Antibody-mimetic molecule

Abstract

To provide a novel antibody-mimetic molecule that binds to monomeric SOD1.SOLUTION: An antibody-mimetic molecule comprises a polypeptide consisting of an amino acid sequence encoding human-derived fibronectin type III domain (FN3) with at least one amino acid mutation. The antibody-mimetic molecule has affinity for monomeric Cu / Zn-superoxide dismutase (SOD1).SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to antibody mimetic molecules. [Background technology]

[0002] Superoxide dismutase (SOD) is an oxidoreductase that decomposes reactive oxygen species generated within cells. Among them, Cu / Zn-superoxide dismutase (SOD1) consists of a homodimer, with copper and zinc ions bound to each subunit. These metal ions contribute to structural stabilization, and the copper ion acts as the catalytic center of the redox reaction.

[0003] Previous studies have reported that misfolded SOD1 accumulates in spinal motor neurons in some patients with familial amyotrophic lateral sclerosis (ALS). Although the mechanism of its onset remains largely unknown, the leading theory is that ALS develops through a structural change from a stable dimer to a monomer that is prone to aggregation, triggered by oxidative stress or dissociation of metal ions. In light of this background, there is a need to develop a probe that recognizes monomeric SOD1, with the hope of contributing to elucidating the pathology of ALS and applying it to diagnostic tools.

[0004] Currently, monomeric SOD1 can be detected by the SEDI antibody, a peptide polyclonal antibody against the dimer junction interface sequence of SOD1, but this antibody has problems with low reproducibility due to lot-to-lot differences.Furthermore, because the antibody requires disulfide bonds (SS bonds) for structural formation, it cannot be used inside cells under a reducing environment. Summary of the Invention [Problem to be solved by the invention]

[0005] An objective of the present invention is to provide a novel antibody-mimetic molecule that binds to monomeric SOD1. [Means for solving the problem]

[0006] The present inventors have conducted extensive research to solve the above problems, and have succeeded in developing a novel antibody mimetic molecule (monobody) that has the ability to bind to monomeric SOD1. The present invention has been completed through further research based on these findings, and includes the following aspects.

[0007] Section 1. An antibody-mimetic molecule that contains a polypeptide consisting of an amino acid sequence in which at least one amino acid is mutated in the amino acid sequence encoding human fibronectin type III domain (FN3), and has the ability to bind to monomeric copper / zinc-superoxide dismutase (SOD1). Section 2. Item 2. The antibody mimetic molecule according to item 1, which binds to monomeric SOD1 with a Kd of 6500 nM or less to which FN3 does not bind with a Kd of 6500 nM or less. Section 3. Item 3. The antibody mimetic molecule according to item 1 or 2, which binds to dimeric SOD1 with a Kd of 650 μM or more. Section 4. (a) the amino acid sequence represented by SEQ ID NO:2 (the 23rd amino acid is D or Y; The 78th amino acid is Y or F; or (b) the amino acid sequence represented by SEQ ID NO:2 (the 23rd amino acid is D or Y; The 78th amino acid is Y or F. Amino acid sequence with 80% or more identity to Item 4. The antibody mimetic molecule according to any one of Items 1 to 3, comprising a polypeptide consisting of: Section 5. An amino acid sequence represented by any one of SEQ ID NOs: 3 to 7; or An amino acid sequence that has 80% or more identity with any of these amino acid sequences 5. The antibody mimetic molecule according to any one of items 1 to 4, comprising a polypeptide consisting of: Section 6. 6. The antibody mimetic molecule according to any one of items 1 to 5, comprising a polypeptide consisting of an amino acid sequence shown in any one of SEQ ID NOs: 3 to 7. Section 7. A method for detecting monomeric SOD1, comprising the step of detecting monomeric SOD1 in a sample using the antibody mimetic molecule according to any one of Items 1 to 6. Section 8. Item 8. The detection method according to Item 7, wherein in the detection step, the antibody mimetic molecule is crosslinked with monomeric SOD1 in the sample. Section 9. Item 10. A kit for detecting monomeric SOD1, comprising the antibody mimetic molecule according to any one of Items 1 to 6. A nucleic acid molecule encoding the antibody mimetic molecule according to any one of items 1 to 6. Section 11. An expression vector comprising the nucleic acid molecule according to item 10. Section 12. A nucleic acid library comprising the nucleic acid molecule according to item 10. Section 13. Item 13. A peptide display library derived from the nucleic acid library according to Item 12. A method for producing an antibody mimetic molecule, comprising a polypeptide consisting of an amino acid sequence in which at least one amino acid is mutated in an amino acid sequence encoding FN3, and binding with a Kd of 6500 nM or less to monomeric SOD1 to which FN3 does not bind with a Kd of 6500 nM or less, comprising: (1) obtaining a polypeptide display library by performing DNA shuffling on nucleic acids encoding polypeptides constituting a polypeptide display library consisting of an amino acid sequence in which at least one amino acid has been mutated in an amino acid sequence encoding FN3; (2) contacting a display library of the polypeptides obtained in the step (1) with monomeric SOD1 and selecting at least one polypeptide that binds to monomeric SOD1 with a Kd of 10,000 nM or less as the antibody mimetic molecule; and (3) contacting monomeric SOD1 with a display library of polypeptides consisting of an amino acid sequence in which at least one amino acid has been mutated in the amino acid sequence encoding the at least one polypeptide selected in the step (2) and selecting at least one polypeptide that binds to monomeric SOD1 with a Kd of 6500 nM or less as the antibody mimetic molecule. A manufacturing method comprising: Section 15. Item 15. An antibody mimetic molecule obtainable by the production method described in Item 14. Effect of the Invention

[0008] According to the present invention, it is possible to provide a novel antibody-mimetic molecule that binds to monomeric SOD1. [Brief description of the drawings]

