SINGLE-DOMAIN ANTIBODY BINDING TO proBNP OR ANTIGEN-BINDING FRAGMENT THEREOF

Abstract

To provide a single-domain antibody binding to proBNP or an antigen-binding fragment thereof.SOLUTION: A single-domain antibody binding to proBNP or an antigen-binding fragment thereof, comprising (a) a CDR3 consisting of an amino acid sequence represented by ANQKGA (SEQ ID NO:1); (b) a CDR3 consisting of an amino acid sequence represented by SNGAA (SEQ ID NO:2); (c) a CDR3 consisting of an amino acid sequence represented by SNGTA (SEQ ID NO:3); (d) a CDR3 consisting of an amino acid sequence represented by SNGVA (SEQ ID NO:4); or (e) a CDR3 consisting of an amino acid sequence represented by KAEIVGRDY (SEQ ID NO:5).SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present disclosure relates to single domain antibodies or antigen-binding portions thereof that bind to proBNP. [Background technology]

[0002] BNP (brain natriuretic peptide BNP) is a cardiac hormone derived from proBNP. When the ventricle is under stress, pre-proBNP, consisting of 134 amino acids, is synthesized. Pre-proBNP is then processed to form proBNP (1-108 amino acids), consisting of 108 amino acids. ProBNP is then further cleaved into biologically active BNP (77-108 amino acids, also known as BNP32) and inactive NT-proBNP (1-76 amino acids). This results in a one-to-one relationship between BNP and NT-proBNP. The synthesis of proBNP within cardiomyocytes and the release of BNP and NT-proBNP into the circulatory system are physiologically triggered by cardiac wall stress resulting from increased blood pressure and blood volume. More than 100 million BNP samples are measured annually worldwide, and 16 million samples in Japan. BNP is being used to treat a wide range of conditions, from heart failure and heart disease (Non-Patent Document 1).

[0003] Single-domain antibodies are smaller than conventional antibody molecules such as IgG and have a single antigen-binding domain. While antibodies typically have four protein subunits, single-domain antibodies have only one of these subunits. Single-domain antibodies have smaller molecular weights than conventional antibody molecules, and have advantages such as the ability to produce antibodies using Escherichia coli. Furthermore, VHH antibodies, which are antibodies with a single variable domain (VH domain), are a type of unique immunoglobulin found in some animals of the camelid and llama families. A method for obtaining VHH antibodies is disclosed in Non-Patent Document 2. Non-Patent Document 2 describes a method for efficiently obtaining VHH antibodies by performing CDR shuffling using a phage display library. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Discovery and Clinical Application of BNP, Naoto Minamino, Kenji Samukawa, Journal of the Japanese Circulation Society, Cardiology Specialist, Vol. 25, No. 2, August 2017 [Non-patent document 2] An alpaca single-domain antibody(VHH) phage display library constructed by CDR shuffling provided high-affinity VHHs against desired protein antigens. Int Immunol. 2022 Jul 26;34(8):421-434 Summary of the Invention [Problem to be solved by the invention]

[0005] The present disclosure relates to providing single domain antibodies or antigen-binding portions thereof that bind to proBNP. [Means for solving the problem]

[0006] The present inventors have found that single domain antibodies containing CDR3s with specific amino acids in their constructs exhibit reactivity with proBNP.

[0007] That is, the present invention provides the following: [Section 1] (a) CDR3 consisting of the amino acid sequence represented by ANQKGA (SEQ ID NO: 1); (b) CDR3 consisting of the amino acid sequence represented by SNGAA (SEQ ID NO: 2); (c) CDR3 consisting of the amino acid sequence represented by SNGTA (SEQ ID NO: 3); (d) CDR3 consisting of the amino acid sequence represented by SNGVA (SEQ ID NO: 4); or (e) CDR3 consisting of the amino acid sequence represented by KAEIVGRDY (SEQ ID NO: 5); A single domain antibody or antigen-binding portion thereof that binds to proBNP, comprising: [Section 2] Item 1, a single domain antibody or an antigen-binding portion thereof, wherein the CDR1 and CDR2 are those of a VHH antibody derived from a camelid or a humanized mouse. [Section 3] Item 3. A single domain antibody or antigen-binding portion thereof according to Item 2, wherein CDR1 and CDR2 are those of a VHH antibody derived from a camelid. [Section 4] Item 4. The single domain antibody or antigen-binding portion thereof according to any one of Items 1 to 3, wherein CDR1 consists of an amino acid sequence containing at least one amino acid substitution, deletion, or addition selected from the group consisting of ERTFSRYA (SEQ ID NO: 6), GRSFSPYA (SEQ ID NO: 7), and NFMINRFD (SEQ ID NO: 8). [Section 5] Item 5. The single domain antibody or antigen-binding portion thereof according to any one of Items 1 to 4, wherein CDR2 consists of an amino acid sequence containing at least one amino acid substitution, deletion, or addition selected from the group consisting of ISWSGINT (SEQ ID NO: 9), ISWSGGST (SEQ ID NO: 10), and ITKISTST (SEQ ID NO: 11). [Section 6] (a) CDR1 consisting of the amino acid sequence represented by ERTFSRYA (SEQ ID NO: 6), CDR2 consisting of the amino acid sequence represented by ISWSGINT (SEQ ID NO: 9), and CDR3 consisting of the amino acid sequence represented by ANQKGA (SEQ ID NO: 1); (b) CDR1 consisting of the amino acid sequence represented by GRSFSPYA (SEQ ID NO: 7), CDR2 consisting of the amino acid sequence represented by ISWSGGST (SEQ ID NO: 10), and CDR3 consisting of the amino acid sequence represented by SNGAA (SEQ ID NO: 2); (c) CDR1 consisting of the amino acid sequence represented by ERTFSRYA (SEQ ID NO: 6), CDR2 consisting of the amino acid sequence represented by ISWSGINT (SEQ ID NO: 9), and CDR3 consisting of the amino acid sequence represented by SNGTA (SEQ ID NO: 3); (d) CDR1 consisting of the amino acid sequence represented by ERTFSRYA (SEQ ID NO: 6), CDR2 consisting of the amino acid sequence represented by ISWSGINT (SEQ ID NO: 9), and CDR3 consisting of the amino acid sequence represented by SNGVA (SEQ ID NO: 4); or (e) CDR1 consisting of the amino acid sequence represented by NFMINRFD (SEQ ID NO: 8), CDR2 consisting of the amino acid sequence represented by ITKISTST (SEQ ID NO: 11), and CDR3 consisting of the amino acid sequence represented by KAEIVGRDY (SEQ ID NO: 5); A single domain antibody or antigen-binding portion thereof that binds to proBNP, comprising: [Section 7] A nucleic acid encoding the single domain antibody or antigen-binding portion thereof according to any one of items 1 to 6. [Section 8] A vector comprising the nucleic acid according to Item 7. [Section 9] A host cell comprising the vector according to item 8. [Effects of the Invention]

[0008] According to the present disclosure, there can be provided a single domain antibody or antigen-binding portion thereof that binds to proBNP. [Brief explanation of the drawings]

[0009] [Figure 1] FIG. 1 shows a sequence comparison of the VHH antibodies of the 2C10 group. [Figure 2] FIG. 2 shows a comparison of the sequences of VHH antibodies in the 1C2 and 3F12 groups. [Figure 3] FIG. 3 shows the sequence of the 3H7 VHH antibody. [Figure 4] FIG. 4 shows a sequence comparison of the obtained VHH antibodies. [Figure 5] FIG. 5 shows the ELISA results for the 2C10 group. [Figure 6] FIG. 6 shows the results of ELISA for groups 1C2 and 3F12. [Figure 7] Figure 7 shows the Biacore results. [Figure 8] FIG. 8 shows the vector map of pPANA01. DETAILED DESCRIPTION OF THE INVENTION

[0010] In a first aspect, the present disclosure relates to a single domain antibody or antigen-binding portion thereof that binds to proBNP, comprising a CDR region consisting of a particular amino acid sequence.

