WO2022079606A1

WO2022079606A1 - Sars cov-2 neutralizing antibodies

Info

Publication number: WO2022079606A1
Application number: PCT/IB2021/059363
Authority: WO
Inventors: Gaily KIVI; Erkki JURONEN; Andres Männik; Kristiina KURG; Anu PLANKEN; Eva-Maria TOMBAK; Denis KAINOV; Eva Zusinaite; Mart USTAV SR.; Mart USTAV JR.
Original assignee: Icosagen Cell Factory Oü
Priority date: 2020-10-12
Filing date: 2021-10-12
Publication date: 2022-04-21

Abstract

Novel neutralizing SARS-CoV-2 S1 binding antibodies are provided here. The antibodies have low IC50 values and are suitable to be used separately or in combination to neutralize SARS-CoV-2 viral infection. The antibodies have potential for both a preventive and therapeutic drug against COVID19 and could be administered either intravenously or locally as a nebulized molecule or a nasal spray for direct delivery to mucosal tissue in various human immunoglobulin isotype frameworks.

Description

Title: SARS CoV-2 neutralizing antibodies

Priority

This application claims priority of US provisional application number 63/090576 filed on October 12, 2020, the contents of which are incorporated herein by reference.

Sequence listing

This application contains a sequence listing provided in computer readable format.

Field of invention

This application relates to novel antibodies and specifically to novel SARS CoV-2 neutralizing antibodies.

Background

COVID 19-pandemic is a pandemic of coronavirus disease 2019 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). SARS-CoV-2 is a novel virus infecting human beings: first human cases of COVID-19 were identified in Wuhan, China, in December 2019.

By now the virus has infected more than 33 million people and at least 998,200 people have died. The virus has been detected in nearly every country. The novel coronavirus has had devastating global health, as well as economic consequences and even if there are several vaccine candidates under development the treatment and prevention of the viral infection are still to be developed.

Recent studies on SARS-CoV-2 have shown that this coronavirus can bind to the alveolar pneumocytes in human lungs through their interaction with the angiotensin I and converting enzyme 2 (ACE2) surface receptor. ACE2 is an enzyme attached to the cell membranes of cells located in the lungs, arteries, heart, kidney, and intestines. As a transmembrane protein, ACE2 serves as the main entry point into cells for some coronaviruses, including SARS-CoV-2. More specifically, the binding of the spike S1 protein of SARS-CoV-2 to the enzymatic domain of ACE2 on the surface of cells results in endocytosis and translocation of both the virus and the enzyme into endosomes located within cells.

Neutralizing antibodies can block the entry of a pathogen into the cell and thus prevent the pathogen from infecting the body. As ACE2 is the main entry point for SARS-Cov-2, finding efficient neutralizing antibodies that could block the entry seems a promising approach for developing prophylactic and therapeutic means to fight against the pandemic.

Summary of the invention

Accordingly, it is an object of this invention to provide efficient neutralizing antibodies binding to the S1 -domain of the Spike protein of SARS-CoV 2 virus and competing with binding of ACE2.

According to certain aspects, the present invention provides neutralizing antibodies and antibody compositions that are suitable for administration to a subject having SARS-Cov-2 infection, or at risk of SARS-Cov-2 infection, in an amount and according to a schedule sufficient to prevent or reduce the virus infection.

According to certain aspects, the present invention provides antibodies suitable for use as a passive vaccine against the SARS-CoV-2 comprising at least one antibody of the invention and a pharmaceutically acceptable carrier. According to certain aspects, the present invention provides antibodies suitable for use as a nasal spray to provide prophylactic treatment against SARS-CoV-2 infection.

According to certain aspects, the present invention provides an antibody-based pharmaceutical composition comprising an effective amount of one of more of the isolated SARS-CoV-2 antibodies described herein for providing a prophylactic or therapeutic treatment to reduce infection of the SARS-CoV- 2.

It is an object of this invention to provide an isolated SARS-CoV-2 S1 -domain binding antibody or fragment thereof, wherein the antibody comprises a heavy chain variable region (VH) comprising the VH CDR 1 -3 according to SEQ ID NOs 95-97, respectively, and a light chain variable region (VL) comprising the VL CDR1 -3 of SEQ ID NOs 89-91 , respectively.

According to certain aspects the isolated SARS-CoV-2 S1 -domain binding antibody or fragment thereof, wherein the antibody comprises the VH protein according to SEQ ID NO: 44 or 72 and VL protein according to SEQ ID NO: 48 or 74, respectively.

It is an object of this invention to provide an isolated SARS-CoV-2 S1 -domain binding antibody or fragment thereof, wherein the antibody comprises a heavy chain variable region VH according to SEQ ID NO: 60 and light chain variable region VL according to SEQ ID NO: 62, or VH according to SEQ ID NO: 64 and VL according to SEQ ID NO: 66, or VH according to SEQ ID NO: 68 and VL according to SEQ ID NO: 70.

It is an object of this invention to provide a composition comprising the isolated SARS-CoV-2 S1 -domain binding antibody comprising a heavy chain variable region (VH) comprising the VH CDR 1 -3 according to SEQ ID Nos: 95-97, respectively, and a light chain variable region (VL) comprising the VL CDR1 -3 of SEQ ID Nos: 89-91 , respectively.

