Fluorescent VHH antibody

Abstract

To provide a fluorescent VHH antibody that is useful for highly sensitive detection of an antigen such as a virus and a method for using the same.SOLUTION: There is provided a fusion of a VHH antibody with a fluorescent protein in which the VHH antibody is bound to the fluorescent protein either directly or via a peptide linker, wherein the fluorescent protein is selected from the following proteins 1) and 2) and mutants thereof. 1) A protein comprising the amino acid sequence represented by SEQ ID NO: 2. 2) A protein comprising the amino acid sequence represented by SEQ ID NO: 4.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This invention relates to a fusion of a VHH antibody with a fluorescent protein and its use. [Background technology]

[0002] Infectious diseases, particularly viral infections, can cause cold-like symptoms as well as severe symptoms such as pneumonia, hepatitis, and encephalitis, and pose a perpetual threat to humanity. In recent years, influenza viruses have wreaked havoc worldwide, sometimes causing pandemics due to the emergence of new strains of influenza with antigenic changes. Furthermore, in 2019, SARS-CoV-2 emerged, causing an acute respiratory disease (COVID-19), affecting not only life and health but also economic activity and social functions.

[0003] To diagnose the virus, in addition to detecting viral genes using PCR (polymerase chain reaction) (PCR test), antigen tests are also used, which use specific antibodies to detect viral structural protein fragments.Antigen tests have the advantage of not requiring expensive equipment or labor, and can diagnose whether or not a person is infected within a few minutes.

[0004] Generally, antibodies (immunoglobulins; Ig) produced after contact with an antigen during an infection are composed of light and heavy chains, but camelids are known to produce heavy-chain antibodies that lack light chains. A single domain containing the variable region of a heavy-chain antibody can function as an antibody by itself and is called a VHH antibody.

[0005] Because VHH antibodies have a molecular weight one-tenth that of IgG antibodies, they can bind to epitopes that conventional IgG cannot bind to due to structural issues. Furthermore, they can bind to the surfaces of many glycosylated virus particles, providing a wide range of potential target molecules. Furthermore, VHH antibodies are acid- and heat-resistant, and unlike IgG, they do not require production in cultured cells; they can be produced in E. coli, yeast, and other organisms. These advantages make them easy to mass-produce and purify. Furthermore, because VHH antibodies are composed of a single peptide chain, their function can be easily modified using protein engineering or chemical modification techniques, making them suitable for the creation of antibody-drug conjugates (ADCs).

[0006] The present inventors have previously succeeded in obtaining VHH antibodies with high affinity for SARS-CoV-2 (Patent Documents 1 to 3). Furthermore, Non-Patent Document 1 reports that SARS-CoV-2-infected cultured cells can be detected by the fluorescent antibody method using a fluorescent VHH antibody in which a fluorescent protein such as mNeonGreen is conjugated to a VHH antibody that binds to SARS-CoV-2. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] PCT / JP2021 / 017106 [Patent Document 2] Patent application 2021-085698 [Patent Document 3] Patent application 2021-029050 [Non-patent literature]

[0008] [Non-Patent Document 1] Laura Jo Sherwood and Andrew Hayhurst. (2021), Toolkit for Quickly Generating and Characterizing Molecular Probes Specific for SARS-CoV-2 Nucleocapsid as a Primer for Future Coronavirus Pandemic Preparedness., ACS Synth Biol. 2021 Feb 19;10(2):379-390. doi: 10.1021 / acssynbio.0c00566. Epub 2021 Feb 3. Summary of the Invention [Problem to be solved by the invention]

[0009] The present invention relates to providing fluorescent VHH antibodies that are useful for highly sensitive detection of antigens such as viruses, and methods for using the same. [Means for solving the problem]

[0010] The present inventors have discovered that a fusion product of a VHH antibody and a specific fluorescent protein binds to an antigen without inhibiting the affinity between the antigen and the VHH antibody, and is useful for highly sensitive detection of the antigen.

[0011] That is, the present invention relates to the following (1) to (5). (1) A VHH antibody-fluorescent protein fusion product in which a VHH antibody and a fluorescent protein are linked directly or via a peptide linker, wherein the fluorescent protein is selected from the following proteins 1) to 2) and their mutants: 1) A protein consisting of the amino acid sequence shown in SEQ ID NO: 2 2) A protein consisting of the amino acid sequence shown in SEQ ID NO: 4 (2) A method for producing the fusion protein, comprising the step of culturing cells carrying a nucleic acid encoding the fusion protein described in (1) above. (3) A method for detecting an antigen in a sample, which comprises the step of contacting the fusion construct described in (1) above with the sample. (4) A method for multimerizing a VHH antibody, which comprises expressing the VHH antibody as the fusion product described in (1). (5) A method for improving the antigen-binding activity of a VHH antibody, which comprises expressing the VHH antibody as the fusion complex described in (1). [Effects of the Invention]

[0012] According to the present invention, antigens such as viruses in a sample can be detected easily and with high sensitivity. [Brief explanation of the drawings]

[0013] [Figure 1] Integrity of VHH antibody-fluorescent protein fusions by SDS-PAGE. [Figure 2] Degree of fluorescent chromophore formation in VHH antibody-fluorescent protein fusions. [Figure 3] Specificity of staining by VHH antibody-fluorescent protein fusions on infected cells. [Figure 4] Fluorescent signal intensity of three types of VHH antibody-fluorescent protein. [Figure 5] Staining of lyophilized VHH antibody-fluorescent protein fusions. [Figure 6] Comparison of signal intensities between E0=KikG and E9=KikG. [Figure 7] E9 = Stability of KikG fluorescent signal over time. [Figure 8] Confocal microscopy of cells contained in a SARS-CoV-2 clinical specimen (saliva sample). [Figure 9] N10 = Staining of SARS-CoV-2 clinical specimens with VHH antibody N10, which recognizes the KikG-mediated nucleocapsid. [Figure 10] Comparison of antibody staining with fluorescent VHHs and indirect immunofluorescence staining. [Figure 11]Binding activity of E9=KikG, E9=AzaleaB5, or E9=mAchilles to the Omicron strain S-protein trimer or RBD measured by surface plasmon resonance. The black line shows the binding / dissociation curve based on the measured raw data, and the gray line shows the fitting curve. DETAILED DESCRIPTION OF THE INVENTION

[0014] All patents, non-patent documents, and other publications cited herein are hereby incorporated by reference in their entirety.