[0011] Single-domain antibodies are smaller than conventional antibody molecules such as IgG and have a single antigen-binding domain. While antibodies typically have four protein subunits, single-domain antibodies have only one of these domains, such as a single heavy chain variable region. Examples of single-domain antibodies include VHH antibodies, shark VNAR (variable region of immunoglobulin new antigen receptor (IgNAR)), antibody fragments containing all or part of the VH / VL domains of antibodies, such as single-domain antibodies artificially created from human antibody VH or VL. In the present disclosure, single-domain antibodies are preferably VHH antibodies. VHH antibodies are antibodies with a single variable domain (VH domain). VHH antibodies are a type of unique immunoglobulin found in some camelids and llamas. A method for obtaining VHH antibodies is disclosed in Non-Patent Document 2. Non-Patent Document 2 discloses a method for efficiently obtaining VHH antibodies by performing CDR shuffling using a phage display library.

[0012] The single-domain antibody or antigen-binding portion thereof of the present disclosure has, from the N-terminus to the C-terminus, the following structural domains that can bind: N-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-C. CDRs are complementarity-determining regions, or antigen-recognition sites with variable sequences. FRs are framework regions, which refer to highly conserved regions other than the complementarity-determining regions in the variable region of an antibody molecule. Structural domains such as CDRs can be determined by those skilled in the art from the amino acid sequence of a binding protein using appropriate software. For example, structural domains such as CDRs can be analyzed using IMGT / V-QUEST, selecting IgG under Vicugna pacos (alpaca) Receptor type or locus under Species, and entering the gene sequence (bp).

[0013] The single domain antibody or antigen-binding portion thereof of the present disclosure can bind to proBNP. Since proBNP includes NT-proBNP and BNP32, an antibody that binds to NT-proBNP or BNP32 can also bind to proBNP. Thus, the single domain antibody or antigen-binding portion thereof of the present disclosure may bind to NT-proBNP, BNP32, or proBNP.

[0014] In the present disclosure, the binding strength of a single domain antibody or its antigen-binding portion to an antigen can be evaluated by methods known to those skilled in the art. More specifically, it can be evaluated by determining the dissociation constant Kd using a method using the antigen portion of proBNP and a surface plasmon resonance evaluation device (SPR method). It can also be evaluated by a method using, for example, an ELISA method in which the antigen is immobilized. The binding strength of a single domain antibody or its antigen-binding portion to an antigen may also be evaluated by immunochromatography, biolayer interferometry (BLI), which measures intermolecular interactions in a label-free manner and measures the dissociation constant Kd, isothermal titration calorimetry (ITC), which determines the binding constant Ka, thermodynamic parameters, and molar binding ratio, microscale thermophoresis (MST), which analyzes interactions between various substances such as compounds, proteins, peptides, and nucleic acids and measures affinity (Kds), flow cytometry, which disperses microparticles in a fluid, passes the fluid through a thin stream, and optically analyzes individual particles, and Western blotting, which transfers proteins separated by electrophoresis to a membrane and detects the presence of the protein using an antibody against the protein.

[0015] The form of the antibody of the present disclosure is not limited as long as it can bind to an antigen. It may be used as a single domain antibody such as a VHH antibody, or as a multimer, e.g., a dimer, in which a single-chain antibody, a heavy-chain antibody, or a variable region fragment is linked via a peptide linker or the like, or as a multimer in which a variable region fragment is linked to one or more variable region fragments with different antigen specificities. The antibody of the present disclosure may also be humanized. Humanized antibodies can be administered to humans and therefore can be used as pharmaceuticals. The antibody of the present disclosure may also be conjugated to other polypeptides or may have amino acid modifications introduced therein, so long as it can bind to an antigen. For example, the single domain antibody or antigen-binding portion thereof of the present disclosure may be conjugated to an Fc region. The single domain antibody or antigen-binding portion thereof of the present disclosure may have neutralizing activity by being conjugated to an Fc region. The single domain antibody or antigen-binding portion thereof of the present disclosure may also be conjugated to a known polypeptide, such as a linker, His tag, or FLAG tag.

[0016] The method for producing the antibodies of the present disclosure is not particularly limited and can be easily produced using techniques known in the art. For example, they can be produced by combining solid-phase peptide synthesis with native chemical ligation (NCL) or by genetic engineering. However, a preferred method involves incorporating nucleic acids encoding the antibodies of the present disclosure into an appropriate vector, introducing this into host cells, and producing the antibodies as recombinant antibodies. Based on the disclosure of Non-Patent Document 2, VHH antibodies may also be produced while maintaining the CDR3.

[0017] The antigen-binding portion refers to the site of an antibody molecule that binds to a specific antigen. The antigen-binding portion specifically binds to the antigen through interaction with the antigen. Those skilled in the art can recognize antigen-binding activity using appropriate methods such as ELISA (enzyme-linked immunosorbent assay) and SPR (surface plasmon resonance).

[0018] In the present disclosure, a CDR region consisting of a specific amino acid sequence relates to the CDR3 region of an antibody. A region consisting of a further specific amino acid sequence relates to CDR1, CDR2, or both CDR1 and 2 of an antibody. While the importance of each of the CDR1-3 regions has been demonstrated in the past, the present disclosure provides antibodies having a variety of CDR1 or 2 while maintaining specific antigen recognition by having a specific CDR3.

[0019] In the present disclosure, a CDR3 consisting of a specific amino acid sequence is (a) CDR3 consisting of the amino acid sequence represented by ANQKGA (SEQ ID NO: 1); (b) CDR3 consisting of the amino acid sequence represented by SNGAA (SEQ ID NO: 2); (c) CDR3 consisting of the amino acid sequence represented by SNGTA (SEQ ID NO: 3); (d) CDR3 consisting of the amino acid sequence represented by SNGVA (SEQ ID NO: 4); (e) a CDR3 consisting of the amino acid sequence represented by KAEIVGRDY (SEQ ID NO: 5); or (f) CDR3 consisting of the amino acid sequence represented by YARIQSTTWGRDY (SEQ ID NO: 12).

[0020] In the present disclosure, CDR1 or CDR2 is not particularly limited, so long as the single-domain antibody or its antigen-binding portion having the CDR3 of the present disclosure binds to proBNP. In one embodiment of the present disclosure, CDR1 and CDR2 are those of a VHH antibody derived from a camelid or a humanized mouse. The animal from which the CDR1 and CDR2 of the VHH antibody are derived is, for example, a camelid or a humanized mouse. Preferably, the CDR1 and CDR2 of the VHH antibody are those of a VHH antibody derived from a camelid. Methods for producing VHH antibodies comprising CDR3 using CDR1 and CDR2 derived from a camelid are known to those skilled in the art (Non-Patent Document 2). For example, Non-Patent Document 2 describes the following: alpaca-derived peripheral blood lymphocytes were used as a source of VHH genes, and the alpaca was immunized with human serum albumin (HSA); cDNA was prepared using mRNA extracted from the peripheral lymphocytes of the immunized alpaca; a VHH gene fragment containing N-terminal CDR1 and CDR2 was amplified with a primer set, and a VHH gene fragment containing C-terminal CDR3 was amplified with a primer set; these amplified products were linked using overlapping PCR to obtain full-length VHH gene fragments, and CDR shuffling was performed; and a phage display library was constructed using the obtained full-length VHH gene fragments.

