Antibody for detecting influenza a virus and use thereof

Abstract

The present invention relates to an antibody for detecting influenza A virus and use thereof. An antibody or a functional fragment thereof, according to an aspect, can specifically detect influenza A virus, and, in particular, specifically binds to subtypes H3N2 and H1N1 of influenza A virus and can be effectively used for early diagnosis or prognosis prediction of influenza A virus infection.

Description

Antibody for detecting influenza A virus and its use

[0001] This invention relates to an antibody for detecting influenza type A virus and its use.

[0002]

[0003] Influenza, commonly known as the flu, is an infectious disease of birds and mammals caused by RNA viruses of the Orthomyxoviridae family (influenza viruses), whose antigenicity changes continuously. Of the three groups of influenza viruses (A, B, and C), Type A viruses are negative sense single-stranded RNA viruses with an 8-segment genome and are further classified into 16 types of hemagglutinin (HA) and 9 types of neuraminidase (NA). The viral HA and NA proteins are envelope glycoproteins and are major antigens that direct the humoral immune response. Two subtypes of influenza A (H3N2 and H1N1) have been circulating in human populations for 30 to 40 years.

[0004] Recently, the new influenza A H1N1 (swine flu) virus was declared a global pandemic by the World Health Organization (WHO). An influenza pandemic occurs when a new species of influenza virus is transmitted from other animal species to humans. In the 20th century, three influenza pandemics occurred—the 1918 Spanish flu (H1N1), the 1957 Asian flu (H2N2), and the 1968 Hong Kong flu (H3N2)—killing tens of millions of people.

[0005] Rapid detection of the influenza virus is essential for prompt patient management and the implementation of public health alerts. Antiviral agents such as zanamivir or oseltamivir (Tamiflu®) can significantly reduce the severity and duration of influenza. Furthermore, rapid treatment reduces the spread of transmission and facilitates effective public health management. Therefore, there is an urgent need to be able to rapidly test for the virus in public places such as airports, railways, and bus stops.

[0006] Influenza detection methods such as viral culture, reverse transcription polymerase chain reaction (RT-PCR), and point-of-care testing (POCT) are time-consuming and require facilities that are not easily accessible in some affected areas. Viral culture, considered the "gold standard," is impractical for clinical decision-making due to the delays in obtaining results. RT-PCR, which is more sensitive than viral culture for influenza detection, is not widely available clinically and is expensive. POCT should not be a routine test for influenza antigens but can be helpful at the onset of symptoms or before deciding to initiate antiviral therapy. Additionally, rapid influenza antigen detection tests (rapid tests), such as enzyme-linked immunosorbent assay (ELISA), erythrocyte agglutination inhibition, microneutralization, and agar gel precipitation, are relatively inexpensive and can provide timely information for clinical decision-making. However, rapid testing requires the development of specific antibodies capable of rapidly detecting and identifying the flu.

[0007] Therefore, the inventors recognized this need and completed the present invention to solve the problems of existing detection methods.

[0008]

[0009] One aspect is to provide an antibody for detecting influenza A virus or a functional fragment thereof.

[0010] Another aspect is to provide a composition for detecting influenza type A virus comprising the above antibody or a functional fragment thereof.

[0011] Another aspect is to provide a kit for detecting influenza A virus comprising the above antibody or a functional fragment thereof.

[0012] Another aspect is to provide a biochip for detecting influenza A virus comprising the above antibody or a functional fragment thereof.

[0013] Another aspect is to provide a method for detecting influenza A virus using the above antibody or a functional fragment thereof.

[0014] Another aspect is to provide a method for providing information on the diagnosis of influenza A virus infection using the above antibody or a functional fragment thereof.

[0015] Another aspect is to provide a polynucleotide encoding the aforementioned antibody or a functional fragment thereof.

[0016] Another aspect is to provide a recombinant vector containing the above-mentioned polynucleotide.

[0017] Another aspect is to provide recombinant cells containing the above vector.

[0018]

[0019] One aspect provides an antibody for detecting influenza type A virus or a functional fragment thereof comprising VH CDR1 having the amino acid sequence of SEQ ID NO. 1; VH CDR2 having the amino acid sequence of SEQ ID NO. 2; VH CDR3 having the amino acid sequence of SEQ ID NO. 3; VL CDR1 having the amino acid sequence of SEQ ID NO. 4; VL CDR2 having the amino acid sequence of SEQ ID NO. 5; and VL CDR3 having the amino acid sequence of SEQ ID NO. 6.

[0020] The above antibody or its functional fragment may comprise VH CDR1 having a sequence having at least 90% homology with the amino acid sequence of SEQ ID NO. 1; VH CDR2 having a sequence having at least 90% homology with the amino acid sequence of SEQ ID NO. 2; VH CDR3 having a sequence having at least 90% homology with the amino acid sequence of SEQ ID NO. 3; VL CDR1 having a sequence having at least 90% homology with the amino acid sequence of SEQ ID NO. 4; VL CDR2 having a sequence having at least 90% homology with the amino acid sequence of SEQ ID NO. 5; and VL CDR3 having a sequence having at least 90% homology with the amino acid sequence of SEQ ID NO. 6.

[0021] In one embodiment, the influenza A virus may be a subtype H3N2 or H1N1.

[0022] In one embodiment, the antibody or its functional fragment may be capable of simultaneously detecting influenza A virus subtypes H3N2 and H1N1.

[0023] In one embodiment, the antibody or its functional fragment may be selected from the group consisting of IgG, Fab, Fab', F(ab')2, xFab, scFab, dsFv, Fv, scFv, scFv-Fc, scFab-Fc, diabody (scFv2, diabody), minibody, scAb, dAb (domain antibody, VH or VL), Fc-scFv, scFv-Fc-scFv, IgG-scFv, scFv-IgG, and combinations thereof. Specifically, the antibody or its functional fragment may be scFv, scFv-Fc, diabody, or minibody.

[0024] In this specification, a “Fab fragment” consists of one light chain and one heavy chain comprising only a variable region and CH1. The heavy chain of the Fab molecule cannot form disulfide bonds with other heavy chain molecules. scFab is two Fab molecules connected by a flexible linker.

