RU2769223C1 - Means and method for therapy and emergency prevention of diseases caused by the sars-cov-2 virus based on a recombinant antibody and a humanized monoclonal antibody - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: inventions group relates to the field of biotechnology, immunology and virology. Means and methods for the treatment and emergency prevention of diseases caused by various strains of the severe acute respiratory syndrome virus SARS-CoV-2 are described. An agent has been created that contains a recombinant antibody having a CDR1 represented by the amino acid sequence of SEQ ID NO:1, a CDR2 represented by the amino acid sequence of SEQ ID NO:2, a CDR3 represented by the amino acid sequence of SEQ ID NO:3, as well as an Fc fragment of human IgG1 and a humanized monoclonal antibody having a light chain CDR1 represented by SEQ ID NO:4, light chain CDR2 represented by SEQ ID NO:5, light chain CDR3 represented by SEQ ID NO:6, heavy chain CDR1 represented by SEQ ID NO:7, heavy chain CDR2 represented by SEQ ID NO:8, heavy chain CDR3 represented by SEQ ID NO:9. Also disclosed is a method for the treatment and emergency prevention of diseases caused by various strains of the severe acute respiratory syndrome virus SARS-CoV-2 by the created agent.

EFFECT: inventions group provides for the creation of an effective agent with virus-neutralizing activity and protective activity against the SARS-CoV-2 virus, including variants that cause concern (Alpha, Beta, Gamma, Delta).

5 cl, 10 dwg, 13 tbl, 12 ex

Description

Technical field

SUBSTANCE: group of inventions relates to the field of biotechnology, immunology and virology and concerns a means and method for therapy and emergency prevention of diseases caused by various strains of SARS-CoV-2 severe acute respiratory syndrome virus.

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) received FDA emergency approval for the treatment of mild to moderate COVID-19 in 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, Østergaard 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 (CN112500480A, 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 (CN111825762A, pub. 12/27/2020), 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 viral infection and preparation of reagents or kits for testing the SARS-COV-2 virus.

The patent (CN112094342A, published 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 diagnosis SARS-CoV-2 infections.

A solution is known (CN112062839A, pub. 12/11/2020), which describes the creation 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.

There is also a solution (CN112062840A, published on 12/11/2020) providing 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 treating and/or diagnosing an infection caused by the SARS-CoV-2 coronavirus.

The patent (CN111303279A, pub. 06/19/2020) 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 above described nucleic acid molecule. 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 variants Alpha, Beta, Gamma, Delta (variants of concern), in addition, single-domain antibodies are rapidly excreted from the body due to low molecular weight. mass and thus require repeated administration.

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 an effective agent with virus-neutralizing activity and protective activity against the SARS-CoV-2 virus, including variants that cause concern (Alpha, Beta, Gamma, Delta).

The specified technical problem is solved by the fact that an agent for the treatment and emergency prevention of diseases caused by the SARS-CoV-2 virus has been obtained, containing a recombinant antibody having a CDR1 represented by the amino acid sequence of SEQ ID NO:1, CDR2 represented by the amino acid sequence of SEQ ID NO:2 , CDR3 represented by the amino acid sequence SEQ ID NO:3, as well as the Fc fragment of human IgG1 (component 1 - GamP2C5) and a humanized monoclonal antibody having a light chain CDR1 represented by the sequence SEQ ID NO:4, a light chain CDR2 represented by the sequence SEQ ID NO:5, Light chain CDR3 represented by SEQ ID NO:6, Heavy chain CDR1 represented by SEQ ID NO:7, Heavy chain CDR2 represented by SEQ ID NO:8, Heavy chain CDR3 represented by SEQ ID NO :9 (component 2 - GamXRH19).

In this case, the recombinant antibody may have the amino acid sequence of SEQ ID NO:10.

Also, a humanized monoclonal antibody may have a heavy chain sequence of SEQ ID NO:11 and a light chain sequence of SEQ ID NO:12.

At the same time, the technical problem is solved by the fact that a method has been created 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 created agent.

Brief description of the drawings

In FIG. 1 shows the electropherogram of the analysis of the recombinant GamP2C5 antibody under denaturing conditions.

1 - molecular weight marker

2 – sample of recombinant GamP2C5 antibody

In FIG. 2 is an electrophoregram of the analysis of the recombinant GamP2C5 antibody under non-denaturing conditions.

1 - molecular weight marker

2 – sample of recombinant GamP2C5 antibody

In FIG. 3 shows a schematic representation of the amino acid sequence of GamP2C5

1 – glycine-serine linker;

2 -sequence of a single-domain antibody;

2 - hinged region;

3 - Fc fragment of human IgG1.

In FIG. 4 shows the electrophoregram of the analysis of the humanized monoclonal antibody GamXRH19.

1 – sample of humanized monoclonal antibody GamXRH19 under denatured conditions;

2 – molecular weight marker;

3 – sample of humanized monoclonal antibody GamXRH19 under undenatured conditions;

In FIG. 5 is a schematic representation of a humanized GamXRH19 monoclonal antibody.

In FIG. 6 shows the results of the analysis of the specific activity of the humanized monoclonal antibody GamXRH19 in indirect ELISA.

