Novel antigen-binding fragment complex and expression platform therefor

Abstract

The present invention relates to an antigen-binding fragment complex in which antigen-binding fragments linked to SARAH domains spontaneously form a non-covalent association through the SARAH domains, and to an expression vector for producing the antigen-binding fragment complex to express same. The antigen-binding fragment complex of the present invention can be efficiently produced in bacteria in a structure that specifically binds to various antigens, and can maintain a stable form.

Description

Novel antigen-binding fragment complex and its expression platform

[0001] Cross-citation with related applications

[0002] This application claims the benefit of priority from Korean Patent Application No. 10-2024-0115317, filed August 27, 2024, the entire contents of which are incorporated herein by reference.

[0003]

[0004] Technology field

[0005] The present invention relates to an antigen-binding fragment complex comprising a SARAH domain and a platform for expressing the same.

[0006]

[0007] Antigen-binding fragments possess antigen recognition function while possessing smaller molecular weights and simpler structures than full-length antibodies, making them useful in the development of diagnostic and therapeutic biological agents. In particular, fragments recombined around the antigen-binding site of antibodies, called Fab (fragment antigen-binding) or scFv (single-chain variable fragment), are capable of specific binding to various antigens, and their production in Escherichia coli (hereinafter referred to as E. coli) using recombinant technology has been attempted.

[0008] Among these, scFv has the advantage of being easily expressed in E. coli, as it has a structure in which the VH (variable heavy) and VL (variable light) domains are connected to a single polypeptide through a linker. However, its own stability and binding affinity are low, and problems such as denaturation at high temperatures or aggregation during storage have been frequently reported.

[0009] Meanwhile, Fab fragments are known to be structurally more stable and have superior antigen-binding affinity than scFvs, as they contain not only the VH and VL domains but also the CH1 and CL domains, which are the constant regions of each. However, Fab fragments have the disadvantage of being difficult to induce dimerization, and because the two chains are noncovalently linked, intracellular expression and purification are difficult, and there are problems with low expression yields. In particular, if the dimer structure is unstable or easily dissociated, reliable signal detection is difficult in Fab-based diagnostic kits, so stable Fab dimer formation is a very important factor in practical kit applications. To improve the structural instability of Fab fragments, methods of incorporating artificial dimerization motifs, such as leucine zippers, have been used. However, even in this case, problems such as nonspecific dimer formation, increased binding to non-target antigens, and increased complexity of the production process have been reported. Furthermore, because these motifs are artificially inserted into the antibody structure, concerns about immunogenicity exist.

[0010] Therefore, a new antigen-binding fragment platform is needed that simultaneously ensures ease of expression and structural stability of antigen-binding fragments, while also inducing spontaneous dimerization. In particular, there is a growing technological demand for antigen-binding fragments that can be stably produced in bacterial expression systems while maintaining specific antigen binding affinity.

[0011] Under this technical background, the present applicant designed an antigen-binding fragment platform that induces stable dimerization while maintaining the antigen-binding ability of the existing Fab structure by fusing the SARAH domain to the light chain constant region (C-terminus) of the Fab fragment, and in particular, completed the present invention by confirming through experiments that the platform can be stably expressed and purified at a high yield even in an E. coli expression system and can be applied to various antigens.

[0012]

[0013] One object of the present invention is to provide an antigen-binding fragment complex in which a monomer including an antigen-binding fragment spontaneously forms a complex, and an expression vector for expressing the same.

[0014] Another object of the present invention is to provide an expression vector for producing an antigen-binding fragment complex that can be utilized as a platform. The present invention provides an expression vector for producing an antigen-binding fragment complex that spontaneously forms a complex when a user inserts a base sequence encoding the antigen-binding fragment into a cloning site and expresses the antigen-binding fragment.

[0015]

[0016] In order to achieve the above object, one aspect of the present invention provides an antigen-binding fragment complex, comprising: a first monomer comprising a first antigen-binding fragment and a SARAH (Sav / Rassf / Hpo) domain linked to the first antigen-binding fragment; and a second monomer comprising a second antigen-binding fragment and a SARAH (Sav / Rassf / Hpo) domain linked to the second antigen-binding fragment; wherein the first monomer and the second monomer are linked via the SARAH (Sav / Rassf / Hpo) domain.

[0017] In addition, in order to achieve the above purpose, another aspect of the present invention provides a polynucleotide encoding the first monomer, second monomer or antigen-binding fragment complex and an expression vector comprising the same.

[0018] In addition, in order to achieve the above purpose, another aspect of the present invention provides a transformant in which the expression vector is introduced into a host cell.

[0019] In addition, in order to achieve the above object, another aspect of the present invention provides an expression vector for producing an antigen-binding fragment complex, comprising: a first gene construct including a first cloning site into which a base sequence encoding a first target protein can be introduced, and a base sequence encoding a SARAH domain, which is linked to the 3'-terminus of the first cloning site so as to be expressed by being fused to the C-terminus of the first target protein; and a second gene construct including a second cloning site into which a base sequence encoding a second target protein can be introduced; wherein the first target protein is a fusion protein of a light chain variable region and a light chain constant region of a first antigen-binding fragment, and the second target protein is a fusion protein of a heavy chain variable region and a heavy chain constant region of the first antigen-binding fragment.

[0020]

[0021] The antigen-binding fragment complex of the present invention is a stable complex that forms spontaneously without covalent bonding by fusing a SARAH (Sav / Rassf / Hpo) domain to the antigen-binding fragment. Furthermore, the antigen-binding fragment complex of the present invention exhibits high expression efficiency and easy purification even in bacterial, particularly Escherichia coli (E. coli) expression systems, compared to existing Fab fragments. The expression vector for producing the antigen-binding fragment complex provided by the present invention can be utilized as a platform, allowing users to easily and conveniently produce an antigen-binding fragment complex with a stable structure that binds to a target antigen.

[0022] However, the effects of the present invention are not limited to the effects mentioned above, and other effects not mentioned will be clearly understood by those skilled in the art from the description below.

[0023]

[0024] Figure 1 is a schematic diagram showing the manufacturing process of an antigen binding fragment and a kit.

[0025] Figure 2a is a diagram showing the construction of a bIFN-γ expression vector containing MBP, TEV cleavage sequence, and His-tag, and the expressed bIFN-γ.

[0026] Figure 2b is a diagram showing the bIFN-γ purification process using a HisTrap column.

[0027] Figure 2c shows the results of SDS-PAGE analysis according to the loading amount of bIFN-γ.

[0028] Figure 3a is a diagram showing the sequence of anti-bIFN-γ antibodies (1H8, B3) selected through phage display.

[0029] Figure 3b is a graph showing indirect ELISA absorbance curves for bIFN-γ and BSA according to antibody concentration.

[0030] Figure 4 shows the results of SDS-PAGE analysis comparing the degree of expression availability of anti-bIFN-γ Fab domains (VH-CH1 and VL-CL).

[0031] Figure 5a illustrates the structure of a vector designed for expression of a Fab fragment.

[0032] Figure 5b is an SDS-PAGE result analyzing the total protein (T), soluble (S), and insoluble (I) fractions after expressing the Fab fragment expressed using the vector of Figure 5a at different temperatures of 37°C, 30°C, and 25°C.

[0033] The left drawing of Fig. 5c shows the result of primary purification of the Fab fragment expressed with the vector of Fig. 5a through metal affinity chromatography (IMAC), and the right drawing shows the result of separating VL-CL and VH-CH1 through anion exchange chromatography after TEV cleavage.

