RU2771288C1 - STRAIN OF HYBRID ANIMAL CELLS MUS MUSCULUS 2E1B5 - PRODUCER OF A MONOCLONAL ANTIBODY AGAINST THE RECEPROT-BINDING DOMAIN OF PROTEIN S OF THE SARS-CoV-2 VIRUS - Google Patents

Abstract

FIELD: biotechnology.SUBSTANCE: invention relates to the field of biotechnology, in particular, to a strain of hybrid cultured animal cells Mus musculus 2E1B5. Hybrid clone of 2E1B5 cells was produced by fusion of mouse myeloma cells of the Sp2/0-Ag-14 line with splenocytes of mice of the Balb/c line, immunised intramuscularly with the recombinant receptor-biniding domain of protein S of the SARS-CoV-2 virus. Hybridoma 2E1B5 synthesises an IgG1-class monoclonal antibody specifically interacting with the recombinant receptor-binding domain of protein S of the SARS-CoV-2 virus, full-length S protein of the SARS-CoV-2 virus, and inactivated SARS-CoV-2 virus. The antibody exhibits virus-neutralising activity and is capable of neutralising the SARS-CoV-2 virus in vitro. The resulting antibody can be used in diagnostic studies in order to detect an antigen and specific antiviral antibodies and to create medicinal products against the SARS-CoV-2 virus.EFFECT: invention expands the range of means for producing monoclonal antibodies against protein S of the SARS-CoV-2 virus.1 cl, 1 dwg, 1 tbl, 5 ex

Description

The invention relates to the field of biotechnology, in particular to hybridoma technology, and concerns the production of a monoclonal antibody (MCA) to the receptor-binding domain of the S protein of the SARS-CoV-2 virus using a new hybrid animal cell clone (hybridoma). The invention can be used to create diagnosticums and immunochemical test systems for determining the presence of the SARS-CoV-2 virus in biological samples or antibodies to the S protein of the SARS-CoV-2 virus. The invention can be used to create drugs against the SARS-CoV-2 virus.

The SARS-CoV-2 virus belongs to the Coronaviridae family of the Betacoronavirus genus. The virus was first isolated at the end of 2019 in Wuhan (China) [1]. On March 11, 2020, the spread of an acute respiratory infection caused by the SARS-CoV-2 virus was recognized by WHO as a pandemic [2]. By the end of January 2021, the pandemic covers the whole world, the number of cases exceeds 100 million people, more than 2 million have died [3]. The most common symptoms of COVID-19 include fever, dry cough, fatigue, loss of smell or taste. In some patients, SARS-CoV-2 infection is accompanied by pneumonia, including severe pneumonia. The incubation period averages from 2 to 6 days [4].

The SARS-COV-2 virion consists of 4 structural proteins: S, M, E, and N. The N-protein is associated with the RNA of the virus, forming the nucleocapsid. The remaining 3 proteins form an envelope, while the largest of them, the S protein, which binds to the ACE2 cell receptor, plays the main role in infection. The S protein is also the main target for neutralizing antibodies against the virus [5].

The blood plasma of recovered Covid-19 patients has been used for therapy since the beginning of the epidemic [6]. Monoclonal antibodies with virus-neutralizing activity against SARS-CoV-2 are also considered as candidates for the role of antiviral drugs. In addition, MCA can be used in diagnostic studies to detect antigen and specific antiviral antibodies.

Hybridoma technology for obtaining monoclonal antibodies to human viral infections is widely known. For example, antibodies to hepatitis C virus [7], pandemic influenza virus A/IIV-Moscow/01/09 (H1N1)sw1 [8], Ebola virus proteins [9, 10], and other pathogens were obtained using this technology. .

Currently, there are several drugs based on monoclonal antibodies to the S protein of the SARS-CoV-2 virus that are in preclinical trials. For this purpose, human monoclonal antibodies obtained from recovered patients with Covid-19 are used, for example, P2C-1F11 [11], CB6-LALA [12], C002 [13], nAB cc12.1 [14], COVA1-18 [15] , COV2-2196 [16] and mouse antibodies, for example REGN-10987 [17]. Some antibodies to the SARS-CoV-1 S protein also neutralize the SARS-COV-2 virus, such as human CR3022 [18], S309 [19], 47D11-H2L2 [20], and mouse H014 antibodies [21]. However, expanding the MCA arsenal to antigens of the SARS-CoV-2 virus remains an urgent task.

