WO2024084667A1

WO2024084667A1 - Method for detecting progranulin

Info

Publication number: WO2024084667A1
Application number: PCT/JP2022/039163
Authority: WO
Inventors: 良介山内; 宏規小林
Original assignee: 大塚製薬株式会社
Priority date: 2022-10-20
Filing date: 2022-10-20
Publication date: 2024-04-25
Also published as: WO2024085222A1

Abstract

Provided is a method for detecting progranulin, characterized by use of an antibody that specifically binds to the P domain or the G domain of progranulin, and an antibody that specifically binds to the E domain of progranulin.

Description

Methods for detecting progranulin

This application relates to a method for detecting progranulin.

Progranulin (PGRN) is a neurotrophic factor expressed mainly in neurons and microglia, and has various neuroprotective effects (anti-inflammatory effects, promotion of neuronal survival and neurite growth, involvement in normal lysosomal function, etc.). Mutations in the Granulin (GRN) gene, which encodes PGRN, have been confirmed in several neurodegenerative dementias, including frontotemporal dementia (FTD), frontotemporal lobar degeneration (FTLD), and Alzheimer's disease (AD). It has also been reported that haploinsufficiency induced by heterozygous mutations in the GRN gene reduces PGRN concentrations in peripheral blood and cerebrospinal fluid, and has been found to be one of the main causes of FTLD (Non-Patent Document 1).

PGRN consists of seven and a half conserved granulin (GRN) domains linked by short linker regions. The GRN domains are named from N-terminus to C-terminus as follows: paragranulin (P domain, 18-47aa), GRN1 (G domain, 58-113aa), GRN2 (F domain, 123-179aa), GRN3 (B domain, 206-261aa), GRN4 (A domain, 281-336aa), GRN5 (C domain, 364-417aa), GRN6 (D domain, 442-496aa) and GRN7 (E domain, 518-573aa). Here, the P domain is approximately half the length of the other granulin domains. Extracellularly secreted PGRN is cleaved into GRN by several proteases, including neutrophil elastase (NE), matrix metalloproteinase 9 (MMP9), MMP12, MMP14, proteinase 3 (PR3 and PRTN3), and ADAMTS7 (a disintegrin and metalloproteinase with thrombospondin 7).

PGRN and GRN are known to have opposing inflammatory functions. Full-length PGRN suppresses the inflammatory effects of TNF-α, while GRN induces them. In addition, full-length PGRN has been reported to promote process outgrowth in neurons. Microglia (especially reactive microglia), which are immune cells resident in the brain, produce and secrete high levels of PGRN. In a rat cerebral ischemia model, the amount of PGRN and NE activity in microglia increase early after cerebral ischemia, leading to the production of GRN. It has been reported that inhibition of NE suppresses the increase in GRN production and inflammatory cytokines, suggesting that increased PGRN cleavage by NE (production of GRN) is involved in the inflammatory response.

The purpose of this application is to provide a method for detecting progranulin.

The present application provides a method for detecting progranulin, which is characterized by using an antibody that specifically binds to the P domain or G domain of progranulin, and an antibody that specifically binds to the E domain of progranulin.

The present application also provides a method for selecting a patient with a neurodegenerative disease caused by the granulin gene, the method comprising the steps of:
(1) detecting Progranulin in a sample derived from a subject by the method of the present application; and (2) evaluating the amount of Progranulin detected in step (1).

The present application also provides a method for evaluating the effectiveness of a treatment for a patient with a neurodegenerative disease, comprising the step of detecting progranulin in a sample from the patient with a neurodegenerative disease before and after the treatment using the method of the present application.

The present application also provides a method for screening a substance for treating a neurodegenerative disease, comprising the steps of:
(1) contacting progranulin, a progranulin-degrading substance, and a candidate substance;
(2) detecting progranulin by the method according to any one of claims 1 to 3; and (3) evaluating the effectiveness of a candidate substance against a neurodegenerative disease.

The present application also provides a combination of an antibody that specifically binds to the P domain or G domain of Progranulin and an antibody that specifically binds to the E domain of Progranulin.

The present application also provides a kit for detecting progranulin, comprising an antibody that specifically binds to the P domain or G domain of progranulin, and an antibody that specifically binds to the E domain of progranulin.

The present application also provides an antibody that specifically binds to the P domain of Progranulin, comprising a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO:1, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO:2, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO:3, and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO:4, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO:5, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:6.

The present application also provides an antibody that specifically binds to the G domain of Progranulin, comprising a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO:11, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO:12, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO:13, and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO:14, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO:15, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:16.

The present application also provides an antibody that specifically binds to the G domain of Progranulin, comprising a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO:21, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO:22, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO:23, and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO:24, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO:25, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:26.

The present application also provides an antibody that specifically binds to the E domain of Progranulin, comprising a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 30, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 31, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 32, and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 33, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 34, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 35.

The present application provides a highly specific method for detecting progranulin.

Schematic diagram of the progranulin (PGRN) protein. Purification of degraded PGRN by CBB staining. A schematic diagram of purified degraded PGRN is shown (Fig. 2a). It can be seen that the molecular weight decreases with the deletion of domains (Fig. 2b). PGRN-FL: full-length PGRN; PGRN-ΔP: PGRN lacking the P domain; PGRN-ΔPG: PGRN lacking the P and G domains; PGRN-ΔE: PGRN lacking the E domain; PGRN-ΔPE: PGRN lacking the P and E domains; PGRN-ΔPGE: PGRN lacking the P, G, and E domains. The results of epitope mapping of each antibody using cleaved PGRN. Figure 3e is a schematic diagram showing the domains recognized by each antibody. Schematic diagram for specifically detecting full-length PGRN (FL-PGRN). Two types of antibodies specific to the termini of PGRN are used. Standard curves for each assay in ELISA. Standard curves for each assay in SPFS (surface plasmon-field enhanced fluorescence spectroscopy). Detection of full-length and cleaved PGRN using commercially available PGRN ELISA kits (Adipogen and Biovendor). Figures 8a, 8b, and 8c show the reaction specificity to full-length PGRN in ELISA, and Figures 8d, 8e, and 8f show the reaction specificity to full-length PGRN in SPFS. Detection of PGRN concentrations in plasma of Alzheimer's disease patients or healthy subjects by SPFS using assay No. 2. Figures 10a and 10c show the calibration curves of Assay No. 2 in SPFS using monkey PGRN recombinant antigen, and Figures 10b and 10d show the detection of PGRN concentrations in commercially available monkey plasma and cerebrospinal fluid in SPFS using Assay No. 2.

In this disclosure, when a numerical value is accompanied by the term "about," it is intended to include a range of ±10% of that value. For example, "about 20" is intended to include "18-22." A range of numerical values includes all values between and at the endpoints. "About" in reference to a range applies to both endpoints of the range. Thus, for example, "about 20-30" is intended to include "18-33."

In this disclosure, amino acid residues are represented by the following abbreviations:
Ala or A: Alanine
Arg or R: Arginine
Asn or N: Asparagine
Asp or D: Aspartic acid
Cys or C: Cysteine
Gln or Q: Glutamine
Glu or E: glutamic acid
Gly or G: glycine
His or H: histidine
Ile or I: Isoleucine
Leu or L: Leucine
Lys or K: Lysine
Met or M: methionine
Phe or F: phenylalanine
Pro or P: Proline
Ser or S: serine
Thr or T: Threonine
Trp or W: Tryptophan
Tyr or Y: Tyrosine
Val or V: Valine

Method for detecting Progranulin The present application provides a method for detecting Progranulin, characterized by using an antibody that specifically binds to the P domain or G domain of Progranulin, and an antibody that specifically binds to the E domain of Progranulin.

