains one or more amino acid substitutions (e.g., conservative amino acid substitutions), deletions or insertions that collectively total no more than four amino acids; (B) one or more light chain complementary determining regions (CDRLs) selected from the group consisting of: (i) a CDRL1 selected from the CDRLIs in SEQ ID NOs:62, 64, 66, 68, 70, 72, 74, 76, 78, 80 , 82, 84, 86, 88, 90, 92, 94, 96, 98, 100 and 102; (ii) a CDRL2 selected from the CDRL2s in SEQ ID NOs:62, 64, 66, 68, 70, 72, 74, 76, 78, 80 , 82, 84, 86, 88, 90, 92, 94, 96, 98, 100 and 102; (iii) a CDRL3 selected from the CDRL3s in SEQ ID NOs:62, 64, 66, 68, 70, 72, 74, 76, 78, 80 , 82, 84, 86, 88, 90, 92, 94, 96, 98, 100 and 102; and optionally (iv) a CDRL of (i), (ii) and (iii) that contains one or more, e.g., one, two, three, four or more, amino acid substitutions (e.g., conservative amino acid substitutions), deletions or insertions that collectively total no more than four amino acids; or (C) one or more heavy chain CDRHs of (A) and one or more light chain CDRLs of (B). In one embodiment, the total number of amino acid substitutions, deletions, or insertions is no more than two amino acids per CDR. In another embodiment, the amino acid substitutions are conservative substitutions.

In another embodiment, the antigen-binding protein comprises at least one or two CDRH of any of the above-mentioned (A) and at least one or two CDRL of any of the above- mentioned (B). In yet another embodiment, the antigen-binding protein comprises (i) at least three CDRH of any of the above-mentioned (A), where the three CDRHs include CDRH1, a CDRH2 and a CDRH3, and (ii) at least three CDRL of any of the above-mentioned (B), where the three CDRLs include CDRL1 , a CDRL2 and a CDRL3. In additional embodiments, the antigen-binding proteins described above comprise a first amino acid sequence comprising at least one CDRH and a second amino acid sequence comprising at least one CDRL. In one embodiment, the first and the second amino acid sequences are covalently bonded to each other.

In another embodiment of the first aspect of the invention, the antigen-binding protein includes a CDRH1, a CDRH2 and a CDRH3. In one embodiment, CDRH1 comprises SEQ ID NO:7, CDRH2 comprises SEQ ID NO:8 and CDRH3 comprises SEQ ID NO:9. In another embodiment, CDRH1 comprises SEQ ID NO:13, CDRH2 comprises SEQ ID NO:14 and CDRH3 comprises SEQ ID NO:15. In another embodiment, CDRH1 comprises SEQ ID NO:19, CDRH2 comprises SEQ ID NQ:20 and CDRH3 comprises SEQ ID NO:21.

In another embodiment of the first aspect of the invention, the antigen-binding protein includes a CDRL1 sequence, a CDRL2 sequence and a CDRL3 sequence. In one embodiment, CDRL1 comprises SEQ ID NQ:10, CDRL2 comprises SEQ ID NO:11 and CDRL3 comprises SEQ ID NO: 12. In another embodiment, CDRL1 comprises SEQ ID NO:16, CDRL2 comprises SEQ ID NO:17 and CDRL3 comprises SEQ ID NO:18. In another embodiment, CDRL1 comprises SEQ ID NO:22, CDRL2 comprises SEQ ID NO:23 and CDRL3 comprises SEQ ID NO:24.

In another embodiment of the first aspect of the invention, the antigen-binding protein includes a CDRL1 sequence, a CDRL2 sequence, a CDRL3 sequence, a CDRH1 sequence, a CDRH2 sequence and a CDRH3 sequence. In one embodiment, CDRL1 comprises SEQ ID NQ:10, CDRL2 comprises SEQ ID NO:11, CDRL3 comprises SEQ ID NO:12, CDRH1 comprises SEQ ID NO:7, CDRH2 comprises SEQ ID NO:8 and CDRH3 comprises SEQ ID NO:9. In another embodiment, CDRL1 comprises SEQ ID NO:16, CDRL2 comprises SEQ ID NO:17, CDRL3 comprises SEQ ID NO:18, CDRH1 comprises SEQ ID NO:13, CDRH2 comprises SEQ ID NO:14 and CDRH3 comprises SEQ ID NO:15. In another embodiment, CDRL1 comprises SEQ ID NO:22, CDRL2 comprises SEQ ID NO:23, CDRL3 comprises SEQ ID NO:24, CDRH1 comprises SEQ ID NO:19, CDRH2 comprises SEQ ID NQ:20 and CDRH3 comprises SEQ ID NO:21.

In another embodiment of the second aspect of the invention, the antigen-binding protein includes a CDRH1, a CDRH2 and a CDRH3. In one embodiment, CDRH1 comprises the CDRH1 of SEQ ID NO:37, the CDRH2 comprises the CDRH2 of SEQ ID NO:37 and CDRH3 comprises the CDRH3 of SEQ ID NO:37. In another embodiment, CDRH1 comprises the CDRH1 of SEQ ID NO:39, the CDRH2 comprises the CDRH2 of SEQ ID NO:39 and CDRH3 comprises the CDRH3 of SEQ ID NO:39. In another embodiment, CDRH1 comprises the CDRH1 of SEQ ID NO:41, the CDRH2 comprises the CDRH2 of SEQ ID NO:41 and CDRH3 comprises the CDRH3 of SEQ ID NO:41. In another embodiment, CDRH1 comprises the CDRH1 of SEQ ID NO:43, the CDRH2 comprises the CDRH2 of SEQ ID NO:43 and CDRH3 comprises the CDRH3 of SEQ ID NO:43. In another embodiment, CDRH1 comprises the CDRH1 of SEQ ID NO:45, the CDRH2 comprises the CDRH2 of SEQ ID NO:45 and CDRH3 comprises the CDRH3 of SEQ ID NO:45. In another embodiment, CDRH1 comprises the CDRH1 of SEQ ID NO:47, the CDRH2 comprises the CDRH2 of SEQ ID NO:47 and CDRH3 comprises the CDRH3 of SEQ ID NO:47.

In another embodiment of the second aspect of the invention, the antigen-binding protein includes a CDRL1 sequence, a CDRL2 sequence and a CDRL3 sequence. In one embodiment, CDRL1 comprises the CDRL1 of SEQ ID NO:38, the CDRL2 comprises the CDRL2 of SEQ ID NO:38 and CDRL3 comprises the CDRL3 of SEQ ID NO:38. In another embodiment, CDRL1 comprises the CDRL1 of SEQ ID NQ:40, the CDRL2 comprises the CDRL2 of SEQ ID NQ:40 and CDRL3 comprises the CDRL3 of SEQ ID NQ:40. In another embodiment, CDRL1 comprises the CDRL1 of SEQ ID NO:42, the CDRL2 comprises the CDRL2 of SEQ ID NO:42 and CDRL3 comprises the CDRL3 of SEQ ID NO:42. In another embodiment, CDRL1 comprises the CDRL1 of SEQ ID NO:44, the CDRL2 comprises the CDRL2 of SEQ ID NO:44 and CDRL3 comprises the CDRL3 of SEQ ID NO:44. In another embodiment, CDRL1 comprises the CDRL1 of SEQ ID NO:46, the CDRL2 comprises the CDRL2 of SEQ ID NO:46 and CDRL3 comprises the CDRL3 of SEQ ID NO:46. In another embodiment, CDRL1 comprises the CDRL1 of SEQ ID NO:48, the CDRL2 comprises the CDRL2 of SEQ ID NO:48 and CDRL3 comprises the CDRL3 of SEQ ID NO:48.

In another embodiment of the second aspect of the invention, the antigen-binding protein includes a CDRL1 sequence, a CDRL2 sequence, a CDRL3 sequence, a CDRH1 sequence, a CDRH2 sequence and a CDRH3 sequence. In one embodiment, CDRL1 comprises the CDRL1 of SEQ ID NO:38, CDRL2 comprises the CDRL2 of SEQ ID NO:38, CDRL3 the CDRL3 of SEQ ID NO:38, CDRH1 comprises the CDRH1 of SEQ ID NO:37, CDRH2 comprises the CDRH2 of SEQ ID NO:37 and CDRH3 comprises the CDRH3 of SEQ ID NO:37. In another embodiment, CDRL1 comprises the CDRL1 of SEQ ID NQ:40, CDRL2 comprises the CDRL2 of SEQ ID NQ:40, CDRL3 the CDRL3 of SEQ ID NQ:40, CDRH1 comprises the CDRH1 of SEQ ID NO:39, CDRH2 comprises the CDRH2 of SEQ ID NO:39 and CDRH3 comprises the CDRH3 of SEQ ID NO:39. In another embodiment, CDRL1 comprises the CDRL1 of SEQ ID NO:42, CDRL2 comprises the CDRL2 of SEQ ID NO:42, CDRL3 the CDRL3 of SEQ ID NO:42, CDRH1 comprises the CDRH1 of SEQ ID NO:41, CDRH2 comprises the CDRH2 of SEQ ID NO:41 and CDRH3 comprises the CDRH3 of SEQ ID NO:41. In another embodiment, CDRL1 comprises the CDRL1 of SEQ ID NO:44, CDRL2 comprises the CDRL2 of SEQ ID NO:44, CDRL3 the CDRL3 of SEQ ID NO:44, CDRH1 comprises the CDRH1 of SEQ ID NO:43, CDRH2 comprises the CDRH2 of SEQ ID NO:43 and CDRH3 comprises the CDRH3 of SEQ ID NO:43. In another embodiment, CDRL1 comprises the CDRL1 of SEQ ID NO:46, CDRL2 comprises the CDRL2 of SEQ ID NO:46, CDRL3 the CDRL3 of SEQ ID NO:46, CDRH1 comprises the CDRH1 of SEQ ID NO:45, CDRH2 comprises the CDRH2 of SEQ ID NO:45 and CDRH3 comprises the CDRH3 of SEQ ID NO:45. In another embodiment, CDRL1 comprises the CDRL1 of SEQ ID NO:48, CDRL2 comprises the CDRL2 of SEQ ID NO:48, CDRL3 the CDRL3 of SEQ ID NO:48, CDRH1 comprises the CDRH1 of SEQ ID NO:47, CDRH2 comprises the CDRH2 of SEQ ID NO:47 and CDRH3 comprises the CDRH3 of SEQ ID NO:47.

In another embodiment of the third aspect of the invention, the antigen-binding protein includes a CDRH1, a CDRH2 and a CDRH3. In one embodiment, CDRH1 comprises the CDRH1 of SEQ ID NO:49, the CDRH2 comprises the CDRH2 of SEQ ID NO:49 and CDRH3 comprises the CDRH3 of SEQ ID NO:49. In another embodiment, CDRH1 comprises the CDRH1 of SEQ ID NO:51, the CDRH2 comprises the CDRH2 of SEQ ID NO:51 and CDRH3 comprises the CDRH3 of SEQ ID NO:51. In another embodiment, CDRH1 comprises the CDRH1 of SEQ ID NO:53, the CDRH2 comprises the CDRH2 of SEQ ID NO:53 and CDRH3 comprises the CDRH3 of SEQ ID NO:53. In another embodiment, CDRH1 comprises the CDRH1 of SEQ ID NO:55, the CDRH2 comprises the CDRH2 of SEQ ID NO:55 and CDRH3 comprises the CDRH3 of SEQ ID NO:55. In another embodiment, CDRH1 comprises the CDRH1 of SEQ ID NO:57, the CDRH2 comprises the CDRH2 of SEQ ID NO:57 and CDRH3 comprises the CDRH3 of SEQ ID NO:57. In another embodiment, CDRH1 comprises the CDRH1 of SEQ ID NO:59, the CDRH2 comprises the CDRH2 of SEQ ID NO:59 and CDRH3 comprises the CDRH3 of SEQ ID NO:59.

In another embodiment of the second aspect of the invention, the antigen-binding protein includes a CDRL1 sequence, a CDRL2 sequence and a CDRL3 sequence. In one embodiment, CDRL1 comprises the CDRL1 of SEQ ID NQ:50, the CDRL2 comprises the CDRL2 of SEQ ID NQ:50 and CDRL3 comprises the CDRL3 of SEQ ID NQ:50. In another embodiment, CDRL1 comprises the CDRL1 of SEQ ID NO:52, the CDRL2 comprises the CDRL2 of SEQ ID NO:52 and CDRL3 comprises the CDRL3 of SEQ ID NO:52. In another embodiment, CDRL1 comprises the CDRL1 of SEQ ID NO:54, the CDRL2 comprises the CDRL2 of SEQ ID NO:54 and CDRL3 comprises the CDRL3 of SEQ ID NO:54. In another embodiment, CDRL1 comprises the CDRL1 of SEQ ID NO:56, the CDRL2 comprises the CDRL2 of SEQ ID NO:56 and CDRL3 comprises the CDRL3 of SEQ ID NO:56. In another embodiment, CDRL1 comprises the CDRL1 of SEQ ID NO:58, the CDRL2 comprises the CDRL2 of SEQ ID NO:58 and CDRL3 comprises the CDRL3 of SEQ ID NO:58. In another embodiment, CDRL1 comprises the CDRL1 of SEQ ID NQ:60, the CDRL2 comprises the CDRL2 of SEQ ID NQ:60 and CDRL3 comprises the CDRL3 of SEQ ID NQ:60.

In another embodiment of the second aspect of the invention, the antigen-binding protein includes a CDRL1 sequence, a CDRL2 sequence, a CDRL3 sequence, a CDRH1 sequence, a CDRH2 sequence and a CDRH3 sequence. In one embodiment, CDRL1 comprises the CDRL1 of SEQ ID NQ:50, CDRL2 comprises the CDRL2 of SEQ ID NQ:50, CDRL3 the CDRL3 of SEQ ID NQ:50, CDRH1 comprises the CDRH1 of SEQ ID NO:49, CDRH2 comprises the CDRH2 of SEQ ID NO:49 and CDRH3 comprises the CDRH3 of SEQ ID NO:49. In another embodiment, CDRL1 comprises the CDRL1 of SEQ ID NO:52, CDRL2 comprises the CDRL2 of SEQ ID NO:52, CDRL3 the CDRL3 of SEQ ID NO:52, CDRH1 comprises the CDRH1 of SEQ ID NO:51, CDRH2 comprises the CDRH2 of SEQ ID NO:51 and CDRH3 comprises the CDRH3 of SEQ ID NO:51. In another embodiment, CDRL1 comprises the CDRL1 of SEQ ID NO:54, CDRL2 comprises the CDRL2 of SEQ ID NO:54, CDRL3 the CDRL3 of SEQ ID NO:54, CDRH1 comprises the CDRH1 of SEQ ID NO:53, CDRH2 comprises the CDRH2 of SEQ ID NO:53 and CDRH3 comprises the CDRH3 of SEQ ID NO:53. In another embodiment, CDRL1 comprises the CDRL1 of SEQ ID NO:56, CDRL2 comprises the CDRL2 of SEQ ID NO:56, CDRL3 the CDRL3 of SEQ ID NO:56, CDRH1 comprises the CDRH1 of SEQ ID NO:55, CDRH2 comprises the CDRH2 of SEQ ID NO:55 and CDRH3 comprises the CDRH3 of SEQ ID NO:55. In another embodiment, CDRL1 comprises the CDRL1 of SEQ ID NO:58, CDRL2 comprises the CDRL2 of SEQ ID NO:58, CDRL3 the CDRL3 of SEQ ID NO:58, CDRH1 comprises the CDRH1 of SEQ ID NO:57, CDRH2 comprises the CDRH2 of SEQ ID NO:57 and CDRH3 comprises the CDRH3 of SEQ ID NO:57. In another embodiment, CDRL1 comprises the CDRL1 of SEQ ID NQ:60, CDRL2 comprises the CDRL2 of SEQ ID NQ:60, CDRL3 the CDRL3 of SEQ ID NQ:60, CDRH1 comprises the CDRH1 of SEQ ID NO:59, CDRH2 comprises the CDRH2 of SEQ ID NO:59 and CDRH3 comprises the CDRH3 of SEQ ID NO:59.

