KR102333496B1 - Antigen binding protein with improved DNA-hydrolyzing activity - Google Patents

Abstract

The present invention relates to an antigen-binding protein with improved hydrolytic activity, and more particularly, through amino acid substitution and/or addition of the antigen-binding protein, DNA hydrolytic activity and thermal stability can be improved.

Description

Antigen binding protein with improved DNA-hydrolyzing activity

The present invention relates to an antigen-binding protein having improved hydrolytic activity.

Antibodies are proteins produced by immune responses of vertebrates, and are immune proteins that inactivate or eliminate the action of an antigen by specifically recognizing and binding to a specific site of the antigen. An antigen binding site is formed from a light chain variable region (VL) and a heavy chain variable region (VH) of an antibody, and research is being conducted to perform the function of the antibody using this.

In particular, single chain variable fragment (scFv) has a small size compared to general antibodies, so it has a high penetration rate into tissues or cancer tissues, can be mass-produced in various expression systems including E. coli system, and various molecular biological modifications (modification) is possible. In addition, scFv has the advantage of reducing production time and cost.

Under this background, it is necessary to develop a method for improving the utility of antigen-binding proteins including scFv. As an antiviral agent for animal cell infection viruses (Korea Patent No. 0890463), there are studies on the 3D8 scFv protein that has antiviral activity against exogenous nucleic acid chains but is not cytotoxic to self-derived nucleic acids, but in order to increase industrial utility , There is a need for research on antigen-binding proteins with improved DNA hydrolysis capacity and thermal stability while retaining DNA binding capacity.

Korean Patent No. 0890463 (2009.03.18.)

An object of the present invention is to provide an antigen-binding protein with improved DNA hydrolytic activity and thermal stability.

An object of the present invention is to provide a scFv mutant with improved DNA hydrolytic activity and thermal stability.

An object of the present invention is to provide an antiviral composition comprising the antigen-binding protein or scFv variant according to the present invention.

1. In the antigen-binding protein comprising the amino acid sequence shown in SEQ ID NO: 1, it further comprises 1 to 6 amino acid residues at the C terminus; It contains at least one amino acid substitution selected from the group consisting of Q5V, P9A and E62D and at least one amino acid substitution selected from the group consisting of S218R, Q220E and E222G among the amino acids of the antigen-binding protein represented by SEQ ID NO: 1, and 1 at the C terminus and no more than 6 amino acid residues; 1 to 6 amino acid residues at the C-terminus are selected from Gin, Ala or Gly.

2. The antigen-binding protein according to the above 1, wherein 1 to 6 amino acid residues at the C terminus are 3 to 5 amino acids selected from Gin, Ala or Gly.

3. The antigen-binding protein according to the above 1, wherein the sequence of 1 to 6 amino acid residues at the C terminus is QAGQ.

4. The antigen binding protein of 1 above, wherein the amino acid substitutions include Q5V, P9A and E62D.

5. The antigen binding protein of 1 above, wherein the amino acid substitutions include S218R, Q220E and E222G.

6. The antigen binding protein of 1 above, wherein the amino acid substitutions include Q5V, P9A, E62D, S218R, Q220E and E222G.

7. The antigen-binding protein according to the above 1, comprising the VH represented by SEQ ID NO: 2 and the VL represented by SEQ ID NO: 3.

8. The antigen-binding protein according to the above 1, comprising the amino acid sequence represented by SEQ ID NO: 4.

9. The antigen-binding protein according to the above 1, comprising the VH represented by SEQ ID NO: 5 and the VL represented by SEQ ID NO: 6.

10. The antigen-binding protein according to the above 1, comprising the amino acid sequence represented by SEQ ID NO: 7.

11. The antigen-binding protein of 1 above, wherein the antigen-binding protein is an scFv.

12. A composition for hydrolysis of nucleic acids comprising the antigen-binding protein according to any one of items 1 to 11 above.

13. An antiviral composition comprising the antigen-binding protein according to any one of items 1 to 11 above.

14. A pharmaceutical composition for the treatment or prevention of viral infection, comprising the antigen-binding protein according to any one of items 1 to 11 above.

15. A feed additive comprising the antigen-binding protein according to any one of items 1 to 11 above.

16. A nucleic acid molecule encoding the antigen-binding protein according to any one of items 1 to 11 above.

17. An expression vector comprising the nucleic acid according to item 16 above.

18. A host cell comprising the expression vector according to item 17 above.

The present invention can provide an antigen-binding protein with improved DNA hydrolytic activity and thermal stability.

The present invention can provide scFv variants with improved DNA hydrolytic activity and thermal stability.

The present invention may provide an antiviral composition comprising the antigen binding protein or scFv variant according to the present invention.

1 is a FRET-based DNA hydrolytic activity analysis result for DNA of a 3D8 scFv mutant according to an embodiment of the present invention.
2 is a FRET-based DNA hydrolysis activity analysis result for comparing the hydrolysis activity of the 3D8 scFv mutant according to an embodiment of the present invention according to temperature and time.

Hereinafter, the present invention will be described in detail.

The present invention provides antigen binding proteins comprising the amino acid sequences described in the present invention.

In the present invention, the antigen-binding protein may include an immunoglobulin or a fragment thereof. Preferably, the antigen-binding protein of the present invention is an immunoglobulin G (IgG)-derived fragment, and in one embodiment, the immunoglobulin G fragment is a variant of the 3D8 antibody comprising the amino acid sequence shown in SEQ ID NO: 1.

Immunoglobulin is a generic term for proteins that play an important role in immunity and act as antibodies among serum components. A pair of ~70,000 H chains (heavy chains) are disulfide bonds, and are classified into IgG, IgA, IgM, IgD and IgE according to the types of H chains γ, α, μ, δ, and ε, respectively.