[0015] Herein, the identity of an amino acid sequence or a nucleotide sequence is calculated by the Lipman-Pearson method (Science, 1985, 227:1435-1441). Specifically, it is calculated by performing analysis using the homology analysis (Search homology) program of the genetic information processing software GENETYX Ver. 12, with the unit size to compare (ktup) set to 2.

[0016] As used herein, the term "amino acid residue" refers to the 20 amino acid residues that make up proteins: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine ​​(Cys or C), glutamine (Gln or Q), glutamic acid (Glu or E), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Val or V).

[0017] In this specification, amino acid positions and variant descriptions are designated using the accepted IUPAC single-letter amino acid abbreviations as follows: That is, an amino acid at a given position is represented as [amino acid, position], an amino acid "substitution" is represented as [original amino acid, position, substituted amino acid], an amino acid "deletion" is represented as [original amino acid, position, Δ], and an amino acid "insertion" is represented as They are represented as [original amino acid, position, original amino acid, inserted amino acid], and variants containing multiple modifications are represented by a plus sign ("+").

[0018] The VHH antibody-fluorescent protein fusion of the present invention is a VHH antibody-fluorescent protein fusion in which a VHH antibody and a fluorescent protein are linked directly or via a peptide linker, and the fluorescent protein is selected from the following proteins 1) to 2) and their mutants. 1) A protein consisting of the amino acid sequence shown in SEQ ID NO: 2 2) A protein consisting of the amino acid sequence shown in SEQ ID NO: 4

[0019] The term "VHH antibody" refers to a molecule obtained by excising the antigen-binding domain of a heavy-chain antibody without the light chain. VHH antibodies are small single-domain antibodies that have three antigen-binding loops (antigen complementarity-determining regions; CDRs), similar to the heavy chains of conventional antibodies. In the present invention, VHH antibodies may be antibodies having affinity for any antigen, but are preferably VHH antibodies having affinity for viruses. Here, viruses may be of any type, regardless of the type of nucleic acid (RNA or DNA) and whether they are enveloped or not. Examples include influenza viruses, which have RNA as their nucleic acid; coronaviruses; SARS-CoV; SARS-CoV-2; respiratory syncytial virus; mumps virus; Lassa virus; dengue virus; rubella virus; human immunodeficiency virus; norovirus; poliovirus; echovirus; hepatitis A virus; hepatitis E virus; rhinovirus; astrovirus; rotavirus; coxsackievirus; enterovirus; sapovirus; human herpesviruses, which have DNA as their nucleic acid; vaccinia virus; hepatitis B virus; adenovirus; B19 virus; papovavirus; and human papillomavirus.

[0020] Examples of VHH antibodies with affinity for SARS-CoV-2 include the following antibodies or peptides A (Patent Document 1), B (Patent Document 2), and C (Patent Document 3) described in Patent Documents 1 to 3. A: An antibody that binds to SARS-CoV-2, having one or more structural domains including CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 9 or an amino acid sequence in which one amino acid in said amino acid sequence has been substituted with another amino acid, CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 10 or an amino acid sequence in which one amino acid in said amino acid sequence has been substituted with another amino acid, and CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 11 or an amino acid sequence in which one amino acid in said amino acid sequence has been substituted with another amino acid. Specific examples include CoVHH1 (SEQ ID NO: 12, referred to as "E0" in the Examples below).

[0021] B: A peptide that binds to SARS-CoV-2 and has one or more structural domains including a CDR3 consisting of an amino acid sequence shown in any of SEQ ID NOs: 13 to 21 or an amino acid sequence in which at least one amino acid in the amino acid sequence has been replaced with another amino acid. Preferably, the peptide further comprises a CDR1 consisting of an amino acid sequence shown in any of SEQ ID NOS: 22 to 30 or an amino acid sequence in which at least one amino acid in the amino acid sequence has been substituted with another amino acid, and a CDR2 consisting of an amino acid sequence shown in any of SEQ ID NOS: 31 to 39 or an amino acid sequence in which at least one amino acid in the amino acid sequence has been substituted with another amino acid. More preferably, a peptide that binds to SARS-CoV-2 is selected from 1) to 9), and has one or more structural domains each including CDR1, CDR2, and CDR3, and consists of an amino acid sequence shown in any of SEQ ID NOs: 13 to 39 below, or an amino acid sequence in which at least one amino acid in the amino acid sequence has been substituted with another amino acid.

[0022] [Table 1]

[0023] Specifically, examples include VHH-COVE1 (sequence number 40), VHH-COVE2 (sequence number 41), VHH-COVE3 (sequence number 42), VHH-COVE4 (sequence number 43), VHH-COVE5 (sequence number 44), VHH-COVE6 (sequence number 45), VHH-COVE7 (sequence number 46), VHH-COVE8 (sequence number 47), and VHH-COVE9 (sequence number 48, referred to as "E9" in the examples described below).

[0024] C: An antibody that binds to SARS-CoV-2 and has one or more structural domains containing the CDRs shown in (a) or (b) below. (a) CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 49 or an amino acid sequence in which one amino acid in the amino acid sequence has been substituted with another amino acid, CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 50 or an amino acid sequence in which one amino acid in the amino acid sequence has been substituted with another amino acid, and CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 51 or an amino acid sequence in which one amino acid in the amino acid sequence has been substituted with another amino acid. (b) CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 52 or an amino acid sequence in which one amino acid in the amino acid sequence has been substituted with another amino acid, CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 53 or an amino acid sequence in which one amino acid in the amino acid sequence has been substituted with another amino acid, and CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 54 or an amino acid sequence in which one amino acid in the amino acid sequence has been substituted with another amino acid. Specific examples include CoVHH-N1 (SEQ ID NO: 55, referred to as "N1" in the Examples below), CoVHH-N2 (SEQ ID NO: 56), and the like.