Y-axis - optical units, OD450nm

The abscissa axis is the concentration of the GamXRH19 antibody, µg/mL

- уровень сигнала антитела GamXRH19 на бычий сывороточный альбумин (отрицательный контроль);

- signal level of GamXRH19 antibody to bovine serum albumin (negative control);

- уровень сигнала антитела GamXRH19 на RBD;

- signal level of GamXRH19 antibody on RBD;

In FIG. 7 shows the HPLC chromatogram of the GamP2C5 recombinant antibody sample in buffer no. 2.

Y-axis - units of optical density 280nm, mAu

The abscissa axis is the peak retention time, min

In FIG. 8 shows the HPLC chromatogram of the GamXRH19 recombinant antibody sample in buffer #2.

Y-axis - units of optical density 280nm, mAu

The abscissa axis is the peak retention time, min

In FIG. Figure 9 shows survival results of animals treated with recombinant GamP2C5 antibody (component 1) and humanized XRH19 monoclonal antibody (component 2) and animals in the placebo group for 15 days after infection with SARS-CoV-2 variant Delta.

Y-axis - survival, %

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

- мыши, смесь компонента 1 и 2 (10 мг/мл) спустя 1 час после заражения вирусом;

- mice, mixture of components 1 and 2 (10 mg/ml) 1 hour after infection with the virus;

- мыши, смесь компонента 1 и 2 (10 мг/мл) спустя 6 часов после заражения вирусом;

- mice, a mixture of components 1 and 2 (10 mg/ml) 6 hours after infection with the virus;

- мыши, смесь компонента 1 и 2 (20 мг/мл) спустя 1 час после заражения вирусом;

- mice, a mixture of components 1 and 2 (20 mg/ml) 1 hour after infection with the virus;

- мыши, смесь компонента 1 и 2 (20 мг/мл) спустя 6 часов после заражения вирусом;

- mice, a mixture of components 1 and 2 (20 mg/ml) 6 hours after infection with the virus;

- мыши, получившие фосфатно-солевой буфер (PBS);

- mice treated with phosphate-buffered saline (PBS);

In FIG. Figure 10 shows the survival results of animals injected with the recombinant GamP2C5 antibody (component 1) and the humanized monoclonal antibody XRH19 (component 2) and animals from the placebo group for 15 days after infection with the SARS-CoV-2 virus of the hCoV-19/Russia/Moscow_PMVL- strain. 1/2020.

Y-axis - survival, %

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

- мыши, получившие смесь компонента 1 и 2 (10 мг/мл) спустя 1 час после заражения вирусом;

- mice receiving a mixture of components 1 and 2 (10 mg/ml) 1 hour after infection with the virus;

- мыши, получившие смесь компонента 1 и 2 (20 мг/мл) спустя 1 час после заражения вирусом;

- mice receiving a mixture of components 1 and 2 (20 mg/ml) 1 hour after infection with the virus;

- мыши, получившие фосфатно-солевой буфер (PBS);

- mice treated with phosphate-buffered saline (PBS);

Implementation of the invention.

The technical task of the claimed group of inventions is to create a drug for the treatment and emergency prevention of diseases caused by the SARS-CoV-2 virus based on monoclonal and single-domain antibodies and their modifications. Monoclonal and single-domain antibodies with high specificity for the RBDS 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. At the same time, monoclonal and single-domain antibodies as agents for therapy COVID 19 has both advantages and disadvantages. Single-domain antibodies are able to interact with hard-to-reach antigen epitopes due to long antigen-binding regions (CDRs), however, they are rapidly eliminated from the body due to their low molecular weight, and are also devoid of Fc-dependent functions. However, these shortcomings can be effectively eliminated by attaching a human IgG Fc fragment to single-domain antibodies and thus obtaining a recombinant antibody.

For this, a tool based on a recombinant antibody (GamP2C5) and a humanized monoclonal antibody (GamXRH19) that specifically binds to the RBD domain S of the SARS-CoV-2 virus protein has been developed, which has virus-neutralizing and protective activity against various strains of the SARS-CoV-2 virus.

To obtain the recombinant GamP2C5 antibody, the alpaca was immunized with the 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 RBD-specific antibodies in the animal's blood serum. Further, peripheral blood mononuclear cells were isolated from animals, from which RNA was obtained. Based on RNA, cDNA was synthesized. On the matrix obtained cDNA, nested PCR was set, which allows amplifying the sequences of single-domain antibodies. Thus, a library of amplicons of single-domain antibody sequences was obtained.

It is known to those skilled in the art that not only mononuclear cells, but also whole blood, lymph nodes, spleen, thymus, or other cells and organs of an immunized animal expressing single domain antibodies can be used for the purposes of the present invention. It is also known to those skilled in the art that synthetic libraries of single domain antibody sequences can be used for the purposes of the present invention.

Antibody selection was performed by phage display using antigen panning (RBD). Those skilled in the art will recognize that other selection approaches such as panning on viral particles, panning in a biotinylated antigen solution, panning using competitive binding and elution are possible for the purposes of the present invention. It is also known to those skilled in the art that other selection methods, such as ribosome display, yeast display, may be used to carry out the present invention. As a result of selection, a single-domain antibody was selected that specifically binds to the RBD domain of the S protein of the SARS-CoV-2 virus. In subsequent experiments, it was shown that this antibody has virus-neutralizing activity.

It is known to those skilled in the art that, for the purposes of the present invention, the sequences of SARS-CoV-2 RBD-specific single-domain antibodies may contain amino acid substitutions that do not affect the secondary and tertiary structure of single-domain antibodies and do not affect their ability to bind and neutralize the RBD of the SARS-CoV-2 virus. Moreover, since only the antigen-binding loops (CDR domains) of single-domain antibodies 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 the 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 at least 70% with the proposed sequences, can be used to implement the claimed invention.