Immunoglobulin G is composed of one pair of L chains with a molecular weight of about 25,000 and one pair of H chains with a molecular weight of about 50,000, with a total molecular weight of about 150,000. It accounts for 70% and crosses the placenta and has various types of antibody activity.

"3D8" as used in the present invention is a protein comprising the amino acid sequence represented by SEQ ID NO: 1, which is a type of cell-penetrating antibody, which has nucleic acid hydrolysis ability, and is an antibody having antiviral and anticancer effects.

As used herein, "antibody", or "antigen binding protein" is used interchangeably and includes a number of antibodies as described herein, including the antibodies described herein as well as antibody derivatives, fragments and mimetics. can have a format.

On the other hand, fragments of immunoglobulins can be produced by recombinant DNA technology or by enzymatic cleavage or chemical cleavage of an intact antibody. The fragment can be easily changed and used by a person skilled in the art as long as it maintains the ability to bind to a nucleic acid chain and its resolution, and the fragment is, for example, Fv, Fab, Fab', F(ab') ₂ , scFv or a diabody, or a combination thereof. In one embodiment, the immunoglobulin fragment of the present invention is an scFv.

Fv fragments have the VL and VH domains of one arm of an antibody, and Fab fragments are monovalent fragments with VL, VH, CL and CH1 domains; F(ab')2 fragments are bivalent fragments with two Fab fragments linked by disulfide bonds at the hinge region.

In scFv, VH and VL are linked by a linker to form a continuous protein chain, and it is generally known that the scFv has superior antigen binding ability than VH or VL alone. In this case, any linker that binds VH and VL may be used as long as it is a linker commonly used in the art. In one embodiment, the linker peptide may include Gly, Ser, Ala or Thr. In the present invention, linkers include glycine-serine polymers, glycine-alanine polymers, including (GS)n, (GSGGS)n, (GGGGS)n, and (GGGS)n (wherein n is an integer from 1 to 4); alanine-serine polymers and other flexible linkers. More specifically, in an embodiment of the present invention, (GGGGS) ₃ linker is used.

A diabody is an antibody fragment in which scFvs having the same or different antigen-binding specificities form a dimer, and an antibody fragment having bivalent antigen-binding activity to the same antigen or specific antigen-binding activity to different antigens.

As used herein, "variant" or "variant protein", or "protein variant" refers to a protein that differs from that of the parent protein by at least one amino acid modification. Protein variants have at least one amino acid modification compared to the parent protein, but protein variants are not so varied that they are not aligned with the parent protein using conventional sequence alignment programs. In general, the protein variants described herein (eg, scFv variants, etc.) are at least 75, 80, 85, 90, 91, 92, 93 with the parent protein when using a commonly used sequence alignment program such as ClustalW. , 94, 95, 96, 97, 98 or 99% identical.

On the other hand, amino acid modification of the present invention is meant to include addition, substitution and/or deletion of amino acids. Antigen binding proteins of the invention may contain amino acid additions, substitutions and/or deletions.

As used herein, "amino acid substitution" or "substitution" means that an amino acid at a specific position in the parent polypeptide sequence is replaced with a different amino acid. For example, when glutamic acid, which is the 272th amino acid in the antigen-binding protein of the present invention, is replaced with tyrosine, it is expressed as E272Y.

As used herein, "amino acid addition" or "addition" means that an amino acid sequence is added at a specific position in the parent polypeptide sequence. In one embodiment, modifications that add or delete amino acid residues can be made at the N-terminus or C-terminus of the VH or VL. For example, 1, 2, 3, 4, 5, or 6 amino acid residues may be added to the C terminus of the VL.

As used herein, a "protein" comprises at least two covalently linked amino acids, including polypeptides, oligopeptides and peptides. In addition, the antibodies of the present invention may contain one or more side chains or ends of synthetic derivatization, glycosylation, PEGylation, circular permutation, cyclization, linkers to other molecules, fusions to proteins or protein domains, and a peptide tag or label.

The antigen-binding protein of the present invention may include the amino acid sequence set forth in SEQ ID NO: 1, and may further include 1 to 6 amino acid residues at the C terminus.

The 1 to 6 amino acid residues at the C terminus are amino acids selected from Gin, Ala or Gly, and in another embodiment, the antigen-binding protein of the present invention has 3 to 5 amino acids at the C terminus of the amino acid sequence set forth in SEQ ID NO: 1 residues, and in another embodiment 4 amino acid residues at the C terminus. In one embodiment, the sequence of the amino acid residue is QAGQ.

An antigen binding protein of the invention may comprise one or more amino acid substitutions. The amino acid substitution comprises at least one amino acid substitution selected from the group consisting of Q5V, P9A and E62D and at least one amino acid substitution selected from the group consisting of S218R, Q220E and E222G. In one embodiment, the amino acid substitutions include Q5V, P9A and E62D. Also, in one embodiment, the amino acid substitutions include S218R, Q220E and E222G. In one embodiment according to the present invention, the amino acid substitution includes Q5V, P9A, E62D, S218R, Q220E and E222G.

The antigen-binding protein of the present invention may include a VH represented by SEQ ID NO: 2 and a VL represented by SEQ ID NO: 3. In addition, the antigen-binding protein of the present invention may include a VH represented by SEQ ID NO: 5 and a VL represented by SEQ ID NO: 6.

The antigen-binding protein of the present invention may include the amino acid sequence represented by SEQ ID NO: 4. In addition, the antigen-binding protein of the present invention may include the amino acid sequence represented by SEQ ID NO: 7.

The antigen-binding protein of the present invention has the ability to bind DNA, and has improved hydrolysis capacity and thermal stability compared to the parent antigen-binding protein.