In another embodiment of the fourth aspect of the invention, the antigen-binding protein includes a CDRH1, a CDRH2 and a CDRH3. In one embodiment, CDRH1 comprises the CDRH1 of SEQ ID NO:61, the CDRH2 comprises the CDRH2 of SEQ ID NO:61 and CDRH3 comprises the CDRH3 of SEQ ID NO:61. In another embodiment, CDRH1 comprises the CDRH1 of SEQ ID NO:63, the CDRH2 comprises the CDRH2 of SEQ ID NO:63 and CDRH3 comprises the CDRH3 of SEQ ID NO:63. In another embodiment, CDRH1 comprises the CDRH1 of SEQ ID NO:65, the CDRH2 comprises the CDRH2 of SEQ ID NO:65 and CDRH3 comprises the CDRH3 of SEQ ID NO:65. In another embodiment, CDRH1 comprises the CDRH1 of SEQ ID NO:67, the CDRH2 comprises the CDRH2 of SEQ ID NO:67 and CDRH3 comprises the CDRH3 of SEQ ID NO:67. In another embodiment, CDRH1 comprises the CDRH1 of SEQ ID NO:69, the CDRH2 comprises the CDRH2 of SEQ ID NO:69 and CDRH3 comprises the CDRH3 of SEQ ID NO:69. In another embodiment, CDRH1 comprises the CDRH1 of SEQ ID NO:71 , the CDRH2 comprises the CDRH2 of SEQ ID NO:71 and CDRH3 comprises the CDRH3 of SEQ ID NO:71. In another embodiment, CDRH1 comprises the CDRH1 of SEQ ID NO:73, the CDRH2 comprises the CDRH2 of SEQ ID NO:73 and CDRH3 comprises the CDRH3 of SEQ ID NO:73. In another embodiment, CDRH1 comprises the CDRH1 of SEQ ID NO:75, the CDRH2 comprises the CDRH2 of SEQ ID NO:75 and CDRH3 comprises the CDRH3 of SEQ ID NO:75. In another embodiment, CDRH1 comprises the CDRH1 of SEQ ID NO:77, the CDRH2 comprises the CDRH2 of SEQ ID NO:77 and CDRH3 comprises the CDRH3 of SEQ ID NO:77. In another embodiment, CDRH1 comprises the CDRH1 of SEQ ID NO:79, the CDRH2 comprises the CDRH2 of SEQ ID NO:79 and CDRH3 comprises the CDRH3 of SEQ ID NO:79. In another embodiment, CDRH1 comprises the CDRH1 of SEQ ID NO:81, the CDRH2 comprises the CDRH2 of SEQ ID NO:81 and CDRH3 comprises the CDRH3 of SEQ ID NO:81. In another embodiment, CDRH1 comprises the CDRH1 of SEQ ID NO:83, the CDRH2 comprises the CDRH2 of SEQ ID NO:83 and CDRH3 comprises the CDRH3 of SEQ ID NO:83. In another embodiment, CDRH1 comprises the CDRH1 of SEQ ID NO:85, the CDRH2 comprises the CDRH2 of SEQ ID NO:85 and CDRH3 comprises the CDRH3 of SEQ ID NO:85. In another embodiment, CDRH1 comprises the CDRH1 of SEQ ID NO:87, the CDRH2 comprises the CDRH2 of SEQ ID NO:87 and CDRH3 comprises the CDRH3 of SEQ ID NO:87. In another embodiment, CDRH1 comprises the CDRH1 of SEQ ID NO:89, the CDRH2 comprises the CDRH2 of SEQ ID NO:89 and CDRH3 comprises the CDRH3 of SEQ ID NO:89. In another embodiment, CDRH1 comprises the CDRH1 of SEQ ID NO:91, the CDRH2 comprises the CDRH2 of SEQ ID NO:91 and CDRH3 comprises the CDRH3 of SEQ ID NO:91. In another embodiment, CDRH1 comprises the CDRH1 of SEQ ID NO:93, the CDRH2 comprises the CDRH2 of SEQ ID NO:93 and CDRH3 comprises the CDRH3 of SEQ ID NO:93. In another embodiment, CDRH1 comprises the CDRH1 of SEQ ID NO:95, the CDRH2 comprises the CDRH2 of SEQ ID NO:95 and CDRH3 comprises the CDRH3 of SEQ ID NO:95. In another embodiment, CDRH1 comprises the CDRH1 of SEQ ID NO:97, the CDRH2 comprises the CDRH2 of SEQ ID NO:97 and CDRH3 comprises the CDRH3 of SEQ ID NO:97. In another embodiment, CDRH1 comprises the CDRH1 of SEQ ID NO:99, the CDRH2 comprises the CDRH2 of SEQ ID NO:99 and CDRH3 comprises the CDRH3 of SEQ ID NO:99. In another embodiment, CDRH1 comprises the CDRH1 of SEQ ID NQ:101, the CDRH2 comprises the CDRH2 of SEQ ID NQ:101 and CDRH3 comprises the CDRH3 of SEQ ID NQ:101.

In another embodiment of the second aspect of the invention, the antigen-binding protein includes a CDRL1 sequence, a CDRL2 sequence and a CDRL3 sequence. In one embodiment, CDRL1 comprises the CDRL1 of SEQ ID NO:62, the CDRL2 comprises the CDRL2 of SEQ ID NO:62 and CDRL3 comprises the CDRL3 of SEQ ID NO:62. In another embodiment, CDRL1 comprises the CDRL1 of SEQ ID NO:64, the CDRL2 comprises the CDRL2 of SEQ ID NO:64 and CDRL3 comprises the CDRL3 of SEQ ID NO:64. In another embodiment, CDRL1 comprises the CDRL1 of SEQ ID NO:66, the CDRL2 comprises the CDRL2 of SEQ ID NO:66 and CDRL3 comprises the CDRL3 of SEQ ID NO:66. In another embodiment, CDRL1 comprises the CDRL1 of SEQ ID NO:68, the CDRL2 comprises the CDRL2 of SEQ ID NO:68 and CDRL3 comprises the CDRL3 of SEQ ID NO:68. In another embodiment, CDRL1 comprises the CDRL1 of SEQ ID NQ:70, the CDRL2 comprises the CDRL2 of SEQ ID NQ:70 and CDRL3 comprises the CDRL3 of SEQ ID NQ:70. In another embodiment, CDRL1 comprises the CDRL1 of SEQ ID NO:72, the CDRL2 comprises the CDRL2 of SEQ ID NO:72 and CDRL3 comprises the CDRL3 of SEQ ID NO:72. In another embodiment, CDRL1 comprises the CDRL1 of SEQ ID NO:74, the CDRL2 comprises the CDRL2 of SEQ ID NO:74 and CDRL3 comprises the CDRL3 of SEQ ID NO:74. In another embodiment, CDRL1 comprises the CDRL1 of SEQ ID NO:76, the CDRL2 comprises the CDRL2 of SEQ ID NO:76 and CDRL3 comprises the CDRL3 of SEQ ID NO:76. In another embodiment, CDRL1 comprises the CDRL1 of SEQ ID NO:78, the CDRL2 comprises the CDRL2 of SEQ ID NO:78 and CDRL3 comprises the CDRL3 of SEQ ID NO:78. In another embodiment, CDRL1 comprises the CDRL1 of SEQ ID NQ:80, the CDRL2 comprises the CDRL2 of SEQ ID NQ:80 and CDRL3 comprises the CDRL3 of SEQ ID NQ:80. In another embodiment, CDRL1 comprises the CDRL1 of SEQ ID NO:82, the CDRL2 comprises the CDRL2 of SEQ ID NO:82 and CDRL3 comprises the CDRL3 of SEQ ID NO:82. In another embodiment, CDRL1 comprises the CDRL1 of SEQ ID NO:84, the CDRL2 comprises the CDRL2 of SEQ ID NO:84 and CDRL3 comprises the CDRL3 of SEQ ID NO:84. In another embodiment, CDRL1 comprises the CDRL1 of SEQ ID NO:86, the CDRL2 comprises the CDRL2 of SEQ ID NO:86 and CDRL3 comprises the CDRL3 of SEQ ID NO:86. In another embodiment, CDRL1 comprises the CDRL1 of SEQ ID NO:88, the CDRL2 comprises the CDRL2 of SEQ ID NO:88 and CDRL3 comprises the CDRL3 of SEQ ID NO:88. In another embodiment, CDRL1 comprises the CDRL1 of SEQ ID NQ:90, the CDRL2 comprises the CDRL2 of SEQ ID NQ:90 and CDRL3 comprises the CDRL3 of SEQ ID NQ:90. In another embodiment, CDRL1 comprises the CDRL1 of SEQ ID NO:92, the CDRL2 comprises the CDRL2 of SEQ ID NO:92 and CDRL3 comprises the CDRL3 of SEQ ID NO:92. In another embodiment, CDRL1 comprises the CDRL1 of SEQ ID NO:94, the CDRL2 comprises the CDRL2 of SEQ ID NO:94 and CDRL3 comprises the CDRL3 of SEQ ID NO:94. In another embodiment, CDRL1 comprises the CDRL1 of SEQ ID NO:96, the CDRL2 comprises the CDRL2 of SEQ ID NO:96 and CDRL3 comprises the CDRL3 of SEQ ID NO:96. In another embodiment, CDRL1 comprises the CDRL1 of SEQ ID NO:98, the CDRL2 comprises the CDRL2 of SEQ ID NO:98 and CDRL3 comprises the CDRL3 of SEQ ID NO:98. In another embodiment, CDRL1 comprises the CDRL1 of SEQ ID NQ:100, the CDRL2 comprises the CDRL2 of SEQ ID NQ:100 and CDRL3 comprises the CDRL3 of SEQ ID NQ:100. In another embodiment, CDRL1 comprises the CDRL1 of SEQ ID NQ:102, the CDRL2 comprises the CDRL2 of SEQ ID NQ:102 and CDRL3 comprises the CDRL3 of SEQ ID NQ:102.

In another embodiment of the second aspect of the invention, the antigen-binding protein includes a CDRL1 sequence, a CDRL2 sequence, a CDRL3 sequence, a CDRH1 sequence, a CDRH2 sequence and a CDRH3 sequence. In one embodiment, CDRL1 comprises the CDRL1 of SEQ ID NO:62, CDRL2 comprises the CDRL2 of SEQ ID NO:62, CDRL3 the CDRL3 of SEQ ID NO:62, CDRH1 comprises the CDRH1 of SEQ ID NO:61 , CDRH2 comprises the CDRH2 of SEQ ID NO:61 and CDRH3 comprises the CDRH3 of SEQ ID NO:61. In another embodiment, CDRL1 comprises the CDRL1 of SEQ ID NO:64, CDRL2 comprises the CDRL2 of SEQ ID NO:64, CDRL3 the CDRL3 of SEQ ID NO:64, CDRH1 comprises the CDRH1 of SEQ ID NO:63, CDRH2 comprises the CDRH2 of SEQ ID NO:63 and CDRH3 comprises the CDRH3 of SEQ ID NO:63. In another embodiment, CDRL1 comprises the CDRL1 of SEQ ID NO:66, CDRL2 comprises the CDRL2 of SEQ ID NO:66, CDRL3 the CDRL3 of SEQ ID NO:66, CDRH1 comprises the CDRH1 of SEQ ID NO:65, CDRH2 comprises the CDRH2 of SEQ ID NO:65 and CDRH3 comprises the CDRH3 of SEQ ID NO:65. In another embodiment, CDRL1 comprises the CDRL1 of SEQ ID NO:68, CDRL2 comprises the CDRL2 of SEQ ID NO:68, CDRL3 the CDRL3 of SEQ ID NO:68, CDRH1 comprises the CDRH1 of SEQ ID NO:67, CDRH2 comprises the CDRH2 of SEQ ID NO:67 and CDRH3 comprises the CDRH3 of SEQ ID NO:67. In another embodiment, CDRL1 comprises the CDRL1 of SEQ ID NQ:70, CDRL2 comprises the CDRL2 of SEQ ID NQ:70, CDRL3 the CDRL3 of SEQ ID NQ:70, CDRH1 comprises the CDRH1 of SEQ ID NO:69, CDRH2 comprises the CDRH2 of SEQ ID NO:69 and CDRH3 comprises the CDRH3 of SEQ ID NO:69. In another embodiment, CDRL1 comprises the CDRL1 of SEQ ID NO:72, CDRL2 comprises the CDRL2 of SEQ ID NO:72, CDRL3 the CDRL3 of SEQ ID NO:72, CDRH1 comprises the CDRH1 of SEQ ID NO:71 , CDRH2 comprises the CDRH2 of SEQ ID NO:71 and CDRH3 comprises the CDRH3 of SEQ ID NO:71 . . In another embodiment, CDRL1 comprises the CDRL1 of SEQ ID NO:74, CDRL2 comprises the CDRL2 of SEQ ID NO:74, CDRL3 the CDRL3 of SEQ ID NO:74, CDRH1 comprises the CDRH1 of SEQ ID NO:73, CDRH2 comprises the CDRH2 of SEQ ID NO:73 and CDRH3 comprises the CDRH3 of SEQ ID NO:73. In another embodiment, CDRL1 comprises the CDRL1 of SEQ ID NO:76, CDRL2 comprises the CDRL2 of SEQ ID NO:76, CDRL3 the CDRL3 of SEQ ID NO:76, CDRH1 comprises the CDRH1 of SEQ ID NO:75, CDRH2 comprises the CDRH2 of SEQ ID NO:75 and CDRH3 comprises the CDRH3 of SEQ ID NO:75. In another embodiment, CDRL1 comprises the CDRL1 of SEQ ID NO:78, CDRL2 comprises the CDRL2 of SEQ ID NO:78, CDRL3 the CDRL3 of SEQ ID NO:78, CDRH1 comprises the CDRH1 of SEQ ID NO:77, CDRH2 comprises the CDRH2 of SEQ ID NO:77 and CDRH3 comprises the CDRH3 of SEQ ID NO:77. In another embodiment, CDRL1 comprises the CDRL1 of SEQ ID NQ:80, CDRL2 comprises the CDRL2 of SEQ ID NQ:80, CDRL3 the CDRL3 of SEQ ID NQ:80, CDRH1 comprises the CDRH1 of SEQ ID NO:79, CDRH2 comprises the CDRH2 of SEQ ID NO:79 and CDRH3 comprises the CDRH3 of SEQ ID NO:79. In another embodiment, CDRL1 comprises the CDRL1 of SEQ ID NO:82, CDRL2 comprises the CDRL2 of SEQ ID NO:82, CDRL3 the CDRL3 of SEQ ID NO:82, CDRH1 comprises the CDRH1 of SEQ ID NO:81, CDRH2 comprises the CDRH2 of SEQ ID NO:81 and CDRH3 comprises the CDRH3 of SEQ ID NO:81. In another embodiment, CDRL1 comprises the CDRL1 of SEQ ID NO:84, CDRL2 comprises the CDRL2 of SEQ ID NO:84, CDRL3 the CDRL3 of SEQ ID NO:84, CDRH1 comprises the CDRH1 of SEQ ID NO:83, CDRH2 comprises the CDRH2 of SEQ ID NO:83 and CDRH3 comprises the CDRH3 of SEQ ID NO:83. In another embodiment, CDRL1 comprises the CDRL1 of SEQ ID NO:86, CDRL2 comprises the CDRL2 of SEQ ID NO:86, CDRL3 the CDRL3 of SEQ ID NO:86, CDRH1 comprises the CDRH1 of SEQ ID NO:85, CDRH2 comprises the CDRH2 of SEQ ID NO:85 and CDRH3 comprises the CDRH3 of SEQ ID NO:85. In another embodiment, CDRL1 comprises the CDRL1 of SEQ ID NO:88, CDRL2 comprises the CDRL2 of SEQ ID NO:88, CDRL3 the CDRL3 of SEQ ID NO:88, CDRH1 comprises the CDRH1 of SEQ ID NO:87, CDRH2 comprises the CDRH2 of SEQ ID NO:87 and CDRH3 comprises the CDRH3 of SEQ ID NO:87. In another embodiment, CDRL1 comprises the CDRL1 of SEQ ID NQ:90, CDRL2 comprises the CDRL2 of SEQ ID NQ:90, CDRL3 the CDRL3 of SEQ ID NQ:90, CDRH1 comprises the CDRH1 of SEQ ID NO:89, CDRH2 comprises the CDRH2 of SEQ ID NO:89 and CDRH3 comprises the CDRH3 of SEQ ID NO:89. In another embodiment, CDRL1 comprises the CDRL1 of SEQ ID NO:92, CDRL2 comprises the CDRL2 of SEQ ID NO:92, CDRL3 the CDRL3 of SEQ ID NO:92, CDRH1 comprises the CDRH1 of SEQ ID NO:91, CDRH2 comprises the CDRH2 of SEQ ID NO:91 and CDRH3 comprises the CDRH3 of SEQ ID NO:91. . In another embodiment, CDRL1 comprises the CDRL1 of SEQ ID NO:94, CDRL2 comprises the CDRL2 of SEQ ID NO:94, CDRL3 the CDRL3 of SEQ ID NO:94, CDRH1 comprises the CDRH1 of SEQ ID NO:93, CDRH2 comprises the CDRH2 of SEQ ID NO:93 and CDRH3 comprises the CDRH3 of SEQ ID NO:93. In another embodiment, CDRL1 comprises the CDRL1 of SEQ ID NO:96, CDRL2 comprises the CDRL2 of SEQ ID NO:96, CDRL3 the CDRL3 of SEQ ID NO:96, CDRH1 comprises the CDRH1 of SEQ ID NO:95, CDRH2 comprises the CDRH2 of SEQ ID NO:95 and CDRH3 comprises the CDRH3 of SEQ ID NO:95. In another embodiment, CDRL1 comprises the CDRL1 of SEQ ID NO:98, CDRL2 comprises the CDRL2 of SEQ ID NO:98, CDRL3 the CDRL3 of SEQ ID NO:98, CDRH1 comprises the CDRH1 of SEQ ID NO:97, CDRH2 comprises the CDRH2 of SEQ ID NO:97 and CDRH3 comprises the CDRH3 of SEQ ID NO:97. In another embodiment, CDRL1 comprises the CDRL1 of SEQ ID NQ:100, CDRL2 comprises the CDRL2 of SEQ ID NQ:100, CDRL3 the CDRL3 of SEQ ID NQ:100, CDRH1 comprises the CDRH1 of SEQ ID NO:99, CDRH2 comprises the CDRH2 of SEQ ID NO:99 and CDRH3 comprises the CDRH3 of SEQ ID NO:99. In another embodiment, CDRL1 comprises the CDRL1 of SEQ ID NQ:102, CDRL2 comprises the CDRL2 of SEQ ID NQ:102, CDRL3 the CDRL3 of SEQ ID NQ:102, CDRH1 comprises the CDRH1 of SEQ ID NO:101 , CDRH2 comprises the CDRH2 of SEQ ID NO:101 and CDRH3 comprises the CDRH3 of SEQ ID NO:101.

In any of the above-mentioned CDR-sequence-defined embodiments, the antigen-binding protein may be, for example, a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human (e.g., fully human) antibody, a humanized antibody, a chimeric antibody, a multi-specific antibody, or an antigen binding fragment thereof. Further, the antibody fragment of the antigen-binding proteins may be a Fab fragment, a Fab' fragment, an F(ab')2 fragment, an Fv fragment, a diabody, or a single-chain antibody molecule. For example, the antigenbinding protein may be a human monoclonal antibody and may be, e.g., an lgG1-, lgG2-, lgG3-, lgG4, IgM, lgA1 or lgA2-type antibody. Antibodies that carry mutations in the CH regions that reduce or abolish effector functions, increase chemical stability and serum halflive. Further, the antigen-binding proteins may be neutralizing antigen-binding proteins, e.g., may have an ICsoof <100 nM, <50 nM, <10 nM, <1 nM, <0.5 nM, or <0.1 nM in a neutralization assay with SARS-CoV-2 with <1 nM or less for preferred antibodies.