[0021] In the present disclosure, camelids from which VHH antibodies are derived include, but are not limited to, camels, alpacas, and llamas. Preferably, the camelid from which VHH antibodies are derived is an alpaca.

[0022] In one embodiment of the present disclosure, CDR1 or CDR2 consists of a specific amino acid sequence. For example, CDR1 consists of ERTFSRYA (SEQ ID NO: 6), GRSFSPYA (SEQ ID NO: 7), NFMINRFD (SEQ ID NO: 8), or GSRFSIYA (SEQ ID NO: 13), or CDR2 consists of ISWSGINT (SEQ ID NO: 9), ISWSGGST (SEQ ID NO: 10), ITKISTST (SEQ ID NO: 11), or ITSGGNT (SEQ ID NO: 14). In another embodiment, CDR1 or CDR2 may consist of an amino acid sequence containing at least one or more amino acid substitutions, deletions, or additions compared to the corresponding specific amino acid sequence. For example, in the present disclosure, CDR1 may consist of an amino acid sequence of ERTFSRYA (SEQ ID NO: 6) and containing 1, 2, 3, 4, 5, 6, 7, or 8 substitutions, deletions, or additions, or CDR2 may consist of an amino acid sequence of ISWSGINT (SEQ ID NO: 9) and containing 1, 2, 3, 4, 5, 6, 7, or 8 substitutions, deletions, or additions. In yet another embodiment, CDR1 or CDR2 may have 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity with the corresponding specific amino acid sequence.

[0023] In one embodiment of the present disclosure, CDR1 is (a) ERTFSRYA (SEQ ID NO: 6), The first E can be replaced by G or K (E1G, E1K); The second R can be replaced by M, K, or F (R2M, R2K, R2F); The third T can be replaced by an I (T3I); The fourth F may be replaced by Y or S (F4Y, F4S); The fifth S may be replaced by N, R, or D (S5N, S5R, S5D); or The sixth R may be substituted with H, S, G, or D (R6H, R6S, R6G, R6D); It is okay to (b) GRSFSPYA (SEQ ID NO: 7), The first G can be replaced by E or V (G1E, G1V); The second R can be replaced by G (R2G); The third S can be replaced by a T (S3T); The sixth P may be replaced by R, L, or H (P6R, P6L, P6H); or The seventh Y may be replaced by S (Y7S); That's fine.

[0024] In one embodiment of the present disclosure, CDR2 is (a) ISWSGINT (SEQ ID NO: 9), The second S can be replaced by G (S2G); The third W can be replaced by S (W3S); The fourth S can be replaced by I (S4I); The fifth G can be replaced by a D (G5D); The sixth I can be replaced by E or G (I6E, I6G); The seventh N may be replaced by R (N7R); or The eighth T may be replaced by I (T8I); It is okay to (b) ISWSGGST (SEQ ID NO: 10), The sixth G may be replaced by I (G6I); or The seventh S may be replaced by N (S7N); It is okay to or (c) ITKISTST (SEQ ID NO: 11), The fourth I may be replaced with V (I4V).

[0025] In one embodiment of the disclosure, a VHH antibody or antigen-binding portion thereof comprises: (a) CDR1 consisting of the amino acid sequence represented by ERTFSRYA (SEQ ID NO: 6), CDR2 consisting of the amino acid sequence represented by ISWSGINT (SEQ ID NO: 9), and CDR3 consisting of the amino acid sequence represented by ANQKGA (SEQ ID NO: 1); (b) CDR1 consisting of the amino acid sequence represented by GRSFSPYA (SEQ ID NO: 7), CDR2 consisting of the amino acid sequence represented by ISWSGGST (SEQ ID NO: 10), and CDR3 consisting of the amino acid sequence represented by SNGAA (SEQ ID NO: 2); (c) CDR1 consisting of the amino acid sequence represented by ERTFSRYA (SEQ ID NO: 6), CDR2 consisting of the amino acid sequence represented by ISWSGINT (SEQ ID NO: 9), and CDR3 consisting of the amino acid sequence represented by SNGTA (SEQ ID NO: 3); (d) CDR1 consisting of the amino acid sequence represented by ERTFSRYA (SEQ ID NO: 6), CDR2 consisting of the amino acid sequence represented by ISWSGINT (SEQ ID NO: 9), and CDR3 consisting of the amino acid sequence represented by SNGVA (SEQ ID NO: 4); (e) CDR1 consisting of the amino acid sequence represented by NFMINRFD (SEQ ID NO: 8), CDR2 consisting of the amino acid sequence represented by ITKISTST (SEQ ID NO: 11), and CDR3 consisting of the amino acid sequence represented by KAEIVGRDY (SEQ ID NO: 5); or (f) CDR1 consisting of the amino acid sequence represented by GSRFSIYA (SEQ ID NO: 13), CDR2 consisting of the amino acid sequence represented by ITSGGNT (SEQ ID NO: 14), and CDR3 consisting of the amino acid sequence represented by YARIQSTTWGRDY (SEQ ID NO: 12); Includes.

[0026] In one embodiment of the present disclosure, the VHH antibody or antigen-binding portion thereof is not particularly limited as long as it has CDR3 and binds to proBNP, but examples thereof include: QVQLVESGGGLVQAGGSLRLSCTASERTFSRYAMGWFRRAPGKEREFVAGISWSGINTRYADSVKGRFTISRDNSKNTLYLQMNNLKPEDTAVYYCANQKGARGQGTQVTVSS (SEQ ID NO: 15); QVQLVESGGGLVQAGGSLRLSCSASGRTFSHYAMGWFRQAPGKEREFVAGISWSGERTDYEDSVKGRFTISRDNAKNTVYLQMNNLKPEDTAVYYCANQKGARGQGTQVTVSS (SEQ ID NO: 16); ELQLVESGGGLVQPGGSLRLSCQASGRSFSPYAMGWFRQAPHKEREFVAGISWSGGSTKYSDAVKGRFTISRDNAKNTLYLQMNNLNPKDTAIYYCSNGAARGQGTQVTVSS (SEQ ID NO: 17); QVQLVESGGGLVQAGGSLRLTCTASERTFSRYAMGWFRRAPGKEREFVAGISWSGINTRYADSVKGRFTISRDNAKITLYLQMNNLNPKDTAIYYCSNGTARGQGTQVTVSS (SEQ ID NO: 18); QVQLVESGGGLVQAGGSLRLSCTASERTFSRYAMGWFRRAPGKEREFVAGISWSGINTRYADSVKGRFTISRDNAKNTLYLQMNNLNPKDTAIYYCSNGVARGQGTQVSVSS (SEQ ID NO: 19); or QVQLVESGGGLVEPGGSLRLSCAASNFMINRFDMAWYRQAPGKEREWVATITKISTSTRYSQSVEGRFTIYRENHENTVYLQMNSLRPDDTGVYFCKAEIVGRDYWGQGTQVTVSS (SEQ ID NO: 20).