According to certain aspects the composition additionally comprises one or more of the antibodies comprising a heavy chain variable region VH according to SEQ ID NO: 60 and light chain variable region VL according to SEQ ID NO: 62, or VH according to SEQ ID NO: 64 and VL according to SEQ ID NO: 66, or VH according to SEQ ID NO: 68 and VL according to SEQ ID NO: 70.

According to certain aspects the composition is a pharmaceutical composition, comprising a pharmaceutically acceptable carrier.

According to certain aspects the composition is a nasal spray.

It is an object of this invention to provide a method to alleviate symptoms of COVID 19 disease, the method comprising administering to a subject an effective amount of the pharmaceutical composition.

It is an object of this invention to provide an isolated nucleic acid encoding the VH comprising the VH CDR 1 -3 according to SEQ ID NOs 95-97, respectively and /or the VL comprising the VL CDR1 -3 of SEQ ID NOs 89-91 , respectively.

It is an object of this invention to provide an isolated nucleic acid encoding the VH according to SEQ ID NO: 44 or 72 and VL according to SEQ ID NO: 48 or 74, respectively.

It is an object of this invention to provide an isolated nucleic acid encoding the VH according to SEQ ID NO: 60 and/or light chain variable region VL according to SEQ ID NO: 62, or VH according to SEQ ID NO: 64 and/or VL according to SEQ ID NO: 66, or VH according to SEQ ID NO: 68 and/or VL according to SEQ ID NO: 70.

Short description of the figures

Figure 1 illustrates the infection cycle of SARS-CoV-2. SARS-CoV-2 uses angiotensin converting enzyme 2 (ACE2) receptor to enter the host cell in which the virus then replicates. Virus inserts its RNA into the host cell, where it utilizes the host cell to produce new viral proteins and replicate the RNA genome. Viral proteins are glycosylated and transported to the host cell membrane, where the assembled particles bud from the host cell. Neutralizing antibodies can bind to spike S1 protein and thereby prevent or impair its capability to bind to ACE-2 receptor.

Figure 2a-d shows analytical Size-Exclusion Chromatography of SARS-CoV-2 neutralizing antibody clones, a) clone 23G7; b) clone 23E2, c) clone 8A12, d) clone 42B7.

Figure 3A-D: A) shows binding sensograms of SARS CoV-2 antibodies: a) clone 8A12 sample 12#22; b) clone 23E2, sample #81 ; c) clone 23G7, sample #86, d) clone 42B7, sample #140 to the S1 -domain of SARS-Cov-2;

B) shows a summary table of binding parameters of the SARS CoV-2 antibodies of clones 8A12, 23G7, 23E2 and 42B7;

C) shows interferometry sensorgrams of SARS CoV-2 antibodies of clone 23G7 and clone 23G7.1 to the trimeric S-ectodomain protein of SARS-Cov-2

D) shows a summary table of binding parameters of SARS CoV-2 antibodies 23G7 and 23G7.1

Figure 4 shows in-tandem bio-layer interferometry binning assay of SARS-CoV-2 RBD binding antibodies indicating the RBD binding antibodies belong to three independent epitope bins.

Figure 5a-d shows ACE2 blocking assay by bio-layer interferometry, a) shows ACE2 binding of clone 8A12 antibodies; b) shows ACE2 binding of clone 23G7 antibodies; c) shows ACE 2 binding of clone 23E2 antibodies; d) shows ACE2 binding of 42B7 antibodies. All tested antibodies except 8A12 is able to block ACE2 binding.

Figure 6 shows ACE binging inhibition assay by ELISA. Samples from Clones 23G7, 23E2, 8A12 and 42B7 were tested. Various concentrations of antibodies were used to treat immobilized S-protein followed by detection with biotinylated ACE2 and Strepavidin-HRP.

Figure 7A-B: A) shows SARS CoV-2 live virus neutralization assay. VERO E6 cells were treated with hCoV-19/Norway/Trondheim-E9/2020 at moi 0.1 together with various concentrations of SARS-CoV-2 antibodies of clones 8A12, 23G7 and 23E2, respectively. Cell viability was measured 72hours post-infection. B) shows live virus assay results measuring final antibody concentration where no cytopathic effects could be detected. Detailed description of the invention

Definitions

As used here, “antibody” is a polypeptide that specifically binds and recognizes an antigen such as S protein of SARS-Cov2 virus or an antigenic fragment thereof.

A "monoclonal antibody" is an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic epitope.

“Vaccine” as used here means a pharmaceutical composition that induces a prophylactic or therapeutic immune response in a subject. In some cases, the immune response is a protective immune response. Typically, a vaccine induces an antigen-specific immune response to an antigen of a pathogen, for example a viral pathogen, such as SARS-Cov-2. A vaccine may include a polynucleotide (such as a nucleic acid encoding a disclosed antigen), or a peptide or polypeptide (such as a disclosed antigen). In one example a vaccine induces an immune response that reduces the severity of the symptoms associated with SARS-Cov 2 infection and/or decreases the viral load compared to a control. In another non-limiting example, a vaccine induces an immune response that reduces and/or prevents SARS-CoV-2 infection compared to a control.

The term "disease" refers to any alternation in state of the body or of some of the organs, interrupting or disturbing the performance of the functions and/or causing symptoms such as discomfort, dysfunction, distress, or even death to the person afflicted or those in contact with a person.

