KR101561665B1 - Specific antibody for ha protein of a/h1n1 influenza virus, and hybridoma producing the same - Google Patents

Abstract

The present invention relates to an antibody which is specifically combined to HA protein of an influenza A/H1N1 virus, a method for detecting an influenza A virus by using the antibody, a kit for the method, a hybridoma cell line producing the antibody, and a composition for treating influenza using the hybridoma producing the antibody. The antibody is selected from a group comprising: (i) a monoclonal antibody produced by hybridoma cell line 4E3 (Accession No. KCLRF-BP-00343); (ii) a monoclonal antibody produced by hybridoma cell line 5E9 (Accession No. KCLRF-BP-00345); and (iii) a monoclonal antibody produced by hybridoma cell line 12D12 (Accession No. KCLRF-BP-00342).

Description

H1N1 INFLUENZA VIRUS, AND HYBRIDOMA PRODUCING THE SAME, which is an antibody specific to the HA protein of A / H1N1 influenza virus and a hybridoma cell line producing the same,

(I) a monoclonal antibody produced by hybridoma cell line 4E3 (Accession No. KCLRF-BP-00343); (ii) a monoclonal antibody produced by hybridoma cell line 5E9 (Accession No. KCLRF-BP-00345); And (iii) a monoclonal antibody produced by a hybridoma cell line 12D12 (Accession No. KCLRF-BP-00342), which specifically binds to the HA protein of influenza A virus, A method for detecting influenza A virus, a kit for the method, a hybridoma cell line producing the antibody, and a composition for treating influenza comprising the antibody and a base sequence of a complementary crystal zone (CDR) of a light chain and a heavy chain .

Influenza A or B viruses cause pandemic disease almost every year in all countries and are the leading cause of death. In the United States, pandemic influenza is known to cause illness in about 20% of people, with an average annual death toll of 20,000 and 114,000 inpatients.

Influenza viruses are RNA enveloped viruses with a diameter of about 125 nm. Influenza viruses consist essentially of an internal nucleocapsid or core of ribonucleic acid (RNA) associated with a nuclear protein surrounded by a lipid bilayer structure and a viral envelope with an external glycoprotein. The inner layer of the viral envelope is composed mainly of substrate proteins and the outer layer is composed mostly of host-derived lipid material. Influenza viruses include two surface antigens, the glycoprotein neuraminidase (NA) and hemagglutinin (HA), which appear as spikes of 10-12 nm in length on the surface of the particles. It is these surface proteins, in particular hemagglutinin, that determine the antigen specificity of the influenza subtype. The virus is classified according to the serologic characteristics of the HA and NA subtype of the host species, segregation area and year, serial number, and influenza A of origin, and 18 HA subtypes (H1-H18) and 11 NA subtypes (H16) was reported (Webster RG et al., Evolution and ecology of influenza viruses. Microbiol. Rev. 1992: 56: 152-179; Fouchier RA et al., Characterization of a Novel Influenza A Virus Hemagglutinin Subtype Obtained from Black-Headed Gulls, J. Virol., 2005: 79: 2814-2822). All HA and NA subtypes have been able to infect aquatic birds, but three HA subtypes (H1, H2 and H3) and two NA subtypes (N1 and N2) have been established in humans since 1918 .

Influenza A virus continues to undergo antigenic changes (Wiley D, Skehel J. The structure and function of the hemagglutinin membrane glycoprotein of influenza virus, Ann. Rev. Biochem. 1987: 56: 365-394). The lack of effective proofreading by viral RNA polymerases results in high-rate transcription errors, which can lead to amino acid substitutions in the surface glycoprotein. The segmented viral genome allows for various antigenic changes. When two influenza viruses infect a host cell at the same time, genetic reassortment can create new viruses with new surface or internal proteins. These antigenic changes, referred to as 'drift' and 'shift', are unpredictable and ultimately induce the emergence of new influenza viruses and allow these viruses to escape the immune system, It can have a significant impact on the outbreak that occurs. These genetic characteristics have created new virus variants that are responsible for pandemics in humans.