[0027] In another aspect of the disclosure, the VHH antibody or antigen-binding portion thereof has a CDR3 consisting of the amino acid sequence YARIQSTTWGRDY (SEQ ID NO: 12), e.g. QVQLVESGGGLVQPGGSLRLSCAASGSRFSIYAMGWYRQAPGKQRERVARITSGGNTNYADSVKGRFTISRDNAKNTVSLQMNSLKAEDTAVYYCYARIQSTTWGRDYWGQGTQVNVVS (SEQ ID NO: 21), Includes.

[0028] In one embodiment of the present disclosure, the VHH antibody or its antigen-binding portion is not particularly limited as long as it has a CDR3 and binds to proBNP, but may have, for example, 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity to the amino acid sequence set forth in any of SEQ ID NOs: 15-21. In another embodiment of the present disclosure, the VHH antibody or its antigen-binding portion is not particularly limited as long as it has a CDR3, a predetermined CDR1 or CDR2, and binds to proBNP, but may have, for example, 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 15-21.

[0029] As used herein, "sequence identity" refers to the percentage of identical bases or amino acids at corresponding positions in two or more sequences (nucleotide sequences or amino acid sequences) when the sequences are aligned, taking into account gaps and insertions, to maximize sequence identity. Methods for determining identity are designed to maximize identity between the aligned sequences. Methods for determining identity between two sequences include, but are not limited to, BLASTP, BLASTN, FASTA, etc. Identity between two sequences can also be determined using DNA SIS (Hitachi Software Engineering Co., Ltd.) or GENETYX (Genetyx Inc.). Alternatively, for short peptides, identity can be determined simply by comparing the sequences. Those skilled in the art can determine identity between sequences using the methods described above.

[0030] Mutations can be introduced by those skilled in the art using known techniques as appropriate. For example, errors in the PCR amplification process can be utilized to introduce mutations. Other examples include genetic engineering techniques such as site-directed mutagenesis, chemical synthesis methods such as the phosphate triester method and the phosphate amidite method, and combinations thereof. More specifically, DNA synthesis can be performed by chemical synthesis using the phosphoramidite method or the triester method, or on a commercially available automated oligonucleotide synthesizer.

[0031] One embodiment of the present disclosure is a nucleic acid encoding a single domain antibody or an antigen-binding portion thereof. The nucleic acid is preferably DNA, and a nucleic acid encoding the corresponding protein may be optimized appropriately depending on the host. Those skilled in the art can appropriately incorporate the nucleic acid encoding the protein into an appropriate vector and introduce it into an appropriate host cell to produce the protein of interest.

[0032] The host cell is not particularly limited as long as it is capable of expressing the target protein from the encoding nucleic acid, and various cells can be used, such as bacteria such as Escherichia coli, yeast, fungi, insect cells, and mammalian cells.

[0033] Expression vectors suitable for various host cells can be used to express antibodies. Plasmid vectors and viral vectors can be used. The vectors contain a replication origin, selection marker, and promoter appropriate for each vector, and may also contain an enhancer, transcription termination sequence (terminator), ribosome binding site, polyadenylation signal, and other elements as needed. Furthermore, to facilitate purification of the expressed polypeptide, the expression vector may contain a nucleotide sequence for fusion expression of a FLAG tag, His tag, HA tag, GST tag, or other tag. Examples of gene transfer methods include lipofection, calcium phosphate, polymer-based methods, electroporation, and particle gun technology. These methods can be selected appropriately depending on the type of host to be transferred and the gene to be transferred.

[0034] Expressed antibodies can be recovered from cultured cells using known methods. For example, cells are collected by centrifugation, suspended in a buffer solution, and the cells or bacteria are disrupted by ultrasound, lysozyme, and / or freeze-thawing, followed by centrifugation to obtain a soluble extract. The target antibody can be isolated from the resulting extract using known separation and purification methods. Examples of methods that can be used include those that utilize specific affinity, such as affinity chromatography; those that utilize differences in molecular weight, such as SDS-PAGE; those that utilize differences in charge, such as ion exchange chromatography; those that utilize solubility, such as salting out and solvent precipitation; those that utilize differences in hydrophobicity, such as dialysis, ultrafiltration, gel filtration, and reversed-phase high-performance liquid chromatography; and those that utilize differences in isoelectric point, such as isoelectric focusing.

[0035] Another aspect of the present disclosure relates to a kit for detecting proBNP or an antigenic portion thereof, comprising a single domain antibody or antigenic portion thereof of the present disclosure. The single domain antibody or antigenic portion thereof of the present disclosure can bind to proBNP and can therefore be used to detect binding to proBNP or an antigenic portion thereof.

[0036] Yet another aspect of the present disclosure relates to a complex comprising a single domain antibody or antigen-binding portion thereof of the present disclosure, wherein the single domain antibody or antigen-binding portion thereof is bound to at least one of a carrier and a labeling substance. The single domain antibody or antigen-binding portion thereof of the present disclosure may be used after being immobilized on a carrier.

[0037] The shape and material of the solid phase carrier are not particularly limited, as long as it is a carrier insoluble in the solvent in the reaction system of the antigen-antibody reaction. Examples of the shape of the solid phase carrier include plates, beads, disks, tubes, filters, and thin films. Examples of the material of the solid phase carrier include polymers such as polyethylene terephthalate, cellulose acetate, polycarbonate, polystyrene, and polymethyl methacrylate; metals such as gold, silver, and aluminum; and glass. Known methods such as physical adsorption, covalent bonding, ionic bonding, and crosslinking can be used to bind the antibody to the solid phase carrier.

[0038] Examples of labeling substances that can be used include fluorescent substances, luminescent substances, dyes, enzymes, radioactive substances, etc. Methods for binding the antibody to the labeling substance include known methods such as physical adsorption, covalent bonding, ionic bonding, and crosslinking.

[0039] In a detection method using an antibody of the present disclosure, a complex containing the antibody is contacted with an analyte, and a change in a physical quantity based on an antigen-antibody reaction between the analyte in the analyte and the antibody in the complex is detected. Examples of such physical quantities include luminescence intensity, chromaticity, light transmittance, turbidity, absorbance, and radiation dose. Specific examples of detection methods include known methods such as enzyme immunoassay, immunochromatography, latex agglutination, radioimmunoassay, fluorescent immunoassay, and surface plasmon resonance. When using fluorescence spectroscopy using localized surface plasmon resonance (LSPR) of metal nanoparticles, it is important to bring the metal nanoparticles into close proximity, so it is preferable to bring the metal nanoparticles into close proximity through an antigen-antibody reaction. Therefore, when using fluorescence spectroscopy using localized surface plasmon resonance of metal nanoparticles, it is preferable that the single domain antibody of the present disclosure, or its antigen-binding portion, have a small molecular weight. For example, single domain antibodies or antigen-binding portions thereof of the present disclosure have a molecular weight of about 600,000 Da or less, about 500,000 Da or less, about 400,000 Da or less, about 300,000 Da or less, about 200,000 Da or less, about 190,000 Da or less, about 180,000 Da or less, about 170,000 Da or less, and preferably about 200,000 Da or less.

[0040] Yet another aspect of the present disclosure relates to a detection device comprising the single domain antibody or antigen-binding portion thereof of the present disclosure and a detection unit, wherein the detection unit detects a change in a physical quantity based on an antigen-antibody reaction between proBNP or its antigen-binding portion in a test sample and the single domain antibody or its antigen-binding portion. The detection device of the present disclosure includes a detection unit for detecting any of the physical quantities that change based on the antigen-antibody reaction. The detection unit is composed of known devices such as various photometers, spectroscopes, and dosimeters.