[0034] Figure 5d shows the results of selectively purifying a Fab fragment expressed with the vector of Figure 5a using a CH1-specific binding resin.

[0035] Figure 6a is a schematic diagram showing the structure of a vector designed for expression of a Fab-ZIP fragment.

[0036] Figure 6b is an SDS-PAGE result analyzing the total protein (T), soluble (S), and insoluble (I) fractions after expressing the Fab-ZIP fragment expressed using the vector of Figure 6a at different temperatures of 37°C, 30°C, and 25°C.

[0037] The left drawing of Fig. 6c shows the result of primary purification of the Fab-ZIP fragment expressed with the vector of Fig. 6a by metal affinity chromatography (IMAC) using a HisTrap column, and the right drawing shows the result of separating VL-CL-ZIP and VH-CH1-ZIP through anion exchange chromatography after TEV protease treatment.

[0038] Figure 6d shows the results of purifying the Fab-ZIP fragment expressed with the vector of Figure 6a using CH1-specific binding resin after TEV treatment.

[0039] Figure 7a is a schematic diagram illustrating the configuration of a vector designed for expression of an antigen-binding fragment complex.

[0040] Figure 7b is an SDS-PAGE result analyzing the total protein (T), soluble (S), and insoluble (I) fractions after expressing the antigen-binding fragment complex expressed using the vector of Figure 7a at different temperatures of 37°C, 30°C, and 25°C.

[0041] The left drawing of Fig. 7c is an SDS-PAGE result showing the result of the first purification using a HisTrap column of an antigen-binding fragment complex expressed using the vector of Fig. 7a, and the right drawing is an image showing the SDS-PAGE result of the result of selective high-purity purification using a CH1 affinity column after TEV cleavage.

[0042] Figure 7d shows the results of analyzing the antigen-binding fragment complex before and after TEV treatment using native PAGE.

[0043] Figure 7e shows the SEC-HPLC analysis results of the purified antigen-binding fragment complex.

[0044] Figures 8a to 8c show the vector construction, purification process, and SDS-PAGE results of the produced antigen-binding fragments for the production of Fab, Fab-ZIP, and antigen-binding fragment complexes.

[0045] Figure 8d shows the results of an indirect ELISA comparing the binding affinity of Fab, Fab-ZIP, and antigen-binding fragment complexes to bIFN-γ.

[0046] Figure 8e is a graph measuring the stability (retention of antigen binding ability) of antigen binding fragments after 2 or 6 months of storage at -20°C.

[0047] Figure 9a is an image showing the results of Western blot analysis under reducing conditions (+DTT) and non-reducing conditions (-DTT) after expressing the heavy and light chain constant regions (VH-CH1 and VL-CL) of antibodies, either singly or in combination.

[0048] Figure 9b is an image showing the extent to which 6HFh8-VL-CL and VL-CL bind specifically or non-specifically to BSA and bIFN-γ at different concentrations using ELISA.

[0049] Figure 10 is an image showing the results of Sandwich ELISA to determine the degree of specific or non-specific binding of Fab, Fab-ZIP, and antigen-binding fragment complexes to BSA and bIFN-γ.

[0050] Figure 11a is a TEM image of gold nanoparticles (AuNPs), and Figure 11b is a graph showing the UV-vis spectrum of AuNPs bound to a complex of AuNPs and antigen-binding fragments.

[0051] Figure 12a (i) is an image of a probe bound to an antigen-binding fragment complex at different concentrations, Figure 12a (ii) is an image of the resulting product after centrifugation, and Figure 12b is the result of treating the centrifuged product with a membrane in which bIFN-γ is dispersed.

[0052] Figure 13 shows the results of an experiment confirming the detection limit of bIFN-γ according to the concentration of a probe that binds AuNPs to an antigen-binding fragment complex.

[0053] Figure 14a is a schematic diagram showing the design of a detection system through T and C lines on an LFIA kit, Figure 14b is an image showing the meaning of the test results using an LFIA kit, Figure 14c is a result of confirming the bIFN-γ detection limit concentration of the LFIA kit, and Figure 14d is a graph showing the result of quantitatively analyzing the T-line intensity (absorbance) according to the bIFN-γ concentration using an LFIA kit.

[0054] Figure 15a shows the results of evaluating the detection limit concentration of bIFN-γ by applying the LFIA kit to an FBS sample to which bIFN-γ has been added, and Figure 15b is a graph showing the results of quantitatively analyzing the T-line intensity (absorbance) according to the bIFN-γ concentration in an FBS sample to which bIFN-γ has been added.

[0055] Figure 16a is an SDS-PAGE image showing the results of expression and purification of an anti-human gelsolin antigen-binding fragment complex, and Figure 16b is a graph showing the extent to which the purified antigen-binding fragment complex binds to BSA and human gelsolin through absorbance.

[0056] Figure 17a is an SDS-PAGE image showing the results of expression and purification of an anti-human PAI-I antigen-binding fragment complex, and Figure 17b is a graph showing the extent to which the purified anti-human PAI-I antigen-binding fragment complex binds to BSA and human PAI-I through absorbance.

[0057]

[0058] First, the terms used in the specification of the present invention are explained.

[0059] The term "antibody" as used herein refers to an immunoglobulin molecule having a structure in which one light chain is connected to each of two heavy chains connected to each other by a disulfide bond, and a multimer thereof. The light chain comprises two regions, namely, one variable region (light chain variable region, VL) and one constant region (light chain constant region, CL), and the heavy chain comprises four regions, namely, one variable region (heavy chain variable region, VH) and three constant regions (heavy chain constant regions, CH; CH1, CH2, and CH3). The constant regions of the light and heavy chains play a role in imparting biological properties such as binding between light and / or heavy chains, secretion, complement binding, and binding to Fc receptors (FcR), and the variable regions of the light and heavy chains determine recognition and binding specificity for antigens. In particular, the binding specificity of an antibody is due to the structural complementarity between the antigen binding site and the epitope. Since the antibody binding site is mainly composed of residues derived from three hypervariable regions called complementarity determining regions (CDRs) included in the variable regions of the heavy and light chains, the complementarity determining regions are referred to as amino acid sequences that define the binding specificity of an antibody. The three CDRs are arranged between four relatively well-conserved regions called framework regions (FRs), which are arranged and connected in the following order from the N-terminus to the C-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. At this time, the three CDRs included in the heavy chain variable region are referred to as CDR1-H, CDR2-H, and CDR3-H, respectively, and the three CDRs included in the light chain variable region are referred to as CDR1-L, CDR2-L, and CDR3-L, respectively.

[0060] The term "antigen-binding fragment" as used herein refers to a portion of an intact antibody, particularly any polypeptide or glycoprotein comprising an antigen-binding site or variable region of an intact antibody. Such antigen-binding fragments can be produced by recombinant DNA techniques or by enzymatically or chemically decomposing intact antibodies, and examples of such antigen-binding fragments include, but are not limited to, Fv, Fab, F(ab')2, Fab', dsFv, (dsFv)2, scFv, sc(Fv)2, diabodies, and the like, as well as bispecific and multispecific antibodies formed therefrom.