[0025] On the other hand, the fluorescent protein according to the present invention is a protein selected from the following 1) to 2) or a mutant thereof: 1) A protein consisting of the amino acid sequence shown in SEQ ID NO: 2 2) A protein consisting of the amino acid sequence shown in SEQ ID NO: 4

[0026] By "fluorescent protein" is meant a protein that is fluorescent and exhibits a certain fluorescence when illuminated with light of the appropriate excitation wavelength (fluorescence activity). The protein consisting of the amino acid sequence shown in SEQ ID NO: 2 in 1) is derived from Favia speciosa, a coral of the family Asteridae, and is called "KikG" (EMBO Rep (2005) 6:233-238). The protein is encoded by a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 1. The excitation and emission spectra of KikG show maxima at 507 and 517 nm, respectively. KikG forms a rigid tetrameric structure, which allows it to be used as a highly dispersible tag overall. Variants of the fluorescent protein include proteins that have at least 90%, preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, and more preferably at least 99% identity to the amino acid sequence shown in SEQ ID NO: 2, and have fluorescent activity similar to that of the protein. An amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 2 includes, for example, an amino acid sequence in which one or more amino acids have been deleted, substituted, added, or inserted relative to the amino acid sequence shown in SEQ ID NO: 2. Here, "having the same fluorescent activity as the protein" means, for example, having a maximum fluorescent wavelength within the range of the maximum fluorescent wavelength of the protein ±5 nm.

[0027] Specific examples of mutants of the protein consisting of the amino acid sequence shown in SEQ ID NO: 2 include proteins consisting of the amino acid sequence shown in SEQ ID NO: 2 with the following amino acid substitutions: D62H M40V+D62H+I198M ·M10I+L12V+M40V+V60A+D62H+Y119N+P144S+R197L+I198M

[0028] 2) The protein consisting of the amino acid sequence shown in SEQ ID NO: 4 is a modified version of a fluorescent protein isolated from the coral Azalea spp., and is designated "AzaleaB5" (bioRxiv, doi: https: / / doi.org / 10.1101 / 2020.03.30.015156). This protein is encoded by a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 3. The excitation and emission spectra of AzaleaB5 show maxima at 574 nm and 596 nm, respectively. AzaleaB5 forms a dimer. Variants of the fluorescent protein include proteins that have at least 90%, preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, and more preferably at least 99% identity to the amino acid sequence shown in SEQ ID NO: 4, and have fluorescent activity similar to that of the protein. An amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 4 includes, for example, an amino acid sequence in which one or more amino acids have been deleted, substituted, added, or inserted relative to the amino acid sequence shown in SEQ ID NO: 4. Furthermore, "having the same fluorescent activity as the protein" means, for example, having a maximum fluorescent wavelength within the range of the maximum fluorescent wavelength of the protein ±5 nm.

[0029] An example of a mutant of the protein consisting of the amino acid sequence shown in SEQ ID NO: 4 is a protein consisting of the amino acid sequence shown in SEQ ID NO: 4 but with the following amino acid substitutions (D108E+C119S+C155S+C177V).

[0030] In the VHH antibody-fluorescent protein fusion of the present invention, the VHH antibody is linked to the fluorescent protein directly or via a peptide linker, and the VHH antibody may be linked to either the N-terminus or C-terminus of the fluorescent protein. A preferred embodiment is one in which the VHH antibody is linked to the N-terminus of the fluorescent protein via a peptide linker.

[0031] A peptide linker refers to a linker consisting of a peptide in which amino acids are linked in a linear chain, and its amino acid length is generally 5 to 50 amino acids, preferably 13 to 30 amino acids, and more preferably 15 to 30 amino acids. There are no particular limitations on the amino acid sequence constituting the peptide linker, and it is suitably designed so as to maintain the functions of the VHH antibody and fluorescent protein in the VHH antibody-fluorescent protein fusion product. In the present invention, for example, GGGGSGGGSGGGGS ((GGGGS)3; SEQ ID NO: 5) is preferably used.

[0032] The VHH antibody-fluorescent protein fusion of the present invention may further comprise a tag. Examples of tags include peptide tags for protein isolation / purification, such as a histidine tag (HHHHHH (SEQ ID NO: 6)), a FLAG tag (DYKDDDDK (SEQ ID NO: 7)), and a Strep tag (WSHPQFEK (SEQ ID NO: 8)). The tag can be linked, for example, to the N-terminus or C-terminus of the VHH antibody-fluorescent protein fusion of the present invention, directly or via a peptide linker (e.g., GGG).

[0033] The VHH antibody-fluorescent protein fusion protein of the present invention can be produced by genetic recombination techniques. Specifically, the VHH antibody-fluorescent protein fusion protein can be produced by culturing Escherichia coli carrying nucleic acid (DNA (e.g., cDNA) or RNA (e.g., mRNA)) encoding the VHH antibody-fluorescent protein fusion protein of the present invention.

[0034] Production by genetic recombination methods can be carried out, for example, by a method comprising inserting DNA encoding the VHH antibody-fluorescent protein fusion of the present invention into an appropriate expression vector, introducing the vector into an appropriate host cell, culturing the resulting cells (transformants), and recovering the desired VHH antibody-fluorescent protein fusion from within the cells or from extracellular fluid.

[0035] DNA encoding the VHH antibody-fluorescent protein fusion protein of the present invention can be obtained, for example, by synthesizing DNA encoding each component (VHH antibody, fluorescent protein, peptide linker, peptide tag) by PCR using appropriate primers and ligating them with a ligase using standard methods. Alternatively, DNA encoding the VHH antibody-fluorescent protein fusion protein of the present invention can be chemically synthesized using standard methods.