As used herein the term "in need thereof" means having a disease, being diagnosed with a disease, or being in need of preventing a disease. A subject in need thereof can be a subject in need of treating or preventing a disease. As used herein, the terms "treat," "treatment," "treating," refer to therapeutic treatments, wherein the object is to reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of an infectious disease such as COVID 19- disease. The term "treating" includes reducing or alleviating at least one adverse effect or symptom of an infectious disease. Treatment is generally "effective" if one or more symptoms or clinical markers are reduced. Alternatively, treatment is "effective" if the progression of a disease is reduced or halted. That is, "treatment" includes not just the improvement of symptoms or markers, but also a cessation of, or at least slowing of, progress or worsening of symptoms compared to what would be expected in the absence of treatment. Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptoms, diminishment of extent of disease, stabilized (i.e. , not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, remission (whether partial or total), and/or decreased mortality. For example, treatment is considered effective if the condition is stabilized, or the progression of COVID 19 disease is slowed or halted. The term "treatment" of a disease also includes providing relief from the symptoms or side-effects of the disease (including palliative treatment).

As used herein, the term "prevent" or "prevention" refers to stopping, hindering, and/or slowing down an infectious disease. In one embodiment, "prevent" is synonymous with "inhibit".

As used herein, the term "administering," refers to the placement of a compound as disclosed herein into a subject by a method or route which results in at least partial delivery of the agent at a desired site. Pharmaceutical compositions comprising the compounds disclosed herein can be administered by any appropriate route which results in an effective treatment in the subject.

As used herein, a "subject" means a human. The terms, "individual," "patient" and "subject" are used interchangeably herein.

The terms "increased", "increase", or "enhance" are all used herein to generally mean an increase by a statically significant amount; the terms "increased", "increase", or "enhance", mean an increase of at least 10% as compared to a reference level, for example an increase of at least about 10%, at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level.

The terms "decrease", "reduce", "reduction", or "inhibit" are all used herein generally to mean a decrease by a statistically significant amount. For example, "decrease", "reduce", "reduction", or "inhibit" means a decrease by at least 10% as compared to a reference level, for example a decrease by at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% decrease (e.g. absent level or non-detectable level as compared to a reference level), or any decrease between 10-100% as compared to a reference level. In the context of a marker or symptom is meant a statistically significant decrease in such level. The decrease can be, for example, at least 10%, at least 20%, at least 30%, at least 40% or more, and is preferably down to a level accepted as within the range of normal for an individual without a given disease.

As used here, the term "pharmaceutically acceptable" refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

As used here, the term "pharmaceutically acceptable carrier" means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material necessary or used in formulating an active ingredient or agent for delivery to a subject. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient.

As used herein, the term "in combination" refers to the use of more than one prophylactic and/or therapeutic agent simultaneously or sequentially and in a manner that their respective effects are additive or synergistic.

“Composition” as used herein may be a single or a combination of antibodies disclosed herein, which can be the same or different, in order to prophylactically or therapeutically treat the progression SARS Cov-2 infection after administration. Such combinations can be selected according to the desired immunity or effect.

The antibody-based pharmaceutical composition of the present invention may be formulated by any number of strategies known in the art (e.g., see McGoff and Scher, 2000, Solution Formulation of Proteins/Peptides: In McNally, E. J., ed. Protein Formulation and Delivery. New York, N.Y.: Marcel Dekker; pp. 139-158; Akers and Defilippis, 2000, Peptides and Proteins as Parenteral Solutions. In: Pharmaceutical Formulation Development of Peptides and Proteins. Philadelphia, Pa.: Talyor and Francis; pp. 145-177; Akers, et al., 2002, Pharm. Biotechnol. 14:47-127).

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) emerged at the end of 2019 and was shortly thereafter sequenced. SARS-CoV-2 appears to have originated in bats from which closely related viruses and viral sequences have been identified. SARS-CoV-2 belongs to the sarbecovirus subgenus and is closely related to SARS-CoV which was responsible for an epidemic in 2002-2003 resulting in 8,098 cases and 774 fatalities worldwide. The lack of pre-existing immunity to SARS-CoV- 2 due to its divergence from the circulating endemic coronaviruses, and its efficient human-to-human transmission has resulted in the ongoing COVID-19 pandemic which has already caused more than 33 million infections and over million fatalities as of late-September 2020. SARS-CoV-2 infection starts upon attachment of the viral trimeric transmembrane spike (S) glycoprotein via a receptor-binding domain (RBD) to angiotensin-converting enzyme 2 glycoprotein (ACE2), leading to membrane fusion and entry into host cells c.f. Figure 1 . As for other coronaviruses, SARS-CoV- 2 spike protein is the main target of neutralizing antibodies (Abs) and a focus of vaccine design and therapeutic targeting strategies. Although vaccine development programs are progressing rapidly, large-scale manufacturing and administration to a large enough population for achieving community protection will probably take several months. Prophylactic and/or therapeutic anti-viral drugs could allow overcome the gap before safe and efficient vaccines become widely available and will continue to be useful in unvaccinated individuals or those who respond poorly to vaccination. Unlike controversial FDA-approved convalescent plasma, a treatment that takes whole plasma from a recovered person, and infuses it into another patient with the disease, monoclonal antibody therapy uses a much more targeted approach, specifically neutralizing SARS-CoV-2 infectivity. Therefore, SARS-CoV-2- targeting antibodies are really valuable as they are supposed to be an important bridge to a vaccine and a hedge in the event vaccines are delayed or work inefficiently. Similar to vaccines, antibody treatments are also being developed to prevent SARS-CoV-2 infection, particularly in people who are at high risk and who might have been exposed to the virus through close contact with an already infected person. These antibodies are designed to neutralize the virus in patients who are sick or offer short-term immunity to people who have been exposed. Passive immunization with neutralizing anti-SARS-CoV-2 antibody could be especially valuable for certain populations that are suffering the most: the elderly, the immunocompromised, patients in nursing homes and long-term care facilities. Signs and symptoms of COVID-19 may appear two to 14 days after exposure. This time after exposure and before having symptoms is called the incubation period. Common signs and symptoms can include fever, cough, tiredness. Other symptoms can include shortness of breath or difficulty breathing, muscle aches, chills, sore throat, runny nose, headache, chest pain. The severity of COVID-19 symptoms can range from very mild to severe. Some people may have only a few symptoms, and some people may have no symptoms at all. Some people may experience worsened symptoms, such as worsened shortness of breath and pneumonia, about a week after symptoms start.