[0061] The Fab has a structure having variable regions of the light and heavy chains, a constant region of the light chain, and the first constant region (CH1 domain) of the heavy chain, and has one antigen-binding site. The Fab' is different from the Fab in that it has a hinge region at the C-terminus of the heavy chain CH1 domain. The F(ab')2 can be produced by covalent or non-covalent bonding between the hinge regions of the Fab'. The Fv refers to the minimum antibody fragment having only a heavy chain variable region and a light chain variable region. A two-chain Fv has a heavy chain variable region and a light chain variable region connected by a non-covalent bond, and a single-chain Fv has a heavy chain variable region and a light chain variable region connected by a covalent bond, generally via a peptide linker, or directly connected at the C-terminus, so that it can form a dimer-like structure like a two-chain Fv. The above antigen binding fragment can be produced using a protein hydrolytic enzyme or through genetic recombination technology, but is not limited thereto.

[0062] The linker may be a peptide linker and may have a length of about 10 to 25 amino acids. For example, the linker may include a hydrophilic amino acid such as glycine (G) and / or serine (S). The linker may include, for example, (GS)n, (GGS)n, (GSGGS)n or (GnS)m (wherein n and m are each 1 to 10), for example, but not limited to, (GnS)m (wherein n and m are each 1 to 10).

[0063]

[0064] Hereinafter, the present invention will be described in detail.

[0065]

[0066] 1. Novel antigen-binding fragment complex

[0067] One aspect of the present invention provides a novel antigen-binding fragment complex.

[0068] The antigen-binding fragment complex of the present invention may be formed by combining two or more monomers. In this case, each monomer may have a SARAH (Sav / Rassf / Hpo) domain linked to an antigen-binding fragment. Accordingly, the antigen-binding fragment complex of the present invention may be expressed as (antigen-binding fragment)2-SARAH, and when the antigen-binding fragment is Fab, it may be expressed as Fab2-SARAH.

[0069] More specifically, the antigen-binding fragment complex of the present invention may be one in which a first monomer and a second monomer are linked via a SARAH (Sav / Rassf / Hpo) domain, and the first monomer may include a first antigen-binding fragment and a SARAH (Sav / Rassf / Hpo) domain linked to the first antigen-binding fragment, and the second monomer may include a second antigen-binding fragment and a SARAH (Sav / Rassf / Hpo) domain linked to the second antigen-binding fragment. That is, in the present specification, the first antigen-binding fragment may mean an antigen-binding fragment included in the first monomer, and the second antigen-binding fragment may mean an antigen-binding fragment included in the second monomer.

[0070] At this time, the target antigens binding to each of the first and second antigen-binding fragments may be identical or different, and preferably, they may be identical.

[0071] In addition, the types of the first and second antigen-binding fragments may be Fab, F(ab')2, scFv (single-chain variable fragment), dsFv (disulfide-stabilized Fv), di-scFv (diabody), sdAb (single-domain antibody), or minibody, respectively. The types of the first and second antigen-binding fragments may be the same or different, and are preferably Fab, but are not limited thereto as long as they can bind to an antigen.

[0072] The SARAH domain linked to the first and second antigen-binding fragments may be linked to the heavy chain constant region or the light chain constant region of each of the first antigen-binding fragment and the second antigen-binding fragment, respectively, preferably linked to the light chain constant region, and more preferably linked to the C-terminus of the light chain constant region.

[0073] Accordingly, the antigen-binding fragment complex of the present invention may be in a form in which a SARAH domain linked to the C-terminus of the light chain constant region of the first antigen-binding fragment and a SARAH domain linked to the C-terminus of the light chain constant region of the second antigen-binding fragment are linked to each other.

[0074] The SARAH domain is a domain commonly found in Salvador (Sav), Ras Association Domain Family (RASSF), and Hippo (Hpo) proteins, and is a functional structural unit that can noncovalently bind to each other through specific interactions between SARAH (Sav / Rassf / Hpo) domains to form dimers, oligomers, or complexes.

[0075] Specifically, the SARAH domain included in the monomer of the present invention may have an anti-parallel coiled-coil structure, and the complementary anti-parallel structure may induce binding between SARAH domains, and may be derived from Mst1, Mst2, RASSF1, RASSF5 proteins, but is not limited thereto as long as it is a protein or enzyme having a SARAH domain.

[0076] Therefore, the monomers forming the antigen-binding fragment complex of the present invention include SARAH domains having complementary antiparallel structures, thereby enabling them to form dimers, oligomers or complexes through non-covalent dimerization without covalent bonding.

[0077] The first antigen-binding fragment and the second antigen-binding fragment may each include a light chain portion and a heavy chain portion, and the light chain portion may include two regions, namely, one variable region (light chain variable region, VL) and one constant region (light chain constant region, CL), and the heavy chain may include two regions, namely, one variable region (heavy chain variable region, VH) and one constant region (heavy chain constant region, CH).

[0078] The first and second antigen-binding fragments of the present invention may include, for example, a light chain variable region, a light chain constant region, a heavy chain variable region and / or a heavy chain constant region of an antibody derived from a human or other mammal, and the light chain variable region, the light chain constant region, the heavy chain variable region and the heavy chain constant region of an antibody derived from a human or mammal may be used without limitation in type or amino acid sequence, as long as the first and second antigen-binding fragments do not inhibit the formation of a complex and expression in a bacterial system.

[0079] Therefore, the amino acid sequences of the light chain variable region, light chain constant region, heavy chain variable region, and heavy chain constant region described above may include variants having different sequences by deletion, insertion, substitution, or a combination thereof of amino acid residues, within a range that does not affect the structure, function, activity, etc. of the polypeptide including them. In addition, the amino acid sequences may include amino acids that have undergone conventional modifications known in the art, and the amino acid modifications may be, for example, phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, etc.

[0080] Additionally, the amino acid sequences described above may include conservative substitutions in which amino acid residues are replaced with amino acid residues having similar side chains. For example, conservative substitutions include substitutions among amino acid residues having basic side chains, such as lysine, arginine, and histidine. In addition, amino acid residues having acidic side chains, such as aspartic acid and glutamic acid; amino acid residues having uncharged polar side chains, such as glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine; amino acid residues having nonpolar side chains, such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; amino acid residues having β-branched side chains, such as threonine, valine, and isoleucine; Substitutions among amino acid residues with aromatic side chains, such as tyrosine, phenylalanine, tryptophan, and histidine, are also conservative substitutions.

[0081] In an embodiment of the present invention, a 6HFh8 tag was attached to the N-terminus of each of the heavy and light chains, and the light and heavy chains were expressed under different individual promoters. The antigen-binding fragment complex containing the produced SARAH domain was confirmed to exhibit specific affinity for bIFN-γ as a result of an indirect ELISA analysis performed with BSA and bIFN-γ as targets, and it was confirmed that the affinity and stability were excellent even after long-term storage at -20°C.

[0082] In the examples of the present invention, the expressed monomers spontaneously formed dimers without separate covalent bonds through the interaction of the SARAH domains and assembled into an antigen-binding fragment complex. The manufactured antigen-binding fragment complex was confirmed to exhibit high specificity and excellent binding affinity for bIFN-γ without nonspecific reaction to BSA as a result of indirect ELISA analysis using bIFN-γ and BSA as target antigens. In addition, it was observed that the binding activity was maintained even after long-term storage of the manufactured antigen-binding fragment complex at -20°C for more than 2 months, confirming that the antigen-binding fragment of the present invention is structurally stable.

[0083]

[0084] 2. A base sequence encoding a novel antigen-binding fragment complex and an expression vector expressing the same.