In particular, HA is the most important antigen in defining the serological specificity of different influenza viruses. The 75-80 kD protein contains a large number of antigenic determinants, some of which are specific for some viruses, and other determinants are common to most HA molecules. Influenza viruses cause epidemics at an infection rate as high as 40% over almost a year, usually over a month or more (6 weeks) each year, and cause a variety of disease states, from latent infections through mild upper respiratory infections to severe viral pneumonia. A typical influenza epidemic may increase the incidence of pneumonia and lower respiratory disease, the severity of which may vary mainly in the age of the host, its immune status and the site of infection.

As an antigen for the production of antibodies against influenza virus, recombinant antigens mainly used for animal cells or via the baculovirus system are used. Korean Patent Application No. 2013-7014167 discloses a new vaccine against A / H1N1 pandemic influenza virus, etc. However, since the recombinant antigen production method used in the past has a high production cost, production and supply are not smooth Moreover, due to the high price, researchers could not easily buy it. Accordingly, there is a need for a simple and economical method for producing an antigen and an antibody / virus detection kit using the same.

Since the influenza antigen undergoes a secondary mutation through glycosylation after its expression, most of the recombinant antigens are produced using animal cells or the baculovirus system. However, such a system has a disadvantage in that it is not easy to produce and supply due to high production cost, and it can not be easily purchased by researchers at a high price.

Accordingly, the present inventors produced antibodies against hemagglutinin using a virus that is not a recombinant antigen and established hybridomas.

However, the problems to be solved by the present invention are not limited to the above-mentioned problems, and other problems not mentioned can be clearly understood by those skilled in the art from the following description.

A first aspect of the invention is a kit comprising: (i) a monoclonal antibody produced by hybridoma cell line 4E3 (Accession No. KCLRF-BP-00343); (ii) a monoclonal antibody produced by hybridoma cell line 5E9 (Accession No. KCLRF-BP-00345); And (iii) a monoclonal antibody produced by hybridoma cell line 12D12 (Accession No. KCLRF-BP-00342), and specifically binds to the HA protein of influenza A / H1N1 virus .

A second aspect of the present application is a method for detecting a protein comprising the steps of: a) contacting a sample with an antibody according to the first aspect of the invention; b) detecting influenza A / H1N1 virus in the sample, comprising detecting the reaction of the antibody and the virus.

A third aspect of the invention relates to a kit for detecting influenza A / H1N1 virus in a sample, comprising an antibody according to the first aspect of the invention attached to a substrate, and a detectably labeled secondary antibody.

A fourth aspect of the present invention is a kit comprising: (i) a hybridoma cell line 4E3 (Accession No. KCLRF-BP-00343); (ii) hybridoma cell line 5E9 (Accession No. KCLRF-BP-00345); And (iii) a hybridoma cell line 12D12 (Accession No. KCLRF-BP-00342).

A fifth aspect of the present invention relates to a composition for treating influenza comprising an antibody according to the first aspect of the present application and a pharmaceutically acceptable excipient and using the base sequence of the light chain and heavy chain complementarity determining region (CDR) of the antibody will be.

In the case of using recombinant antigens, in order to solve production and supply problems due to high unit price, it is possible to produce antibodies by using influenza virus, establish a hybridoma cell line for production of monoclonal antibodies thereof, Thereby preparing a kit for the detection of influenza A / H1N1 virus, and a composition for treating influenza using the base sequence of the light chain and heavy chain complementarity determining region (CDR) of the antibody.