This invention provides novel neutralizing anti-SARS-CoV-2 antibodies. The invention is now described with examples and references to the figures.

Human donors and peripheral blood mononuclear cell (PBMC) samples used for obtaining of the antibodies recognizing the S1 protein of SARS-CoV-2. Human sample collection and further processing were approved by Research Ethics Committee of the University of Tartu (No. 305/T-1 , 07.04.2020). Monoclonal antibodies recognizing S1 protein ( SEQ ID NO:6) of the SARS-CoV-2 virus or, more specifically, receptor binding domain (RBD) (SEQ ID NO: 2 or 4) of the S1 protein were isolated from human subjects diagnosed with COVID-19 by detection of the SARS-CoV-2 virus RNA in nasal swab sample by RT-PCR. Peripheral blood mononuclear cells (PBMCs) were collected 2-4 weeks after diagnosis and at this time the donors were recovered from the infection and had no acute COVID-19 infection symptoms.

Venous blood samples were collected and PBMCs were separated into BD Vacutainer® CPT™ Cell Preparation Tubes containing the cell separation medium (BD Biosciences, Cat No.362753) according to manufacturer’s instructions. The collected PBMCs were stored under liquid nitrogen up to further processing for the isolation of the monoclonal antibodies.

Isolation of S1/RBD binding human monoclonal antibodies using Hybrifree technology

Monoclonal antibodies recognizing SARS-Cov-2 S1 protein were isolated using Hybrifree technology (Kivi G, et al., BMC Biotechnol. 2016; 16:2 incorporated herein by reference). Briefly, the frozen PBMCs was thawed, washed and collected into 10 ml of RPMI1640 supplemented with penicillin/streptomycin and 10 % of heat inactivated fetal bovine serum. Then the cells were seeded into a 100 mm cell culture dish and incubated ~1 h at 37 °C in an 8 % CO2 atmosphere. Then, free- floating cells (the fraction enriched for B-cells and separated from plastic-adherent cells, e.g. macrophages) were collected, viable cell count was determined and the cells were transferred into the capture medium (RPMI1640 supplemented with 0.5 % BSA and 0.1 % NaN₃).

For capture or panning of the B-cells that express SARS-CoV-2 S1 protein specific antibodies on their surface, MaxiSorp™ surface 96-wells (Thermo Fisher Scientific, US) were coated with the recombinant S1 subunit (SEQ ID NO:6) of the spike protein (SEQ ID NO:8) or RBD domain (SEQ ID NO:4) of the S1 and blocked for 1 h with 2 % BSA in PBS. One hundred microliters of cell suspension containing 1 x 10⁴ live cells in capture medium were loaded into a single well. The plate was centrifuged (200 x g, 5 min) and incubated for 45-60 min. The medium was discarded, and loosely attached cells were removed by washing 4-5 times with PBS. Finally, the plastic-bound cells were lysed and subjected to total RNA isolation and cDNA synthesis using oligo-T primer. The cDNA synthesis was conducted using SuperScript™ IV First-Strand Synthesis System (Thermo Fischer Scientific Catalog number: 18091050) and oligo d(T)2o primer supplied with the kit.

The cDNAs of antibody VH and VL regions were amplified using cocktails of forward primers that bind to FR1 regions of VH or VL kappa and VLJambda light chain coding sequences and were designed to maximally cover the variability of the human VH and VL sequences. In each reaction, single reverse primer was used that binds to the beginning of the constant region encoding sequence of the human IgG heavy chains, kappa light chain or lambda light chain, respectively.

The VH forward primers (5’-3’) were according to SEQ ID NO:9 - SEQ ID NO: 22.

The VH reverse primer (5’-3’) was according to SEQ ID NO:23.

The VL kappa forward primers (5’- 3’) were according to SEQ ID NO 24-33.

The VL kappa reverse primer was according to SEQ ID NO:34.

The VL lambda forward primers (5’-3’) were according to SEQ ID NO: 35-41 and SEQ ID NO:98.