[0085] One aspect of the present invention provides an expression vector capable of expressing an antigen-binding fragment complex.

[0086] More specifically, one aspect of the present invention provides a polynucleotide encoding a first monomer, a second monomer, or an antigen-binding fragment complex, an expression vector comprising the nucleotide, and a transformant in which the expression vector is introduced into a host cell. The polynucleotide may be DNA or RNA, and contains genetic information, enabling expression of a protein encoded by the polynucleotide, preferably an antigen-binding fragment complex.

[0087] Descriptions of the first monomer, first antigen-binding fragment, second monomer, second antigen-binding fragment, and antigen-binding fragment complex of the present invention are the same as those described in '1. Novel antigen-binding fragment complex' and are therefore omitted.

[0088] In order to express the antigen-binding fragment complex of the present invention in the above host cell, the present invention provides a polynucleotide encoding all or part of the antigen-binding fragment complex.

[0089] The sequence of the polynucleotide encoding all or part of the above antigen-binding fragment complex can be designed depending on the target antigen to which the first or second antigen-binding fragment is intended to bind.

[0090] In one embodiment of the present invention, a polynucleotide encoding a light chain variable region and a light chain constant region of a first antigen-binding fragment of the present invention and a gene encoding a heavy chain variable region and a heavy chain constant region of a second antigen-binding fragment of the present invention may be provided. The genes encoding the variable and constant regions of the light and heavy chains may undergo various modifications in the coding region as long as the amino acid sequence expressed from the coding region is not changed, and various mutations may also be made in a portion excluding the coding region as long as the expression of the gene is not affected. Consequently, the genes of the variable and constant regions may undergo mutations by substitution, deletion, insertion, or a combination thereof of one or more nucleic acid bases, as long as they encode a protein having an activity equivalent thereto, and such mutant genes are also included in the scope of the present invention.

[0091] In addition, another aspect of the present invention provides an expression vector comprising a polynucleotide encoding all or part of the antigen-binding fragment complex of the present invention, and a transformant in which the expression vector is introduced into a host cell.

[0092] The above expression vector may include an optimized base sequence depending on the type of organism to be introduced and expressed and the expression system of the organism, such as transcription or translation. This is due to the degeneracy of codons, and thus, various combinations of nucleotide sequences capable of encoding the expressed protein may exist, all of which are included within the scope of the present invention. The modification of the polynucleotide according to the above codon optimization may be determined depending on the type of host cell to which the antigen-binding fragment complex of the present invention is to be expressed and applied.

[0093] A transformant can be produced by transforming the expression vector according to the present invention into any one appropriate host cell selected from the group consisting of bacteria, yeast, Escherichia coli, fungi, plant cells, and animal cells, depending on the purpose of expression. For example, the host cell may be selected from the group consisting of Bacillus subtilis, Pseudomonas fluorescens, Lactococcus lactis, Clostridium thermocellum, Streptomyces, Bacillus megaterium, Rhodococcus erythropolis, Corynebacterium glutamicum, Pichiapastoris, Saccharomyces cerevisiae, Candida utilis, Schizosaccharomyces pombe, Hansenula polymorpha, Yarrowia The host cell may be Yarrowialipolytica, Candida boidnii, Cryptococcus albidus, preferably Escherichia coli (E. coliBL21(DE3), DH5α, etc.) or yeast cells (Saccharomyces genus, Pichia genus, etc.), more preferably E. coli (E. coliBL21(DE3), DH5α, etc.), but is not limited thereto as long as it is a cell into which the expression vector of the present invention can be introduced. At this time, a person skilled in the art can easily select an appropriate culture method and medium conditions, etc., depending on the type of host cell, from known techniques in the field.

[0094] For example, the gene or polynucleotide of the present invention may be a gene modified to be optimized for bacteria, and more specifically, may be modified to be optimized to be suitable for expression and function from E. coli.

[0095] The expression vector of the present invention may be a recombinant expression vector and comprises a gene encoding all or part of the antigen-binding fragment complex of the present invention, wherein the gene may be operably linked to a promoter.

[0096] The above expression vector includes, but is not limited to, a plasmid vector, a cosmid vector, a bacteriophage vector, and a viral vector.

[0097] The above expression vector can appropriately combine expression control sequences such as a promoter, terminator, enhancer, etc., or sequences for secretion, etc., according to the purpose, depending on the type of host cell in which all or part of the antigen-binding fragment complex of the present invention is to be expressed or produced.

[0098] The above expression vector may additionally include a selection marker for selecting a host cell into which the vector has been introduced, and if it is a replicable expression vector, may include an origin of replication.

[0099] In addition, the polynucleotide encoding the antigen-binding fragment complex or the expression vector may include a sequence for facilitating purification of the expressed protein, and specifically, a gene encoding a tag for separation and purification may be linked to a gene encoding all or part of the antigen-binding fragment complex of the present invention so as to be operable. At this time, the tag for separation and purification may be GST, poly-Arg, FLAG, histidine-tag (His-tag), c-myc, 6HFh8 tag, etc., used alone, or two or more of them may be sequentially linked and used.

[0100] For example, the polynucleotide encoding the antigen-binding fragment complex or the expression vector may include a base sequence encoding a 6HFh8 tag and a linker sequence, and the antigen-binding fragment complex of the present invention expressed in a host cell may have the 6HFh8 tag linked by a linker.

[0101] The gene encoding all or part of the antigen-binding fragment complex of the present invention can be cloned through a restriction enzyme cleavage site, and when a gene encoding a protein cleavage enzyme recognition site is used in the vector, it can be linked in frame with a gene encoding all or part of the antigen-binding fragment of the present invention, so that when all or part of the antigen-binding fragment complex of the present invention is obtained and then cleaved with a protein cleavage enzyme, all or part of the antigen-binding fragment complex of the present invention in its original form can be produced.

[0102] Additionally, a recombinant expression vector containing a gene encoding all or part of the antigen-binding fragment complex of the present invention may be introduced into the transformant of the present invention.

[0103] In addition, the present invention provides a method for producing an antigen-binding fragment complex, which comprises a step of culturing the transformant.

[0104] The method for introducing a recombinant expression vector for producing a transformant of the present invention can use a known technique, such as a heat shock method or an electric shock method.

[0105] Since the protein expressed from the transformant is the antigen-binding fragment complex of the present invention, the antigen-binding fragment complex of the present invention can be easily mass-produced by mass-cultivating the transformant and expressing the gene.

[0106] In an embodiment of the present invention, an anti-human gelsolin antigen-binding fragment complex and an anti-human PAI-I antigen-binding fragment complex were prepared using the same method as the method for preparing an anti-bIFN-γ antigen-binding fragment complex including a SARAH domain, and the purification and antigen-binding specificity were confirmed. As a result, it was confirmed that an excellent antigen-binding fragment complex can be prepared using the platform of the present invention for antigens other than the bIFN-γ antigen.

[0107]

[0108] 3. Expression vector platform for generating antigen-binding fragment complexes

[0109] One embodiment of the present invention provides a vector platform in which expressed monomers spontaneously form complexes.

[0110] The above vector platform is an expression vector for producing an antigen-binding fragment complex, and can introduce a sequence for expressing a desired target protein by the user, including a cloning site.

[0111] Specifically, the expression vector for producing the antigen-binding fragment complex comprises two or more gene constructs, each of which comprises a cloning site into which a base sequence encoding a target protein can be introduced.

