RU2765731C1 - Humanized monoclonal antibody specifically binding to rbd s of the protein of the sars-cov-2 virus, agent and method for therapy and emergency prevention of diseases caused by sars-cov-2 virus - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: group of inventions relates to biotechnology, immunology and virology. Humanized monoclonal antibody is created, that specifically binds to RBD S of the protein of the SARS-CoV-2 virus and has virus-neutralizing activity, as well as an agent and a method for therapy and emergency prevention of diseases caused by SARS-CoV-2 virus. Humanized monoclonal antibody has the heavy chain amino acid sequence SEQ ID NO: 1 and the light chain amino acid sequence SEQ ID NO: 2. Also disclosed is a method for therapy and emergency prevention of viral infection SARS-CoV-2.

EFFECT: group of inventions extends the range of agents having virus-neutralizing activity and protective activity against various strains of the SARS-CoV-2 virus.

3 cl, 7 dwg, 7 tbl, 10 ex

Description

Technical field

The group of inventions relates to the field of biotechnology, immunology and virology. A humanized monoclonal antibody has been created that specifically binds to the RBD S protein of the SARS-CoV-2 virus and has virus-neutralizing activity; a means and method for the treatment and emergency prevention of diseases caused by the SARS-CoV-2 virus have also been proposed.

prior art.

On December 31, 2019, the World Health Organization (WHO) received information about an outbreak of pneumonia of unknown etiology in the city of Wuhan (People's Republic of China). The causative agent of the disease was a single-stranded RNA-containing SARS-CoV-2 virus belonging to the Coronaviridae family, to the Beta-CoV B line.

Coronavirus SARS-CoV-2 can be transmitted by airborne droplets, airborne dust, contact, fecal-oral methods, as well as through contaminated objects and surfaces (fomites), through blood, from mother to child and from animals to humans (Mechanisms of transmission of the SARS virus -CoV-2 and their implications for prevention choices, Research Summary, 9 July 2020 WHO). In a few months, the virus spread around the world, and in January 2020, WHO declared the epidemic associated with SARS-CoV-2 an international health emergency, and in March 2020 described the spread of the disease as a pandemic.

The disease that SARS-CoV-2 causes has been given its own name, COVID-19. This is a potentially severe acute respiratory infection that can be mild or severe, and is accompanied by complications such as pneumonia, acute respiratory distress syndrome, acute respiratory failure, acute heart failure, acute renal failure, septic shock, cardiomyopathies. , etc. Currently, the number of cases of COVID-19 is more than 185 million people, and more than 4 million people have died, and these numbers continue to grow. Clearly, there is an urgent need worldwide for the development of safe and effective means of prevention and treatment.

In addition, since the end of 2020, new variants of the SARS-CoV-2 virus began to appear, containing point amino acid substitutions, which make it possible to acquire partial or complete resistance to the immune response developed against the original version of the virus. One of these new options B.1. 617.2 Delta, isolated for the first time in India, is able to induce the formation of cellular syncytium, which additionally helps to escape from virus-neutralizing antibodies.

One of the promising approaches to the treatment of coronavirus infection is the use of antibodies against certain antigenic determinants of the virus. This is due to two main reasons: the high specificity of antibodies and the manufacturability of their industrial production. (V.S. Smirnov, Areg A. Totolyan. Some possibilities of immunotherapy for coronavirus infection. Infection and Immunity 2020, Vol. 10, No. 3, pp. 446-458).

Currently, more than 50 clinical trials related to monoclonal antibodies are being conducted around the world (Deb P, Molla MMA, Saif-Ur-Rahman KM. An update to monoclonal antibody as a therapeutic option against COVID-19. Biosaf Health. 2021 Apr;3( 2):87-91 doi: 10.1016/j.bsheal.2021.02.001 Epub 2021 Feb 10. PMID: 33585808; PMCID: PMC7872849). (https://www.sciencedirect.com/science/article/pii/S2590053621000197#s0005).

Currently, two drugs based on monoclonal antibodies are approved for emergency use. On November 21, 2020, the U.S. Food and Drug Administration (FDA) issued an Emergency Use Authorization (EUA) for REGEN-COV (Regeneron Pharmaceuticals), for the treatment of mild to moderate COVID-19 in people aged twelve years of age or older weighing at least 40 kg (88 lb) who have positive direct testing for the SARS-CoV-2 virus and who are at high risk of developing severe COVID-19. This includes those aged 65 and over or those with certain chronic conditions. REGEN-COV consists of two monoclonal antibodies, casirivimab (REGN10933) and imdevimab (REGN10987), that bind to non-overlapping epitopes of the SARS-CoV-2 S protein. The results of clinical studies of this drug showed that it is able to reduce the viral load, with a large effect in patients in whom the immune response has not yet been initiated or who initially had a high viral load (Weinreich DM, Sivapalasingam S, Norton T, Ali S, Gao H, Bhore R, Musser BJ, Soo Y, Rofail D, Im J, Perry C, Pan C, Hosain R, Mahmood A, Davis JD, Turner KC, Hooper AT, Hamilton JD, Baum A, Kyratsous CA, Kim Y, Cook A, Kampman W, Kohli A, Sachdeva Y, Graber X, Kowal B, DiCioccio T, Stahl N, Lipsich L, Braunstein N, Herman G, Yancopoulos GD, Trial Investigators REGN-COV2, a Neutralizing Antibody Cocktail, in Outpatients with Covid-19. N Engl J Med. 2021 Jan 21; 384(3):238-251. doi: 10.1056/NEJMoa2035002. Epub 2020 Dec 17. PMID: 33332778; PMCID: PMC7781102).

Another monoclonal antibody drug is bamlanivimab (LY-CoV555,

developer: Eli Lilly) has received FDA emergency clearance for the treatment of mild to moderate COVID-19 in non-hospitalized adults and children. However, the results of clinical studies are mixed. Among hospitalized patients with COVID-19, co-administration of bamlanivimab with remdesivir was not effective. (ACTIV-3/TICO LY-CoV555 Study Group, Lundgren JD, Grund B, Barkauskas CE, Holland TL, Gottlieb RL, Sandkovsky U, Brown SM, Knowlton KU, Self WH, Files DC, Jain MK, Benfield T, Bowdish ME , Leshnower BG, Baker JV, Jensen JU, Gardner EM, Ginde AA, Harris ES, Johansen IS, Markowitz N, Matthay MA, ∅stergaard L, Chang CC, Davey VJ, Goodman A, Higgs ES, Murray DD, Murray TA, Paredes R, Parmar MKB, Phillips AN, Reilly C, Sharma S, Dewar RL, Teitelbaum M, Wentworth D, Cao H, Klekotka P, Babiker AG, Gelijns AC, Kan VL, Polizzotto MN, Thompson BT, Lane HC, Neaton JD A Neutralizing Monoclonal Antibody for Hospitalized Patients with Covid-19 N Engl J Med 2021 Mar 11;384(10):905-914 doi: 10.1056/NEJMoa2033130 Epub 2020 Dec 22 PMID: 33356051; PMCID: PMC7781100) . Clinical studies in outpatients showed that only one of the three doses of the study drug (2800 mg LY-CoV555) significantly accelerated the natural decrease in viral load. (Chen P, Nirula A, Heller B, Gottlieb RL, Boscia J, Morris J, Huhn G, Cardona J, Mocherla B, Stosor V, Shawa I, Adams AC, Van Naarden J, Custer KL, Shen L, Durante M, Oakley G, Schade AE, Sabo J, Patel DR, Klekotka P, Skovronsky DM; (3):229-237.doi:10.1056/NEJMoa2029849.Epub 2020 Oct 28.PMID:33113295;PMCID:PMC7646625).

