WO2022138911A1

WO2022138911A1 - HUMAN ANTI-SARS-CoV2 VIRUS ANITBODY

Info

Publication number: WO2022138911A1
Application number: PCT/JP2021/048176
Authority: WO
Inventors: 長▲崎▼洋司; 健石井; 康司小檜山; 輝人安居; 武春南谷; 亮太大坪; 亘神谷
Original assignee: 国立大学法人東京大学; 国立研究開発法人医薬基盤・健康・栄養研究所; 国立大学法人群馬大学; 長▲崎▼洋司
Priority date: 2020-12-25
Filing date: 2021-12-24
Publication date: 2022-06-30
Also published as: JP2024022702A

Abstract

The present invention addresses the problem of providing a recombinant antibody, a recombinant antibody derivative, or a drug composition including one or more of such antibodies which can be used to treat SARS-CoV2 virus infection by neutralizing infection by a SARS-CoV2 virus amplified inside the body due to a SARS-CoV2 virus infection.　As a result of rigorous examination, the inventors of the present invention have discovered a plurality of cells that produce antibodies against the SARS-CoV2 virus from bodies which have been infected with and recovered from the SARS-CoV2 virus in the past, and have shown that the aforementioned problem can be solved by making, from the aforementioned cells, antibodies or antibody derivatives that have the ability to bind to one or more SARS-CoV2 viruses and have the action of suppressing amplification of the SARS-CoV2 virus or neutralizing SARS-CoV2 virus infection.

Description

Human anti-SARS-CoV2 virus antibody

In the present invention, in an individual infected with SARS-CoV2 virus infection, a recombinant antibody or a recombinant antibody derivative for neutralizing the infection of the virus, or infection with SARS-CoV2 virus containing such an antibody is medium. With respect to providing a pharmaceutical composition for reconciliation.

Since the confirmation of the new coronavirus infection COVID-19 due to infection with the new coronavirus and SARS-CoV2 virus from 2019 to 2020, 195,880 infected people, of which 2,873 have died, in Japan. In the United States, 17,655,591 infected people, of which 316,159 died, and worldwide, 76,288,108 infected people, of which 1,685,650 died (thickness as of December 20, 2020). According to the official announcement by the Ministry of Labor).

SARS-CoV2 invades cells through receptors on the surface of human cells, replicates using enzymes derived from viral genes (RNA polymerases that do not exist in humans), and (3) human cells. It is amplified in infected individuals by repeating the cycle of making proteins and enzymes inside, proliferating, and (4) going out of cells and spreading to other normal cells. In addition, it is known that when it becomes severe, it causes an excessive immune reaction called a cytokine storm and causes severe respiratory failure called acute respiratory distress syndrome (ARDS).

Many of the antiviral drugs that have already been approved for use against the SARS-CoV2 virus and are still under development are (1) invasion, (2) replication, and (3) proliferation of this virus. , (4) Target one of the diffusion processes.

For example, remdesivir is an antiviral drug that was originally under development as a therapeutic drug for Ebola hemorrhagic fever, but it is expected to have the effect of inhibiting RNA polymerase and suppressing viral replication, and SARS-has already been developed in the United States, Europe, and Asia. Its use for CoV2 infection was urgently approved (Non-Patent Document 1). However, the interim results of clinical trials conducted by the World Health Organization (WHO) and its affiliated organizations reported in the latter half of 2020 have not demonstrated improvement effects such as case fatality rate, and side effects. It is said that there are problems with the possibility and the burden on the medical field.

Avigan is an antiviral drug originally approved for new influenza, and is expected to have the effect of inhibiting RNA polymerase and suppressing viral replication. Teratogenicity (both female and male may have an adverse effect on the fetus when taken orally) has been clarified as a side effect of this drug, and there were restrictions on its use, but in the latter half of 2020 In Japan, verification of efficacy and safety is underway (Non-Patent Document 2), but there are reports that no clear efficacy is recognized.

In addition to antiviral drugs, the application of drugs aimed at alleviating the symptoms of pneumonia in severe cases of COVID-19 is being considered, and dexamethasone and actemra are promising. However, these drugs are not intended to be used to prevent SARS-CoV2 infection, but are thought to be used symptomatically.

On the other hand, the development of vaccines against SARS-CoV2 virus is also progressing, and some of them have already reached the stage of application for approval. Since the SARS-CoV2 virus infects cells by binding the spike protein to the ACE2 receptor of the cell and disappears from the outside of the cell, an antibody that recognizes the ACE2 binding site of this spike protein can exert the ability to neutralize the infection. It is believed that there are many cases. However, as a result of measuring the antibody against SARS-CoV2 virus present in the blood of those who recovered from SARS-CoV2 virus infection, in many recoverers, IgG antibody disappeared from the blood within a few months after recovery. There is also a report that there is (Non-Patent Document 3), and there is debate as to whether the effect of the vaccine is sustained.

As a flow of development of treatment methods different from these developments, convalescent plasma was administered to a small number of patients in China, Singapore, South Korea, and the United States from around March 2020, suggesting its effectiveness. It has been reported. Based on the same idea, studies on the safety and efficacy of treatment using COVID-19 recoverer plasma have begun in Japan (Non-Patent Document 4). Such serum therapy or plasma therapy using serum or plasma collected from a patient in the convalescent period from SARS-CoV2 virus infection is a serum from blood provided by a patient who has recovered after being infected with SARS-CoV2 virus. Alternatively, it is a classic treatment of preparing serum and administering it to patients infected with the SARS-CoV2 virus. This treatment method is based on the premise that an antibody (neutralizing antibody) that inhibits SARS-CoV2 virus infection is present in the blood of patients who have recovered from SARS-CoV2 virus infection.

However, in order to adopt serum therapy or plasma therapy, blood provided by a patient who has recovered after being infected with the SARS-CoV2 virus is required, and there is a problem of stable supply due to a shortage of blood raw materials. There are safety issues specific to blood products.

The present invention is a recombinant antibody or recombinant antibody that can be used for the treatment of SARS-CoV2 virus infection by neutralizing SARS-CoV2 virus infection that is amplified in vivo by SARS-CoV2 virus infection. It is an object of the present invention to provide a recombinant antibody derivative or a pharmaceutical composition containing one or a plurality of such antibodies.

As a result of diligent studies, the inventors of the present invention have found a plurality of cells that produce antibodies against SARS-CoV2 virus from individuals who have been infected and recovered with SARS-CoV2 virus in the past, and converted them into SARS-CoV2 virus. On the other hand, the above-mentioned problems can be solved by producing an antibody or an antibody derivative which has a binding property and has an action of suppressing the amplification of SARS-CoV2 virus or neutralizing the infection of SARS-CoV2 virus. showed that.