In the present application, progranulin may be from any species, typically mammals (e.g., human, mouse, rat, hamster, rabbit, cat, dog, cow, sheep, monkey, etc.). Among these, mouse, rat or human is preferred, and human is particularly preferred. The amino acid sequences of progranulin derived from various biological species can be easily obtained using publicly known databases. Representative amino acid sequences of human and mouse progranulin are registered as NP_002078 and NP_032201, respectively.

In this disclosure, the term "antibody" is used to include various antibody structures, such as monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, and human antibodies. The species of the antibody is not particularly limited, and examples include antibodies derived from mice, rats, rabbits, goats, llamas, and humans. The antibody of the present application is, for example, a monoclonal antibody.

In the present disclosure, the term "antibody" also includes molecules that contain a portion of an antibody as a component and retain the ability to bind to an antigen. For example, but not limited to, the antibody heavy and light chain variable regions ( _VH and _VL ), F(ab') ₂ , Fab', Fab, Fv, disulphide-linked FV (sdFv), Single-Chain FV (scFV), Fab3, Diabody, Triabody, Tetrabody, Minibody, Bis-scFv, (scFv) ₂ -Fc, intact-IgG, and polymers thereof are included in the antibody of the present application.

The immunoglobulin class of an antibody is determined based on the heavy chain constant region. Immunoglobulin classes include IgA, IgD, IgE, IgG, and IgM, and the corresponding heavy chains are called α chain, δ chain, ε chain, γ chain, and μ chain, respectively. Immunoglobulin classes can be further classified into subclasses (isotypes), for example, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The immunoglobulin class and subclass of the antibody in this specification are not particularly limited. In one embodiment, the immunoglobulin class is IgG. The light chain of an antibody can be divided into κ chain and λ chain based on its constant region, but the antibody in this specification may have either a κ chain or a λ chain.

An antibody variable region is usually composed of three complementarity determining regions (CDRs) flanked by four framework regions (FRs). In this specification, the amino acid positions assigned to the CDRs and frameworks of an antibody variable region are defined according to Kabat (see Sequences of Proteins of Immunological Interest, National Institute of Health, Bethesda, Md., (1987) and (1991)). Antibody numbering according to Kabat can be performed using known tools such as Abnum (http://www.bioinf.org.uk/abs/abnum/).

In the present application, antibodies that specifically bind to the P, G, or E domain of Progranulin are not particularly limited and can be produced by known methods. For example, anti-Progranulin antibodies can be produced by general methods using Progranulin as an immunogen, and antibodies that bind to each domain can be screened from the produced anti-Progranulin antibodies. Alternatively, antibodies that specifically bind to each domain can be produced using a peptide containing the P, G, or E domain of Progranulin as an immunogen.

Monoclonal antibodies can be obtained by known methods, such as producing hybridomas that produce antibodies, or using genetic engineering techniques to produce expression vectors containing antibody genes and expressing them in cells. Hybridomas that secrete monoclonal antibodies can be produced according to the method described in Kohler et al., Nature 256:495, 1975. First, an immunogen is mixed with a suitable substance for enhancing antigenicity (e.g., keyhole limpet hemocyanin or bovine serum albumin, etc.) and, if necessary, an immunostimulant (Freund's complete or incomplete adjuvant, etc.), and a non-human mammal such as a rat, mouse, rabbit, goat, or horse is immunized with the mixture. Usually, the animal is immunized multiple times at intervals of 3 to 10 days, and 1 to 100 μg of the immunogen peptide is administered. Next, immunocompetent cells (cells capable of producing antibodies in the immunized animal) are collected from the immunized animal after multiple immunizations, and fused with myeloma cells (e.g., cells derived from a mammal such as a mouse, rat, guinea pig, hamster, rabbit, or human) that are not capable of producing autoantibodies. The polyethylene glycol method, electric fusion method, or the like is used for cell fusion. Furthermore, cells that have been successfully fused are selected based on the selection marker possessed by the fused cells, and the reactivity of the antibodies produced by the selected cells against the immunogen is confirmed by ELISA, radioimmunoassay, fluorescent antibody method, or the like, to obtain a hybridoma that produces the desired monoclonal antibody. The monoclonal antibody can be isolated from the culture supernatant obtained by culturing the obtained hybridoma in vitro. It can also be cultured in vivo, such as in ascites of a mouse, rat, guinea pig, hamster, or rabbit, and isolated from the ascites.

Monoclonal antibodies can also be obtained by cloning the antibody genes from the obtained hybridomas, incorporating them into an appropriate expression vector, and expressing them in host cells, as described below (P.J.Delves., ANTIBODY PRODUCTION ESSENTIAL TECHNIQUES., 1997 WILEY; P.Shepherd and C.Dean., Monoclonal Antibodies., 2000 OXFORD UNIVERSITY PRESS; J.W.Goding., Monoclonal Antibodies: principles and practice., 1993 ACADEMIC PRESS). Furthermore, using transgenic animal production techniques, transgenic animals (e.g., cows, goats, sheep, or pigs) in which the gene for the desired antibody has been incorporated into their endogenous genes can be produced, and monoclonal antibodies derived from the antibody gene can be obtained from the milk of the transgenic animals.

As host cells, for example, eukaryotic cells such as animal cells, plant cells, and fungal cells can be used. Examples of animal cells include mammalian cells (e.g., CHO, COS, NIH3T3, myeloma, BHK (baby hamster kidney), HeLa, and Vero), amphibian cells (e.g., Xenopus oocytes), and insect cells (e.g., Sf9, Sf21, and Tn5). Examples of fungal cells include yeast (e.g., Saccharomyces genus, e.g., Saccharomyces cerevisiae), filamentous fungi (e.g., Aspergillus genus, e.g., Aspergillus niger), and the like. Prokaryotic cells such as Escherichia coli (E. coli) (e.g., JM109, DH5α, and HB101), and Bacillus subtilis can also be used as host cells. Vectors can be introduced into host cells by, for example, the calcium phosphate method, the DEAE-dextran method, electroporation, lipofection, etc.

The obtained monoclonal antibody can be purified by an appropriate combination of methods well known in the art, such as chromatography using a protein A column, ion exchange chromatography, hydrophobic chromatography, ammonium sulfate precipitation, gel filtration, affinity chromatography, etc.

The specific binding of each of the obtained antibodies to the P, G, or E domain of Progranulin can be confirmed by ELISA, fluorescent antibody technique, radioimmunoassay (RIA), BIACORE ^{(registered trademark)} , surface plasmon resonance assay, Biolayer Interferometry assay, etc.

In one embodiment, an antibody that specifically binds to the P domain of Progranulin comprises a heavy chain variable region comprising a heavy chain CDR1 comprising the sequence of SEQ ID NO:1, a heavy chain CDR2 comprising the sequence of SEQ ID NO:2, and a heavy chain CDR3 comprising the sequence of SEQ ID NO:3, and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO:4, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO:5, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:6.