[0112] When the base sequences encoding the first and second target proteins that the user wishes to produce are introduced into the expression vector for producing an antigen-binding fragment complex of the present invention and expressed, the first target protein and the second target protein having a SARAH domain linked to the C-terminus can be expressed, and the SARAH domain linked to the C-terminus of the first target protein can spontaneously form a dimer or complex through the SARAH domain linked to another protein.

[0113] For example, an expression vector for producing an antigen-binding fragment complex of the present invention may include a first cloning site into which a base sequence encoding a first target protein can be introduced, and a first gene construct including a base sequence encoding a SARAH domain, which is linked to the 3'-terminus of the first cloning site so as to be expressed by being fused to the C-terminus of the first target protein; and a second gene construct including a second cloning site into which a base sequence encoding a second target protein can be introduced. In this case, the first target protein may be a protein in which the light chain variable region and the light chain constant region of the first antigen-binding fragment are fused, and the second target protein may be a protein in which the heavy chain variable region and the heavy chain constant region of the first antigen-binding fragment are fused.

[0114] Descriptions of the first monomer, first antigen-binding fragment, second monomer, second antigen-binding fragment, antigen-binding fragment complex, polynucleotide, gene, expression vector, and transformant of the present invention are the same as those described in '1. Novel antigen-binding fragment complex and 2. Base sequence encoding novel antigen-binding fragment complex and expression vector expressing the same', and therefore are omitted.

[0115] In addition, the expression vector for producing the antigen-binding fragment complex of the present invention may include multiple promoters, and the first gene construct and the second gene construct may each be operated by independent promoters.

[0116] According to another embodiment of the present invention, the first and second gene constructs may further comprise a 6Fh8 tag and a TEV protease recognition sequence, respectively. The 6Fh8 tag facilitates purification of the expressed protein, and the TEV protease cleavage site facilitates tag removal after purification of the target protein, thereby advantageously securing a highly pure protein.

[0117] As an example of an expression vector for producing an antigen-binding fragment complex of the present invention, a first gene construct included in the expression vector for producing an antigen-binding fragment complex may include, in the 5' to 3' direction, a promoter, a 6HFh8 tag, a linker, a TEV protease recognition sequence, a first cloning site into which a base sequence encoding a first target protein can be introduced, a base sequence encoding a SARAH domain linked so as to be fused to the C-terminus of the first target protein and expressed, and a terminator sequence, and a second gene construct may include, in the 5' to 3' direction upstream or downstream of the first gene construct, a promoter, a 6HFh8 tag, a linker, a TEV protease recognition sequence, a second cloning site into which a base sequence encoding a second target protein can be introduced, and a terminator sequence. At this time, the first target protein may be a protein in which the light chain variable region and the light chain constant region of the first antigen-binding fragment are fused, and the second target protein may be a protein in which the heavy chain variable region and the heavy chain constant region of the first antigen-binding fragment are fused.

[0118] In addition, the expression vector for producing an antigen-binding fragment complex of the present invention may further include third and fourth gene constructs. In this case, the expression vector for producing an antigen-binding fragment complex of the present invention may further include a third cloning site into which a base sequence encoding a third target protein can be introduced, and a third gene construct including a base sequence encoding a SARAH domain, which is linked to the 3'-terminus of the third cloning site so as to be expressed by being fused to the C-terminus of the third target protein; and a fourth gene construct including a fourth cloning site into which a base sequence encoding a fourth target protein can be introduced. In this case, the third target protein may be a protein in which the light chain variable region and the light chain constant region of the second antigen-binding fragment are fused, and the fourth target protein may be a protein in which the heavy chain variable region and the heavy chain constant region of the second antigen-binding fragment are fused.

[0119] As an example of an expression vector for producing an antigen-binding fragment complex of the present invention, the expression vector for producing an antigen-binding fragment complex includes first to fourth gene constructs, and the third gene construct may include, in the 5' to 3' direction, a promoter, a 6HFh8 tag, a linker, a TEV protease recognition sequence, a third cloning site into which a base sequence encoding a third target protein can be introduced, a base sequence encoding a SARAH domain linked so as to be fused to the C-terminus of the third target protein and expressed, and a terminator sequence, and the fourth gene construct may include, in the 5' to 3' direction upstream or downstream of the third gene construct, a promoter, a 6HFh8 tag, a linker, a TEV protease recognition sequence, a fourth cloning site into which a base sequence encoding a fourth target protein can be introduced, and a terminator sequence. At this time, the third target protein may be a protein in which the light chain variable region and the light chain constant region of the second antigen-binding fragment are fused, and the fourth target protein may be a protein in which the heavy chain variable region and the heavy chain constant region of the second antigen-binding fragment are fused.

[0120] In one embodiment, the first genetic construct and the second genetic construct each include genetic information constituting a light chain and a heavy chain of the first antigen-binding fragment, and the light chain expressed by the first genetic construct and the heavy chain expressed by the second genetic construct can bind to each other to form a monomer comprising the antigen-binding fragment, which can be defined as a “first monomer,” and the first monomers can form a complex with each other or with another monomer comprising a SARAH domain.

[0121] For example, the light chain and heavy chain expressed by the first genetic construct and the second genetic construct may combine to form a first monomer, and the first monomer and another first monomer may form a complex via the SARAH domain, and the light chain and heavy chain expressed by the third genetic construct and the fourth genetic construct may combine to form a complex with the second monomer. In addition, the second monomers may form a complex with each other.

[0122] The expression vector for producing an antigen-binding fragment complex of the present invention serves as a platform, and when a user inserts a base sequence encoding a target protein into a cloning site and expresses it, a structure that spontaneously binds by the SARAH domain can be formed, thereby producing an antigen-binding fragment complex capable of binding to a single or multiple target antigens.

[0123] Therefore, by using the expression vector for producing an antigen-binding fragment complex of the present invention, the user can conveniently design a complex suitable for a single or multiple targets by inserting the base sequence of the desired antigen-binding fragment. The expressed monomers spontaneously associate with each other by the SARAH domain to form a structurally stable complex, and can also be implemented in a bispecific or multispecific form capable of binding to multiple targets. In addition, when using the expression vector for producing an antigen-binding fragment complex of the present invention, the expression efficiency of the antigen-binding fragment complex is excellent, and the 6Fh8 purification tag and TEV cleavage sequence can be selectively included, so that the purification of the protein is excellent, and a highly pure antigen-binding fragment complex can be secured.

[0124]

[0125] 4. Kit containing a novel antigen-binding fragment complex

[0126] Another aspect of the present invention provides a kit for antigen detection.

[0127] The antigen detection kit of the present invention comprises the antigen binding fragment complex of the present invention.

[0128] The description of the antigen-binding fragment complex of the present invention is the same as that described in '1. Novel antigen-binding fragment complex', so it is omitted.

[0129] The antigen detection kit of the present invention may be a lateral flow immunoassay (LFIA), an enzyme-linked immunosorbent assay (ELISA), a chemiluminescent immunoassay (CLIA), a fluorescence immunoassay (FIA), an immunoblot assay kit (Western Blot Kit), a flow cytometry antibody kit, a surface plasmon resonance (SPR) kit, a bio-layer interferometry (BLI) kit, or an antibody microarray kit, but is not limited thereto as long as it is a kit capable of binding to and detecting a target antigen.

[0130] For example, the antigen detection kit of the present invention can detect a target antigen by including the antigen binding fragment complex.