[0025] In this specification, the “Fab’ fragment” includes a region between the CH1 and CH2 domains of a heavy chain in addition to the Fab fragment, and can form a disulfide bond between the two heavy chains of two molecules of the Fab’ fragment that produce an F(ab’)2 molecule.

[0026] In this specification, the “F(ab')2 fragment” comprises two light chains and two heavy chains comprising a variable region, a portion of the CH1 and CH2 domains as mentioned above, and accordingly comprises two intrachain disulfide bonds. Thus, the F(ab')2 fragment is composed of two Fab' fragments, and the two Fab' fragments meet each other by a disulfide bond between them.

[0027] In this specification, "cross-Fab fragment," "xFab fragment," or "crossover Fab fragment" refers to a Fab fragment in which the variable or constant regions of the heavy chain and the light chain are exchanged. Two different chain compositions of the crossover Fab molecule are possible. The variable regions of the Fab heavy chain and the light chain may be exchanged. That is, the crossover Fab molecule comprises a peptide chain composed of a light chain variable region (VL) and a heavy chain constant region (CH1), and a peptide chain composed of a heavy chain variable region (VH) and a light chain constant region (CL). This crossover Fab molecule is also referred to as CrossFab(VLVH). Meanwhile, when the Fab heavy chain and the constant region of the light chain are exchanged, the crossover Fab molecule comprises a peptide chain composed of a heavy chain variable region (VH) and a light chain constant region (CL), and a peptide chain composed of a light chain variable region (VL) and a heavy chain constant region (CH1). This crossover Fab molecule is also referred to as CrossFab(CLCH1).

[0028] In this specification, "single-strand Fab fragment" or "scFab" is a polypeptide composed of an antibody heavy chain variable domain (VH), an antibody constant domain 1 (CH1), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL), and a linker. The antibody domains and the linker have one of the following sequences from the N-terminus to the C-terminus: a) VH-CH1-linker-VL-CL, b) VL-CL-linker-VH-CH1, c) VH-CL-linker-VL-CH1, or d) VL-CH1-linker-VH-CL; the linker is a polypeptide of at least 30 amino acids, preferably 32 to 50 amino acids. The single-strand Fab fragment is stabilized through a natural disulfide bond between the CL domain and the CH1 domain. In addition, these single-chain Fab molecules can be further stabilized by the creation of interchain disulfide bonds through the insertion of cysteine ​​residues (e.g., position 44 of the variable heavy chain and position 100 of the variable light chain according to Kabat numbering).

[0029] In this specification, a “Fv region” is a fragment of an antibody that includes each variable region of the heavy chain and light chain, but does not include a constant region. sdFV is a form in which the heavy chain and light chain are connected by disulfide bonds. scFv is a form in which single-stranded variable regions (scFv) of the heavy chain and light chain are connected through a flexible linker. scFv-Fc is a form in which Fc is connected to scFv and may be referred to as a maxibody. The minibody is a form in which CH3 is connected to scFv. The diabody comprises two molecules of scFv. The scFv contained in the antibody may include heavy chain variable regions and light chain variable regions in any order. For example, the scFv included in the antibody may include a heavy chain variable region and a light chain variable region in the direction from the N-terminus to the C-terminus, optionally a peptide linker between them, or alternatively, the antibody may include a light chain variable region and a heavy chain variable region in the direction from the N-terminus to the C-terminus, and optionally a peptide linker between them.

[0030] The above term "peptide linker" may comprise any 1 to 100, 2 to 50, or 10 to 25 amino acids, and any type of amino acid may be included without any limitation. The peptide linker may, for example, comprise Gly, Asn, and / or Ser residues, and may also comprise neutral amino acids such as Thr and / or Ala. An amino acid sequence suitable for the peptide linker may be known in the relevant art. Meanwhile, the length of the peptide linker may be varied within a range that does not affect the function of the antibody. For example, the peptide linker may be formed by comprising a total of about 1 to about 100, about 2 to about 50, or about 5 to about 25 of one or more selected from the group consisting of Gly, Asn, Ser, Thr, and Ala. In one embodiment, the peptide linker may be (G4S)n, (SG4)n, or G4(SG4)n, where "n" is generally a number between 1 and 10, typically 2 to 4. The peptide linker may be, for example, GGGGS, GGGGSGGGGS, SGGGGSGGGG, GGGGSGGGGSGGGG, GSPGSSSSGS, GGGGSGGGGSGGGGS, GSGSGSGS, GSGSGNGS, GGSGSGSG, GGSGSG, GGSG, GGSGNGSG, GGNGSGSG, or GGNGSG.

[0031] In this specification, a “short-chain antibody (scAb)” is a single polypeptide chain comprising one variable region of a heavy chain or a light chain constant region, wherein the heavy chain and the light chain variable region are connected by a flexible linker. Reference to U.S. Patent No. 5,260,203 may be made to the short-chain antibody, which is disclosed herein by reference.

[0032] In this specification, “domain antibody (dAb)” is an immunologically functional immunoglobulin fragment comprising only a variable region of a heavy chain or a variable region of a light chain.

[0033] In this specification, “whole-length IgG” is defined as essentially comprising complete IgG, but does not necessarily possess all the functions of complete IgG. Whole-length IgG comprises two heavy chains and two light chains. Each chain comprises constant (C) and variable (V) regions, which can be degraded into domains designated as CH1, CH2, CH3, VH and CL, VL.

[0034] In one embodiment, the Fc may include an amino acid sequence derived from a human, mouse, chicken, monkey, or camel.

[0035] In one embodiment, the antibody or its functional fragment may be a monoclonal antibody or a polyclonal antibody.

[0036] In this specification, “antibody” means any isotype of intact immunoglobulin or an intact antibody or antigen-binding fragment for binding to a target antigen. Examples include chimeric, humanized, fully human, and bispecific antibodies or antigen-binding fragments thereof. The antibody itself is a type of antigen-binding protein. The intact antibody generally comprises at least two full-length heavy chains and two full-length light chains, but in some cases, antibodies naturally found in camels may comprise only heavy chains. The antibody or its antigen-binding fragment may be derived from a single source or a chimera. The chimeric antibody comprises portions derived from two different types of antibodies, which are described in more detail below. The antibody or its antigen-binding fragment may be produced by a hybridoma, recombinant DNA technology, or enzymatic or chemical cleavage of an intact antibody. Unless otherwise noted, the term antibody in this specification includes derivatives, variants, fragments, and mutants thereof, examples thereof as described below.