In one embodiment, an antibody that specifically binds to the P domain of Progranulin comprises a heavy chain variable region comprising a sequence having 80% or more, 85% or more, 90% or more, or 95% or more sequence identity with the sequence of SEQ ID NO: 7 or 8, and a light chain variable region comprising a sequence having 80% or more, 85% or more, 90% or more, or 95% or more sequence identity with the sequence of SEQ ID NO: 9 or 10. In one embodiment, an antibody that specifically binds to the P domain of Progranulin comprises a heavy chain variable region comprising a sequence in which 0, 1, 2, 3, 4, or 5 amino acids are deleted, substituted, or added in the sequence of SEQ ID NO: 7 or 8, and a light chain variable region comprising a sequence in which 0, 1, 2, 3, 4, or 5 amino acids are deleted, substituted, or added in the sequence of SEQ ID NO: 9 or 10. These embodiments include antibodies that have no modifications in the CDRs of the heavy chain variable region and light chain variable region, specifically, antibodies that specifically bind to the P domain of Progranulin, comprising a heavy chain variable region that includes a heavy chain CDR1 that includes the amino acid sequence of SEQ ID NO:1, a heavy chain CDR2 that includes the amino acid sequence of SEQ ID NO:2, and a heavy chain CDR3 that includes the amino acid sequence of SEQ ID NO:3, and a light chain variable region that includes a light chain CDR1 that includes the amino acid sequence of SEQ ID NO:4, a light chain CDR2 that includes the amino acid sequence of SEQ ID NO:5, and a light chain CDR3 that includes the amino acid sequence of SEQ ID NO:6.

In one embodiment, an antibody that specifically binds to the P domain of Progranulin comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7 or 8 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 9 or 10. In one embodiment, an antibody that specifically binds to the P domain of Progranulin comprises a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 7 or 8 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 9 or 10.

In one embodiment, an antibody that specifically binds to the G domain of Progranulin comprises a heavy chain variable region comprising a heavy chain CDR1 comprising the sequence of SEQ ID NO: 11, a heavy chain CDR2 comprising the sequence of SEQ ID NO: 12, and a heavy chain CDR3 comprising the sequence of SEQ ID NO: 13, and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 14, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 15, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 16.

In one embodiment, an antibody that specifically binds to the G domain of Progranulin comprises a heavy chain variable region comprising a sequence having 80% or more, 85% or more, 90% or more, or 95% or more sequence identity with the sequence of SEQ ID NO: 17 or 18, and a light chain variable region comprising a sequence having 80% or more, 85% or more, 90% or more, or 95% or more sequence identity with the sequence of SEQ ID NO: 19 or 20. In one embodiment, an antibody that specifically binds to the G domain of Progranulin comprises a heavy chain variable region comprising a sequence in which 0, 1, 2, 3, 4, or 5 amino acids are deleted, substituted, or added in the sequence of SEQ ID NO: 17 or 18, and a light chain variable region comprising a sequence in which 0, 1, 2, 3, 4, or 5 amino acids are deleted, substituted, or added in the sequence of SEQ ID NO: 19 or 20. These embodiments include antibodies that have no modifications in the CDRs of the heavy chain variable region and the light chain variable region, specifically, antibodies that specifically bind to the G domain of Progranulin, comprising a heavy chain variable region that includes a heavy chain CDR1 that includes the amino acid sequence of SEQ ID NO: 11, a heavy chain CDR2 that includes the amino acid sequence of SEQ ID NO: 12, and a heavy chain CDR3 that includes the amino acid sequence of SEQ ID NO: 13, and a light chain variable region that includes a light chain CDR1 that includes the amino acid sequence of SEQ ID NO: 14, a light chain CDR2 that includes the amino acid sequence of SEQ ID NO: 15, and a light chain CDR3 that includes the amino acid sequence of SEQ ID NO: 16.

In one embodiment, an antibody that specifically binds to the G domain of Progranulin comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 17 or 18 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 19 or 20. In one embodiment, an antibody that specifically binds to the G domain of Progranulin comprises a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 17 or 18 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 19 or 20.

In another embodiment, an antibody that specifically binds to the G domain of Progranulin comprises a heavy chain variable region comprising a heavy chain CDR1 comprising the sequence of SEQ ID NO:21, a heavy chain CDR2 comprising the sequence of SEQ ID NO:22, and a heavy chain CDR3 comprising the sequence of SEQ ID NO:23, and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO:24, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO:25, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:26.

In one embodiment, an antibody that specifically binds to the G domain of Progranulin comprises a heavy chain variable region comprising a sequence having 80% or more, 85% or more, 90% or more, or 95% or more sequence identity with the sequence of SEQ ID NO: 27, and a light chain variable region comprising a sequence having 80% or more, 85% or more, 90% or more, or 95% or more sequence identity with the sequence of SEQ ID NO: 28 or 29. In one embodiment, an antibody that specifically binds to the G domain of Progranulin comprises a heavy chain variable region comprising a sequence in which 0, 1, 2, 3, 4, or 5 amino acids are deleted, substituted, or added in the sequence of SEQ ID NO: 27, and a light chain variable region comprising a sequence in which 0, 1, 2, 3, 4, or 5 amino acids are deleted, substituted, or added in the sequence of SEQ ID NO: 28 or 29. These embodiments include antibodies that have no modifications in the CDRs of the heavy chain variable region and the light chain variable region, specifically, antibodies that specifically bind to the G domain of Progranulin, comprising a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO:21, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO:22, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO:23, and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO:24, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO:25, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:26.

In one embodiment, an antibody that specifically binds to the G domain of Progranulin comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 27 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 28 or 29. In one embodiment, an antibody that specifically binds to the G domain of Progranulin comprises a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 27 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 28 or 29.

In one embodiment, an antibody that specifically binds to the E domain of Progranulin comprises a heavy chain variable region comprising a heavy chain CDR1 comprising the sequence of SEQ ID NO: 30, a heavy chain CDR2 comprising the sequence of SEQ ID NO: 31, and a heavy chain CDR3 comprising the sequence of SEQ ID NO: 32, and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 33, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 34, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 35.

In one embodiment, an antibody that specifically binds to the E domain of Progranulin comprises a heavy chain variable region comprising a sequence having 80% or more, 85% or more, 90% or more, or 95% or more sequence identity with the sequence of SEQ ID NO: 36, and a light chain variable region comprising a sequence having 80% or more, 85% or more, 90% or more, or 95% or more sequence identity with the sequence of SEQ ID NO: 37 or 38. In one embodiment, an antibody that specifically binds to the E domain of Progranulin comprises a heavy chain variable region comprising a sequence in which 0, 1, 2, 3, 4, or 5 amino acids are deleted, substituted, or added in the sequence of SEQ ID NO: 36, and a light chain variable region comprising a sequence in which 0, 1, 2, 3, 4, or 5 amino acids are deleted, substituted, or added in the sequence of SEQ ID NO: 37 or 38. These embodiments include antibodies that have no modifications in the CDRs of the heavy chain variable region and the light chain variable region, specifically, antibodies that specifically bind to the E domain of Progranulin, comprising a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 30, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 31, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 32, and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 33, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 34, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 35.

In one embodiment, an antibody that specifically binds to the E domain of Progranulin comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 36 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 37 or 38. In one embodiment, an antibody that specifically binds to the E domain of Progranulin comprises a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 36 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 37 or 38.