[0131] The above lateral flow immunoassay (LFIA) kit is a diagnostic device configured to allow a sample solution to flow on a solid support by capillary action, and when the sample solution contains an antigen, the antigen contained in the sample solution binds to an antigen-binding fragment or antigen-binding fragment complex contained in the kit, thereby enabling rapid and easy detection of the target antigen.

[0132] The above LFIA kit may include a sample pad, a conjugate pad, a reaction membrane, an absorbent pad, a backing card, and may additionally include instructions for use.

[0133] The sample pad of the LFIA kit of the present invention is the area where the sample to be tested is first administered. When using the kit of the present invention, the examiner drips or contacts the sample containing the substance to be analyzed onto the sample pad. After the sample is administered onto the sample pad, it moves toward the binding pad by capillary action. The binding pad contains an antigen-binding fragment bound to a marker particle such as a gold nanoparticle (AuNP), and the antigen-binding fragment can specifically bind to the substance to be analyzed.

[0134] In one embodiment of the present invention, an antigen-binding fragment complex may be used as the antigen-binding fragment included in the binding pad, which enables antigen detection through specific binding to a target antigen present in a sample.

[0135] When a test target sample comes into contact with the binding pad, the antibody, antigen-binding fragment, or antigen-binding fragment complex on the binding pad dissolves, and if the target antigen is present in the test target sample, the target antigen and the antibody or antigen-binding fragment form an immune complex and then migrate along the reaction membrane. The reaction membrane includes a test line and a control line, and if an immune complex is present, a visually observable color change (e.g., a red line) is induced in the test line. The control line is used to verify whether the kit is functioning normally.

[0136] The LFIA kit has the advantage of enabling point-of-care testing and is characterized by high specificity and a relatively short analysis time.

[0137] The LFIA kit of the present invention has superior sensitivity and reliability compared to an LFIA kit using a single Fab or scFv structure by introducing an antigen-binding fragment complex.

[0138]

[0139] Hereinafter, the present invention will be described in detail by examples.

[0140] However, the following examples specifically illustrate the present invention, and the content of the present invention is not limited by the following examples.

[0141]

[0142] [Example 1]

[0143] Design and production of the antigen-binding fragment of the present invention

[0144] An antigen-binding fragment complex was produced through Examples 1-1 and 1-2, and the process is schematically illustrated in Figure 1.

[0145] Example 1-1. Selection of target antigen and antibody

[0146] To produce an antigen-binding fragment complex, bovine interferon gamma (bIFN-γ) was selected as the target antigen, and antibodies that can specifically bind to it were screened and selected.

[0147] First, an expression vector containing the bIFN-γ gene was constructed, and the expressed peptide was designed to include malE, a maltose-binding protein (MBP), a TEV protease cleavage site, bIFN-γ, and a six-histidine sequence (6ХHis) in the direction from the N-terminus to the C-terminus (Fig. 2a).

[0148] The constructed expression vector was introduced into E. coli, and culture and protein expression were induced. The cultured E. coli were harvested and disrupted to extract the protein. As a first purification step, metal affinity chromatography based on the 6ХHis tag was performed using a HisTrap column. Through this, the MBP-TEV-(bIFN-γ)-His fusion protein was bound to the column, and the remaining impurities were removed (Fig. 2b, left). After purification by metal affinity chromatography, the MBP tag was cleaved by treatment with TEV protease, and then the (bIFN-γ)-His protein was isolated with high purity by anion exchange chromatography (Fig. 2b, right). To confirm the quality of the purified bIFN-γ protein, SDS-PAGE analysis was performed, and a clearer band appeared as the loading amount (2 μL, 5 μL, and 10 μL) increased (Fig. 2c). The band was located at a molecular weight of approximately 17–20 kDa, which is consistent with the theoretical molecular weight of bIFN-γ, confirming that bIFN-γ was produced and purified with high purity.

[0149] Afterwards, binding screening was performed against a phage display library using purified bIFN-γ as an antigen, and phages that bind to bIFN-γ were identified. As a result, 1H8 antibody and B3 antibody were selected as antibodies with high binding affinity to bIFN-γ.

[0150] To determine whether the 1H8 and B3 antibodies specifically bind to the antigen and determine their antigen affinity, the 1H8 and B3 antibodies were purified and subjected to indirect ELISA. bIFN-γ was used as the antigen, and BSA was used as the control. As a result, the 1H8 antibody exhibited a rapid saturation response curve in the concentration range of 0 to 3 nM, and there was almost no reaction to BSA. In contrast, the B3 antibody exhibited a curve that increased slowly in the concentration range of 0 to 80 nM, and showed a relatively low binding response (Fig. 3b).

[0151] Kinetic analysis was performed additionally to measure the dissociation constant (Kd) of each antibody, and the 1H8 antibody was found to have a Kd of 8.633 Х 10 -11 M, B3 antibodies are 1.421 Х 10 -8 It was shown as M. This means that the 1H8 antibody has an antigen binding affinity that is more than 100 times higher than that of B3. Accordingly, in the following examples, Fab and antigen-binding fragment complexes using the Fab were produced based on the VH and VL sequences of the 1H8 antibody, and their expression and purification, structural stability, and antigen-binding ability were evaluated.

[0152] Example 1-2. Design and fabrication of antigen-binding fragment complexes

[0153] A monomer linking the SARAH (Salvador / RASSF / Hippo) domain to the anti-bIFN-γ Fab antigen-binding fragment was designed as follows.

[0154] First, the heavy chain domain (VH-CH1) and light chain domain (VL-CL) of the selected 1H8 antibody were expressed using an Escherichia coli expression system, respectively. To compare the solubility and expression efficiency of each domain after expression, SDS-PAGE analysis was performed at culture temperatures of 37°C, 30°C, and 25°C. As a result, both fragments showed the highest soluble expression at 25°C. In particular, the VL-CL domain (Fig. 4, left) showed relatively higher solubility than the VH-CH1 (Fig. 4, right).

[0155] The SARAH domain was fused to the C-terminus of the VL-CL domain, which exhibits excellent solubility. SARAH is a domain derived from human Mst1 (Mammalian sterile 20-like kinase 1) and can induce spontaneous non-covalent dimerization through an anti-parallel coiled-coil structure. Consequently, a monomer consisting of VL-CL-SARAH and VH-CH1 was prepared, and a complex was prepared in which these monomers non-covalently dimerized through the SARAH domain.

[0156] As a control, Fab-ZIP constructs were also constructed by fusing acidic ZIP and basic ZIP domains to the VL-CL and VH-CH1 domains, respectively, to induce dimerization. Both constructs were tagged with a 6HFh8 tag at the N-terminus to improve purification and expression efficiency, and each domain was expressed using an expression vector designed to be expressed under a separate promoter (Fig. 6a).

[0157]

[0158] [Example 2]

[0159] Evaluation of stability and antigen binding ability of the antigen-binding fragment complex of the present invention

[0160] Example 2-1. Expression, purification, and confirmation of dimeric structure formation of the antigen-binding fragment complex of the present invention in E. coli.

[0161] Each construct produced in Example 1 was expressed in Escherichia coli (E. coli), and then primary purification was performed through metal affinity chromatography using a HisTrap column, and the 6HFh8 tag was removed by treatment with TEV (tobacco etch virus) protease. Subsequently, to improve the purification purity, CH1 affinity resin, a CH1 domain-specific binding resin, was performed, resulting in the acquisition of highly pure antigen-binding fragments (Fig. 7b).