More specifically, the present application provides the following aspects in order to solve the above-mentioned problems:
[1] An antibody or antibody derivative that does not contain blood-derived components and has a binding property to the spike protein constituting the SARS-CoV2 virus and has an effect of neutralizing the infection of the SARS-CoV2 virus;
[2] The antibody or antibody derivative according to [1], wherein the antibody is a human antibody;
[3] The antibody or antibody derivative according to [1] or [2] produced by genetic recombination;
[4] The antibody derivative is selected from humanized antibody variants selected from humanized antibodies, chimeric antibodies, polyvalent antibodies, and multispecific antibodies or functional fragments thereof, [1] to [3]. The antibody or antibody derivative according to any of the above;
[5] The antibody or antibody derivative according to [4], wherein the functional fragment is F (ab') 2.
[6] Heavy chains contain complementarity determining regions, CDR1 (GFTFRNYA, SEQ ID NO: 1), CDR2 (IWYDGSNK, SEQ ID NO: 2), CDR3 (ARDQGFGDNYYYYGMDV, SEQ ID NO: 3).
The light chain comprises the complementarity determining regions, CDR1 (ALPKEY, SEQ ID NO: 4), CDR2 (KDS, SEQ ID NO: 5), CDR3 (QLADSSMHYVV, SEQ ID NO: 6).
The antibody or antibody derivative according to any one of [1] to [5];
[7] Of the amino acid sequence of SEQ ID NO: 7 or the amino acid sequence of SEQ ID NO: 7, the heavy chain variable region VH domain is CDR1 (SEQ ID NO: 1), CDR2 (SEQ ID NO: 2), And the antibody or antibody according to any one of [1] to [6], which comprises an amino acid sequence containing substitution, insertion, or deletion of one or several amino acids in a portion other than CDR3 (SEQ ID NO: 3). Derivatives;
[8] Of the amino acid sequence of SEQ ID NO: 8 or the amino acid sequence of SEQ ID NO: 8, the light chain variable region VL domain is CDR1 (SEQ ID NO: 4), CDR2 (SEQ ID NO: 5), And contains amino acid sequences containing substitutions, insertions, or deletions of one or several amino acids in parts other than CDR3 (SEQ ID NO: 6).
The antibody or antibody derivative according to any one of [1] to [7];
[9] Of the amino acid sequence of SEQ ID NO: 9 or the amino acid sequence of SEQ ID NO: 9, the heavy chain is CDR1 (SEQ ID NO: 1), CDR2 (SEQ ID NO: 2), and CDR3 (SEQ). ID NO: The antibody or antibody derivative according to any one of [1] to [8], which comprises an amino acid sequence containing substitution, insertion, or deletion of one or several amino acids in a portion other than 3);
[10] The light chain is
Amino acid sequence of SEQ ID NO: 10 or amino acid sequence of SEQ ID NO: 10 other than CDR1 (SEQ ID NO: 4), CDR2 (SEQ ID NO: 5), and CDR3 (SEQ ID NO: 6) Of the amino acid sequence containing substitutions, insertions, or deletions of one or several amino acids in a portion, or the amino acid sequence of SEQ ID NO: 11, or the amino acid sequence of SEQ ID NO: 11, CDR1 (SEQ ID NO: 4) ), CDR2 (SEQ ID NO: 5), and amino acid sequences containing substitutions, insertions, or deletions of one or several amino acids in parts other than CDR3 (SEQ ID NO: 6).
The antibody or antibody derivative according to any one of [1] to [9], which comprises.
[11] A pharmaceutical composition for neutralizing SARS-CoV2 virus infection, which comprises the antibody or antibody derivative according to any one of [1] to [10];
[12] The pharmaceutical composition according to [11], which comprises in combination with another antibody or antibody derivative.

In the case of conventional serum or plasma therapy for SARS-CoV2 virus, serum or plasma containing anti-SARS-CoV2 virus antibody produced from human blood is administered, but there are various problems peculiar to blood products. is doing. By using the recombinant antibody or recombinant antibody derivative of the present invention, all the problems caused by blood products can be solved. In addition, these antibodies can also be used as research reagents, diagnostic agents and the like.

FIG. 1 is a diagram showing the results of examining whether or not the SARS-CoV2 virus-reactive clone obtained in Example 1 has neutralizing activity against infection of cells with the SARS-CoV2 virus. FIG. 2 is a diagram showing the confirmed results of the reactivity of the purified recombinant 7G7.1 antibody with the SARS-CoV2 virus spike protein. FIG. 3 is a diagram showing the results of examining the neutralizing activity of SARS-CoV2 virus infection in vitro of the purified recombinant 7G7.1 antibody. FIG. 4 is a diagram showing the results of ELISA in which the reactivity of the purified recombinant 7G7.2 antibody to the SARS-CoV2 virus spike protein was confirmed. FIG. 5-1 is a diagram showing the results of surface plasmon resonance (SPR) confirming the binding affinity of the purified recombinant 7G7.1 antibody and 7G7.2 antibody to the SARS-CoV2 virus spike protein. FIG. 5-2 is a diagram showing the results of surface plasmon resonance (SPR) in which the binding affinity of the control antibody to the SARS-CoV2 virus spike protein was confirmed. FIG. 6 is a diagram showing the results of examining the neutralizing activity of SARS-CoV2 virus infection in vitro of the purified recombinant 7G7.2 antibody in comparison with the 7G7.1 antibody. FIG. 7 is a diagram showing that the purified recombinant 7G7.2 antibody significantly suppresses SARS-CoV2 infection and has neutralizing activity against SARS-CoV2 in vivo. FIG. 8 is a diagram showing the analysis results of the binding region of the SARS-CoV2 virus spike protein of the purified recombinant 7G7.2 antibody.

The present invention, in one embodiment, is an antibody or antibody that does not contain a blood-derived component and has a binding property to a spike protein constituting the SARS-CoV2 virus and has an action of neutralizing the infection of the SARS-CoV2 virus. Derivatives can be provided. The present invention relates to an individual infected with or suspected of being infected with SARS-CoV2 virus by suppressing the amplification of SARS-CoV2 virus in the body or neutralizing the infection with SARS-CoV2 virus. It can be used to prevent and treat the new coronavirus infection (COVID-19) caused by viral infection.

<Antibody or antibody derivative>
The antibody or antibody derivative used in the present invention is intended to solve various problems (particularly, infectivity problems and immunity problems) peculiar to blood preparations used in serum therapy and plasma therapy. Does not contain pathogens such as hepatitis B virus, hepatitis C virus, and human immunodeficiency virus (HIV) contained in blood (which may be mixed in blood preparations), and is contained in blood. It is characterized by the absence of blood-derived components that may cause immune abnormalities in the administered individual, such as antigenic proteins and antibodies that bind to human proteins.

As an antibody that does not contain blood-derived components, an antibody prepared by obtaining immortalized cells derived from antibody-producing cells under conditions that do not contain blood-derived components and from the cells under culture conditions that do not contain blood-derived components. , A recombinant antibody that can be prepared by recombinantly expressing the antibody protein using the DNA defining the antibody protein obtained from the immortalized cell can be used.

That is, in one embodiment, the antibody in the present invention is an antibody-producing cell by obtaining and immortalizing a cell clone that produces an antibody against the SARS-CoV2 virus from the blood of an individual that has been infected and recovered with the SARS-CoV2 virus in the past. Obtained by obtaining immortalized cells derived from the above, and selecting those having an action of suppressing the amplification of SARS-CoV2 virus or neutralizing the infection of SARS-CoV2 virus in vivo from the immortalized antibody-producing cells. be able to.