In any of the above-mentioned CDR-sequence-defined embodiments, the antigen-binding protein may specifically bind to a domain, e.g., the trimeric ectodomain of the Spike protein or NTD, RBD, S1 , S2 of the CoV-2 Spike protein.

In any of the above-mentioned CDR-sequence-defined embodiments, the antigen-binding protein may specifically bind to domains, e.g., the (membrane-bound) CoV-2 spike protein, the trimeric ectodomain of, NTD, RBD, S1 , S2, of the CoV-2 Spike protein, preferably to the NTD, RBD, S1 , S2, of the CoV-2 Spike protein with a KD <1 pM, <100 nM, <10 nM, or <5 nM, < 1 nM or < 0.1 nM, e.g., as determined using an Elisa binding assay and analyzed.

In any of the above-mentioned CDR-sequence-defined embodiments, the antigen-binding proteins that compete for binding of ACE-2 to RBD of the CoV-2 Spike protein specifically bind to RBD of the CoV-2 Spike protein with a KD <1 pM, <100 nM, <10 nM, or <5 nM, < 1 nM or < 0.1 nM, e.g., as determined using an Elisa or FACS binding assay and analyzed, for example, using methods described in Rathanaswami et al., Biochemical and Biophysical Research Communications 334 (2005) 1004-1013.

In any of the above-mentioned CDR-sequence-defined embodiments, the antigen-binding proteins that compete for binding to RBD of the CoV-2 Spike protein have an EC50 of <100 nM, <10 nM, <1 nM, <0.5 nM or <0.1 nM in an Elisa-based hACE-2 binding competition assay with immobilized CoV-2 spike. In any of the above-mentioned CDR-sequence-defined embodiments, the antigen-binding proteins that compete for binding to RBD of the CoV-2 Spike protein may be neutralizing antigen-binding proteins, e.g. may have an ICsoof <100 nM, <50 nM, <10 nM, <1 nM, <0.5 nM, or <0.1 nM in a neutralization assay with SARS-CoV-2, with <1 nM or less for preferred antibodies.

In any of the above-mentioned CDR-sequence-defined embodiments, the antigen-binding proteins that compete for binding to a subdomain of the CoV-2 Spike protein, e.g. S1, NTD or another subdomain different from RBD, bind to a subdomain of the CoV-2 Spike protein, e.g. S1, NTD or another subdomain different from RBD, with a KD <1 pM, <100 nM, <10 nM, or <5 nM, < 1 nM or < 0.1 nM, e.g., as determined using an Elisa or FACS binding assay and analyzed, for example, using methods described in Rathanaswami et al., Biochemical and Biophysical Research Communications 334 (2005) 1004-1013.

In any of the above-mentioned CDR-sequence-defined embodiments, the antigen-binding proteins that compete for binding to a subdomain of the CoV-2 Spike protein, e.g. S1, NTD or another subdomain different from RBD, may be neutralizing antigen-binding proteins, e.g. may have an ICsoof <100 nM, <50 nM, <10 nM, <1 nM, <0.5 nM, or <0.1 nM in a neutralization assay with SARS-CoV-2, with <1 nM or less for preferred antibodies.

In other of the above-mentioned CDR-sequence-defined embodiments, antigen-binding proteins do not bind to RBD, but nevertheless, neutralize CoV-2 infection, e.g. has an ICso of <100 nM, <50 nM, <10 nM, <1 nM, <0.5 nM, or <0.1 nM in a neutralization assay with SARS-CoV-2 with <1 nM or less for preferred antibodies. Such antibodies preferably bind to NTD.

Another set of embodiments includes antigen-binding proteins that include one or a combination of CDRs having the consensus sequences described below and bind the CoV-2 Spike protein. In one aspect, the CDRs from the various groups may be mixed and matched in any particular antigen-binding protein that binds to the CoV-2 Spike protein, e.g. the membrane-bound form of the CoV-2 spike protein, RBD or other domains of the CoV-2 Spike protein. In another aspect, the antigen-binding protein comprises heavy and light chain CDRs that are derived from the same phylogenetically-related group of antibody clones. Exemplary CDR consensus sequences are as follows:

VHC Consensus

CDR1 : GYGMH (SEQ ID NO:19), wherein G (at position 1) may be G or may be substituted by S or an amino acid selected from CDR2: VIWYDGSNQYYADSVKG (SEQ ID NO:20), wherein Q may be Q or may be substituted by K or an amino acid selected from

CDR3: ETVDGMDV (SEQ ID NO:21).

VLC Consensus

CDR1 : RARQDINNYLA (SEQ ID NO:22).

CDR2: AASSLLS (SEQ ID NO:23).

CDR3: LQHNSYPYT (SEQ ID NO:24).

In any of the above-mentioned consensus sequence-defined embodiments, the antigen-binding protein may be, for example, an AVIMER polypeptide, a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human (e.g., fully human) antibody, a humanized antibody, a chimeric antibody, a multi-specific antibody, or an antigen binding fragment thereof. Further, the antibody fragment of the antigen-binding proteins may be a Fab fragment, a Fab' fragment, an F(ab')2 fragment, an Fv fragment, a diabody, or a single-chain antibody molecule. For example, the antigen-binding protein may be a human monoclonal antibody and may be, e.g., an lgG1-, lgG2-, lgG3-, lgG4-, IgM-, lgA1- or lgA2 -type antibody and antibodies that carry mutations in the CH regions that reduce or abolish effector functions, increase chemical stability and serum half-live. Further, the antigen-binding proteins may be neutralizing antigen-binding proteins, e.g., may have an ICso of <100 nM <50 nM, <10 nM, <1 nM, <0.5 nM, or <0.1 nM in a neutralization assay with SARS-CoV-2 with <1 nM or less for preferred antibodies.

Some of the antigen-binding proteins described comprise a heavy chain variable region (VH) sequence that has at least 80%, 85%, and 90% or 95% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs:1 , 3 and 5, 37, 39, 41 , 43, 45, 47, 49, 51 , 53, 55, 57 and 59, 61 , 63, 65, 67, 69, 71 , 73, 75, 77, 79, 81 , 83, 85, 87, 89, 91 , 93, 95, 97, 99 and 101. Some of the antigen-binding proteins described comprise a light chain variable region (VL) sequence that has at least 80%, 85%, and 90% or 95% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs:2, 4 and 6, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58 and 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100 and 102. Some of the antigen-binding proteins described comprise a VH sequence that has at least 80%, 85%, 90% or 95% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs:1 , 3 and 5, 37, 39, 41 , 43, 45, 47, 49, 51 , 53, 55, 57 and 59, 61 , 63, 65, 67, 69, 71 , 73, 75, 77, 79, 81 , 83, 85, 87, 89, 91 , 93, 95, 97, 99 and 101. and a Vi that has at least 80%, 85%, 90% or 95% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs:2, 4 and 6, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58 and 60,62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100 and 102. In some embodiments, the antigen-binding proteins comprise (A) a heavy chain variable region (VH) comprising a sequence (i) selected from the group consisting of SEQ ID NOs:1 , 3 and 5, 37, 39, 41 , 43, 45, 47, 49, 51 , 53, 55, 57 and 59, 61 , 63, 65, 67, 69, 71 , 73, 75, 77, 79, 81 , 83, 85, 87, 89, 91 , 93, 95, 97, 99 and 101. or (ii) as defined by (i) and containing one or more (e.g., five, ten, fifteen or twenty) amino acid substitutions (e.g., conservative amino acid substitutions), deletions or insertions; (B) a VL comprising a sequence (iii) selected from the group consisting of SEQ ID NOs:2, 4 and 6, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58 and 60,62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100 and 102 or (iv) as defined by (iii) containing one or more (e.g., five, ten, fifteen or twenty) amino acid substitutions (e.g., conservative amino acid substitutions), deletions or insertions; or (C) a VH of (A) and a L of (B). In some embodiments, the antigen-binding proteins comprise a heavy chain variable region (VH) comprising a sequence selected from the group consisting of SEQ ID NOs:1 , 3 and 5, 37, 39, 41 , 43, 45, 47, 49, 51 , 53, 55, 57 and 59, 61 , 63, 65, 67, 69, 71 , 73, 75, 77, 79, 81 , 83, 85, 87, 89, 91 , 93, 95, 97, 99 and 101 and a VL comprising a sequence selected from the group consisting of SEQ ID NOs:2, 4 and 6, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58 and 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100 and 102.

In any of the above-mentioned Vr and VH sequence defined embodiments, the antigen-binding protein may be, for example, a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human (e.g., fully human) antibody, a humanized antibody, a chimeric antibody, a multi-specific antibody, or an antigen binding fragment thereof. Further, the antibody fragment of the antigen-binding proteins may can be a Fab fragment, a Fab' fragment, an F(ab')2 fragment, an Fv fragment, a diabody or a single-chain antibody molecule; and maybe, for example, a human monoclonal antibody, e.g., an lgG1-, lgG2-, lgG3-, or lgG4-, IgM-, lgA1-, lgA2-type antibody or antibodies that carry mutations in the CH regions that reduce or abolish effector functions, increase chemical stability, and serum half-live. Further, the antigen-binding proteins may be neutralizing antigen-binding proteins, e.g. may have an ECso of <100 nM, <50 nM, <10 nM, <1 nM, <0.5 nM, or <0.1 nM in a neutralization assay with SARS-CoV-2 with <1 nM or less for preferred antibodies.

In any of the above-mentioned VL and VH sequence defined embodiments, the antigenbinding protein may specifically bind to domains, e.g., the membrane-bound CoV-2 spike protein, the trimeric ectodomain of, NTD, RBD, S1 , S2, of the CoV-2 Spike protein, preferably to the NTD, RBD, S1 , S2, of the CoV-2 Spike protein. In any of the above-mentioned VL and VH sequence defined embodiments, the antigenbinding protein may specifically bind to domains, e.g., the (membrane-bound) CoV-2 spike protein, the trimeric ectodomain of, NTD, RBD, S1 , S2, of the CoV-2 Spike protein, preferably to the NTD, RBD, S1 , S2, of the CoV-2 Spike protein with a KD <1 pM, <100 nM, <10 nM, or <5 nM, < 1 nM or < 0.1 nM, e.g., as determined using an Elisa binding assay and analyzed.

In any of the above-mentioned Viand H sequence defined embodiments, the antigen-binding proteins that compete for binding of ACE-2 to RBD of the CoV-2 Spike protein specifically bind to RBD of the CoV-2 Spike protein with a KD <1 pM, <100 nM, <10 nM, or <5 nM, < 1 nM or < 0.1 nM, e.g., as determined using an Elisa or FACS binding assay and analyzed, for example, using methods described in Rathanaswami et al., Biochemical and Biophysical Research Communications 334 (2005) 1004-1013.

In any of the above-mentioned Viand VH sequence defined embodiments, the antigen-binding proteins that compete for binding to RBD of the CoV-2 Spike protein have an EC50 of <100 nM, <10 nM, <1 nM, <0.5 nM or <0.1 nM in an Elisa-based hACE-2 binding competition assay with immobilized CoV-2 spike.

In any of the above-mentioned Viand VH sequence defined embodiments, the antigen-binding proteins that compete for binding to RBD of the CoV-2 Spike protein may be neutralizing antigen-binding proteins, e.g. may have an ICsoof <100 nM, <50 nM, <10 nM, <1 nM, <0.5 nM, or <0.1 nM in a neutralization assay with SARS-CoV-2, with <1 nM or less for preferred antibodies.

In any of the above-mentioned Viand VH sequence defined embodiments, the antigen-binding proteins that compete for binding to a subdomain of the CoV-2 Spike protein, e.g. S1, NTD or another subdomain different from RBD, bind to a subdomain of the CoV-2 Spike protein, e.g. S1, NTD or another subdomain different from RBD, with a KD <1 pM, <100 nM, <10 nM, or <5 nM, < 1 nM or < 0.1 nM, e.g., as determined using an Elisa or FACS binding assay and analyzed, for example, using methods described in Rathanaswami et al., Biochemical and Biophysical Research Communications 334 (2005) 1004-1013.

In any of the above-mentioned Viand VH sequence defined embodiments, the antigen-binding proteins that compete for binding to a subdomain of the CoV-2 Spike protein, e.g. S1, NTD or another subdomain different from RBD, may be neutralizing antigen-binding proteins, e.g. may have an ICsoof <100 nM, <50 nM, <10 nM, <1 nM, <0.5 nM, or <0.1 nM in a neutralization assay with SARS-CoV-2, with <1 nM or less for preferred antibodies. In any of the above-mentioned Vi and H sequence defined embodiments, antigen-binding proteins do not bind to RBD, but nevertheless, neutralize CoV-2 infection, e.g. has an ICso of <100 nM, <50 nM, <10 nM, <1 nM, <0.5 nM, or <0.1 nM in a neutralization assay with SARS-CoV-2 with <1 nM or less for preferred antibodies. Such antibodies preferably bind to NTD.

In one aspect, the antigen-binding proteins comprise a heavy chain sequence that comprises a sequence having at least 80%, 85%, 90% or 95% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs:31-33. Some of the antigenbinding proteins described comprise a light chain sequence that comprises a sequence having at least 80%, 85%, 90% or 95% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs:34-36. Some of the antigen-binding proteins comprise a heavy chain sequence that comprises a sequence having at least 80%, 85%, 90% or 95% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 31-33, and a light chain sequence that comprises a sequence having at least 80%, 85%, 90% or 95% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 34-36. In some embodiments, the antigen-binding proteins comprise (A) a heavy chain comprising a sequence (i) selected from the group consisting of SEQ ID NOs: 31-33, or (ii) as defined by (i) and containing one or more (e.g., five, ten, fifteen or twenty) amino acid substitutions (e.g., conservative amino acid substitutions), deletions or insertions; (B) a light chain comprising a sequence (iii) selected from the group consisting of SEQ ID NOs: 34-36, or (iv) as defined by (iii) containing one or more (e.g., five, ten, fifteen or twenty) amino acid substitutions (e.g., conservative amino acid substitutions), deletions or insertions; or (C) a heavy chain of (A) and a light chain of (B). In some embodiments, the antigen-binding proteins comprise a heavy chain comprising a sequence selected from the group consisting of SEQ ID NOs: 31- 33 and a light chain comprising a sequence selected from the group consisting of SEQ ID NOs: 34-36.

In any of the above-mentioned light and heavy chain sequence defined embodiments, the antigen-binding protein may comprise the specified heavy and/or light chain sequence, but with an additional signal peptide, a different signal peptide or with no signal peptide. In any of the above-mentioned light and heavy chain sequence defined embodiments, the antigen-binding protein may be, for example, a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human (e.g., fully human) antibody, a humanized antibody, a chimeric antibody, a multi-specific antibody, or an antigen binding fragment thereof. Further, the antibody fragment of the antigen-binding proteins may can be a Fab fragment, a Fab' fragment, an F(ab')2 fragment, an Fv fragment, a diabody or a single-chain antibody molecule; and maybe, for example, a human monoclonal antibody, e.g., an lgG1-, lgG2-, lgG3-, or lgG4-, IgM-, lgA1-, lgA2-type antibody or antibodies that carry mutations in the CH regions that reduce or abolish effector functions, increase chemical stability, and serum half-live. Further, the antigen-binding proteins may be neutralizing antigen-binding proteins, e.g. may have an ICso of <100 nM, <50 nM, <10 nM, <1 nM, <0.5 nM, or <0.1 nM in a neutralization assay with SARS-CoV-2 with <1 nM or less for preferred antibodies.

In any of the above-mentioned light and heavy chain sequence-defined embodiments, the antigen-binding protein may specifically bind domains of the Spike CoV-2, e.g., RBD, S1 , S2, trimeric ectodomain, preferably RBD, with a KD <1 pM, <100 nM, <10 nM, or <5 nM, < 1 nM or < 0.1 nM, e.g., as determined using an Elisa binding. In any of the above-mentioned light and heavy chain sequence-defined embodiments, the antigen-binding protein may have an EC50 of <100 nM <10 nM, <1 nM, <0.5 nM or <0.1 nM in an hACE-2 binding competition assay, e.g., in flow cytometry-based l-ACE-2 binding competition assay to membranes from cells expressing CoV-2 Spike protein or Elisa-based assays with immobilized recombinant Spike protein and subdomains (e.g., trimeric ectodomain, NTD, RBD, S1 and S2)

In a further aspect, also provided are nucleic acid polynucleotides that encode any of the CoV-2 Spike protein antigen-binding proteins that either neutralize CoV-2 and bind to either RBD or other parts of the CoV-2 spike protein as summarized above. In one embodiment, the polynucleotide comprises a sequence selected from the group consisting of SEQ ID NOs:25-30. In another embodiment, the polynucleotide comprises a sequence that is about 80%, 85%, 90% or 95% or more identical to a sequence selected from the group consisting of SEQ ID NOs:25-30.

In some embodiments, the nucleic acid molecules encoding any format of antibodies carrying the sequences are operably-linked to a control sequence and can be delivered in liposomes or viruses such as AAV, measles, lentiviruses, etc. In related embodiments, the polynucleotides are incorporated into a vector for expression in bacteria, insect cells, plant cells, yeast and mammalian cells.

Also included are cell lines transformed with expression vectors comprising polynucleotides as described above. In a related aspect, also provided are expression vectors and host cells transformed or transfected with the expression vectors that comprise the aforementioned nucleic acid molecules that encode CoV-2 Spike protein antigen-binding proteins, preferably CoV-2 neutralizing antigen-binding proteins described above In another aspect, also provided is a method of preparing the antigen-binding proteins that includes the step of preparing the antigen-binding protein from a host cell that secretes the antigen-binding protein. In some embodiments, the antigen-binding protein is generated using an immunogen comprising the entire soluble trimeric spike protein. In yet another aspect, a pharmaceutical composition is provided comprising at least one of the antigenbinding proteins summarized above and a pharmaceutically acceptable excipient. In one embodiment, the pharmaceutical composition may comprise an additional active agent that is selected from the group consisting of a radioisotope, radionuclide, a toxin, or a therapeutic and a chemotherapeutic group neutralizing the processing of CoV-2 proteins or replication of CoV-2.