The best effect was observed with combination therapy with two monoclonal antibodies: bamlanivimab and etsevimab. (Gottlieb RL, Nirula A, Chen P, Boscia J, Heller B, Morris J, Huhn G, Cardona J, Mocherla B, Stosor V, Shawa I, Kumar P, Adams AC, Van Naarden J, Custer KL, Durante M, Oakley G, Schade AE, Holzer TR, Ebert PJ, Higgs RE, Kallewaard NL, Sabo J, Patel DR, Klekotka P, Shen L, Skovronsky DM Effect of Bamlanivimab as Monotherapy or in Combination With Etesevimab on Viral Load in Patients With Mild to Moderate COVID-19: A Randomized Clinical Trial JAMA 2021 Feb 16;325(7):632-644 doi: 10.1001/jama.2021.0202 PMID: 33475701; PMCID: PMC7821080).

In addition, a promising direction for the therapy and emergency prevention of infectious diseases is the use of single-domain antibodies (nanoantibodies), which can be found in nature in representatives of the camelid family. Due to their relatively small size, single domain antibodies have favorable biophysical properties and are cheaper to produce than standard monoclonal antibodies. They can be produced using prokaryotic or eukaryotic expression systems. Their small size, as well as long complementarity-determining regions of the heavy chain, allow them to target concave epitopes. In addition, nanoantibodies can be sprayed and delivered directly to the lungs of a Covid-19 patient using an inhaler, which is a better alternative to classical antibodies administered intravenously (Ram Sasisekharan. Preparing for the Future - Nanobodies for Covid-19? April 22, 2021 N Engl J Med 2021; 384:1568-1571 DOI: 10.1056/NEJMcibr2101205).

There are currently no single-domain antibody drugs approved for use in the treatment of COVID-19.

A solution is known (CN 112500480 A, pub. 03/16/2021), in which variants of a single-domain antibody against the SARS-CoV-2 virus, the corresponding expression vector, as well as a cell line capable of expressing the above variants of a single-domain antibody, and a method for obtaining variants of this antibody are developed .

A solution is known (CN 111825762 A, pub. 10/27/2020), in which several variants of nanoantibodies to the RBD S domain of the SARS-COV-2 virus protein were developed, as well as a method for using nanoantibody variants for the preparation of drugs for inhibiting the SARS-COV- 2 and preparation of reagents or kits for testing the SARS-COV-2 virus.

The patent (CN 112094342 A, pub. 12/18/2020) presents a bi-epitope specific antibody derived from alpaca, or an antigen-binding fragment thereof, that binds to the RBD domain of SARS-CoV-2 with high affinity, which can be used for prevention, treatment and / or diagnosing SARS-CoV-2 infection.

A solution is known (CN 112062839 A, pub. 12/11/2020), which describes the creation of a nanoantibody to the S protein of SARS-CoV-2, as well as a method of using it to prepare a reagent for the treatment and / or diagnosis of an infection caused by the coronavirus SARS-CoV- 2.

There is also a known solution (CN 112062840 A, pub. 12/11/2020), which provides for the development of a nanoantibody to the SARS-CoV-2 S protein, as well as a method for using it to prepare a reagent for the treatment and / or diagnosis of an infection caused by the SARS-CoV-2 coronavirus .

The patent (CN 111303279 A, published September 19, 2020) describes the creation of a humanized single-domain antibody against SARS-CoV-2 coronavirus, a nucleic acid molecule encoding a single-domain antibody, as well as the creation of an expression cassette, a recombinant vector, a recombinant bacterium or a transgenic cell line, containing the nucleic acid molecule described above. In addition, a method has been developed for using a single-domain antibody or engineered nucleic acid molecule or engineered expression cassette, recombinant vector, recombinant bacterium, or transgenic cell line in the manufacture of a product that can be used to prevent and/or treat diseases caused by infection with the novel coronavirus SARS-CoV- 2; as well as to suppress infection with the novel coronavirus SARS-CoV-2. In this case, the product may: communicate with the new coronavirus SARS-CoV-2; detect binding of the novel coronavirus SARS-CoV-2; bind to the S protein of the novel coronavirus SARS-CoV-2; detect the S protein of the new SARS-CoV-2 coronavirus. A pharmaceutical composition has also been developed containing the created single-domain antibody and a pharmaceutically acceptable excipient, diluent or carrier.

This solution, as the closest to the claimed one, was chosen by the authors of the patent for the prototype. However, the disadvantage of the prototype is the lack of data on the possibility of using the obtained antibodies for the treatment of a disease caused by the SARS-CoV-2 virus of other variants (for example, the Delta variant), in addition, single-domain antibodies are rapidly excreted from the body due to low molecular weight and, thus require multiple injections.

Thus, in the prior art there is a need to create a drug for the treatment and emergency prevention of a disease caused by the severe acute respiratory syndrome virus SARS-CoV-2, devoid of this disadvantage.

Disclosure of the essence of the invention

The technical objective of the claimed group of inventions is to expand the arsenal of agents for therapy and emergency prevention of diseases caused by the SARS-CoV-2 virus.

The technical result consists in creating a humanized monoclonal antibody with virus-neutralizing activity and protective activity against various strains of the SARS-CoV-2 virus. And also the technical result is to create an effective method for the treatment or emergency prevention of diseases caused by the SARS-CoV-2 virus, which consists in the introduction of an effective agent, including the developed humanized monoclonal antibody.

This technical problem is solved by the fact that a humanized monoclonal antibody has been created for the treatment and emergency prevention of diseases caused by the SARS-CoV-2 virus, and has the amino acid sequence of the heavy chain SEQ ID NO: 1 and the amino acid sequence of the light chain SEQ ID NO: 2.

In addition, an agent for the treatment and emergency prevention of diseases caused by the SARS-CoV-2 virus has been created, containing the developed humanized monoclonal antibody in an effective amount, as well as a pharmaceutically acceptable carrier and/or diluent.

A method has also been developed for the treatment or emergency prevention of diseases caused by the SARS-CoV-2 virus, which consists in introducing a humanized monoclonal antibody in an effective amount into the body of mammals.

Brief description of the drawings

In FIG. 1 shows an electrophoregram of the analysis of a humanized XRH19 monoclonal antibody.