In yet another embodiment, mRNA from an immortalized antibody-producing cell having the effect of suppressing the amplification of SARS-CoV2 virus selected by the method described above or neutralizing the infection of SARS-CoV2 virus according to a well-known method. It is also possible to prepare as a recombinant antibody by obtaining a DNA sequence defining an antibody protein through the acquisition of the antibody and the preparation of cDNA and expressing it in a mammalian expression system containing no blood-derived component via a vector. ..

In the present invention, derivatives of these antibodies can also be used. As the antibody derivative that can be used in the present invention, for example, a humanized antibody variant selected from a humanized antibody, a chimeric antibody, a polyvalent antibody, and a multispecific antibody or a functional fragment thereof can be used. Yes, but not limited to these. Of these, as the functional fragment, for example, F (ab') 2 can be used, but the functional fragment is not limited thereto.

Derivatives of these antibodies can be produced according to a method well known in the art after the antibody is obtained.

The antibody or antibody derivative obtained by the above-mentioned method has binding property to SARS-CoV2 virus, but in the present invention, among those having binding property to SARS-CoV2 virus. Further, screening is performed based on having an action of suppressing amplification of SARS-CoV2 virus or neutralizing infection of SARS-CoV2 virus, and an antibody or antibody derivative having the neutralizing action is provided. do.

Although a plurality of types of antibodies actually obtained are shown in the examples described later, the present invention is not limited to these.

<SARS-CoV2 virus-derived antigen targeted by the antibody or its derivative>
The SARS-CoV2 virus-derived antigen targeted by the antibody or derivative thereof is one or a combination of enveloped protein, membrane protein, nucleocapsid protein, and spike protein, which are constituents of SARS-CoV2 virus. May be. It is preferable to use a protein (for example, envelope protein, spike protein) existing (that is, exposed on the surface) outside the SARS-CoV-2 virus to which the antibody or its derivative is easily bound as an antigen.

The proteins constituting the SARS-CoV-2 virus that can be targeted by the antibody or its derivative in the present invention are obtained based on the complete genomic sequence of the SARS-CoV-2 virus already registered in the public database. It is something that can be done. In the present invention, the SARS-CoV-2 virus reference genome sequence NC_045512 registered in the NCBI RefSeq database is used from the viewpoint of representativeness, and the SARS-CoV-2 virus specified based on this reference genome sequence is used. It is preferable to use the proteins constituting the above.

<Action of antibody or antibody derivative>
The antibody or antibody derivative of the present invention binds to the above-mentioned SARS-CoV-2 virus-derived antigen, and as a result, suppresses infection / amplification of SARS-CoV2 virus or neutralizes SARS-CoV2 virus infection. Anything that has such an action can be used to achieve the object of the present invention. Since the antibody or antibody derivative of the present invention is intended to be used as a therapeutic agent for SARS-CoV2 virus, it is required to have such an action.

The above action of an antibody or antibody derivative causes cells that can cause infection with SARS-CoV2 virus (eg, VERO cells) to be infected with SARS-CoV2 virus in vitro in the presence of the antibody or antibody derivative. It can be identified by whether or not SARS-CoV2 virus infection suppression is observed in cells.

<Example of antibody of the present invention>
In the present invention, blood can be collected from an individual who has been infected with the SARS-CoV2 virus in the past and recovered, and an antibody against the SARS-CoV2 virus obtained from the blood can be used. Specifically, cells producing an antibody that binds to the above-mentioned SARS-CoV2 virus-derived antigen are collected from an individual that has been infected with the SARS-CoV2 virus in the past and recovered as described above <antibody or antibody derivative>. As for the obtained antibody or antibody derivative, the action of suppressing the amplification of SARS-CoV2 virus or neutralizing the infection of SARS-CoV2 virus was investigated as described above <Action of antibody or derivative thereof>. As a result, a plurality of antibodies and cells producing the antibodies were obtained.

Under such circumstances, as a concrete example examined in the examples,
Heavy chain complementarity determining regions, including CDR1 (GFTFRNYA, SEQ ID NO: 1), CDR2 (IWYDGSNK, SEQ ID NO: 2), CDR3 (ARDQGFGDNYYYYGMDV, SEQ ID NO: 3)
Contains light chain complementarity determining regions, CDR1 (ALPKEY, SEQ ID NO: 4), CDR2 (KDS, SEQ ID NO: 5), CDR3 (QLADSSMHYVV, SEQ ID NO: 6);
Antibodies or derivatives thereof can be provided.

Such antibodies or antibody derivatives include:
The heavy chain variable region VH domain is the amino acid sequence of SEQ ID NO: 7 or the amino acid sequence of SEQ ID NO: 7, CDR1 (SEQ ID NO: 1), CDR2 (SEQ ID NO: 2), and CDR3 (. An antibody or antibody derivative containing an amino acid sequence comprising substitution (eg, conservative substitution), insertion, or deletion of one or several amino acids in a portion other than SEQ ID NO: 3).
The light chain variable region VL domain is the amino acid sequence of SEQ ID NO: 8 or the amino acid sequence of SEQ ID NO: 8, CDR1 (SEQ ID NO: 4), CDR2 (SEQ ID NO: 5), and CDR3 (. An antibody or antibody derivative containing an amino acid sequence comprising a substitution (eg, conservative substitution), insertion, or deletion of one or several amino acids in a portion other than SEQ ID NO: 6).
It can also be expressed as.

In the following examples herein, as a specific antibody,
・ 7G7.1 clone:
Of the amino acid sequence of SEQ ID NO: 9 or the amino acid sequence of SEQ ID NO: 9, the heavy chain is CDR1 (SEQ ID NO: 1), CDR2 (SEQ ID NO: 2), and CDR3 (SEQ ID NO: 1). Contains an amino acid sequence containing one or several amino acid substitutions (eg, conservative substitutions), insertions, or deletions in portions other than 3), and the light chain is the amino acid sequence of SEQ ID NO: 10, or SEQ. Substitution of one or several amino acids in the amino acid sequence of ID NO: 10 other than CDR1 (SEQ ID NO: 4), CDR2 (SEQ ID NO: 5), and CDR3 (SEQ ID NO: 6). For example, containing an amino acid sequence containing a conservative substitution), insertion, or deletion,
Antibodies or antibody derivatives, and 7G7.2 clones (replacement of the constant region of the light chain Igλ chain of the 7G7.1 clone with the constant region of the Igk chain):
Of the amino acid sequence of SEQ ID NO: 9 or the amino acid sequence of SEQ ID NO: 9, the heavy chain is CDR1 (SEQ ID NO: 1), CDR2 (SEQ ID NO: 2), and CDR3 (SEQ ID NO: 1). Contains an amino acid sequence containing one or several amino acid substitutions (eg, conservative substitutions), insertions, or deletions in portions other than 3), and the light chain is the amino acid sequence of SEQ ID NO: 11, or SEQ. Substitution of one or several amino acids in the amino acid sequence of ID NO: 11 other than CDR1 (SEQ ID NO: 4), CDR2 (SEQ ID NO: 5), and CDR3 (SEQ ID NO: 6). For example, containing an amino acid sequence containing a conservative substitution), insertion, or deletion,
Antibodies or antibody derivatives were made.