Binding ability refers to the ability of a protein to recognize and bind to a nucleic acid chain such as DNA or RNA. It is bound by the positive charges of the positively charged residues among the amino acids constituting the protein and the negative charges of the phosphate backbone of DNA or RNA.

Hydrolysis capacity refers to the ability of a protein to hydrolyze and cut nucleic acid chains such as DNA or RNA, and the expression catalytic capacity is also used.

Thermal stability refers to the property of maintaining DNA hydrolytic activity when heat is applied to the antigen-binding protein of the present invention.

The present invention may provide a composition for hydrolysis of nucleic acids comprising the antigen-binding protein or scFv according to the present invention.

The composition for hydrolysis of nucleic acids of the present invention can be used for decomposing nucleic acids or viruses that may be included in the manufacture of feeds, foodstuffs, health functional foods, favorite foods, processed foods, cosmetics and detergents.

In the present invention, the nucleic acid may be a nucleic acid encoding a viral genomic nucleic acid or a viral protein as an exogenous nucleic acid.

The composition for hydrolysis of nucleic acids of the present invention may contain at least one active ingredient having an antiviral effect together with a nucleic acid hydrolysis antigen-binding protein.

The composition for hydrolysis of nucleic acids of the present invention may contain various additives such as isotonic agents, excipients, diluents, thickeners, stabilizers, buffers, and preservatives, depending on the type of use. The compounding quantity of these additives can be suitably set according to the usage form of the composition for nucleic acid hydrolysis.

The compositions of the present invention may be formulated in a variety of forms, including liquid, semi-solid and solid dosage forms, including liquid solutions (eg, injectable, infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes and It may take the form of a suppository. The preferred form depends on the intended mode of use. The compositions of the present invention may take the form of suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulating agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be presented in powder form for use with a suitable vehicle, eg, sterile, pyrogen-free, prior to use.

The present invention may provide an antiviral composition comprising the antigen-binding protein or scFv according to the present invention.

The antiviral composition of the present invention may include a pharmaceutically acceptable carrier.

The pharmaceutically acceptable carrier means any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. Examples of pharmaceutically acceptable carriers include water, saline, phosphate buffered saline, dextrose, glycerol and ethanol, and the like, and mixtures thereof. In many cases, it will be desirable to include isotonic agents such as sugars, polyhydric alcohols such as mannitol, sorbitol or sodium chloride in the composition. Examples of additions of pharmaceutically acceptable substances are wetting agents or minor amounts of auxiliary substances that prolong and enhance the shelf life or efficacy of the antibody, such as wetting or emulsifying agents, preservatives or buffers.

The compositions of the present invention may be formulated in a variety of forms, including liquid, semi-solid and solid dosage forms, including liquid solutions (eg, injectable, infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes and It may take the form of a suppository. Preferred forms depend on the intended mode of administration and treatment. The compositions of the present invention may take the form of suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulating agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be presented in powder form for use with a suitable vehicle, eg, sterile, pyrogen-free, prior to use.

The pharmaceutical composition of the present invention may be formulated in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, or sterile injection solutions according to conventional methods, respectively. have. In detail, when formulating, it can be prepared using a diluent or excipient such as a filler, a weight agent, a binder, a wetting agent, a disintegrant, a surfactant, etc. commonly used. Solid preparations for oral administration include, but are not limited to, tablets, pills, powders, granules, capsules, and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. It may be prepared by adding various excipients, for example, wetting agents, sweetening agents, fragrances, preservatives, etc., in addition to liquids for oral use and liquid paraffin. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations and tasks. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used. As the base of the suppository, Witepsol, Macrosol, Tween 61, cacao butter, laurin oil, glycerogelatin, etc. may be used.

The present invention may provide a pharmaceutical composition for treating or preventing viral infection comprising the antigen-binding protein or scFv according to the present invention.

In the present invention, the virus is not limited to a specific type of virus, and may be a dsDNA-based virus, an ssDNA-based virus or an RNA-based virus. Specifically, it can be applied to adenovirus, herpesvirus, mamavirus, parvovirus, reovirus, rotavirus or retrovirus, and the like, and is not particularly limited as long as the antigen-binding protein or scFv of the present invention exhibits antiviral activity.

"Treatment" as used in the present invention means an activity of improving or favorably changing symptoms caused by a viral infection.

As used herein, “prevention” means prevention of the onset, recurrence or transmission of a disease or disorder, or one or more symptoms caused by the disease/disorder, including prophylactic treatment for a potential candidate. can

A suitable dosage of the pharmaceutical composition of the present invention varies depending on the patient's condition and weight, the severity of the disease, the drug form, and time, but may be appropriately selected by those skilled in the art.

The present invention may provide a feed additive comprising the antigen-binding protein or scFv according to the present invention.

The feed additive of the present invention may include general basic feed fed according to the type of livestock. These basic feeds are all known in the art, and all of the necessary components and compositions and suitable composition ratios according to the type of livestock are commercially available, so anyone skilled in the art will be able to very easily manufacture or purchase them. Specifically, the basic feed may include some or all of a starch-containing material, a protein-containing material, a fat-containing material, a vitamin-containing material, a mineral-containing material, an antioxidant material, an antibiotic, etc. suitable for the type of livestock.

The present invention may provide a nucleic acid molecule encoding the antigen binding protein. In the present invention, the nucleic acid molecule encoding the antigen-binding protein is meant to include a nucleic acid molecule of an immunoglobulin or a fragment thereof that has the ability to bind and degrade a nucleic acid chain. According to one embodiment, the nucleic acid molecule of the present invention may be a nucleic acid molecule encoding a fragment derived from a variant of the 3D8 antibody. As is known in the art, a nucleic acid encoding a component of the invention may be incorporated into an expression vector depending on the host cell used to produce the antigen binding protein of the invention.