The objective of the invention is the creation of a stable strain of a hybrid clone of cells that produces a virus-neutralizing MCA to the receptor-binding domain of the protein S of the SARS-CoV-2 virus, for the subsequent construction of highly specific test systems based on them for determining the antigen of the SARS-CoV-2 virus or antibodies to this virus, as well as for the possible use of this mAb as therapeutic drugs for the treatment of the disease caused by the SARS-CoV-2 virus.

The technical result of the invention is to create a monoclonal antibody (MAB) to the receptor-binding domain of the S protein of the SARS-CoV-2 virus and to expand the arsenal of hybridoma strains that produce MCA to SARS-CoV-2 virus antigens.

The specified technical result is achieved by obtaining a new hybrid clone of Mus museums 2E1B5 cultured cells, which is a producer of MCA to the S protein of the SARS-CoV-2 virus. The resulting MCA has specificity and affinity for virus-neutralizing epitopes of the S protein, which, in turn, makes it possible to determine the viral antigen in biological fluids of humans and animals. The neutralizing properties of MCA make it possible to consider it as a candidate therapeutic drug for the prevention and treatment of Covid-19. The claimed strain is deposited in the Collection of transplantable cell cultures of the Federal State Budgetary Institution "National Research Center for Epidemiology and Microbiology named after Honorary Academician N.F. Gamaleya” of the Ministry of Health of the Russian Federation, number 01-1/21.

The hybrid clone of cultured Mus musculus 2E1B5 cells is characterized by the following properties.

Species affiliation:

Mus muscle

How to obtain a strain:

The strain was obtained by fusion of Sp2/0-Ag-14 mouse myeloma cells with spleen cells of Balb/c mice immunized with intraperitoneal injection of the recombinant receptor-binding domain of SARS-CoV-2 virus S protein. The first immunization - with complete Freund's adjuvant, the second immunization - 2 weeks after the first, with incomplete Freund's adjuvant, booster immunization 2 weeks after the second, 3 days before the selection of splenocytes, all immunizations at 100.0 μg of protein / mouse. Hybridoma selection is carried out on RPMI-1640 nutrient medium (Sigma, USA) with the addition of HAT (Hypoxanthine-Aminopterin-Thymidine) Media Supplement (Hybri-Max®, Sigma, USA). The strain synthesizes monoclonal antibodies (MABs) that specifically interact in enzyme-linked immunosorbent assay (ELISA) with the receptor-binding domain of the S protein of the SARS-CoV-2 virus.

Cultural properties of the strain:

The hybrid cells grow as a suspension in vitro or as an ascitic tumor in the peritoneal cavity after injection into syngeneic Balb/c mice.

Cultivation of the strain in vitro:

RPMI-1640 medium (Sigma, USA) containing 20% fetal calf serum (FCS), 4.5 g/l glucose, 3 mM glutamine, 0.2 U/ml insulin, 60 μg is used as a growth medium. /ml gentamicin. Cells are cultured at 37°C in an atmosphere containing 5% CO ₂ . For cultivation, plastic plates or vials with a volume of 50 and 250 ml (Greiner, Germany) are used. The nature of growth is a stationary suspension. The passaging frequency is 2 days. Multiplicity of sieving 1:2-1:4.

Cultivation of the strain in the animal body in vivo:

One day before the introduction of hybridoma cells, Balb/c mice are pretreated intraperitoneally with Freund's incomplete adjuvant (Sigma, USA) at a dose of 0.5 ml/animal. Next, primed animals are injected into the abdominal cavity with hybrid cells of the strain at a dose of 5-10×10 ⁶ cells. Ascitic fluids containing MCA are obtained 7-15 days after administration.

Biotechnological characteristics of the strain (productivity):

- titer of MCA in the culture fluid when grown in vitro - 1.5×10 ⁴ (in ELISA);

- titer of MCA in ascitic fluid when grown in vivo - 8×10 ⁵ (in ELISA).

Conditions for storing and maintaining the viability of the strain:

The strain is preserved by cryopreservation in liquid nitrogen. 24 hours before freezing, hybridoma cells are passaged with a sieving ratio of 1:2. Sedimented by centrifugation (2000 rpm, 10 min) cells are suspended in a cryoprotective medium (90% FST, 10% DMSO) and dispensed into ampoules at a concentration of 3-5 million/ml, 1.0 ml each. Incubated for 24 hours at minus 70°C, then transferred to liquid nitrogen (minus 196°C).

Recovery after defrosting:

Rapid thawing of the strain is carried out at 37°C. Hybridoma cells are diluted 10 times with growth medium without serum, sedimented by centrifugation at 2000 rpm for 10 minutes and resuspended in growth medium containing 20% fetal calf serum at a concentration of 3-4×10 ⁵ cells/cm ³ . The viability of the restored cells is 70-80% and is determined by the differential staining of cells using a 0.4% solution of trypan blue, pH=7.2-7.3.