<Pharmaceutical composition>
The present invention provides, in one embodiment, a pharmaceutical composition comprising the antibody or antibody derivative described above for suppressing amplification of SARS-CoV2 virus or neutralizing SARS-CoV2 virus infection. Can be done. This pharmaceutical composition is intended as an antiviral drug against SARS-CoV2 virus or for the purpose of preventing and treating the onset of symptoms caused by SARS-CoV2 virus in individuals infected with or suspected of being infected with SARS-CoV2 virus. And can be used.

Since the antibody or antibody derivative of the present invention contained in this pharmaceutical composition is not derived from a blood raw material, this pharmaceutical composition is a risk factor (for example, hepatitis B virus) that may be contaminated in blood preparations. , Hepatitis C virus, viruses such as human immunodeficiency virus (HIV), and blood-derived components that may cause immune abnormalities in administered individuals such as antigenic proteins and antibodies that bind to human proteins) It is characterized by not including.

The pharmaceutical composition in the present invention may contain one type of the above-mentioned antibody or antibody derivative, or may contain a plurality of types.

Hereinafter, the present invention will be specifically shown with reference to examples. The examples shown below do not limit the invention in any way.

Example 1: Isolation of antibody using modified EBV method In this example, a cell clone that produces an antibody against SARS-CoV2 virus was obtained by the EBV method using Epstein-Barr virus (EBV), and there. Experiments were performed with the aim of isolating the antibody from.

EBV is a dsDNA virus belonging to the subfamily Gammaherpesvirinae and is involved in various cancers. It is known to mainly infect B cells, and when human B cells are infected with EBV in vitro, the B cells can proliferate continuously and transform into immortalized human lymphoblastoid cells (LCL). Are known.

(1-1) Isolation of PBMCs and differentiation into LCL Blood was collected from donors (n = 1) who had recovered from SARS-CoV2 virus infection in the past (obtained from the National Hospital Organization Kyushu Medical Center), and Lymphoprep (Abbott). Peripheral blood mononuclear cells (PBMC, lymphocytes / monocytes) were collected using Diagnostics Technologies AS). Negative selection was performed by adding Anti-human IgM magnetic beads (Miltenyi) to the cell suspension and removing the cells, and cells not expressing IgM on the cell surface were collected.

Negatively selected cells were harvested and incubated with EBV (B95-8 strain) at 37 ° C. for 1 hour. After incubation, cells were collected by centrifugation and suspended in LCL culture medium. 200 μL of this cell suspension (10 ⁴ cells / well) was inoculated on a 96-well plate (U-bottom delta: NUNC) and then cultured for 2 weeks to differentiate into immortalized human lymphoblast-like cells (LCL). Was done.

The LCL culture medium used in this example is 15% FBS (SIGMA), L-glutamine (GIBCO), Penicillin / streptomycin (Nacalai tesque), Sodium Pyruvate (Nacalai tesque), 2-Mercaptoethanol (GIBCO), Non-essential. RPMI medium (Nacalai tesque) containing amino acids (GIBCO), K3 (GeneDesign), cyclosporin (Novartis Pharma), and various additives was used. The same operation was performed twice.

(1-2) Screening for SARS-CoV2 virus-reactive clones Add 1 μg / mL of SARS-CoV2 virus spike protein (SARS-CoV2 Spike ECD protein (GenScript)) to a 96-well plate for ELISA at 50 μL / well and solidify. After symmetry, 50 μL / well of LCL culture supernatant (which may contain anti-SARS-CoV2 virus IgG) was added to carry out an antigen-antibody binding reaction. After that, Goat Anti-Human IgG-AP (SouthernBiotech) was used as the secondary antibody, and Phosphatase substrate (SIGMA) was used as the color-developing substrate, and the absorbances at 405 nm and 650 nm were measured.

As a result, 10 clones in the first screening and 18 clones in the second screening, a total of 28 clones were obtained as clones producing an antibody having binding to the SARS-CoV2 virus spike protein.

Example 2: In vitro functional analysis (SARS-CoV2 virus infection neutralization experiment using Vero cells)
In this example, an experiment was conducted for the purpose of analyzing the in vitro function and the in vivo function of the SARS-CoV2 virus-reactive clone obtained in Example 1.

The SARS-CoV2 virus spike protein is thought to play a central role in binding to receptors on the surface of target cells and subsequent cell invasion. Therefore, it is considered that inhibition of binding of the peplomer protein to the receptor on the cell surface is important for the neutralizing activity of SARS-CoV2 virus infection. Therefore, among the clones obtained in Example 1, in Example 1. It was investigated whether the obtained 28 SARS-CoV2 virus-reactive clones had the activity of neutralizing the infection of SARS-CoV2 virus into cells.

One day before the infection experiment, Vero cells (1 x 10 ⁵ cells / ml) were prepared with cell culture medium D10 (DMEM (High Glucose), 10% FBS, penicillin / streptomycin added) per well, and each was placed on a 96-well plate. 100 μL was dispensed, 1 × 10 ⁴ cells were seeded and cultured at 37 ° C. in a 5% CO ₂ atmosphere for 18 to 20 hours.

On the day of the infection experiment, cell culture medium R15 (RPMI1640, 15% FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, non-) was added to 30 μL of the LCL culture supernatant for each of the 28 clones obtained in Example 1. (Essential amino acid, 2-mercaptomethanol, penicillin / sprepmaici added) was added in an amount of 70 μL to make a total volume of 100 μL. To this, 1 μL of 1 × 10 ⁵ TCID ₅₀ / mL SARS-CoV2 virus suspension was added so that the infected MOI was 0.01, and the total volume was 101 μL. The virus resuspension was incubated at 37 ° C. for 1 hour.

In Vero cell culture, remove culture D10 (100 μL), add the total amount of virus resuspension (101 μL) for each of the 28 clones, and incubate for 1 hour at 37 ° C. in a 5% CO ₂ atmosphere. , Cells were infected with SARS-CoV2 virus. Then, the virus resuspension was removed, 100 μL of cell culture medium D1 (DMEM (High Glucose), 1% FBS, penicillin / streptomycin added) was added, and the cells were cultured at 37 ° C. in a 5% CO ₂ atmosphere for 3 days. ..

After 3 days, the cell culture solution D1 was removed, 100 μL of 10% neutral buffered formaldehyde solution was added and fixed, CPE (cytopathic effect) was observed, and the SARS-CoV2 virus-reactive clone obtained in Example 1 was observed. Was examined whether SARS-CoV2 virus has neutralizing activity against cell infection. CPE was determined by counting the number of dead cells.

The screening of this example was performed in two parts, and the results of each are shown in FIGS. 1A and 1B. As a result, of the 28 clones of antibodies bound to the SARS-CoV2 virus spike protein, 7 clones of 7G7.1, # 8, # 10, # 18, # 20, # 26, and # 28 were neutralized against infection. The sum activity was positive (Fig. 1).

Example 3: Functional analysis of recombinant antibody In this example, the antibody gene was cloned for one clone found to have neutralizing activity against infection of SARS-CoV2 virus cells in Example 2. , An experiment was conducted for the purpose of producing a recombinant antibody.