The present invention may provide an expression vector comprising the nucleic acid. The expression vector may comprise an expression control sequence operably linked to the nucleic acid.

As used herein, "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is bound. For example, the vector is a plasmid, i.e., a circular double-stranded piece of DNA to which additional DNA segments can be ligated. Further, the vector includes a viral vector, ie, a vector in which additional DNA segments can be ligated into the viral genome. The vector can be autonomously replicated in a host cell into which the vector has been introduced. For example, it may be a bacterial vector and an episomal mammalian vector having a bacterial origin of replication. In addition, the vector (eg, non-episomal mammalian vector) can be integrated into the genome of a host cell upon introduction into the host cell, thereby being replicated along with the host genome. In addition, any vector is capable of expressing a gene operably linked to the vector.

As used herein, "expression control sequence" refers to a polynucleotide sequence to which a coding sequence is ligated to express and process the coding sequence. Expression control sequences include appropriate transcription initiation, termination, promoter, enhancer sequences, efficient RNA processing signals such as splicing and polyadenylation signals, sequences stabilizing cytoplasmic mRNA, sequences increasing translation efficiency (i.e., Kozak ( Kozak) consensus sequence) and sequences that enhance the stability of the protein and, if desired, also include sequences that promote protein secretion. The properties of such an expression control sequence vary depending on the host organism. In prokaryotes, the expression control sequence generally includes a promoter, a ribosome binding site, and a transcription termination sequence. In eukaryotes, the expression control sequence is a promoter. and transcription termination sequences. In the present invention, the expression control sequence includes at least all components whose presence is essential for the expression and processing process, and may include additional components advantageous in its existence, for example, a leader sequence and a fusion partner sequence.

The present invention can provide a host cell comprising the expression vector.

As used herein, the term "host cell" is a cell that can be used to express a nucleic acid, eg, a nucleic acid of the present invention. The host cell may be a prokaryote, eg, E. coli, or a eukaryote, eg, a unicellular eukaryote (eg, yeast or other fungus), a plant cell (eg, tobacco or tomato plant cell). , animal cells (eg, human cells, monkey cells, hamster cells, rat cells, mouse cells, or insect cells) or hybridomas. Examples of host cells include the COS-7 cell line of monkey kidney cells (ATCC CRL 1651) (see Gluzman et al., 1981, Cell 23:175), L cells, C127 cells, 3T3 cells (ATCC CCL 163), Chinese Hamster Ovary (CHO) cells or derivatives thereof such as Veggie CHO and related cell lines grown in serum-free medium (see Rasmussen et al., 1998, Cytotechnology 28:31) or CHO lineage DX- deficient in DHFR B11 (see Urlaub et al., 1980, Proc. Natl. Acad. Sci. USA 77:4216-20), HeLa cells, BHK (ATCC CRL 10) cell line, CV1 derived from the African green monkey kidney cell line CV1 /EBNA cell line (ATCC CCL 70) (see McMahan et al., 1991, EMBO J. 10:2821), human embryonic kidney cells such as 293, 293 EBNA or MSR 293, human epithelial A431 cells, human Colo205 cells, Other transformed primate cell lines, normal aneuploid cells, cell lines derived from in vitro cultures of primary tissues, primary explants, HL-60, U937, HaK or Jurkat cells are included. Typically, the host cell is a cultured cell capable of being transformed or transfected with a polypeptide-encoding nucleic acid, after which the nucleic acid can be expressed in the host cell.

Hereinafter, examples will be given to describe the present invention in detail.

Example

Experimental Example 1. Preparation of 3D8 scFv variants

1-1. Rationale for making 3D8 scFv variants

The 3D8 scFv variant is Ewert S1, Hoegger A, Pluckthun A. Structure-based improvement of the biophysical properties of immunoglobulin VH domains with a generalizable approach. Biochemistry. 2003 Feb 18;42(6):1517-28.; Nieba L1, Honegger A, Krebber C, Pluckthun A. Disrupting the hydrophobic patches at the antibody variable/constant domain interface: improved in vivo folding and physical characterization of an engineered scFv fragment. Protein Eng. It was prepared based on the antibody structure described in 1997 Apr;10(4):435-44. More specifically, in Example 1, Gln-Ala-Gly-Gln was added to the C terminus of the wild-type 3D8 scFv represented by SEQ ID NO: 1. Example 2 replaces amino acids with Q5V, P9A, and E62D at positions expected to increase protein yield among the variant sequences of Example 1, and C-terminal hydrophobic amino acid residues expected to be exposed to the external environment Ser218, Gln220, and Glu222 phosphorus were substituted with Arg, Glu, and Gly, which are amino acid residues with hydrophilic properties to increase protein solubility, respectively. The scFv of the amino acid sequence represented by SEQ ID NO: 1 was used as Comparative Example 1. Comparative Example 2 substituted amino acids with Q5V, P9A, and E62D at positions expected to increase protein yield in the amino acid sequence of Example 1, and substituted amino acid residues at positions expected to prevent aggregation of the VH domain ( K38R, K67R, and T87R). Comparative Example 3 is a hydrophobicity of Val18, Tyr27, Leu70, Ser72, Ala79, Leu83, Leu137, Ser160, and Leu164, respectively, to improve solubility by well forming a core in the scFv in the amino acid sequence of Comparative Example 2 The amino acid residues of the properties of Leu, Phe, Ile, Arg, Leu, Met, Ile, Ala, and Ile were substituted (Ewert S1, Huber T, Honegger A, Pluckthun A. Biophysical properties of human antibody variable domains. J Mol Biol (2003 Jan 17;325(3):531-53.).

The amino acid sequence of the 3D8 scFv mutant according to the above is shown in Table 1, and the nucleic acid sequence for expressing it is shown in Table 2.