Other aspects further provide methods either preventing the CoV-2 infection in healthy individuals or for treating or reducing severity of a condition associated with SARS-CoV-2 infection in a patient, comprising administering to a healthy proband or patient a sufficient amount in the form of at least one antigen-binding protein or any form of RNA or DNA encoding the antigen-binding domain summarized above. In one embodiment, the condition is an acute respiratory disease (e.g., sore throat, cough (usually dry cough), shortness of breath, chest pain), fever, loss of the sense of smell and/or taste, headache, general weakness, malaise, muscle aches, sniffles, a gastrointestinal symptom (e.g., nausea, vomiting, diarrhea, abdominal pain), myocarditis, meningoencephalitis and Kawasaki-like symptoms in children.

Definitions

The term "polynucleotide" or "nucleic acid" includes both single-stranded and doublestranded nucleotide polymers. The nucleotides comprising the polynucleotide can be ribonucleotides or deoxyribonucleotides or a modified form of either type of nucleotide. Said modifications include base modifications such as bromouridine and inosine derivatives, ribose modifications such as 2',3'-deoxyribose, and internucleotide linkage modifications such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate and phosphoroamidate.

For purposes of this disclosure, it should be understood that "a nucleic acid molecule comprising" a particular nucleotide sequence does not encompass intact chromosomes. "Nucleic acid molecules” "comprising" specified nucleic acid sequences may include, in addition to the specified sequences, coding sequences for up to ten or even up to twenty other proteins or portions thereof, or may include operably linked regulatory sequences that control the expression of the coding region of the recited nucleic acid sequences, and/or may include vector sequences. Unless specified otherwise, the left-hand end of any single-stranded polynucleotide sequence discussed herein is the 5' end; the left-hand direction of double-stranded polynucleotide sequences is referred to as the 5' direction. The direction of 5' to 3' addition of nascent RNA transcripts is referred to as the transcription direction; sequence regions on the DNA strand having the same sequence as the RNA transcript that are 5' to the 5' end of the RNA transcript are referred to as "upstream sequences;" sequence regions on the DNA strand having the same sequence as the RNA transcript that are 3' to the 3' end of the RNA transcript are referred to as "downstream sequences."

The term "control sequence" refers to a polynucleotide sequence that can affect the expression and processing of coding sequences to which it is ligated. The nature of such control sequences may depend upon the host organism. In particular embodiments, control sequences for prokaryotes may include a promoter, a ribosomal binding site, and a transcription termination sequence. For example, control sequences for eukaryotes may include promoters comprising one or a plurality of recognition sites for transcription factors, transcription enhancer sequences, and transcription termination sequence. "Control sequences" can include leader sequences and/or fusion partner sequences.

The term "vector" means any molecule or entity (e.g., nucleic acid, plasmid, bacteriophage or virus) used to transfer protein-coding information into a host cell. “Vectors” include, but are not limited to DNA vehicles used to introduce DNA or RNA into cells, e.g, adenovirus, cow pox virus, measle virus, lentivirus, adeno-asscoated virus). Such vectors may transfer RNA or DNA encoding an antigen-binding protein of the present invention into cells. Such vectors that transfer RNA or DNA encoding an antigen-binding protein of the present invention into cells may be used for therapeutic purposes, e.g. for passive DNA- or RNA- immunization.

The term "expression vector" or "expression construct" refers to a vector that is suitable for transformation of a host cell and contains nucleic acid sequences that direct and/or control (in conjunction with the host cell) expression of one or more heterologous coding regions operatively linked thereto. An expression construct may include, but is not limited to, sequences that affect or control transcription, translation, and, if introns are present, affect RNA splicing of a coding region operably linked thereto.

As used herein, "operably linked" means that the components to which the term is applied are in a relationship that allows them to carry out their inherent functions under suitable conditions. For example, a control sequence in a vector that is "operably linked" to a proteincoding sequence is ligated thereto so that expression of the protein-coding sequence is achieved under conditions compatible with the transcriptional activity of the control sequences.

The term "host cell" means a cell that has been transformed, or is capable of being transformed, with a nucleic acid sequence and thereby expresses a gene of interest. The term includes the progeny of the parent cell, whether or not the progeny is identical in morphology or in genetic make-up to the original parent cell, so long as the gene of interest is present.

The term "transduction" means the transfer of genes from one bacterium to another, usually by bacteriophage. "Transduction" also refers to the acquisition and transfer of eukaryotic cellular sequences by replication-defective retroviruses.

The term "transfection" means the uptake of foreign or exogenous DNA by a cell, and a cell has been "transfected" when the exogenous DNA has been introduced inside the cell membrane. A number of transfection techniques are well known in the art and are disclosed herein. See, e.g., Graham et al., 1973, Virology 52:456; Sambrook et al., 2001 , Molecular Cloning: A Laboratory Manual, supra, Davis et al., 1986, Basic Methods in Molecular Biology, Elsevier; Chu et al., 1981 , Gene 13:197. Such techniques can be used to introduce one or more exogenous DNA moieties into suitable host cells.

The term "transformation" refers to a change in a cell's genetic characteristics, and a cell has been transformed when it has been modified to contain new DNA or RNA. For example, a cell is transformed where it is genetically modified from its native state by introducing new genetic material via transfection, transduction, or other techniques. Following transfection or transduction, the transforming DNA may recombine with that of the cell by physically integrating into a chromosome of the cell, or maybe maintained transiently as an episomal element without being replicated, or may replicate independently as a plasmid. A cell is considered to have been "stably transformed" when the transforming DNA is replicated with the division of the cell.

The terms "polypeptide" or "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms also apply to amino acid polymers in which one or more amino acid residues is an analog or mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. The terms can also encompass amino acid polymers that have been modified, e.g., by the addition of carbohydrate residues to form glycoproteins, or by phosphorylation. Polypeptides and proteins can be produced by a naturally-occurring and non-recombinant cell, or it is produced by a genetically- engineered or recombinant cell, and comprise molecules having the amino acid sequence of the native protein or molecules having deletions from, additions to, and/or substitutions of one or more amino acids of the native sequence. The terms "polypeptide" and "protein" specifically encompass antigen-binding proteins, e.g., against the entire spike protein, the ectodomain, the NTD, S1 or S2 subunits and the RBD, antibodies, or sequences that have deletions from, additions to, and/or substitutions of one or more amino acids of an antigenbinding protein. The term "polypeptide fragment" refers to a polypeptide that has an aminoterminal deletion, a carboxyl-terminal deletion, and/or an internal deletion as compared with the full-length protein. Such fragments may also contain modified amino acids as compared with the full-length protein. In certain embodiments, fragments are about five to 500 amino acids long. For example, fragments may be at least 5, 6, 8, 10, 14, 20, 50, or 70 amino acids long. Useful polypeptide fragments include immunologically functional fragments of antibodies, including binding domains. In the case of an RBD or other regions of the CoV-2 Spike protein-binding antibody, useful fragments include but are not limited to a CDR region, a variable domain of a heavy or light chain, a portion of an antibody chain or just its variable domain including two CDRs, and the like.

A "variant" of a polypeptide (e.g., an antigen-binding protein, or an antibody) comprises an amino acid sequence wherein one or more amino acid residues are inserted into, deleted from and/or substituted into the amino acid sequence relative to another polypeptide sequence. Variants include fusion proteins.

A "derivative" of a polypeptide is a polypeptide (e.g., an antigen-binding protein, or an antibody) that has been chemically modified in some manner distinct from insertion, deletion, or substitution variants, e.g., via conjugation to another chemical moiety.

The term "naturally occurring" as used throughout the specification in connection with biological materials such as polypeptides, nucleic acids, host cells, and the like, refers to materials which are found in nature.

An "antigen-binding protein" as used herein means a protein that binds explicitly a specified target antigen, such as RBD or other parts of the CoV-2 Spike protein.

An antigen-binding protein is said to "specifically bind" its target when the dissociation constant (KD) is <10^-6 M. The antibody specifically binds the target antigen with "high affinity" when the KD is <1x 10'⁸ M. In one embodiment, the antibodies will bind to RBD or other subdomains of the CoV-2 Spike protein (e.g., the soluble ectodomain, NTD, RBD, S1, and S2, preferably RBD, or the CoV-2 Spike protein (e.g membrane-bound spike protein) with a KD <5x 10'⁷; in another embodiment, the antibodies will bind with a KD <1x 10'⁷; in another embodiment, the antibodies will bind with a KD <5X 10'⁸; in another embodiment, the antibodies will bind with a KD <1X 10'⁸; in another embodiment, the antibodies will bind with a KD <5x 10'⁹; in another embodiment, the antibodies will bind with a KD <1x 10'⁹; in another embodiment, the antibodies will bind with a KD <5X 10'¹°; in another embodiment, the antibodies will bind with a KD <1X 10^-10.

Throughout all embodiments of the invention, an antigen-binding protein that “inhibits the binding of CoV-2 Spike Protein, e.g. RBD to human ACE-2 (hACE-2) enzyme” is preferably an antigen-binding protein that competes for binding to RBD of the CoV-2 Spike protein with an EC50 of <100 nM in an Elisa-based hACE-2 binding competition assay with immobilized CoV-2 spike or subdomains of it (e.g., membrane-bound spike protein, the soluble ectodomain, NTD, RBD, S1 , and S2, preferably RBD). More preferred is an antigen-binding protein that has an ECso of <10 nM, even more preferred <1 nM, most preferred <0.1 nM. Other assays established in the art may also be used.

Throughout all embodiments of the invention, an antigen-binding protein that “inhibits the binding of CoV-2 to human ACE-2 (hACE-2) enzyme” preferably is an antigen-binding protein that has an EC50 of <100 nM in an hACE-2 flow cytometric analysis to membranes from cells expressing the complete CoV-2 Spike protein. More preferred is an antigenbinding protein that has an ECso of <10 nM, even more preferred an ICso of <1 nM, most preferred an ECso of <0.1 nM. Other assays established in the art may also be used.

Throughout all embodiments of the invention, an antigen-binding protein that “neutralizes CoV- 2 infection of cells” is capable of preventing CoV-2 from infecting a cell. Preferably, an antigenbinding protein that “neutralizes CoV-2 infection of cells” is an antigen-binding protein, that has an ICso of <100 nM in a neutralization assay with SARS-CoV-2. Preferred is an antigen-binding protein that has an ICso of <10 nM in a neutralization assay with SARS-CoV-2.

More preferred is an antigen-binding protein that has an ICso of <10 nM, even more prefered 1 nM or most preferred ICso of < 0.1 nM in a neutralization assay with SARS-CoV-2. A preferred neutralization assay is performed with Vero E6 cells as described in this specification. Neutralization assays established in the art may also be used.

"Antigen binding region" means a protein, or a portion of a protein, that binds explicitly a specified antigen. For example, that portion of an antigen-binding protein that contains the amino acid residues that interact with an antigen and confer on the antigen-binding protein its specificity and affinity for the antigen is referred to as "antigen binding region." An antigenbinding region typically includes one or more "complementary determining regions" ("CDRs"). Certain antigen-binding regions also include one or more "framework" regions. A "CDR" is an amino acid sequence that contributes to antigen-binding specificity and affinity." Framework" regions can aid in maintaining the proper conformation of the CDRs to promote binding between the antigen-binding region and an antigen.

In certain aspects, recombinant antigen-binding proteins that bind RBD or other parts of the CoV-2 Spike protein are provided. In this context, a "recombinant protein" is a protein made using recombinant techniques, /.e., through the expression of a recombinant nucleic acid as described herein. Methods and techniques for the production of recombinant proteins are well known in the art.

The term "antibody" refers to an intact immunoglobulin of any isotype, or an antigen binding fragment thereof that can compete with the intact antibody for specific binding to the target antigen, and includes, for instance, chimeric, humanized, fully human, and bispecific antibodies. An "antibody" as such is a species of an antigen-binding protein. An intact antibody generally will comprise at least two full-length heavy chains and two full-length light chains, but in some instances may include fewer chains such as antibodies naturally occurring in camelids, which may comprise only heavy chains. Antibodies may be derived solely from a single source or maybe "chimeric," that is, different portions of the antibody may be derived from two different antibodies, as described further below. The antigenbinding proteins, antibodies, or binding fragments may be produced in hybridomas, by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies. Unless otherwise indicated, the term "antibody" includes, in addition to antibodies comprising two full-length heavy chains and two full-length light chains, derivatives, variants, fragments, and mutations thereof, examples of which are described below.

The term "light chain" includes a full-length light chain and fragments thereof having sufficient variable region sequence to confer binding specificity. A full-length light chain includes a variable region domain, VL, and a constant region domain, CL. The variable region domain of the light chain is at the amino-terminus of the polypeptide. Light chains include kappa chains and lambda chains.

The term "heavy chain" includes a full-length heavy chain and fragments thereof having sufficient variable region sequence to confer binding specificity. A full-length heavy chain includes a variable region domain, VH, and three constant region domains, CH1 , CH2, and CH3. The VH domain is at the amino-terminus of the polypeptide, and the CH domains are at the carboxyl-terminus, with the CH3 being closest to the carboxy-terminus of the polypeptide. Heavy chains may be of any isotype, including IgG (including lgG1 , lgG2, lgG3 and lgG4 subtypes), IgA (including lgA1 and lgA2 subtypes), IgM and IgE and IgG variants carrying mutations that abolish effector functions, increase stability and serum half-live. The term "signal sequence", “leader sequence” or “signal peptide” refers to a short (3-60 amino acids long) peptide chain that directs the transport of a protein. Signal peptides may also be called targeting signals, signal sequences, transit peptides, or localization signals. Some signal peptides are cleaved from the protein by signal peptidase after the proteins are transported, such that the biologically active form of the protein (e.g., an antigen-binding protein as described herein) is the cleaved, shorter form. Accordingly, terms such as “antibody comprising a heavy chain...”, “antibody comprising a light chain...”, etc., where the antibody is characterized as having a heavy and/or light chain with a particular identified sequence, are understood to include antibodies having the specifically identified sequences, antibodies having the specifically identified sequences except that the signal sequences are replaced by different signal sequences, as well as antibodies having the identified sequences, minus any signal sequences.

The term “antigen-binding fragment” (or simply ‘fragment”) of an antibody or immunoglobulin chain (heavy or light chain), as used herein, comprises a portion (regardless of how that portion is obtained or synthesized) of an antibody that lacks at least some of the amino acids present in a full-length chain but which is capable of specifically binding to an antigen. Such fragments are biologically active in that they bind specifically to the target antigen and can compete with other antigen-binding proteins, including intact antibodies, for specific binding to a given epitope. In one aspect, such a fragment will retain at least one CDR present in the full- length light or heavy chain, and in some embodiments will comprise a single heavy chain and/or light chain or portion thereof. These biologically active fragments may be produced by recombinant DNA techniques or may be produced by enzymatic or chemical cleavage of antigen-binding proteins, including intact antibodies. Immunologically functional immunoglobulin fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, domain antibodies and single-chain antibodies, and may be derived from any mammalian source, including but not limited to human, mouse, rat, camelid or rabbit. It is contemplated further that a functional portion of the antigen-binding proteins disclosed herein, for example, one or more CDRs, could be covalently bound to a second protein or to a small molecule to create a therapeutic agent directed to a particular target in the body, possessing bifunctional therapeutic properties, or having a prolonged serum half-life.

A “Fab fragment” is comprised of one light chain and the CH1 and the variable region of one heavy chain. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule. Depending on the Ig class, an “Fc” region contains two heavy chain fragments comprising either CH1 and CH2 domains of an IgG, IgA or IgD antibody, or CH1, CH2 and CH3 domains of IgM or IgE. The two heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interactions of the CH3 domains.

A “Fab¹ fragment” contains one light chain and a portion of one heavy chain that contains the VH domain and the CH1 domain and also the region between the CH1 and CH2 domains, such that an interchain disulfide bond can be formed between the two heavy chains of two Fab' fragments to form an F(ab')2 molecule.

An “F(ab')2 fragment” contains two light chains and two heavy chains containing a portion of the constant region between the CH1 and CH2 domains, such that an interchain disulfide bond is formed between the two heavy chains. An F(ab')2 fragment thus is composed of two Fab' fragments that are held together by a disulfide bond between the two heavy chains.

The “Fv region” comprises the variable regions from both the heavy and light chains, but lacks the constant regions.

“Single-chain antibodies” are Fv molecules in which the heavy and light chain variable regions have been connected by a flexible linker to form a single polypeptide chain, which forms an antigen-binding region. Single chain antibodies are discussed in detail in International Patent Application Publication No. WO 88/01649 and the United States Patent No. 4,946,778 and No. 5,260,203.

The term “neutralizing antigen-binding protein” or “neutralizing antibody” refers to an antigen-binding protein or antibody, respectively, that binds to a ligand (e.g., the RBD of the spike protein or other parts of the spike protein) and prevents infection of cells by the wildtype CoV-2 virus or by viral vectors pseudotyped with the CoV-2 spike protein. In assessing the binding and specificity of an antigen-binding protein, e.g., an antibody or immunologically functional antigen binding fragment thereof, an antibody or fragment may substantially inhibit binding of a ligand to its binding partner when an excess of antibody reduces the quantity of binding partner bound to the ligand by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99% or more (as measured in an in vitro competitive binding assay). In the case of an RBD of the CoV-2 Spike protein-binding protein, such a neutralizing molecule will diminish the ability of RBD of the CoV-2 Spike protein to bind hACE-2. However, neutralizing antigen-binding proteins in some embodiments of the interaction interact with parts of the CoV-2 spike that differ from RBD and, therefore, e.g. do not compete with ACE-2 for RBD nor CoV-2 spike protein binding.