1 shows a sample of a humanized XRH19 monoclonal antibody under denatured conditions;

2 - molecular weight marker;

3 shows a sample of a humanized XRH19 monoclonal antibody under undenatured conditions;

In FIG. 2 is a schematic representation of a humanized XRH19 monoclonal antibody.

In FIG. 3 shows the results of the analysis of the specific activity of the humanized monoclonal antibody XRH19 in indirect ELISA.

Y-axis - optical units, OD450nm;

Abscissa axis - XRH19 antibody concentration, μg/ml;

- уровень сигнала антитела XRH19 на бычий сывороточный альбумин (отрицательный контроль);

- signal level of XRH19 antibody to bovine serum albumin (negative control);

- уровень сигнала антитела XRH19 на RBD.

- XRH19 antibody signal level on RBD.

In FIG. 4 shows the HPLC chromatogram of the XRH19 humanized monoclonal antibody sample in buffer #2.

The y-axis - units of optical density 280nm, mAu;

The abscissa axis is the peak retention time, min.

In FIG. Figure 5 shows the results of the survival of animals that were injected with the humanized monoclonal antibody XRH19 and animals from the placebo group for 15 days after infection with the SARS-CoV-2 virus of the hCoV-19/Russia/Moscow_PMVL-1/2020 strain.

Y-axis - survival, %;

The abscissa axis is the time after infection of animals with the SARS-CoV-2 virus, days;

- мыши, получившие гуманизированное моноклональное антитело XRH19 (10 мг/кг) спустя 1 час после заражения вирусом;

- mice receiving humanized monoclonal antibody XRH19 (10 mg/kg) 1 hour after infection with the virus;

- мыши, получившие гуманизированное моноклональное антитело XRH19 (20 мг/кг) спустя 1 час после заражения вирусом;

- mice receiving humanized monoclonal antibody XRH19 (20 mg/kg) 1 hour after infection with the virus;

- мыши, получившие фосфатно-солевой буфер (PBS) спустя 1 час после заражения вирусом.

mice treated with phosphate buffered saline (PBS) 1 hour after virus infection.

In FIG. 6 shows the results of the analysis of the change in weight of animals that were injected with humanized monoclonal antibody XRH19 and animals from the placebo group within 15 days after infection with the SARS-CoV-2 variant Delta virus. At each time point for each group of animals, the weight is presented as an arithmetic mean.

The y-axis is the change in the weight of animals from the initial one, %;

The abscissa axis is the time after infection of animals with the SARS-CoV-2 virus, days;

- мыши, получившие гуманизированное моноклональное антитело XRH19 10 мг/кг спустя 1 час после заражения вирусом;

mice treated with humanized monoclonal antibody XRH19 10 mg/kg 1 hour after infection with the virus;

- мыши, получившие гуманизированное моноклональное антитело XRH19 10 мг/кг спустя 6 часов после заражения вирусом;

mice treated with humanized monoclonal antibody XRH19 10 mg/kg 6 hours after infection with the virus;

- мыши, получившие гуманизированное моноклональное антитело XRH19 20 мг/кг спустя 1 час после заражения вирусом;

- mice receiving humanized monoclonal antibody XRH19 20 mg/kg 1 hour after infection with the virus;

- мыши, получившие гуманизированное моноклональное антитело XRH19 20 мг/кг спустя 6 часов после заражения вирусом;

- mice receiving humanized monoclonal antibody XRH19 20 mg/kg 6 hours after infection with the virus;

- мыши, получившие фосфатно-солевой буфер (PBS) спустя 1 часов после заражения вирусом;

- mice treated with phosphate-buffered saline (PBS) 1 hour after infection with the virus;

- мыши, получившие фосфатно-солевой буфер (PBS) спустя 6 часов после заражения вирусом.

mice treated with phosphate buffered saline (PBS) 6 hours after virus infection.

In FIG. 7 shows the survival results of animals treated with the humanized monoclonal antibody XRH19 and animals in the placebo group for 15 days after infection with the SARS-CoV-2 variant Delta virus.

Y-axis - survival, %;

The abscissa axis is the time after infection of animals with the SARS-CoV-2 virus, days;

- мыши, получившие гуманизированное моноклональное антитело XRH19 10 мг/кг спустя 1 час после заражения вирусом;

mice treated with humanized monoclonal antibody XRH19 10 mg/kg 1 hour after infection with the virus;

- мыши, получившие гуманизированное моноклональное антитело XRH19 10 мг/кг спустя 6 часов после заражения вирусом;

mice treated with humanized monoclonal antibody XRH19 10 mg/kg 6 hours after infection with the virus;

- мыши, получившие гуманизированное моноклональное антитело XRH19 20 мг/кг спустя 1 час после заражения вирусом;

- mice receiving humanized XRH19 monoclonal antibody 20 mg/kg 1 hour after infection with the virus;

- мыши, получившие гуманизированное моноклональное антитело XRH19 20 мг/кг спустя 6 часов после заражения вирусом;

- mice receiving humanized monoclonal antibody XRH19 20 mg/kg 6 hours after infection with the virus;

- мыши, получившие фосфатно-солевой буфер (PBS) спустя 1 часов после заражения вирусом;

- mice treated with phosphate-buffered saline (PBS) 1 hour after infection with the virus;

- мыши, получившие фосфатно-солевой буфер (PBS) спустя 6 часов после заражения вирусом.

mice treated with phosphate buffered saline (PBS) 6 hours after virus infection.

Implementation of the invention.

The technical task of the claimed group of inventions is the creation of a humanized monoclonal antibody for the treatment and emergency prevention of diseases caused by the SARS-CoV-2 virus. Monoclonal antibodies with high specificity for the RBD S protein of the SARS-CoV-2 virus and virus-neutralizing activity are considered as promising agents for the treatment and emergency prevention of COVID 19.

For this, a humanized monoclonal antibody (XRH19) was developed that specifically binds to the RBD domain S of the SARS-CoV-2 virus protein, has virus-neutralizing and protective activity against various strains of the SARS-CoV-2 virus, and has the heavy chain amino acid sequence SEQ ID NO: 1 and the light chain amino acid sequence of SEQ ID NO:2. A humanized monoclonal antibody can be used for the treatment and emergency prevention of COVID 19.

To obtain a humanized XRH19 monoclonal antibody, Balb/c mice were immunized with recombinant RBD of the SARS-CoV-2 virus obtained in the CHO cell line and purified by metal affinity and size exclusion chromatography. The effectiveness of immunization was confirmed by assessing the titer of antibodies specific to RBD in the blood serum of mice. Next, splenocytes were isolated from mice, which were then fused with the mouse myeloma line Sp2/0-Ag-14, thus obtaining hybridomas. Hybridoma selection was performed on RPMI-1640 nutrient medium (Sigma, USA) supplemented with HAT Media Supplement (Hybri-Max®, Sigma, USA). The selection of clones producing antibodies specific to RBD was carried out by the method of solid-phase ELISA.

It is known to those skilled in the art that, in order to carry out the present invention, it is possible to use not only splenocytes, but also whole blood, lymph nodes of cells and organs of an immunized animal expressing monoclonal antibodies. It is also known to those skilled in the art that synthetic libraries of monoclonal antibody sequences can be used to carry out the present invention.