FIG. 1 (a) is an image showing the virus titer used in one embodiment of the present invention, FIG. 1 (b) is an image obtained by confirming a gene through RT-PCR for a virus vaccine strain used in one embodiment of the present invention, In the example, plaque analysis was used to check the virus titers used.
FIG. 2 is a result of immunological induction in a mouse according to one embodiment of the present invention and then examining the serum thereof by the HI method.
FIG. 3 is a table showing cross-reaction test results of hybridomas produced according to one embodiment of the present invention.
FIG. 4 is a table showing a result of isotyping test of a hybridoma produced according to an embodiment of the present invention.
FIG. 5 is a table showing ELISA results of antibodies produced from hybridomas produced according to one embodiment of the present invention. FIG.
FIG. 6 is a table showing HI potency analysis results of an antibody produced from a hybridoma produced according to an embodiment of the present invention.

Hereinafter, embodiments and examples of the present invention will be described in detail with reference to the accompanying drawings, which will be readily apparent to those skilled in the art to which the present invention pertains.

It should be understood, however, that the present invention may be embodied in many different forms and is not limited to the embodiments and examples described herein. In order to clearly illustrate the present invention, parts not related to the description are omitted, and similar parts are denoted by like reference characters throughout the specification.

Throughout this specification, when an element is referred to as "including " an element, it is understood that the element may include other elements as well, without departing from the other elements unless specifically stated otherwise.

As used herein, the terms "about," " substantially, "and the like are used herein to refer to or approximate the numerical value of manufacturing and material tolerances inherent in the stated sense, Accurate or absolute numbers are used to prevent unauthorized exploitation by unauthorized intruders of the mentioned disclosure. Also, throughout the present specification, the phrase " step "or" step "does not mean" step for.

Throughout this specification, the term "combination thereof" included in the expression of the machine form means one or more combinations or combinations selected from the group consisting of the constituents described in the expression of the machine form, And the like.

Throughout this specification, the description of "A and / or B" means "A or B, or A and B".

Throughout this specification, "antibody" refers to any immunoglobulin that binds to a specific antigen, such as a monoclonal antibody, polyclonal antibody, multispecific or bispecific antibody. Whole antibodies include two heavy chains and two light chains. Each heavy chain consists of a variable region and first, second and third constant regions, while each light chain consists of one variable region and one constant region. The antibody has a " Y "shape, and the stem of the Y consists of a second and a third constant region of two heavy chains joined together through a disulfide bond. Each arm of the Y consists of a single variable heavy zone and a first constant zone coupled to a variable and constant zone of a single light chain. The variable regions of the light and heavy chains are responsible for antigen binding. In both light and heavy chains, the variable region comprises three highly variable loops, commonly referred to as complementarity determining regions (CDRs) (light chain (L) CDRs include LCDR1, LCDR2 and LCDR3, As defined in Kabat, et al., Sequences of Proteins of Immunological Interest, Fifth Edition (1991), vol. 1-3, NIH Publication 91-3242, Bethesda Md. being). The three CDRs are interposed between adjacent flanking stretches known as framework regions (FRs) that are more highly conserved than the CDRs and form the framework supporting the hypervariable loops. The constant regions of heavy and light chains are not involved in antigen binding, but exhibit various effects and functions.

Hereinafter, the antibody, the method for detecting influenza A virus using the antibody, the kit for the method, the hybridoma cell producing the antibody, and the base sequence of the light chain and heavy chain complementarity determining region (CDR) The composition for treating influenza will be described in detail with reference to the embodiments and examples and the drawings. However, the present invention is not limited to these embodiments and examples and drawings.

A first aspect of the invention is a kit comprising: (i) a monoclonal antibody produced by hybridoma cell line 4E3 (Accession No. KCLRF-BP-00343); (ii) a monoclonal antibody produced by hybridoma cell line 5E9 (Accession No. KCLRF-BP-00345); And (iii) a monoclonal antibody produced by a hybridoma cell line 12D12 (Accession No. KCLRF-BP-00342), and which specifically binds to the HA protein of the influenza A / H1N1 virus .

For example, the antibody may be, but is not limited to, those generated from a mouse inoculated with an influenza vaccine strain A / California / 07/09.

Influenza vaccine A / California / 07/09 (A / H1N1) is a WHO-designated 2009 pandemic virus that is used as a vaccine from the 2010/2011 season until May 2015.