Recombinant GamP2C5 antibody, which is a single-domain antibody modified with the Fc fragment of human immunoglobulin G1, specifically binding to the RBD domain S of the SARS-CoV-2 virus protein, having virus-neutralizing and protective activity, having the amino acid sequence SEQIDNO:10. This antibody was obtained by expressing the nucleotide sequence of the recombinant antibody in CHO-producing cells.

The nucleotide sequences of the recombinant GamP2C5 antibody were genetically engineered based on the nucleotide sequence of the single domain antibody and the nucleotide sequence of the human immunoglobulin G1 Fc fragment. It is known to those skilled in the art that for purposes of the present invention, the nucleotide sequence encoding a recombinant antibody may differ based on the principle of degeneracy of the genetic code. Thus, all nucleotide sequences encoding the amino acid sequence of the recombinant antibody (SEQIDNO:10) can be used to implement the claimed invention.

Using genetic engineering methods, a producer cell expressing the recombinant GamP2C5 antibody (SEQIDNO:10) was obtained. Thus, a producer cell based on the CHO cell line expressing the recombinant GamP2C5 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, a technological scheme was developed for cultivating the cell-producer of the recombinant GamP2C5 antibody in 200-liter wave bioreactors. It is known to those skilled in the art that any other type and size of bioreactors can be used to culture the producer cell for purposes of the present invention. Thus, all technological schemes for cultivating the cell-producer of the recombinant GamP2C5 antibody can be used to implement the claimed invention.

Also, a technological scheme has been developed for the chromatographic purification of the recombinant GamP2C5 antibody using protein A affinity chromatography, anion exchange chromatography, and multimodal chromatography. It is known to those skilled in the art that in order to carry out the present invention, various technological schemes using various sorbents can be used to purify antibodies. Thus, all technological schemes for the purification of the recombinant GamP2C5 antibody can be used to implement the claimed invention.

To obtain a humanized monoclonal antibody GamXRH19, 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 (SEQIDNO:11 heavy chain and SEQIDNO:12 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 sites (CDR domains) are involved in antigen binding, as is known to those skilled in the art, any immunoglobulin or its analogue containing the same amino acid sequences of CDR domains can be used to implement the claimed 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 whose homology is at least 70% with the proposed sequences can be used to implement the claimed invention.

The humanized monoclonal antibody GamXRH19 (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 nucleotide sequences of the heavy and light chains of the humanized monoclonal antibody GamXRH19 were genetically 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 GamXRH19 (SEQIDNO: 11 heavy chain and SEQIDNO: 12 light chain) can be used to practice the present invention.

Using genetic engineering methods, a producer cell expressing the humanized monoclonal antibody GamXRH19 (SEQIDNO:11 heavy chain and SEQIDNO:12 light chain) was obtained. Thus, a producer cell based on the CHO cell line expressing the humanized monoclonal antibody GamXRH19 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.

The pharmacokinetics of single-domain antibodies and their modifications has been studied. It has been demonstrated that the modification of single-domain antibodies and their oligomers with the Fc fragment of human IgG1 makes it possible to increase the time of their circulation in the body from several hours to several weeks.

Thus, the authors of the claimed invention obtained antibody variants that specifically bind to the RBDS protein of the SARS-CoV-2 virus, have virus-neutralizing activity and have improved pharmacokinetics compared to single-domain antibodies.

In addition, an optimal formulation of the GamP2C5 antibody (component 1) and GamXRH19 antibody (component 2) agent was developed to provide the greatest stability.

Thus, the authors of the patent obtained a tool based on antibodies that specifically bind to the RBDS protein of the SARS-CoV-2 virus, intended for the treatment and emergency prevention of COVID-19.

In addition, the authors of the claimed invention have developed a method for the treatment and emergency prevention of diseases caused by the SARS-CoV-2 virus, which consists in introducing into the body of mammals in an effective amount of any of the developed antibody variants.

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 RBDS of the SARS-CoV-2 virus protein was obtained.

Example 2 Preparation of recombinant GamP2C5 antibody

Alpaca (Lama pacos - a member of the Cameliedae family) were immunized with the received RBD (100 μg per immunization) 5 times at intervals of 10-14 days. 7 days after the last immunization, 50.0 ml of peripheral blood was taken from alpacas and mononuclear cells were isolated on a ficoll gradient of 1.077 (Paneko, Russia). Isolated mononuclear cells were used for total RNA isolation with Trizol reagent (Invitrogene, USA) according to the manufacturer's protocol. The isolated RNA was used as a template for cDNA synthesis with random primers and SuperScriptIII reversease (Invitrogene, USA). Nested PCR was used on the matrix obtained from cDNA, allowing amplification of the sequences of single-domain antibodies. For this procedure, high fidelity polymerase Q5 (NEB, UK) and specific primers containing restriction sites at the ends were used. Further, the amplicons were digested with restriction enzymes and cloned into a phagemid vector digested at the same sites. Then, E. coli cells (TG1 strain) were transformed with phagemid vectors obtained by cloning.

A suspension of bacterial clone-transformants of E. coli cells (TG1 strain) was used for the production of recombinant bacteriophages using the helper bacteriophage M13KO7 (NEB, UK) according to the manufacturer's protocol. Recombinant bacteriophages were precipitated by polyethylene glycol (PEG/NaCl) precipitation. Thus, a library of recombinant bacteriophages with a titer of 10 ¹² transducing units/ml of suspension was obtained.