In one embodiment, an antibody that specifically binds to the P domain of Progranulin comprises a heavy chain variable region comprising a heavy chain CDR1 comprising the sequence of SEQ ID NO: 1, a heavy chain CDR2 comprising the sequence of SEQ ID NO: 2, and a heavy chain CDR3 comprising the sequence of SEQ ID NO: 3, and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 6; or
An antibody that specifically binds to the G domain of Progranulin comprises a heavy chain variable region comprising a heavy chain CDR1 comprising the sequence of SEQ ID NO: 11, a heavy chain CDR2 comprising the sequence of SEQ ID NO: 12, and a heavy chain CDR3 comprising the sequence of SEQ ID NO: 13, and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 14, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 15, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 16, or an antibody that specifically binds to the G domain of Progranulin comprises a heavy chain variable region comprising a heavy chain CDR1 comprising the sequence of SEQ ID NO: 21, a heavy chain CDR2 comprising the sequence of SEQ ID NO: 22, and a heavy chain CDR3 comprising the sequence of SEQ ID NO: 23, and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 24, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 25, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 26, and An antibody that specifically binds to the E domain of Progranulin comprises a heavy chain variable region comprising a heavy chain CDR1 comprising the sequence of SEQ ID NO: 30, a heavy chain CDR2 comprising the sequence of SEQ ID NO: 31, and a heavy chain CDR3 comprising the sequence of SEQ ID NO: 32, and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 33, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 34, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 35.

"Sequence identity" is determined by comparing two sequences aligned in an optimal state over the entire region of the sequences to be compared. Here, the sequences to be compared may have additions or deletions (e.g., gaps, etc.) in the optimal alignment of the two sequences. Sequence identity can be calculated using programs such as FASTA, BLAST, and CLUSTAL W provided in public databases (e.g., DDBJ (http://www.ddbj.nig.ac.jp)). Alternatively, it can be determined using commercially available sequence analysis software (e.g., Vector NTI ^{(registered trademark)} software, GENETYX ^{(registered trademark)} ver. 12).

The detection method of the present application is not particularly limited as long as an antibody that specifically binds to the P domain or G domain of Progranulin and an antibody that specifically binds to the E domain of Progranulin are used. In certain embodiments, the detection method of the present application may be an ELISA, such as a sandwich ELISA, an EIA (enzyme immunoassay), a CLEIA (chemiluminescent enzyme immunoassay), a RIA (radio immunoassay), an ECLIA (electrochemiluminescence immunoassay), a TRFIA (time-resolved fluorescence immunoassay), a lateral flow immunoassays, a latex immunoturbidimetric assay, an SPR (surface plasmon resonance) assay, or a SPFS (surface plasmon-field enhanced fluorescence spectroscopy), e.g. a sandwich ELISA or a SPFS. The detection method of the present application may be performed using commercially available equipment. For example, when the detection method of the present application is a sandwich ELISA, Simoa (Quanterix) may be used. Also, when the detection method of the present application is an ECLIA, MESO SECTOR (MESO SCALE DIAGNOSTICS, LLC) may be used.

In one embodiment, the method of the present application uses an antibody that specifically binds to the P domain of Progranulin and an antibody that specifically binds to the E domain of Progranulin. When the method of the present application uses an antibody that specifically binds to the P domain of Progranulin and an antibody that specifically binds to the E domain of Progranulin, only full-length Progranulin can be detected. In another embodiment, the method of the present application uses an antibody that specifically binds to the G domain of Progranulin and an antibody that specifically binds to the E domain of Progranulin. When the method of the present application uses an antibody that specifically binds to the G domain of Progranulin and an antibody that specifically binds to the E domain of Progranulin, only full-length Progranulin and Progranulin lacking the P domain can be detected.

In one embodiment, the method of the present application comprises the steps of:
(1) providing a first antibody adsorbed to a solid phase;
(2) contacting progranulin with the first antibody adsorbed to a solid phase;
(3) contacting the Progranulin with a second antibody, wherein the second antibody comprises a label, and (4) detecting the label.
Here, the first antibody and the second antibody are one and the other of an antibody that specifically binds to the P domain or G domain of Progranulin and an antibody that specifically binds to the E domain of Progranulin.

Step (1) may be carried out, for example, as follows: A 96-well microtiter plate is coated with anti-human progranulin antibody for at least about 10 hours, more preferably overnight, at about 4-20°C, more preferably at about 4-8°C. The diluent may be sodium carbonate, pH 8-12, more preferably pH 9-10. After incubation, the plate is washed at least once with a suitable washing solution, specifically a buffer (e.g., PBS, PBS-T, TBS, or TBS-T) containing a surfactant (e.g., Tween 20, Triton X-100). The plate on which the antibody is immobilized is contacted with a blocking solution to suppress unwanted binding. Examples of blocking agents that may be used include bovine serum albumin (BSA), egg albumin, casein, gelatin, and cow's milk protein. The blocking treatment is carried out at about 20-40°C for about 1-4 hours, or at about 4-8°C overnight.

Step (2) may be carried out, for example, as follows: the specimen sample is contacted (incubated) under appropriate conditions (e.g., temperature: 20°C to 40°C, more preferably 25°C to 38°C, time: 5 to 120 minutes, more preferably 30 to 60 minutes) for Progranulin in the sample to bind to the anti-human Progranulin antibody on the plate. The specimen sample may be diluted with a diluent (e.g., buffer solution), and the buffer solution may be appropriately selected from phosphate buffer, Tris-HCl buffer, citrate buffer, phosphate-citrate buffer, carbonate buffer, borate buffer, succinate buffer, acetate buffer, etc. The buffer solution may contain NaCl, a surfactant (e.g., Tween 20, Triton X-100), and/or a preservative (e.g., PROCLIN300, sodium azide) as necessary.

Step (3) may be carried out, for example, as follows: After incubation, the plate is washed one or more times with a washing solution to remove any substances that have not been immobilized on the plate. The labeled anti-human progranulin antibody is contacted under appropriate conditions (e.g., temperature: 20°C to 40°C, more preferably 25°C to 38°C, time: 5 to 120 minutes, more preferably 30 to 60 minutes). The plate is washed one or more times with a washing solution to remove any substances that have not been immobilized on the plate.

Step (4) may be carried out, for example, as follows: The signal from the label is quantified by a method according to the label attached to the anti-human Progranulin antibody. For example, a method according to the label is avidin or streptavidin-peroxidase and 3,3',5,5'-tetramethylbenzidine for an antibody labeled with biotin. Photometry is performed using a microplate reader together with an antigen and/or a filter as necessary. Before, during, or after quantifying the Progranulin immobilized on the plate, a standard curve (calibration curve) may be prepared using a blank and a standard substance (standard solution) depending on the quantification method used.

The above-mentioned antibodies can be used as the antibody that specifically binds to the P domain or G domain of Progranulin and the antibody that specifically binds to the E domain of Progranulin. In one embodiment, one of the first antibody and the second antibody is an antibody that specifically binds to the E domain of Progranulin, comprising a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 30, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 31, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 32, and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 33, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 34, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 35; and the other of the first antibody and the second antibody is an antibody that specifically binds to the P domain of Progranulin, comprising a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 3, and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 6;
an antibody that specifically binds to the G domain of Progranulin comprising a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 11, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 12, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 13, and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 14, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 15, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 16; or an antibody that specifically binds to the G domain of Progranulin comprising a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 21, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 23, and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 24, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 25, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 26.