[0036] Vectors include, but are not limited to, plasmids, phages, cosmids, phagemids, viruses, etc. Plasmid vectors include, but are not limited to, plasmids derived from Escherichia coli (e.g., pET17b, pET22b(+), pBR322, pBR325, pUC118, pUC119, pUC18, pUC19, pBluescript, etc.), plasmids derived from Bacillus subtilis (e.g., pUB110, pTP5, etc.), and plasmids derived from yeast (e.g., YEp13, YCp50, etc.). Phage vectors include, but are not limited to, T7 phage display vectors (T7Select10-3b, T7Select1-1b, T7Select1-2a, T7Select1-2b, T7Select1-2c, etc. (Novagen)) and λ phage vectors (Charon4A, Charon21A, EMBL3, EMBL4, λgt10, λgt11, λZAP, λZAPII, etc.). Viral vectors include, but are not limited to, animal viruses such as retroviruses, adenoviruses, adeno-associated viruses, vaccinia viruses, and Sendai viruses, as well as insect viruses such as baculoviruses. Cosmid vectors include, but are not limited to, Lorist6, Charomid9-20, and Charomid9-42. Known phagemid vectors include, but are not limited to, pSKAN, pBluescript, pBK, and pComb3H.

[0037] A vector may contain regulatory sequences that enable expression of a DNA of interest, selection markers for selecting vectors containing the DNA of interest, and a multicloning site for inserting the DNA of interest. Such regulatory sequences include promoters, enhancers, terminators, SD sequences or ribosome binding sites, replication origins, and poly(A) sites. Examples of selectable markers that may be used include ampicillin resistance genes, neomycin resistance genes, kanamycin resistance genes, and dihydrofolate reductase genes.

[0038] Host cells for introducing vectors include bacteria such as Escherichia coli and Bacillus subtilis, yeast cells, insect cells, animal cells (e.g., mammalian cells), and plant cells, but in the present invention, Escherichia coli is preferably used as the host cell. Transformation or transfection of these host cells can be performed using methods such as the calcium phosphate method, electroporation, lipofection, particle gun method, and PEG method.

[0039] Transformed cells are cultured according to conventional methods used for culturing host cells. For example, the culture medium for microorganisms such as Escherichia coli and yeast cells contains carbon sources, nitrogen sources, inorganic salts, and other substances that can be utilized by the host microorganism. To facilitate recovery of the VHH antibody-fluorescent protein fusion of the present invention, it is preferable to secrete the expressed VHH antibody-fluorescent protein fusion extracellularly. This can be achieved by attaching DNA encoding a peptide sequence that enables secretion of the VHH antibody-fluorescent protein fusion from the cell to the 5' end of the DNA encoding the VHH antibody-fluorescent protein fusion. The fusion peptide translocated to the cell membrane is cleaved by a signal peptidase, resulting in the secretion and release of the desired VHH antibody-fluorescent protein fusion into the medium. Alternatively, VHH antibody-fluorescent protein fusions accumulated intracellularly can also be recovered. In this case, the cells are physically or chemically disrupted, and the desired VHH antibody-fluorescent protein fusion is recovered using protein purification techniques.

[0040] The produced VHH antibody-fluorescent protein fusion can be recovered or purified by standard methods, such as chromatography (e.g., gel filtration chromatography, ion exchange column chromatography, affinity chromatography, reverse-phase column chromatography, HPLC), ammonium sulfate fractionation, ultrafiltration, and immunoadsorption. As described above, when the VHH antibody-fluorescent protein fusion of the present invention has a purification tag such as a histidine tag, the VHH antibody-fluorescent protein fusion can be purified from cells or culture medium using the purification tag. For example, when the VHH antibody-fluorescent protein fusion has a histidine tag, the VHH antibody-fluorescent protein fusion can be purified by immobilized metal affinity chromatography (IMAC).

[0041] As described above, KikG forms a tetrameric structure and AzaleaB5 forms a dimeric structure, and therefore, it can be said that VHH antibodies can be multimerized by expressing them as VHH antibody-fluorescent protein fusions. Furthermore, multimerization of such VHH antibodies can improve the binding activity to antigens, particularly multimeric antigens. Therefore, the present invention also provides a method for multimerizing a VHH antibody by expressing the VHH antibody as a VHH antibody-fluorescent protein fusion, and a method for improving the binding activity of a VHH antibody to an antigen by expressing the VHH antibody as a VHH antibody-fluorescent protein fusion.

[0042] The VHH antibody-fluorescent protein fusion of the present invention thus obtained can be used as a fluorescently labeled antibody to detect the presence of an antigen in a sample. Therefore, the present invention provides a method for detecting an antigen in a sample, which comprises the step of contacting the VHH antibody-fluorescent protein fusion with the sample. Specifically, the presence of an antigen in a sample is detected by contacting the VHH antibody-fluorescent protein fusion protein of the present invention with the sample to cause an antigen-antibody reaction, and measuring the fluorescence of the reaction product (antigen-antibody complex). This method allows direct detection of the antigen, does not require any additional steps, and enables antigen detection with high sensitivity in a shorter time.

[0043] Here, the sample may be any biological sample such as tracheal swab, nasal swab, oral swab, pharyngeal swab, nasal wash, nasal aspirate, nasal mucus / nasal blow, saliva, sputum, tears, blood, serum, urine, feces, tissue, cells, tissue or cell fragments, or a sample collected from a solid surface to which antigens such as viruses may be attached, such as a doorknob or toilet bowl. In particular, when detecting SARS-CoV-2 in a sample using the VHH antibody-fluorescent protein fusion of the present invention, it is possible to detect SARS-CoV-2 with high sensitivity comparable to that of PCR, whether the sample used is saliva or virus-infected cells.

[0044] The contact between the VHH antibody-fluorescent protein fusion and the sample may be any amount that allows for sufficient antigen-antibody reaction, and the concentration, contact amount, and contact time of the VHH antibody-fluorescent protein fusion may be appropriately determined. For example, when detecting SARS-CoV-2, a VHH antibody-fluorescent protein fusion diluted in the same blocking solution may be added to a sample that has been previously subjected to a blocking treatment, and the mixture may be allowed to react at room temperature for one to several hours.

[0045] Fluorescence measurement is performed by, but is not limited to, irradiating the reaction product with excitation light having a certain wavelength range to cause the fluorescent substance to emit light, and measuring the intensity of the collected light or the number of photons using a light-receiving element such as a camera in a fluorescence microscope or a confocal laser microscope, thereby identifying the antigen contained in the sample. [Example]

[0046] The present invention will be described in more detail below using examples, but the technical scope of the present invention is not limited to these examples.