[0037] In this specification, the terms “antibody fragment” or “antigen-binding fragment” are parts of antibodies such as scFv-Fc, F(ab')2, F(ab)2, Fab', Fab, Fv, scFv, etc., as used herein. Regardless of structure, the antibody fragment binds to the same antigen recognized by the intact antibody. The terms “antibody fragment” include aptamers, enantiomers, scFv, dAb, diabodies, and maxibadiates, etc. The terms “antibody fragment” also include any synthetic or genetically engineered protein that acts similarly to an antibody by binding to a specific antigen and forming a complex.

[0038] “Single-chain variable fragment” or “v” refers to a fusion protein of variable regions of the heavy chain (VH) and light chain (VL) of an immunoglobulin. In one embodiment, the regions may be connected by a short linker peptide of 10 to about 25 amino acids. The linker may be glycine-rich for flexibility, as well as serine or threonine-rich for solubility, and may connect the N-terminus of the VH and the C-terminus of the VL, or vice versa. Additionally, the linker may contain a mutation. This protein retains the specificity of the original immunoglobulin even with the removal of the constant region and the introduction of the linker. scFv molecules are known in the art, for example, as described in U.S. Patent 5,892,019.

[0039] Both the light chain and the heavy chain are divided into regions of structural and functional homology. The terms “invariant” and “variable” are used functionally. In this regard, it can be seen that the variable domains of both the light chain (VL) and heavy chain (VH) parts determine antigen recognition and specificity.

[0040] The variable region allows the antibody to selectively recognize an epitope on the antigen and bind specifically to it. That is, the antibody's VL domain and VH domain, or a subset of the complementarity determining region (CDR), are combined to form the variable region that defines the three-dimensional antigen-binding site. This quaternary structure forms the antigen-binding site present at each N-terminus of Y. More specifically, the antigen-binding site is defined by three CDRs on the VH and VL chains, respectively (i.e., CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3).

[0041] In naturally occurring antibodies, the six “complementary determining regions” or “CDRs” present in each antigen-binding domain are short, non-contiguous sequences of amino acids that are specifically positioned to form the antigen-binding domain because the antibody assumes a three-dimensional configuration in an aqueous environment. The remaining amino acids within the antigen-binding domain, called “framework” regions, exhibit less intermolecular variability. The framework regions mostly adopt the β-sheet structure, and the CDRs form loops that connect the β-sheet structures and, in some cases, form part of them. Thus, the framework regions act to form a scaffold that positions the CDRs in the correct orientation through non-covalent interactions between chains. The antigen-binding domain formed by the positioned CDRs defines a surface complementary to the epitope on the immunoreactive antigen. This complementary surface facilitates the non-covalent binding of the antibody to its cognate epitope. Since the amino acids including the CDR and framework regions are each precisely defined, any given heavy chain or light chain variable region can be easily identified by those skilled in the art.

[0042] Where there are two or more definitions of a term used and / or accepted in the field, the definition of said term used herein is intended to include all such meanings unless explicitly stated otherwise. A specific example is the use of the term “Complementary Determining Region” (“CDR”) to describe non-adjacent antigen combination sites found within said variable regions of both heavy and light chain polypeptides. A number of methods may be used to define the CDR. Current technology utilizes various numbering systems with different definitions of CDR length and position. For example, the Kabat numbering system is based on sequence alignment and predicts the CDR using a “variability parameter” of a given amino acid position (the number of different amino acids at a given position divided by the frequency of the most frequently occurring amino acid at that position). On the other hand, the Chothia numbering system is a structure-based numbering scheme in which antibody determination structures are aligned when loop structures are defined as the CDR. The Martin numbering system focuses on the structural alignment of different framework regions of unusual lengths. The IMGT numbering system is a standardized numbering system based on the alignment of sequences from a complete reference gene database containing the entire immunoglobulin superfamily. Honneger's numbering system (AHo's) is based on the structural alignment of the 3D structure of variable regions and infers framework and CDR lengths using structurally conserved Cα positions.

[0043] When numbered according to the Kabat numbering system, residues in VL are around 24-34 (L1), 50-56 (L2), and 89-97 (L3), and in VH are around 31-35 (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al. Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991); When numbered according to the Chothia numbering system, residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in VL, and 26-32 (H1), 52-56 (H2), and 95-101 (H3) in VH (Chothia and Lesk. J. Mol. Biol. 1987, 196, 901-917); When numbered according to the IMGT numbering system, residues 27-38 (L1), 56-65 (L2), and 105-120 (L3) in VL, and 27-38 (H1), 56-65 (H2), and 105-120 (H3) in VH (Lefranc et al. Nucl. Acids Res. 1999, 27, 209-212; Ruiz et al. Nucl. Acids Res. 2000, 28, 219-221); When numbered according to Honneger's numbering system (AHo's), the positions in VL are 28, 36 (L1), 63, 74-75 (L2) and 123 (L3) and in VH are 28, 36 (H1), 63, 74-75 (H2) and 123 (H3) [Honneger and Plunkthun (Mol. Biol. 2001, 309, 657-670)].

[0044] Those skilled in the art will recognize that the definition of CDR will vary depending on the method used. Any method of defining CDR is considered in conjunction with the sequences disclosed herein.

[0045] In this specification, the terms “antigen” or “immunogen” mean a molecule or part of a molecule to which, for example, an antigen-binding protein (e.g., an antibody or an immunologically functional antigen-binding fragment thereof) can bind and which can be used in the production of an antibody capable of binding to an antigen in animals. The antigen may comprise one or more epitopes capable of interacting with different antibodies or fragments thereof. In one embodiment, the antigen may be an influenza A virus, specifically, an influenza A virus subtype H3N2 or H1N1.