(3-1) Acquisition of antibody gene In this example, RNA was directly extracted from the LCL prepared in Example 1 without producing a hybridoma, and an antibody was obtained by the RT-PCR method using the RNA. Gene cloning was performed.

The antibody genes (heavy chain, light chain) of 7G7,1 clones among the clones that showed neutralizing activity against infection in Example 2 in the in vitro functionality test of Example 2 (2-1) were cloned. , An expression vector was constructed. That is, total RNA was extracted from the LCL of the 7G7.1 clone using miRNeasy Microkit (QIAGEN) according to the attached protocol.

SMART cDNA Library Construction Kit (TAKARA) capable of amplifying 5'full-length cDNA using the total RNA extracted from the above method as a template for isolating the heavy chain IgG1 gene and the light chain IgL gene of the antibody gene. A reverse transcription reaction was carried out according to the attached protocol to prepare cDNA.

Using the cDNA synthesized by the above method as a template, two PCRs (1st PCR, 2nd PCR) were performed using KODFX (TOYOBO). In 1st PCR, 1 μl of cDNA reverse transcribed from each LCL was used as a template, and the following forward and reverse primers (Primer1) and reverse primer (Primer2) were used as primers, and after 2 minutes at 94 ° C, “98 ° C. 10 seconds, 55 ° C., 30 seconds, 68 ° C., 2 minutes] for 35 cycles, and finally 68 ° C., 3 minutes.

For 2nd PCR, 0.5 μl of the extension product of 1st PCR was used as a template, and the following forward and reverse primers (Primer3) and reverse primer (Primer4) were used as primers. After 2 minutes at 94 ° C, “98” The PCR reaction was carried out by 35 cycles of ° C. for 10 seconds, 60 ° C. for 30 seconds, 68 ° C. for 2 minutes, and finally at 68 ° C. for 3 minutes.

The PCR sample was subjected to QIAquick Purification Kit (QIAGEN), and unreacted primers and enzymes were removed to obtain a purified product.

The purified PCR purified product was treated with a restriction enzyme, separated and purified by agarose gel electrophoresis, and then incorporated into a vector. The pQEFIP vector (a retroviral vector in which the CMV promoter is replaced with the human EF1alpha promoter with pQCXIP (TAKARA) as the backbone) is used for the preparation of the vector containing the heavy chain gene, and the pQEFIN vector is used for the preparation of the vector containing the light chain gene. (A retrovirus vector in which the CMV promoter was replaced with the human EF1alpha promoter with pQCXIN (TAKARA) as the backbone) was used. The PCR product was incorporated into the vector using Ligation high (TOYOBO) to transform competent cells (DH-5alpha: Nippon Gene).

From Escherichia coli cultured in ampicillin-containing LB liquid medium, plasmid was extracted using NucleoSpin Plasmad EasyPure (MACHEREY-NAGEL) according to the attached protocol.

(3-2) Expression of recombinant antibody Expi 293F cells (Thermo) using the heavy chain and light chain expression plasmid vectors obtained by the method of Example 3 (3-1) using the Expi 293 Expression System (Thermo Fisher Scientific). Fisher Scientific) was transiently co-transfected to express and secrete recombinant antibody.

80 μL of ExpiFectamine 293 reagent and heavy and light chain plasmids (15 μg each) were mixed with 1.5 mL of Opti-MEM I (gibco) and allowed to stand at room temperature for 5 minutes. After standing, the two solutions were combined and allowed to stand at room temperature for 20 minutes, and added to 4.5 to 5.5 × 10 ⁶ cells / mL (30 mL) of cells cultured in a 125 mL flask (Corning), and swirled (37 ° C., 8% CO ₂ ). After culturing for 18 to 20 hours, 50 μL of Expi Fectamine 293 Transfection Enhancer 1 and 1.5 mL of Expi Fectamine 293 Transfection Enhancer 2 were added, and the cells were swirled for 5 to 6 days.

Recombinant antibody expressed by the expression system using Expi293F cells was purified using Protein G affinity chromatography (GE Hitrap protein GHP (1 mL)).

(3-3) Confirmation of reactivity of purified recombinant 7G7.1 antibody to SARS-CoV2 virus spike protein In order to examine the reactivity of purified recombinant antibody to SARS-CoV2 virus spike protein, the above-mentioned (1-2) ) Was used for ELISA. To examine the reactivity to SARS-CoV2 virus spike protein, 96 holes coated with 1 μg / mL SARS-CoV2 virus spike protein (SARS-CoV2 Spike ECD protein (GenScript)) at 50 μL / well on a 96-well plate. Using a plate for ELISA, purified recombinant antibody was added at 0.001, 0.01, 0.1, 1, 10 μg / mL as the primary antibody, and Goat Anti-Human IgG-AP was used as the secondary antibody. As the negative control, a human-derived antibody (hereinafter referred to as a control antibody) isolated by the inventors was used.

The result is shown in figure 2. As shown in this figure, it was confirmed that the purified recombinant 7G7.1 antibody binds to the SARS-CoV2 virus spike protein in a dose-dependent manner.

(3-4) In vitro functional analysis of purified recombinant 7G7.1 antibody Purified 7G7. Infection inhibitory activity of SARS-CoV2 virus was confirmed in the SARS-CoV2 virus infection neutralization experiment using Vero cells of Example 2. A luciferase assay was performed on one antibody to examine the neutralizing activity of SARS-CoV2 virus infection in cells.

On the day of the infection experiment, the cell culture solution D1 (DMEM (High Glucose), 1% FBS, penicillin / streptomycin) was adjusted to 10, 3, 1, 0.3, 0.1, 0.03, 0.01, 0.003, 0 μg / mL. Addition) was used to prepare 250 μL of each concentration solution. To this, 25 μL of SARS-CoV2 virus suspension introduced with a 1 × 10 ⁵ TCID ₅₀ / mL luciferase gene was added so as to have an infectious MOI of 0.1, and the total volume was 275 μL. The virus resuspension was incubated at 37 ° C. for 1 hour.

In Vero cell culture, culture medium D10 was removed, 100 μL of virus resuspension was added, and the cells were cultured for 1 hour in a 5% CO ₂ atmosphere at 37 ° C., and the cells were infected with SARS-CoV2 virus. Then, the virus resuspension was removed, 100 μL of cell culture medium D1 (DMEM (High Glucose), 1% FBS, penicillin / streptomycin added) was added, and the cells were cultured at 37 ° C. in a 5% CO ₂ atmosphere for 24 hours. ..

After 24 hours, the cell culture solution D1 was removed, and 50 μL of Passive lysis buffer was added to lyse the infected cells. 25 μL of infected cell lysate and 25 μL of substrate solution were mixed, and the luminescence of luciferase was measured. There is a positive correlation between luminescence intensity and viral infection.

The results are shown in Figure 3. 7G7.1 antibody can reduce virus-induced cell infection to 50% or less at a concentration of 0.01 μg / ml, and dose-dependently reduce virus-induced cell infection at higher concentrations. Revealed.

Example 4: Modification and functional analysis of recombinant 7G7.1 antibody In this example, an experiment was conducted for the purpose of modifying the recombinant 7G7.1 antibody produced in Example 3 and confirming its activity.