[0041] Yet another aspect of the present disclosure relates to a method for detecting proBNP or an antigenic portion thereof in a sample, comprising the steps of contacting a test sample with a single domain antibody or antigenic portion thereof of the present disclosure, and detecting a change in a physical quantity based on an antigen-antibody reaction between proBNP or an antigenic portion thereof in the test sample and the single domain antibody or antigenic portion thereof.

[0042] As used herein, "about" means within a range of ±10%, preferably ±5%.

[0043] The present disclosure will be specifically and in detail explained below by showing examples, but the examples are used to illustrate the present disclosure and are not intended to limit the present disclosure. [Example]

[0044] [Example 1] antigen Phage display was used to synthesize genes to increase the molecular weight of the antigen by linking the antigen itself to the phage for efficient binding and to ensure sufficient epitope content. To synthesize the gene, we first designed the precursor monomeric pro-BNP gene (hereafter referred to as 1xpro-BNP) and synthesized it at Eurofins Genomics. Monomeric BNP was digested with restriction enzymes, and then ligated to another restriction enzyme-digested BNP fragment at the digestion site to synthesize 2xpro-BNP and 3xpro-BNP. The resulting gene was inserted into a vector and transformed into DH10B Escherichia coli. Protein expression and purification were performed using IPTG (isopropyl-β-D-thiogalactopyranoside) induction to utilize the lac promoter. Purification was performed using the Ni-NTA (nickel-nitrilotriacetic acid) method, a simple and efficient purification method widely used. The BNP antigens obtained are as follows: (AA sequence: 6xHis-1xpro-BNP) MGSSHHHHHHSSGLVPRGSHMHPLGSPGSASDLETSGLQEQRNHLQGKLSELQVEQTSLEPLQESPRPTGVWKSREVATEGIRGHRKMVLYTLRAPRSPKMVQGSGCFGRKMDRISSSSGLGCKVLRRH (SEQ ID NO: 22) (AA sequence: 6xHis-2xpro-BNP) MGSSHHHHHHSSGLVPRGSHMHPLGSpgsasdletsglqeqrnhlqgklselqveqtsleplqesprptgvwksrevategirghrkmvlytlraprspkmvqgsgcfgrkmdrissssglgckvlrrhgsPGSASDLETSGLQEQRNHLQGKLSELQVEQTSLEPLQESPRPTGVWKSREVATEGIRGHRKMVLYTLRAPRSPKMVQGSGCFGRKMDRISSSSGLGCKVLRRH (SEQ ID NO: 23) (AA sequence: 6xHis-3xpro-BNP) MGSSHHHHHHSSGLVPRGSHMHPLGSpgsasdletsglqeqrnhlqgklselqveqtsleplqesprptgvwksrevategirghrkmvlytlraprspkmvqgsgcfgrkmdrissssglgckvlrrhgspgsasdletsglqeqrnhlqgklselqveqtsleplqesprptgvwksrevategirghrkmvlytlraprspkmvqgsgcfgrkmdrissssglgckvlrrhgsPGSASDLETSGLQEQRNHLQGKLSELQVEQTSLEPLQESPRPTGVWKSREVATEGIRGHRKMVLYTLRAPRSPKMVQGSGCFGRKMDRISSSSGLGCKVLRRH (SEQ ID NO: 24)

[0045] [Example 2] Phage display Phages were screened from a phage library using the phage display method. The antigen, proBNP, the precursor of BNP, is small, approximately 12 kDa (BNP is 3.5 kDa, NT-proBNP is 8.5 kDa), and there was a possibility that it would be buried in the blocking agent used during screening, or that the recovery of hidden epitopes would be impaired. Therefore, by using the multimerized antigen 3xpro-BNP, we were able to efficiently screen and obtain antibodies.

[0046] In the present disclosure, a biopanning method was used in the screening process. Biopanning is a method that allows the enrichment of phage populations that bind to a target antigen by binding a library to the target antigen and then performing a selection procedure. In this disclosure, screening was performed using trimeric pro-BNP (3xpro-BNP) as the antigen, and phages that bind were recovered. The binding ability of the recovered phages was confirmed by ELISA (Enzyme-Linked Immunosorbent Assay). Those that were confirmed to bind were named 1C2, 3F12, 2C10, and 3H7. The recovered phages were confirmed to have almost no binding to cTnT (to determine whether they bind to the His tag) or HSA (to determine whether they nonspecifically adsorb). Based on their respective binding avidity to proBNP and NT-BNP, 1C2, 3F12, and 2C10 bound to 1xproBNP, while 3H7 was thought to have binding avidity to NT-BNP (data not shown). Among those whose binding activity was confirmed, VHH-3xFLAG tag-6xHis tag fusion proteins of 1C2, 3F12, 2C10, and 3H7 were purified.

[0047] The amino acid sequence of the binding protein of the phage gene whose binding ability was confirmed is shown below. The underlined portion indicates the CDR3 sequence, and the shaded portion indicates the domain structure. DYKDHDGDYKDHDIDYKDDDDK (SEQ ID NO: 25) is 3xFLAG. (AA sequence: Anti-BNP-1C2) [ka] (SEQ ID NO: 26) (AA sequence: Anti-BNP-3F12) [ka] (SEQ ID NO: 27) (AA sequence: Anti-BNP-2C10) [ka] (SEQ ID NO: 28) (AA sequence: Anti-BNP-3H7) [ka] (SEQ ID NO: 29)

[0048] [Example 3] CDR3-preserving library CDR3-preserved libraries were prepared for 1C2, 3F12, and 2C10, and antibodies were obtained that had different CDR1 and CDR2 from the original antibodies while maintaining CDR3. The CDR3-preserved libraries were prepared based on the method described in Non-Patent Document 2. Specifically, the CDR3-preserved libraries were prepared as follows.

[0049] (N-terminal gene recombination) The genes obtained by screening were combined with diverse alpaca-derived genes using the method described in Non-Patent Document 2. The following primers were used for the C-terminal halves of the obtained VHH genes and the N-terminal halves of the diverse alpaca-derived genes. Alp-VHH-F: 5'-CTGCTCCTCGCGGCCCAGCCGGCCATGGCTSAGKTGCAGCTCGTGGAGTC-3' (SEQ ID NO: 30) Alp-Shinge-R: 5'-TTTTGCTCTGCGGCCGCAGAGGCCGTGGGGTCTTCGCTGTGGTGCG -3' (SEQ ID NO: 31) Alp-Linge-R: 5'- TTTGCTCTGCGGCCGCAGAGGCCGATTGTGGTTTTGGTGTCTTGGG -3' (SEQ ID NO: 32) Alp-overlap-R: 5'-GAATCGGCCCTTCACGGA-3' (SEQ ID NO: 33) Alp-overlap-F: 5'-TCCGTGAAGGGCCGATTC-3' (SEQ ID NO: 34)

[0050] Restriction enzyme digestion of vector and insert The vector and insert were each treated with a restriction enzyme under the following conditions (r Cut smart (B6004S) manufactured by BioLab, and Sfi I (SD3505) manufactured by BioLab). JPEG2025146217000006.jpg44112 Purification (GP kit): GP wash 1 / 2, 1 mL, column (1 insert, 3 vectors), eluted with GP3 30 uL. The restriction enzyme digestion solution was collected in the tube below the column. GP wash 1 / 2 was added to the tube and mixed. All of the solution in the tube was collected and placed in columns 1-3, followed by centrifugation (8000 rpm, 10 seconds), and the liquid in the tube was discarded. 600 μL of GP2 was added to the column and centrifuged (8000 rpm, 10 seconds). The liquid in the tube was discarded, the lid was opened, and the column was centrifuged again (13000 rpm, 10 minutes). 30 μL of GP3 was added to the column and eluted by centrifugation (13,000 rpm, 10 seconds).