The VL lambda reverse primer was according to SEQ ID NO: 42.

Using the ligase independent cloning strategy, VH and VL PCR products retrieved from the same capture reaction were both exactly joined with: (i) the RSV LTR promoter (SEQ ID NO:75) and synthetic 5’ intron (SEQ ID NO 76) as well as mouse immunoglobulin heavy chain secretion leader peptide cDNA (SEQ ID NO:77) at the 5’ end and with human lgG1 constant domain cDNA (SEQ ID NO: 78) at the 3’ end for the VH fragment; and (ii) the CMV IE promoter (SEQ ID NO: 79) linked with the leader sequence from herpes virus TK gene (SEQ ID NO: 80) as well as mouse immunoglobulin light chain secretion leader peptide cDNA (SEQ ID :81) at the 5’ end and with human immunoglobulin kappa (SEQ ID: 82) or lambda (SEQ ID: 83) light chain constant domain cDNA at the 3’ end, for the VL fragment. The reaction creates just natural joining between variable and constant domain in the human IgG 1 heavy and kappa (if the VL was amplified with kappa specific primers) or lambda (kappa if the VL was amplified with lambda specific primers) light chain, respectively. The final product resulting from the cloning reaction is the pQMCF IgG shuttle expression vector (SEQ ID NOs 84; 85) containing ampicillin resistance gene for selective growth of transformed E. coli and separate mammalian expression cassettes for the lgG1 heavy and light chain, respectively. It is to be understood that the promoters, introns, secretion signals, and constant domains used in this example are just one combination of heterologous elements that can be used to express and produce the antibodies with particular variable domain. A person skilled in the art would be able to find other promoters and signal elements that would work also for this purpose.

Transformation of the cloning reactions to competent E. coli cells and the bacterial culture was propagated in liquid media. Then purification of the plasmid DNA products from the propagated bacteria resulted library pools of the antibody expressing vectors. In principle, such library pools are VH/VL combinatory libraries of limited numbers of VH and VL sequences retrieved from the same capture reaction and linked pairwise in the single expression vector molecule.

The efficiency of the antigen-specific IgG reconstruction from VH and VL combinations was initially analyzed via the transfection of library pools. The DNA was transfected into Chinese hamster ovary cell line CHOEBNALT85 (Icosagen Cell Factory, Estonia) and 48-72 h later the culture supernatants were assessed by ELISA for the secretion of IgG molecules that specifically recognize S1 protein (SEQ ID NO:6) of the SARS-CoV-2, thus indicating the presence of the desired VH/VL combinations in the library.

Next, the library pools that showed the clearly positive signal were split to individual clones by back-transformation into competent E. coli cells and picking individual bacterial colonies - each containing single type of the plasmid with unique VH and VL combination. Then, specific S1 -binding determination by ELISA was repeated using the supernatants of CHO cells transfected with plasmid DNA preparations derived from single clones instead of library pools. Finally, the VH and VL sequences were identified by sequencing of the VH and VL insertions of the positive plasmid clones. The sequence confirmed antibody expressing clones were recombinantly expressed in CHO cells followed by affinity purification and buffer exchange procedures. Figure 2a-d shows analytical size exclusion chromatography results of four selected neutralizing antibody clones. The purified antibodies were used to determine the binding affinities of the developed anti SARS-CoV-2 Spike protein antibodies. In Figure 3A-D, A) shows binding sensograms of the four selected antibody clones and the kinetic analysis to determine the Kon, Kdiss, Kd of the developed antibodies was conducted by using Bio-Layer-Interferometry (PALL Biosciences). The obtained binding kinetic properties against the S1 Protein SEQ ID NO:6 are shown in B of Figure 3A-D. In figure 3A-D, C shows binding sensograms of SARS CoV-2 antibodies of clones 23G7 and 23G7.1 . Summary table for binding parameters for these two clones is shown in part D of Figure 3A-D.

Identifying ACE2 blocking clones by bio-layer interferometry and ELISA based competition assays.

As antibodies capable of ACE2 blocking have been characterized to elicit virus neutralizing capacity (see Figure 1 ), we performed competition binding assays by bio-layer interferometry (BLI) and ELISA. For BLI assays we loaded the sensor tip with the SARS-CoV-2 specific antibody from the above antibody clones, followed by a first binding event of S1 -domain protein. This was followed by dipping the sensor in ACE2 containing buffer for a determining a secondary binding event. As demonstrated in figure 4 antibody clones 23G7, 23E2 and 42B7 were able to block the secondary binding event, indicating that they are competing with ACE2 for binding with the RBD. 8A12 was not able to block ACE2 binding.

To confirm these results, we performed an ELISA based competition assay (Figure 5a-d, Figure 6). S-protein (SEQ ID NO:8) was coated at 4ug/ml on a MaxiSorp high- capacity binding ELISA plate (Thermo Fisher Scientific, US). S-protein was incubated with antibodies from various clones at various concentrations of the antibodies, followed by washes and incubation with biotinylated ACE2-Fc and detection by Strepavidin-HRP. Concentration dependent blocking of ACE2-Fc was determined for the antibodies that was in correlation with data obtained with BLI assays. Antibody 8A12 binds to the S1 domain but does not bind to the RBD and does not block ACE2-Fc binding at any concentration.