The selection of specific recombinant bacteriophages was carried out by panning on the antigen of the original bacteriophage library. For this, recombinant RBD (example 1) 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. Then a suspension of 10 ¹¹ recombinant bacteriophages was added and incubated for 1 hour at room temperature. Unbound bacteriophages were washed with a colloidal 0.1% solution of Tween-20 detergent in phosphate buffer. The bound bacteriophages were eluted with a trypsin solution (0.1 mg/ml). The eluted bacteriophages were used to transduce E. coli TG1 cells, which were then plated on agar plates at a dilution to allow isolation of individual colonies. The resulting colonies were used to build up the bacterial mass and isolate plasmid DNA. Plasmids were sequenced to determine the nucleotide sequences encoding single domain antibodies. 8 individual sequences were determined.

For the production of single-domain antibodies to rapidly dividing E. coli TG1 cells obtained from the colonies at the previous stage, 1.0 μM isopropyl thiogalactopyranoside (IPTG) (Helikon, Russia) was added, after 4 hours the bacterial mass was used to isolate recombinant proteins on a HisTrap FF column 5 ml (GE Healthcare Life Sciences, USA) using the AKTA start chromatographic system (GE Healthcare Life Sciences, USA), according to the manufacturer's protocol.

Purified single-domain antibodies were further studied in the virus-neutralization reaction. The SARS-CoV-2 virus neutralization test was performed in the variant of a constant dose of the virus - dilution of a sample of single-domain antibodies. Samples of single-domain antibodies were prepared in DMEM culture medium with 2% inactivated fetal bovine serum, then mixed with 100 PFU of SARS-CoV-2 virus, incubated for 1 hour at 37°C, and added to Vero E6 cells. Initial concentrations of single-domain antibody preparations were 0.5-1 mg/ml. Final dilutions of single domain antibodies were 1/20-1/1280. 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 titer of single-domain antibodies, their highest dilution was taken, at which the cytopathic effect is suppressed in 2 out of 3 wells.

Data on the virus-neutralizing activity of the selected antibodies are presented in Table 1.

Table 1. Virus-neutralizing activity of the obtained single-domain antibodies.

Name of the antibody sample Virus neutralizing activity, ng/ml p2g1 single domain antibody 332 p2c5 single domain antibody 664 p5f8 single domain antibody 295 p2h1 single domain antibody 8300 p2h2 single domain antibody 12500 p3d3 single domain antibody 4300 Single domain antibody p2d1 9310 Single domain antibody p2d5 1900

As can be seen from the presented data, all 8 samples had a pronounced virus-neutralizing activity (working concentration from 1 to 12.5 µg/ml), of which 3 samples had the most pronounced virus-neutralizing activity (working concentration from less than 1 µg/ml). Three samples of single-domain antibodies with the most pronounced virus-neutralizing activity were selected for further studies.

Interaction constants (KD) of single-domain antibodies p2c5, p5f8 and p2g1 were determined by detecting changes in surface plasmon resonance parameters on a Biacore3000 instrument (General Electric, Sweden). To do this, recombinant RBD was covalently immobilized on the surface of the dextran matrix of the CM5 chip (General Electric, Sweden), and then various concentrations of the obtained single-domain antibodies were passed over the chip surface. Data processing and calculation of constants were carried out automatically using the Bioevaluation program (General Electric, Sweden). The data obtained are presented in table 2

Table. 2. Equilibrium dissociation constant of the obtained single-domain antibodies with recombinant RBD.

Name of the antibody sample Equilibrium dissociation constant p2g1 single domain antibody 8.2*10e ^-9 M p2c5 single domain antibody 2.8*10e ^-9 M p5f8 single domain antibody 3.5*10e ^-9 M

Further, based on the virus-neutralization data and equilibrium dissociation constants, the p2c5 clone was taken for further work to obtain a recombinant GamP2C5 antibody. The recombinant antibody is a single-domain antibody modified with a human immunoglobulin Fc fragment. This modification of single-domain antibodies will improve their pharmacokinetic properties by adding the human IgG1 Fc fragment and its correct glycosylation in eukaryotic producer cells. In addition, modification with an Fc fragment will allow antibodies to activate the complement system and bind to Fc receptors on the surface of immunocompetent cells.

At the first stage of the work, we developed the design of the amino acid sequence of a recombinant antibody (SEQIDNO:10), which is a single-domain antibody modified with the Fc fragment of human immunoglobulin G1. Based on the amino acid sequence, the nucleotide sequences of recombinant antibodies 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 CHOGro system (MirusBio, 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 preparation of the recombinant GamP2C5 antibody was determined by vertical polyacrylamide gel electrophoresis under non-denaturing conditions and denaturing conditions (Fig. 2 and Fig. 1). A schematic representation of the recombinant GamP2C5 antibody is shown in FIG. 3.

Further, using the analysis of literature data and methods of bioinformatics, the sequences of CDR1-CDR3 were identified in the sequence of the recombinant GamP2C5 antibody.

Thus, as a result of the work carried out, a recombinant GamP2C5 antibody with the amino acid sequence SEQIDNO:10 was obtained, and the sequences CDR1, CDR2, CDR3 with the amino acid sequences SEQIDNO:1, SEQIDNO:2 and SEQIDNO:3, respectively, were identified.