[0051] The vector (pPANA01_SfiI) and inserter (VHH_SfiI) were ligated at a vector:VHH ratio of 1:6 (16°C, 3 hours) under the following conditions (the vector map of pPANA01 is shown in Figure 8). After ligation, the product was precipitated with isopropanol and then dried. (Ligation High was manufactured by TOYOBO.) JPEG2025146217000007.jpg3248

[0052] (Transformation into E. coli) The resulting precipitate was dissolved in 5 μL of DW and electroporated by applying (1.5 kV, 200 Ω, 25 μF) to a JM109 (manufactured by Takara Bio Inc.) The electroporated E. coli was plated on a 2YTGA plate and cultured overnight at 30°C.

[0053] (phage rescue) E. coli recovery 4 mL of 2YTA was added to an overnight culture plate of E. coli, the solution was spread evenly, and the plate was left to stand for 15 minutes. The plate was then collected with a conical rod and placed in a centrifuge tube. Infection with helper phage 100 mL of 2YTGA and 500 μL of E. coli solution were added to an Erlenmeyer flask. The mixture was cultured with shaking at 30°C for 1-1.5 hours. 50 mL of the 100 mL mixture was placed in a centrifuge tube, and 2 mL of M13KO7 was added. The mixture was infected in an incubator at 37°C for 45 minutes. After centrifugation (4,000 rpm, 10 minutes), the supernatant was discarded. The precipitate was collected with 200 mL of 2YTAK and placed in an Erlenmeyer flask. The mixture was cultured with shaking at 30°C until the next morning.

[0054] (phage enrichment) Polyethylene Glycol Concentrate The 20% PEG solution was prepared by mixing 500 mL of Milli-Q, 100 g of PEG (8,000), and 73 g of NaCl, then autoclaved and cooled with stirring. After incubation, the culture was centrifuged (10,000 rpm, 20 minutes), and 40 mL of the supernatant was collected. 10 mL of 20% PEG was added, mixed by inversion, and allowed to stand on ice for 1 hour. The culture was further centrifuged (10,000 rpm, 15 minutes, with the brake set to minimum to prevent shaking), and the supernatant was slowly discarded from the side opposite the pellet. The rotor was then rotated again and centrifuged (10,000 rpm, 15 minutes, with the pellet facing inward). The solution was removed using a pipettor, and 2 mL of PBS was added. The mixture was vortexed thoroughly and allowed to stand on ice for 1 hour. Vortexing was continued occasionally during this period. The mixture was vortexed and centrifuged (10,000 rpm, 10 minutes). The supernatant was slowly collected without shaking the centrifuge tube and used as a phage library solution (stored at -80°C).

[0055] (Biopanning (screening)) 1 st Panning The day before, 3 mL of 1xpro BNP 30 μg / mL (in PBS(Az)) solution was added to an immunotube and immobilized. 70 mL of 2YT was mixed with 70 μL of TG-1 to prepare the TG-1 solution, which was then incubated with shaking at 37°C for approximately 2-3 hours. 3% skim milk solution (in PBS / Az) and 100 mM triethylamine solution were prepared. The immunotube was washed three times with PBS / Az. It was then blocked with 3% skim milk (PBS / Az) for at least 1 hour. It was then washed three times with PBS / Az. 3 mL of 3% skim milk (PBS / Az), 100 μL of library solution, and 100 μL of 10 mg / mL BSA solution were added, mixed by inversion on a rotator for 10 minutes, and then allowed to stand for 80 minutes. The tube was then washed ten times with PBS-T. 1 mL of 100 mM triethylamine was added, vortexed (degrees: 7), and the cells were collected in a tube containing 1 mL of a pH 5.6 Tris solution (neutralizing solution). 1 mL of triethylamine was added again, vortexed, and collected. 12 mL of the cultured TG-1 was added, mixed, and incubated (37°C). 100 μL of the solution was seeded onto a pre-warmed 2xTGA plate. Plates 2-4 were centrifuged, excess supernatant discarded, and the pelleted solution was vortexed and seeded in three equal portions. Then, the plate was cultured overnight at 30°C.

[0056] (phage rescue) Three mL of 2YTGA was added to each of three pellet-seeded plates, and the E. coli cells were scraped off with a cone rod and collected in a 15 mL centrifuge tube. 20 mL of 2YTGA solution and 0.5 mL (target OD value = 0.2) of E. coli solution were placed in a 50 mL centrifuge tube and cultured with shaking at 30°C for 2 hours. 0.6 mL of M13KO7 was added to infect 15 μL of culture (MOI = 20). The tube was left to stand at 37°C for 40 minutes. After centrifugation (4,000 rpm, 10 minutes), the supernatant was discarded, and 20-25 mL of 2YTAK was added. The tube was cultured overnight with shaking at 30°C with the lid slightly ajar. The next morning, the culture was centrifuged (8,000 rpm, 5 minutes), and the supernatant was collected and used as a phage solution (2 nd to panning).

[0057] (2 nd Panning (1xBNP) The immunotube, on which the antigen had been immobilized the day before, was washed three times with PBS. It was blocked with 3% skim milk (PBS(Az)) for 1.5 h and washed three times with PBS. 3 mL of 3% skim milk and phage solution (1 st 500 μL of rescue solution was added, the tube was sealed with parafilm, and the reaction was carried out on a rotator for 10 minutes, followed by 80 minutes of standing time. The solution was discarded and the tube was thoroughly washed with PBS-T. 1 mL of 100 mM triethylamine was added and vortexed. 1 mL of Tris solution, pH 5.6, was placed in a 5 mL tube, and the triethylamine solution was transferred to the neutralizing solution. 1 mL of triethylamine was added again, vortexed, and the tube was recovered. 900 μL of pre-cultured TG-1 was added to 100 μL of neutralizing solution in an Eppendorf tube and infected. After mixing, the plate was incubated (37°C, 45 minutes). 10 μL of the infected solution was plated on one plate and 100 μL on two plates of a pre-warmed 2YTGA plate. The plate was then cultured overnight at 30°C.

[0058] (Single colony recovery) 2YTGA was added to a 96-well plate at 200 μL / well. E. coli colonies were harvested from the cultured plate using a sterilized toothpick and individually inoculated into wells. The plates were cultured with shaking at 30°C for 4 hours. Helper phage (M13KO7) diluted 50-fold with 2YT was added to a new plate at 100 μL / well. The cultured E. coli solution was added at 50 μL / well and allowed to infect in an incubator at 37°C for 30 minutes. The plate was centrifuged at 2,000 rpm for 20 minutes, and the supernatant was discarded. 2YTAK was added at 200 μL / well and the plates were cultured with shaking at 30°C overnight.