Strain stability:

The stability of antibody production by hybrid cells is maintained for 70 passages in cell culture and 5 passages in BALB/c mice.

Contamination:

Bacteria and fungi were not found in the cultures during long-term observation and inoculation on standard nutrient media (meat peptone agar - to detect bacteria and Sabouraud agar - for fungi). Mycoplasma infection was not detected (Mycoplasma Stain Kit test system, Flow Lab.).

The karyotype of hybrid cells and the modal number of chromosomes were not determined.

Characterization of the produced mAbs to the receptor-binding domain of the S protein of the SARS-CoV-2 virus:

- target - receptor-binding domain of the S protein of the SARS-CoV-2 virus;

- the average interaction constant for the resulting MCA is 8.2e-9 ± 1.6e-9 M.

- Isotype of produced immunoglobulins - IgG1 (ELISA, test system "Mouse-Hybridoma-Subtyping Kit" ("Sigma", USA).

Brief description of the drawings.

Figure 1 shows an example of immunoblotting using mAb obtained using strain 2E1B5, demonstrating the detection of the receptor binding domain: 1 - negative control (bovine serum albumin), 2 - molecular weight marker, 3 - sample of the S protein receptor-binding domain.

The invention is illustrated but not limited to the following.

Example 1. Obtaining the claimed hybridoma strain.

Balb/c mice weighing 18-20 g are immunized 3 times with the recombinant receptor-binding domain of the S protein of the SARS-CoV-2 virus. The antigen was obtained in a cell culture of Chinese hamster CHO (Chinese hamster ovary). The interval between immunizations is 2 weeks. Animals are injected with antigen intraperitoneally (100 μg/mouse), with complete Freund's adjuvant (first immunization) or with incomplete Freund's adjuvant (second and third immunizations). A booster dose (100.0 μg/mouse) is administered without adjuvant intraperitoneally 3 days prior to spleen isolation.

3 days after the injection of the booster dose of the antigen, a suspension of mouse spleen cells is obtained by the standard method in a chilled growth medium without serum.

For fusion, a continuous mouse myeloma cell line Sp2/0-Ag-14 is used, which is defective in the HGPRT gene and does not produce its own immunoglobulins. Hybridization is carried out according to the classical method of Köhler and Milstein. The suspension of spleen cells and myeloma cells are mixed in a ratio of 5:1, respectively. After confluence, the cells are resuspended in selective medium supplemented with HAT, introduced into 96-well panels with a feeder layer of mouse peritoneal macrophages (5×10 ⁶ cells/ml, 50 μl/lun) at a concentration of 1.5×10 ⁵ cells per well in a volume of 0.1 ml. Panels with cells are incubated at 37°C in an atmosphere of 5% CO ₂ .

The replacement of the medium in the wells with hybridoma clones is carried out every 2-3 days, using the medium with HAT within 14 days after fusion. For selection of a hybrid producing MCA of a given specificity, culture fluids are taken from the wells with growing colonies. When clones of hybrid cells occupy at least 50% of the surface of the well, they are tested for the presence of antibodies to the receptor-binding domain of the S protein of the SARS-CoV-2 virus in an enzyme-linked immunosorbent assay. Clones that have found specific activity are recloned by the method of limiting dilutions in 96-well panels with a feeder layer of mouse peritoneal macrophages (5×10 ⁶ cells/ml, 50 μl/moon). Supernatants from wells containing recloned hybridomas are again tested for the presence of antibodies and their specificity.

Example 2. Obtaining the claimed MCA.

Screening of MCA given specificity and immunological activity is carried out by indirect ELISA. To do this, 0.1 ml of the antigen (recombinant receptor-binding domain of the S protein of the SARS-CoV-2 virus) is introduced into the wells of immunological microplates (Greiner, Germany) at a working dilution in 0.1 M carbonate-bicarbonate buffer, pH 9, 5. Microplates are incubated for 18 hours at 4°C. Free plastic areas are blocked with 1% Top Block (Yuro, Switzerland) in 0.01 M phosphate-buffered saline (PBS). Incubated for 30 minutes at 37°C and contribute to the wells of 0.1 ml of the culture liquid MCA in various dilutions. The microplates are incubated for 1 hour at 37°C and a peroxidase conjugate of anti-mouse IgG antibodies (Sigma, USA) is added at the working dilution. Incubate 1 hour at 37°C. After each step, unbound reagents are washed with PBS with the addition of 0.1% Tween-20 (PBS). Tetramethylbenzidine (TMB) is used as a chromogen. The color intensity in the wells is determined after stopping the reaction with 1M H ₂ SO ₄ on an iMark spectrophotometer (Bio-rad, USA) at a wavelength of 450 nm.