Example 3. Obtaining a humanized monoclonal antibody GamXRH19.

BALB/c mice (10 females, 5–6 weeks of age, 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 scheme 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 (washing buffer) and antibodies against mouse immunoglobulins labeled with horseradish peroxidase (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 the ELISA method, 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 into the wells of the tablet 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 dilution of RBD protein labeled with horseradish peroxidase (Faizyme, South Africa) were added to the wells and incubated for 30 minutes at 37 C. After washing 5 times with wash buffer, the reaction was manifested using chromogen 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 SuperScriptIVTMfirst-strandsynthesissystem 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 3.

Table. 3. 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

Further, the amino acid sequences of the variable domains of the heavy and light chains of the XR19 antibody were humanized using the bioinformatic resource Abisysdatabase (http://www.abysis.org/abysis/). The resulting amino acid sequences of the humanized XR19 monoclonal antibody (GamXRH19) are shown in Table 4.

Table 4. 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

Further, using literature data analysis and bioinformatics methods in the light and heavy chain sequences of the GamXRH19 antibody, the sequences of CDR1-CDR3 were identified.

Next, the amino acid sequence design of the full length heavy and light chains of the humanized GamXRH19 antibody was developed. The humanized heavy amino acid sequence is shown in SEQIDNO:11 and the humanized light amino acid sequence is shown in SEQIDNO:12. Based on the amino acid sequences, the nucleotide sequences of the GamXRH19 antibody were obtained, which were synthesized by 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 CHOGro system (MirusBio, 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. 4). A schematic representation of the humanized GamXRH19 monoclonal antibody is shown in FIG. 5.

Thus, as a result of the work performed, a humanized monoclonal antibody GamXRH19 was obtained, having the amino acid sequence of heavy and light chains SEQIDNO:11 and SEQIDNO:12, respectively. In addition, the light chain CDR1, CDR2, CDR3 sequences of the GamXRH19 antibody having the amino acid sequences of SEQIDNO:4, SEQIDNO:5 and SEQIDNO:6, respectively, and the heavy chain CDR1, CDR2, CDR3 sequences of the GamXRH19 antibody having the amino acid sequences of SEQIDNO:7, SEQIDNO:8 and SEQIDNO:9 respectively.

Example 4. Study of the specific activity of the humanized monoclonal antibody GamXRH19 in indirect ELISA.

To determine the specific activity of the GamXRH19 antibody, an indirect ELISA method. For this, the recombinant RBDS of the SARS-CoV-2 virus protein 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 GamXRH19 monoclonal antibody was added. Each dilution was made in 3 repetitions, incubated for 1 hour at 37 ⁰ C with weak stirring. Then, a 3-fold washing with a 0.1% solution of Tween-20 detergent in phosphate buffer was carried out. 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 shown in Fig. 6. The GamXRH19 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 GamXRH19 antibody in indirect ELISA was studied and its minimum working concentration was determined.

Example 5 Determination of the affinity constant of the humanized monoclonal antibody GamXRH19.

Interaction constants (KD) of the humanized monoclonal antibody GamXRH19 were determined by detecting changes in surface plasmon resonance parameters on a Biacore3000 instrument (General Electric, Sweden). For this, the recombinant RBDS 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 GamXRH19 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 GamXRH19 antibody to the recombinant RBDS protein of the SARS-CoV-2 virus was demonstrated.

Example 6. Determination of the virus-neutralizing activity of the humanized monoclonal antibody GamXRH19 against various strains of the SARS-CoV-2 virus.

The purpose of this experiment was to evaluate the ability of the developed humanized monoclonal antibody GamXRH19 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. The virus-neutralizing antibody titer was taken as the highest dilution 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 5.

Table. 5. Virus-neutralizing titers of the humanized monoclonal antibody GamXRH19 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.1.1.7 (hCoV-19/Netherlands/NoordHolland_20432/2020, VOC 202012/01) Alpha, V1 does not neutralize in vitro B.1.351 (hCoV-19/Russia/SPE-RII-27029S/2021) Beta, V2 does not neutralize in vitro B.1.1.28/P.1 (hCoV-19/Netherlands/NoordHolland_10915/2021)Gamma, V3 does not neutralize in vitro 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 monoclonal antibody GamXRH19 against the SARS-CoV-2 virus, including the Delta variant of concern.

Example 7. Determination of the virus-neutralizing activity of the recombinant GamP2C5 antibody against various strains of the SARS-CoV-2 virus.

The purpose of this experiment was to evaluate the ability of the developed recombinant GamP2C5 antibody to neutralize the SARS-CoV-2 virus of various strains. At the first stage of the work, samples of the recombinant GamP2C5 antibody were prepared in the 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. The virus-neutralizing antibody titer was taken as the highest dilution 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 6.

Table. 6. Virus-neutralizing titers of the recombinant GamP2C5 antibody 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) 10 ng/ml B.1.1.7 (hCoV-19/Netherlands/NoordHolland_20432/2020, VOC 202012/01) Alpha, V1 10 ng/ml B.1.351 (hCoV-19/Russia/SPE-RII-27029S/2021) Beta, V2 20 ng/ml B.1.1.28/P.1 (hCoV-19/Netherlands/NoordHolland_10915/2021)Gamma, V3 40 ng/ml B.1.617.2 (T19R G142D E156G F157del R158del L452R T478K D614G P681R D950N) Delta does not neutralize in vitro

Thus, this example demonstrates a pronounced virus-neutralizing activity of the recombinant GamP2C5 antibody against the SARS-CoV-2 virus, including the feared Alpha, Beta, and Gamma variants.