In this application, the term "solid phase" refers to a plate (e.g., a microwell plate), a tube, beads (e.g., plastic beads and magnetic beads), a support for chromatography (e.g., water-absorbent substrates such as nitrocellulose membranes and Sepharose), a membrane (e.g., nitrocellulose membranes and PVDF membranes), a gel (e.g., polyacrylamide gel), a metal membrane (e.g., gold membrane), and the like, and a plate is used, for example. The adsorption method may be a covalent bond, an ionic bond, a physical adsorption, and the like, and is not particularly limited, but a covalent bond and/or a physical adsorption is preferable because it can obtain sufficient binding strength. Adsorption to the solid phase may be directly bonded to the solid phase, or may be indirectly adsorbed to the solid phase using a substance known per se. In addition, in order to suppress nonspecific adsorption or nonspecific reactions, a phosphate buffer solution such as bovine serum albumin (BSA), egg albumin, casein, gelatin, or cow's milk protein may be brought into contact with the solid phase to block the surface portion of the solid phase that is not coated with the antibody.

In step (3), examples of the "label" include fluorescent substances (FITC, rhodamine, etc.), radioactive substances ( ^125I , ^32P , ^35S , ^14C , ^3H , etc.), enzymes (alkaline phosphatase, peroxidase, glucose oxidase, β-galactosidase, etc.), colored particles (metal colloid particles, colored latex, etc.), biotin, etc.

The present application also provides a method for selecting a patient with a neurodegenerative disease whose causative gene is the granulin gene, comprising the steps of:
(1) detecting Progranulin in a sample derived from a subject by the method of the present application; and (2) evaluating the amount of Progranulin detected in step (1).

In the present application, examples of neurodegenerative diseases include frontotemporal dementia (FLD), frontotemporal lobar degeneration (FTLD), Alzheimer's dementia, Parkinson's disease, amyotrophic lateral sclerosis (ALS), neuronal ceroid lipofuscinosis (NCL), and age-related hippocampal sclerosis (HS-aging), such as frontotemporal dementia (FLD), frontotemporal lobar degeneration (FTLD), Alzheimer's dementia, or Parkinson's disease.

In step (1), progranulin is detected in a sample derived from a subject by the method of the present application. The subject is an animal, typically a mammal (e.g., human, monkey, mouse, rat, guinea pig, hamster, rabbit, cat, dog, sheep, pig, cow, horse, goat, etc.), preferably a rodent (e.g., mouse, rat) or a primate (e.g., human, monkey), more preferably a human. The sample is not particularly limited as long as it contains progranulin, but may be plasma, cerebrospinal fluid, whole blood, or serum, for example, plasma or cerebrospinal fluid.

In step (2), the amount of progranulin detected can be evaluated by comparing it with a control. If the amount of progranulin detected is, for example, decreased compared to the control, the subject can be selected as a patient with a neurodegenerative disease in which the granulin gene is the causative gene. The control can be, for example, a healthy subject.

Method for evaluating the effectiveness of treatment for a patient with a neurodegenerative disease The present application also provides a method for evaluating the effectiveness of treatment for a patient with a neurodegenerative disease, comprising detecting progranulin in a sample from a patient with a neurodegenerative disease before and after treatment using the method of the present application.

Patients with neurodegenerative diseases include animals, typically mammals (e.g., humans, monkeys, mice, rats, guinea pigs, hamsters, rabbits, cats, dogs, sheep, pigs, cows, horses, goats, etc.), preferably rodents (e.g., mice, rats) or primates (e.g., humans, monkeys), more preferably humans. Samples are not particularly limited as long as they contain progranulin, but include plasma, cerebrospinal fluid, whole blood, and serum, for example, plasma or cerebrospinal fluid.

The effectiveness of a treatment for a patient with a neurodegenerative disease can be evaluated by comparing the amount of progranulin detected in a sample from a patient with a neurodegenerative disease before and after treatment. For example, if the amount of progranulin detected in a sample from a patient with a neurodegenerative disease after treatment is increased compared to the amount of progranulin detected in a sample from a patient with a neurodegenerative disease before treatment, the treatment can be evaluated as effective.

The present application also provides a method for screening a substance for treating a neurodegenerative disease , comprising the steps of:
(1) contacting progranulin, a progranulin-degrading substance, and a candidate substance;
(2) detecting progranulin by the method of the present application; and (3) evaluating the effectiveness of a candidate substance against a neurodegenerative disease.

The progranulin-degrading substance is not particularly limited as long as it can degrade progranulin into granulin, and examples include proteases such as neutrophil elastase (NE), matrix metalloproteinase 9 (MMP9), MMP12, MMP14, proteinase 3 (PR3 and PRTN3), and ADAMTS7 (a disintegrin and metalloproteinase with thrombospondin 7).

The "candidate substance" may be any substance that can be a candidate for treating a neurodegenerative disease, and there is no particular limit to the type. Candidate substances include all substances, such as proteins, amino acids, nucleic acids, lipids, carbohydrates, low molecular weight compounds, and inorganic compounds. Candidate substances that are typically purified or isolated can be used, but they may also be unpurified or unisolated crude products. Candidate substances may be provided in the form of a compound library, a nucleic acid library, a random peptide library, or the like, or may be provided as natural products.

In step (3), the effectiveness of the candidate substance can be evaluated by comparing the amount of progranulin with a control. For example, if the amount of progranulin detected in step (2) is increased compared to the control, the candidate substance can be selected as a substance effective in treating a neurodegenerative disease. The control can be, for example, the amount of progranulin detected when steps (1) and (2) are performed under the same conditions except for the absence of the candidate substance.

Kits The present application also provides a kit for detecting Progranulin, comprising an antibody that specifically binds to the P domain or G domain of Progranulin, and an antibody that specifically binds to the E domain of Progranulin.

Examples of antibodies used in this embodiment are as described above. In one embodiment, an antibody that specifically binds to the P domain of Progranulin comprises a heavy chain variable region comprising a heavy chain CDR1 comprising the sequence of SEQ ID NO: 1, a heavy chain CDR2 comprising the sequence of SEQ ID NO: 2, and a heavy chain CDR3 comprising the sequence of SEQ ID NO: 3, and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 6, or
An antibody that specifically binds to the G domain of Progranulin comprises a heavy chain variable region comprising a heavy chain CDR1 comprising the sequence of SEQ ID NO: 11, a heavy chain CDR2 comprising the sequence of SEQ ID NO: 12, and a heavy chain CDR3 comprising the sequence of SEQ ID NO: 13, and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 14, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 15, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 16, or an antibody that specifically binds to the G domain of Progranulin comprises a heavy chain variable region comprising a heavy chain CDR1 comprising the sequence of SEQ ID NO: 21, a heavy chain CDR2 comprising the sequence of SEQ ID NO: 22, and a heavy chain CDR3 comprising the sequence of SEQ ID NO: 23, and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 24, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 25, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 26, and An antibody that specifically binds to the E domain of Progranulin comprises a heavy chain variable region comprising a heavy chain CDR1 comprising the sequence of SEQ ID NO: 30, a heavy chain CDR2 comprising the sequence of SEQ ID NO: 31, and a heavy chain CDR3 comprising the sequence of SEQ ID NO: 32, and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 33, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 34, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 35.

In one embodiment, the kit of the present application includes an antibody that specifically binds to the P domain of progranulin and an antibody that specifically binds to the E domain of progranulin. When the kit of the present application includes an antibody that specifically binds to the P domain of progranulin and an antibody that specifically binds to the E domain of progranulin, only full-length progranulin can be detected by the kit of the present application. In another embodiment, the kit of the present application includes an antibody that specifically binds to the G domain of progranulin and an antibody that specifically binds to the E domain of progranulin. When the kit of the present application includes an antibody that specifically binds to the G domain of progranulin and an antibody that specifically binds to the E domain of progranulin, only full-length progranulin and progranulin lacking the P domain can be detected by the kit of the present application.