[0047] Production Example 1: Preparation of VHH antibody-fluorescent protein fusion protein (1) Construction of E. coli expression plasmid The cDNA of the VHH antibody, E0 (SEQ ID NO: 12), was amplified by PCR and ligated into the BamHI / The KikG cDNA was amplified by PCR and cloned into the HindIII / SalI site of pBSC4 / E0 (pBSC4 / E0 = KikG). Between the EcoRI and HindIII sites of pBSC4, a linker (GGGGS)3 (denoted "=", G: glycine, S: Since the base sequence corresponding to the nucleotide sequence of the nucleotide ... Other VHH-fluorescent protein fusion constructs were constructed using pRSETB / E0=KikG as a template. KikG was excised from pRSETB / E0=KikG by restriction enzyme digestion with HindIII and SalI and replaced with PCR-amplified cDNAs of fluorescent proteins (AzaleaB5, Achilles, EGFP). Furthermore, PCR-amplified cDNAs of VHH antibodies (E9 (SEQ ID NO: 48) and N10 (SEQ ID NO: 55)) were replaced with E0 using restriction enzymes (BamHI / EcoRI) or In-Fusion reagent (Clontech). The amino acid sequences of the VHH-fluorescent protein fusion proteins used to prepare each VHH-fluorescent protein fusion protein and the nucleotide sequences encoding them are shown below.

[0048] [Table 2]

[0049] (2) Transformation and cultivation of E. coli The constructed VHH antibody-fluorescent protein fusion expression plasmid was transformed into competent cells (JM109(DE3)) by heat shock. The transformed E. coli was plated on an agar medium (LB broth) containing ampicillin and cultured at 37°C for approximately 16 hours. One of the formed colonies was inoculated into LB broth (25 ml) containing ampicillin and cultured with shaking at room temperature (approximately 23°C) for 4 days.

[0050] (3) Purification of VHH antibody-fluorescent protein fusion E. coli cultured with shaking was harvested using a centrifuge and suspended in PBS. After adding protease inhibitors and lysozyme, the cells were disrupted by freezing in liquid nitrogen and thawing under running water approximately three times. The cells were further disrupted using a sonicator, and the genome was sheared. The disrupted solution was centrifuged to remove debris, and 1 mL of Ni-NTA agarose resin (Qiagen, cat. #30230) was added to the supernatant at a ratio of 1 mL per 25 mL of E. coli culture and stirred at 4°C for approximately 1 hour. The Ni-NTA agarose resin was packed into a column, washed with PBS and PBS containing 10 mM imidazole, and then eluted with PBS containing 300 mM imidazole. The eluted protein solution was passed through a gel filtration column PD-10, where the buffer was replaced with 150 mM KCl, 50 mM HEPES-KOH (pH 7.4). The concentration of the purified VHH antibody-fluorescent protein fusion was determined by the Bradford assay using bovine serum albumin (Quick Start Bovine Serum Albumin Standard (BIO-RAD, cat. #500-0206)) as a standard protein. Protein integrity was examined by SDS-PAGE using a 10% (weight / ml) acrylamide-0.33% (weight / ml) bisacrylamide cross-linked gel. The degree of chromophore formation was confirmed by measuring the absorption spectrum using an absorption spectrophotometer (U-3310 (Hitachi High-Tech Science)) against a background of 150 mM KCl, 50 mM HEPES-KOH (pH 7.4).

[0051] (4) Results The results for protein integrity are shown in Figure 1. Additionally, the results for chromophore formation of the fluorescent proteins are shown in Figure 2. Based on the mobility of the main band on SDS-PAGE in Figure 1, the molecular weight of the target protein was predicted (according to the molecular design) for all VHH antibody-fluorescent protein fusions shown in the figure. Furthermore, it was confirmed that recombinant proteins (VHH antibody-fluorescent protein) could be produced efficiently and stably while suppressing degradation, and that a high degree of purification was achieved. This is also evident from the fact that the absorption spectra indicating chromophore formation, shown in Figure 2, showed the characteristic spectra of KikG or AzaleaB5 for all VHH antibody-fluorescent protein fusions. From the above, it was considered that the stable formation of molecules that met the molecular design was supported.

[0052] Test Example 1: Fluorescence observation of SARS-CoV-2 infected cultured cells using VHH antibody-fluorescent protein fusion (1) Method We used SARS-CoV-2-infected VeroE6 / TMPRSS2 cells (Toshiki Ebisudani et al., Cell Rep. 2021 Jun 8;35(10):109218) and uninfected cells (35 mm glass-bottom dishes (IWAKI) or 96-well glass-bottom plates (Matsunami)). The protease TMPRSS2 is required for cleaving the spike protein on the viral membrane and converting it to its active form when the SARS-CoV-2 virus binds to ACE2 (the SARS-CoV-2 virus receptor) present on the host cell membrane. Like ACE2, it is present on the host cell membrane. VeroE6 / TMPRSS2 cells were pre-transfected with the TMPRSS2 gene for the purpose of experimentally inducing infection, as TMPRSS2 is not present in native Vero E6 cells. The cells were blocked with blocking solution (3% BSA, 1% Triton-X / 120 mM NaCl, 50 mM PIPES-NaOH (pH 6.8)) for 1 hour at room temperature. After incubation with 10 μg / ml VHH antibody-fluorescent protein fusions (E0 = KikG, E0 = EGFP, E0 = Achilles) diluted in blocking solution for 1 hour at room temperature, the cells were stained and then washed three times with PBS (pH 7.8). The fluorescence of experimentally infected cultured cells (Vero E6 / TMPRSS2 cells) was observed using the fluorescence microscope system described below. The microscope system and imaging conditions are described below.