The term “compete”, when used in the context of antigen-binding proteins that may bind the same region on a target antigen, means competition between antigen-binding proteins is determined by an assay in which the antigen-binding protein (e.g., antibody or immunologically functional antigen binding fragment thereof) under test prevents or inhibits specific binding of a reference antigen-binding protein (e.g., a ligand, or a reference antibody) to a common antigen (e.g., RBD or another site of the CoV-2 Spike protein or an antigen binding fragment thereof). Any of a number of competitive binding assays can be used, for example, solid-phase direct or indirect radioimmunoassay (RIA), solid-phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay (see, e.g., Stahli et al., 1983, Methods in Enzymology 9:242-253); solid-phase direct biotin-avidin EIA (see, e.g., Kirkland et al., 1986, J. Immunol. 137:3614-3619) solid-phase direct labeled assay, solid-phase direct labeled sandwich assay (see, e.g., Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press); solid-phase direct label RIA using 1-125 label (see, e.g., Morel et al., 1988, Molec. Immunol. 25:7-15); solid-phase direct biotin-avidin EIA (see, e.g., Cheung, et al., 1990, Virology 176:546-552); and direct labeled RIA (Moldenhauer et al., 1990, Scand. J. Immunol. 32:77-82) or fluorescence-based flow cytometry. Such an assay may involve the use of purified antigen bound to a solid surface or cells bearing either of these, an unlabeled test antigen-binding protein and a labeled reference antigen-binding protein. Competitive inhibition may be measured by determining the amount of label bound to the solid surface or cells in the presence of the test antigen-binding protein. Antigen-binding proteins identified by competition assay (competing antigen-binding proteins) include antigen-binding proteins binding to the same epitope as the reference antigen-binding proteins and antigen-binding proteins binding to an adjacent epitope sufficiently proximal to the epitope bound by the reference antigen-binding protein for steric hindrance to occur. Usually, when a competing antigen-binding protein is present in excess, it will inhibit specific binding of a reference antigen-binding protein to a common antigen by at least 40%, 45%, 50%, 55%, 60%, 65%, 70% or 75%. In some instances, binding is inhibited by at least 80%, 85%, 90%, 95%, or 97% or more. Competitive inhibition may also be measured by immobilizing a reference antigen-binding protein to a substrate, e.g., a “sensor chip”, capturing antigen on the substrate via binding to the reference antibody, and assaying whether a different antigenbinding protein (a competing antigen-binding protein) can additionally bind to the antigen. An example of the latter competitive binding assay employs a Biacore analysis. The term “antigen” or “immunogen” refers to a molecule or a portion of a molecule capable of being bound by a selective binding agent, such as an antigen-binding protein (including, e.g., an antibody or immunological functional antigen binding fragment thereof), and additionally capable of being used in an animal to produce antibodies capable of binding to that antigen. An antigen may possess one or more epitopes that are capable of interacting with different antigen-binding proteins, e.g., antibodies.

The term “epitope” is the portion of a molecule that is bound by an antigen-binding protein (for example, an antibody). The term includes any determinant capable of specifically binding to an antigen-binding protein, such as an antibody or to a T-cell receptor. An epitope can be contiguous or non-contiguous (e.g., (i) in a single-chain polypeptide, amino acid residues that are not contiguous to one another in the polypeptide sequence but that within in the context of the molecule are bound by the antigen-binding protein, or (ii) in a multimeric protein. In certain embodiments, epitopes may be mimetic in that they comprise a three-dimensional structure that is similar to an epitope used to generate the antigen-binding protein, yet comprise none or only some of the amino acid residues found in that epitope used to generate the antigen-binding protein. Most often, epitopes reside on proteins, but in some instances may reside on other kinds of molecules, such as nucleic acids. Epitope determinants may include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and may have specific three- dimensional structural characteristics, and/or specific charge characteristics. Generally, antibodies specific for a particular target antigen will preferentially recognize an epitope on the target antigen in a complex mixture of proteins and/or macromolecules.

The term “identity” refers to a relationship between the sequences of two or morepolypeptide molecules or two or more nucleic acid molecules, as determined by aligning and comparing the sequences. “Percent identity” means the percent of identical residues between the amino acids or nucleotides in the compared molecules and is calculated based on the size of the smallest of the molecules being compared. For these calculations, gaps in alignments (if any) must be addressed by a particular mathematical model or computer program (/.e., an “algorithm”). Methods that can be used to calculate the identity of the aligned nucleic acids or polypeptides include those described in Computational Molecular Biology, (Lesk, A. M., ed.), 1988, New York: Oxford University Press; Biocomputing Informatics and Genome Projects, (Smith, D. W., ed.), 1993, New York: Academic Press; Computer Analysis of Sequence Data, Part I, (Griffin, A. M., and Griffin, H. G., eds.), 1994, New Jersey: Humana Press; von Heinje, G., 1987, Sequence Analysis in Molecular Biology, New York: Academic Press; Sequence Analysis Primer, (Gribskov, M. and Devereux, J., eds.), 1991, New York: M. Stockton Press; and Carillo et al., 1988, SIAM J. Applied Math. 48:1073. In calculating percent identity, the sequences being compared are aligned in a way that gives the most significant match between the sequences. The computer program used to determine percent identity is the GCG program package, which includes GAP (Devereux et al., 1984, Nucl. Acid Res. 12:387; Genetics Computer Group, University of Wisconsin, Madison, Wl). The computer algorithm GAP is used to align the two polypeptides or polynucleotides for which the percent sequence identity is to be determined. The sequences are aligned for optimal matching of their respective amino acid or nucleotide (the “matched span”, as determined by the algorithm). A gap opening penalty (which is calculated as 3x the average diagonal, wherein the “average diagonal” is the average of the diagonal of the comparison matrix being used; the “diagonal” is the score or number assigned to each perfect amino acid match by the particular comparison matrix) and a gap extension penalty (which is usually 1/10 times the gap opening penalty), as well as a comparison matrix such as PAM 250 or BLOSUM 62, are used in conjunction with the algorithm. In certain embodiments, a standard comparison matrix (see, Dayhoff et al., 1978, Atlas of Protein Sequence and Structure 5:345-352 for the PAM 250 comparison matrix; Henikoff et al., 1992, Proc. Natl. Acad. Sci. U.S.A. 89:10915-10919 for the BLOSUM 62 comparison matrix) is also used by the algorithm.

Recommended parameters for determining percent identity for polypeptides or nucleotide sequences using the GAP program are the following:

Algorithm: Needleman et al., 1970; J. Mol. Biol. 48:443-453;Comparison matrix: BLOSUM 62 from Henikoff et al., 1992, supra, Gap Penalty: 12 (but with no penalty for end gaps) Gap Length Penalty: 4 Threshold of Similarity: 0

Specific alignment schemes for aligning two amino acid sequences may result in the matching of only a short region of the two sequences, and this small aligned region may have very high sequence identity even though there is no significant relationship between the two full-length sequences. Accordingly, the selected alignment method (GAP program) can be adjusted if so desired to result in an alignment that spans at least 50 contiguous amino acids of the target polypeptide.

As used herein, “substantially pure” means that the described species of the molecule is the predominant species present, that is, on a molar basis it is more abundant than any other individual species in the same mixture. In certain embodiments, a substantially pure molecule is a composition wherein the object species comprises at least 50% (on a molar basis) of all macromolecular species present. In other embodiments, a substantially pure composition will comprise at least 80%, 85%, 90%, 95%, or 99% of all macromolecular species present in the composition. In other embodiments, the object species is purified to essential homogeneity wherein contaminating species cannot be detected in the composition by conventional detection methods, and thus, the composition consists of a single detectable macromolecular species.

The term “treating” refers to any indicia of success in the treatment or amelioration of an injury, pathology or condition, including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the injury, pathology or condition more tolerable to the patient; slowing in the rate of degeneration or decline; making the final point of degeneration less debilitating; improving a patient’s physical or mental well-being. The treatment or amelioration of symptoms can be based on objective or subjective parameters; including the results of a physical examination, neuropsychiatric exams, and/or a psychiatric evaluation. For example, certain methods presented herein successfully treat migraine headaches either prophylactically or as an acute treatment, decreasing the frequency of migraine headaches, decreasing the severity of migraine headaches, and/or ameliorating a symptom associated with migraine headaches.

Particular antigen-binding proteins described herein are antibodies or are derived from antibodies. In certain embodiments, the polypeptide structure of the antigen-binding proteins is based on antibodies, including, but not limited to, monoclonal antibodies, bispecific antibodies, minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as “antibody mimetics”), chimeric antibodies, humanized antibodies, human antibodies, antibody fusions (sometimes referred to herein as “antibody conjugates”), and fragments thereof. The various structures are further described hereinbelow.

The antigen-binding proteins provided herein have been demonstrated to bind to the CoV-2 Spike protein.

The antigen-binding proteins that are disclosed herein have a variety of utilities. Some of the antigen-binding proteins, for instance, are useful in specific binding assays, affinity purification of CoV-2 Spike protein and in screening, assays to identify other antagonists of RBD of the CoV-2 Spike protein. Some of the antigen-binding proteins are useful for inhibiting the binding of hACE-2 to the CoV-2 Spike protein.

CoV-2 spike Binding Proteins A variety of selective binding agents useful for detecting the CoV-2 spike protein or regulating the activity of CoV-2 are provided. These agents include, for instance, antigen-binding proteins that contain an antigen binding domain (e.g., single chain antibodies, domain antibodies, immunoadhesin, and polypeptides with an antigen binding region) and specifically bind to RBD or another epitope of the CoV-2 Spike protein. Some of the agents, for example, are useful in inhibiting the binding of hACE-2 to RBD of the CoV-2 Spike protein, and can thus be used to inhibit CoV-2 infection.

In general, the antigen-binding proteins that are provided typically comprise one or more CDRs as described herein (e.g., 1 , 2, 3, 4, 5 or 6). In some instances, the antigen-binding protein comprises (a) a polypeptide structure and (b) one or more CDRs that are inserted into and/or joined to the polypeptide structure. The polypeptide structure can take a variety of different forms. For example, it can be or comprise the framework of a naturally occurring antibody, or fragment or variant thereof, or maybe completely synthetic in nature. Examples of various polypeptide structures are further described below.

CDR sequences of the sequence listing were determined by the Kabat nomenclature. Other nomenclatures are well known to the skilled person and may be used as well.

In certain embodiments, the polypeptide structure of the antigen-binding proteins is an antibody or is derived from an antibody, including, but not limited to, monoclonal antibodies, bispecific antibodies, minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as “antibody mimetics”), chimeric antibodies, humanized antibodies, antibody fusions (sometimes referred to as “antibody conjugates”), and portions or fragments of each, respectively. In some instances, the antigen-binding protein is an immunological fragment of an antibody (e.g., a Fab, a Fab’, an F(ab’)2, or an scFv). The various structures are further described and defined herein.

In embodiments where the antigen-binding protein is used for preventive or therapeutic applications, an antigen-binding protein can inhibit, interfere with or modulate one or more biological activities of the CoV-2 Spike protein. In this case, an antigen-binding protein binds specifically to and/or substantially inhibits binding of the CoV-2 Spike protein, e.g. the RBD, to hACE-2 when an excess of antibody reduces the quantity of RBD of the CoV-2 Spike protein bound to hACE-2, or vice versa, by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99% or more (for example by measuring binding in an in vitro competitive binding assay). An antigen-binding protein can also bind to parts of the CoV-2 spike protein other than RBD, and either neutralize alone or enhances the activity of neutralizing RBD binding proteins antigen-binding protein by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99% or more (for example by measuring binding in an in vitro competitive binding assay).

Monoclonal Antibodies

The antigen-binding proteins that are provided include monoclonal antibodies that bind to RBD or other parts of the CoV-2 Spike protein. Monoclonal antibodies may be produced using any technique known in the art, e.g., by single cell sequencing techniques or immortalizing spleen cells harvested from the transgenic animal after completion of the immunization schedule. The spleen cells can be immortalized using any technique known in the art, e.g., by fusing them with myeloma cells to produce hybridomas. Myeloma cells for use in hybridoma-producing fusion procedures preferably are non-antibody-producing, have high fusion efficiency, and enzyme deficiencies that render them incapable of growing in certain selective media which support the growth of only the desired fused cells (hybridomas). Examples of suitable cell lines for use in mouse fusions include Sp-20, P3-X63/Ag8, P3-X63- Ag8.653, NS1/1.Ag 4 1 , Sp210-Ag14, FO, NSO/U, MPC-11 , MPC11-X45-GTG 1.7 and S194/5XXO Bui; examples of cell lines used in rat fusions include R210.RCY3, Y3-Ag 1.2.3, IR983F and 4B210. Other cell lines useful for cell fusions are U-266, GM1500-GRG2, LICR- LON-HMy2 and UC729-6. In some instances, a hybridoma cell line is produced by immunizing an animal (e.g., a transgenic animal having human immunoglobulin sequences) with the CoV-2 Spike protein immunogen; harvesting spleen cells from the immunized animal; fusing the harvested spleen cells to a myeloma cell line, thereby generating hybridoma cells; establishing hybridoma cell lines from the hybridoma cells, and identifying a hybridoma cell line that produces an antibody that binds CoV-2 Spike protein (e.g., as described in Examples 1-3, below). Such hybridoma cell lines, and anti-CoV-2 Spike protein monoclonal antibodies produced by them, are aspects of the present application.

Monoclonal antibodies secreted by a hybridoma cell line can be purified using any technique known in the art. Hybridomas or mAbs may be further screened to identify mAbs with particular properties, such as the ability to bind cells expressing the CoV-2 Spike protein.

Fully Human Antibodies

Fully human antibodies are also provided. Methods are available for making fully human antibodies specific for a given antigen without exposing human beings to the antigen (“fully human antibodies”). One specific means provided for implementing the production of fully human antibodies is the "humanization" of the mouse humoral immune system. Introduction of human immunoglobulin (Ig) loci into mice in which the endogenous Ig genes have been inactivated is one means of producing fully human monoclonal antibodies (mAbs) in mouse, an animal that can be immunized with any desirable antigen. Using fully human antibodies can minimize the immunogenic and allergic responses that can sometimes be caused by administering mouse or mouse-derived mAbs to humans as therapeutic agents.

Fully human antibodies can be produced by immunizing transgenic animals (usual mice) that are capable of producing a repertoire of human antibodies in the absence of endogenous immunoglobulin production. Antigens for this purpose typically have six or more contiguous amino acids, and optionally are conjugated to a carrier, such as a hapten. See, e.g., Jakobovits et al., 1993, Proc. Natl. Acad. Sci. USA 90:2551-2555; Jakobovits et al., 1993, Nature 362:255-258; and Bruggermann et al., 1993, Year in Immunol. 7:33.

Using the hybridoma technology, antigen-specific human mAbs with the desired specificity can be produced and selected from the transgenic mice, such as those described above. Such antibodies may be cloned and expressed using a suitable vector and host cell, or the antibodies can be harvested from cultured hybridoma cells.

Preparing of Antigen-binding proteins

Non-human antibodies that are provided can be, for example, derived from any antibodyproducing animal, such as a mouse, rat, rabbit, goat, donkey, or non-human primate (such as monkey (e.g., cynomolgus or rhesus monkey) or ape (e.g., chimpanzee)). Non-human antibodies can be used, for instance, in in vitro cell culture and cell-culture based applications, or any other application where an immune response to the antibody does not occur or is insignificant, can be prevented, is not a concern, or is desired. In certain embodiments, the antibodies may be produced by immunizing animals using methods known in the art,

Monoclonal antibodies (mAbs) can be produced by a variety of technigues, including conventional monoclonal antibody methodology, e.g., the standard somatic cell hybridization technigue of Kohler and Milstein, 1975, Nature 256:495. Alternatively, other technigues for producing monoclonal antibodies can be employed, for example, the viral or oncogenic transformation of B-lymphocytes. One suitable animal system for preparing hybridomas is the murine system, which is a very well established procedure. Immunization protocols and technigues for isolation of immunized splenocytes for fusion are known in the art, and comparative approaches are described in the Examples, below. For such procedures, B cells from immunized mice are typically fused with a suitable immortalized fusion partner, such as a murine myeloma cell line. If desired, rats or other mammals besides can be immunized instead of mice and B cells from such animals can be fused with the murine myeloma cell line to form hybridomas. Alternatively, a myeloma cell line from a source other than a mouse may be used. Fusion procedures for making hybridomas also are well known.

The single-chain antibodies may be provided e.g. by linking heavy and light chain variable domain (Fv region) fragments via an amino acid bridge (short peptide linker), resulting in a single polypeptide chain. Such single-chain Fvs (scFvs) may be prepared by fusing DNA encoding a peptide linker between DNAs encoding the two variable domain polypeptides (VL and VH). The resulting polypeptides can fold back on themselves to form antigen-binding monomers, or they can form multimers (e.g., dimers, trimers, or tetramers), depending on the length of a flexible linker between the two variable domains (Kortt et al., 1997, Prot. Eng. 10:423; Kortt et al., 2001, Biomol. Eng. 18:95-108). By combining different Vi and VH - comprising polypeptides, one can form multimeric scFvs that bind to different epitopes (Kriangkum et al., 2001 , Biomol. Eng. 18:31-40). Techniques developed for the production of single-chain antibodies include those described in U.S. Pat. No. 4,946,778; Bird, 1988, Science 242:423; Huston et al., 1988, Proc. Natl. Acad. Sci. U.S.A. 85:5879; Ward et al., 1989, Nature 334:544, de Graaf et al., 2002, Methods Mo! Bio!. 178:379-387. Single chain antibodies derived from antibodies provided herein include, but are not limited to scFvs comprising the variable domain combinations of the heavy and light chain variable regions provided herein. Antibodies provided herein that are of one subclass can be changed to antibodies from a different subclass using subclass switching methods. Thus, IgG or other antibodies may be derived from an IgM antibody, for example, and vice versa. Such techniques allow the preparation of new antibodies that possess the antigen-binding properties of a given antibody (the parent antibody) but also exhibit biological properties associated with an antibody isotype or subclass different from that of the parent antibody. Recombinant DNA techniques may be employed. Cloned DNA encoding particular antibody polypeptides may be employed in such procedures, e.g., DNA encoding the constant domain of an antibody of the desired isotype. See, e.g., Lantto et al., 2002, Methods Mol. Biol. 178:303-316.