Example 8 Preparation of the formulation of component 1 (recombinant GamP2C5 antibody).

A drug for the treatment and emergency prevention of COVID-19 disease based on the recombinant GamP2C5 antibody (component 1) was obtained by selecting a formulating buffer. For comparison, various physiological buffer systems were used, presented in Table 7.

Table 7. 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 recombinant GamP2C5 antibody 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 8. Examples of HPLC analysis chromatograms are shown in FIG. 7.

Table. 8. Percentage of target and impurity peaks of the recombinant GamP2C5 antibody (component 1) in various buffer solutions.

Name Buffer #1 Buffer #2 Buffer #3 Buffer #4 Percentage of GamP2C5 target peak (component 1) 93.29% 98.43% 92.37% 92.31% Percentage of sum of GamP2C5 impurity peaks (component 1) 6.71% 1.57% 7.63% 7.69%

As a result, it was demonstrated that the greatest stability of the 1st component is observed in buffer No. 2 Sodium phosphate 20 mM, Sodium chloride 150 mM, Polysorbate 80 0.05%, pH 7.2.

Thus, in this example, an agent based on the recombinant GamP2C5 antibody (component 1) was obtained and the optimal buffer composition was selected.

Example 9 Formulation of component 2 (humanized monoclonal antibody GamXRH19).

A drug for the treatment and emergency prevention of COVID-19 disease based on the humanized monoclonal antibody GamXRH19 (component 2) was obtained by selecting a formulating buffer. For comparison, various physiological buffer systems were used, presented in Table 9.

Table 9. 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 ⁰ C for a week and then analyzed for their stability and purity using HPLC. The results of the analysis are presented in table 10. Examples of HPLC analysis chromatograms are shown in Fig.8.

Table. 10. Percentage of target and impurity peaks of the humanized monoclonal antibody GamXRH19 (component 2) in various buffer solutions.

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

As a result, it was demonstrated that the greatest stability of component 2 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 the humanized monoclonal antibody GamXRH19 (component 2) was obtained and the optimal buffer composition was selected.

Example 10. Determination of the virus-neutralizing activity of an agent based on the recombinant GamP2C5 antibody (component 1) and the humanized monoclonal antibody GamXRH19 (component 2).

The purpose of this experiment was to evaluate the ability of each of the components of the agent and their mixture in the optimal buffer solution to neutralize the SARS-CoV-2 virus of various strains. The methodology for this analysis is described in examples 6 and 7. As a result, the following working virus-neutralizing concentrations of the drug components were determined, presented in table 11.

Table. 11. Virus-neutralizing titers of the recombinant GamP2C5 antibody against the SARS-CoV-2 virus of various strains.

SARS-CoV-2 virus strain Minimum virus-neutralizing concentration of component 1 Minimum virus-neutralizing concentration of component 2 Minimum virus-neutralizing concentration of the mixture of components wuhan 10 ng/ml 40 ng/ml 10 ng/ml Alpha, V1 10 ng/ml does not neutralize in vitro 150 ng/ml Beta, V2 20 ng/ml does not neutralize in vitro 150 ng/ml Gamma V3 40 ng/ml does not neutralize in vitro 40 ng/ml Delta does not neutralize 10 ng/ml 40 ng/ml

Thus, this example demonstrated a pronounced virus-neutralizing activity of an agent based on the recombinant GamP2C5 antibody (component 1) and the humanized monoclonal antibody GamXRH19 (component 2) against various SARS-CoV-2 strains, including variants, AlphaV1, BetaV2, GammaV3, Delta. In addition, component 1 was shown to have virus-neutralizing activity against the AlphaV1, BetaV2 and GammaV3 variants of concern, and component 2 against the Delta variants of concern.

Example 11. A method for the treatment and emergency prevention of COVID-19 disease using an agent based on the recombinant GamP2C5 antibody (component 1) and the humanized monoclonal antibody GamXRH19 (component 2).

Therapeutic and emergency prophylaxis efficacy of the recombinant GamP2C5 antibody (component 1) and the humanized monoclonal GamXRH19 antibody (component 2), as well as their mixtures, was evaluated in an ACE2 transgenic mouse model of infection caused by the SARS-CoV-2 virus. 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 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 a monoclonal antibody preparation or placebo was administered, according to Table 12.

Table 12. Design of an experimental study of the effectiveness of the recombinant GamP2C5 antibody (component 1) and the humanized monoclonal antibody GamXRH19 (component 2), as well as their mixtures.

A drug Dose of the drug Route of administration Time of administration Infection intranasally at a dose of TCID50 Efficiency mark Mix of components 1 and 2 10 mg/kg i/b 1 hour after infection 10 ⁵ 10 pieces. Survival analysis Mix of components 1 and 2 10 mg/kg i/b 6 hours after infection 10 ⁵ 10 pieces. Mix of components 1 and 2 20 mg/kg i/b 1 hour after infection 10 ⁵ 10 pieces. Mix of components 1 and 2 20 mg/kg i/b 6 hours after infection 10 ⁵ 10 pieces. placebo - i/b 1 hour after infection 10 ⁵ 10 pieces.