In one embodiment, one or both of the antibodies included in the kit may be fluorescently labeled or magnetically labeled. In addition, one of the antibodies included in the kit may be adsorbed to a solid phase. The contents of such a kit are not particularly limited, but may include reagents and devices used to detect progranulin. For example, the kit may include a secondary antibody for detecting the antibody, a magnet if the antibody is magnetically labeled, etc. The kit may also include buffer solutions, reaction vessels, instructions, etc.

Antibodies and Antibody Combinations The present application also provides antibodies that specifically bind to the P domain, G domain, or E domain of Progranulin. Examples of each antibody are as described above.

The present application also provides a combination of an antibody that specifically binds to the P domain or G domain of Progranulin, and an antibody that specifically binds to the E domain of Progranulin. Examples of each antibody are as described above. In one embodiment, the antibody combination of the present application is a combination of an antibody that specifically binds to the P domain of Progranulin, and an antibody that specifically binds to the E domain of Progranulin. In another embodiment, the antibody combination of the present application is a combination of an antibody that specifically binds to the G domain of Progranulin, and an antibody that specifically binds to the E domain of Progranulin.

The following provides further details with examples, but the present invention is not limited to these examples.

Preparation of degraded PGRN Based on the full-length PGRN shown in Figure 1, degraded PGRN (Figure 2a) with domain deletion was prepared. Specifically, vectors encoding each degraded progranulin with 6X histidine tag attached to the C-terminus were introduced into HEK 293 cells by lipofection. After culturing for about 2 weeks, the supernatant was collected by centrifugation, and the target protein was adsorbed onto the column by passing it through HisPur (trademark) Ni-NTA Resin (Thermo Fisher SCIENTIFIC) equilibrated with 250 mM imidazole solution. The column was washed with a sufficient amount of 20 mM imidazole solution, and the target protein was purified by eluting with 250 mM imidazole solution. Each purified degraded PGRN was confirmed by CBB staining (Figure 2b). It was confirmed that the molecular weight of each domain-deleted PGRN decreased according to the deletion of the domain.

Epitope mapping of each PGRN antibody The method for producing anti-human progranulin antibodies is described below. Human progranulin was bound to RIBI adjuvant (Sigma-Aldrich) and administered to mice multiple times. Spleen cells (B cells) of immunized mice were fused with myeloma cells by cell fusion to produce immortalized cells (hybridomas), and hybridomas producing antibodies capable of binding to human progranulin were selected. The obtained hybridomas were cultured, and the supernatant was adsorbed to rProteinA sepharose Fast Flow (cytiva), followed by elution with sodium citrate buffer to purify anti-human progranulin antibodies.

Using each purified cleaved form of PGRN, we evaluated which domain of PGRN the PGRN antibodies #10B2, #2B3, #8G5, and #4A7 recognize. ELISA was performed for each cleaved form of PGRN using PGRN antibodies #10B2, #2B3, #8G5, and #4A7. If the PGRN antibody has an epitope region in the domain missing in cleaved form of PGRN, the antibody reaction to cleaved form of PGRN disappears.

Specifically, each degraded form of progranulin was diluted to 1 μg/mL with sodium carbonate solution adjusted to pH 9.6, and 100 μL was added to a 96-well microtiter plate and incubated at 4°C for 18 hours for immobilization. After that, the plate was washed once with a washing solution (D-PBS(-) containing 0.05% Tween 20), and 300 μL of 10 mM ethanolamine was added to inactivate the reactive groups, followed by incubation at 4°C for 18 hours. The ethanolamine solution was removed from the plate, which was then dried in a dry room and stored at 4°C until use.

The antibody for epitope mapping was diluted to 1 μg/mL with sample diluent, and 100 μL of each was added to each well and incubated at 25°C for 1 hour. Next, each well was washed three times with 350 μL of washing solution, and 100 μL of 5000-fold diluted HRP-labeled anti-mouse IgG antibody (Thermo Fisher SCIENTIFIC) was added to each well and incubated at 25°C for 1 hour. Finally, each well was washed three times with washing solution, and 100 μL of 3,3',5,5'-tetramethylbenzidine (TMB) color development solution was added and allowed to develop for 15 minutes at room temperature. After color development, 100 μL of 1N diluted sulfuric acid was added to the wells to stop the reaction, and the absorbance at 450 nm (reference wavelength 650 nm) (Abs (450-650 nm)) was measured using a microplate reader Vmax (Molecular Devices).

PGRN antibody #10B2 was confirmed to be reactive to full-length PGRN and PGRN lacking the E domain, but its reactivity to PGRN lacking the P domain was lost (Figure 3a). Therefore, PGRN antibody #10B2 was shown to be an antibody that recognizes the P domain.

PGRN antibodies #2B3 and #4A7 were confirmed to be reactive to full-length PGRN and PGRN lacking the P domain, E domain, or P and E domains, but were not reactive to PGRN lacking the G domain (Fig. 3b: 2B3, Fig. 3c: 4A7). Thus, PGRN antibodies #2B3 and #4A7 were shown to be antibodies that recognize the G domain.

PGRN antibody #8G5 was confirmed to be reactive to full-length PGRN and PGRN lacking the P domain or the P and G domains, but was not reactive to PGRN lacking the E domain (Fig. 3d). Therefore, PGRN antibody #8G5 was shown to be an antibody that recognizes the E domain.

These results indicate that PGRN antibody #10B2 recognizes the P domain, #2B3 and #4A7 recognize the G domain, and #8G5 recognizes the E domain (Fig. 3e). Therefore, we investigated combinations of antibodies that can specifically detect full-length PGRN and PGRN lacking the P domain. The combinations of capture and detection antibodies in assays No. 1, 2, and 3, as well as the target PGRNs, are shown in Table 1.

Measurement by ELISA Measurement by ELISA was performed as follows, using the reaction steps shown in Figure 4. PGRN antibody #8G5 was diluted to 5 μg/mL with 1X PBS, added to a 96-well microtiter plate at 100 μL, and incubated at 4°C for 18 hours for immobilization. After washing once with washing solution (D-PBS(-) containing 0.05% Tween20), 300 μL of blocking solution (D-PBS(-) containing 1% bovine serum albumin (BSA) and 5% D-sorbitol) was added, and incubated at 4°C for 18 hours. The blocking solution was removed from the plate, which was then dried in a dry room and stored at 4°C until use.

Human progranulin recombinant protein prepared with sample dilution solution was added to each well at 100 μL each and incubated at 25°C for 1 hour. Next, each well was washed three times with 350 μL of washing solution, and then 100 μL of biotin-labeled anti-human progranulin antibody was added to each well and incubated at 25°C for 1 hour. Next, each well was washed three times with 350 μL of washing solution, and then 100 μL of 20,000-fold diluted HRP-labeled streptavidin (Thermo Fisher SCIENTIFIC) was added to each well and incubated at 25°C for 1 hour. Finally, each well was washed three times with washing solution, and then 100 μL of 3,3',5,5'-tetramethylbenzidine (TMB) color development solution was added and the color was developed at room temperature for 15 minutes. After color development, 100 μL of 1N diluted sulfuric acid was added to the wells to stop the reaction, and the absorbance at 450 nm (reference wavelength 650 nm) (Abs (450-650 nm)) was measured using a microplate reader Vmax (Molecular Devices).