[0046] In this specification, “amino acid” includes the general meaning as understood in the art. The 20 naturally occurring amino acids and their abbreviations are as commonly used in the art (Immunology-A Synthesis, 2nd Edition, ES Golub and DR Green, eds., Sinauer Associates: Sunderland, Mass. 1991). The amino acids include typical amino acids, stereoisomers of the 20 typical amino acids (D-amino acids), non-natural amino acids, such as α-,α-disubstituted amino acids, N-alkyl amino acids, and other non-typical amino acids. Examples of non-typical amino acids include 4-hydroxyproline, γ-carboxyglutamate, ε-trimethyllysine, ε-acetylysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, σ-N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline). In polypeptide marks used herein, as is commonly used in the art, the left side of the sequence represents the amino terminus and the right side represents the carboxy terminus.

[0047] In this specification, “polypeptide” or “protein” refers to a polymer of amino acid residues and is used interchangeably herein. It also includes polymers of naturally occurring amino acid residues as well as polymers of their analogues or mimetics. Furthermore, said polypeptide or protein may include modifications such as the addition of carbohydrates for phosphorylation or glycosylation. Moreover, said polypeptide or protein may be produced in recombinant or naturally occurring cells. Furthermore, said polypeptide or protein may include wild-type sequences or those in which parts of said amino acid sequences have deletions, additions, and / or substitutions. Additionally, said polypeptide or protein may be an antibody, or an antigen-binding fragment, in which one or more amino acids have been deleted, added, and / or substituted in the sequence. Furthermore, “polypeptide fragment” refers to a polypeptide having amino-terminal deletions, carboxyl-terminal deletions, and / or internal deletions compared to the full-length protein. This fragment may also contain amino acids modified relative to the full-length protein. In one embodiment, the fragment may have a length of about 5 to 500 amino acids, e.g., at least 5, 6, 8, 10, 14, 20, 50, 70, 100, 110, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850 or more amino acid lengths.

[0048] The antibody and / or antigen-binding fragment disclosed herein may include “variants,” and since the variant may refer to a polypeptide in which one or more amino acid residues are inserted, deleted, added, and / or substituted from the polypeptide sequence, it may include a fusion polypeptide by linking to another polypeptide as long as the desired biological activity and / or structure of the antibody and / or antigen-binding fragment is maintained. Additionally, the variant may include a modification by proteolytic cleavage, phosphorylation, and / or other post-translational modifications that maintains the biological activity of the antibody and / or antigen-binding fragment disclosed herein. The above variant may have sequence identity of 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more with respect to the sequence of the antibody or its antigen-binding fragment disclosed in this specification, for example, approximately 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, or 80%.

[0049] One aspect relates to an antibody that binds to influenza type A virus, specifically a recombinant antibody that specifically binds to subtypes H3N2, H1N1, or their antigen-binding fragments. In this aspect, the “recombinant protein” is a protein produced using recombinant technology, that is, through the expression of recombinant nucleic acid according to one embodiment. Methods and techniques for producing recombinant proteins are widely known in the art.

[0050] The above antibody or its functional fragment may include various functional groups at the N-terminus or C-terminus. As examples of the various functional groups, the N-terminus functional group may be any one selected from the group consisting of free amines, acetylation, biotin, and fluorophores, and the C-terminus functional group may be any one selected from the group consisting of free acid, amidation, biotin, and fluorophores, but is not limited thereto. Furthermore, the antibody according to the present invention may be labeled for detection or identification and may be labeled with any one selected from the group consisting of chromogenic enzymes, radioisotopes, chromophores, luminescent substances, and fluorescent substances, but is not limited thereto.

[0051] The above-mentioned chromogenic enzyme may be, for example, peroxidase or alkaline phosphatase, and the above-mentioned radioactive isotope may be, for example, 124I, 125I, 111In, 99mTc, 32P, 35S, and the above-mentioned luminescent material or fluorescent material may be, for example, FITC, RITC, rhodamine, Texas Red, fluorescein, phycoerythrin, quantum dots, etc.

[0052] The above antibody can be used for the diagnosis and prognosis of influenza A virus.

[0053] Another aspect provides a composition for detecting influenza type A virus comprising the above antibody or a functional fragment thereof.

[0054] Another aspect provides a composition for detecting influenza type A virus comprising the above antibody or a functional fragment thereof.

[0055] The above antibody or its functional fragment is as described above.

[0056] The above-mentioned kit for detecting influenza A virus may include one or more other component compositions or devices suitable for the analysis method of peptides for detecting influenza A virus, and may additionally include a buffer or reaction solution that maintains the structure or physiological activity of the antibody stably. In addition, to maintain stability, it may be provided in a state maintained at 4°C.

[0057] In order to facilitate the identification, detection, and quantification of the antibody according to one embodiment bound to the influenza A virus, the antibody included in the kit above may be provided in a labeled state as described above. Meanwhile, if the antibody of the present invention is provided unlabeled, the kit according to one embodiment may additionally include a component for exploring the degree of binding of the antibody to the influenza A virus in vitro or in vivo. The component may be a known compound for labeling the antibody, or a secondary antibody against the antibody and a reagent for detecting the same for exploration through an antigen-antibody reaction. The kit according to one embodiment may be used to detect the influenza A virus from a sample obtained from various subjects, for example, an individual infected with the influenza A virus, or to confirm whether there is an infection with the virus.

[0058] Another aspect provides a biochip for detecting influenza A virus comprising the above antibody or a functional fragment thereof.

[0059] The above antibody or its functional fragment is as described above.

[0060] The above antibody or its functional fragment can be specifically immobilized on various chips that can be used as biochips (gold, silver, magnetic bead, silica, graphene, carbon nanotube, etc.). However, the features of the present invention are related to the features of the aforementioned antibody, and manufacturing the antibody into a biochip in this manner is a part that can be done according to known technology.

[0061] Another aspect provides a method for detecting influenza A virus, comprising the steps of: treating a sample with the antibody or a functional fragment thereof; and detecting the reaction of the antibody or the functional fragment thereof.

[0062] The above antibody or its functional fragment is as described above.