[0053] (Widefield Fluorescence Microscope System) Microscope system: Microscope: Inverted IX83 (Olympus), Light source: X-cite XYLIS (EXCELITAS), Camera: ORCA-Fusion (Hamamatsu Photonics), Control software: cellSens Dimension (Olympus) Objective lens: UPLXAPO20x / NA 0.8 (Olympus) Fluorescent mirror unit: VHH antibody-fluorescent protein fusion (E0=KikG, E0=EGFP, E0=Achilles): U-FBNA (Olympus) Excitation light intensity: 5% Exposure time: 1 second

[0054] (2) Results The results shown in Figure 3 demonstrate that staining with the VHH antibody-fluorescent protein fusion was not observed in uninfected cells and was specific to infected cells. From the results shown in Figure 4, when comparing three fluorescent proteins fused to VHH antibodies, E0=KikG, E0=EGFP, and E0=Achilles, it was found that E0=KikG strongly stained infected cells and enabled highly sensitive detection of infected cells. While PCR only reveals the average infection status of suspected infected samples, it is possible to distinguish between infected and non-infected states for each cell individually, and it is thought that it is possible to detect the degree of infection for each cell individually from the fluorescence intensity of the staining.

[0055] Test Example 2: Staining of freeze-dried VHH antibody-fluorescent protein fusions in saliva from patients infected with SARS-CoV-2 Based on the findings of the staining performance of VHH antibody-fluorescent protein fusions in cultured cells confirmed in Test Example 1, clinical samples were used to confirm whether freeze-drying affects the stability of protein molecules. (1) Method 500 μL of the sample suspended in 20 mL of CellPrep System Reagent for Gynecological and Oral Use (cat. no. 518-111458) was transferred to a microtube and centrifuged at 15,000 rpm (20,954 x g) for 20 minutes. The supernatant was removed, and approximately 3 μL of the pellet suspension was applied to a silane-coated glass slide (Water Edge Polished Frosted 1106, Muto Chemicals). After confirming that the cellular components had been air-dried to a semi-moist state and fixed to the slide glass, the slide was fixed in 99.5% ethanol (Fujifilm Wako Pure Chemical Industries) for 30 minutes at room temperature. Wash twice with PBS (pH 7.8) and add blocking solution (3% BSA, 1% Triton X-10). Blocking was carried out with nX / 120 mM NaCl, 50 mM PIPES-NaOH (pH 6.8) for 1 hour at room temperature. 10 μg / mL VHH antibody-fluorescent protein fusion diluted in blocking solution was The reaction was carried out at room temperature for 1 hour. The cells were washed three times with PBS (pH 7.8), and the cell nuclei were extracted with DAPI (10 μg / mL in The sections were stained with PBS (Fujifilm Wako Pure Chemical Industries, cat. no. 340-07971) for 5 minutes at room temperature. After washing three times with PBS, the sections were embedded in a cover glass using PBS as an embedding medium, and observed under a wide-field fluorescence microscope under the same conditions as in Test Example 1.

[0056] (1) Method 100 μL of purified VHH antibody-fluorescent protein fusion E0 = KikG was dispensed into a 1.5 mL microtube and frozen in liquid nitrogen. The sample was then freeze-dried for 24 hours using a freeze dryer FDU-830 (EYELA). The lyophilized sample was sealed with parafilm and stored at room temperature. Immediately before use, the sample was reconstituted by adding 100 μL of purified water. (2) Results The results are shown in Figure 5. A solution of the VHH antibody-fluorescent protein fusion protein E0=KikG, purified, then rapidly frozen in liquid nitrogen and stored at -80°C (fresh probe), and the same protein solution, freeze-dried and stored at room temperature under dry conditions (freeze-dried probe), showed comparable signal intensities. Therefore, it was thought that the VHH antibody-fluorescent protein fusion protein retained almost complete functionality even after freeze-drying.

[0057] Test Example 3 Comparison of staining signal intensity of cells infected with E0=KikG and E9=KikG We examined whether signal intensity would change by fusing different VHH antibodies, E0=KikG and E9=KikG, to KikG. As a result of staining infected cells using the method of Test Example 1, it was confirmed that E9=KikG had a higher signal intensity and maintained specificity for infected cells (FIG. 6). Considering the results of chromophore formation in Figure 2, it was thought that the difference in signal intensity was not due to stress on the formation of higher-order molecular structure between the fused VHH antibody, but rather due to differences in the amount of binding of the VHH antibody-fluorescent protein fusion protein depending on the difference in affinity of the VHH antibody for the antigen.

[0058] Test Example 4: Stability of fluorescent signal from VHH antibody-fluorescent protein fusion Saliva samples from patients infected with SARS-CoV-2 were stained with the VHH antibody-fluorescent protein fusion E9=KikG, and the fluorescence intensity was observed daily in the same field of view over a 3-day period (observation was performed at room temperature and storage was performed at 4°C). As a result, it was confirmed that the intensity of the fluorescent signal of E9=KikG remained stable for at least 3 days after staining (FIG. 7). The KikG-fused E9 VHH antibody maintained a stable and strong signal even after storage for several days, and was thought to remain stably bound to the antigen without detaching from it.