The ELISA standard curves for each assay are shown in Figure 5 (Figure 5a: ELISA assay No. 1 (#10B2 x #8G5), Figure 5b: ELISA assay No. 2 (#2B3 x #8G5), Figure 5c: ELISA assay No. 3 (#4A7 x #8G5)). It was demonstrated that each assay was capable of detecting human progranulin recombinant protein.

Measurement by Surface Plasmon Excitation Enhanced Fluorescence Spectroscopy Measurement by surface plasmon excitation enhanced fluorescence spectroscopy (SPFS) was carried out as follows.
(Preparation of sensor chip)
After plasma cleaning of a plastic transparent support, a gold thin film was formed on its surface by sputtering. The gold thin film had a thickness of 42-47 nm. The transparent support with the metal thin film obtained by the above process was reacted with an ethanol solution of 11-amino-1-undecanethiol adjusted to 1 mM to form a SAM on the surface of the gold thin film. Next, the transparent support with the SAM obtained by the above process was reacted with 1 mg/mL of carboxymethyl dextran (CMD) with a molecular weight of about 500,000 to adsorb and fix CMD to the surface of the SAM. Next, the transparent support with the CMD solid-phase layer obtained by the above process was reacted with MES buffered saline containing 50 mM N-hydroxysuccinimide (NHS) and 100 mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) as a water-soluble carbodiimide (WSC) to activate the carboxy groups in the CMD solid-phase layer. The monoclonal antibody was then immobilized on the CMD by reacting it with an anti-PGRN monoclonal antibody solution (30 μg/mL) for 20 minutes. In addition, a stabilizer containing 1% by mass of bovine serum albumin (BSA) was reacted to prevent nonspecific adsorption. The transparent plastic support with the anti-PGRN monoclonal antibody immobilized thereon was joined to a microchannel forming member to prepare a sensor chip.

(Preparation of fluorescently labeled antibodies)
Anti-PGRN antibodies #10B2, #2B3, and #4A7 were fluorescently labeled using Alexa Fluor (trademark) 647 Protein Labeling Kit (Invitrogen). The procedure followed the protocol attached to the kit. In order to remove unreacted antibodies and unreacted fluorescent dyes, the solution was purified using an ultrafiltration membrane (Nihon Millipore) to obtain an Alexa Fluor 647-labeled anti-human progranulin antibody solution. The obtained fluorescently labeled anti-human progranulin antibody solution was stored at 4°C after protein quantification.

(SPFS Fluorescence Immunoassay)
First washing step: A washing solution was sent to the sensor chip flow path to wash the surface of the flow path before the sample reaction. Specifically, a buffer solution containing 0.5% Tween 20 was used as the washing solution.
First reaction step: A sample diluted with a specimen diluent was sent to a sensor chip flow path on which an anti-human progranulin antibody immobilized area was formed, and a reaction was carried out.
Second washing step: A washing solution was sent to the flow path of the sensor chip to remove unreacted sample from the flow path.
Blank measurement step: With the flow path filled with sample dilution solution, the sensor chip was irradiated with laser light with a wavelength of 635 nm. The intensity of the fluorescence was measured with a photodiode installed above the measurement area. This measurement value was used as the base value before the reaction with the fluorescently labeled antibody.
Second reaction step: A fluorescently labeled anti-human progranulin antibody solution was circulated through the sensor chip flow path, and a reaction was carried out.
Third washing step: A sample dilution solution was sent to the flow channel of the sensor chip to remove unreacted fluorescently labeled antibody solution from the flow channel.
Signal measurement step: With the flow path filled with the sample dilution solution, the sensor chip was irradiated with laser light having a wavelength of 635 nm. The intensity of the amount of fluorescence was measured with a photodiode installed above the measurement area. The difference between the measured amount of fluorescence and the base value measured in the blank measurement step was calculated.
The reaction times in the first and second reaction steps were adjusted appropriately depending on the target sample.

The standard curves for SPFS from each assay are shown in Figure 6 (Figure 6a: SPFS assay No. 1 (#10B2 x #8G5), Figure 6b: SPFS assay No. 2 (#2B3 x #8G5), Figure 6c: SPFS assay No. 3 (#4A7 x #8G5)).

Confirmation of Specificity for Full-Length PGRN by Commercially Available PGRN ELISA Kits Full-length PGRN and each of the cleaved forms of PGRN were detected using commercially available PGRN ELISA kits (Adipogen and Biovendor).

　The reactivity of Adipogen's Progranulin ELISA kit to degraded human progranulin recombinant protein was evaluated according to the protocol attached to the kit, as follows. Degraded human progranulin was diluted using the attached dilution buffer, and 100μL was added to each well and incubated at 37℃ for 1 hour. Next, after washing three times with 300μL of the attached washing buffer, 100μL of the detection antibody diluted 1000-fold was added to each well and incubated at 37℃ for 1 hour. After washing three times with 300μL of washing buffer, 100μL of the attached STREP-HRP diluted 200-fold was added to each well and incubated at 37℃ for 1 hour. Finally, after washing five times with 300μL of washing buffer, 100μL of TMB solution was added and the color was developed at room temperature for 10 minutes. After color development, 100 μL of Stopping Solution was added to the wells to stop the reaction, and the absorbance at 450 nm (reference wavelength 650 nm) (Abs (450-650 nm)) was measured using a microplate reader.

For Biovendor's Progranulin ELISA kit, the reactivity to degraded human Progranulin recombinant protein was evaluated according to the protocol provided with the kit, as follows: 50 μL of antibody conjugate was added to each well, followed by 50 μL of sample diluted in dilution buffer and incubated at 25°C for 1 hour. Next, the wells were washed five times with 300 μL of the provided washing buffer, after which 100 μL of enzyme conjugate EK was added to each well and incubated at 25°C for 30 minutes. After that, the wells were washed five times with 300 μL of the provided washing buffer, and 100 μL of the provided substrate solution S was added to each well and incubated at 25°C for 30 minutes. Finally, 100 μL of the included Stopping Solution SL was added to stop the reaction, and the absorbance at 450 nm (reference wavelength 650 nm) (Abs (450-650 nm)) was measured using a microplate reader.

The commercially available kits were confirmed to be reactive to full-length PGRN and all degraded forms of PGRN (Figure 7a: Adipogen, Figure 7b: Biovendor). Therefore, it was demonstrated that the two commercially available kits do not have specificity for full-length PGRN.

Confirmation of full-length PGRN specificity by each assay Full-length PGRN and each degraded form of PGRN were detected using ELISA assay No. 1, 2, and 3, and SPFS assay No. 1, 2, and 3. Specifically, for ELISA, full-length progranulin or degraded progranulin was adjusted to 4 ng/mL using 1XPBS containing 0.25% by mass of Tween (registered trademark) 20. Then, the specificity to full-length progranulin was evaluated for the three assays by performing measurements according to the above-mentioned "measurement by ELISA". In addition, for SPFS, full-length progranulin and degraded progranulin were adjusted to 1 ng/mL using 1XPBS containing 0.05% by mass of Tween (registered trademark) 20. These samples were measured according to the above-mentioned "measurement by surface plasmon excitation enhanced fluorescence spectroscopy", and the specificity to full-length progranulin was evaluated for the three assays. The reaction time of SPFS was 7.2 minutes for the first reaction step and 2 minutes for the second reaction step.