[0063] The above detection may be performed by one or more methods selected from the group consisting of cyclic voltammetry, square wave voltammetry, electrochemical impedance spectroscopy, quartz crystal microbalance, surface plasmon resonance, lateral flow assay (LFA) / variable flip angle (VFA), enzyme-linked immunoassay, fluorescence resonance energy transfer, and surface-enhanced Raman spectroscopy.

[0064] In addition, the sample may be obtained from one or more selected from the group consisting of tissue, cell, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid, stool, urine, rectal smear, oropharyngeal smear, nasopharyngeal smear, and nasal lavage fluid.

[0065] Another aspect provides a method for providing information on the diagnosis of influenza A virus infection, comprising the steps of: treating a sample with the antibody or a functional fragment thereof; and detecting the reaction of the antibody or the functional fragment thereof.

[0066] The above antibody or its functional fragment is as described above.

[0067] The term “diagnosis” includes determining an individual’s susceptibility to a specific disease or condition or its cause, determining whether an individual currently has a specific disease or condition, determining the prognosis of an individual with a specific disease or condition, or therametrics (e.g., monitoring an individual’s condition to provide information on therapeutic efficacy).

[0068] Another aspect provides a polynucleotide encoding the above antibody or a functional fragment thereof.

[0069] The above antibody or its functional fragment is as described above.

[0070] Another aspect provides a recombinant vector containing the above polynucleotide.

[0071] Another aspect provides a recombinant cell containing the above vector.

[0072] In this specification, “polynucleotide” or “nucleic acid” comprises a single or double-stranded nucleotide polymer. The nucleotides comprising such polynucleotides may be ribonucleotides or deoxyribonucleotides or modified forms thereof.

[0073] In this specification, “isolated nucleic acid molecule” means DNA or RNA or mRNA, cDNA or a combination thereof of genomic origin, which is all or part of which is associated with said polynucleotides that are not associated with polynucleotides that exist in nature or are not found in nature. For the purposes of the present invention, said nucleic acid molecule containing a specific nucleic acid sequence does not contain an intact chromosome. Instead, said isolated nucleic acid molecule containing a specific nucleic acid sequence may include at least a few additional protein-coding sequences in addition to its specific sequence, or may additionally include a regulatory sequence and / or vector for the expression of the specific nucleic acid sequence.

[0074] In this specification, the term “regulatory sequence” refers to a polynucleotide sequence that can be operably linked to influence the expression and processing of a coding sequence. The characteristics of the regulatory sequence may be influenced by the type of host. For example, a regulatory sequence applicable to prokaryotic cells may include a promoter, sometimes an operator, a ribosome binding site, and a transcription termination sequence. In eukaryotic cells, the regulatory sequence may include a promoter comprising multiple recognition sites, a transcription enhancer, a polyadenylation sequence, and a transcription termination sequence. The regulatory sequence may further include a reader sequence and / or a fusion partner sequence.

[0075] In this specification, “vector” refers to any molecule used to deliver a nucleic acid molecule encoding a protein to a host cell, and includes, for example, nucleic acids, plasmids, bacteriophages, or viruses.

[0076] In this specification, “expression vector” or “recombinant vector” refers to a vector suitable for the transformation of a host cell and comprises a nucleic acid sequence operably linked to the expression vector to regulate the expression of a heterologous sequence encoding a target protein. This expression vector may also include sequences that can be operably linked to the encoding sequence and may include sequences that affect transcription, translation, and RNA splicing where introns are present.

[0077] In this specification, “operably connected or linked” means that the nucleic acid sequence to be linked is positioned so that it can perform a targeting function under appropriate conditions. For example, in a vector comprising a coding sequence and a regulatory sequence, if the transcription of the coding sequence is influenced by the regulatory sequence under appropriate conditions, it is operably connected.

[0078] In this specification, “host cell” means a cell capable of expressing a target gene that is transformed or to be transformed by a target nucleic acid sequence. The term includes the offspring of a host cell that expresses the target gene, regardless of the identity, morphology, and genetic composition of the host cell.

[0079] In this specification, “transduction” generally refers to the transfer of nucleic acids from one bacterium to another by bacteriophages. For example, it includes the transfer of nucleic acids into eukaryotic cells using non-replicating retroviruses.

[0080] In this specification, “transfection” means that a cell obtains foreign or exogenous DNA, in which case the DNA is introduced into the cell through the cell membrane. This may be understood by referring to methods known in the art, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012), Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates.

[0081] In this specification, “transformation” refers to a change in the genetic characteristics of a cell that has been modified to include new DNA or RNA. For example, a cell may be transformed as its genetic characteristics change by introducing new genetic material through transduction, transfection, or other techniques. DNA transformed by methods such as transduction or transfection may exist physically integrated into the cell's chromosomes, exist temporarily in the form of an episome without replication, or exist as a replicable plasmid. When the transformed DNA is replicated by the division of the host cell, it is considered to have been stably transformed.

[0082]

[0083] Antibodies or functional fragments thereof according to a specific pattern can specifically detect influenza A virus, and in particular, by specifically binding to influenza A subtypes H3N2 and H1N1, they can be effectively used for early diagnosis of influenza A virus infection or prognosis prediction.

[0084]

[0085] Figure 1 is a schematic diagram showing a method for building a pazimid library.

[0086] Figure 2 is a schematic diagram showing a panning method.

[0087] Figure 3 is a graph confirming affinity in each panning round using cyclophage ELISA (white bars; BSA coating, black bars; inactivated H3N2 HK fluA virus coating).

[0088] Figure 4 is a graph confirming scFv-phages binding to H3N2 using a monoclonal phage ELISA after randomly selecting 94 clones. A12 is the control group.

[0089] Figure 5 is a graph confirming selected A4 phage clones by scFv phage ELISA. fd-S is another target phage clone, and M13 is a helper phage. (White bars; BSA coating, black bars; inactivated H3N2 HK fluA virus coating).

[0090] Figure 6 is a graph showing that selected A4 phage clones do not bind to influenza B virus but bind to H3N2 and H1N1 (white bars; control scFv phage, black bars; selected A4 scFv phage).

[0091] Figure 7 shows the nucleotide and amino acid sequences of scFv MA4.