As a result of the selection and selection of clones, a hybridoma clone was selected that produces the mouse monoclonal antibody XR19, which is specific to RBD and has a pronounced virus-neutralizing and protective activity against the SARS-CoV-2 virus, including the Delta variant. Next, sections of the genome of the selected hybridoma clone encoding the antigen-binding regions (CDR domains) of the XR19 antibody were sequenced and used to create a humanized monoclonal antibody GamXRH19. Thus, the nucleotide sequences encoding the humanized monoclonal antibody GamXRH19 (SEQ ID NO: 1 heavy chain and SEQ ID NO: 2 light chain) were obtained.

It is known to those skilled in the art that, for the purposes of the present invention, the sequences of monoclonal antibodies specific to the RBD of the SARS-CoV-2 virus may contain amino acid substitutions that do not affect the secondary and tertiary structure of the monoclonal antibodies and do not affect their ability to bind and neutralize the RBD of the SARS-CoV-2 virus. Moreover, since only antigen-binding regions (CDR domains) are involved in antigen binding, as is known to those skilled in the art, any immunoglobulin or analogue thereof containing the same amino acid sequences of CDR domains can be used to practice this invention. Moreover, sequences of CDR domains may contain substitutions/deletions/insertions of amino acids, which, when paired with amino acid sequences, provide homology of at least 70% and do not qualitatively affect the ability to bind to the antigen. Thus, all immunoglobulins or their analogues containing sequences of CDR domains, the homology of which is not less than 70% with the proposed sequences can be used for the implementation of this invention.

The humanized monoclonal antibody XRH19 (human IgG1 isotype), which specifically binds to the RBD domain S of the SARS-CoV-2 virus protein and has virus-neutralizing and protective activity, was obtained by expressing the nucleotide sequence of the heavy and light chains in CHO-producing cells.

The heavy and light chain nucleotide sequences of the humanized monoclonal antibody XRH19 were engineered from the nucleotide sequence of human immunoglobulin G1 and the nucleotide sequences of the antigen-binding regions of the XR19 antibody. Those skilled in the art will recognize that, for purposes of the present invention, the nucleotide sequence encoding a humanized monoclonal antibody may differ based on the principle of degeneracy of the genetic code. Thus, all nucleotide sequences encoding the amino acid sequence of the humanized monoclonal antibody XRH19 (SEQ ID NO: 1 heavy chain and SEQ ID NO: 2 light chain) can be used to implement the present invention.

Using genetic engineering methods, a producer cell containing nucleic acids was obtained with the nucleotide sequences of the humanized XRH19 monoclonal antibody (SEQ ID NO:3 heavy chain and SEQ ID NO:4 light chain) and expressing the humanized XRH19 monoclonal antibody (SEQ ID NO: 1 heavy chain and SEQ ID NO: 2 light chain). Thus, a producer cell based on the CHO cell line expressing the humanized XRH19 monoclonal antibody was obtained. Those skilled in the art will recognize that other eukaryotic expression systems can be used as a producer for the purposes of the present invention.

In addition, the authors of the patent have developed a method for the treatment and emergency prevention of diseases caused by the SARS-CoV-2 virus (including the Delta variant), which consists in systemically administering an effective amount of humanized XRH19 monoclonal antibody into the body of mammals.

The implementation of the invention is confirmed by the following examples.

Example 1. Obtaining an immunogen - receptor-binding domain (RBD) S protein of the SARS-CoV-2 virus.

At the first stage of the work, an immunogen was obtained - the receptor-binding domain (RBD) S protein of the SARS-CoV-2 virus. For this, the amino acid sequence of the receptor-binding domain (RBD) of the surface Spike protein of the SARS-CoV-2 virus (a.a. 319 - 541, UniProtKB: locus SPIKE_SARS2, accession P0DTC2) was modified from the N-terminus with the signal peptide of alkaline phosphatase SEAP (MLLLLLLGLRLRLQLSLGI ) and from the C-terminus with a glycine-serine linker and histidine tag sequence (GSHHHHHHHHH). Based on the amino acid sequence, a nucleotide sequence was obtained, which was synthesized by Evrogen CJSC. After that, the nucleotide sequence of the resulting polypeptide was cloned into a plasmid for transient expression in mammalian cells. Next, the CHO cell culture was tariffed with the obtained plasmid using the CHO Gro kit (Mirus Bio, USA) according to the manufacturer's protocol. The cells were then cultured in Erlenmeyer flasks for 10 days. After this period, the culture liquid was clarified by centrifugation at 5000g. The receptor binding domain (RBD) was purified by metal affinity chromatography on an AKTA start system (GE Healthcare Life Sciences, USA) using 5 ml HisTrap FF columns (GE Healthcare Life Sciences, USA) according to the manufacturer's protocol. Additional purification and replacement of the buffer with 20 mM sodium phosphate, 500 mM sodium chloride (pH=7.2) was performed on an XK 26/100 column (GE Healthcare Life Sciences, USA) packed with Superdex 200 pg sorbent (GE Healthcare Life Sciences, USA). Thus, as a result of the work performed, a purified recombinant RBD S protein of the SARS-CoV-2 virus was obtained.

Example 2. Obtaining monoclonal antibody XR19.

BALB/c mice (10 females, 5-6 weeks old, weighing 15-20 g) were immunized with the preparation of the recombinant RBD of the SARS-CoV-2 virus obtained in example 1. The immunization schedule included 4 subcutaneous injections of the antigen with an injection interval of 21 days. The dose of the drug was 50 mcg per injection. 18 days after the last injection, blood was taken from the paraorbital sinus and the titer of specific antibodies was determined using enzyme-linked immunosorbent assay. To do this, the tablets were sensitized with antigen in 50 mm carbonate-bicarbonate buffer with a pH of 9.6. Mouse blood samples were diluted by 10-fold titration in ELISA buffer (0.1 M phosphate-buffered saline supplemented with 1% bovine serum albumin (Serva, Germany) and 0.1% Tween 20 (Serva, Germany) and incubated in the plate wells for 30 minutes at 37° C. The wells were washed with three additions of 0.9% sodium chloride with the addition of 0.1% Tween 20 (wash buffer) and horseradish peroxidase-labeled anti-mouse immunoglobulin antibodies (Cat. No. AS302-HRP, Khema, Russia) were added. ) at the working dilution for 30 minutes at 37° C. After washing 5 times with wash buffer, a chromogen-substrate mixture (Cat. No. R055, Khema, Russia) was added to the wells for 15 minutes, the reaction was stopped with a 5% sulfuric acid solution, and the optical density at 450 nm on a HyPo plate reader (Biosan, Latvia) An antibody titer of at least 1:100,000 was considered satisfactory.

Next, the animals with the highest level of antibody production 20 days after immunization received an intra-abdominal injection (booster dose) of the antigen in the amount of 50 μg. On the fourth day after the booster injection, the spleen was aseptically removed, splenocytes were obtained by perfusion with RPMI1640 medium (Sigma-Aldrich, USA).