[0009] [Figure 1] FIG. 1 shows the three-dimensional structures and mutation sites of two types of SOD1 mutants (SOD1(MM) and SOD1(FG)). [Diagram 2]This figure shows the results of analysis of two types of SOD1 mutants (SOD1(MM) and SOD1(FG)) using a size exclusion chromatography-multiple angle light scattering detection system (SEC-MALS). SOD1 protein (50 μM) was injected into an HPLC system (Prominence, Shimadzu Corporation) at a flow rate of 0.5 mL / min, and separated in MN buffer using a gel filtration column (TSKgel G2000SW, Tosoh Corporation). The absolute molar mass of the eluate was estimated using a multi-angle static light scattering detector (miniDAWN TREOS, WYATT Technology) installed downstream of the HPLC system. (A) E,E-SOD1 is SOD1 lacking metal ions (copper ions and zinc ions), which means that the above experiment was performed in the absence of these metal ions. MN buffer containing 5 mM EDTA was used as the running buffer. (B) E,E-SOD1+Zn2+ means that E,E-SOD1 and an equivalent amount of ZnSO4 were incubated in MN buffer at 37°C for 30 minutes and then subjected to the above experiment. (C) E,E-SOD1+Cu2+ means that E,E-SOD1 and an equivalent amount of CuSO4 were incubated in MN buffer at 37°C for 30 minutes and then subjected to the above experiment. [Diagram 3] FIG. 1 shows the amino acid sequences and structures of the monobody side library. [Figure 4]The figure shows the amino acid sequences (B) and binding properties (C and D) of the antibody mimetic molecules (Mb(S1), Mb(S2), Mb(S3), and Mb(S4)) obtained by selection using the monobody side library (A) for monomeric or dimeric SOD1. The binding properties for monomeric or dimeric SOD1 were evaluated using the yeast surface display method as previously reported ([Tanaka, S.-i. et al., Monobody-mediated alteration of enzyme specificity. Nat. Chem. Biol. 11 (2015) 762-764.] and [Tanaka, S.-i. et al., Monobody-mediated alteration of lipase substrate specificity. ACS Chem. Biol. 13 (2018) 1487-1492.]). The Kd value was determined from a plot of median fluorescence intensity versus SOD1 concentration using a 1:1 binding model using SigmaPlot software (Systat Software). [Diagram 5]Figure 1 shows the results of biolayer interferometry (BLI) analysis of the interaction of Mb(S4) or Mb(S2) with SOD1 protein (SOD1(MM) (A, D), SOD1(WT) (B, E), and SOD1(FG) (C, F)) using a BLItz apparatus (Sartorius). A streptavidin-coated biosensor was immersed in a BLI assay buffer (pH 7.4) containing 50 mM Tris, 150 mM NaCl, and 0.02% Tween® 20 for at least 10 minutes. To analyze the apo-state SOD1 protein, 1 mM EDTA was further added to the BLI assay buffer. The biosensor was placed in the BLItz apparatus and immersed in a BLI assay buffer containing 5 μM Mb(S4) for 120 seconds, and washed with the BLI assay buffer for 60 seconds. The binding assay consisted of three steps: an initial baseline step (60 s) with BLI assay buffer, an association step (120 s) with 20, 50, or 100 μM SOD1 in BLI assay buffer, and a dissociation step (120 s) with BLI assay buffer. The dissociation constant (Kd), association rate constant (ka), and dissociation rate constant (kd) were estimated by globally fitting the observed sensorgrams to a 1:1 binding model using BLItz Pro software version 1.2. [Figure 6]Figure 1 shows that chemical cross-linking improves the detection stability of the complex between Mb(S4) and monomeric SOD1. SOD1 protein (15 μM) was mixed with 15 μM Mb(S4) in MN buffer, and the cross-linker DSG (Disuccinimidyl glutarate, Thermo Fisher Scientific) or BS3 (Bis(sulfosuccinimidyl)suberate disodium salt, Dojindo Laboratories, Ltd.) dissolved in water was added (final concentration 1 mM). After incubation at room temperature for 30 min, 1 M Tris (pH 8) was added (final concentration of Tris, 50 mM) to stop the cross-linking reaction. The sample was further mixed with Laemmli sample buffer containing β-mercaptoethanol and analyzed by SDS-PAGE using a 12.5% ​​polyacrylamide gel. The gel was stained with Coomassie Brilliant Blue R-250. [Figure 7] This figure shows the results of detection of monomeric SOD1 by Mb(S4) using E. coli lysate or yeast lysate expressing SOD1 protein. E. coli lysate or yeast lysate expressing SOD1 protein (total protein 10 μg) was mixed with 15 μM Mb(S4) in MN buffer, chemically crosslinked in the same manner as above, and then analyzed by SDS-PAGE. Proteins separated on the gel by SDS-PAGE were electroblotted onto a PVDF membrane and examined by Western blotting using a polyclonal antibody against SOD1 (#GTX100554, GeneTex). [Figure 8]FIG. 1 shows a schematic diagram (A) of an ELISA kit for detecting monomeric SOD1 using Mb(S4) and a diagram (B) showing the reactivity of the kit with SOD1 protein (SOD1(WT), SOD1(MM), and SOD1(FG)). Recombinant SOD1 protein (0.01-1.0 μM) was mixed with Mb(S4) in MN buffer. BS3 was added to a final concentration of 1 mM, and the mixture was incubated at room temperature for 30 minutes, after which Tris was added to a final concentration of 50 mM to stop the reaction. The mixture was applied to wells of a Pierce® NeutrAvidin® coated high-capacity plate (Thermo Scientific) and pre-washed with a washing buffer (25 mM Tris (pH 7.2) containing 150 mM NaCl, 0.1% BSA, and 0.05% Tween® 20). After 1 hour of incubation, the wells were washed with washing buffer and incubated for 30 minutes with polyclonal anti-SOD1 antibody (#GTX100554, GeneTex) diluted 1:300 in washing buffer. The wells were then washed with washing buffer and incubated for 30 minutes with goat anti-rabbit IgG (H+L) secondary antibody (#32460, Invitrogen) diluted 1:300 in washing buffer. The wells were washed with washing buffer and a substrate solution (100 mM sodium citrate (pH 5.0) containing 0.4 g / L o-phenylenediamine and 0.012% H2O2) was added. The reaction was stopped by adding hydrogen chloride, and the absorbance at 490 nm was measured using a plate reader Epoch (BioTek). [Figure 9] The top photo shows the results of Western blot using Mb(S4), which detects monomeric SOD1 in Neuro2a lysate. The middle photo shows the results of Western blot using Mb(sh). The bottom photo shows the thiol-disulfide state of SOD1 in cell lysate. The experiment was repeated at least twice, and reproducible results were obtained. Mb(sh) represents SOD1 and unbound Mb (negative control). [Figure 10]FIG. 1 shows the results of affinity analysis of Mb(S4_Y53S / Y54K) and Mb(MMcAvi_03) for SO1(FG) using yeast surface display and FACS. [Figure 11] FIG. 1 shows the results of an analysis of the specificity of Mb(MMcAvi_03) and Mb(FG_03) to monomeric SOD1 using BLI. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] 1. Antibody mimetic molecules of the invention The antibody mimetic molecule of the present invention comprises a polypeptide consisting of an amino acid sequence in which at least one amino acid has been mutated in the amino acid sequence encoding human fibronectin type III domain (FN3) (herein, human FN3 may be referred to as "hFN3"), and has the ability to bind to monomeric copper / zinc-superoxide dismutase (Cu / Zn-superoxide dismutase; SOD1).