Such an antibody can be produced by immunogenicity and antibody purification methods well known in the art.

For example, the antibody may be used to inhibit an increase in influenza virus titer, to reduce influenza virus titer, to reduce influenza virus replication or proliferation, or to reduce one or more symptoms or complications associated with influenza virus infection in a subject But may not be limited thereto.

For example, the antibody may be a monoclonal antibody, and the monoclonal antibody may be prepared by a hybridoma method well known in the art.

According to one embodiment of the invention, the antibody may be selected from but not limited to IgG, IgA, IgM, IgE, and IgD isotypes.

According to one embodiment of the invention, the IgG isotypes may be selected from IgG _1, IgG _2, IgG ₃ and IgG _4, but may not be limited thereto.

According to one embodiment of the disclosure, the antibody may be, but not limited to, inhibiting influenza virus infection, influenza virus replication or influenza virus replication of cells in vitro or in vivo.

According to one embodiment of the invention, the influenza A / H1N1 virus may be but is not limited to the human influenza A / H1N1 virus.

A second aspect of the present application is a method for detecting a protein comprising the steps of: a) contacting a sample with an antibody according to the first aspect of the invention; b) detecting the reaction of said antibody with the virus. < Desc / Clms Page number 5 >

Although a detailed description of the parts overlapping with the first aspect of the present application is omitted, the description of the first aspect of the present application may be applied to the second aspect of the present invention.

The method of detecting influenza virus may be by any method of virus detection known in the art.

According to one embodiment of the present application, the sample may be selected from the group consisting of tissue, cells, nasal mucus, throat secretions, blood and saliva, but may not be limited thereto.

According to one embodiment of the invention, the reaction may be detected by enzymatic colorimetry, fluorescence analysis or chemiluminescence assay, but may not be limited thereto.

According to one embodiment of the invention, the influenza A / H1N1 virus may be, but is not limited to, a human A / H1N1 influenza virus.

A third aspect of the invention provides a kit for detecting influenza A / H1N1 virus in a sample, comprising an antibody according to the first aspect of the invention attached to a substrate, and a detectably labeled secondary antibody have.

Although a detailed description of the parts overlapping with the first aspect and the second aspect of the present application is omitted, the description of the first and second aspects of the present application will be applied equally to the third aspect of the present invention .

For example, the kit can be used to treat (prevent or treat), inhibit, prevent, or sensitize one or more symptoms or complications associated with an influenza virus infection of a subject by one or more influenza species / isolates or subtypes Or to reduce the progression, severity, frequency, duration or likelihood of such symptoms or complications. In other embodiments, the kit may comprise a product, such as an aerosol, spray or squeeze bottle, or other delivery means suitable for inhalation or nasal administration to a subject.

For example, the kit or product may comprise an antiviral agent (e.g., antibody or drug) or a substance that inhibits one or more symptoms or complications associated with influenza infection.

According to one embodiment of the present invention, the secondary antibody may specifically bind to the influenza A / H1N1 virus but may not be limited thereto.

According to one embodiment of the invention, the influenza A / H1N1 virus may be, but is not limited to, a human A / H1N1 influenza virus.

A fourth aspect of the present invention is a kit comprising: (i) a hybridoma cell line 4E3 (Accession No. KCLRF-BP-00343); (ii) hybridoma cell line 5E9 (Accession No. KCLRF-BP-00345); And (iii) a hybridoma cell line 12D12 (Accession No. KCLRF-BP-00342).

Although the detailed description of the parts overlapping with the first aspect to the third aspect of the present application is omitted, the description of the first to third aspects of the present invention is applied equally to the fourth aspect of the present invention .