Moreover, techniques for deriving antibodies having different properties (/.e., varying affinities for the antigen to which they bind) are also known. One such technique, referred to as chain shuffling, involves displaying immunoglobulin variable domain gene repertoires on the surface of a filamentous bacteriophage, often referred to as phage display. Chain shuffling has been used to prepare high-affinity antibodies to the hapten 2-phenyloxazol-5- one, as described by Marks et al., 1992, BioTechnoloqy 10:779.

CoV-2 spike and in particular, RBD-specific antigen-binding proteins may be further modified in various ways. For example, if they are to be used for preventive or therapeutic purposes, they may be conjugated with polyethylene glycol (pegylated) to prolong the serum half-life or to enhance protein delivery. Alternatively, the V region of the subject antibodies or fragments thereof may be fused with the Fc region of a different antibody molecule. The Fc region used for this purpose may be modified so that it does not bind complement, thus reducing the likelihood of inducing cell lysis in the patient when the fusion protein is used as a therapeutic agent. In addition, the subject antibodies or functional fragments thereof may be conjugated with human serum albumin to enhance the serum half-life of the antibody or antigen binding fragment thereof. Another useful fusion partner for the antigen-binding proteins or fragments thereof is transthyretin (TTR). TTR has the capacity to form a tetramer; thus, an antibody-TTR fusion protein can form a multivalent antibody, which may increase its binding avidity.

Methods of Expressing Antigen-binding proteins

Expression systems and constructs in the form of plasmids, expression vectors, transcription or expression cassettes that comprise at least one polynucleotide as described above are also provided herein, as well host cells comprising such expression systems or constructs.

The antigen-binding proteins provided herein may be prepared by any of a number of conventional technigues. For example, CoV-2 spike protein antigen-binding proteins. e.g. binding to RBD, may be produced by expression systems, using any technigue known in the art. See, e.g., Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Kennet et al. (eds.) Plenum Press, New York (1980); and Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1988).

Antigen-binding proteins can be expressed in hybridoma cell lines (e.g., in particular antibodies may be expressed in hybridomas) or in cell lines other than hybridomas. Expression constructs encoding the antibodies can be used to transform a mammalian, insect or microbial host cell. Transformation can be performed using any known method for introducing polynucleotides into a host cell, including, for example packaging the polynucleotide in a virus or bacteriophage and transducing a host cell with the construct by transfection procedures known in the art, as exemplified by the United States Patent No. 4,399,216; No. 4,912,040; No. 4,740,461 ; No. 4,959,455. The optimal transformation procedure used will depend upon which type of host cell is being transformed. Methods for introduction of heterologous antigen-binding protein-encoding polynucleotides, e.g., DNA or RNA, into mammalian cells are well known in the art and include, but are not limited to, dextran-mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, mixing nucleic acid with positively-charged lipids, and direct microinjection of the DNA into nuclei.

A host cell, when cultured under appropriate conditions, synthesizes an antigen-binding protein that can subsequently be collected from the culture medium (if the host cell secretes it into the medium) or directly from the host cell producing it (if it is not secreted). The selection of an appropriate host cell will depend upon various factors, such as desired expression levels, polypeptide modifications that are desirable or necessary for the activity (such as glycosylation or phosphorylation) and ease of folding into a biologically active molecule.

Mammalian cell lines available as hosts for expression are well known in the art and include, but are not limited to, immortalized cell lines available from the American Type Culture Collection (ATCC), including but not limited to Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), and a number of other cell lines. In certain embodiments, cell lines may be selected through determining which cell lines have high expression levels and constitutively produce antigen-binding proteins with CoV-2 Spike protein binding properties. In another embodiment, a cell line from the B cell lineage that does not make its own antibody but has a capacity to make and secrete a heterologous antibody can be selected.

Use of Human CoV-2 Spike- and RBD-specific Antigen-Binding Proteins for Diagnostic and Therapeutic Purposes

Antigen-binding proteins are useful for detecting CoV-2 Spike protein, e.g. RBD or other subunits, in biological samples and identification of cells or tissues that produce CoV-2 Spike protein or subunits thereof. For instance, the CoV-2 spike protein antigen-binding proteins can be used in diagnostic assays, e.g., binding assays to detect and/or quantify CoV-2 Spike protein, or subunits thereof like RBD, expressed in a tissue or cell. Antigen-binding proteins suitable for diagnosis may be, but do not have to be neutralizing antibodies.

Antigen-binding proteins that specifically bind to CoV-2 spike or RBD of the CoV-2 Spike protein can also be used as a way to prevent COVID-19 in, e.g., healthy risk groups and immunocompromised individuals, and in the treatment of diseases related to CoV-2 in a patient in need thereof. In addition, CoV-2 spike and RBD antigen-binding proteins can be used to inhibit RBD of the CoV-2 Spike protein from forming a complex with hACE-2, thereby modulating the biological activity of the CoV-2 in a cell or tissue. Examples of activities that can be modulated either by directly blocking virus infection or indirectly by suppressing collateral damages, e.g., cause an uncontrolled immune response or cell damage. Symptoms that can be treated include but are not limited to symptoms accompanied by the CoV-2 induced COVID-19 disease. These are sore throat, cough (usually dry cough), shortness of breath, chest pain), fever, loss of the sense of smell and/or taste, headache, general weakness, malaise, muscle aches, sniffles, a gastrointestinal symptom (e.g., nausea, vomiting, diarrhea, abdominal pain), myocarditis, meningoencephalitis and Kawasaki-like symptoms in children. Antigen-binding proteins that bind to CoV-2 Spike protein thus can modulate and/or block interaction with other binding compounds and, as such, may have therapeutic use in ameliorating diseases related to CoV-2.

Indications

A disease or condition associated with CoV-2 Spike protein includes any disease or condition whose onset in a patient is caused by, at least in part, the interaction of the CoV-2 Spike protein, e.g. the RBD, with hACE-2 or another not yet identified host protein.

Examples of diseases and conditions that can be treated with the antigen-binding proteins described herein include sore throat, cough (usually dry cough), blood clotting, shortness of breath, chest pain), fever, loss of the sense of smell and/or taste, headache, general weakness, malaise, muscle aches, sniffles, a gastrointestinal symptom (e.g., nausea, vomiting, diarrhea, abdominal pain), myocarditis, meningoencephalitis and Kawasaki-like symptoms in children) and collateral tissue damages caused by cell death of non-infected cells and an uncontrolled pathology immune response.

In particular, antigen-binding proteins described herein can be used as a passive vaccine or passive immunization agent, and/or as a prophylactic treatment means administered, e.g., daily, weekly, biweekly, monthly, bimonthly, biannually, etc.) to prevent or reduce the frequency and/or severity of symptoms, e.g., sore throat, cough (usually dry cough), blood clotting, shortness of breath, chest pain), fever, loss of the sense of smell and/or taste, headache, general weakness, malaise, muscle aches, sniffles, a gastrointestinal symptom (e.g., nausea, vomiting, diarrhea, abdominal pain), myocarditis, meningoencephalitis and Kawasaki-like symptoms in children) and collateral tissue damages caused by cell death of non-infected cells and an uncontrolled pathology immune response associated with CoV-2 infection.

The antigen-binding protein of the present invention can be used to treat or protect by passive immunization humans and animals. The antigen-binding protein can be used as proteins or can be transferred either delivered naked, in vesicles or in a vector to a subject as RNA or DNA encoding the antigen-binding protein or as DNA and RNA naked or packed in nanoparticles consisting of metals, lipids, carbohydrates or derivates of them

Diagnostic Methods

The Cov-2 spike protein-specific antigen-binding proteins, regardless of whether they neutralize or not, described herein can be used for diagnostic purposes to detect, diagnose, or monitor diseases and/or conditions associated with CoV-2 infection. Also provided are methods for the detection of the presence of CoV-2 Spike protein or its subdomains, e.g. the RBD, the NTD, trimeric ectodomain, S1 , S2, in a sample using classical immunohistological methods known to those of skill in the art (e.g., Tijssen, 1993, Practice and Theory of Enzyme Immunoassays, Vol 15 (Eds R.H. Burdon and P.H. van Knippenberg, Elsevier, Amsterdam); Zola, 1987, Monoclonal Antibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc.); Jalkanen et al., 1985, J. Cell. Biol. 101 :976-985; Jalkanen et al., 1987, J. Cell Biol. 105:3087-3096). The detection of CoV-2 Spike protein can be performed in vivo or in vitro.

Diagnostic applications provided herein include the use of the antigen-binding proteins to detect the expression of CoV-2 Spike protein and binding of other molecules to CoV-2 Spike protein. Examples of methods useful in the detection of the presence of CoV-2 spike protein of the CoV-2 Spike protein include immunoassays, such as the enzyme-linked immunosorbent assay (ELISA), fluorescence-based flow cytometry, the radioimmunoassay (RIA), histo-chemical of fluorescence-microscopy of tissue.

For diagnostic applications, the antigen-binding protein typically will be labeled with a detectable labeling group. Suitable labeling groups include, but are not limited to, the following: radioisotopes or radionuclides (e.g., ³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y, "Tc, ¹¹¹ In, ¹²⁵l, ¹³¹l), fluorescent groups (e.g., FITC, rhodamine, lanthanide phosphors), enzymatic groups (e.g., horseradish peroxidase, p-galactosidase, luciferase, alkaline phosphatase), chemiluminescent groups, biotinyl groups, or predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags). In some embodiments, the labeling group is coupled to the antigen-binding protein via spacer arms of various lengths to reduce potential steric hindrance. Various methods for labeling proteins are known in the art and may be used.

Methods of Treatment: Pharmaceutical Formulations, Routes of Administration Methods of using the antigen-binding proteins are also provided.

In some embodiments, an antigen-binding protein is provided to a patient. The antigenbinding protein inhibits the binding of ACE-2 to RBD of the CoV-2 Spike protein.

In some embodiments, a combination of at least two different antigen-binding proteins of the invention is provided to a patient. Both can be neutralizing antigen-binding proteins, or a neutralizing antigen-binding proteins may be combined with non-neutralizing antigen-binding proteins. Preferably, the non-neutralizing antigen-binding proteins enhance the therapeutic effect of the neutralizing antibodies.

Pharmaceutical compositions that comprise a therapeutically effective amount of one or a plurality of the antigen-binding proteins and a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative, and/or adjuvant are also provided. In addition, methods of treating a patient, e.g., for sore throat, cough (usually dry cough), blood clotting, shortness of breath, chest pain), fever, loss of the sense of smell and/or taste, headache, general weakness, malaise, muscle aches, sniffles, a gastrointestinal symptom (e.g., nausea, vomiting, diarrhea, abdominal pain), myocarditis, meningoencephalitis and Kawasaki-like symptoms in children) and collateral tissue damages caused by cell death of non-infected cells and an uncontrolled pathology immune response, by administering such pharmaceutical composition are included. The term “patient” includes human patients. However, the antibodies can also be used in the treatment of animals that are infected by CoV-2, e.g., primates, non-human primates, dogs, cats, minks, bats and others.

Acceptable formulation materials are nontoxic to recipients at the dosages and concentrations employed. In specific embodiments, pharmaceutical compositions comprising a therapeutically effective amount of CoV-2 spike protein antigen-binding proteins, e.g. RBD binding, are provided.

In certain embodiments, acceptable formulation materials preferably are nontoxic to recipients at the dosages and concentrations employed. In certain embodiments, the pharmaceutical composition may contain formulation materials for modifying, maintaining or preserving, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or penetration of the composition. In such embodiments, suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite or sodium hydrogen-sulfite); buffers (such as borate, bicarbonate, Tris-HCI, citrates, phosphates or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediamine tetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (such as glucose, mannose or dextrins); proteins (such as serum albumin, gelatin or immunoglobulins); coloring, flavoring and diluting agents; emulsifying agents; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; saltforming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such as glycerin, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate, triton, tromethamine, lecithin, cholesterol, tyloxapal); stability enhancing agents (such as sucrose or sorbitol); tonicity enhancing agents (such as alkali metal halides, preferably sodium or potassium chloride, mannitol sorbitol); delivery vehicles; diluents; excipients and/or pharmaceutical adjuvants. See, REMINGTON’S PHARMACEUTICAL SCIENCES, 18” Edition, (A.R. Genrmo, ed.), 1990, Mack Publishing Company.

In certain embodiments, the optimal pharmaceutical composition will be determined by one skilled in the art depending upon, for example, the intended route of administration, delivery format and desired dosage. See, for example, REMINGTON’S PHARMACEUTICAL SCIENCES, supra. In certain embodiments, such compositions may influence the physical state, stability, rate of in vivo release and rate of in vivo clearance of the antigen-binding proteins disclosed. In certain embodiments, the primary vehicle or carrier in a pharmaceutical composition may be either aqueous or non-aqueous in nature. For example, a suitable vehicle or carrier may be water for injection, physiological saline solution, or artificial cerebrospinal fluid, possibly supplemented with other materials common in compositions for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles. In specific embodiments, pharmaceutical compositions comprise Tris buffer of about pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5, and may further include sorbitol or a suitable substitute. In certain embodiments, RBD antigen-binding protein compositions may be prepared for storage by mixing the selected composition having the desired degree of purity with optional formulation agents (REMINGTON’S PHARMACEUTICAL SCIENCES, supra) in the form of a lyophilized cake or an aqueous solution. Further, in certain embodiments, the CoV-2 spike protein (e.g. RBD) antigen-binding protein may be formulated as a lyophilizate using appropriate excipients such as sucrose.

The pharmaceutical compositions can be selected for parenteral delivery. Alternatively, the compositions may be selected for inhalation or for delivery through the digestive tract, such as orally. The preparation of such pharmaceutically acceptable compositions is within the skill of the art.

The formulation components are present, preferably in concentrations that are acceptable to the site of administration. In certain embodiments, buffers are used to maintain the composition at physiological pH or at a slightly lower pH, typically within a pH range of from about 5 to about 8.

When parenteral administration is contemplated, the therapeutic compositions may be provided in the form of a pyrogen-free, parenterally acceptable aqueous solution comprising the desired RBD of the CoV-2 Spike protein-binding protein in a pharmaceutically acceptable vehicle. A particularly suitable vehicle for parenteral injection is sterile distilled water in which the CoV-2 spike protein (e.g. RBD) antigen-binding protein is formulated as a sterile, isotonic solution, properly preserved. In certain embodiments, the preparation can involve the formulation of the desired molecule with an agent, such as injectable microspheres, bio-erodible particles, polymeric compounds (such as polylactic acid or polyglycolic acid), beads or liposomes, that may provide controlled or sustained release of the product which can be delivered via a depot injection. In certain embodiments, hyaluronic acid may also be used, having the effect of promoting sustained duration in the circulation. In certain embodiments, implantable drug delivery devices may be used to introduce the desired antigen-binding protein.

Certain pharmaceutical compositions are formulated for inhalation. In some embodiments, CoV-2 spike protein (e.g. RBD) antigen-binding proteins are formulated as a dry, inhalable powder. In specific embodiments, CoV-2 spike protein (e.g. RBD) antigen-binding protein inhalation solutions may also be formulated with a propellant for aerosol delivery. In certain embodiments, solutions may be nebulized. Pulmonary administration and formulation methods, therefore, are further described in International Patent Application No. PCT/US94/001875, which describes pulmonary delivery of chemically modified proteins. Some formulations can be administered orally. CoV-2 spike protein antigen-binding protein that are administered in this fashion can be formulated with or without carriers customarily used in the compounding of solid dosage forms such as tablets and capsules. In certain embodiments, a capsule may be designed to release the active portion of the formulation at the point in the gastrointestinal tract when bioavailability is maximized and pre-systemic degradation is minimized. Additional agents can be included to facilitate absorption of the CoV-2 spike protein protein antigen-binding protein. Diluents, flavorings, low melting point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents, and binders may also be employed.

Some pharmaceutical compositions comprise a sufficient quantity of one or a plurality of CoV-2 spike protein antigen-binding proteins in a mixture with non-toxic excipients that are suitable for the manufacture of tablets. By dissolving the tablets in sterile water, or another appropriate vehicle, solutions may be prepared in unit-dose form. Suitable excipients include, but are not limited to, inert diluents, such as calcium carbonate, sodium carbonate or bicarbonate, lactose, or calcium phosphate; or binding agents, such as starch, gelatin, or acacia; or lubricating agents such as magnesium stearate, stearic acid, or talc.

Additional pharmaceutical compositions will be evident to those skilled in the art, including formulations involving CoV-2 spike protein antigen-binding proteins in sustained- or controlled-delivery formulations. Techniques for formulating a variety of other sustained- or controlled-delivery means, such as liposome carriers, bio-erodible microparticles or porous beads and depot injections, are also known to those skilled in the art. See, for example, International Patent Application No. PCT/US93/00829, which describes the controlled release of porous polymeric microparticles for the delivery of pharmaceutical compositions. Sustained-release preparations may include semipermeable polymer matrices in the form of shaped articles, e.g., films, or microcapsules. Sustained release matrices may include polyesters, hydrogels, polylactides (as disclosed in U.S. Patent No. 3,773,919 and European Patent Application Publication No. EP 058481), copolymers of L-glutamic acid and gamma ethyl-L-glutamate (Sidman et al., 1983, Biopolymers 2:547-556), poly (2-hydroxyethyl- methacrylate) (Langer et al., 1981 , J. Biomed. Mater. Res. 15:167-277 and Langer, 1982, Chem. Tech. 12:98-105), ethylene vinyl acetate (Langer et al., 1981 , supra) or poly-D(-)-3- hydroxybutyric acid (European Patent Application Publication No. EP 133,988). Sustained release compositions may also include liposomes that can be prepared by any of several methods known in the art. See, e.g., Eppstein et al., 1985, Proc. Natl. Acad. Sci. U.S.A. 82:3688-3692; European Patent Application Publication Nos. EP 036,676; EP 088,046 and EP 143,949. Pharmaceutical compositions used for in vivo administration are typically provided as sterile preparations. Sterilization can be accomplished by filtration through sterile filtration membranes. When the composition is lyophilized, sterilization using this method may be conducted either before or following lyophilization and reconstitution. Compositions for parenteral administration can be stored in lyophilized form or a solution. Parenteral compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.