[0011] In the present invention, the term "mutation" refers to, for example, substitution, deletion, insertion, and the like of amino acid residues.

[0012] In the antibody mimetic molecule of the present invention, the number of mutated amino acids in the amino acid sequence (SEQ ID NO: 1) constituting hFN3 is not particularly limited as long as it has monomeric SOD1 binding ability, but is at least 1. The lower limit of the number of mutated amino acids can be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, etc. Among them, the lower limit of the number of mutated amino acids is preferably 21, and more preferably 24.

[0013] In the antibody mimetic molecule of the present invention, the mutated amino acid can be a surface amino acid and / or an internal amino acid of the structure. The surface amino acid of the structure refers to an amino acid exposed on the surface of the three-dimensional structure of hFN3. The internal amino acid of the structure refers to an amino acid facing inward of the three-dimensional structure of hFN3.

[0014] hFN3 contains a loop structure and a β-sheet structure. In the antibody mimetic molecule of the present invention, the mutated amino acid may be contained in the loop region of hFN3, may be contained in the β-sheet region, or may be contained in a region other than these. When there are two or more mutated amino acids, they may be contained in only one of these multiple regions, may be contained in two or more types of regions, or may be contained in all regions.

[0015] More specifically, hFN3 contains two β-sheet structures composed of β-strands A, B, and E, or β-strands C, D, F, and G, and seven loop structures, AB loop, BC loop, CD loop, DE loop, EF loop, FG loop, and GA loop. In the antibody mimetic molecule of the present invention, the mutated amino acid may be contained in the loop region of hFN3, may be contained in the β-sheet region, or may be contained in a region other than these, as described above, but it is preferable that the mutation is contained in at least one or more regions of β-strand C, β-strand D, CD loop, DE loop, and FG loop, and it is more preferable that the mutation is contained in all regions of β-strand C, β-strand D, CD loop, DE loop, and FG loop.

[0016] Such mutations do not affect the higher order structure of hFN3, except for partial conformational changes that contribute to its monomeric SOD1 binding ability.

[0017] Due to the partial conformational changes that accompany the introduction of such mutations, the antibody mimetic molecule of the present invention is able to bind to monomeric SOD1 to which hFN3 does not bind or binds less strongly before the mutations are introduced.

[0018] The binding affinity of the antibody mimetic molecules of the present invention to monomeric SOD1 can be represented by the dissociation constant, Kd.

[0019] The method for analyzing the binding affinity of the antibody mimetic molecule of the present invention to monomeric SOD1 is not particularly limited, and for example, the Kd value can be calculated by fluorescence-activated cell sorting (FACS) analysis.

[0020] The antibody mimetic molecule of the present invention preferably binds to monomeric SOD1 to which hFN3 does not bind with a Kd of 6500 nM or less, more preferably binds to monomeric SOD1 to which FN3 does not bind with a Kd of 6000 nM or less, and even more preferably binds to monomeric SOD1 to which FN3 does not bind with a Kd of 5500 nM or less.

[0021] The antibody mimetic molecule of the present invention preferably binds to dimeric SOD1 with a Kd of 650 μM or more, more preferably binds to dimeric SOD1 with a Kd of 700 μM or more, and even more preferably binds to dimeric SOD1 with a Kd of 750 μM or more.

[0022] The antibody mimetic molecule of the present invention may have the above-mentioned monomeric SOD1 binding ability or dimeric SOD1 binding ability independently, or may have the above-mentioned monomeric SOD1 binding ability and dimeric SOD1 binding ability in combination.

[0023] The antibody mimetic molecule of the present invention includes a polypeptide consisting of an amino acid sequence represented by SEQ ID NO: 2 (the 23rd amino acid is D or Y; the 78th amino acid is Y or F) or an amino acid sequence having 80% or more, preferably 90% or more, more preferably 95% or more, and even more preferably 98% or more identity to the amino acid sequence represented by SEQ ID NO: 2 (the 23rd amino acid is D or Y; the 78th amino acid is Y or F). In the antibody mimetic molecule of the present invention, at least one of the amino acids at positions 25 to 30 and / or 75 to 86 in the amino acid sequence represented by SEQ ID NO: 2 may be substituted with another amino acid, deleted, or at least one amino acid may be added to this region. Examples of such mutants include polypeptides consisting of the amino acid sequences represented by SEQ ID NOs: 6 and 7. The antibody mimetic molecule of the present invention may comprise a polypeptide consisting of an amino acid sequence having 80% or more, preferably 90% or more, more preferably 95% or more, and even more preferably 98% or more identity to an amino acid sequence in which the amino acids at positions 25 to 30 and / or the amino acids at positions 75 to 86 in the amino acid sequence shown in SEQ ID NO: 2 are substituted with the corresponding amino acids in SEQ ID NO: 6, or to an amino acid sequence in which the amino acids at positions 25 to 30 and / or the amino acids at positions 75 to 86 in the amino acid sequence shown in SEQ ID NO: 7 are substituted with the corresponding amino acids in SEQ ID NO: 7.

[0024] Here, in an amino acid sequence having 80% or more, preferably 90% or more, more preferably 95% or more, and even more preferably 98% or more identity with the amino acid sequence shown in SEQ ID NO: 2 (the 23rd amino acid is D or Y; the 78th amino acid is Y or F), the 3rd, 6th, 7th, 30th, 31st, 33rd, 44th, 45th, 47th, 49th, 53rd, 54th, 75th, 76th, 77th, 78th, 79th, 80th, 81st, 82nd, 83rd, 84th, 85th, 86th, 87th, 88th, 89th, 90th, and 91st amino acids of the amino acid sequence shown in SEQ ID NO: 2 are preferably maintained in terms of monomeric SOD1 binding ability or dimeric SOD1 binding ability. The antibody mimetic molecule of the present invention may be further modified to improve solubility, if necessary. An example of such a polypeptide is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 5 (in which mutations have been introduced into the 53rd and 54th amino acids).