Epitope binning assays

In order to determine if the obtained anti-S protein antibodies enable simultaneous binding to the S-protein we performed an in-tandem protocol binning experiment by BLI (Fig. 4). Seven clones of antibodies were selected for the assays. The S1 protein was captured to the sensor followed by capture of the 1 ^st mAb. Subsequently the sensor was dipped in a buffer containing the second competing antibody. A secondary binding event with the competing mAb indicates non-competing binding of the tested antibody pair. As evidenced in Figure 4, the RBD specific antibodies belong to 3 independent epitope bins. Antibody 23G7 belongs to an independent epitope bin by blocking of all other tested RBD specific antibodies. Antibody clones 31 B9, 13A4 and 38G5 belong to a second overlapping epitope bin. Antibodies 23E2, 42B7 and 42H8 belong to the third overlapping epitope bin. As antibody 8A12 does not bind the RBD but binds the S1 domain outside of the RBD (data not shown) it is evident that in total we are able to target 4 independent epitope bins located on the S1 domain. Based on this experiment there is potential to use these antibodies as a combinational therapy option as a subset of these antibodies can bind to the S1 protein simultaneously and potentially neutralize the SARS-CoV-2 infection.

IgG subtype determination of the selected antibodies

According to the method used to obtain the antibodies as described above, the VH sequences were amplified from PBMCs using reverse primer (SEQ ID NO: 23) that binds to very beginning of the CH1 domain of the heavy chain constant region that is identical for all known human IgG subclasses (i.e. IgG 1 , lgG2, lgG3 , lgG4). Later, during the vector cloning process in construction of the library pools, the amplified VH sequences are artificially linked to human lgG1 constant region present in the vector backbone. Thus, according to the method and the used vector, all antibodies obtained were initially produced and screened as human IgG 1 subtype antibodies and the initial sequence analysis of the VH region could not reveal the original IgG subtype (determined by heavy chain constant region) of the discovered antibody into which it belonged in human donor. However, it is well known that each subclass has a unique profile with respect to antigen binding, immune complex formation, complement activation, triggering of effector cells, half-life, and placental transport. Thus, we determined the original subclass belongings of the selected antibodies 23E2, 23G7 and 18B6. For this, the fragment of the antibody coding sequence including the unique c-terminal half of the VH domain and N-terminal part of the IgG heavy chain constant domain was amplified from the cDNA reaction prepared from capture reaction that resulted of each particular antibody clone. To amplify the coding sequence of the selected antibody linked with any IgG constant region, the PCR was using very specific forward primer (SEQ ID NO 9-22) that binds unique sequence of VH CDR2 of the antibody of interest together with reverse primer (SEQ ID NO:23) that binds to CH2 domain of all known human IgG subclasses (i.e. lgG1 , lgG2, lgG3, lgG4). The sequence differences in the region from the beginning of the constant domain to the reverse primer binding site in CH2 domain are sufficient for unambiguous determination of the IgG subclass (i.e. IgG 1 , lgG2, lgG3, lgG4) of the particular antibody. On the other hand, the identity of amplified unique sequences from CDR2 to the end of the VH confirms that the VH-CH1-CH2 fragment here was amplified from the very same cDNA that resulted the initially obtaining clones 23E2, 23G7 or 18B6.

The amplified fragments were cloned into the pJetl .2 cloning vector (available at ThermoFisher Scientific Cat. Nos. K1231 , K1232 Pub. No. MAN0012707 Rev. B.01 ) and 6-8 individual clones were sequenced for each antibody. The sequence analysis clearly revealed that all those antibodies originally belonged to lgG1 subclass in human donors.

Sequence analysis of the selected monoclonal antibodies

Table 1 . Characterization of antibody clones 23G7

Table 2. Characterization of clone 23G7 CDR sequences:

Table 3. Characterization of clone 23E2

Table 4. Characterization of clone 42B4

Table 5. Characterization of clone 8A12

The primary sequence analysis of the selected antibodies was conducted using IgBLAST tool (US National Library of Medicine) https://www.ncbi. nlm.nih.gov/igblast/). The analysis using VH and VL cDNA data revealed that:

(i) 23G7 VH is most close to human germline sequence IGHV1 -2*04 (97.3% identity); and 23G7 VL (lambda) is most close to human germline gene sequence IGLV2-14*01 (98.6% identity);

(ii) 23E2 VH is most close to human germline sequence IGHV3-53*04 (99.0% identity); and 23E2 VL (kappa) is most close human germline gene sequence IGKV1 -9*01 (99.3% identity);

(iii) 8A12 VH is most close to human germline sequence IGHV1 -24*01 (97.6% identity).; and 8A12 VL (kappa) is most close to human germline gene sequence IGKV2-24*01 (98.7% identity).

(iv) 42B7 VH is most close to human germline sequence IGHV3-66*02 (95.9% identity); and 42B7 VL (kappa) is most close to human germline gene sequence IGKV1 -39*01 (100% identity).