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

In FIG. 9 shows the survival data of animals after infection with SARS-CoV-2 variant Delta. As a result of the study, it was shown that a single systemic administration of a mixture of two components of the drug at doses 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 variant virus. 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 developed agent based on the recombinant GamP2C5 antibody (component 1) and the humanized monoclonal antibody GamXRH19 (component 2) can be used for the treatment and emergency prevention of the disease caused by the SARS-CoV-2 virus, including the Delta variant. .

Example 12. A method for the treatment and emergency prevention of COVID-19 disease caused by various strains of the SARS-CoV-2 virus using an agent based on the recombinant GamP2C5 antibody (component 1) and the humanized monoclonal antibody GamXRH19 (component 2).

To assess the breadth of therapeutic properties of the agent based on the recombinant GamP2C5 antibody (component 1) and the humanized monoclonal antibody GamXRH19 (component 2), we used the SARS-CoV-2 virus of the hCoV-19/Russia/Moscow_PMVL-1/2020 strain, isolated in 2020 at the Federal State Budgetary Institution "NITsEM them. 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 a monoclonal antibody preparation or placebo was administered, according to Table 13.

Table 13. Design of an experimental study of the breadth of the protective activity of the agent based on the recombinant GamP2C5 antibody (component 1) and the humanized monoclonal antibody GamXRH19 (component 2), as well as their mixtures.

A drug Dose of the drug Route of administration Time of administration Infection intranasally at a dose of TCID50 Efficiency mark Mix of components 1 and 2 10 mg/kg i/b 1 hour after infection 10 ⁵ 5 pieces. Survival analysis Mix of components 1 and 2 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. 10 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 of a mixture of two components of the drug at doses of 10 and 20 mg/kg to animals one hour after infection allows protecting animals from infection caused by the SARS-CoV-2 virus of the hCoV-19/Russia/Moscow_PMVL-1 strain. /2020.

Thus, it can be concluded that the developed agent based on the recombinant GamP2C5 antibody (component 1) and the humanized monoclonal antibody GamXRH19 (component 2) can be used for the treatment and emergency prevention of the disease caused by the SARS-CoV-2 virus of various variants and strains.

Industrial Applicability

All the above examples confirm the effectiveness of the created drug based on the recombinant GamP2C5 antibody (component 1) and the humanized monoclonal antibody GamXRH19 (component 2) for the treatment and emergency prevention of the disease caused by the SARS-CoV-2 virus, including the feared Alpha, Beta variants. , Gamma and Delta, and its industrial applicability.

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SEQUENCE LIST

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

<120> An agent and method for the treatment and emergency prevention of diseases caused by the SARS-CoV-2 virus based on a recombinant antibody and a humanized monoclonal antibody.

SEQID NO:1.

<160> 4

<170> BISSAP1.3.6

<210> 1

<211> 8

<212>PRT

<213>ArtificialSequence

<220>

<223>CDR1 amino acid sequence of recombinant GamP2C5 antibody

<400> 1

Gly Tyr Thr Tyr Cys Ser Tyr Asp

fifteen

SEQ ID NO:2.

<210> 2

<211> 8

<212>PRT

<213>ArtificialSequence

<220>

<223>CDR2 amino acid sequence of recombinant GamP2C5 antibody

<400>2

Ile Ile Arg Arg Asp Gly Ser Thr

fifteen

SEQIDNO:3.

<210> 3

<211> 15

<212>PRT

<213>ArtificialSequence

<220>

<223>CDR3 amino acid sequence of recombinant GamP2C5 antibody

<400>3

Lys Ser Trp Ala Cys Ser Ser Gly Glu Tyr Leu Tyr Gln Gly Asp

1 5 10 15

SEQIDNO:4.

<210> 4

<211> 6

<212>PRT

<213>ArtificialSequence

<220>

<223>amino acid sequence of the light chain CDR1 of the humanized monoclonal antibody GamXRH19

<400>4

Glu Asn Ile Tyr Gly Val

fifteen

SEQIDNO:5.

<210> 5

<211> 3

<212>PRT

<213>ArtificialSequence

<220>

<223>amino acid sequence of the light chain CDR2 of the humanized monoclonal antibody GamXRH19

<400>5

Gly Ala Thr

one

SEQIDNO:6.

<210> 6

<211> 9

<212>PRT

<213>ArtificialSequence

<220>

<223>amino acid sequence of the light chain CDR3 of the humanized monoclonal antibody GamXRH19

<400>6

Gln Asn Val Leu Ser Ser Pro Arg Thr

fifteen

SEQIDNO:7.

<210> 7

<211> 8

<212>PRT

<213>ArtificialSequence

<220>

<223>amino acid sequence of the heavy chain CDR1 of the humanized monoclonal antibody GamXRH19

<400>7

Gly Tyr Ala PheThrAsn Tyr Leu

fifteen

SEQIDNO:8.

<210>8

<211> 8

<212>PRT

<213>ArtificialSequence

<220>

<223>amino acid sequence of the heavy chain CDR2 of the humanized monoclonal antibody GamXRH19

<400>8

Gly Tyr Ala PheThrAsn Tyr Leu

fifteen

SEQIDNO:9.

<210>9

<211> 10

<212>PRT

<213>ArtificialSequence

<220>

<223>amino acid sequence of the heavy chain CDR3 of the humanized monoclonal antibody GamXRH19

<400>9

Ala Thr Tyr Tyr Arg Tyr Asp Asp Ala Tyr

1 5 10

SEQIDNO:10.