1-2. 3D8 scFv variant protein expression and purification

A PIg20 expression vector containing a Pho A leader sequence signal and a protein A tag at the C-terminus was used to express scFv variant proteins with either VH or VL linked by a linker (translated as (GGGGS) _{3 ).} The scFv mutant protein was expressed in Escherichia coli BL21(DE3) pLysE cells, and purified from the lysed bacterial culture supernatant through IgG-Sepharose column (GE Healthcare) chromatography. Proteins were concentrated using a Vivaspin 20 (Sartorius Stedim Biotech) centrifuge with a molecular cutoff of 10,000 Da. The protein concentration was determined using the extinction coefficient at 280 nm calculated from each amino acid sequence.

Experimental Example 2. Analysis of DNA binding force between 3D8 ScFv variants using surface plasmon resonance (SPR) assay

The kinetics of protein-protein interaction was measured at 25°C using a Biacore T200 SPR biosensor (GE Healthcare). Measured SPR values are expressed in arbitrary response units. For the measurement of protein-DNA interaction, ds-(dN) ₄₀ labeled with biotin at the 3'-end was used as a substrate. To immobilize the substrate on the stramtavidin-coated sensor chip (GE Healthcare), the substrate (10 mM HEPES, pH 7.4, 250 mM NaCl, 3 mM EDTA, and 0.005% surfactant P20) was added to the HBS-EP buffer. nM) was injected onto the flow cell at a rate of 5 μl/min or more to show a fixed level of 50-100 response units. The 3D8 scFv mutant diluted in HBS-EP buffer was injected onto the flow cell at a rate of 50 µl/min for 3 minutes, and then flowed again with HBS-EP buffer at a rate of 50 µl/min for 3 minutes. dissociation was observed. All kinetic parameters were determined by nonlinear regression analysis according to a 1:1 binding model using the BIAevaluation version 3.2 software provided by the manufacturer. The dissociation constant K _D was calculated according to the formula _{K D} = k _off /k _{on , where k off} and k _on are the dissociation and association rate constants, respectively. The results are shown in Table 3.

Referring to Table 3, it can be seen that the binding capacity of most of the 3D8 scFv variants is not significantly different compared to that of the wild type 3D8 scFv.

Experimental Example 3. Measurement of DNA hydrolysis activity between 3D8 scFv mutants

In order to confirm the DNA hydrolytic activity of the 3D8 scFv mutant, a DNA degradation analysis method based on the fluorescence resonance energy transfer (FRET) principle was performed.

A synthetic DNA fragment (5'-FAM-CGATGAGTGCCATGGATATAC-BHQ-3') of 21 bases in length labeled with 6-carboxyfluorescin (6-FAM) at the 5'-end and a black hole quencher (BHQ) at the 3'-end was synthesized. A DNA substrate containing 3D8 scFv variants (100 μl, final seeding 500 nM) or 1U DNase I were treated in 96-black well plates containing 100 μl of DNA substrate. Immediately after addition to the DNA substrate, the fluorescence intensity was read in real time at 37°C for 6 hours or more in a fluorescence detector (FLUOstar Microplate Reader). Each reaction was carried out in a final volume of 200 μl using TBS-Mg buffer (100 mM Tris-HCl, 150 mM NaCl, 0.05% Tween 20 and 2 mM MgCl _{2 pH 7.4).} DNase I reaction was performed in DNase I buffer (10 mM Tris-HCL, 2.5 mM MgCl ₂ , and 0.5 mM CaCl ₂ , pH 7.6). The results are shown in FIG. 1 and Table 4.

Referring to FIG. 1 , it was confirmed that Examples 1 and 2 according to the present invention increased DNA hydrolytic activity compared to the wild type, whereas Comparative Examples 2 and 3 decreased, and in particular, Comparative Example 3 had DNA hydrolysis activity. It was confirmed that it was removed.

3D8 scFv hydrolytic activity Measurement result after 1 h Measurement result after 3h Example 1 198.2 117.2 Example 2 191 119.1 Comparative Example 1 (wt) 100 100 Comparative Example 2 55.6 71.1 Comparative Example 3 0 0

In addition, in order to evaluate the effect of temperature and time on the DNA hydrolysis activity of scFv mutant proteins, proteins dissolved in PBS were pre-incubated for less than 30 minutes at each temperature, and then the proteins were placed in a 96-black well plate containing a DNA substrate. added. The results are shown in FIG. 2 and Table 5. Referring to FIG. 2 , it can be seen that when heat of 60° C. and 65° C. is applied, the hydrolysis activity of Examples 1 and 2 is maintained higher than that of Comparative Example 1 (wt). The results are expressed as relative values when the activity of Comparative Example 1 (wt) is 100% at room temperature.

3D8 scFv room temperature 60℃/30min 65/10 min 65/15 min Example 1 117.2 120.8 48.0 55.4 Example 2 119.1 117.0 23.9 19.0 Comparative Example 1 (wt) 100 111.6 23.8 13.0 Comparative Example 2 71.1 16.1 0.0 0.0 Comparative Example 3 0.0 0.0 0.0 0.0