[0025] Furthermore, the antibody mimetic molecule of the present invention includes an amino acid sequence of any one of SEQ ID NOs: 3 to 7; or a polypeptide consisting of an amino acid sequence having 80% or more, preferably 90% or more, more preferably 95% or more, and even more preferably 98% or more identity to any one of these amino acid sequences.

[0026] Here, in an amino acid sequence having 80% or more, preferably 90% or more, more preferably 95% or more, and even more preferably 98% or more identity to any of the amino acid sequences of SEQ ID NOs: 3 to 7, it is preferable that the amino acids at positions 3, 6, 7, 30, 31, 33, 44, 45, 47, 49, 53, 54, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90 and 91 of the amino acid sequence represented by SEQ ID NO: 3 or 4 are maintained in terms of monomeric SOD1 binding ability or dimeric SOD1 binding ability.

[0027] Furthermore, the antibody mimetic molecule of the present invention includes a polypeptide consisting of the amino acid sequence of any one of SEQ ID NOs:3-7.

[0028] As used herein, "identity" refers to the degree of identity when the sequences being compared are appropriately aligned, and can be calculated using, for example, analysis software such as BLAST or FASTA with default parameters.

[0029] The antibody mimetic molecules of the present invention may be preferably used in the diagnosis of ALS.

[0030] 2. Detection Method of the Present Invention The detection method of the present invention is a method for detecting monomeric SOD1, which comprises the step of detecting monomeric SOD1 in a sample using the above-described antibody-mimetic molecule of the present invention.

[0031] In the detection method of the present invention, detection and measurement of monomeric SOD1 may be performed using any immunoassay method, such as immunoelectrophoresis, Western blotting, agglutination, solid-phase immunoassay, etc. Examples of agglutination methods include immunoturbidimetry and immunonephelometry, etc. Examples of solid-phase immunoassays include radioimmunoassay and enzyme-labeled immunoassay, etc.

[0032] Examples of samples to be subjected to the detection method of the present invention include biological samples such as tissues, cells, crushed or lysed tissues or cells, saliva, blood, serum, urine, feces, etc., or purified products thereof.

[0033] The detection method of the present invention may include crosslinking the antibody mimetic molecule with the monomeric SOD1 in the sample in the above-mentioned detection step. Here, crosslinking refers to chemical crosslinking.

[0034] The crosslinking agent is not particularly limited, and examples thereof include disuccinimidyl glutarate (DSG), bis(sulfosuccinimidyl) suberate (BS3), N-hydroxysuccinimide (NHS), disuccinimidyl suberate (DSS), dithiobis(succinimidyl propionate) (DSP), dithiobis(sulfosuccinimidyl propionate) (DTSSP), ethylene glycol bis(succinimidyl succinate) (EGS), ethylene glycol bis(sulfosuccinimidyl succinate) (sulfo-EGS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo-DST), bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone (BSOCOES), bis[2-(sulfosuccinimidooxycarbonyloxy)ethyl]sulfone (sulfo-BSOCOES), and the like.

[0035] 3. Detection kit of the present invention The detection kit of the present invention is a kit for detecting monomeric SOD1, which comprises the above-described antibody-mimetic molecule of the present invention.

[0036] In the detection kit of the present invention, the detection and measurement of monomeric SOD1 may be performed using any of the above-mentioned immunoassay methods, preferably using a solid-phase immunoassay method, and more preferably using an enzyme-linked immunoassay method, which includes various enzyme-linked immunosorbent assays (ELISAs), such as direct adsorption ELISA, sandwich ELISA, and competitive ELISA.

[0037] The detection kit of the present invention can contain, in addition to the above-described antibody mimetic molecule of the present invention, reagents and instruments necessary for detection, such as a solid phase carrier, an antibody, a buffer solution, and a reaction stop solution.

[0038] In the detection kit of the present invention, the antibody mimetic molecule of the present invention may be directly or indirectly immobilized on a solid phase carrier. Examples of the solid phase include testing devices such as well plates, microplates, strips, sticks, tubes, beads, microbeads, and membranes. Among them, Pierce (registered trademark) NeutrAvidin (registered trademark) Coated Plates (manufactured by Thermo Scientific) can be preferably used. The antibody mimetic molecule of the present invention can be immobilized on a solid phase carrier by a method known in the art.

[0039] The detection kit of the present invention may contain the antibody mimetic molecule of the present invention and an antibody different from the antibody mimetic molecule. The antibody mimetic molecule of the present invention and the antibody bind to different sites on the antigen. The antibody may be labeled with a labeling substance to facilitate detection and measurement of monomeric SOD1, or the detection kit of the present invention may further contain a known secondary antibody labeled with a labeling substance.

[0040] The labeling substance is not particularly limited, and examples thereof include enzymes such as horseradish peroxidase (HSP) and alkaline phosphatase; biotin; fluorescent dyes such as rhodamine, FITC, and Cy; fluorescent proteins such as phycoerythrin (PE) and allophycocyanin (APC); chemiluminescent substances such as luminol; radioisotopes; agar; agarose; gelatin; magnetic particles; metal colloid particles such as gold, silver, copper, iron, platinum, palladium, and mixtures thereof; and particles obtained by labeling particles made of natural or synthetic polymers (e.g., latex, rubber, polyethylene, polypropylene, polystyrene, styrene-butadiene copolymer, polyvinyl chloride, polyvinyl acetate, polyacrylamide, polymethacrylate, styrene-methacrylate copolymer, etc.), glass, or inorganic materials (e.g., silica, kaolin, talc, silica-alumina, alumina, barium sulfate, etc.) with dye molecules, fluorescent molecules, magnetic particles, etc.

[0041] The detection kit of the present invention can be used as a diagnostic tool for ALS.

[0042] 4. Nucleic Acid Molecules of the Invention The nucleic acid molecules of the invention are nucleic acid molecules that encode the antibody mimetic molecules of the invention described above.

[0043] 5. Expression Vector of the Present Invention The expression vector of the present invention is an expression vector comprising the nucleic acid molecule encoding the antibody mimetic molecule of the present invention described above. More specifically, the expression vector of the present invention is an expression vector in which the nucleic acid molecule of the present invention described above is linked under the control of a suitable promoter.

[0044] The expression vector of the present invention is not particularly limited, but for example, in the case of an E. coli expression system, a pBlueScript vector (manufactured by Agilent Technologies), a pUC vector (manufactured by TaKaRa), a pET vector (manufactured by Novagen), a pCold vector (manufactured by TaKaRa), etc. can be used. In the case of a yeast expression system, a pYD1 vector (manufactured by Invitrogen), a pPICZ vector (manufactured by Invitrogen), etc. can be used.