The hybridomas can be prepared by a hybridoma preparation method well known in the art. In the hybridoma preparation method, the mouse or any suitable host animal is immunized with one or more injections of an immunizing agent and, where appropriate, an adjuvant. Typically, the immunizing agent and / or adjuvant may be injected into the host animal by multiple subcutaneous or intraperitoneal injections. It may be useful to conjugate to a protein known to be immunogenic in a host animal that will immunize the immunizing agent, for example, serum albumin or soybean trypsin inhibitor. Examples of antigenic adjuvants that may be used include Freund's complete antigen adjuvant and MPL-TDM. After immunization, the host animal produces a lymphocyte capable of producing or producing an antibody that will specifically bind to the antigen used for immunization. Alternatively, the lymphocytes may be immunized in vitro. The desired lymphocytes may be collected and fused with myeloma cells or the like using a suitable fusing agent, such as polyethylene glycol, to form hybridoma cells, but the present invention is not limited thereto.

A fifth aspect of the invention provides an antibody according to the first aspect of the invention; And a pharmaceutically acceptable excipient, and can provide a composition for treating influenza using the base sequence of the light chain and heavy chain complementarity determining region (CDR) of the antibody.

Although the detailed description of the parts overlapping with the first to fourth aspects of the present application has been omitted, the description of the first to fourth aspects of the present application is not limited to the fifth aspect of the present invention, .

For example, the pharmaceutically acceptable excipient may be a preservative, a dispersant, a surface active agent, an emulsifier, a buffer, a solvent, a diluent, a liquid vehicle, a dispersing or suspending aid, an isotonic agent, But are not limited to, those selected from the group consisting of solid binders, lubricants, and combinations thereof.

For example, the antibodies included in the influenza therapeutic compositions herein may inhibit infection of cells by one or more influenza virus species in vitro or in vivo. For example, the antibody may inhibit, prevent, or reduce the activity of one or more influenza virus species or an increase in the amount of influenza virus protein. For example, the antibody may protect a subject from infection by one or more influenza virus species or reduce the susceptibility of the subject to the infection.

For example, the composition for treating influenza can be used to prevent an increase in virus titer, reduce viral titer, decrease viral replication or proliferation, or prevent viral infection , But may not be limited thereto. ≪ RTI ID = 0.0 > [0050] < / RTI >

For example, the symptom or complication may be chill, fever, cough, sore throat, nasal obstruction, obstruction, nasal infection, sinus infection, systemic disease, headache, fatigue, pneumonia, bronchitis, ear infection, But the present invention is not limited thereto.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

[Example]

Virus purification

A / California / 07/09 was used as an antigen virus strain. In the production of viruses, fertilized eggs were used to obtain high productivity. The antiviral response index used was confirmed by HA analysis (Fig. 1A) and confirmed by RT-PCR (Fig. 1B). In addition, the virus vaccine strain proliferated using fertilized eggs was confirmed by plaque analysis (FIG. 1C).

The viruses produced by using 50 fertilized eggs exhibited a titer of 64-256HA and a total of 500 ml was recovered. The virus was centrifuged at a high speed and concentrated 150 times.

The recovered virus was precipitated with a high-speed centrifuge (32 Ti, Backman) at 25,000 rpm for 2 hours. After removing the supernatant, 200 μl of PBS per tube (pH 7.4) Lt; 0 > C, o / n and used as an antigen.

Mouse immunization using virus antigens

The mice were injected with the influenza virus prepared in the above example as an antigen to induce an immune response. Blood samples were collected after each immunization, and immunity was confirmed by ELISA and HI (Hemaglutinin Inhibition) test. After the third boost, the immunological level was confirmed (FIG. 2), and a hybridoma was produced.

Hybridoma screening and clonal screening

B cells obtained from immunized mice were fused with myeloma cells to produce hybridomas. The resulting hybridoma cells were subjected to HI test to obtain 14 clones in primary and secondary selection. Among them, five clones that did not cross-react with H3N2 and B type influenza virus were obtained, followed by third round monoclonalization (Fig. 3). Of the clones, 4E3, 5E9 and 12D12 were finally screened for A / H1N1 monoclonal antibody candidates.