(4-1) Modification of 7G7.1 antibody (7G7.2) and expression of recombinant antibody The 7G7.1 antibody obtained in Example 3 (3-1) has IgG1 for the heavy chain and Igλ for the light chain. However, it was decided to modify the constant region of the light chain Igλ by replacing it with that of Igk. That is, for the heavy chain, the plasmid obtained in (3-1) above is used as it is, and for the light chain, the constant region of the Igλ gene contained in the Igλ plasmid obtained in (3-1) is used. A modified antibody was prepared by substituting the specified portion with a separately prepared portion of the Igk constant region.

Using the human Igk expression plasmid vector (self-prepared product) as a template, the fragment containing the human Igk constant region was PCR-amplified (using TOYOBO KOD plus neo) (reaction conditions: 94 ° C for 2 minutes). → [98 ℃ ・ 10 seconds, 58 ℃ ・ 30 seconds, 68 ℃ ・ 4.5 minutes] × 30 cycles → 68 ℃ ・ 5 minutes).

On the other hand, the 7G7.1 antibody Igλ2 light chain expression plasmid vector prepared in Example 3 (3-1) was used as a template, and the following primers were used to make the 7G7.1 antibody light chain (Igλ2) variable. PCR-amplified fragment containing region (using TOYOBO KOD plus neo) (Reaction conditions: 94 ° C for 2 minutes → [98 ° C for 10 seconds, 58 ° C for 30 seconds, 68 ° C for 30 seconds] × 30 cycles → 68 ℃ ・ 3 minutes).

Each of the two PCR products (human Igk constant region and variable region of 7G7.1 antibody (Igλ2)) obtained in the above step was treated with the restriction enzyme DpnI (NEB) at 37 ° C for 30 minutes and agarose gel. After separation by gel electrophoresis, DNA was extracted from the gel.

The obtained two types of DNA were mixed and treated with NEBuilder HiFi DNA Assembly Master Mix (NEB) at 50 ° C for 15 minutes, and each fragment was bound. This bound DNA was transformed into Escherichia coli according to a conventional method to obtain a single colony, and a 7G7.2 light chain expression plasmid vector was obtained.

The heavy chain expression plasmid vector obtained by the method of Example 3 (3-1) and the light chain expression plasmid vector obtained by the above method (Igλ2 constant region replaced with Igk constant region) were used. The recombinant antibody was expressed and purified in the same manner as in Example (3-2). Specifically, these expression plasmid vectors were transiently co-transfected into Expi293F cells (Thermo Fisher Scientific) using the Expi293 Expression System (Thermo Fisher Scientific) to express and secrete recombinant antibodies. .. The obtained modified antibody is hereinafter referred to as 7G7.2 antibody.

(4-2) Analysis of the binding strength of the 7G7.1 antibody and the 7G7.2 antibody to the antigen The binding strength of the 7G7.2 antibody obtained in (4-1) above to the antigen is determined by the antigen of the 7G7.1 antibody. Responsiveness to SARS-CoV2 virus spike protein was confirmed by comparison with the binding strength to SARS-CoV2 virus spike protein. The comparison of the bond strength was measured by the ELISA method in the same manner as the ELISA method described in Example 3 (3-3). As a negative control, a control antibody was used.

The results are shown in Fig. 4. As shown in this figure, it was confirmed that the 7G7.2 antibody also showed dose-dependent binding to the antigen as in the case of the 7G7.1 antibody.

(4-3) Affinity analysis of 7G7.1 antibody and 7G7.2 antibody by surface plasmon resonance (SPR) Binding of 7G7.1 antibody and 7G7.2 antibody obtained in this example to antigen Affinity was confirmed by surface plasmon resonance (SPR) analysis. As a negative control, a control antibody was used.

Biacore T200 (Cytiva) was used for SPR analysis, and the operation of the equipment and the preparation of experimental reagents followed the Biacore T200 version 2 Japanese instruction manual.

PBS-T (1 x Phsophate Buffered Saline, 0.05% Tween 20) was prepared as a running buffer, filtered through a 0.22 μm filter before use, and the running buffer was used at room temperature in all the following steps.

Using Anti-Human IgG (Fc) antibody (0.5 mg / mL) as the primary antibody and Human Antibody Capture Kit (Cytiva), the metal of Series Sensor Chip CM5 (Cytiva) (hereinafter referred to as CM5 sensor chip). The primary antibody was immobilized on a thin film substrate by an amine coupling method (AmineCouplingKit, Cytiva). That is, Anti-Human IgG (Fc) antibody (primary antibody, 0.5 mg / mL) was diluted to 25 μg / mL using the Immobilization buffer that is an accessory of Human Antibody Capture Kit, and the amount of primary antibody immobilized on the substrate. Diluted Anti-Human IgG (Fc) antibody was sent onto the substrate at a flow rate of 10 μL / min for 360 seconds so that the amount of antibody per flow cell was approximately 10,000 RU (Response unit), and the primary antibody was immobilized on the substrate. ..

Three types of test antibodies (7G7.1 antibody, 7G7.2 antibody and control antibody) at the following concentrations:
7G7.1 antibody: 1.0 μg / mL
7G7.2 antibody: 0.8 μg / mL
Control antibody: 0.8 μg / mL
Each test antibody was immobilized on the primary antibody on the substrate at a flow rate of 30 μL / min, an analysis temperature of 25 ° C., and a ligand immobilization time of 60 seconds. The immobilization conditions are such that the antibody concentration and the liquid feeding time are adjusted so that the amount of antibody immobilized on the substrate is about 150 RU.

Next, an antigen protein (SARS-CoV2 Spike protein (ECD, His & Flag Tag)) dissolved in a running buffer on a substrate on which three types of antibodies (7G7.1 antibody, 7G7.2 antibody and control antibody) were immobilized. ) (GenScript, Z03481) (135 kDa) adjusted to 0.625 nM, 1.25 nM, 2.5 nM, 5 nM, 10 nM, 20 nM, 40 nM, 80 nM in order, and the antibody addition time: 240 seconds, SPR measurement was performed with an analysis dissociation time of 900 seconds. After each SPR measurement for each concentration of antigen protein was completed, a Regeneration solution (3 M MgCl ₂ ) was sent to remove each test antibody immobilized on the CM5 sensor chip substrate.

The results are shown in Table 5 and Fig. 5 below. KD is a parameter for evaluating antibody affinity and is calculated from the binding rate constant ka and the dissociation rate constant kd (KD = kd / ka). Rmax is the theoretical maximum amount of antigen bound to an antibody.

The antigen protein (SARS-CoV2 Spike protein) bound to 7G7.1 antibody and 7G7.2 antibody with almost the same affinity (KD values of 2.04 nM and 1.55 nM, respectively) (Table 5) (Fig. 5). Each parameter shown in this table was calculated using the 1: 1 binding model of Biacore T200 Evaluation Software ver.2.0 based on the results shown in FIGS. 1 to 3. In general, the KD value of antibodies used as pharmaceuticals is about 10 ^-9 to 10 ^-10 M, indicating that these antibodies have sufficient affinity as anti-human SARS-CoV2 antibodies.