[0045] 6. Nucleic Acid Library of the Present Invention The nucleic acid library of the present invention is a nucleic acid library comprising nucleic acid molecules encoding the above-described antibody mimetic molecules of the present invention.

[0046] The nucleic acid library of the present invention provides the peptide display library of the present invention described below.

[0047] 7. Peptide Display Library of the Present Invention The peptide display library of the present invention is a peptide display library derived from a nucleic acid library containing a nucleic acid molecule encoding the above-mentioned antibody mimetic molecule of the present invention. Specifically, the peptide display library of the present invention is a display library of a polypeptide consisting of an amino acid sequence in which at least one amino acid is mutated in the amino acid sequence encoding hFN3. As a method for introducing mutations to prepare the display library of the present invention, for example, the Kunkel method, the QuikChange method, the Inverse PCR method, the Overlap-extension method, and artificial gene synthesis can be used.

[0048] The peptide display library of the present invention can be used for the selection of the antibody mimetic molecules of the present invention.

[0049] 8. Methods for Producing Antibody Mimics of the Invention The method for producing an antibody mimetic molecule of the present invention comprises a polypeptide consisting of an amino acid sequence in which at least one amino acid is mutated in the amino acid sequence encoding FN3, and the antibody mimetic molecule binds with a Kd of 6500 nM or less to monomeric SOD1 to which FN3 does not bind with a Kd of 6500 nM or less, the method comprising the steps of: (1) obtaining a polypeptide display library by performing DNA shuffling on nucleic acids encoding polypeptides constituting a polypeptide display library consisting of an amino acid sequence in which at least one amino acid has been mutated in an amino acid sequence encoding FN3; (2) contacting a display library of the polypeptides obtained in the step (1) with monomeric SOD1 and selecting at least one polypeptide that binds to monomeric SOD1 with a Kd of 10,000 nM or less as the antibody mimetic molecule; and (3) A production method comprising the steps of contacting monomeric SOD1 with a display library of polypeptides consisting of an amino acid sequence in which at least one amino acid has been further mutated in the amino acid sequence encoding at least one of the polypeptides selected in step (2) and selecting at least one polypeptide that binds to monomeric SOD1 with a Kd of 6,500 nM or less as the antibody mimetic molecule.

[0050] In step (1), the number of mutated amino acids in the amino acid sequence encoding FN3 is not particularly limited, but is at least 1. The lower limit of the number of mutated amino acids can be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, etc. Among them, the lower limit of the number of mutated amino acids is preferably 21.

[0051] In step (3), the method for introducing at least one further mutation into the amino acid sequence encoding the at least one polypeptide selected in step (2) is not particularly limited, and examples thereof include site-directed mutagenesis, random mutagenesis, etc. Examples of random mutagenesis include error-prone PCR, DNA shuffling, etc. Among these, error-prone PCR is preferred.

[0052] In step (3), the number of mutations to be introduced into the amino acid sequence encoding at least one type of polypeptide selected in step (2) is not particularly limited, but is preferably 1 to 3.

[0053] In the present specification, nucleic acid means deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). DNA is not particularly limited, and examples thereof include double-stranded DNA, single-stranded DNA, plasmid DNA, genomic DNA, cDNA, etc. RNA is not particularly limited, and examples thereof include mRNA, rRNA, tRNA, miRNA, siRNA, etc. The nucleic acid in step (1) is preferably a plasmid.

[0054] By carrying out steps (1), (2), and (3), an antibody mimetic molecule having improved binding affinity to monomeric SOD1 can be obtained.

[0055] In this specification, the term "comprising" includes "consisting substantially of" and "consisting of." In addition, the present disclosure includes any combination of the constituent elements described in this specification.

[0056] In addition, the various characteristics (properties, structures, functions, etc.) described in each embodiment of the present disclosure above may be combined in any way to specify the subject matter included in the present disclosure. In other words, the present disclosure includes all subject matter consisting of all combinations of each combinable characteristic described in this specification. EXAMPLES

[0057] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these.

[0058] Example 1: Preparation of monomeric SOD1 As models of monomeric SOD1, two SOD1 mutants (SOD1(MM) and SOD1(FG)) were generated (Fig. 1). It has been reported that E,E-SOD1 is monomerized by the reduction of the intramolecular disulfide bond between Cys57 and Cys146 ([F. Arnesano et al., The unusually stable quaternary structure of human SOD1 is controlled by both metal occupancy and disulfide status, J Biol Chem 279 (2004) 47998-48003.] and [Y. Furukawa et al., Oxygen-induced maturation of SOD1: a key role for disulfide formation by the copper chaperone CCS, EMBO J 23 (2004) 2872-2881.]). In SOD1(MM), all four cysteine ​​residues (Cys6, Cys57, Cys111, and Cys146) are replaced by serine residues, and four Zn 2+ Two of the ligands, a histidine residue (His80) and an aspartic acid residue (Asp80), and Cu 2+ and Zn 2+ In SOD1(FG), the histidine residue (His63), which is a bridging ligand that binds to both SOD1 and SOD1, is replaced by an alanine residue. In addition, compared to wild-type SOD1, SOD1(FG) has a phenylalanine residue (Phe50) and a glycine residue (Gly51) located at the dimer junction interface of SOD1 replaced by glutamic acid residues.

[0059] Analysis using a size exclusion chromatography-multiple angle light scattering detection system (SEC-MALS) demonstrated that the two SOD1 mutants (SOD1(MM) and SOD1(FG)) exhibited excellent anti-Cu binding activity. 2+ and Zn 2+We confirmed that it exists in a monomeric form regardless of the presence or absence of .

[0060] Example 2: Preparation of a monobody capable of binding to monomeric SOD1 To generate a monobody that recognizes monomeric SOD1, the monobody side library (Fig. 3) was used for selection against biotinylated SOD1(MM) with an N-terminal His tag followed by an N-terminal Avi tag. After four rounds of library selection by phage display, enriched clones were subjected to gene shuffling, and the resulting gene pool was subjected to yeast surface display for further selection. As a result, two monobodies (Mb(S1) and Mb(S2)) that showed strong binding to biotinylated SOD1(MM) were identified (Fig. 4). Mb(S2) showed binding to both biotinylated SOD1(MM) and biotinylated SOD1(WT) with an N-terminal His tag followed by an N-terminal Avi tag, whereas Mb(S1) showed binding to biotinylated SOD1(MM) but not to biotinylated SOD1(WT). This demonstrated that Mb(S1) specifically binds to monomeric SOD1.