<110> novelgen <120> Antigen binding protein with improved DNA-hydrolyzing activity <130> 19P06024 <160> 14 <170> KoPatentIn 3.0 <210> 1 <211> 248 <212> PRT <213> Artificial Sequence <220> <223> 3D8 scFv <220> <221> PEPTIDE <222> (1)..(248) <223> wild type 3D8 scFv (Comparison 1) <400> 1 Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Val Met His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Ala Tyr Lys Arg Gly Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Ser Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125 Gly Ser Gly Gly Gly Gly Ser Asp Leu Val Met Ser Gln Ser Pro Ser 130 135 140 Ser Leu Ala Val Ser Ala Gly Glu Lys Val Thr Met Ser Cys Lys Ser 145 150 155 160 Ser Gln Ser Leu Phe Asn Ser Arg Thr Arg Lys Asn Tyr Leu Ala Trp 165 170 175 Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Trp Ala 180 185 190 Ser Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser 195 200 205 Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu 210 215 220 Ala Val Tyr Tyr Cys Lys Gln Ser Tyr Tyr His Met Tyr Thr Phe Gly 225 230 235 240 Ser Gly Thr Lys Leu Glu Ile Lys 245 <210> 2 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> 3D8 scFv <220> <221> PEPTIDE <222> (1)..(120) <223> Example 1 VH <400> 2 Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Val Met His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Ala Tyr Lys Arg Gly Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Ser Val Thr Val Ser Ser 115 120 <210> 3 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> 3D8 scFv <220> <221> PEPTIDE <222> (1)..(123) <223> Example 1 VL <400> 3 Asp Leu Val Met Ser Gln Ser Pro Ser Ser Leu Ala Val Ser Ala Gly 1 5 10 15 Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Phe Asn Ser 20 25 30 Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Lys Gln 85 90 95 Ser Tyr Tyr His Met Tyr Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile 100 105 110 Lys Gln Ala Gly Gln His His His His His His 115 120 <210> 4 <211> 258 <212> PRT <213> Artificial Sequence <220> <223> 3D8 scFv <220> <221> PEPTIDE <222> (1)..(258) <223> Example 1 fl <400> 4 Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Val Met His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Ala Tyr Lys Arg Gly Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Ser Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125 Gly Ser Gly Gly Gly Gly Ser Asp Leu Val Met Ser Gln Ser Pro Ser 130 135 140 Ser Leu Ala Val Ser Ala Gly Glu Lys Val Thr Met Ser Cys Lys Ser 145 150 155 160 Ser Gln Ser Leu Phe Asn Ser Arg Thr Arg Lys Asn Tyr Leu Ala Trp 165 170 175 Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Trp Ala 180 185 190 Ser Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser 195 200 205 Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu 210 215 220 Ala Val Tyr Tyr Cys Lys Gln Ser Tyr Tyr His Met Tyr Thr Phe Gly 225 230 235 240 Ser Gly Thr Lys Leu Glu Ile Lys Gln Ala Gly Gln His His His His 245 250 255 His His <210> 5 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> 3D8 scFv <220> <221> PEPTIDE <222> (1)..(120) <223> Example 2 VH <400> 5 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Val Met His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Asp Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Ala Tyr Lys Arg Gly Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Ser Val Thr Val Ser Ser 115 120 <210> 6 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> 3D8 scFv <220> <221> PEPTIDE <222> (1)..(123) <223> Example 2 VL <400> 6 Asp Leu Val Met Ser Gln Ser Pro Ser Ser Leu Ala Val Ser Ala Gly 1 5 10 15 Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Phe Asn Ser 20 25 30 Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Arg Val Glu Ala Gly Asp Leu Ala Val Tyr Tyr Cys Lys Gln 85 90 95 Ser Tyr Tyr His Met Tyr Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile 100 105 110 Lys Gln Ala Gly Gln His His His His His His 115 120 <210> 7 <211> 258 <212> PRT <213> Artificial Sequence <220> <223> 3D8 scFv <220> <221> PEPTIDE <222> (1)..(258) <223> Example 2 fl <400> 7 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Val Met His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Asp Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Ala Tyr Lys Arg Gly Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Ser Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125 Gly Ser Gly Gly Gly Gly Ser Asp Leu Val Met Ser Gln Ser Pro Ser 130 135 140 Ser Leu Ala Val Ser Ala Gly Glu Lys Val Thr Met Ser Cys Lys Ser 145 150 155 160 Ser Gln Ser Leu Phe Asn Ser Arg Thr Arg Lys Asn Tyr Leu Ala Trp 165 170 175 Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Trp Ala 180 185 190 Ser Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser 195 200 205 Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Val Glu Ala Gly Asp Leu 210 215 220 Ala Val Tyr Tyr Cys Lys Gln Ser Tyr Tyr His Met Tyr Thr Phe Gly 225 230 235 240 Ser Gly Thr Lys Leu Glu Ile Lys Gln Ala Gly Gln His His His His 245 250 255 His His <210> 8 <211> 258 <212> PRT <213> Artificial Sequence <220> <223> 3D8 scFv <220> <221> PEPTIDE <222> (1)..(258) <223> Comparison 2 fl <400> 8 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Val Met His Trp Val Arg Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Asp Lys Phe 50 55 60 Lys Gly Arg Ala Thr Leu Thr Ser Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Ala Tyr Lys Arg Gly Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Ser Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125 Gly Ser Gly Gly Gly Gly Ser Asp Leu Val Met Ser Gln Ser Pro Ser 130 135 140 Ser Leu Ala Val Ser Ala Gly Glu Lys Val Thr Met Ser Cys Lys Ser 145 150 155 160 Ser Gln Ser Leu Phe Asn Ser Arg Thr Arg Lys Asn Tyr Leu Ala Trp 165 170 175 Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Trp Ala 180 185 190 Ser Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser 195 200 205 Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu 210 215 220 Ala Val Tyr Tyr Cys Lys Gln Ser Tyr Tyr His Met Tyr Thr Phe Gly 225 230 235 240 Ser Gly Thr Lys Leu Glu Ile Lys Gln Ala Gly Gln His His His His 245 250 255 His His <210> 9 <211> 258 <212> PRT <213> Artificial Sequence <220> <223> 3D8 scFv <220> <221> PEPTIDE <222> (1)..