On the other hand, Rmax (a parameter indicating the reaction when the maximum amount of antigen protein is bound to the test antibody immobilized on the CM5 sensor chip substrate) is almost the same value for the 7G7.1 antibody and the 7G7.2 antibody (the parameter showing the reaction when the antigen protein is bound to the maximum amount). It was 164.1 RU and 151.3 RU, respectively), whereas it was about 1/10 (15.24 RU) of the control antibody used as a comparative control (Table 5), so the 7G7.1 antibody and 7G7.2 antibody were It was shown to have a high affinity for the antigenic protein compared to the negative control.

(4-4) In vitro functional analysis of purified recombinant 7G7.2 antibody The purified 7G7.2 antibody prepared in (4-1) above was subjected to a luciferase assay to neutralize SARS-CoV2 virus infection in cells. The activity was examined. The measurement of the neutralizing activity was examined by performing a luciferase assay by the same method as in Example 3 (3-4) except that the purified 7G7.2 antibody was used instead of the purified 7G7.1 antibody.

The results are shown in Fig. 6. Although the 7G7.2 antibody is less active than the 7G7.1 antibody, it reduces viral infection of cells to less than 50% at a concentration of 0.03 μg / ml, much more than the 7G7.1 antibody. It was clarified that the infection of cells by the virus can be reduced in a dose-dependent manner even at the concentration.

(4-5) In vivo functional analysis of modified antibody 7G7.2 antibody The above 7G7.2 antibody was subjected to a luciferase assay to examine the neutralizing activity of SARS-CoV2 virus infection to cells.

The SARS-CoV2 virus spike protein is thought to play a central role in binding to receptors on the surface of target cells and subsequent cell invasion. Therefore, it is considered that inhibition of binding of the peplomer protein to the receptor on the cell surface is important for the neutralizing activity of SARS-CoV2 virus infection. Therefore, among the clones obtained in Example 1, in Example 1. It was investigated whether the obtained SARS-CoV2 virus-reactive clone had the activity of neutralizing infection with SARS-CoV2 virus.

As an experimental animal, a 3-week-old male Syrian hamster Slc: Syrian (SLC) was used. Immediately before the infection experiment, inhalation anesthesia was performed using isoflurane for animals (MSD Animal Health). Then, 4 mg / kg of recombinant human anti-SARS-CoV2 antibody 7G7.2 antibody or negative control antibody was administered intravenously.

Subsequently, 10 ³ TICD 50/20 μL SARS-CoV2 suspension was nasally administered. On the 4th day after nasal infection, the lungs were dissected and the lungs were collected. A part of the lung was separated for virus copy count measurement and TCID50 measurement.

Regarding the number of virus copies, 1 mL of TRIzol Reagent (Thermo Fisher Scientific) was added to the separated lung tissue and physically crushed using a bead-type crusher to extract RNA. The amount of RNA per sample was 10 ng, and the measurement was performed by the real-time one-step RT-PCR method using the TaqMan probe according to the Pathogen Detection Manual 2019-nCoV (National Institute of Infectious Diseases).

On the other hand, for TCID50, the separated lung tissue was weighed, 0.5 mL PBS was added, and the tissue was physically crushed using a bead crusher, and the supernatant was collected after centrifugation. The supernatant was diluted 500-fold and further diluted 10-fold in 7 steps. The day before, 1 × 10 ⁴ cells were seeded in 100 μL of cell culture medium D10 (DMEM (High Glucose), 10% FBS, penicillin / streptomycin added) per well on a 96-well plate, and 37 ° C, 5% CO ₂ atmosphere. Vero cells cultured under 18 to 20 hours were prepared. Cells were prepared in 4 wells for each sample, and 50 μL of serially diluted lung tissue disruption solution was added to Vero cell culture medium and cultured at 37 ° C. in a 5% CO ₂ atmosphere for 4 days. The cytopathic effect in each well was observed and TCID50 / g was calculated. The Behrens-Karber method was used for the calculation.

The results are shown in Fig. 7. From the virus copy count and TCID50 measurement results, the 7G7.2 antibody-administered group significantly suppressed SARS-CoV2 infection compared to the negative control antibody-administered group. From this result, it was shown that the 7G7.2 antibody has a neutralizing activity against SARS-CoV2 even in vivo.

Example 5: Determination of binding region of modified 7G7.2 antibody In this example, an experiment was conducted to determine the binding region of the purified 7G7.2 antibody prepared in Example 4 above.

SARS-CoV2 Spike protein Extra Cellular Domain (ECD) (13 to 1208 amino acids), S1 (13 to 685 amino acids), S2 (686 to 1208 amino acids), N-terminal Domain (NTD) (13 to 318 amino acids), Receptor About BindingDomain (RBD) (319-541 amino acids), NTD + RBD (13-541 amino acids), S1ΔRBD (fusion protein of 13-318 amino acids and 542-685 amino acids), S1 (542-685) (542-685 amino acids) The DNA sequence in which the Flag-6 × His tag protein was fused to the C-terminal was amplified by the PCR method, introduced into the pFastBacBip vector, and cloned. These cloning vectors were introduced into insect cells Sf9 cells, and the culture supernatant was collected to collect the antigen. Bovine serum albumin (BSA) was used as the antigen for negative control.

These culture supernatants were diluted 10-fold, 50 μL was added to each well of a 96-well plate for ELISA, and the cells were solid-phased. Next, after blocking, 50 μL of 1 μg / mL recombinant human anti-SARS-CoV2 antibody 7G7.2 antibody or negative control antibody 8A7 antibody was added. Goat Anti-Human IgG-Ap was used as a secondary antibody, the substrate was reacted, and the absorbance at 405 nm was measured.

The results are shown in Fig. 8. 7G7.2 antibody is Extra Cellular Domain (ECD) (13 to 1208 amino acids), S1 (13 to 685 amino acids), S1ΔRBD (fusion protein of 13 to 318 amino acids and 542 to 685 amino acids), NTD + RBD (13 to 541 amino acids). These results indicate that the 7G7.2 antibody is likely to bind to the S1 domain of the spike protein, especially the NTD domain.

Example 6: Sequence analysis of recombinant antibody In this example, sequence analysis of two types of antibodies, recombinant 7G7.1 antibody obtained in Example 3 and recombinant 7G7.2 antibody variant obtained in Example 4 The purpose was to do.