[0162] Meanwhile, Fab-ZIP fragments were expressed in the forms of 6HFh8-VH-CH1-ZIPB and 6HFh8-VL-CL-ZIPA, and 100% soluble expression was confirmed at 25°C (Fig. 6b). Subsequently, high-purity proteins were obtained through the same HisTrap purification, 6HFh8 tag removal, and CH1 column purification processes (Fig. 6c).

[0163] Vectors were expressed in the VL-CL-SARAH and VH-CH1 forms, respectively, and were produced soluble at optimal expression conditions of 25°C (Fig. 7c). SDS-PAGE, native PAGE, and SEC-HPLC analyses were performed to analyze the structural properties of the antigen-binding fragment complex obtained after purification.

[0164] Native PAGE and SEC-HPLC analysis results showed that the antigen-binding fragment complex of the present invention was observed as a dimer band only by mixing the VH-CH1 and VL-CL fragments, confirming that a spontaneous non-covalent dimer structure was formed through the SARAH domain (Fig. 7C). In addition, SEC-HPLC analysis showed that the main peak of the antigen-binding fragment complex was detected at a position ahead of BSA with a molecular weight of 70 kDa, confirming that the complex was a dimer with a molecular weight in the range of approximately 66-140 kDa (Fig. 7E).

[0165] Through the above results, it was confirmed that the antigen-binding fragment complex produced in this example can be effectively expressed and purified in an E. coli system, and in particular, that the monomer can spontaneously form a dimer through the SARAH domain.

[0166] Example 2-2. Confirmation of antigen binding specificity and stability of anti-bIFN-γ antigen-binding fragment complexes.

[0167] We evaluated whether the antigen-binding fragment complex specifically binds to bovine interferon-γ (bIFN-γ) and confirmed whether the antigen recognition function is maintained even after long-term storage.

[0168] First, indirect ELISA was performed on anti-bIFN-γ Fab, Fab-ZIP, and antigen-binding fragment complexes, respectively. bIFN-γ was used as the antigen, and BSA was set as a negative control. The analysis results showed that the antigen-binding fragment complex exhibited higher absorbance for bIFN-γ than Fab and Fab-ZIP, but lower response to BSA, confirming high antigen specificity (Fig. 8).

[0169] For the Fab-ZIP fragment, reactivity to bIFN-γ was enhanced, but binding to BSA was increased. To confirm this, the VH-CH1 and VL-CL fragments were individually expressed and then mixed, and native PAGE analysis was performed. This confirmed the formation of unintended homodimers. This suggests that the Fab-ZIP fragment may be responsible for non-specific antigen binding (Fig. 9a).

[0170] In addition, as can be seen in FIG. 9b and FIG. 10, it was confirmed that all of the tagged VL-CL, VL-CL, Fab, and Fab-ZIP non-specifically bound to BSA, but it was confirmed that the antigen-binding fragment complex of the present invention (Fab2-SARAH) specifically bound to bIFN-γ.

[0171] To compare the structural stability of the antigen-binding fragment complexes, Fab, Fab-ZIP, and antigen-binding fragment complex (Fab2-SARAH) were diluted in PBS to a concentration of approximately 50 nM and stored at -20°C for 2 and 6 months, respectively, and then analyzed again for bIFN-γ by ELISA. As a result, after 6 months, the Fab fragment had almost lost its binding ability to bIFN-γ, and the reactivity of Fab-ZIP also decreased to less than about 1 / 5. In contrast, the antigen-binding fragment complex maintained the same level of antigen-binding ability as before storage. This confirmed that the antigen-binding fragment complex structure is an antigen-binding fragment with high antigen specificity and excellent long-term stability (Fig. 8e).

[0172] Example 2-3. Evaluation of LFIA-based antigen detection ability of anti-bIFN-γ antigen-binding fragment complexes

[0173] We confirmed that the antigen-binding fragment complex can detect bovine interferon-γ (bIFN-γ) through a Lateral Flow Immunoassay (LFIA) system, and performed optimization of detection sensitivity and conditions.

[0174] First, nanoparticles were conjugated to the antigen-binding fragment complex of the present invention for application to the LFIA detection system. Specifically, gold nanoparticles (AuNPs) with a size of approximately 28 nm were synthesized through the citric acid reduction method, and the purified antigen-binding fragment complexes were prepared at various concentrations (0.5, 1.0, 1.25, 1.5, and 2.0 μg / mL) and conjugated to AuNPs, respectively. Then, each complex was applied to the bIFN-γ-doped NC membrane to confirm the reaction. As a result, the antigen-binding fragment complex showed the most stable and uniform dispersion characteristics at 1.25 μg / mL, and aggregation of the complex was observed at concentrations of 1.5 μg / mL and 2.0 μg / mL (Fig. 12a). Accordingly, 1.25 μg / mL was set as the optimal protein concentration.

[0175] Afterwards, in order to confirm the change in detection sensitivity according to the absorbance concentration (OD 530) of gold nanoparticles, a bIFN-γ detection experiment was performed by adjusting the OD value to 2, 4, and 8. As a result, it was confirmed that the detection limit was 103 ng / mL under the condition of OD 530 = 2, and a maximum of 102 ng / mL could be detected under OD 530 = 4 or 8. Considering the background signal and reaction stability, the condition of OD 530 = 4 was determined to be optimal (Fig. 13).

[0176] Based on the optimal conditions (antigen-binding fragment complex concentration 1.25 μg / mL, OD 530 = 4), various concentrations of bIFN-γ (0, 1, 5, 10, 50, 102, 103, 104 ng / mL) were applied to the NC membrane, and a detection experiment was performed using the LFIA kit. After 30 minutes, the T line (detection line) and C line (normal operation confirmation line) were observed, and the T line was clearly confirmed up to a concentration of 50 ng / mL, confirming that the detection limit was 50 ng / mL (Fig. 14c).

[0177] In addition, to confirm the possibility of detection in an actual serum environment, a mock sample was prepared by adding bIFN-γ to FBS (fetal bovine serum) and applying the same LFIA kit. As a result, a T line was clearly formed even at a concentration of 5 ng / mL, and it was confirmed that the antigen-binding fragment complex could effectively recognize and detect the target antigen even in serum (Fig. 15a).

[0178] Through the above experimental results, it was confirmed that the antigen-binding fragment complex of the present invention functions appropriately in the AuNP-based LFIA system and can be utilized as a practical in vitro diagnostic antigen-binding fragment platform even under biological sample conditions while maintaining high sensitivity and specificity.

[0179]

[0180] [Example 3]

[0181] Verification of the possibility of expressing antigen-binding fragment complexes specific to various target antigens using the expression vector for producing antigen-binding fragment complexes of the present invention.

[0182] Example 3-1. Production of anti-human gelsolin antigen-binding fragment complex using an expression vector for producing antigen-binding fragment complex.

[0183] In order to confirm whether antigen-binding fragment complexes for various target antigens can be produced using the expression vector for producing antigen-binding fragment complexes of the present invention, human gelsolin was selected as the target antigen, and an antigen-binding fragment complex that specifically binds to it was produced.