[0092] Figure 8a confirms the expression of scFv MA4 by Coomassie blue staining (M4; MA4, l; control library I-4, M; marker).

[0093] Figure 8b shows the confirmation of scFv MA4 by Western blotting (M; marker, l; supernatant of MA4 culture at 30°C for 6 hours, ll; supernatant of MA4 culture at 30°C for 20 hours, lll; supernatant of purified MA4 culture at 30°C for 6 hours).

[0094] Figure 9a is a graph confirming the affinity of expressed scFv MA4 for H3N2 by scFv phage ELISA (white bars; PBS, gray bars; other scFv proteins, black bars; expressed MA4 scFv protein).

[0095] Figure 9b is a graph confirming the affinity of expressed scFv MA4 for H1N1 and H3N2 by scFv phage ELISA (white bars; PBS, gray bars; other scFv proteins, black bars; expressed MA4 scFv protein).

[0096]

[0097] The following examples will be explained in more detail. However, these examples are intended to illustrate one or more specific examples, and the scope of the present invention is not limited to these examples.

[0098]

[0099] Example 1. Construction of a recombinant scFv phagemide library

[0100] The recombinant scFv phagemid library was constructed to contain random variable region segments amplified from H3N2 HK_flu A virus-immunized mouse cDNA via PCR using VH and Vk-specific primers. After construction by polymerase chain reaction, mouse cDNA (provided by KRICT, Korea) was immunized using PCR I and PCR II primers. The PCR reaction was performed in 1 cycle of 5 min at 95°C, followed by 30 cycles; and 1 cycle of 30 sec at 94°C, 30 sec at 60°C, 10 min at 72°C, and 10 min at 4°C. The PCR primers are shown in Table 1. Each VH and Vk PCR product was verified by 0.8% gel electrophoresis. PCR products of the correct size, approximately 370 bp (VH) and 350 bp (Vk), were obtained after amplification from the first-strand cDNA. The amplified VH and Vk segments were combined with synthetic oligonucleotides containing random CDRs with amino acid sequence modifications to generate complete VH and Vk genes. The phagemid library construction phase consisted of four steps. First, a monoclone from the Tomlinson library was incubated overnight at 37°C. Next, plasmid DNA was prepared using the maxi prep kit (Qiagen, Germany) and digested with Nco l / Xho l restriction enzymes (NEB, Germany). Subsequently, calf intestinal alkaline phosphatase (CIP) was applied. Second, each amplified VH and Vk segment was digested with either Nco l / Xho l or Sal l / Not l restriction enzymes. Third, the VH segment was ligated to a plasmid and used to generate multiple copies of the digested plasmid by transforming the E. coli XL1-blue strain. This was then digested with Sal l / Not l restriction enzymes and treated with CIP. Finally, the Vk segment was connected to the reconstructed plasmid.These were cloned into vector pIT2 as scFvs fused to the N-terminus of M13 minor coat protein III. The phagemid library was inserted into E. coli XL1-Blue by electroporation. This contains approximately 1 x 10⁶. 4 Canine independent clones were included. To rescue phagemid particles from the recombinant library, bacteria taken from the library stock (approx. 50 µl) were inoculated and incubated with shaking at 37°C for 2 hours until the optical density at 600 nm (OD600) reached 0.5. Cells infected with VCS M13 helper phage were incubated in a water bath at 37°C for 30 minutes without shaking. The cells were then replaced with culture medium. They were then amplified with 20% (w / v) polyethylene glycol 8000 and 2.5 M NaCl and stored at 4°C. The titer of the prepared library, measured in ampicillin-resistant colony-forming units (CFU), was 1.3 x 10⁶ per ml. 9 was (Fig. 1).

[0101]

[0102] PCR l 프라이머서열번호VH 프라이머VH1_FAGG TSM ARC TGC AGS AGT CWG G7VH1_RTGA GGA GAC GGT GAC CGT GGT CCC TTG GCC CC8Vk 프라이머Vk1_MF_1GAC ATT GTG ATG WCA CAG TCT CCR T9Vk1_MF_2GAT RTT GTG RTG ACY CAR ACT CCA10Vk1_MF_3RAC ATT GTG CTG ACM CAR TCT CCW G11Vk1_MF_4SAA AWT GTK CTS ACC CAG TCT CCA12Vk1_MF_5GAY ATY MAG ATG ACM CAG TCT MCA13Vk1_MF_6CAA ATT GTT CTC WCC CAG TCT CCA14Vk1_MFGAC ATT GAG CTC ACC CAG TCT CCA15Vk1_MR_1 / 4CCG TTT GAT TTC CAG CTT GGT GCC16Vk1_MR_2 / 4CCG TTT TAT TTC CAG CTT GGT CCC17Vk1_MR_3 / 4CCG TTT TAT TTC CAA CTT TGT CCC18Vk1_MR_4 / 4CCG TTT CAG CTC CAG CTT GGT CCC19PCR ll 프라이머VH 프라이머VH2_N / X_FCGG CCA TGG CCG AGG TSM ARC TGC AGS20VH2_N / X_RTCC ACC GCT CGA GAC TGA GGA GAC GGT21Vk 프라이머Vk2_SaF_1 / 3GGC GTG TCG ACG RAC ATT GTG MTG WCA CAR T22Vk2_SaF_2GGC GTG TCG ACG GAT RTT GTG RTG ACY CAR ACT CCA23Vk2_SaF_4GGC GTG TCG ACG SAA AWT GTK CTS ACC CAG TCT CCA24Vk2_SaF_5GGC GTG TCG ACG GAY ATY MAG ATG ACM CAG TCT MCA25Vk2_SaF_6GGC GTG TCG ACG CAA ATT GTT CTC WCC CAG TCT CCA26Vk2_NotR_1ATG TGC GGC CGCCCG TTT GAT TTC CAG CTT GGT GCC27Vk2_NotR_2ATG TGC GGC CGC CCG TTT TAT TTC CAG CTT GGT CCC28Vk2_NotR_3ATG TGC GGC CGC CCG TTT TAT TTC CAA CTT TGT CCC29Vk2_NotR_4ATG TGC GGC CGC CCG TTT CAG CTC CAG CTT GGT CCC30