[0059] Test Example 5: High-resolution observation of SARS-CoV-2 clinical specimens using VHH antibody-fluorescent protein fusions with a confocal microscope Quadruple staining was performed using E9=KikG, which shows a strong fluorescent signal when staining S-protein, a VHH antibody (N10=AzaleaB5) that recognizes nucleocapsids fused with the fluorescent protein AzaleaB5, nuclear staining with DAPI, and a pan-cytokeratin antibody to stain oral epithelial cells. (1) Method 500 μL of the sample suspended in 20 ml of Cellprep System Reagent for Gynecological and Oral Use (cat. no.: 518-111458) was dispensed into a microtube and stored at 15,000°C. The mixture was centrifuged at 0 rpm (20,954 x g) for 20 minutes. The supernatant was removed, and approximately 3 μL of the pellet suspension was The cells were then applied to a silane-coated slide (Water Edge Polished Frost 1106, Muto Chemicals). After air-drying to confirm that the semi-wetted cellular components were fixed to the slide, they were fixed in 99.5% ethanol (Fujifilm Wako Pure Chemicals) for 30 minutes at room temperature. After washing twice with PBS (pH 8), blocking was carried out with a blocking solution (3% BSA, 1% Triton-X / 120 mM NaCl, 50 mM PIPES-NaOH (pH 6.8)) for 1 hour at room temperature. 10 μg / mL VHH antibody-fluorescent protein fusion (E N10 = KikG and N10 = AzaleaB5) were reacted for 1 hour at room temperature. After the VHH antibody-fluorescent protein fusion reaction, the cells were washed twice with PBS (pH 7.8) and then re-fixed with 4% PFA / PBS (pH 7.8) for 10 minutes at room temperature. After this, the cells were washed three times with PBS (pH 7.8), and a mouse monoclonal anti-pan-cytokeratin antibody (clone: ​​AE1 / AE3, BioLegend, code: 914204), a marker for squamous epithelial cells, was diluted with 0.1% (wt / vol) TritonX-100 / PBS (pH 7.8) to a final concentration of 1.25 μg / ml and incubated for 1 hour in a room temperature The reaction was carried out at room temperature. After washing three times with PBS (pH 7.8), Alexa Fluor 633-labeled goat anti-mouse IgG (H+L)-F(ab')2 fragment (Thermo Fisher / Invitrogen, code: A21053) diluted with 0.1% (wt / vol) Triton X-100 / PBS (pH 7.8) to a final concentration of 2 μg / ml The mixture was stirred and reacted at room temperature for 1 hour. After washing three times with PBS (pH 7.8), the cell nuclei were stained with DAPI (1 mg / mL in PBS, Fujifilm Wako Pure Chemical Industries, cat. no. 340-07971) diluted to 2 μg / mL in PBS (pH 7.8) (5 minutes, room temperature). After washing three times with PBS, the cells were embedded in a cover glass using PBS as an embedding medium, and the exfoliated cells contained in the clinical specimen of saliva (naturally excreted) were subjected to fluorescence observation using an inverted confocal laser scanning microscope. The observation and imaging conditions for fluorescence observation using a confocal laser scanning microscope were as follows.

[0060] (confocal laser scanning microscope system) Microscope: Inverted FV3000 (Olympus) 1. Objective lens (40x): UPLFLN-40XO (Olympus, Oil, NA: 1.30, WB: 0.2mm) 2. Laser line: Use a 10% ND filter DAPI detection: 405nm (excitation), fluorescence detection range: 430-470nm KikG detection: 488nm (excitation), fluorescence detection range: 500-540nm Azalea detection: 561 nm (excitation), fluorescence detection range 570-611 nm. Pan-cytokeratin (squamous epithelial cells, Alexa Fluor 633) detection: 640 nm (excitation), fluorescence detection range 650-750 nm. 3. Scan XY scan: Mode: Sequential Line Scan speed 4.0μsec / pixel ·z-direction: z-step width: 1.5 μm, number of optical sections: 5 to 8 4. Image size: 512 x 512 5. Pinhole diameter: 158 μm 6. Digital zoom: 1.0x to 3x The observation conditions for infected and non-infected samples were the same. The xy images for each observation are shown as projection images superimposed in the z direction.

[0061] (2) Results Microscopic images are shown in Figure 8. E9=KikG, which recognizes the S protein, enabled clear visualization of cells in infected specimens with a strong fluorescent signal. In contrast, fluorescent signals were barely detectable in uninfected cells. Similarly, N10=AzaleaB5, which recognizes the nucleocapsid and was stained at the same time, also enabled visualization of cells in infected specimens with a relatively strong signal. Furthermore, because S protein staining signals were observed even in pan-cytokeratin antibody-negative cells, it is possible that cells other than squamous epithelial cells in the oral cavity may also be infected. These are thought to be inflammatory cells responding to inflammation caused by infection.

[0062] Test Example 6 N10 = Staining of SARS-CoV-2 clinical specimens with KikG To examine whether KikG exhibits a strong fluorescent signal even when the type of VHH antibody is changed, nucleocapsids were stained using N10=KikG, a VHH antibody that recognizes nucleocapsids fused to KikG, and E9=AzaleaB5, a VHH antibody that recognizes S-proteins fused to AzaleaB5. In addition, nuclear staining with DAPI and quadruple staining using a pan-cytokeratin antibody were performed to stain oral epithelial cells, and the results were observed.

[0063] (1) Method Clinical specimen samples were prepared in the same manner as in Test Example 5 and observed under a confocal microscope under the same conditions. (2) Results Microscopic images are shown in Figure 9. The VHH antibody N10 (N10 = KikG), which recognizes the KikG-fused nucleocapsid, enabled clear visualization of cells in infected specimens with a stronger fluorescent signal than the N10 = AzaleaB5 antibody shown in Figure 8. In contrast, the fluorescent signal was barely detectable in uninfected cells. N10=KikG showed a signal intensity higher than N10=AzaleaB5, and clearer images were obtained, suggesting that fusion with KikG works effectively without imposing a burden on molecule formation, even if the VHH antibody is different. This is also thought to depend on the physical properties of the KikG molecule itself and the fact that KikG forms a tetramer.

[0064] Reference example: Comparison of antibody staining using fluorescent VHH antibodies and conventional indirect fluorescent antibody staining (1) Method Clinical specimens (oral swab samples) were mounted on glass slides using the same method as in Example 5 and stained by antibody staining with E9 KikG (corresponding to direct immunofluorescence). Alternatively, staining was performed by indirect immunofluorescence using a mouse anti-Spike monoclonal antibody (GeneTex, clone 1A9, code: GTX632604) that recognizes S protein as the primary antibody and Alexa Fluor 488-labeled anti-mouse IgG (H+L) (Thermo Fisher, code: A11001) as the secondary antibody. For indirect immunofluorescence, the primary antibody was used at a final concentration of 17 μg / mL, and the secondary antibody at a final concentration of 2 μg / mL. Each reaction step was performed at two different temperatures: 30 minutes at room temperature followed by another 30 minutes at 37°C. In addition, each specimen was stained with DAPI (final concentration 1 μg / mL), mounted, and observed under a wide-field fluorescence microscope to compare the results of E9=KikG antibody staining with those of indirect immunofluorescence.