The procedure for fusion of immune splenocytes and SP2/0-Ag14 myeloma cells was carried out according to the method proposed by G.Kohler and C.Milstein modified by De St.Fazekas, S.Groth and D.Scheidegger. A 50% solution of polyethylene glycol with a molecular weight of 1450 (Sigma-Aldrich, USA) was used as an inducer of cell fusion. Hybrid cells were cultured in vitro in a CO2 incubator at 37°C and 5% carbon dioxide. Growth medium RPMI-1640 containing 2 mM L-glutamine supplemented with 20% fetal calf serum (Thermo Scientific, USA) and 80 mg/L gentamicin (Russia) was used for cultivation. For selection of hybrid cells, HAT (Sigma-Aldrich, USA) containing hypoxanthine (H), aminopterin (A), and thymidine (t) in working dilution was added to the growth medium.

Primary screening of hybrid cultures was carried out starting from the 10th day after hybridization. The specific activity of antibody production of hybrid cultures was determined in the culture fluid without dilution by ELISA, similarly to that described in the section "Monitoring of the immune response" three times with an interval of 2-3 days, depending on the intensity of cell growth. Samples were considered positive if the optical density in the wells with the studied samples was 0.5 higher than the optical density in the control wells with the cultivation medium.

Cloning of cultures of hybrid cells was carried out by the method of limiting dilutions at the calculated seed concentration of one cell per well. The production activity of individual clones was determined, starting from the 10th day, twice with an interval of three days by enzyme immunoassay according to the scheme described above. After the isolation of positive clones, their cells were expanded in sufficient quantity, the samples were frozen in RPMI growth medium containing 20% fetal calf serum and 8% dimethyl sulfoxide (Serva, Germany) and stored in a container with liquid nitrogen.

The cultural and secretory properties of the resulting clones were studied during in vitro cultivation in 24-well plates (Flow Laboratories, UK) for ten passages.

After cloning, culture fluid samples from monoclonal cultures were studied by competitive ELISA. The recombinant ACE2 receptor protein (Vaxine, Australia) was adsorbed in the wells of the plate at a concentration of 0.5 μg/ml in 0.1 M phosphate buffer, pH 7.2 for 12-16 hours at a temperature of +4 C. After a single wash with wash buffer (see Control of the immune response), 50 μl of culture fluid samples and 50 μl of a dilution of RBD protein labeled with horseradish peroxidase (Faizyme, South Africa) were added to the wells and incubated for 30 minutes at 37 C. substrate mixture and photometry at 450 nm. The culture medium was placed in the control well instead of the culture fluid sample. Selected cultures that gave the maximum inhibition of the interaction of ACE2 and RBD-peroxidase (not less than 90%). The secondary screening procedure was repeated for selected clones at all stages of obtaining monoclonal antibodies in order to obtain antibodies, as a result, the monoclonal antibody XR19 was chosen, which gives the maximum specific activity of inhibition.

Next, sequencing of the selected hybridoma clone was performed to determine the nucleotide sequences of the coding. For this, XR19 hybridoma cells were lysed with 1 ml of TRIzol reagent (Thermofisher scientific) followed by total RNA isolation. 5 μg of total RNA was used for cDNA synthesis using the SuperScript IV TM first-strand synthesis system kit (Invitrogen). The heavy and light chain variable domain sequences were amplified by PCR using a panel of primers specifically annealing at the beginning and end of mouse variable domain sequences. The obtained PCR products were sequenced by Sanger on the Genetic Analyzer 3500 Applied Biosystems.

Thus, the sequences of the variable domains of the heavy and light chains of the XR19 antibody were obtained, presented in table 1.

Table. 1. Nucleotide and amino acid sequences of the heavy and light chain variable domains of the XR19 antibody.

Nucleotide sequence of the heavy chain variable domain CAGGTGCAACTGCAACAGAGTGGAGCTGAACTTGTCAGACCTGGCACCTCTGTGAAGGTGAGCTGCAAAGCATCAGGCTATGCCTTCACCAACTACCTGATCGAGTGGGTGAAGCAGAGACCAGGACAAGGTCTCGAGTGGATTGGGATGATCAATCCTGGCAGTGGAGTGACCAACTACGATGAGAAGTTCAAAGGGAAAGCCACACTGACTGCCGACAAGAGCTCTAGCACAGTCTACATGCAGTTGAGCAGTCTGACCTCTGATGACTCAGCAGTGTACTTCTGTGCCACCTACTACAGGTATGACGATGCCTACTGGGGACAGGGCACCCTGGTCACAGTGAGCGCT Amino acid sequence of the heavy chain variable domain QVQLQQSGAELVRPGTSVKVSCKASGYAFTNYLIEWVKQRPGQGLEWIGMINPGSGVTNYDEKFKGKATLTADKSSSTVYMQLSSLTSDDSAVYFCATYYRYDDAYWGQGTLVTVSA Nucleotide sequence of the light chain variable domain GACATTCAGATGACACAGTCTCCTGCCAGTCTGTCAGCCAGCGTTGGAGAGACTGTGACCATCACCTGTGGTGCAAGCGAGAACATCTATGGCGTTCTGAACTGGTACCAGAGGAAGCAAGGCAAGTCTCCACAGTTGCTGATCTATGGAGCTACCAACCTTGCTGATGGCATGAGTAGCAGGTTCTCAGGCAGTGGCTCAGGCAGACAGTACAGTCTGAAGATCAGCTCACTTCATCCTGACGATGTTGCCACCTACTACTGTCAGAACGTGCTCTCCAGT Amino acid sequence of the light chain variable domain DIQMTQSPASLSASVGETVTITCGASENIYGVLNWYQRKQGKSPQLLIYGATNLADGMSS

Thus, in this example, a mouse monoclonal antibody XR19 was obtained, which specifically binds to the RBD S protein of the SARS-CoV-2 virus.

Example 3 Humanization of the mouse monoclonal antibody XR19.

Next, the amino acid sequences of the variable domains of the heavy and light chains of the XR19 antibody were humanized using the bioinformatics resource Abisys database (http://www.abysis.org/abysis/). The resulting amino acid sequences of the humanized XR19 monoclonal antibody (GamXRH19) are shown in Table 2.

Table 2. Humanized nucleotide and amino acid sequences of the heavy and light chain variable domains of the GamXRH19 antibody.

Humanized amino acid sequence of the heavy chain variable domain QVQLQQSGELVKPGASVKVSCKASGYAFTNYLIEWVKQAPGQGLEWIGMINPGSGVTNYDEKFKGRVTLTADKSTSTVYMQLSSLTSDTAVYFCATYYRYDDAYWGQGTLVTVSA Humanized light chain variable domain amino acid sequence DIQMTQSPSSLSASVGDRVTITCGASENIYGVLNWYQRKPGKSPKLLIYGATNLADGMSSRFSGSGSGRQYTLTISSLQPEDFVATYYCQNVLS

Thus, in this example, a mouse monoclonal antibody was humanized and the heavy and light chain amino acid sequences of the humanized XRH19 monoclonal antibody were obtained.