[0061] To improve the binding affinity of Mb(S1) to monomeric SOD1, we identified two more monobodies (Mb(S3) and Mb(S4)) using error-prone PCR and directed evolution (Fig. 4). Mb(S3) and Mb(S4) showed higher binding affinity than Mb(S1) while maintaining the specificity for monomeric SOD1.

[0062] The interaction of Mb(S4) or Mb(S2) with SOD1 protein (SOD1(MM), SOD1(WT), and SOD1(FG)) was analyzed by biolayer interferometry (BLI) using a BLItz instrument. The results are shown in Figure 5.

[0063] In the interaction between Mb (S4) and SOD1 (MM), the binding increased in a concentration-dependent manner during the binding phase, whereas the binding rapidly dissociated during the dissociation phase (Fig. 5A). The dissociation constant Kd value was 1.6 × 10 -4 M, and the association and dissociation rate constants are (2.8±1.3)×10 4 M -1 s -1 and (4.4±1.7)×10 -1 s -1 On the other hand, the binding between Mb(S4) and SOD1(WT) was significantly weaker than that between Mb(S4) and SOD1(MM) (Fig. 5B), and was comparable to the results of the analysis of SOD1(WT) in the absence of Mb(S4). This suggested that SOD1(WT) was nonspecifically adsorbed onto the biosensor, and that the interaction between Mb(S4) and dimeric SOD1 was significantly weaker than that with monomeric SOD1. In addition, in the interaction between Mb(S4) and SOD1(FG), a rapid increase in binding was observed in the binding phase, and a rapid decrease in binding was observed in the dissociation phase (Fig. 5C). However, the binding of SOD1(FG) gradually increased in the binding phase and did not return to the baseline in the dissociation phase. This phenomenon was also observed in the analysis of SOD1(FG) in the absence of Mb(S4), and was therefore considered to be due to nonspecific adsorption of SOD1(FG) to the biosensor. Therefore, although it was difficult to quantitatively analyze the interaction between Mb(S4) and SOD1(FG), it was thought that the two rapidly bind and dissociate in the interaction between Mb(S4) and SOD1(FG). These results demonstrated that Mb(S4) specifically binds to monomeric SOD1, but it was found that the complex of Mb(S4) and monomeric SOD1 was difficult to stably detect by conventional methods such as pull-down assays.

[0064] The interaction between Mb(S2) and SOD1(MM) had a lower Kd value (1.8 × 10 -6 M), which corresponds to a faster association rate constant ((6.5±1.6)×10 4 M -1 s -1) and a slower dissociation rate constant ((1.2±0.38)×10 -1 s -1 ) (Fig. 5D). On the other hand, unlike Mb(S4), Mb(S2) also interacted with SOD1(WT), and the association and dissociation rate constants of the interaction between Mb(S2) and SOD1(MM) were orders of magnitude slower than those of the interaction between Mb(S2) and SOD1(MM) ((3.3 ± 0.89) × 10, respectively). 3 M -1 s -1 and (1.7±0.40)×10 -2 s -1 ), Kd value (5.0 × 10 -6 The Kd value of Mb(S2) was comparable to that observed for the interaction between Mb(S2) and SOD1(MM) (Fig. 5E). Mb(S2) also appeared to bind to SOD1(FG) (Fig. 5F), but the interaction between Mb(S2) and SOD1(FG) was difficult to quantitatively analyze due to nonspecific adsorption of SOD1(FG) to the biosensor. These results indicate that Mb(S2) binds to both monomeric and dimeric SOD1.

[0065] Example 3: Improvement of stability of Mb(S4)-monomeric SOD1 complex by chemical crosslinking In order to realize more stable detection of the complex of Mb(S4) and monomeric SOD1, we attempted to detect the complex by chemically crosslinking it with a crosslinker. Disuccinimidyl glutarate (DSG) or bis(sulfosuccinimidyl) suberate (BS3) was used as the crosslinker. The results are shown in Figure 6. A band was observed at about 30 kDa in the mixture of Mb(S4) and monomeric SOD1 (SOD1(MM) or SOD1(FG)) by chemical crosslinking with DSG or BS3 (lanes 6, 7, 9, and 10 in Figure 6A). This band was not observed in the absence of monomeric SOD1 (SOD1(MM) or SOD1(FG)) or Mb(S4) (lanes 1, 2, 5, 8, and 11 in Figure 6A). This suggests that the band around 30 kDa represents a complex of cross-linked Mb(S4) and monomeric SOD1. On the other hand, in the mixture of Mb(S4) and SOD1(WT), the band around 30 kDa was significantly weaker than that in the mixture of Mb(S4) and SOD1(MM) or SOD1(FG) (lanes 3 and 4 in Fig. 6A). This suggests that in the mixture of Mb(S4) and SOD1(WT), SOD1(WT) is favorably cross-linked to itself, probably due to the homodimeric conformation of SOD1(WT) (lane 5 in Fig. 6A).

[0066] Moreover, the presence of metal ions did not affect the formation of a cross-linked complex between Mb(S4) and monomeric SOD1 (FIG. 6B).

[0067] It is known that SOD1(WT) is monomerized by reduction of the disulfide bond between Cys57 and Cys146 (F. Arnesano, L. Banci, I. Bertini, M. Martinelli, Y. Furukawa, T. V. O'Halloran, The unusually stable quaternary structure of human SOD1 is controlled by both metal occupancy and disulfide status, J Biol Chem 279 (2004) 47998-48003., Y. Furukawa, A. S. Torres, T. V. O'Halloran, Oxygen-induced maturation of SOD1: a key role for disulfide formation by the copper chaperone CCS, EMBO J 23 (2004) 2872-2881.). We therefore performed a similar experiment by reducing the disulfide bonds in SOD1(WT), and found that in a mixture of Mb(S4) and SOD1(WT), the amount of these cross-linked complexes was significantly increased by reducing the disulfide bonds (Fig. 6C).

[0068] From the above results, it was found that chemical cross-linking of the complex of Mb(S4) and monomeric SOD1 using a cross-linking agent is effective for more stable detection of the complex of Mb(S4) and monomeric SOD1. Therefore, in the following examples, chemical cross-linking treatment using a cross-linking agent (BS3) was also performed.