The CDR sequence boundaries were determined as published previously. (Ehrenmann F., Kaas Q, and Lefranc M.P. Nucleic Acids res., 38: D 301 -D307 (2010). PMID: 19900967; and Ehrenmann, F., Lefranc, M-P- Cold Spring Harbor Protoc., 6:737-749 (2011 ) PMID: 21632775

As the amplification of 23G7 clone VH and VL sequences using the established primer sequence cocktail generated a 5’ FR1 region mutation when comparing to the parental germline sequence, we also generated the corrected FR1 sequence variant of VH and VL of 23G7 identified as VH 23G7.1 (SEQ ID NO: 72) and VL 23G7.1 (SEQ ID NO: 74). The VH 23G7.1 and VL 23G7.1 encoding DNA sequences were codon optimized resulting in the following sequence for VH (SEQ ID: 71 ) and for VL (SEQ ID: 73). Part C of Figure 3A-D demonstrates the comparative binding sensorgram of 23G7 IgG and 23G7.1 IgG to the trimeric Spike ectodomain and part D of Figure 3A-D demonstrates the binding parameters of both antibody variants to the trimeric Spike ectodomain. As seen from parts C and D of Figure 3A-D both antibody variants elicit identical binding properties.

Determination of the epitope type of the selected antibodies The epitopes that are recognized by the antibody in its target protein sequence can be divided to two types: linear and conformational. Linear epitopes consist of continuous residues on a protein sequence. In contrast, conformational epitopes consist of residues that are discontinuous in the protein sequence but come within close proximity to form an antigenic surface on the protein's three-dimensional structure. Thus, only linear epitopes are recognized when the target protein is completely denatured, such as heated in reducing conditions and separated in SDS- PAGE gels.

To determine the type of epitope for the selected antibodies 23E2, 23G7, 8A12, 18B6, the same S1 protein of SARS-CoV-2 that was used for initial ELISA screening and obtaining those antibodies and which is recognized by those antibodies in the ELISA assay were also tested in conditions of completely denatured protein. Briefly, 1 pg of the S1 protein was denatured in 50 pl of SDS-PAGE loading buffer with DTT as reducing agent (2% SDS; 10% glycerol; 0.002% bromphenol blue; 67.5 mM Tris HCI, pH 6.8; 100 mM DTT) by heating the sample 5 minutes at 99 °C. Then the sample was loaded and run on gradient (8-16%) SDS-PAGE gel using 5 pl or 100 ng of S1 per lane. Then, the binding of the antibody was analyzed using Western blot technique with the antibodies of interest as primary antibodies (in concentrations 1 pg/ml and 5 pg/ml for 23E2, 23G7 and 8A12; and 1 pg/ml for 18B6) and anti-human IgG HRP conjugate as the secondary antibody (the same secondary antibody was successfully used for determination of the S1 binding of the same antibodies in ELISA). In this assay none of the selected antibodies showed binding to denatured S1 protein of SARS-CoV-2 (results not shown) indicating that those antibodies recognize conformational and not linear epitope in the target protein.

Cell based virus neutralization assays

In order to determine the ability of the developed antibodies to neutralize viral infection we performed a cell-based virus neutralization assay (part A of Fig. 7A-B). 4 x 10⁴ Vero-E6 cells were seeded per well in 96-well plates. The cells were grown for 24 h in DMEM supplemented with 10% FBS and Pen-Strep. Antibodies were prepared in 5-fold dilutions at 7 different concentrations, starting from 0.04mg/mL in the virus growth medium (VGM) containing 0.2% BSA and Pen-Strep in

DMEM. Virus strain hCoV-19/Norway/Trondheim-E9/2020 obtained from infected patient in Norway and expanded in a BSL3 containment facility, was added to the samples to achieve a moi of 0.1 and incubated for 1 h at 37 °C. 0,1 % DMSO was added to the control wells. The Vero-E6 cells were incubated for 72 h with VGM.

After the incubation period, the medium was removed, and a CellTiter-Glo assay was performed to measure viability.

Part B of Figure 7A-B shows inhibition (neutralizing effect) of antibody clones 8A12, 23G7 and 23E2.

From the obtained cell viability readouts after treatment at various concentrations of the antibodies we were able to determine IC50 values for the developed antibodies. Table 5 below show the IC50 values for each of the clones:

Table 5. IC50 live virus neutralization values of selected antibodies

23G7 had the most potent neutralizing effect of 5.9 ng/ml, followed by 8A12 and 23E2 with IC50 values of 839.8 ng/ml and 1267 ng/ml respectively.

For antibody 42B7 and the IgA isotype antibodies of 23G7.1 we performed a live virus neutralization assay where we identified final antibody concentrations in which treating the virus we could not see any cytopathic effects caused by viral infection, indicating a minimal antibody neutralization titer. In detail, the SARS-CoV-2 (Estonian isolate 3542) was diluted in DMEM/0.2% BSA/Pen-Strep 50 ul/well and added to the serially diluted antibodies. Mixtures were incubated for 1 h at 37C. Suspension of Vero E6 cells was added to the wells of a 96 well tissue culture plate in triplicates (4x 10e4 cells/well) at a final volume 100 ul/well. The virus treated cells were incubated at 37C, 5% CO2 for 5 days. Results of neutralization assay was evaluated microscopically by detecting appearance of cytopathic effect (CPE). The first dilution, where CPE was absent was determined as the neutralizing titer. The results of the neutralization assays is demonstrated in part B of Figure 7A-B. Examples of application

Antibodies are the first line of defense for many mucosal pathogens including respiratory viruses. For both influenza and coronaviruses, the neutralizing IgA response located in the mucosal tissue has been demonstrated to be most efficacious in preventing viral infection. Antibodies have the ability to bind and block cell entry of viral pathogens, thus they are an important class of biologic therapeutic molecules that can be developed recombinantly to both protect against viral infection and neutralize an already occurred infection. Recombinant antibodies that have been cloned from convalescent humans, immunized animals or synthetic antibodies obtained from molecular display libraries can all be an excellent source for antibodies that elicit virus neutralizing properties. These antibodies administered through a systemic intravenous delivery, local delivery using nebulized antibodies for inhalation to the lower respiratory tract or as a nasal spray that can be administered to the upper respiratory tract to generate a virus neutralizing protective biolayer.