[0103] Example 2. Panning against H3N2 Hong Kong flu A

[0104] The triple screening and amplification of conjugated scFv phages were performed by panning using an Immunotube (Nunc, Germany). The panning procedure is illustrated in Figure 2. Immunotubes (Nunc, Germany) were coated with inactivated H3N2 HK diluted 1:100 in PBS and incubated overnight at 4°C. The coated tubes were washed three times and blocked. After shaking off the blocking solution, 1 ml of phage library (typically 1.3 x 10⁶ 12 cfu) and 4 ml of 2% (w / v) skim milk in PBS were added to the tube. In each round, the input phage library titer was 1.3 x 10⁶ 12The concentration was maintained homogeneously at cfu / ml. After incubating the tubes, unbound phages were removed with wash buffer. Washing was performed 10 times with PBS / Tween-20 (0.1% Tween-20 in PBS) and 10 times with PBS. Phages bound to inactivated H3N2_HK were eluted by adding 0.5 ml of elution buffer (1 mg / ml trypsin stock solution in PBS, Sigma) and inverting the tubes at room temperature for 10 minutes using a rotary turntable. The eluted phages were used to infect fresh E. coli XL1-Blue cells. Serial dilutions of the eluted phages were prepared and plated on large square Bio-assay dishes (Nunc, Germany) on LB agar containing 1% glucose, 100 µg / ml ampicillin, and 20 µg / ml tetracycline. Incubated overnight at 37°C.

[0105] The titer of eluted scFv phages was measured by counting the number of grown colonies to monitor the efficiency of the screening step. Strict conditions were used, and screening was performed three times with the number of washing steps increased from 10 to 20. The titer of the amplified scFv phages was estimated as follows. E. coli XL1 Blue was cultured in LB medium at 37°C until an optical density of 600 nm (OD600) = 0.5 was reached. 100 µl of the culture medium was inoculated with 10 µl of serial dilution of the amplified scFv phages, and after incubation at 37°C for 30 minutes without shaking, the cultures were plated onto LB agar containing 1% glucose, 100 µg / ml ampicillin, and 20 µg / ml tetracycline. The number of eluted phages increased by more than 300-fold between rounds 1 and 3 (Table 2). These results demonstrate that the panning was successful, as panning titers should generally increase.

[0106] Each of the obtained phage libraries was tested for specificity against H3N2 HK using a polyclonal phage ELISA (Fig. 3), whereas individual bacterial A4 clones expressing H3N2 HK-responsive phages were selected using a monoclonal phage ELISA (Fig. 4). For the ELISA, micro-96-well plates (Nunc, Germany) were first coated with 10 µg / ml bovine serum albumin (Pierce, Germany) and 10,000,000 plaque-forming units (PFU) / ml of inactivated H3N2, then washed with PBS and immersed in PBS overnight at 4°C. The plates were blocked with 2% (w / v) skim milk in PBS at room temperature for 2 hours. The supernatant containing scFv phages or scFv purified from individual colonies was incubated in the plates at room temperature for 2 hours. After washing the plates with PBS containing 0.1% Tween-20, mouse anti-M13 antibody (P8) or mouse anti-c-myc antibody conjugated with horseradish peroxidase (HRP) was added to the wells as a second antibody. The plates were washed again with PBS containing 0.1% Tween-20, and the peroxidase substrate 3,3',5,5'-tetramethylbenzidine kit (TMB, Pierce, Germany) was added. The reaction was stopped after 5 minutes with a stopping reagent solution (Sigma, Germany). Absorbance (450 nm) was determined using a Thermo microplate reader. The specificity of positive clones was further tested by ELISA using plates coated with four unrelated antigen panels: BSA, H1N1, H3N2, and influenza B. As a result, other phages (helper phage, fd-S phage) did not bind to H3N2 HK, and it was confirmed that the selected A4 phage strongly binds to influenza A virus (Fig. 5). In addition, it was confirmed that the selected A4 phage strongly binds to H1N1 and H3N2 and does not bind to influenza B (Fig. 6).

[0107]

[0108] Panning Round Phage Input (PFU) Phage Recovery (PFU) Input / Recovery Ratio Fold Enrichment 11.3 X 10 12 6.83 X 10 5 1.90 X 10 6 121.3 X 10 12 8.98 X 10 5 1.45 X 10 6 1.3131.3 X 10 12 2.31 X 10 8 5.63 X 10 3 338

[0109]

[0110] Example 3. Identification of selected phage clones and site-specific mutagenesis

[0111] Affinity-selected phage clones were identified through plasmid DNA sequencing. Each phage plasmid DNA was isolated from 3 ml of overnight culture medium of H3N2-specific binding clones. The phage plasmid DNA tested was randomly selected from 94 clones to 24 clones. The nucleotide sequences of the VH and Vk genes of the selected clones were determined using primers pIT2_F) LMB3 (CAG GAA ACA GCT ATG ACC ATG) and pIT2_R) pHEN (CTA TGC GGC CCC ATT CAG ATC). The DNA sequences of the scFv genes were determined. The 22 clones from the sequencing results were identical to the 24 clones. Therefore, A4 clones containing a stop codon were selected. The pIT2 vector used for the selected phage A4 clones was also applied for site-specific mutagenesis. The primers used for A4 mutagenesis were the mutation primers (F 5' - ATT CAC TGG GTG AAG CAG AGT CTT GGA AAG A, R 5' - TCT TT C CAA GAC TCT GCT TCA CCC AGT GAA T). 5 µl of 10x pfu buffer, 0.5 µl of template pDNA (A4 clone), 1.2 µl of F primer and R primer, 1 µl of dNTP mixture, and 1 µl of pfu ultra HF DNA polymerase kit (QuikChange II site-specific mutation kit, STRATAGENE, Germany) were added, and the tank was filled to 50 µl with ddH2O. After 30 seconds at 95°C, 12 cycles of PCR reaction (30 seconds at 95°C, 1 minute at 55°C, 7 minutes at 68°C), and 1 cycle of 5 minutes at 37°C, 1 µl of Dpn was added and incubated at 37°C for 1 hour. After electroporation of the HB2151 strain, site-specific mutations were confirmed by DNA sequencing analysis. The inferred amino acid sequence of mutagenic A4 (MA4) is shown in Figure 7. The predicted molecular weight of the total soluble scFv is approximately 32 kDa.