The DNA sequences of the vectors for expressing the respective antibodies of the recombinant 7G7.1 antibody and the 7G7.2 variant antibody were analyzed, and the amino acid sequences were identified based on the DNA sequences. Both the 7G7.1 antibody and the 7G7.2 antibody have the same heavy chain variable region and light chain variable region. The amino acid sequence of each antibody was as follows:
・ 7G7.1 clone:
Heavy IgG1 chain (SEQ ID NO: 9) (underlined in VH region (SEQ ID NO: 7));
MEFGLSWVFLVALLRGVQC QVQLVESGGGVVQPGRSLRLSCIASGFTFRNYAMYWVRQAPGKGLEWVAVIWYDGSNKFYTDSVKGRFTISRDNSKNSLYLQMNSLRAEDTAVYFCARDQGFGDNYYYYGMDVWGQGTTVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNHYTQKSLSLSPGK
Light chain Igλ 2 chain (SEQ ID NO: 10) (underlined in VL region (SEQ ID NO: 8)):
MAWIPLLLPLLTLCTGSEA SYELTQPVSVSPGQTARISCSGDALPKEYAYWYQQKPGQAPVLVIYKDSERPSGIPERFSGSSSGTTVTLTISGVQAEDEADYYCQLADSSMHYVVFGGGTKLTVL GQPKAAPSVTLFPPSSEELQAKS
7G7.2 clone (replacement of the constant region of the light chain Igλ chain of the 7G7.1 clone with the constant region of the Igk chain):
Heavy chain IgG1 chain (SEQ ID NO: 9);
Light chain (7G7.1 clone light chain Igλ chain constant region replaced with Igk chain constant region) (SEQ ID NO: 11) (VL region (SEQ ID NO: 8) underlined):
MAWIPLLLPLLTLCTGSEA SYELTQPPSVSPGQTARISCSGDALPKEYAYWYQQKPGQAPVLVIYKDSERPSGIPERFSGSSSGTTVTLTISGVQAEDEADYYCQLADSSMHYVVFGGGTKLTVL RTVAAPSVFIFPPSDEQLKSGTASVVC

As a result of analyzing these sequences, it was confirmed that the complementarity determining regions (CDR1 to CDR3) among the variable regions of the heavy chain and the light chain of each clone are as follows:
・ 7G7.1 clone and 7G7.2 clone:
VH chain (SEQ ID NO: 7) (CDR1 (SEQ ID NO: 1), CDR2 (SEQ ID NO: 2), CDR3 (SEQ ID NO: 3) are underlined):
QVQLVESGGGVVQPGRSLRLSCIAS GFTFRNYA MYWVRQAPGKGLEWVAV IWYDGSNK FYTDSVKGRFTISRDNSKNSLYLQMNSLRAEDTAVYFC ARDQGFGDNYYYYGMDV WGQGTTVTVSS
VL chain (SEQ ID NO: 8) (CDR1 (SEQ ID NO: 4), CDR2 (SEQ ID NO: 5), CDR3 (SEQ ID NO: 6) are underlined):
SYELTQPPSVSVSPGQTARISCSGD ALPKEY AYWYQQKPGQAPVLVIY KDS ERPSGIPERFSGSSSGTTVTLTISGVQAEDEADYYC QLADSSMHYVV FGGGTKLTVL.

7G7.1 and 7G7.2 antibody heavy chain complementarity determining regions: CDR1 (GFTFRNYA, SEQ ID NO: 1), CDR2 (IWYDGSNK, SEQ ID NO: 2), and CDR3 (ARDQGFGDNYYYYGMDV, SEQ ID NO: 3) )
Complementarity determining regions of the light chains of 7G7.1 and 7G7.2 antibodies: CDR1 (ALPKEY, SEQ ID NO: 4), CDR2 (KDS, SEQ ID NO: 5), and CDR3 (QLADSSMHYVV, SEQ ID NO: 6). )
Amino acid sequence of heavy chain variable region VH domain of 7G7.1 antibody and 7G7.2 antibody: SEQ ID NO: 7
Amino acid sequence of light chain variable region VL domain of 7G7.1 antibody and 7G7.2 antibody: SEQ ID NO: 8
7G7.1 antibody and 7G7.2 antibody heavy chain full-length amino acid sequence: SEQ ID NO: 9
7G7.1 antibody light chain full length amino acid sequence: SEQ ID NO: 10
7G7.2 antibody light chain full length amino acid sequence: SEQ ID NO: 11
Primer sequence used in Example 3 (3-1): SEQ ID NO: 12 to SEQ ID NO: 18
Primer sequence used in Example 4 (4-1): SEQ ID NO: 19 to SEQ ID NO: 22

Claims

An antibody or antibody derivative that does not contain blood-derived components and has a binding property to the spike protein constituting the SARS-CoV2 virus and has an effect of neutralizing the infection of the SARS-CoV2 virus.
The antibody or antibody derivative according to claim 1, wherein the antibody is a human antibody.
The antibody or antibody derivative according to claim 1 or 2, which is produced by genetic recombination.
13. The antibody or antibody derivative described.
The antibody or antibody derivative according to claim 4, wherein the functional fragment is F (ab') 2.
Heavy chain complementarity determining regions, including CDR1 (GFTFRNYA, SEQ ID NO: 1), CDR2 (IWYDGSNK, SEQ ID NO: 2), CDR3 (ARDQGFGDNYYYYGMDV, SEQ ID NO: 3)
Contains light chain complementarity determining regions, CDR1 (ALPKEY, SEQ ID NO: 4), CDR2 (KDS, SEQ ID NO: 5), CDR3 (QLADSSMHYVV, SEQ ID NO: 6),
The antibody or antibody derivative according to any one of claims 1 to 5.
The heavy chain variable region VH domain is the amino acid sequence of SEQ ID NO: 7 or the amino acid sequence of SEQ ID NO: 7, CDR1 (SEQ ID NO: 1), CDR2 (SEQ ID NO: 2), and CDR3 (. SEQ ID NO: Contains an amino acid sequence containing substitutions, insertions, or deletions of one or several amino acids in parts other than 3).
The antibody or antibody derivative according to any one of claims 1 to 6.
The light chain variable region VL domain is the amino acid sequence of SEQ ID NO: 8 or the amino acid sequence of SEQ ID NO: 8, CDR1 (SEQ ID NO: 4), CDR2 (SEQ ID NO: 5), and CDR3 (. SEQ ID NO: Contains an amino acid sequence containing substitutions, insertions, or deletions of one or several amino acids in parts other than 6).
The antibody or antibody derivative according to any one of claims 1 to 7.
Of the amino acid sequence of SEQ ID NO: 9 or the amino acid sequence of SEQ ID NO: 9, the heavy chain is CDR1 (SEQ ID NO: 1), CDR2 (SEQ ID NO: 2), and CDR3 (SEQ ID NO: 1). Contains an amino acid sequence containing substitutions, insertions, or deletions of one or several amino acids in parts other than 3).
The antibody or antibody derivative according to any one of claims 1 to 8.
The light chain
Amino acid sequence of SEQ ID NO: 10 or amino acid sequence of SEQ ID NO: 10 other than CDR1 (SEQ ID NO: 4), CDR2 (SEQ ID NO: 5), and CDR3 (SEQ ID NO: 6) Of the amino acid sequence containing substitutions, insertions, or deletions of one or several amino acids in a portion, or the amino acid sequence of SEQ ID NO: 11, or the amino acid sequence of SEQ ID NO: 11, CDR1 (SEQ ID NO: 4) ), CDR2 (SEQ ID NO: 5), and amino acid sequences containing substitutions, insertions, or deletions of one or several amino acids in parts other than CDR3 (SEQ ID NO: 6).
The antibody or antibody derivative according to any one of claims 1 to 9, which comprises.
A pharmaceutical composition for neutralizing SARS-CoV2 virus infection, which comprises the antibody or antibody derivative according to any one of claims 1 to 10.
The pharmaceutical composition according to claim 11, which comprises in combination with another antibody or antibody derivative.