(258) <223> Comparison 3 fl <400> 9 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Leu Lys Met Ser Cys Lys Ala Ser Gly Phe Thr Phe Thr Ser Tyr 20 25 30 Val Met His Trp Val Arg Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Asp Lys Phe 50 55 60 Lys Gly Arg Ala Thr Ile Thr Arg Asp Lys Ser Ser Ser Thr Leu Tyr 65 70 75 80 Met Glu Met Ser Ser Leu Arg Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Ala Tyr Lys Arg Gly Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Ser Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125 Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Met Ser Gln Ser Pro Ser 130 135 140 Ser Leu Ala Val Ser Ala Gly Glu Lys Val Thr Met Ser Cys Lys Ala 145 150 155 160 Ser Gln Ser Ile Phe Asn Ser Arg Thr Arg Lys Asn Tyr Leu Ala Trp 165 170 175 Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Trp Ala 180 185 190 Ser Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser 195 200 205 Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu 210 215 220 Ala Val Tyr Tyr Cys Lys Gln Ser Tyr Tyr His Met Tyr Thr Phe Gly 225 230 235 240 Ser Gly Thr Lys Leu Glu Ile Lys Gln Ala Gly Gln His His His His 245 250 255 His His <210> 10 <211> 756 <212> DNA <213> Artificial Sequence <220> <223> 3D8 scFv <220> <221> gene <222> (1)..(756) <223> Example 1 <400> 10 gaagttcagc tgcagcagag cggtccggaa ctggttaaac cgggtgcaag cgttaaaatg 60 agctgtaaag caagcggtta tacctttacc agctatgtta tgcattgggt gaaacagaaa 120 cctggtcaag gtctggaatg gattggttat atcaatccgt ataatgacgg caccaaatac 180 aacgagaaat tcaaaggtaa agcaaccctg accagcgata aaagcagcag caccgcatat 240 atggaactga gcagcctgac cagtgaagat agcgcagttt attactgtgc acgtggtgca 300 tataaacgtg gttatgcaat ggattattgg ggtcagggca ccagcgttac cgttagcagc 360 ggtggtggtg gtagtggtgg cggtggttca ggcggtggcg gtagcgatct ggttatgagc 420 cagagtccga gcagtctggc agttagtgcc ggtgaaaaag ttaccatgag ctgcaaaagc 480 agccagagcc tgtttaatag tcgtacccgt aaaaactatc tggcatggta tcagcagaaa 540 ccgggacaga gcccgaaact gctgatttat tgggcaagca cccgtgaaag cggtgttccg 600 gatcgtttta ccggtagcgg tagtggcacc gattttaccc tgacaattag cagcgttcag 660 gcagaggatc tggcagtgta ttattgtaaa cagagctact atcatatgta tacctttggc 720 agcggtacga aactggaaat caaacaggca ggtcag 756 <210> 11 <211> 756 <212> DNA <213> Artificial Sequence <220> <223> 3D8 scFv <220> <221> gene <222> (1)..(756) <223> Example 2 <400> 11 gaagttcagc tggttcagag cggtgcagaa ctggttaaac cgggtgcaag cgttaaaatg 60 agctgtaaag caagcggtta tacctttacc agctatgtta tgcattgggt gaaacagaaa 120 cctggtcaag gtctggaatg gattggttat atcaatccgt ataatgatgg caccaagtac 180 aacgacaaat tcaaaggtaa agcaaccctg accagcgata aaagcagcag caccgcatat 240 atggaactga gcagcctgac cagtgaagat agcgcagttt attactgtgc acgtggtgca 300 tataaacgtg gttatgcaat ggattattgg ggtcagggca ccagcgttac cgttagcagc 360 ggtggtggtg gtagtggtgg cggtggttct ggcggtggcg gtagcgatct ggttatgagc 420 cagagtccga gcagtctggc agttagtgcc ggtgaaaaag ttaccatgag ctgcaaaagc 480 agccagagcc tgtttaatag tcgtacccgt aaaaactatc tggcatggta tcagcagaaa 540 ccgggacaga gcccgaaact gctgatttat tgggcaagca cccgtgaaag cggtgttccg 600 gatcgtttta ccggtagcgg tagtggcacc gattttaccc tgacaattag ccgtgttgaa 660 gccggtgatc tggcagtgta ttattgtaaa cagagctact atcatatgta tacctttggt 720 agcggcacca aactggaaat caaacaggca ggtcag 756 <210> 12 <211> 744 <212> DNA <213> Artificial Sequence <220> <223> 3D8 scFv <220> <221> gene <222> (1)..(744) <223> Wild type 3D8 scFv (comparison 1) <400> 12 gaagttcagc tgcagcagag cggtccggaa ctggttaaac cgggtgcaag cgttaaaatg 60 agctgtaaag caagcggtta tacctttacc agctatgtta tgcattgggt gaaacagaaa 120 cctggtcaag gtctggaatg gattggttat atcaatccgt ataatgacgg caccaaatac 180 aacgagaaat tcaaaggtaa agcaaccctg accagcgata aaagcagcag caccgcatat 240 atggaactga gcagcctgac cagtgaagat agcgcagttt attactgtgc acgtggtgca 300 tataaacgtg gttatgcaat ggattattgg ggtcagggca ccagcgttac cgttagcagc 360 ggtggtggtg gtagtggtgg cggtggttca ggcggtggcg gtagcgatct ggttatgagc 420 cagagtccga gcagtctggc agttagtgcc ggtgaaaaag ttaccatgag ctgcaaaagc 480 agccagagcc tgtttaatag tcgtacccgt aaaaactatc tggcatggta tcagcagaaa 540 ccgggacaga gcccgaaact gctgatttat tgggcaagca cccgtgaaag cggtgttccg 600 gatcgtttta ccggtagcgg tagtggcacc gattttaccc tgacaattag cagcgttcag 660 gcagaggatc tggcagtgta ttattgtaaa cagagctact atcatatgta tacctttggc 720 agcggtacga aactggaaat caaa 744 <210> 13 <211> 756 <212> DNA <213> Artificial Sequence <220> <223> 3D8 scFv <220> <221> gene <222> (1)..(756) <223> Comparison 2 <400> 13 gaagttcagc tggttcagag cggtgcagaa ctggttaaac cgggtgcaag cgttaaaatg 60 agctgtaaag caagcggtta tacctttacc agctatgtta tgcattgggt tcgtcagaaa 120 cctggtcaag gtctggaatg gattggttat atcaatccgt ataatgatgg caccaagtac 180 aacgacaaat tcaaaggtcg tgcaaccctg accagcgata aaagcagcag caccgcatat 240 atggaactga gcagcctgcg tagcgaagat agcgcagtgt attattgtgc acgtggtgca 300 tataaacgtg gctatgcaat ggattattgg ggtcagggca ccagcgttac cgttagcagc 360 ggtggtggtg gtagtggtgg cggtggttct ggcggtggcg gtagcgatct ggttatgagc 420 cagagtccga gcagtctggc agttagtgcc ggtgaaaaag ttaccatgag ctgcaaaagc 480 agccagagcc tgtttaatag tcgtacccgt aaaaactatc tggcatggta tcagcagaaa 540 ccgggacaga gcccgaaact gctgatttat tgggcaagca cccgtgaaag cggtgttccg 600 gatcgtttta ccggtagcgg tagtggcacc gattttaccc tgacaattag cagcgttcag 660 gcagaggatc tggccgtcta ttattgtaaa cagagctatt atcatatgta tacctttggc 720 agcggcacca aactggaaat caaacaggca ggtcag 756 <210> 14 <211> 756 <212> DNA <213> Artificial Sequence <220> <223> 3D8 scFv <220> <221> gene <222> (1)..(756) <223> Comparison 3 <400> 14 gaagttcagc tggttcagag cggtgcagaa ctggttaaac cgggtgcaag cctgaaaatg 60 agctgtaaag caagcggttt tacctttacc agctatgtta tgcattgggt tcgtcagaaa 120 cctggtcaag gtctggaatg gattggttat atcaatccgt ataatgatgg caccaagtac 180 aacgacaaat tcaaaggtcg tgcaaccatt acacgtgata aaagcagcag caccctgtat 240 atggaaatga gcagcctgcg tagcgaagat agcgcagtgt attattgtgc acgtggtgca 300 tataaacgtg gctatgcaat ggattattgg ggtcagggca ccagcgttac cgttagcagc 360 ggtggtggtg gtagtggtgg cggtggttct ggcggtggcg gtagcgatat tgttatgagc 420 cagagtccga gcagtctggc agttagtgcc ggtgaaaaag ttaccatgtc atgtaaagcc 480 agccagagca tttttaacag tcgtacccgt aaaaactatc tggcatggta tcagcagaaa 540 ccgggacaga gcccgaaact gctgatttat tgggcaagca cccgtgaaag cggtgttccg 600 gatcgtttta ccggtagcgg tagtggcacc gattttaccc tgaccattag cagcgttcag 660 gcagaggatc tggccgtcta ttattgtaaa cagagctatt atcatatgta tacctttggc 720 agcggcacca aactggaaat taaacaggca ggtcag 756