The IgA antibody isotype is the most predominant antibody isotype located in the mucosal tissue and can form a multimeric molecule with up to a decavalent pentameric structure. The anti-SARS-CoV-2 antibody 23G7 described here can be expressed either as hlgG isotype antibody and it’s derivates as well as an IgA isotype molecule while retaining binding characteristics to the SARS-CoV-2 Spike protein. As 23G7 has an extremely low IC50 value as shown here, it is a candidate to neutralize SARS-CoV-2 viral infection, and therefore it has the potential for both a preventive and therapeutic drug against COVID19 that could be administered either intravenously or locally as a nebulized molecule or a nasal spray for direct delivery to mucosal tissue in various human immunoglobulin isotype frameworks.

The nasal cavity and nasopharynx contain some of the highest viral loads in the body, and viral loads are similar in symptomatic and asymptomatic individuals. Therefore, silent spreaders may unknowingly contribute to the exponential growth of disease, as nasal secretions contain spreadable virus, and contagiousness appears to be highest before or shortly after symptom onset. For this reason, nasal applications are specifically potent to mitigate the pandemic. Non limiting examples of intranasal delivery of the antibodies characterized in this application, and especially antibody 23G7, but also antibodies 8A12, 23E2, 42B7, or any combinations thereof could be in form of solution sprays, saline rinses, nanogels, intranasal foams or dissolvable packings, dry nasal powder sprays, nasal ointments.

A pharmaceutically acceptable composition suitable for patient administration will contain an effective amount of the antibody in a formulation which both retains biological activity while also promoting maximal stability during storage within an acceptable temperature range. The pharmaceutical compositions can also include, depending on the formulation desired, pharmaceutically acceptable diluents, pharmaceutically acceptable carriers and/or pharmaceutically acceptable excipients, or any such vehicle commonly used to formulate pharmaceutical compositions for human administration. The diluent is selected so as not to affect the biological activity of the combination. Examples of such diluents are distilled water, physiological phosphate-buffered saline, Ringer's solutions, dextrose solution, and Hank's solution. The amount of an excipient that is useful in the pharmaceutical composition or formulation of this invention is an amount that serves to uniformly distribute the antibody throughout the composition so that it can be uniformly dispersed when it is to be delivered to a subject in need thereof. It may serve to dilute the antibody to a concentration which provides the desired beneficial palliative or curative results while at the same time minimizing any adverse side effects that might occur from too high a concentration.

Claims

24 Claims What is claimed is:

1 . An isolated SARS-CoV-2 S1 domain RBD binding antibody or fragment thereof, wherein the antibody comprises a heavy chain variable region (VH) comprising the VH CDR 1 -3 according to SEQ ID Nos: 95-97, respectively, and a light chain variable region (VL) comprising the VL CDR1 -3 of SEQ ID Nos: 89-91 , respectively.

2. The isolated SARS-CoV-2 S1 RBD binding antibody or fragment thereof according to claim 1 , wherein the antibody comprises the VH according to SEQ ID NO: 44 or 72 and VL according to SEQ ID NO: 48 or 74, respectively.

3. The isolated SARS-CoV-2 S1 RBD binding antibody or fragment thereof according to claim 1 , wherein the antibody has a IC50 live virus neutralization values below 1300, more preferably below 900 and most preferably below 10 ng/ml.

4. An isolated SARS-CoV-2 S1 domain binding antibody or fragment thereof, wherein the antibody comprises a heavy chain variable region VH according to SEQ ID NO: 60 and light chain variable region VL according to SEQ ID NO: 62, or VH according to SEQ ID NO: 64 and VL according to SEQ ID NO: 66 or VH according to SEQ ID NO: 68 and VL according to SEQ ID NO: 70.

5. A composition comprising the isolated SARS-CoV-2 RBD binding antibody according to any of claims 1 -3.

6. A composition comprising the isolated SARS-CoV-2 SI domain binding antibody of claim 4.

7. The composition of claim 5, wherein the composition additionally comprises one or more of the antibodies of claim 4.

8. The composition of any of claims 5 to 7, wherein the composition is a pharmaceutical composition, comprising a pharmaceutically acceptable carrier.

9. The composition of claim 8, wherein the composition is administered intravenously or locally as a nebulized molecule or a nasal spray.

10. A method to alleviate symptoms of COVID 19 disease, the method comprising administering to a subject an effective amount of the composition of any of claims 5-9.

11 . An isolated nucleic acid encoding any of the VHs and /or the VLs of claim 1 or 2.

12. An isolated nucleic acid encoding any of the VHs and /of the VLs of claim 4.