Clones that have shown specific activity for the receptor-binding domain of the S protein of the SARS-CoV-2 virus are recloned by the method of limiting dilutions in 96-well panels with a feeder layer of mouse peritoneal macrophages (5×10 ⁶ cells/ml, 50 μl/moon) . Supernatants from wells containing recloned hybridomas are again tested for the presence of antibodies and their specificity. Positive recloned hybridomas are grown in culture flasks of increasing size (first 25 cm ³ , then 75 cm ³ ) (Greiner, Germany).

Isolation of MCA from culture fluids (CL) is carried out using an AKTA START chromatographic system (GE Healthcare, Sweden) and a HiTrap Protein A HP 1.0 ml column (GE Healthcare, Sweden), according to the manufacturer's protocol. Desalting and replacement of the buffer in the sample purified at the first stage is carried out using an AKTA START chromatographic system (GE Healthcare, Sweden) and a HiTrap desalting 5 ml column (GE Healthcare, Sweden), according to the manufacturer's protocol. After the final stages of purification, 10.0 ml of the MCA preparation with a concentration of 0.7 mg/ml is obtained from 100.0 ml of QOL. The yield of purified MCA with QOL is 70 μg/ml.

Isolation of class G immunoglobulins (Ig) from ascitic fluid (AF) is carried out by precipitation of proteins using caprylic acid (Sigma, USA). AF is diluted with 0.06 M acetate buffer, pH 4.0, caprylic acid (3.3 μl/ml AF) is added, incubated for 30 min at 20°C, centrifuged. The supernatant is filtered through a PVDF filter (0.45 μm) and dialyzed for a day against PBS. Then add a saturated solution of ammonium sulfate (up to 50% saturation), incubate for 18 hours. at 40°C, centrifuged, the precipitate is dissolved in PBS and desalted in columns with semi-dry Sephadex G-25 Medium in PBS. The optical density of IgG solutions is measured on a spectrophotometer (Eppendorf, Germany) at a wavelength of 280 nm. Protein concentration is determined by the formula:

C=D/1.4, where D is the optical density, C is the protein concentration (mg/ml), 1.4 is the optical density of an IgG solution with a concentration of 1.0 mg/ml.

The characteristics of the resulting drug are presented in table 1.

Example 3 Use of mAb for protein S detection by immunoblotting.

Samples of the receptor-binding domain of protein S, obtained in example 1, are separated in a 10-15% gradient polyacrylamide gel (SDS-PAGE) using the Mini-Protein 3 cell apparatus (Bio-Rad, USA). Next, the samples were transferred to a Hybond-P PVDF membrane (GE Healthcare, USA) using Trans-Blot SD Semi-Dry Transfer cell equipment (Bio-Rad, USA) according to the manufacturer's protocol. After transfer, the membrane is blocked with a 5% solution of skimmed milk powder on PBST for one hour at room temperature. Then, the MCA obtained using strain 2E1B5 is added to the blocking solution to a working concentration of 1.0 μg/ml, and the membrane is incubated for an hour at 37°C with gentle stirring. Washing the membrane from unbound antibodies is carried out 5 times with a solution of PSBT. Then incubated in blocking solution with the addition of anti-mouse conjugate (1:2500) ("Sigma", A9044-2ML) and incubated for one hour at 37°C, with gentle agitation. After 5-fold washing with PSBT solution, the membrane is developed with a luminescent substrate solution (ECL) (GE Healthcare, USA) according to the manufacturer's protocol. The results of immunoblotting are detected using an Amersham Imager 600 system (GE Healthcare, USA) according to the manufacturer's instructions. Staining with the obtained MCA allows the detection of the receptor-binding domain of the S protein of the SARS-CoV-2 virus by immunoblotting. An example of an immunoblot result is shown in Fig. one.

Example 4. Determination of the average interaction constant of MCA.

The interaction constant of the resulting MCA with the receptor-binding domain of the S protein obtained in example 1 is determined using the Octet Red 96 molecular interaction detection system (ForteBio, USA) using Ni-NTA (NTA) Dip and Read Biosensors (ForteBio) , USA), according to the manufacturer's recommendations. The receptor-binding domain of protein S is adsorbed onto the above sensors in a single kinetic buffer (ForteBio, USA) at a concentration of 25 μg/ml. The binding of MCA to the antigen presorbed on the sensors is also carried out in a single kinetic buffer (ForteBio, USA) at concentrations of 50, 25, 12.5, 6.25, 3.125, 1.5625, 0.78, and 0 µg/mL, respectively. The baseline and dissociation of the antibody with the antigen are also measured in a single kinetic buffer (ForteBio, USA), according to the manufacturer's recommendations. The Data Analysis 10.0 software is used to calculate the interaction constant of the mAb with the receptor-binding domain of the S protein.