[0059] (ELISA) The day before, 50 μL of 1x pro BNP (8.38 μL / μL) in PBS (Az) was added to an ELISA plate at 10 μg / mL, sealed, and stored in a refrigerator for immobilization. The immobilized 96-well plate was washed three times with 200 μL / well of PBS (Az). Blocking was performed with 200 μL / well of 3% skim milk (1 h). The plate was then washed three times with 200 μL / well of PBS. 50 μL / well of 3% skim milk solution was added, and the plate was then centrifuged (2,000 rpm, 20 min). The supernatant was added as phage solution (50 μL / well) and incubated for 60 min. The plate was washed five times with 200 μL / well of PBST. Mouse anti-M13 antibody was added at 50 μL / well (15 min). The plate was then washed five times with 200 μL / well of PBST. Anti-mouse IgG antibody-HRP diluted 5,000-fold with PBST was added at 50 μL / well (15 minutes). The plate was washed six times with 200 μL / well of PBST. A substrate solution consisting of 10 mL of 0.1 M NaH2PO4, 5 μL of hydrogen peroxide, and 10 mg of o-phenylenediamine dihydrochloride was added at 50 μL / well (10 minutes). After confirming color development, the reaction was stopped by adding 50 μL / well of 1 M sulfuric acid. Absorbance was measured at OD 490 nm using a TECAN.

[0060] (Sequence analysis) Plasmids were recovered from Escherichia coli (TG-1) infected with the phages that had binding activity, and sequence analysis was performed.

[0061] (Cloning, expression and purification) Using the recovered plasmid as a template, DNA was amplified by PCR under the following conditions. The PCR product was electrophoresed and then purified using a GP kit. Primer-F: CTTCCGGCTCGTATGTTGTGTG (SEQ ID NO: 35) Primer-R: CAAAATCACCGGAACCCTATGC (SEQ ID NO: 36) JPEG2025146217000008.jpg49129

[0062] (restriction enzyme treatment) The PCR product obtained after purification was prepared into a solution in the following proportions, incubated (37°C, 60 minutes), and then purified. JPEG2025146217000009.jpg2251

[0063] (ligation) 2 μL each of the eluate, pPANA-01 (SfiI-treated), and Ligation high ver.2 was placed in a PCR tube and pipetted. Then, the tube was incubated at 16°C for 1 hour.

[0064] (Transformation and E. coli cultivation) 2 μL of the ligation solution was added to the E. coli solution and pipetted into a cuvette. The cuvette was shaken well to remove any air bubbles, the outside of the cuvette was wiped thoroughly with a Kimwipe, and the cuvette was then placed in the device. Voltage was applied (15 kV, 200 Ω, 25 μF), 500 μL of SOC medium was added, and the mixture was incubated (37°C, 30 minutes). The mixture was then plated onto a 2YTA plate and incubated (37°C, 12 hours).

[0065] (Cultivation and IPTG induction) Colonies that had confirmed the presence of inserts by colony PCR were inoculated into a culture tube containing 4 mL of 2YTA (shaking culture at 37°C overnight). 200 mL of 2YTA solution was placed in an Erlenmeyer flask, and approximately 1 mL of the culture solution was added and cultured with shaking (37°C, 2 hours). IPTG was added to a final concentration of 100 mM, and the culture was shaken at 30°C for 5 hours.

[0066] (Collection and disruption) The culture solution was placed in a 50 mL centrifuge tube and centrifuged (8000 rpm, 5 minutes). The supernatant was discarded and the culture solution was added (all of the bacteria were collected by centrifugation). As much of the supernatant as possible was discarded. PBS (Az) solution was added to the centrifuge tube up to 45 mL. 45 μL of 5 M imidazole, pH 7.2, was then added. A probe (washed with 70% ethanol and wiped) was then immersed in the centrifuge tube under ice cooling and disrupted under the following conditions. Output control: 5 Duty cycle: 50% Time: 15 minutes x 2 sets

[0067] (Ni-NTA purification) The His60 Ni Super flow Resin solution (PBS / Az) was mixed by slow inversion. 1 mL of the resin solution prepared above was added to the solution recovered from the crushing, and the mixture was mixed by inversion on a rotator (2 h, rotation speed 10). The entire solution was then poured into a column, and two columns were washed with 20 mM imidazole (PBS / Az). 2-4 mL of 300 mM imidazole (PBS / Az) was added to elute the VHH.

[0068] [Example 4] Mutation library construction To demonstrate the importance of preserving the CDR3 region in the single-domain antibodies of the present disclosure or their antigen-binding portions, we attempted to introduce mutations into the VHH genes obtained above. Specifically, we randomly introduced mutations into the gene sequence of a plasmid containing a VHH gene reactive with an antigen using the GeneMorph II Random Mutagenesis kit (Agilent Technologies). PCR was performed with reference to the kit's manual. Furthermore, the following primers were used, with reference to Non-Patent Document 2: Alp-VHH-F: 5'-CTGCTCCTCGCGGCCCAGCCGGCCATGGCTSAGKTGCAGCTCGTGGAGTC-3' (SEQ ID NO: 30) Alp-Shinge-R: 5'-TTTGCTCTGCGGCCGCAGAGGCCGTGGGGTCTTCGCTGTGGTGCG-3' (SEQ ID NO: 31) Alp-Linge-R: 5'-TTTGCTCTGCGGCCGCAGAGGCCGATTGTGGTTTTGGTGTCTTGGG-3' (SEQ ID NO: 32) The amplified products were subjected to the same procedure as in Example 3 above to obtain mutant VHHs.

[0069] The sequences of the VHH antibodies derived from 2C10, 1C2, and 3F12 obtained in Examples 3 and 4 were compared. The domain structures of the obtained VHH antibodies are shown in Figures 1 and 2. Figure 3 shows the sequence of the VHH antibody derived from 3H7.

[0070] [Example 5] According to Example 2-4, phage display, CDR3 preservation, and random mutagenesis were appropriately performed to obtain Anti-pBNP-VHH23-29 capable of binding to pro-BNP. The first antibodies with novel CDR3s were: Anti-pBNP-VHH23: QVQLVESGGGLVQAGGSLRLTCTASERTFSRYAMGWFRRAPGKEREFVAGISWSGINTRYADSVKGRFTISRDNAKITLYLQMNNLNPKDTAIYYCSNGTARGQGTQVTVSS (SEQ ID NO: 18) Anti-pBNP-VHH25: QVQLVESGGGLVQAGGSLRLSCTASERTFSRYAMGWFRRAPGKEREFVAGISWSGINTRYADSVKGRFTISRDNAKNTLYLQMNNLNPKDTAIYYCSNGVARGQGTQVSVSS (SEQ ID NO: 19) Anti-pBNP-VHH27: QVQLVESGGGLVEPGGSLRLSCAASNFMINRFDMAWYRQAPGKEREWVATITKISTSTRYSQSVEGRFTIYRENHENTVYLQMNSLRPDDTGVYFCKAEIVGRDYWGQGTQVTVSS (SEQ ID NO: 20). A comparison of the obtained sequences is shown in FIG.