In certain embodiments, cells expressing a recombinant antigen-binding protein as disclosed herein is encapsulated for delivery (see Invest. Ophthalmol Vis Sci 43:3292-3298, 2002 and Proc. Natl. Acad. Sciences 103:3896-3901, 2006).

In certain formulations, an antigen-binding protein has a concentration of at least 10 mg/ml, 20 mg/ml, 30 mg/ml, 40 mg/ml, 50 mg/ml, 60 mg/ml, 70 mg/ml, 80 mg/ml, 90 mg/ml, 100 mg/ ml or 150 mg/ml. Some formulations contain a buffer, sucrose and polysorbate. An example of a formulation is one containing 50-100 mg/ml of antigen-binding protein, 5-20 mM sodium acetate, 5-10% w/v sucrose, and 0.002 - 0.008% w/v polysorbate. Certain, formulations, for instance, contain 65-75 mg/ml of an antigen-binding protein in 9-11 mM sodium acetate buffer, 8-10% w/v sucrose, and 0.005-0.006% w/v polysorbate. The pH of certain such formulations is in the range of 4.5-6. Other formulations have a pH of 5.0-5.5 (e.g., pH of 5.0, 5.2 or 5.4).

Once the pharmaceutical composition has been formulated, it may be stored in sterile vials as a solution, suspension, gel, emulsion, solid, crystal, or as a dehydrated or lyophilized powder. Such formulations may be stored either in a ready-to-use form or in a form (e.g., lyophilized) that is reconstituted before administration. Kits for producing a single-dose administration unit are also provided. Certain kits contain a first container having a dried protein and a second container having an aqueous formulation. In certain embodiments, kits containing single and multi-chambered pre-filled syringes (e.g., liquid syringes and lyosyringes) are provided. The therapeutically effective amount of an CoV-2 spike protein (e.g. RBD) antigen-binding protein-containing pharmaceutical composition to be employed will depend, for example, upon the therapeutic context and objectives. One skilled in the art will appreciate that the appropriate dosage levels for treatment will vary depending, in part, upon the molecule delivered, the indication for which the CoV-2 spike protein (e.g. RBD) antigen-binding protein is being used, the route of administration, and the size (body weight, body surface or organ size) and/or condition (the age and general health) of the patient. In certain embodiments, the clinician may titer the dosage and modify the route of administration to obtain the optimal therapeutic effect.

A typical dosage may range from about 1 pg/kg to up to about 30 mg/kg or more, depending on the factors mentioned above. In specific embodiments, the dosage may range from 10 pg/kg up to about 30 mg/kg, optionally from 0.1 mg/kg up to about 30 mg/kg, alternatively from 0.3 mg/kg up to about 20 mg/kg. In some applications, the dosage is from 0.5 mg/kg to 20 mg/kg. In some instances, an antigen-binding protein is dosed at 0.3 mg/kg, 0.5mg/kg, 1 mg/kg, 3 mg/kg, 10 mg/kg, or 20 mg/kg. The dosage schedule in some treatment regimes is at a dose of 0.3 mg/kg qW, 0.5mg/kg qW, 1 mg/kg qW, 3 mg/kg qW, 10 mg/kg qW, or 20 mg/kg qW.

The dosing frequency will depend upon the pharmacokinetic parameters of the particular CoV-2 spike protein (e.g. RBD) antigen-binding protein in the formulation used. Typically, a clinician administers the composition until a dosage is reached that achieves the desired effect. The composition may, therefore, be administered as a single dose, or as two or more doses (which may or may not contain the same amount of the desired molecule) over time, or as a continuous infusion via an implantation device or catheter. Appropriate dosages may be ascertained through the use of appropriate dose-response data. In certain embodiments, the antigen-binding proteins can be administered to patients throughout an extended period. Chronic administration of an antigen-binding protein minimizes the adverse immune or allergic response commonly associated with antigen-binding proteins that are not fully human, for example, an antibody raised against a human antigen in a non-human animal, for example, a non-fully human antibody or non-human antibody produced in a non-human species.

The route of administration of the pharmaceutical composition is in accord with known methods, e.g., orally, through injection by intravenous, intraperitoneal, intracerebral (intra- parenchymal), intracerebroventricular, intramuscular, intraocular, intraarterial, intraportal, or intralesional routes; by sustained release systems or by implantation devices. In certain embodiments, the compositions may be administered by bolus injection or continuously by infusion, or by implantation device.

The composition also may be administered locally via implantation of a membrane, sponge or another appropriate material onto which the desired molecule has been absorbed or encapsulated. In certain embodiments, where an implantation device is used, the device may be implanted into any suitable tissue or organ, and delivery of the desired molecule may be via diffusion, timed-release bolus, or continuous administration.

PREFERRED EMBODIMENTS OF THE INVENTION

1. An antigen-binding protein, wherein the antigen-binding protein binds to the CoV-2 Spike protein, e.g. to the receptor-binding domain (RBD) of the CoV-2 Spike protein and/or inhibits the binding of CoV-2 spike protein, e.g. RBD, to human ACE-2 (hACE-2) enzyme.

Preferably the antigen-binding protein inhibits binding of CoV-2 to angiotensinconverting enzyme 2 (ACE-2, hACE-2).

Most preferably, the antigen-binding protein neutralizes CoV-2 infection of cells.

The antigen binding protein may be an antigen binding protein that binds RBD of the CoV-2 Spike protein and neutralizes CoV-2 infection of cells.

The antigen binding protein may be an antigen binding protein that does not bind RBD of the CoV-2 Spike protein and neutralizes CoV-2 infection of cells. Preferably, in this embodiment, the antigen binding protein binds NTD of the CoV-2 Spike protein.

Throughout all embodiments of the invention, an antigen-binding protein that “inhibits the binding of CoV-2 spike protein to human ACE-2 (hACE-2) enzyme” is preferably an antigenbinding protein that competes for binding to RBD of the CoV-2 Spike protein with an EC50 of <100 nM in an Elisa-based hACE-2 binding competition assay with immobilized CoV-2 spike More preferred is an antigen-binding protein that has an ECso of <10 nM, even more preferred <1 nM, most preferred <0.1 nM. Other assays established in the art may also be used.

Throughout all embodiments of the invention, an antigen-binding protein that “neutralizes CoV-2 infection of cells” is an antigen-binding protein, that has an ICso of <100 nM in a neutralization assay with SARS-CoV-2. Preferred is an antigen-binding protein that has an ICso of <10 nM in a neutralization assay with SARS-CoV-2. More preferred is an antigen-binding protein that has an ICso of <1 nM, most preferred ICso of <0.1 nM in a neutralization assay with SARS-CoV-2. A preferred neutralization assay is performed with Vero E6 cells as described in this specification. Neutralization assays established in the art may also be used.

2. The antigen-binding protein of item 1, wherein the antigen-binding protein specifically binds to the CoV-2 Spike protein, e.g. RBD, S1 or NTD of the CoV-2 Spike protein, with a KD <100 nM.

Preferably, the KD is determined using an Elisa binding assay. Other assays established in the art, e.g. a FACS binding assay, may also be used.

3. The antigen-binding protein of item 2, wherein the antigen-binding protein specifically binds to the CoV-2 Spike protein, e.g. RBD, trimeric ectodomain, S1 , S2 or NTD of the CoV-2 Spike protein, with a KD <10 nM, preferably KD <5 nM, more preferably KD <1 nM.

Preferably, the KD is determined using an Elisa binding assay. Other assays established in the art may also be used.

4. The antigen-binding protein of item 1 , 2 or 3, wherein a) the antigen-binding protein has an EC50 of less than 10 nM in an hACE-2 Elisa binding competition assay, or b) the antigen-binding protein has an EC50 of less than 10 nM in an hACE-2 flow cytometric analysis to membranes from cells expressing the complete CoV-2 Spike protein.

5. The antigen-binding protein of item 4, wherein a) the antigen-binding protein has an EC50 of less than 1 nM in an hACE-2 flow cytometric competition assay to membranes from cells expressing the complete CoV-2 Spike protein; or c) the antigen-binding protein has an EC50 of less than 1 nM in an hACE-2 Elisa competition assay to membranes from cells expressing the complete CoV-2 Spike protein. 6. The antigen-binding protein of item 1 , wherein the antigen-binding protein competes for binding of hACE-2 with RBD of the CoV-2 Spike protein, with a reference antibody, said reference antibody comprising (i) a heavy chain variable region comprising a sequence selected from the group consisting of SEQ ID NOs:1 , 3 and 5; and (ii) a light chain variable region comprising a sequence selected from the group consisting of SEQ ID NOs: 2, 4 and 6.

7. The antigen-binding protein of item 6, wherein the reference antibody comprises (i) a heavy chain variable region defined by a sequence selected from the group consisting of SEQ ID NOs:1 and 5 (ii) a light chain variable region defined by a sequence selected from the group consisting of SEQ ID NOs: 4 and 6.

8. The antigen-binding protein of item 7, wherein the reference antibody comprises a heavy chain variable region and a light chain variable region defined by one of the following pairs of sequences:

SEQ ID NO: 1 and SEQ ID NO: 2; SEQ ID NO: 3 and SEQ ID NO: 4; and SEQ ID

NO: 5 and SEQ ID NO: 6;

9. An antigen-binding protein comprising

(A) one or more heavy chain complementary determining regions (CDRHs) selected from the group consisting of: (i) a CDRH1 selected from the group of SEQ ID NO:7. 13 and 19; (ii) a CDRH2 selected from the group consisting of SEQ ID NO:8, 14 and 20 ; (iii) a CDRH3 selected from the group consisting of SEQ ID NO:9, 15 and 21 ; and (iv) a CDRH of (i), (ii) or (iii) that contains one, two, three or four amino acid substitutions, deletions or insertions;

(B) one or more light chain complementary determining regions (CDRLs) selected from the group consisting of: (i) a CDRL1 selected from the group consisting of SEQ ID NQs:10; 16 and 22 (ii) a CDRL2 selected from the group consisting of SEQ ID NOs: 11 , 17 and 23; (iii) a CDRL3 selected from the group consisting of SEQ ID NOs: 12; 18 and 24 and optionally (iv) a CDRL of (i), (ii) and (iii) that contains one, two, three or four amino acid substitutions, deletions or insertions; or (C) one or more heavy chain CDRHs of (A) and one or more light chain CDRLs of

(B). Preferably, the antigen-binding protein inhibits the binding of. RBD of the CoV-2 Spike protein, to human ACE-2 (hACE-2) enzyme. More preferably the antigen-binding protein inhibits binding of CoV-2 to angiotensin-converting enzyme 2 (ACE-2, hACE-2). Most preferably, the antigen-binding protein neutralizes CoV-2 infection of cells.

10. The antigen-binding protein of item 9, wherein the CDRHs are further selected from the group consisting of: (i) a CDRH1 selected from the group consisting of SEQ ID NO:7 and 19; (ii) a CDRH2 selected from the group consisting of SEQ ID NO:8 and 20; (iii) a CDRH3 selected from the group consisting of SEQ ID NO:9 and 21 ; and (iv) a CDRH of (i), (ii) and (iii) that contains one, two or three amino acid substitutions, deletions or insertions.

11 . The antigen-binding protein of item 9, wherein the CDRLs are further selected from the group consisting of: (i) a CDRL1 selected from the group consisting of SEQ ID NO 10 and 22; (ii) a CDRL2 selected from the group consisting of SEQ ID NO: 11 and 23; (iii) a CDRL3 selected from the group consisting of SEQ ID NO 12 and 24; and (iv) a CDRL of (i), (ii) and (iii) that contains one, two, three, or four amino acid substitutions, deletions or insertions.

12. The antigen-binding protein of any of items 9-11 , wherein the antigen-binding protein comprises at least one CDRH and at least one CDRL.

13. The antigen-binding protein of item 12, wherein the antigen-binding protein comprises at least two CDRH and at least two CDRL.

14. The antigen-binding protein of any of items 9-13, wherein the antigen-binding protein comprises a CDRH1 , a CDRH2, a CDRH3, a CDRL1 , a CDRL2 and a CDRL3.

15. An antigen-binding protein comprising a heavy chain variable region (VH) sequence that has at least 90% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs:1 , 3 and 5, 37, 39, 41 , 43, 45, 47, 49, 51 , 53, 55, 57 and 59.

Preferably, the antigen-binding protein binds to RBD. NTD or S1.

In some embodiments, the antigen-binding protein inhibits the binding of RBD, to human ACE-2 (hACE-2) enzyme. More preferably the antigen-binding protein inhibits binding of CoV-2 to angiotensin-converting enzyme 2 (ACE-2, hACE-2).

Most preferably, the antigen-binding protein neutralizes CoV-2 infection of cells. 16. An antigen-binding protein comprising a light chain variable region (VL) sequence that has at least 90% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs:2, 4 and 6, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58 and 60.

Preferably, the antigen-binding protein binds to RBD. NTD or S1.

17. An antigen-binding protein comprising a VH sequence that has at least 90% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 , 3 and 5, 37, 39, 41 , 43, 45, 47, 49, 51 , 53, 55, 57 and 59, and a VL sequence that has at least 90% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4 and 6, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58 and 60.

Preferably, the antigen-binding protein binds to RBD. NTD or S1.

Some embodiments inhibit the binding of RBD, to human ACE-2 (hACE-2) enzyme. More preferably the antigen-binding protein inhibits binding of CoV-2 to angiotensin-converting enzyme 2 (ACE-2, hACE-2).

18. The antigen-binding protein of item 17, comprising a H sequence selected from the group consisting of SEQ ID NOs: 1 , 3 and 5, 37, 39, 41 , 43, 45, 47, 49, 51 , 53, 55, 57 and 59 and a VL sequence selected from the group consisting of SEQ ID NOs: 2, 4 and 6, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58 and 60.

19. The antigen-binding protein of item 18, comprising a heavy chain sequence comprising a sequence selected from the group consisting of SEQ ID NOs: 31 , 32 and 33 and a light chain sequence comprising a sequence selected from the group consisting of SEQ ID NOs: 34, 35 and 36.

20. An antigen-binding protein comprising

(A) one or more heavy chain complementary determining regions (CDRHs) selected from the group consisting of: (i) a CDRH1 having a CDRH1 of any of SEQ ID NOs:37, 39, 41 , 43, 45, 47, 49, 51 , 53, 55, 57, 59; (ii) a CDRH2 having a CDRH2 of any of SEQ ID NOs:37, 39, 41 , 43, 45, 47, 49, 51 , 53, 55, 57, 59; (iii) a CDRH3 having a CDRH3 of any of SEQ ID NOs:37, 39, 41 , 43, 45, 47, 49, 51 , 53, 55, 57, 59; and (iv) a CDRH of (i), (ii) or (iii) that contains one, two, three or four amino acid substitutions, deletions or insertions;

(B) one or more light chain complementary determining regions (CDRLs) selected from the group consisting of: (i) a CDRL1 having a CDRL1 of any of SEQ ID NOs:38,

40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60 (ii) a CDRL2 having a CDRL2 of any of SEQ ID NOs:38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60; (iii) a CDRL3 having a CDRL3 of any of SEQ ID NOs:38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60 and optionally (iv) a CDRL of (i), (ii) and (iii) that contains one, two, three or four amino acid substitutions, deletions or insertions; or (C) one or more heavy chain CDRHs of (A) and one or more light chain CDRLs of (B).

Preferably, the antigen-binding protein neutralizes CoV-2 infection of cells.

21. The antigen-binding protein of any of item 20, wherein the antigen-binding protein comprises at least one CDRH and at least one CDRL.

22. The antigen-binding protein of item 20 or 21 , wherein the antigen-binding protein comprises at least two CDRH and at least two CDRL.

23. The antigen-binding protein of any of items 20-22, wherein the antigen-binding protein comprises a CDRH1 , a CDRH2, a CDRH3, a CDRL1 , a CDRL2 and a CDRL3.

24. The antigen-binding protein of any of items 1 - 23, wherein the antigenbinding protein is selected from the group consisting of a monoclonal antibody, a Fab fragment, a Fab' fragment, an F(ab')2 fragment, an Fv fragment, a diabody, and a single-chain antibody.

25. The antigen-binding protein of item 24, wherein the antigen-binding protein is a monoclonal antibody selected from the group consisting of a fully human antibody, a humanized antibody, and a chimeric antibody.

26.A nucleic acid polynucleotide (e.g. DNA and RNA) that encodes an antigen-binding protein of any of items 1-21. The nucleic acid polynucleotide may comprise any of the sequences of SEQ ID Nos 25-30. Preferably, for therapeutic or prophylatic purposes, the antigen-binding protein can be transferred as RNA or DNA encoding the antigen-binding protein, either delivered naked, in vesicles or in a vector to a subject.

27.A vector comprising a polynucleotide of item 26. The vector is preferably an expression vector or equally preferably a gene shuttle. A gene shuttle is a vector that is capable of transferring RNA or DNA encoding an antigen-binding protein of the present invention into cells or subjects, preferably a human or an animal infected with or at the risk of being infected with CoV-2. Such vectors that transfer RNA or DNA encoding an antigenbinding protein of the present invention into cells may be used for therapeutic purposes, e.g. for passive DNA- or RNA- immunization. Such embodiments encompass mRNA vaccines.

28. A cell line transformed with the expression vector of item 27.

29. A method of making an antigen-binding protein of any of items 1-25, comprising preparing the antigen-binding protein from a host cell that secretes the antigen-binding protein.

30. A pharmaceutical composition comprising an antigen-binding protein of any of items 1-25 and a pharmaceutically acceptable excipient.

31. An antigen-binding protein of any of items 1-25 for use in treating CoV-2 infection or a condition associated with a CoV-2 infection.