[0065] Microscope: IX83 (Olympus) Light source:X-cite XYLIS (EXCELITAS) Objective lens: UPLXAPO20x / NA=0.8 (Olympus) Camera: ORCA-Fusion (Hamamatsu Photonics) Fluorescent mirror unit: VHH antibody-fluorescent protein fusion and AlexaFluor488:U-FBNA (Olympus) Nuclear staining (DAPI): U-FUNA (Olympus) Control software: CellSens Dimension (Olympus) Excitation light: Reduced to 5% using an ND filter Exposure time (VHH antibody-fluorescent protein fusion and Alexa Fluor 488): 1 second

[0066] (2) Results: The results are shown in Figure 10. In the indirect fluorescent antibody staining, strong non-specific fluorescence was remarkably observed on the glass surface where no sample was present, compared to E9=KikG. Compared to conventional indirect antibody staining, staining with E9=KikG was found to enable specific, highly sensitive, and good S / N observation of SARS-CoV-2-infected cells mounted on glass slides.

[0067] Test Example 7 Measurement of antigen binding activity of E9=KikG and E9=AzaleaB5 by surface plasmon resonance method The high detection sensitivity of KikG-fused VHH antibodies was thought to be due to the fact that KikG forms a strong tetrameric structure, which causes the VHH antibodies to also tetramerize, resulting in high binding activity due to the sum of these bonds. To confirm this, we measured the antigen binding activity using surface plasmon resonance. (1) Method The binding activity of fluorescent VHH antibodies against SARS-CoV-2 Omicron strain S-protein trimer or S-protein RBD (Receptor Binding Domain, a part of the S-protein that binds to receptors on host cells, excluding the region required for trimer formation) immobilized on a Series S Sensor Chip CM5 (Cytiva) was measured using a Biacore T200 (Cytiva). Measurements were performed using Wizard in Kinetics / Affinity mode. The temperature was set to 25°C. The running buffer used was 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.05% (v / v) Tween 20 (pH 7.4). 1) Antigen immobilization: The flow rate was set to 10 μL / mL. Equal volumes of 75 mg / mL EDC hydrochloride solution and 11.5 mg / mL NHS solution were mixed and added for 420 seconds to activate the carboxyl groups on the sensor chip surface. SARS-CoV-2 Spike Trimer, His Tag (B.1.1.529 / Omicron) (MALS verified) (His-tagged SARS-CoV-2 Omicron strain S-protein trimer, Acro Biosystems) or SARS-CoV-2 B.1.1.529 (Omicron) Spike RBD Protein (His Tag) (His-tagged SARS-CoV-2 Omicron strain S-protein RBD, Sino Biological) diluted in 10 mM sodium acetate buffer (pH 5.5) was added to immobilize 1800 RU and 360 RU, respectively. Blocking was performed by adding 1 M ethanolamine-HCl (pH 8.5) for 420 seconds. 2) Measurement of binding activity: The flow rate was set to 30 μL / mL, and E9 = KikG, E9 = AzaleaB5, or E9 = mAchilles diluted in running buffer was allowed to interact with the sample for a contact time of 180 seconds and a dissociation time of 420 seconds. 3) Regeneration of the sensor chip surface: The flow rate was set to 30 μL / mL, and 50 mM NaOH aqueous solution was added for 30 seconds.Analysis was performed using a 1:1 binding model using Biacore T200 Evaluation Software (software version 2.0) (Cytiva), and binding activity was calculated.

[0068] (2) Results The acquired sensorgram is shown in Figure 11, and the kinetic parameters calculated from the analysis results are shown in Table 3. The calculated KD value is the sum of the binding activities of the multimerized VHH antibodies and is therefore shown as the "apparent KD value." Compared to E9=mAchilles (SEQ ID NO: 74) fused to the monomeric fluorescent protein mAchilles, E9=KikG and E9=AzaleaB5 showed slower dissociation rates, particularly in E9=KikG. KikG forms a tetramer, while AzaleaB5 forms a dimer, suggesting that multimerization of the VHH antibodies increases the valency of antigen binding. This difference is likely responsible for the high sensitivity of virus visualization using KikG-fused VHH antibodies. Furthermore, when comparing the S-protein trimer with the S-protein RBD, the dissociation rate was even slower for the former. This is likely due to the VHH antibodies binding to each S-protein constituting the trimer. From the above, it was considered that the use of multimerized VHH antibodies is an effective means for detecting viral antigens with multimeric structures.

[0069] [Table 3]

Claims

1. A VHH antibody-fluorescent protein fusion product in which a VHH antibody and a fluorescent protein are linked directly or via a peptide linker, wherein the fluorescent protein is selected from the following proteins 1) to 2) and mutants thereof: 1) A protein consisting of the amino acid sequence shown in SEQ ID NO: 2 2) A protein consisting of the amino acid sequence shown in SEQ ID NO: 4

2. The fusion of claim 1, wherein the VHH antibody is linked to the N-terminus or C-terminus of the fluorescent protein directly or via a peptide linker.

3. The fusion of claim 1, wherein the VHH antibody is linked to the N-terminus of the fluorescent protein via a peptide linker.

4. The fusion of claim 3, wherein the peptide linker is (GGGGS)3 (SEQ ID NO: 5).

5. The fusion protein according to any one of claims 1 to 4, wherein the VHH antibody is a VHH antibody having affinity for a virus.

6. The fusion protein according to any one of claims 1 to 4, wherein the VHH antibody is a VHH antibody having affinity for SARS-CoV-2.

7. The fusion according to any one of claims 1 to 4, wherein the VHH antibody is a VHH antibody against SARS-CoV-2 consisting of the amino acid sequence shown in SEQ ID NO: 12, SEQ ID NO: 48 or SEQ ID NO:

55.

8. A method for producing the fusion protein according to any one of claims 1 to 4, comprising the step of culturing cells carrying a nucleic acid encoding the fusion protein.

9. A method for detecting an antigen in a sample, comprising the step of contacting the fusion protein according to any one of claims 1 to 4 with the sample.

10. 10. The method of claim 9, wherein the antigen is a virus.

11. 10. The method of claim 9, wherein the antigen is SARS-CoV-2.

12. A method for multimerizing a VHH antibody, which comprises expressing the VHH antibody as a fusion protein according to claim 1.

13. A method for improving the antigen-binding activity of a VHH antibody, which comprises expressing the VHH antibody as the fusion protein according to claim 1.

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