A / H1N1 monoclonal antibody candidates were tested for isotype by isotype testing (FIG. 4). As a result of the test, 4E3 was identified as IgG2b, 5E9 as IgG1, and 12D12 as IgG2a. In addition, ELISA test was performed using the antigen used in immunization to confirm the antibody titer (Fig. 5). The results of measuring the antibody HI titer of A / H1N1 monoclonal antibody candidates are shown in FIG. As shown in FIG. 6, it can be seen that the A / H1N1 monoclonal antibodies of the present invention have an excellent effect indicating a high HI titer.

Selected hybridoma cell lines were deposited with the Korean Cell Line Bank (KCLB) on April 6, 2015. Hybridoma cell line 4E3 was deposited with accession number KCLRF-BP-00343, hybridoma cell line 5E9 with accession number KCLRF-BP-00345, and hybridoma cell line 12D12 with accession number KCLRF-BP-00342.

It will be understood by those of ordinary skill in the art that the foregoing description of the embodiments is for illustrative purposes and that those skilled in the art can easily modify the invention without departing from the spirit or essential characteristics thereof. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive. For example, each component described as a single entity may be distributed and implemented, and components described as being distributed may also be implemented in a combined form.

The scope of the present invention is defined by the appended claims rather than the detailed description, and all changes or modifications derived from the meaning and scope of the claims and their equivalents should be interpreted as being included in the scope of the present invention .

Korea Cell Line Research Foundation KCLRFBP00342 20150406 Korea Cell Line Research Foundation KCLRFBP00343 20150406 Korea Cell Line Research Foundation KCLRFBP00345 20150406

Claims (15)

(i) a monoclonal antibody produced by hybridoma cell line 4E3 (Accession No. KCLRF-BP-00343);
(ii) a monoclonal antibody produced by hybridoma cell line 5E9 (Accession No. KCLRF-BP-00345); And
(iii) monoclonal antibody produced by hybridoma cell line 12D12 (Accession No. KCLRF-BP-00342)
, And specifically binds to the HA protein of the influenza A / H1N1 virus.
delete delete The method according to claim 1,
An antibody that inhibits influenza virus infection, influenza virus multiplication or influenza virus replication of cells in vitro or in vivo.
The method according to claim 1,
The influenza A / H1N1 virus is an antibody against human influenza A / H1N1 virus.
The method according to claim 1,
Lt; RTI ID = 0.0 > influenza < / RTI > vaccine strain A / California / 07/09.
a) contacting the sample with the antibody of claim 1;
b) detecting the reaction of the antibody with the virus
Lt; RTI ID = 0.0 > A / H1N1 < / RTI > virus in a sample isolated from human body.
8. The method of claim 7,
The sample isolated from the human body is selected from the group consisting of a tissue isolated from a human body, a cell isolated from a human body, a runny nose separated from a human body, a throat secretion separated from a human body, a blood separated from a human body, How, one.
8. The method of claim 7,
Wherein the reaction is detected by enzymatic colorimetry, fluorescence analysis or chemiluminescence assay.
8. The method of claim 7,
Wherein said influenza A / H1N1 virus is a human A / H1N1 influenza virus.
A kit for detecting an influenza A / H1N1 virus in a sample, comprising the antibody of claim 1 attached to a substrate, and a detectably labeled secondary antibody.
12. The method of claim 11,
Wherein said secondary antibody specifically binds to the influenza A / H1N1 virus.
12. The method of claim 11,
Wherein the influenza A / H1N1 virus is a human influenza A / H1N1 virus.
(i) hybridoma cell line 4E3 (Accession No. KCLRF-BP-00343);
(ii) hybridoma cell line 5E9 (Accession No. KCLRF-BP-00345); And
(iii) Hybridoma cell line 12D12 (Accession No. KCLRF-BP-00342)
Wherein the hybridoma cell line is selected from the group consisting of:
An antibody according to any one of claims 1 to 6; And
A composition for treating an influenza infection, comprising a pharmaceutically acceptable excipient.

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