[0071] The antibodies obtained in Examples 1 to 5 are summarized in the table below. [Table 1]

[0072] [Example 6] Antibody affinity measurement by ELISA The affinity of the obtained antibodies for the 1x proBNP antigen was measured by ELISA. As a preliminary preparation, 50 μL each of 10 μg / mL 1x BNP antigen and HSA solution were added to an ELISA plate and immobilized (refrigerated, sealed, and left for at least 12 hours). The antibody was prepared in PBS to a concentration of 10 μM. The ELISA plate with the immobilized antigen was washed three times with 200 μL / well of PBS, and then blocked by adding 200 μL / well of 3% skim milk-containing PBS and leaving it to stand for at least 30 minutes. After washing three times with 200 μL / well of PBS and adding 50 μL / well of 3% skim milk-containing PBS, the diluted antibody solution was added at 50 μL / well and left to stand for 60 minutes. The plate was washed five times with 200 μL / well of PBS-T, and then a solution of monoclonal anti-FLAG® 2 antibody (6 μL / 6 mL) in PBS-T was added at 50 μL / well and left to stand for 15 minutes. After washing five times with 200 μL / well of PBST, mouse anti-M13 antibody (RePHAGEN) was added at 50 μL / well and allowed to stand for 15 minutes. Anti-mouse IgG antibody-HRP (1030-05, SouthernBiotech) diluted 5,000-fold in PBS-T was added at 50 μL / well and allowed to stand for 10 minutes. After washing six times with 200 μL / well of PBS-T, a substrate solution consisting of 10 mL of 0.1 M NaH2PO4, 5 mL of hydrogen peroxide, and 10 mg of o-phenylenediamine dihydrochloride was added at 50 μL / well and allowed to stand for 20 minutes. After confirming color development, the reaction was stopped by adding 50 μL / well of 1 M sulfuric acid. The absorbance at 490 nm was measured using a plate reader. The results are shown in Tables 4-6. The results showed that by preserving CDR3, VHH antibodies that retain their antigen-binding ability can be obtained even if mutations are present in CDR1 and 2 and the FR region.

[0073] [Example 7] Protein concentration measurement and Biacore (molecular interaction) measurement Protein concentration measurement The VHH antibody concentration was measured using a BCA protein assay kit (Thermo Scientific, #23227). Bovine serum albumin (Thermo Scientific) was diluted with PBS (Takara Bio) to a concentration between 25 and 2000 μg / mL. Each synthesized VHH antibody was diluted with PBS to 200 μg / mL based on the concentration measured using Nanodrop. The rest of the procedure was performed according to the protocol of the BCA protein assay kit. 25 μL of standard or sample solution was placed in a 96-well plate (Greiner). 200 μL of BCA working reagent was added to each well, and the solution was thoroughly mixed for 30 seconds using a plate shaker (M&S Instruments). The plate was covered and incubated at 37°C for 30 minutes (Espec). The plate was then cooled to room temperature. The absorbance at 562 nm was measured using a plate reader (TECAN). A standard curve was prepared using BSA, and the protein concentration was calculated and used as the protein concentration.

[0074] Biacore measurement Immobilization of antigens on C5 chips BNP32 (Peptide Institute) was used as the antigen to measure the molecular interaction between the synthesized VHH antibodies. The Biacore T200 (Cytiva) was used according to the Biacore instructions. BNP32 antigen was prepared as a stock solution at 0.3375 mg / mL in Milli-Q and diluted with 10 mM HEPES (Dojindo) at pH 8.0 to a final concentration of 5 μg / mL. It was then immobilized on a CM5 chip (Cytiva) (minimum immobilization amount: RU 243.6). The carboxyl groups of CM dextran were activated with EDC (N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride) (Cytiva) and NHS (N-hydroxy succinimide) (Cytiva), and the remaining active NHS groups were blocked with ethanolamine hydrochloride (Cytiva). The running buffer used was HBS-P+ (Cytiva).

[0075] Injection of VHH antibodies into a C5 chip The regeneration conditions were determined so that the RU value returned to the baseline and the same response was obtained when the same analyte was injected. 10 mM Glycine-HCl, pH 1.5 was suitable. Five VHH concentrations were prepared using HBS-P+ by 2-fold dilution from 16,000 nM. After preparation, the samples were injected and measured. The analyte addition time was 2 minutes, followed by 4 minutes of dissociation. The samples used were 1C2, 2C10, and 3F12.

[0076] The results are shown in Figure 7. The results confirmed that the antibodies obtained in this disclosure bind to the antigen. [Industrial Applicability]

[0077] According to the present disclosure, there can be provided a single domain antibody or antigen-binding portion thereof that binds to proBNP.

Claims

1. (a) CDR3 consisting of the amino acid sequence represented by ANQKGA (SEQ ID NO: 1); (b) CDR3 consisting of the amino acid sequence SNGAA (SEQ ID NO: 2); (c) CDR3 consisting of the amino acid sequence represented by SNGTA (SEQ ID NO: 3); (d) a CDR3 consisting of the amino acid sequence represented by SNGVA (SEQ ID NO: 4); or (e) CDR3 consisting of the amino acid sequence represented by KAEIVGRDY (SEQ ID NO: 5); A single domain antibody or antigen-binding portion thereof that binds to proBNP, comprising:

2. 2. A single domain antibody or antigen-binding portion thereof according to claim 1, wherein the CDR1 and CDR2 are those of a VHH antibody derived from a camelid or a humanized mouse.

3. 3. A single domain antibody or antigen-binding portion thereof according to claim 2, wherein the CDR1 and CDR2 are the CDR1 and CDR2 of a VHH antibody derived from a camelid.

4. 4. The single domain antibody or antigen-binding portion thereof according to any one of claims 1 to 3, wherein CDR1 consists of an amino acid sequence comprising at least one amino acid substitution, deletion, or addition selected from the group consisting of ERTFSRYA (SEQ ID NO: 6), GRSFSPYA (SEQ ID NO: 7), and NFMINRFD (SEQ ID NO: 8).

5. 5. A single domain antibody or antigen-binding portion thereof according to any one of claims 1 to 4, wherein CDR2 consists of an amino acid sequence comprising at least one amino acid substitution, deletion, or addition selected from the group consisting of ISWSGINT (SEQ ID NO: 9), ISWSGGST (SEQ ID NO: 10), and ITKISTST (SEQ ID NO: 11).

6. (a) CDR1 consisting of the amino acid sequence represented by ERTFSRYA (SEQ ID NO: 6), CDR2 consisting of the amino acid sequence represented by ISWSGINT (SEQ ID NO: 9), and CDR3 consisting of the amino acid sequence represented by ANQKGA (SEQ ID NO: 1); (b) CDR1 consisting of the amino acid sequence represented by GRSFSPYA (SEQ ID NO: 7), CDR2 consisting of the amino acid sequence represented by ISWSGGST (SEQ ID NO: 10), and CDR3 consisting of the amino acid sequence represented by SNGAA (SEQ ID NO: 2); (c) CDR1 consisting of the amino acid sequence represented by ERTFSRYA (SEQ ID NO: 6), CDR2 consisting of the amino acid sequence represented by ISWSGINT (SEQ ID NO: 9), and CDR3 consisting of the amino acid sequence represented by SNGTA (SEQ ID NO: 3); (d) CDR1 consisting of the amino acid sequence represented by ERTFSRYA (SEQ ID NO: 6), CDR2 consisting of the amino acid sequence represented by ISWSGINT (SEQ ID NO: 9), and CDR3 consisting of the amino acid sequence represented by SNGVA (SEQ ID NO: 4); or (e) CDR1 consisting of the amino acid sequence represented by NFMINRFD (SEQ ID NO: 8), CDR2 consisting of the amino acid sequence represented by ITKISTST (SEQ ID NO: 11), and CDR3 consisting of the amino acid sequence represented by KAEIVGRDY (SEQ ID NO: 5); A single domain antibody or antigen-binding portion thereof that binds to proBNP, comprising:

7. A nucleic acid encoding a single domain antibody or antigen-binding portion thereof according to any one of claims 1 to 6.

8. A vector comprising the nucleic acid of claim 7.

9. A host cell comprising the vector of claim 8.

Images