[0069] Example 4: Ex vivo detection of monomeric SOD1 using Mb(S4) To verify whether Mb(S4) can specifically detect monomeric SOD1 even in a mixture containing heterologous proteins, SOD1 protein was overexpressed in SHuffle T7 Competent E. coli (NEW ENGLAND Biolabs), and the lysate was used to perform an experiment to detect SOD1 protein with Mb(S4). The results are shown in Figure 7. In the E. coli lysate expressing monomeric SOD1 (SOD1(FG)), a crosslinked complex between Mb(S4) and SOD1(FG) was detected as a band at about 30 kDa with high intensity, whereas no similar band was detected in the absence of Mb(S4) (Figure 7A). In addition, compared with wild-type SOD1 (SOD1(WT)), the SOD1 mutant (SOD1(C)) in which all four cysteine ​​residues (Cys6, Cys57, Cys111, and Cys146) were replaced with serine residues showed a higher detection rate than the wild-type SOD1 (SOD1(WT)). 4 In SOD1(C S), the intramolecular disulfide bond between Cys57 and Cys146 does not form, thus favoring the monomeric state over the dimeric form. 4 In the lysate of E. coli expressing SOD1(C), Mb(S4) and SOD1(C) were detected in the same manner as in the lysate of E. coli expressing SOD1(FG). 4 The cross-linked complex with SOD1(S) was detected as a band around 30 kDa with strong intensity, whereas no similar band was detected in the absence of Mb(S4) (Fig. 7B). On the other hand, in the E. coli lysate expressing SOD1(WT), the band around 30 kDa was detected with significantly weaker intensity (Fig. 7B). These results indicate that Mb(S4) can specifically detect monomeric SOD1 even in a mixture containing heterologous proteins.

[0070] In addition, ALS-associated SOD1 mutant variants (SOD1 mutants having G37R, G85R, G93R, C111Y or L144F mutations) were overexpressed in SHuffle T7 Competent E. coli (NEW ENGLAND Biolabs) in the same manner as above, and the lysate was used to perform an experiment to detect SOD1 protein with Mb(S4). The results are shown in FIG. 7B. In the E. coli lysate expressing SOD1 mutants having G37R, G85R, G93R or C111Y mutations, the relative intensity of the bands showing the cross-linked complexes between Mb(S4) and each SOD1 mutant was comparable to the relative intensity of the bands showing the cross-linked complexes between Mb(S4) and wild-type SOD1 (SOD1(WT)). On the other hand, in the lysate of E. coli expressing the SOD1 mutant with the L144F mutation (SOD1(L144F)), the relative intensity of the band representing the cross-linked complex between Mb(S4) and SOD1(L144F) was stronger than that of the band representing the cross-linked complex between Mb(S4) and wild-type SOD1 (SOD1(WT)). This result is consistent with a previous report showing the selective instability of the dimer junction interface in SOD1(L144F) (MJ Lindberg et al., Systematically perturbed folding patterns of amyotrophic lateral sclerosis (ALS)-associated SOD1 mutants, Proc Natl Acad Sci USA 102(28) (2005) 9754-9759.). These results also indicate that Mb(S4) can specifically detect monomeric SOD1 in a mixture containing heterologous proteins.

[0071] Furthermore, a detection experiment of SOD1 protein by Mb(S4) was carried out using lysates of yeast (S. cerevisiae) in which the endogenous sod1 gene was replaced with cDNA encoding human wild-type SOD1 (SOD1(WT)) or a variant of a human SOD1 mutant caused by ALS (SOD1 mutant having A4V, G37R, G85R, G93A or I149T mutation). In this experimental system using modified yeast, the modified yeast cells express SOD1 protein at physiological levels, so that the ability of Mb(S4) to detect monomeric SOD1 expressed at physiological levels can be verified, unlike the above-mentioned SOD1 protein overexpression system using E. coli. The results are shown in Figure 7C. In the yeast lysates expressing SOD1 mutants having A4V or I149T mutations (SOD1(A4V) and SOD1(I149T)), bands indicating cross-linked complexes between Mb(S4) and SOD1(A4V) or SOD1(I149T) in the presence of Mb(S4) were detected, whereas in the yeast lysates expressing SOD1 mutants having G37R, G85R, or G93A mutations (SOD1(G37R), SOD1(G85R), and SOD1(G93A)), bands indicating cross-linked complexes between Mb(S4) and SOD1(G37R), SOD1(G85R), or SOD1(G93A) were not detected. This result indicates that A4V and I149T mutations promote monomerization of SOD1. In fact, Ala4 and Ile149 are located at the dimer junction interface of SOD1, while Gly37, Gly85, and Gly93 are located away from the dimer junction interface. It has also been experimentally shown that the dimer of SOD1(A4V) has an increased dissociation constant compared to SOD1(WT) (Y. Furukawa et al., Mutation-dependent polymorphism of Cu,Zn-superoxide dismutase aggregates in the familial form of amyotrophic lateral sclerosis, J Biol Chem 285(29) (2010) 22221-22231.). These results indicate that Mb(S4) can detect the monomerization of SOD1 expressed at physiological levels.

[0072] Example 5: Preparation of a kit for detecting monomeric SOD1 using Mb(S4) An ELISA detection kit for monomeric SOD1 was prepared using Mb(S4) (FIG. 8A). The reactivity of the kit with SOD1 protein (SOD1(WT), SOD1(MM), and SOD1(FG)) was confirmed, and it showed no reactivity with SOD1(WT), but marked reactivity with monomeric SOD1 (SOD1(MM) and SOD1(FG)) (FIG. 8B). This result demonstrated that the kit can be used to detect monomeric SOD1.

[0073] Example 6: Detection of monomeric SOD1 in mouse neuroblastoma cell line Neuro2a lysate using Mb(S4) Neuro2a cell lysates expressing WTSOD1 and various ALS mutants were mixed with 10 μM biotinylated Mb(S4) or Mb(sh) with C-terminal Avi tag in MN buffer, to which the crosslinker BS3 (bis(sulfosuccinimidyl) suberic acid disodium salt, DOJINDO) dissolved in water was added (final concentration 1 mM). After incubation at room temperature for 30 min, 1 M Tris (pH 8) was added to the sample to stop the crosslinking reaction (final concentration of Tris 50 mM). The sample was further mixed with Laemmli sample buffer containing β-mercaptoethanol and subjected to SDS-PAGE using 12.5% ​​polyacrylamide gel. In addition, Western blotting was performed using a polyclonal antibody against SOD1 (GeneTex, No. GTX100554) (References: Amesaka, H.; Hara, M.; Sakai, Y.; Shintani, A.; Sue, K.; Yamanaka, T.; Tanaka, S.; Furukawa, Y. Protein Sci., 33, e4961 (2024).).