Example 4. Obtaining, nucleic acids, producer cells and humanized monoclonal antibody XRH19.

Next, the amino acid sequence design of the full-length heavy and light chains of the humanized XRH19 antibody was developed. The amino acid sequence of the humanized heavy is shown by SEQ ID NO: 1 and the amino acid sequence of the humanized light is shown by SEQ ID NO: 2.

Based on the amino acid sequences, the nucleotide sequences of the XRH19 antibody were obtained, which were synthesized at ZAO Evrogen. The resulting nucleotide sequences were cloned into a vector for expression in eukaryotic cells. Next, CHO cells were transfected with the obtained expression vectors using the CHO Gro system (Mirus Bio, USA) in accordance with the manufacturer's protocol. Cells were cultured in Erlenmeyer flasks for 10 days. After that, the culture liquid was clarified by centrifugation at 5000g. The antibody was purified by affinity chromatography on an AKTA start system (Cytiva, Sweden) using MAbSelect SuRe 1 ml columns (Cytiva, Sweden) according to the manufacturer's protocol. Additional purification and buffer replacement were carried out on an XK 26/100 column (Cytiva, Sweden) packed with a Superdex 200 pg sorbent (Cytiva, Sweden). The purity of the resulting humanized XRH19 monoclonal antibody preparation was determined by vertical polyacrylamide gel electrophoresis under non-denaturing conditions and denaturing conditions (FIG. 1). A schematic representation of the humanized XRH19 monoclonal antibody is shown in FIG. 2.

Thus, as a result of the work performed, nucleic acids were obtained that have nucleotide sequences encoding the heavy and light chains of the humanized XRH19 monoclonal antibody (SEQ ID NO:3 and SEQ ID NO:4), the cell producing the humanized XRH19 monoclonal antibody, and the humanized XRH19 monoclonal antibody , having the final amino acid sequence of the heavy and light chains of SEQ ID NO: 1 and SEQ ID NO: 2.

Example 5. Study of the specific activity of the humanized monoclonal antibody XRH19 in indirect ELISA.

To determine the specific activity of the XRH19 antibody, an indirect ELISA method. For this purpose, the recombinant RBD S protein of the SARS-CoV-2 virus was immobilized in the well of an immunological plate in 50.0 mm carbonate-bicarbonate buffer overnight. Unbound protein was removed and the well was blocked with 5% milk powder in phosphate buffer for 1 hour at room temperature. The wells were then washed with 0.1% Tween-20 detergent solution in phosphate buffer and a solution of 5% milk powder in phosphate buffer containing various dilutions of the humanized XRH19 monoclonal antibody was added. Each dilution was made in 3 repetitions, incubated for 1 hour at 37°C with gentle stirring. Then, a 3-fold wash was performed with a 0.1% solution of Tween-20 detergent in phosphate buffer. After washing, a peroxidase-labeled conjugate solution was added and incubated for 1 hour at 37°C with gentle stirring. Next, a 5-fold wash was carried out with a 0.1% solution of Tween-20 detergent in phosphate buffer. Next, a TMB substrate solution was added and incubated for 10 min at room temperature without stirring, after which the reaction was stopped by adding a sulfuric acid solution and measurements were taken on a spectrophotometer. The results of the study are presented in Fig. 3. The XRH19 antibody has been shown to have specific activity at a concentration of at least 3.3 ng/mL.

Thus, in this example, the specific activity of the XRH19 antibody in indirect ELISA was studied and its minimum working concentration was determined.

Example 6 Determination of the affinity constant of the humanized monoclonal antibody XRH19.

The interaction constants (KD) of the humanized monoclonal antibody XRH19 were determined by detecting changes in surface plasmon resonance parameters on a Biacore3000 instrument (General Electric, Sweden). To do this, the recombinant RBD of the S protein of the SARS-CoV-2 virus was covalently immobilized on the surface of the dextran matrix of the CM5 chip (General Electric, Sweden), and then various concentrations of the resulting XRH19 antibody were passed over the chip surface. Data processing and calculation of constants were carried out automatically using the Bioevaluation program (General Electric, Sweden). As a result, the equilibrium dissociation constant of XRH19 was 4*10 ^-9 M, which demonstrates the high affinity of the antibody for recombinant RBD.

Thus, in this example, the high affinity of the XRH19 antibody for the recombinant RBD of the S protein of the SARS-CoV-2 virus was demonstrated.

Example 7. Determination of the virus-neutralizing activity of the humanized monoclonal antibody XRH19 against various strains of the SARS-CoV-2 virus.

The purpose of this experiment was to evaluate the ability of the developed humanized XRH19 monoclonal antibody to neutralize the SARS-CoV-2 virus of various strains. At the first stage of the work, XRH19 antibody samples were prepared in DMEM culture medium with 2% inactivated fetal bovine serum. Then, the obtained antibody samples were mixed with 100 pfu of the SARS-CoV-2 virus of various strains, incubated for 1 hour at 37°C, and added to Vero E6 cells. Cells were incubated at 37°C in 5% CO2. After 96 hours, the development of the cytopathic effect of the virus on the cell culture was recorded visually by assessing the violation of the cell monolayer. For the virus-neutralizing antibody titer, their highest dilution was taken, at which the cytopathic effect is suppressed in 2 out of 3 wells. As a result, the following working virus-neutralizing concentrations were determined, presented in table 3.

Table. 3. Virus-neutralizing titers of the humanized monoclonal antibody RH19 against the SARS-CoV-2 virus of various strains.

SARS-CoV-2 virus strain Minimum virus-neutralizing concentration SARS-CoV-2 strains B.1.1.1 (PMVL-1, S: D614G; hCoV-19/Russia/Moscow_PMVL-1/2020) 80 ng/ml B.1.617.2 (T19R G142D E156G F157del R158del L452R T478K D614G P681R D950N) Delta 20 ng/ml

Thus, this example demonstrated a pronounced virus-neutralizing activity of the humanized XRH19 monoclonal antibody against the SARS-CoV-2 virus, including the Delta variant.

Example 8 Preparation of an agent containing a humanized XRH19 monoclonal antibody.

An agent for the treatment and emergency prevention of COVID-19 disease based on the humanized monoclonal antibody GamXRH19 was obtained by selecting a formulating buffer. For comparison, various physiological buffer systems were used, presented in Table 4.

Table 4. Composition of buffer systems for product development.

Name Compound Buffer #1 Tris 20mM, Sodium Chloride 150mM, Polysorbate 80 0.05%, pH 7.5 Buffer #2 Sodium Phosphate 20mM, Sodium Chloride 150mM, Polysorbate 80 0.05%, pH 7.2 Buffer #3 Tris 20mM, sodium citrate 150mM, polysorbate 80 0.05%, pH 7.2 Buffer #4 Tris 20mM, glycine 200mM, polysorbate 80 0.05%, pH 7.0

The humanized monoclonal antibody GamXRH19 was transferred to each buffer system using size exclusion chromatography on a Superdex 200 pg sorbent and compressed to a concentration of 20 mg/ml. Next, the samples were incubated at 37°C for a week and then analyzed for their stability and purity using HPLC. The results of the analysis are shown in Table 5. Examples of HPLC analysis chromatograms are shown in FIG. 4.