32. The antigen-binding protein for the use of item 31 , wherein the condition is pneumonia or heart disease or a disease or condition selected from sore throat, cough (e.g. dry cough), blood clotting, shortness of breath, chest pain), fever, loss of the sense of smell and/or taste, headache, general weakness, malaise, muscle aches, sniffles, a gastrointestinal symptom (e.g., nausea, vomiting, diarrhea, abdominal pain), myocarditis, meningoencephalitis and Kawasaki-like symptoms in children) and collateral tissue damages caused by cell death of non-infected cells and an uncontrolled pathology immune response.

33.. The antigen-binding protein for the use of item 31 or 32, wherein treating comprises prophylactic treatment.

34. An antigen-binding protein of any of items 1-25 for use in the passive immunization against CoV-2 infection. In particular, antigen-binding proteins described herein can be used as a passive vaccine or passive immunization agent, and/or as a prophylactic treatment means administered, e.g., daily, weekly, biweekly, monthly, bimonthly, biannually, etc.) to prevent or reduce the frequency and/or severity of symptoms, e.g., sore throat, cough (usually dry cough), blood clotting, shortness of breath, chest pain), fever, loss of the sense of smell and/or taste, headache, general weakness, malaise, muscle aches, sniffles, a gastrointestinal symptom (e.g., nausea, vomiting, diarrhea, abdominal pain), myocarditis, meningoencephalitis and Kawasaki-like symptoms in children) and collateral tissue damages caused by cell death of non-infected cells and an uncontrolled pathology immune response associated with CoV-2 infection.

EXAMPLES

The human monoclonal antibodies described in this patent were isolated by the conventional hybridoma technology from spleen cells of human antibody mice that were immunized with DNA or RNA encoding CoV-2 spike protein and purified CoV-2 spike protein. The mouse line was established by Trianni Inc US 2013/0219535 A1 and had the complete repertoire of human variable region gene segments of immunoglobulin (Ig)heavy (HC) and L chains (LC).

1. Immunization

Trianni mice were immunized intramuscularly by electroporation with a Spike-CoV-encoding DNA plasmid and boosted twice with a recombinant form of the soluble trimeric form of the CoV-2 spike protein (Fig. 1). Fluorescence-based flow cytometry detected in serum from immunized mice antibodies that bound to cell-anchored Spike-CoV-2 but surprisingly not Spike from SARS-CoV (not shown).

2. Isolation of CVR antibodies

Five days after the last immunization, spleen cells were prepared and fused with Sp2/0 hybridoma cells via the PEG method. The fusion mixture was plated in HAT selection medium in 96 well plates. The resulting HAT-resistant hybridomas were named CVR, for Trianni-Erlangen Anti CoV-2 Spike. Supernatants from wells with detectable cell growth were tested for binding using the fluorescence-based flow cytometry assay described in Fig. 1. This analysis identified 21 hybridomas that produce CoV-2-spike binding antibodies CoV (Figs. 2 and 3). Surprisingly, none of the CVR antibodies bound to SARS-CoV or any of the other five human Coronaviruses and SARS-MERS, HCoV-OC43, HCoV-229E, HCoV-NL63 and HCoV-HUK (data not shown). The CVR hybridomas were subcloned by the limiting dilution method.

3. CVR antibodies block hACE-2 binding

To determine whether the CVR antibodies blocked the interaction of the CoV-2 spike with its cognate receptor, we incubated CoV-2-spike transfected 293 T cells with a soluble Myc- tagged hACE-2 and either with CVR antibodies or with control antibodies. This analysis identified CVR224, CVR6 and CVR567 as potent CoV-2 spike blocking antibodies (Figure 6) with EC50 values ranging from 0.1 - 0.3 nM (Figure 6B). Similar EC50 values between 0,52 to 1.14 nM were determined for all three ACE-2-blocking CVR antibodies in an ELISA with plates coated with CoV-2 RBD purified from the serum-free culture medium of HEK293F cells transfected with CoV-2 RBD (Fig. 6C).

4. hACE-2 Blocking CVR antibodies bind to CoV-2 spike protein

Interestingly, all three ACE-2-blocking antibodies reacted in Elisa-based assays with the recombinant soluble extracellular region of trimeric CoV-2 Spike protein called now Spike- ecto (Fig. 3), the RBD (Fig,. 7A, B) and the S1 domain of CoV-2 spike (Fig. 3), but not with the NTD (Fig. 7A) and S2 domain (Fig. 3), indicating that the blocking antibodies detect a region (epitope) in the RBD. Interestingly, none of the other CVR antibodies, all of which bound in the fluorescence-based flow assay to membrane-bound CoV-2 spike (Fig. 2 and 3), reacted in Elisa assays with RBD (Fig. 3 and Fig. 7A). However, some of them bound only to recombinant S1 and Spike-ecto (78, 473, 1082, 1427, 1471 , 1537, 698), only to recombinant S2 (CVR1 , 167), recombinant NTD and Spike-ecto (CVR49, 219, 328, 618, 1209, 1293) and some did not bind to any recombinant spike components (CVR4, 41 , 816). The Elisa spike- non-binder CVR antibodies could be antibodies that recognize carbohydrates on cell membrane-bound CoV-2 spike protein that are absent in the recombinantly produce CoV-2 spike domains.

Surprisingly, all three hACE-2 blocking CVR antibodies that bound in Elisa assays to recombinant RBD detected reduced and denatured recombinant RBDs in Western blot analysis (Fig. 7B), indicating that the three CVR hACE-2 blocking antibodies recognize a linear epitope in the RBD of the CoV-2 spike protein.

5. Virus neutralization

When we tested all CVR antibodies for virus neutralization, we found that the three CVR RBD-binding antibodies that block hACE-2 binding efficiently blocked the infection of Vero E6 cells with the wildtype virus with an IC50s in the range of 0.4 - 0.74 nM (Figure 4B). Surprisingly, we found that the CVR antibodies 49, 19, 328, 618, 1209 and 1293, all of which bound in Elisa to recombinant ecto spike and NTD but not to RBD (Fig. 3 and 7A), also efficiently neutralized CoV-2 infection with IC50 values between 0.01 und 0.22 nM (Figure 4B). This second cluster of neutralizing antibodies are very interesting because they could still neutralize CoV-2 that escape the neutralizing RBD-binding antibodies by mutating the cognate epitope.

6. Affinity measure To estimate the affinity of the neutralizing CVR antibodies, we incubated microtiter wells with immobilized recombinant RBD or ecto spike with increasing amounts of CVR antibodies and determined the number of bound antibodies. The dissociation constant KD, which is a measure for affinity, was determined from the saturation curve in Fig. 8 by using Graphpad Prism. The analysis revealed an impressive KD for the three neutralizing and RBD-binding antibodies 0.1 and 0.2 nM and the six other neutralizing antibodies of 0.04 - 0.05 nM.

7. Sequences of CVR antibodies

To determine the sequences of all antibodies that bound to the CoV-2 spike protein expressed on the surface of transiently transfected HEK cells (Fig. 2), total RNA was isolated from clones or subclones of CVR hybridomas, transcribed into cDNA, ligated to a 5‘ primer, and amplified with a 5‘ race primer and a 3’ primer specific for the respective isotype of the mouse Cgamma region. PCR fragments were gel-purified, eluted and cloned into a standard sequencing plasmid, and 5-10 plasmids were sequenced. The consensus sequences are listed in Appendix I.

Depending on the VH usage and the VDJ joining sequence, some of the hybridomas can be divided into three clusters with clonally related VDJ and VJ sequences. The first cluster contains the three RBD-binding and CoV-2 neutralizing CVR hybridomas (Fig. 11 A). All three clones contain identical V(D)J joining sequences at the H and L chain V region. This indicates that this cluster of neutralizing CVR antibodies originated from the same naive CoV-2-Spike- specific B cells. When compared to a virtually assembled VH and VL region, the VH regions of all three CVR antibodies shared four new amino acids; all of them are in the framework region, and the VH in CVR243 acquired two additional new amino acids one in the CDR1 and one in the CDR3. In contrast, all three VL exons carry six new amino acids, five of them in CDR3 regions. Based on these findings, we conclude that we isolated two strongly neutralizing antibodies that differ in their amino acids sequence in the VH region.

The second cluster contains neutralizing CoV-2 antibodies that bind to NTD but not to RBD. All six clones contain again identical V(D)J joining sequences at the H and L chain V region (Figure 11 B) that are different from cluster 1 antibodies. This indicates that this cluster of neutralizing CVR antibodies also originated from a common naive CoV-2-Spike-specific B cell. When compared to a virtually assembled germline VH and VL region, the VH and VL regions contained numerous amino acid changes in the CDR1 , 2 and 3, and a few changes in the framework regions (Fig. 11 B). Interestingly, all six VH differ in at least one amino acid residue, whereas CVR49, 2918 and 328 carry the same L chain. The same was found for CVR618 and 1293. Based on these findings, we conclude that we isolated six more strongly neutralizing antibodies that differ in their amino acid sequence in their VH region.

The third cluster consists of clonally related hybridomas that secrete S1 -binding, nonneutralizing antibodies. Interestingly, the antibodies utilize a clonally related human VH and a mouse Vlambdax region (SEQ ID NOs.: 49-60).

The other hybridomas produce non-clonal antibodies (SEQ ID NOs.: 61-102) that recognize all the membrane-bound form of CoV-2 spike protein, differ in their reactivity towards recombinant spike proteins and do not neutralize CoV-2. Nevertheless, together with the cluster 3 antibodies, they will be useful for developing diagnostic assays to detect complete viruses in so-called antigen tests or as controls on antibody tests.

8. Production of recombinant fully human CVR antibodies

The provision of therapeutic antibodies requires the production of complete human antibodies and a cell line that is approved by the regulatory authorities. Therefore, we cloned the sequences of the entire VH and VL of all cluster 1 and cluster 2 hybridomas into pcDNA3.1 (+) cloning vectors together with a human Cgammal and human Ckappa region, respectively. Recombinant antibodies were isolated from the serum-free culture supernatant of transiently transfected HEK293T cells and verified for the ability to bind to the CoV-2 spike and neutralizing CoV-2 infection as described in Fig. 2 and Fig. 4, respectively. Figure 9 shows that all recombinant fully human cluster 1 and 2 antibodies bound specifically to 293T cells transfected with CoV-2 spike (Figure 9A). Similarly, all antibodies prevented the infection of Vero-E6 cells with wildtype CoV-2 with IC50 (Fig. 9B) in the range of that seen with the CVR antibodies isolated from serum-free hybridoma medium (Fig. 4B).

9. Further CVR Antibodies

Further antibodies of the invention are shown in tables 1 and 2 below. These antibodies are characterized by SEQ ID NOs 73-102.

SUBSTITUTE SHEET (RULE 26) Table 1

Table 2

They all bind to membrane-bound CoV-2 Spike protein in flow cytometric assays. Some of them bind to the S1, S2 and PBD domains of CoV-2 spike and some neutralize infection of Vero cells with infectious CoV-2 with IC50 of < 350 ng/ml.

SUBSTITUTE SHEET (RULE 26)

Claims

1. An antigen-binding protein, wherein the antigen-binding protein binds to the receptor-binding domain (RBD) of the SARS-CoV-2 (CoV-2) Spike protein and inhibits the binding of RBD to human ACE-2 (hACE-2) enzyme.

2. The antigen-binding protein of claim 1 , wherein the antigen-binding protein specifically binds to the RBD of the CoV-2 Spike protein with a KD ^10 nM as determined using an Elisa binding assay.

3. The antigen-binding protein of claim 1 or 2, wherein a) the antigen-binding protein has an EC50 of less than 10 nM in an hACE-2 flow cytometric competition assay to membranes from cells expressing the complete CoV-2 Spike protein; or b) the antigen-binding protein has an EC50 of less than 10 nM in an hACE-2 Elisa competition assay to membranes from cells expressing RBD of the CoV-2 Spike protein.

4. The antigen-binding protein of claim 1 , 2 or 3, wherein the antigen-binding protein competes for binding of hACE-2 with RBD of the CoV-2 Spike protein with a reference antibody, said reference antibody comprising (i) a heavy chain variable region comprising a sequence selected from the group consisting of SEQ ID NOs:1, 3 and 5; and (ii) a light chain variable region comprising a sequence selected from the group consisting of SEQ ID NOs: 2, 4 and 6.

5. An antigen-binding protein comprising

(A) one or more heavy chain complementary determining regions (CDRHs) selected from the group consisting of: (i) a CDRH1 selected from the group consisting of SEQ ID NO:7. 13 and 19; (ii) a CDRH2 selected from the group consisting of SEQ ID NO:8, 14 and 20 ; (iii) a CDRH3 selected from the group consisting of SEQ ID NO:9, 15 and 21; and (iv) a CDRH of (i), (ii) or (iii) that contains one, two, three or four amino acid substitutions, deletions or insertions;

(B) one or more light chain complementary determining regions (CDRLs) selected from the group consisting of: (i) a CDRL1 selected from the group consisting of SEQ ID NQs:10; 16 and 22 (ii) a CDRL2 selected from the group consisting of SEQ ID NOs: 11, 17 and 23; (iii) a CDRL3 selected from the group consisting of SEQ ID NOs: 12; 18 and 24 and optionally (iv) a CDRL of (i), (ii) and (iii) that contains one, two, three or four amino acid substitutions, deletions or insertions; or (C) one or more heavy chain CDRHs of (A) and one or more light chain CDRLs of (B).

6. The antigen-binding protein of claim 5, wherein the antigen-binding protein comprises a CDRH1, a CDRH2, a CDRH3, a CDRL1, a CDRL2 and a CDRL3.

7. An antigen-binding protein comprising

(a) a heavy chain variable region (VH) sequence that has at least 90% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs:1 , 3 and 5;

(b) a light chain variable region (VL) sequence that has at least 90% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs:2, 4 and 6; or

(c) a VH sequence that has at least 90% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 3 and 5, and a L sequence that has at least 90% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4 and 6.

8. The antigen-binding protein of claim 7, comprising a VH sequence selected from the group consisting of SEQ ID NOs: 1 , 3 and 5 and a VL sequence selected from the group consisting of SEQ ID NOs: 2, 4 and 6.

9. The antigen-binding protein of claim 8, comprising a heavy chain sequence comprising a sequence selected from the group consisting of SEQ ID NOs: 31, 32 and 33 and a light chain sequence comprising a sequence selected from the group consisting of SEQ ID NOs: 34, 35 and 36.

10. An antigen-binding protein, wherein the antigen-binding protein binds to the N- terminal domain (NTD) of the CoV-2 Spike protein.

11. The antigen-binding protein of claim 10, wherein the antigen-binding protein specifically binds to the NTD of the CoV-2 Spike protein with a KD ^10 nM as determined using an Elisa binding assay.

12. An antigen-binding protein, wherein the antigen-binding protein binds to the S1 domain (S1) of the CoV-2 Spike protein.

13. The antigen-binding protein of claim 12, wherein the antigen-binding protein specifically binds to the S1 of the CoV-2 Spike protein with a KD ^10 nM as determined using an Elisa binding assay.

14. An antigen-binding protein, wherein the antigen-binding protein binds to a subdomain of the CoV-2 Spike protein other than RBD, S1 and NTD.

15. The antigen-binding protein of claim 14, wherein the antigen-binding protein specifically binds to the subdomain of the CoV-2 Spike protein with a KD ^10 nM as determined using an Elisa binding assay.

16. An antigen-binding protein comprising

(C) one or more heavy chain complementary determining regions (CDRHs) selected from the group consisting of: (i) a CDRH1 having a CDRH1 of any of SEQ ID NOs:37,

39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59; (ii) a CDRH2 having a CDRH2 of any of SEQ ID NOs:37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59; (iii) a CDRH3 having a CDRH3 of any of SEQ ID NOs:37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59; and (iv) a CDRH of (i), (ii) or (iii) that contains one, two, three or four amino acid substitutions, deletions or insertions;

(D) one or more light chain complementary determining regions (CDRLs) selected from the group consisting of: (i) a CDRL1 having a CDRL1 of any of SEQ ID NOs:38,

17. An antigen-binding protein comprising a heavy chain variable region (VH) sequence that has at least 90% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 37, 39, 41 , 43, 45, 47, 49, 51, 53, 55, 57 and 59.

18. An antigen-binding protein comprising a light chain variable region (VL) sequence that has at least 90% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58 and 60.

19. An antigen-binding protein comprising a VH sequence that has at least 90% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 37, 39, 41 , 43, 45, 47, 49, 51 , 53, 55, 57 and 59, and a VL sequence that has at least 90% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58 and 60.

20. An antigen-binding protein comprising a heavy chain variable region (VH) sequence that has at least 90% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 61 , 63, 65, 67, 69, 71 , 73, 75, 77, 79, 81 , 83, 85,

87, 89, 91 , 93, 95, 97, 99 and 101 .

21. An antigen-binding protein comprising a light chain variable region ( L) sequence that has at least 90% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100 or 102.

22. An antigen-binding protein comprising a VH sequence that has at least 90% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 61 , 63, 65, 67, 69, 71 , 73, 75, 77, 79, 81 , 83, 85, 87, 89, 91 , 93, 95, 97, 99 and 101 , and a VL sequence that has at least 90% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86,

88, 90, 92, 94, 96, 98, 100 or 102.

23. The antigen-binding protein of any of claims 1 - 22, wherein the antigenbinding protein is selected from the group consisting of a monoclonal antibody, a Fab fragment, a Fab' fragment, an F(ab')2 fragment, an Fv fragment, a diabody, and a singlechain antibody.

24. The antigen-binding protein of any of claims 1 - 23, wherein the antigen-binding protein neutralizes CoV-2 infection of cells.

25. A nucleic acid polynucleotide that encodes an antigen-binding protein of any of claims 1-24.

26. An vector comprising a polynucleotide of claim 25, wherein the vector is

(a) an expression vector; or

(b) a gene shuttle.

27. A cell line transformed with the expression vector of claim 26(a).

28. An antigen-binding protein of any of claims 1-24, or the gene shuttle of claim(b) for use in treating CoV-2 infection or a condition associated with a CoV-2 infection.

29. An antigen-binding protein of any of claims 1-24, or the gene shuttle of claim(b) for use in the passive immunization against CoV-2 infection.