[0112]

[0113] Example 4. MA4 Expression and Purification

[0114] To obtain a large amount of purified scFv, 5 ml of overnight activated E. coli HB2151 MA4 culture was transferred to 500 ml of TY (containing 100 μg / ml ampicillin) and shaken at 37°C until OD600=0.8. Then, isopropyl-β-D-thiogalactogalactopyranoside (IPTG, concentration 0.5 mM) was added to the culture and shaken continuously at 30°C for 5 hours. Afterward, centrifuged and filtered at 0.45 μm. In the non-repressed strain E. coli HB2151, the amber stop codon between the c-myc tag and p lll of the scFv clone is recognized as a stop codon, resulting in the production of a soluble scFv fusion protein. The supernatant of the expressed scFv MA4 was selected for expression analysis, and samples extracted according to induction time were subjected to 10% SDS-PAGE electrophoresis stained with Coomassie blue (Fig. 8a). scFv MA4 was purified using the Ni-NTA His-bind resin kit (Qiagen, Germany). The supernatant of the expressed or purified scFv MA4 was subjected to Western blot to determine recognition of the c-myc antibody. The purified MA4 was boiled with 4x loading sample buffer and subjected to 10% SDS-PAGE electrophoresis at 130V for 1 hour and 30 minutes in running buffer (ClearPAGE, Germany). Then, MA4 scFv protein was transferred from the gel to a PVDF membrane (Amersham Pharmacia Biotech, Germany) at 100V for 1 hour in transfer buffer (1L; glycine 2.98 g, Tris base 5.8 g, SDS 0.8 g, methanol 200 ml). Then, the PVDF membrane was blocked with 5% MPBS for 2 hours and then washed with PBS. Next, 10 ml of 1:2,500 mouse mAb HRP-conjugated anti-c-myc mouse antibody 9E10 (Santa Cruz Biotechnology, USA) was incubated with the membrane at room temperature for 2 hours.Finally, the membrane was washed, and the color was developed by adding the staining substrate ECL (GE healthcare, Germany). The purified scFv was evaluated by 10% SDS-PAGE. Proteins were visualized by staining the gel with Coomassie Brilliant Blue R-250. The concentration of the purified scFv was determined by measuring the absorbance value at 595 nm using a Bradford (BIO-RAD, Germany). Western blotting confirmed that the reaction with the HRP-conjugated anti-c-myc mouse antibody 9E10 showed a single positive band at 32 kDa (Fig. 8b).

[0115]

[0116] Example 5. Affinity of scFv MA4

[0117] The affinity and specificity of soluble scFv MA4 proteins were evaluated by ELISA. The supernatant of expressed scFv MA4 specifically bound to H3N2. However, the control scFv I-4 did not bind (Fig. 9a). Purified scFv MA4 was found to bind strongly to influenza A (H3N2 and H1N1). However, it did not bind to other influenza B strains (Fig. 9b). Additionally, purified MA4 bound more strongly than the supernatant of expressed scFv MA4. The concentration of MA4 was found to affect influenza A.

Claims

1. An antibody for detecting influenza type A virus or a functional fragment thereof comprising VH CDR1 having the amino acid sequence of SEQ ID NO. 1; VH CDR2 having the amino acid sequence of SEQ ID NO. 2; VH CDR3 having the amino acid sequence of SEQ ID NO. 3; VL CDR1 having the amino acid sequence of SEQ ID NO. 4; VL CDR2 having the amino acid sequence of SEQ ID NO. 5; and VL CDR3 having the amino acid sequence of SEQ ID NO.

6.

2. The antibody or functional fragment thereof in which the influenza A virus is subtype H3N2 or H1N1.

3. The antibody or functional fragment thereof according to claim 1, wherein the antibody or functional fragment thereof is capable of simultaneously detecting influenza A virus subtypes H3N2 and H1N1.

4. The antibody or functional fragment thereof according to claim 1, wherein the antibody or functional fragment thereof is selected from the group consisting of VH, VL, IgG, Fab, Fab', F(ab')2, crossover Fab, scFab, dsFv, Fv, scFv, scFv-Fc, scFab-Fc, diabody, minibody, scAb, dAb, Fc-scFv, scFv-Fc-scFv, IgG-scFv, scFv-IgG, and combinations thereof.

5. A composition for detecting influenza type A virus comprising the antibody of claim 1 or a functional fragment thereof.

6. A kit for detecting influenza type A virus comprising the antibody of claim 1 or a functional fragment thereof.

7. A biochip for detecting influenza type A virus comprising the antibody of claim 1 or a functional fragment thereof.

8. A step of treating a sample with the antibody of Claim 1 or a functional fragment thereof; and A method for detecting influenza type A virus comprising the step of detecting a reaction of the above antibody or a functional fragment thereof.

9. The method of claim 8, wherein the detection is performed by one or more methods selected from the group consisting of cyclic voltammetry, square wave voltammetry, electrochemical impedance spectroscopy, quartz crystal microbalance, surface plasmon resonance, lateral flow assay (LFA) / variable flip angle (VFA), enzyme-linked immunoassay, fluorescence resonance energy transfer, and surface-enhanced Raman spectroscopy.

10. The method of claim 8, wherein the sample is obtained from one or more selected from the group consisting of tissue, cell, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid, feces, urine, rectal smear, oropharyngeal smear, nasopharyngeal smear, and nasal lavage fluid.

11. A step of treating a sample with the antibody of Claim 1 or a functional fragment thereof; and A method for providing information on the diagnosis of influenza A virus infection, comprising the step of detecting a reaction of the antibody or a functional fragment thereof.

12. A polynucleotide encoding the antibody of Claim 1 or a functional fragment thereof.

13. A recombinant vector comprising a polynucleotide according to claim 12.

14. A recombinant cell comprising a vector according to claim 13.

Images