<210>10

<211>352

<212>PRT

<213>ArtificialSequence

<220>

<223>amino acid sequence of the recombinant GamP2C5 antibody

<400> 10

Glu Val Gln Leu Val Glu Ser GlyGlyGly Ser Val Gln Ala GlyGly

1 5 10 15

Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Tyr Thr Tyr Cys Ser Tyr

20 25 30

Asp Met Ser Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

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

50 55 60

Gly Arg Phe Ala Ile Ser Arg Asp Asn Ala Lys AsnThr Leu Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Glu Pro Glu Asp Thr Ala Met Tyr TyrCys Lys

85 90 95

Ser Trp Ala Cys Ser SerGly Glu Tyr Leu Tyr Gln Gly Asp TrpGly

100 105 110

Gln GlyThr Gln Val Thr Val Ser Ser Glu Pro Lys Ser Cys Asp Lys

115 120 125

Thr His ThrCys Pro ProCys Pro Ala Pro Glu Leu LeuGlyGly Pro

130 135 140

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

145 150 155 160

Arg Thr Pro Glu Val ThrCys Val Val Val Asp Val Ser His Glu Asp

165 170 175

Pro Glu Val Lys PheAsnTrp Tyr Val Asp Gly Val Glu Val His Asn

180 185 190

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

195 200 205

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu AsnGly Lys Glu

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

Cys Leu Val Lys GlyPhe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

275 280 285

Ser AsnGly Gln Pro Glu AsnAsn Tyr Lys ThrThr Pro Pro Val Leu

290 295 300

Asp Ser Asp Gly Ser PhePhe Leu Tyr Ser Lys Leu Thr Val Asp Lys

305 310 315 320

Ser Arg Trp Gln GlnGlyAsn Val Phe Ser Cys Ser Val Met His Glu

325 330 335

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

340 345 350

SEQIDNO:11.

<210>11

<211>447

<212>PRT

<213>ArtificialSequence

<220>

<223>GamXRH19 humanized monoclonal antibody heavy chain amino acid sequence

<400> 11

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 PheThrAsn 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 ThrAsn 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 AspThr Ala Val Tyr PheCys

85 90 95

Ala Thr Tyr Tyr Arg Tyr Asp Asp Ala Tyr TrpGly Gln GlyThr 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 GlyGlyThr Ala Ala Leu GlyCys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser TrpAsn Ser

145 150 155 160

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

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val ValThr Val Pro Ser Ser Ser Ser

180 185 190

Leu GlyThr Gln Thr Tyr Ile CysAsn 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

ThrCys Pro ProCys Pro Ala Pro Glu Leu LeuGlyGly 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 ThrCys Val ValVal Asp Val Ser His Glu Asp Pro Glu

260 265 270

Val Lys PheAsnTrp 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 AsnGly 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 ThrCys Leu

355 360 365

Val Lys GlyPhe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu AsnAsn Tyr Lys ThrThr Pro Pro Val Leu Asp Ser

385 390 395 400

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

405 410 415

Trp Gln GlnGlyAsn 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

SEQIDNO:12.

<210>12

<211>215

<212>PRT

<213>ArtificialSequence

<220>

<223>light chain amino acid sequence of humanized monoclonal antibody GamXRH19

<400> 12

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 ThrCysGly Ala Ser Glu Asn Ile Tyr Gly Val

20 25 30

Leu AsnTrp Tyr Gln Arg Lys Pro Gly Lys Ser Pro Lys Leu Leu Ile

35 40 45

Tyr Gly Ala ThrAsn 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 TyrCys Gln Asn Val Leu Ser Ser Pro

85 90 95

Arg ThrPheGlyGlyGlyThr 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

GlyThr Ala Ser Val ValCys Leu LeuAsnAsnPhe Tyr Pro Arg Glu

130 135 140

Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser GlyAsn 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 SerThr 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 PheAsn Arg Gly Glu Cys

210 215

Claims (5)

1. An agent for the treatment and emergency prevention of diseases caused by the SARS-CoV-2 virus, containing a recombinant antibody having a CDR1 represented by the amino acid sequence SEQ ID NO:1, CDR2 represented by the amino acid sequence SEQ ID NO:2, CDR3 represented by the amino acid sequence SEQ ID NO:3, as well as a human IgG1 Fc fragment, and a humanized monoclonal antibody having a light chain CDR1 represented by the sequence SEQ ID NO:4, a light chain CDR2 represented by the sequence SEQ ID NO:5, a light chain CDR3 represented by the sequence SEQ ID NO:6, Heavy chain CDR1 represented by SEQ ID NO:7, Heavy chain CDR2 represented by SEQ ID NO:8, Heavy chain CDR3 represented by SEQ ID NO:9. 2. An agent for therapy and emergency prevention of diseases caused by the SARS-CoV-2 virus according to claim 1, where the recombinant antibody has the amino acid sequence of SEQ ID NO:10. 3. An agent for the treatment and emergency prevention of diseases caused by the SARS-CoV-2 virus according to claim 1, where the humanized monoclonal antibody has a heavy chain sequence of SEQ ID NO:11 and a light chain sequence of SEQ ID NO:12. 4. An agent for the treatment and emergency prevention of diseases caused by the SARS-CoV-2 virus according to claim 1, where the recombinant antibody has the sequence of SEQ ID NO: 10 and the humanized monoclonal antibody has the heavy chain sequence of SEQ ID NO: 11 and the light chain sequence of SEQ ID NO:12. 5. 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 1-4.

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