As a result of repeated measurements, the average interaction constant (KD)=8.2×10 ^-9 ±1.6×10 ^-9 M was calculated.

Example 5. Evaluation of the virus-neutralizing properties of the resulting MCA.

The neutralization reaction is performed by the micromethod [22] in 96-well plates (Costar, USA) on a transplantable Vero E6 cell culture grown in Eagle's medium supplemented with 5% fetal calf serum. Maintenance medium contains 1% fetal calf serum.

The titer of the virus (SARS-CoV-2 strain: PMVL-12, deposit number EPI_ISL_572398 in the Gisaid database) is determined by the final dilution method by parallel titration in steps of 10 in rows of the tablet, starting from a virus dilution of 1×10 ^-1 and up to 1×10 ^{- 8} . The virus titer is taken as the last dilution in which a cytopathogenic effect (CPE) is noted. The number of 50% cytopathic doses (CPP ₅₀ ) is calculated according to the classical method of Reed and Mench.

The antibody in the neutralization reaction is tested against 100 CPE ₅₀ doses of the virus in 0.1 ml. The antibody is titrated starting at a concentration of 125 µg/ml to 0.014 ng/ml.

The results of the reaction are taken into account on the 3rd, 5th and 7th days after infection of the cells, looking at the wells of the plate in an inverted microscope. The titer (concentration) of antibodies is taken as the highest dilution at which the culture was protected from 100 CPE ₅₀ of the virus (i.e., CPE does not develop). Neutralization experiments are accompanied by control of the working dose of viruses, toxicity, sterility and protective activity of positive specific sera (from people who have recovered from COVID-19).

It was revealed that monoclonal antibodies produced by the 2E1B5 strain have neutralizing activity (122 ng of antibodies neutralize 1000 cytopathic doses of the SARS-CoV-2 virus in 1.0 ml).

Thus, the created 2E1B5 hybridoma synthesizes a monoclonal antibody (MAB) of the IgG1 class, which specifically interacts in ELISA with the recombinant receptor-binding domain of the S protein of the SARS-CoV-2 virus, the full-length S protein of the SARS-CoV-2 virus, and the inactivated SARS virus -CoV-2. The resulting MCA has virus-neutralizing activity and is able to neutralize the SARS-CoV-2 virus in in vitro tests.

The invention can be used to create diagnostic kits and immunochemical test systems to determine the presence of the SARS-CoV-2 virus in biological samples or to detect antibodies to the S protein of the SARS-CoV-2 virus. The neutralizing properties of the resulting MCA make it possible to consider it as a candidate therapeutic drug for the prevention and treatment of Covid-19.

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Florian Krammer / Current Protocols in Microbiology Volume 58, Issue 1 September 2020, p. 1-15.22. An In Vitro Microneutralization Assay for SARS-CoV-2 Serology and Drug Screening // Fatima Amanat Kris M. White Lisa Miorin Shirin Strohmeier Meagan McMahon Philip Meade Wen-Chun Liu Randy A. Albrecht Viviana Simon Luis Martinez-Sobrido Thomas Moran Adolfo

Florian Krammer / Current Protocols in Microbiology Volume 58, Issue 1 September 2020, p. 1-15.

Claims (1)

The strain of hybrid cultured animal cells Mus musculus 2E1B5, deposited in the Collection of transplanted cell cultures of the Federal State Budgetary Institution “National Research Center for Epidemiology and Microbiology named after N.N. N.F. Gamaleya" of the Ministry of Health of Russia, number 01-1/21, - producer of a monoclonal antibody to the receptor-binding domain of the S protein of the SARS-CoV-2 virus, which has an average interaction constant of 8.2 × 10 ^-9 ± 1.6 × 10 ^-9 M , belonging to the IgG1 isotype, capable of specifically interacting with the recombinant receptor-binding domain of the S protein of the SARS-CoV-2 virus, the full-length S protein of the SARS-CoV-2 virus, inactivated by the SARS-CoV-2 virus, which has virus-neutralizing activity and is able to neutralize the SARS virus -CoV-2 in vitro, 122 ng of which neutralizes 1000 cytopathic doses of the SARS-CoV-2 virus in 1.0 ml.

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