When assay No. 1 was used, only full-length PGRN was detected (Fig. 8a: ELISA assay No. 1 (#10B2 x #8G5), Fig. 8d: SPFS assay No. 1 (#10B2 x #8G5)). When SPFS assays No. 2 and 3 were used, only full-length PGRN and PGRN lacking the P domain were detected (Fig. 8b: ELISA assay No. 2 (#2B3 x #8G5), Fig. 8c: ELISA assay No. 1 (#4A7 x #8G5), Fig. 8e: SPFS assay No. 2 (#2B3 x #8G5), Fig. 8f: SPFS assay No. 3 (#4A7 x #8G5)). Therefore, SPFS assays No. 1, 2 and 3 were shown to have high specificity compared to the two commercially available kits.

Measurement of PGRN concentration in plasma of Alzheimer's disease patients and healthy subjects Using SPFS Assay No. 2, the PGRN concentration in plasma of Alzheimer's disease patients or healthy subjects was measured by SPFS. Specifically, the plasma of Alzheimer's disease patients or healthy subjects was diluted 40 times with 1XPBS containing 0.05% by mass of Tween (registered trademark) 20. The PGRN concentration in the sample was measured by performing the measurement according to the above-mentioned "Method of measurement by SPFS". The reaction time was 7.2 minutes for the first reaction step and 2 minutes for the second reaction step.

　Measurements of PGRN concentrations in plasma from 10 healthy individuals and 10 Alzheimer's disease patients showed that PGRN concentrations were significantly higher in the Alzheimer's disease patient group than in the healthy individual group (Figure 9).

Measurement of PGRN concentration in monkey plasma and cerebrospinal fluid In order to measure the monkey PGRN concentration in monkey-derived samples by SPFS, we first performed measurements using monkey PGRN recombinant antigen according to the above-mentioned "SPFS measurement method" to create calibration curves for monkey-derived plasma and cerebrospinal fluid. In this case, taking into account the difference in concentration between each sample, we used different protocols: the first reaction step was 13 minutes and the second reaction step was 2 minutes when measuring monkey plasma, and the first reaction step was 60 minutes and the second reaction step was 1 minute when measuring monkey cerebrospinal fluid, and created calibration curves corresponding to each sample (Figure 10a: calibration curve for monkey plasma, Figure 10c: calibration curve for monkey cerebrospinal fluid). Then, we performed measurements according to the above-mentioned "SPFS measurement method" and calculated the monkey PGRN concentration in the sample by extrapolating the calibration curve (Figures 10b, d). For this measurement, we used SPFS assay No. 2, which was found to be reactive to monkey PGRN recombinant antigen.

Claims

A method for detecting progranulin, characterized by using an antibody that specifically binds to the P domain or G domain of progranulin, and an antibody that specifically binds to the E domain of progranulin.
The method of claim 1, which is an ELISA, EIA, CLEIA, RIA, ECLIA, TRFIA, lateral flow immunoassay, latex immunoturbidimetry, SPR measurement, or SPFS.
The method of claim 2, which is a sandwich ELISA.
(1) providing a first antibody adsorbed to a solid phase;
(2) contacting progranulin with the antibody adsorbed to the solid phase;
(3) contacting the Progranulin with a second antibody, where the second antibody comprises a label; and (4) detecting the label,
The method according to any one of claims 1 to 3, wherein the first antibody and the second antibody are one and the other of an antibody that specifically binds to the P domain or G domain of Progranulin and an antibody that specifically binds to the E domain of Progranulin.
A method for selecting a patient with a neurodegenerative disease caused by the granulin gene, comprising the steps of:
(1) detecting progranulin in a sample derived from a subject by the method according to any one of claims 1 to 4; and (2) evaluating the amount of progranulin detected in step (1).
A method for evaluating the effectiveness of treatment for a patient with a neurodegenerative disease, comprising the step of detecting progranulin in a sample from a patient with a neurodegenerative disease before and after treatment using the method according to any one of claims 1 to 4.
A method for screening for a substance for treating a neurodegenerative disease, comprising the steps of:
(1) contacting progranulin, a progranulin-degrading substance, and a candidate substance;
(2) detecting progranulin by the method according to any one of claims 1 to 4; and (3) evaluating the effectiveness of a candidate substance against a neurodegenerative disease.
An antibody that specifically binds to the P domain of Progranulin comprises a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 3, and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 6, or
An antibody that specifically binds to the G domain of Progranulin comprises a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 11, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 12, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 13, and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 14, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 15, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 16, or an antibody that specifically binds to the G domain of Progranulin comprises a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 21, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 23, and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 24, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 25, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 26, and The method of any one of claims 1 to 7, wherein the antibody that specifically binds to the E domain of Progranulin comprises a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 30, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 31, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 32, and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 33, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 34, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 35.
The method according to any one of claims 1 to 8, which uses an antibody that specifically binds to the P domain of progranulin and an antibody that specifically binds to the E domain of progranulin.
The method according to any one of claims 1 to 8, wherein an antibody that specifically binds to the G domain of Progranulin and an antibody that specifically binds to the E domain of Progranulin are used.
A combination of an antibody that specifically binds to the P domain or G domain of Progranulin, and an antibody that specifically binds to the E domain of Progranulin.
An antibody that specifically binds to the P domain of Progranulin comprises a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 3, and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 6, or
An antibody that specifically binds to the G domain of Progranulin comprises a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 11, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 12, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 13, and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 14, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 15, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 16, or an antibody that specifically binds to the G domain of Progranulin comprises a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 21, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 23, and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 24, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 25, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 26, and The combination described in claim 11, wherein the antibody that specifically binds to the E domain of Progranulin comprises a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 30, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 31, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 32, and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 33, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 34, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 35.
The combination according to claim 11 or 12, which is a combination of an antibody that specifically binds to the P domain of progranulin and an antibody that specifically binds to the E domain of progranulin.
The combination according to claim 11 or 12, which is a combination of an antibody that specifically binds to the G domain of progranulin and an antibody that specifically binds to the E domain of progranulin.
A kit for detecting progranulin, comprising an antibody that specifically binds to the P domain or G domain of progranulin, and an antibody that specifically binds to the E domain of progranulin.
An antibody that specifically binds to the P domain of Progranulin comprises a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 3, and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 6, or
An antibody that specifically binds to the G domain of Progranulin comprises a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 11, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 12, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 13, and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 14, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 15, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 16, or an antibody that specifically binds to the G domain of Progranulin comprises a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 21, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 23, and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 24, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 25, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 26, and The kit described in claim 15, wherein the antibody that specifically binds to the E domain of Progranulin comprises a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 30, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 31, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 32, and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 33, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 34, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 35.
The kit according to claim 15 or 16, comprising an antibody that specifically binds to the P domain of progranulin and an antibody that specifically binds to the E domain of progranulin.
The kit according to claim 15 or 16, comprising an antibody that specifically binds to the G domain of progranulin and an antibody that specifically binds to the E domain of progranulin.
An antibody that specifically binds to the P domain of Progranulin, comprising a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO:1, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO:2, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO:3, and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO:4, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO:5, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:6.
An antibody that specifically binds to the G domain of Progranulin, comprising a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO:11, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO:12, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO:13, and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO:14, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO:15, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:16.
An antibody that specifically binds to the G domain of Progranulin, comprising a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO:21, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO:22, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO:23, and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO:24, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO:25, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:26.
An antibody that specifically binds to the E domain of Progranulin, comprising a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 30, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 31, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 32, and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 33, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 34, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 35.