[0184] The VL and VH domains of the anti-human gelsolin antibody were recombined into the VL-CL-SARAH and VH-CH1 forms, respectively, and cloned into an E. coli expression vector with a 6HFh8 tag attached to the N-terminus. The two domains were expressed under different promoters at 25°C, and the expressed protein was purified to high purity through a CH1 affinity column after primary purification using a HisTrap column and treatment with TEV protease. The results of SDS-PAGE analysis of the purified protein are shown in Fig. 16a. As a result, the LC-S (VL-CL-SARAH) and VH-CH1 bands in Fig. 16a were clearly separated, confirming that the anti-human gelsolin antigen-binding fragment complex was successfully expressed and purified through the expression vector for generating the antigen-binding fragment complex of the present invention.

[0185] To evaluate the antigen binding ability of the purified anti-human gelsolin antigen-binding fragment complex, the binding reaction was analyzed on an ELISA plate immobilized with human gelsolin and BSA, and the result is shown in Fig. 16b. As can be seen in Fig. 16b, an absorbance of approximately twice that of BSA (OD 450 ≈ 0.37) was observed for human gelsolin, confirming specific antigen binding ability (Fig. 16b).

[0186] Through the above results, it was confirmed that an antigen-binding fragment complex for anti-human gelsolin with excellent antigen-binding ability can be produced using the expression vector for producing an antigen-binding fragment complex of the present invention, and thus, it can be utilized as a platform for producing an antigen-binding fragment complex.

[0187] Example 3-2. Production of anti-human PAI-1 antigen-binding fragment complex using an expression vector for producing antigen-binding fragment complex.

[0188] To determine whether the expression vector for generating antigen-binding fragment complexes is applicable to various antibody sequences, human PAI-1 (plasminogen activator inhibitor-1) was selected as the target antigen, and an antigen-binding fragment complex that specifically binds to it was produced.

[0189] The VL and VH domains of the anti-human PAI-1 antibody were recombined into VL-CL-SARAH and VH-CH1 forms, respectively, and cloned into an E. coli expression vector with a 6HFh8 tag attached to the N-terminus. The two fragments were expressed under separate promoters, and expression was induced at 25°C. The expressed protein was first purified using a HisTrap column, and then treated with TEV protease to remove the tag. Next, high-purity purification was performed using a CH1 affinity column, and the SDS-PAGE results are shown in Figure 17a.

[0190] As a result of SDS-PAGE, the LC-S (VL-CL-SARAH) and VH-CH1 bands in Figure 17a were clearly separated, indicating that LC-S (VL-CL-SARAH) and VH-CH1 were successfully expressed and purified under separate promoters, respectively.

[0191] To evaluate the antigen binding ability of the purified anti-human PAI-1 antigen-binding fragment complex, the binding reaction was analyzed on an ELISA plate immobilized with human PAI-1 and BSA, and the result is shown in Fig. 17B. As can be seen in Fig. 17B, an absorbance of approximately 6 times higher for human PAI-1 (OD 450 ≈ 0.65) than that for BSA was observed, confirming specific antigen binding ability (Fig. 17B).

[0192] Through the above results, it was confirmed that an antigen-binding fragment complex recognizing human PAI-1 can also be produced using the expression vector for producing an antigen-binding fragment complex of the present invention.

[0193]

[0194] Although representative embodiments of the present invention have been described above as examples, the scope of the present invention is not limited to the specific embodiments described above, and those skilled in the art will be able to make appropriate changes within the scope described in the claims of the present application.

Claims

1. A first monomer comprising a first antigen-binding fragment and a SARAH (Sav / Rassf / Hpo) domain linked to the first antigen-binding fragment; and A second monomer comprising a second antigen-binding fragment and a SARAH (Sav / Rassf / Hpo) domain linked to the second antigen-binding fragment; Including, An antigen-binding fragment complex wherein the first monomer and the second monomer are linked via a SARAH (Sav / Rassf / Hpo) domain.

2. In claim 1, An antigen-binding fragment complex, wherein the target antigens of the first and second antigen-binding fragments are the same or different from each other.

3. In claim 1, An antigen-binding fragment complex wherein the target antigens of the first and second antigen-binding fragments are identical.

4. In claim 1, An antigen-binding fragment complex, wherein the first and second antigen-binding fragments are each selected from the group consisting of Fab, F(ab')2, scFv (single-chain variable fragment), dsFv (disulfide-stabilized Fv), di-scFv (diabody), sdAb (single-domain antibody), and minibody.

5. In claim 4, An antigen-binding fragment complex, wherein the first and second antigen-binding fragments are each Fab.

6. In claim 1, An antigen-binding fragment complex, wherein the SARAH (Sav / Rassf / Hpo) domain linked to the first and second antigen-binding fragments is linked to the heavy chain constant region or the light chain constant region of each of the first and second antigen-binding fragments, respectively.

7. In claim 1, An antigen-binding fragment complex, wherein the SARAH (Sav / Rassf / Hpo) domains linked to the first and second antigen-binding fragments are linked to the light chain constant regions of each of the first and second antigen-binding fragments, respectively.

8. In claim 5, An antigen-binding fragment complex, wherein the SARAH (Sav / Rassf / Hpo) domains linked to the first and second antigen-binding fragments are linked to the C-terminus of the light chain constant region of each of the first and second antigen-binding fragments, respectively.

9. In claim 1, The antigen-binding fragment complex is an antigen-binding fragment complex formed by non-covalent dimerization of the SARAH domain of the first monomer and the SARAH domain of the second monomer.

10. A polynucleotide encoding the first monomer, second monomer or antigen-binding fragment complex of claim 1.

11. An expression vector comprising the polynucleotide of claim 10.

12. A transformant in which the expression vector of claim 11 is introduced into a host cell.

13. In claim 12, A transformant wherein the host cell is an E. coli cell.

14. A first gene construct comprising a first cloning site into which a base sequence encoding a first target protein can be introduced, and a base sequence encoding a SARAH domain linked to the 3'-terminus of the first cloning site so as to be expressed by being fused to the C-terminus of the first target protein; and A second gene construct comprising a second cloning site into which a base sequence encoding the second target protein can be introduced; Including, The above first target protein is a fusion protein of the light chain variable region and the light chain constant region of the first antigen-binding fragment, An expression vector for producing an antigen-binding fragment complex, wherein the second target protein is a fusion protein of the heavy chain variable region and the heavy chain constant region of the first antigen-binding fragment.

15. In claim 14, The expression vector for producing the above antigen-binding fragment complex comprises two or more promoters, An expression vector for producing an antigen-binding fragment complex, wherein the first gene construct and the second gene construct are expressed by different promoters.

16. In claim 14, An expression vector for producing an antigen-binding fragment complex, wherein the first gene construct and the second gene construct further comprise a base sequence encoding a 6Fh8 tag and a TEV protease, respectively.

17. In claim 14, A third gene construct comprising a third cloning site into which a base sequence encoding a third target protein can be introduced, and a base sequence encoding a SARAH domain linked to the 3'-terminus of the third cloning site so as to be fused to the C-terminus of the third target protein and expressed; and A fourth gene construct comprising a fourth cloning site into which a base sequence encoding the fourth target protein can be introduced; Including more, The third target protein is a fusion protein of the light chain variable region and light chain constant region of the second antigen-binding fragment, An expression vector for producing an antigen-binding fragment complex, wherein the fourth target protein is a fusion protein of the heavy chain variable region and the heavy chain constant region of the second antigen-binding fragment.

18. In claim 17, The expression vector for producing the above antigen-binding fragment complex further comprises two or more promoters, An expression vector for producing an antigen-binding fragment complex, wherein the third gene construct and the fourth gene construct are expressed by different promoters.

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