Table. 5. Percentages of target and impurity peaks of the humanized monoclonal antibody XRH19 in various buffer solutions.

Name Buffer #1 Buffer #2 Buffer #3 Buffer #4 Percentage of Target Peak XRH19 88.8% 95.95% 88.55% 82.61% Percentage of sum of XRH19 impurity peaks 11.2% 4.05% 11.45% 17.39%

As a result, it was demonstrated that the greatest stability of the XRH19 antibody is observed in buffer No. 2: Sodium Phosphate 20mM, Sodium Chloride 150mM, Polysorbate 80 0.05%, pH 7.2.

Thus, in this example, an agent based on a humanized monoclonal antibody XRH19 was obtained and the optimal buffer was selected.

Example 9. A method for the treatment and emergency prevention of COVID-19 disease using a humanized monoclonal antibody XR19.

The therapeutic and emergency prophylactic efficacy of the humanized monoclonal antibody XRH19 was evaluated in an ACE2 transgenic mouse model of SARS-CoV-2 virus infection. These animals were chosen because they are highly susceptible to SARS-CoV-2 infection. The lethality of animals after infection with the SARS-CoV-2 virus is 100%.

We used the SARS-CoV-2 virus of the hCoV-19/Russia/Moscow_PMVL-1/2020 strain, isolated in 2020 at the N.N. N.F. Gamalei” of the Ministry of Health of Russia, stored in the State Collection of Viruses. The infectious titer of the virus was 10 ⁷ TCID50/ml and 3.5x10 ⁷ PFU/ml. Animals were infected with the SARS-CoV-2 virus intranasally at a dose of 10 ⁵ TCID50 per animal, then an agent containing a humanized XRH19 monoclonal antibody or a placebo was administered, according to Table 6.

Table 6. Design of a pilot study of the effectiveness of the humanized monoclonal antibody XRH19.

A drug Dose of the drug Route of administration Time of administration Infection intranasally at a dose of TCID50 Efficiency mark XRH19 10 mg/kg i/b 1 hour after infection 10 ⁵ 5 pieces. Survival Analysis XRH19 20 mg/kg i/b 1 hour after infection 10 ⁵ 5 pieces. placebo - i/b 1 hour after infection 10 ⁵ 5 pieces.

Within 30 days after infection, the survival rate of animals was assessed.

In FIG. Figure 5 shows the survival data of animals after infection with the SARS-CoV-2 virus of the hCoV-19/Russia/Moscow_PMVL-1/2020 strain. As a result of the study, it was shown that a single systemic administration based on a humanized monoclonal antibody XRH19 at a dose of 10 and 20 mg/kg to animals one hour after infection can protect animals from infection caused by the SARS-CoV-2 virus of the hCoV-19/Russia/Moscow_PMVL strain. -1/2020.

Thus, it can be concluded that the humanized monoclonal antibody XRH19 can be used for therapy and emergency prevention of the disease caused by the SARS-CoV-2 virus.

Example 10. A method for the treatment and emergency prevention of COVID-19 disease caused by the SARS-CoV-2 variant Delta virus using a humanized XR19 monoclonal antibody.

We used the SARS-CoV-2 virus of the Delta B.1.617.2 variant (T19R G142D E156G F157del R158del L452R T478K D614G P681R D950N) isolated in 2021 at the N.N. N.F. Gamalei” of the Ministry of Health of Russia, stored in the State Collection of Viruses. The infectious titer of the virus was 10 ⁷ TCID50/ml and 3.5x10 ⁷ PFU/ml. Animals were infected with the SARS-CoV-2 virus intranasally at a dose of 10 ⁵ TCID50 per animal, then the preparation of monoclonal antibodies or placebo was administered, according to Table 7.

Table 7. Design of an experimental study of the efficacy of the humanized monoclonal antibody XRH19 against the SARS-CoV-2 Delta variant virus.

A drug Dose of the drug Route of administration Time of administration Infection intranasally at a dose of TCID50 Efficiency mark XRH19 10 mg/kg i/b 1 hour after infection 10 ⁵ 5 pieces. Analysis of weight dynamics.
Survival Analysis XRH19 20 mg/kg i/b 1 hour after infection 10 ⁵ 5 pieces. XRH19 10 mg/kg i/b 6 hours after infection 10 ⁵ 5 pieces. XRH19 20 mg/kg i/b 6 hours after infection 10 ⁵ 5 pieces. placebo - i/b 1 hour after infection 10 ⁵ 5 pieces. placebo - i/b 6 hours after infection 10 ⁵ 5 pieces.

Within 30 days after infection, the weight and survival of the animals were assessed.

In FIG. Figure 6 shows the data on the dynamics of the weight of animals after infection with the SARS-CoV-2 Delta variant virus. In FIG. 7 shows the survival data of animals after infection with the SARS-CoV-2 variant Delta virus. As a result of the study, it was shown that a single systemic administration of an agent based on a humanized monoclonal antibody XRH19 at a dose of 10 and 20 mg/kg to animals one hour and 6 hours after infection can protect animals from infection caused by the SARS-CoV-2 Delta virus. at the same time, a drop in the weight of animals by more than 10% was not observed. In the control group of animals after infection received a placebo, a decrease in body weight was observed. By day 10, all animals from the control group died.

Thus, it can be concluded that the resulting humanized XRH19 monoclonal antibody can be used for therapy and emergency prevention of a disease caused by the SARS-CoV-2 virus, including the Delta variant.

It is obvious to the specialist that this antibody can be used for the treatment and emergency prevention of diseases caused by the SARS-CoV-2 virus in various ways. In this case, the antibody can be administered subcutaneously, intramuscularly, intravenously, intraperitoneally and intranasally.

Experimental data show that antibodies can be administered to humans at concentrations of 1 to 100 mg/mL.

Industrial Applicability

All the above examples confirm the effectiveness of the created humanized monoclonal antibody with virus-neutralizing activity, therapeutic and protective properties against the SARS-CoV-2 virus of various variants and their industrial applicability.

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SEQUENCE LIST

<110> N.F. Gamaleya Research Center for Epidemiology and Microbiology, Ministry of Health of Russia

<120> Humanized monoclonal antibody that specifically binds

with RBD S protein of SARS-CoV-2 virus, agent and method for therapy and

emergency prevention of diseases caused by the SARS-CoV-2 virus.