Claims (18)

In the antigen-binding protein comprising the amino acid sequence represented by SEQ ID NO: 1, it further comprises 1 to 6 amino acid residues at the C terminus;
It contains at least one amino acid substitution selected from the group consisting of Q5V, P9A and E62D and at least one amino acid substitution selected from the group consisting of S218R, Q220E and E222G among the amino acids of the antigen-binding protein represented by SEQ ID NO: 1, and 1 at the C-terminus and no more than 6 amino acid residues;
1 to 6 amino acid residues at the C-terminus are selected from Gin, Ala or Gly.
The antigen binding protein according to claim 1, wherein 1 to 6 amino acid residues at the C terminus are 3 to 5 amino acids selected from Gin, Ala or Gly.
The antigen binding protein of claim 1, wherein the sequence of 1 to 6 amino acid residues at the C-terminus is QAGQ.
The antigen binding protein of claim 1 , wherein the amino acid substitutions include Q5V, P9A and E62D.
The antigen binding protein of claim 1 , wherein the amino acid substitutions include S218R, Q220E and E222G.
The antigen binding protein of claim 1 , wherein the amino acid substitutions include Q5V, P9A, E62D, S218R, Q220E and E222G.
The antigen-binding protein according to claim 1, comprising a VH represented by SEQ ID NO: 2 and a VL represented by SEQ ID NO: 3.
The antigen-binding protein according to claim 1, comprising the amino acid sequence represented by SEQ ID NO: 4.
The antigen-binding protein according to claim 1, comprising a VH represented by SEQ ID NO: 5 and a VL represented by SEQ ID NO: 6.
The method according to claim 1, comprising the amino acid sequence represented by SEQ ID NO: 7, antigen-binding protein.
The antigen binding protein of claim 1 , wherein the antigen binding protein is an scFv.
A composition for hydrolysis of nucleic acids, comprising the antigen-binding protein according to any one of claims 1 to 11.
12. An antiviral composition comprising the antigen binding protein according to any one of claims 1 to 11.
A pharmaceutical composition for the treatment or prevention of viral infection, comprising the antigen-binding protein according to any one of claims 1 to 11.
A feed additive comprising the antigen binding protein according to any one of claims 1 to 11.
12. A nucleic acid molecule encoding the antigen binding protein according to any one of claims 1 to 11.
An expression vector comprising the nucleic acid molecule according to claim 16 .
A host cell comprising the expression vector according to claim 17 .

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