[0074] To test whether Mb(S4) can detect SOD1 mutants in a heterogeneous protein mixture in vitro, human SOD1 proteins carrying ALS-causing mutations were overexpressed in mouse neuroblastoma cells Neuro2a, and cell lysates were examined by cross-linking with Mb(S4) or the negative control monobody Mb(sh). As shown in Figure 9 (top), a strong band of approximately 30 kDa cross-linked Mb(S4)-SOD1 was confirmed in SOD1(WT) and SOD1(L144F). Next, taking advantage of the difference in electrophoretic mobility of SOD1 with and without disulfide bonds, we investigated the disulfide bond state of human SOD1 protein overexpressed in Neuro2a, and found that there was a small amount of disulfide-reduced SOD1 (especially WT and L144F) (Figure 9, bottom). These results suggest that disulfide formation of SOD1 does not proceed efficiently under the condition of overexpression in Neuro2a, leading to the existence of monomeric SOD1. Therefore, these results suggest that the cross-linking assay using Mb(S4) can detect monomeric SOD1 in cell lysates containing many contaminating substances.

[0075] Example 7: Obtaining the Mb sequences of the modified mutant Mb (S4) (S4_Y53S / Y54K) and two newly obtained types (Mb(MMcAvi_03) and Mb(FG_03)) The soluble expression level of Mb(S4) in E. coli was low, so we created a mutant Mb(S4_Y53S / Y54K) with improved soluble expression level of Mb(S4). Mb(MMcAvi_03) and Mb(FG_03) were obtained by binding screening again using phage display and yeast surface display (SEQ ID NOs: 5 to 7).

[0076] Affinity analysis was performed on Mb(S4_Y53S / Y54K) and Mb(MMcAvi_03) using yeast surface display and fluorescence-activated cell sorting (FACS). The results are shown in Figure 10. Furthermore, we analyzed the specificity of Mb (MMcAvi_03) and Mb (FG_03) for monomeric SOD1 using biolayer interferometry (BLI) with a BLItz device. The results are shown in Figure 11. As shown in Figure 11, the BLI analysis showed a strong signal for interaction with E,E-SOD1 (MM), but the signal intensity was low for interactions with E,E-SOD1 (WT) SS and E,Zn-SOD1 (WT) SS. However, since E,E-SOD1 (WT) SS and E,Zn-SOD1 (WT) SS also contain some monomers, it is considered that monomeric SOD1 was detected by Mb (MMcAvi_03) and Mb (FG_03). In the case of E,Zn-SOD1(WT)SS, the dissociation constant of the SOD1 dimer decreases upon Zn binding, so the proportion of monomeric SOD1 becomes extremely small, resulting in a smaller signal than that of E,E-SOD1(WT)SS.

Claims

1. An antibody-mimetic molecule comprising a polypeptide consisting of an amino acid sequence in which at least one amino acid is mutated in an amino acid sequence encoding a human-derived fibronectin type III domain (FN3), and having a binding ability to monomeric copper / zinc-superoxide dismutase (Cu / Zn-superoxide dismutase; SOD1).

2. The antibody mimetic molecule of claim 1 , which binds to monomeric SOD1 with a Kd of 6500 nM or less, to which FN3 does not bind with a Kd of 6500 nM or less.

3. The antibody mimetic molecule of claim 1 or 2, which binds to dimeric SOD1 with a Kd of 650 μM or more.

4. (a) the amino acid sequence represented by SEQ ID NO:2 (The 23rd amino acid is D or Y; The 78th amino acid is Y or F; or (b) the amino acid sequence represented by SEQ ID NO:2 (The 23rd amino acid is D or Y; The 78th amino acid is Y or F. Amino acid sequence having 80% or more identity with 3. The antibody mimetic molecule of claim 1 or 2, comprising a polypeptide consisting of:

5. An amino acid sequence represented by any one of SEQ ID NOs: 3 to 7; or An amino acid sequence having 80% or more identity with any of these amino acid sequences 3. The antibody mimetic molecule of claim 1 or 2, comprising a polypeptide consisting of:

6. The antibody mimetic molecule according to claim 1 or 2, comprising a polypeptide consisting of an amino acid sequence represented by any one of SEQ ID NOs: 3 to 7.

7. A method for detecting monomeric SOD1, comprising the step of detecting monomeric SOD1 in a sample using the antibody-mimetic molecule according to claim 1 or 2.

8. The detection method according to claim 7 , wherein in the detection step, the antibody-mimicking molecule is crosslinked with monomeric SOD1 in the sample.

9. A kit for detecting monomeric SOD1, comprising the antibody mimetic molecule of claim 1 or 2.

10. A nucleic acid molecule encoding an antibody mimetic molecule according to claim 1 or 2.

11. An expression vector comprising the nucleic acid molecule of claim 10.

12. A nucleic acid library comprising the nucleic acid molecule of claim 10.

13. A peptide display library derived from the nucleic acid library of claim 12.

14. A method for producing an antibody mimetic molecule, comprising a polypeptide consisting of an amino acid sequence in which at least one amino acid is mutated in an amino acid sequence encoding FN3, and binding with a Kd of 6500 nM or less to monomeric SOD1 to which FN3 does not bind with a Kd of 6500 nM or less, comprising: (1) obtaining a polypeptide display library by performing DNA shuffling on nucleic acids encoding polypeptides constituting a polypeptide display library consisting of an amino acid sequence in which at least one amino acid is mutated in an amino acid sequence encoding FN3; (2) contacting the display library of the polypeptide obtained in the step (1) with monomeric SOD1 and selecting at least one polypeptide that binds to monomeric SOD1 with a Kd of 10,000 nM or less as the antibody mimetic molecule; and (3) contacting monomeric SOD1 with a display library of polypeptides consisting of an amino acid sequence in which at least one amino acid has been mutated in the amino acid sequence encoding the at least one polypeptide selected in the step (2), and selecting at least one polypeptide that binds to monomeric SOD1 with a Kd of 6500 nM or less as the antibody mimetic molecule. A manufacturing method comprising:

15. An antibody mimetic molecule obtainable by the production method according to claim 14.

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