<160> 4

<170> BISSAP1.3.6

<210> 1

<211> 447

<212> PRT

<213> Artificial Sequence

<220>

<223> heavy chain amino acid sequence

humanized monoclonal antibody XRH19

<400> 1

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn Tyr

20 25 30

Leu Ile Glu Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Met Ile Asn Pro Gly Ser Gly Val Thr Asn Tyr Asp Glu Lys Phe

50 55 60

Lys Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Asp Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Thr Tyr Tyr Arg Tyr Asp Asp Ala Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His

210 215 220

Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

260 265 270

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 2

<211> 215

<212> PRT

<213> Artificial Sequence

<220>

<223> light chain amino acid sequence

humanized monoclonal antibody XRH19

<400> 2

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Gly Ala Ser Glu Asn Ile Tyr Gly Val

20 25 30

Leu Asn Trp Tyr Gln Arg Lys Pro Gly Lys Ser Pro Lys Leu Leu Ile

35 40 45

Tyr Gly Ala Thr Asn Leu Ala Asp Gly Met Ser Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Arg Gln Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Val Ala Thr Tyr Tyr Cys Gln Asn Val Leu Ser Ser Pro

85 90 95

Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala

100 105 110

Ala Pro Ser Val Phe Ile Phe Pro Ser Asp Glu Gln Leu Lys Ser

115 120 125

Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu

130 135 140

Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser

145 150 155 160

Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu

165 170 175

Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val

180 185 190

Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys

195 200 205

Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 3

<211> 1341

<212> DNA

<213> Artificial Sequence

<220>

<223> nucleotide amino acid sequence of the heavy chain

humanized monoclonal antibody XRH19

<400> 3

CAGGTGCAAC TGCAACAGAG TGGAGCTGAA CTTGTCAAGC CAGGAGCCAG TGTGAAGGTC 60

AGCTGCAAAG CCTCTGGCTA TGCCTTCACC AACTACCTGA TCGAGTGGGT GAAGCAAGCT 120

CCCGGACAAG GCTTAGAGTG GATTGGCATG ATCAATCCAG GCAGTGGAGT CACCAACTAT 180

GATGAGAAGT TCAAAGGCAG AGTGACCCTG ACAGCTGACA AGAGCACCTC CACAGTGTAC 240

ATGCAGCTGA GTAGCCTCAC CTCAGATGAC ACAGCAGTGT ACTTCTGTGC CACCTACTAC 300

AGGTATGACG ATGCCTACTG GGGACAGGGC ACCCTGGTCA CAGTGAGCGC TGCCAGCACC 360

AAGGGTCCCT CCGTGTTTCC ACTTGCTCCC AGCTCTAAGT CCACCTCTGG AGGCACAGCA 420

GCACTTGGCT GCTTGGTGAA GGACTACTTT CCTGAACCAG TGACTGTCTC CTGGAACAGT 480

GGAGCCTTGA CCAGCGGAGT CCACACCTTC CCTGTGTCC TGCAGAGCTC TGGACTGTAC 540

AGTCTCTCTA GTGTCGTGAC AGTCCCCAGC TCCAGCCTTG GCACACAGAC CTACATCTGC 600

AACGTCAATC ACAAACCCAG CAACACCAAA GTGGACAAGA GAGTGGAACC TAAGTCCTGT 660

GACAAGACCC ACACTGTCC ACCTTGTCCT GCTCCAGAGC TGCTTGGAGG TCCAAGCGTG 720

TTCCTGTTTC CTCCCAAACC TAAGGACACC CTGATGATCT CCAGGACACC AGAAGTGACC 780

TGTGTCGTTG TGGACGTCTC TCACGAGGAT CCTGAGGTGA AGTTCAACTG GTATGTGGAT 840

GGTGTGGAGG TCCACAATGC CAAGACCAAA CCCAGAGAGG AACAGTACAA CAGCACCTAC 900

AGAGTCGTGA GTGTGTTGAC CGTGCTGCAT CAAGACTGGC TGAATGGCAA GGAATATAAG 960

TGCAAAGTGA GCAACAAAGC ACTTCCTGCT CCCATCGAGA AGACCATCTC CAAAGCCAAA 1020

GGACAACCTA GAGAACCTCA AGTCTACACC CTGCCACCCT CCAGAGAAGA GATGACCAAA 1080

AACCAAGTGT CTCTGACCTG TCTGTCAA GGCTTCTATC CCTCTGACAT TGCTGTGGAG 1140

TGGGAGAGCA ATGGACAACC TGAGAACAAC TATAAGACCA CACCACCTTGT CCTGGACAGT 1200

GATGGCTCCT TCTTTCTGTA TAGTAAGCTG ACAGTGGACA AGAGCAGGTG GCAGCAAGGC 1260

AATGTCTTCT CCTGCAGTGT GATGCATGAA GCCTTGCACA ACCACTACAC CCAGAAGAGC 1320

CTGTCTTTGT CTCCTGGCAA G 1341

<210> 4

<211> 645

<212> DNA

<213> Artificial Sequence

<220>

<223> nucleotide amino acid sequence of the light chain

Humanized monoclonal antibody XRH19

<400> 4

GACATTCAGA TGACACAGTC TCCTTCCAGT CTGTCAGCCA GCGTTGGAGA CAGAGTGACC 60

ATCACCTGTG GTGCAAGCGA GAACATCTAT GGCGTTCTGA ACTGGTACCA GAGGAAGCCA 120

GGCAAGTCTC CAAAGTTGCT GATCTATGGA GCTACCAACC TTGCTGATGG CATGAGTAGC 180

AGGTTCTCAG GCAGTGGCTC AGGCAGACAG TACACTCTGA CCATCAGCTC ACTTCAACCT 240

GAAGATTTCG TTGCCACCTA CTACTGTCAG AACGTGCTCT CCAGTCCCAG GACCTTTGGA 300

GGTGGCACCA AGCTCGAGAT CAAGAGGACA GTTGCAGCTC CCAGCGTGTT CATCTTTCCA 360

CCCTCTGATG AACAGCTCAA ATCTGGCACA GCCAGTGTGG TGTGCTTGCT GAATAATTTC 420

TATCCGAGAG AAGCCAAGGT CCAGTGGAAG GTGGACAACG CTCTCCAGAG CGGCAATTCT 480

CAAGAGAGCG TGACTGAGCA AGACTCCAAG GACAGCACCT ACTCTCTGAG CTCCACACTG 540

ACCCTGAGCA AAGCAGACTA CGAGAAGCAC AAGGTGTATG CCTGTGAGGT CACCCATCAG 600

GGCTTGAGCT CTCCTGTGAC CAAGTCCTTC AATAGAGGAG AGTGC 645

<---

Claims (3)

1. A humanized monoclonal antibody that specifically binds to the RBD S protein of the SARS-CoV-2 virus, has virus-neutralizing and protective activity against the SARS-CoV-2 virus, having the amino acid sequence of the heavy chain of SEQ ID NO: 1 and the amino acid sequence of the light chain of SEQ ID NO: 2. 2. An agent for the treatment and emergency prevention of diseases caused by the SARS-CoV-2 virus, containing a humanized monoclonal antibody according to claim 1 in an effective amount, as well as a pharmaceutically acceptable carrier and/or diluent. 3. A method for the treatment and emergency prevention of diseases caused by the SARS-CoV-2 virus, which consists in the systemic administration of an effective amount of the agent according to claim 2.

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