JP2023107529A - Pharmaceutical composition for treating or preventing nonalcoholic steatohepatitis - Google Patents

Abstract

To provide a pharmaceutical composition for treating or preventing nonalcoholic steatohepatitis.SOLUTION: The present invention provides a pharmaceutical composition for treating or preventing nonalcoholic steatohepatitis, comprising an anti-prion protein antibody or antibody fragment.SELECTED DRAWING: None

Description

The present invention relates to treatment or prevention of non-alcoholic steatohepatitis.

Prion protein is a protein that is abundantly expressed in the brain, particularly in nerve cells, and accumulation of abnormal prion protein, which has a three-dimensional structure different from that of normal prion protein, is known to cause prion disease. It has been reported that prion proteins are expressed not only in the brain but also in the lungs, albeit at low levels. It is reported that there is However, the function of normal prion protein in vivo is still largely unknown.

Non-alcoholic fatty liver disease (NAFLD) is fatty liver not caused by alcohol, and is classified into non-alcoholic fatty liver (NAFL), which rarely progresses, and progressive non-alcoholic steatohepatitis (NASH). be done. NASH is known to progress from liver fibrosis to liver cirrhosis and liver cancer, and early appropriate therapeutic intervention is required.

WO2016/163354

Chida et al., PLoS Pathog 16(8): e1008823 (p1-29), 2020

The present disclosure aims to provide pharmaceutical compositions for treating or preventing NASH.

The present inventors have found that anti-prion protein antibodies improve serological test values in NASH model animals, suppress inflammation, fat accumulation, and fibrosis in liver tissue, and exhibit anti-inflammatory properties in liver tissue. The inventors have found that M2 macrophages are increased and inflammatory M1 macrophages are decreased, and that these effects are observed both by prophylactic and therapeutic administration, thus completing the present invention.

Thus, in one aspect, the present disclosure provides pharmaceutical compositions comprising anti-prion protein antibodies or antibody fragments for treating or preventing non-alcoholic steatohepatitis (NASH).

The present invention enables treatment or prevention of NASH with anti-prion protein antibodies or antibody fragments.

FIG. 1 shows the schedule of experiments to verify the NASH preventive effect of 38-2 antibody. FIG. 2 shows changes in food intake and body weight of NASH model mice administered with 38-2 antibody. FIG. 3 shows suppression of liver injury by prophylactic administration of 38-2 antibody to NASH model mice. Results of serological tests are shown. FIG. 4 shows suppression of hepatic fat accumulation and inflammation by prophylactic administration of 38-2 antibody to NASH model mice. Representative pictures of HE staining are shown. FIG. 5 shows suppression of hepatic fat accumulation by prophylactic administration of 38-2 antibody to NASH model mice. Representative pictures of Oil Red staining are shown. FIG. 6 shows suppression of hepatic fat accumulation by prophylactic administration of 38-2 antibody to NASH model mice. Adipose accumulation assessed by Oil Red staining is shown. FIG. 7 shows suppression of hepatic fat accumulation by prophylactic administration of 38-2 antibody to NASH model mice. The number of lipid droplets evaluated by Oil Red staining is shown. FIG. 8 shows suppression of liver tissue fibrosis by prophylactic administration of 38-2 antibody to NASH model mice. Representative pictures of Sirius Red staining are shown. FIG. 9 shows suppression of liver tissue fibrosis by prophylactic administration of 38-2 antibody to NASH model mice. The extent of liver tissue fibrosis assessed by Sirius Red staining is shown. FIG. 10 shows suppression of inflammatory M1 macrophages and increase of anti-inflammatory M2 macrophages in liver tissue by prophylactic administration of 38-2 antibody to NASH model mice. FIG. 11 shows the schedule of NASH therapeutic effect verification experiment with 38-2 antibody. FIG. 12 shows suppression of liver injury by therapeutic administration of 38-2 antibody to NASH model mice. Results of serological tests are shown. FIG. 13 shows suppression of hepatic fat accumulation by therapeutic administration of 38-2 antibody to NASH model mice. Representative pictures of HE staining are shown. FIG. 14 shows suppression of hepatic fat accumulation by therapeutic administration of 38-2 antibody to NASH model mice. Representative photographs of Oil Red staining and lipid droplet number and fat accumulation assessed by Oil Red staining are shown. FIG. 15 shows suppression of hepatic fibrosis by therapeutic administration of 38-2 antibody to NASH model mice. Representative photographs of Sirius Red staining and the extent of liver tissue fibrosis assessed by Sirius Red staining are shown.

In one aspect, the present disclosure relates to pharmaceutical compositions comprising anti-prion protein antibodies or antibody fragments for treating or preventing non-alcoholic steatohepatitis (NASH).

As used herein, an antibody means an immunoglobulin molecule that has the ability to bind to an antigen. As used herein, antibodies include monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, multispecific antibodies (eg, bispecific antibodies). Antibody species are not particularly limited, and examples thereof include antibodies derived from mice, rats, rabbits, goats, and humans.

As used herein, the term "antibody fragment" refers to a molecule that contains a portion of an immunoglobulin molecule and has antigen-binding properties. Antibody fragments include antibody heavy and light chain variable regions (V _H and V _L ), F(ab′) ₂ , Fab′, Fab, Fv, Fd, disulfide-linked FV (sdFv), Single-Chain FV (scFV) and polymers thereof.

The immunoglobulin class of antibodies and antibody fragments is not particularly limited. The immunoglobulin classes include IgG, IgM, IgA, IgD, and IgE, and their corresponding heavy chains are the gamma, mu, alpha, delta, and epsilon chains, respectively. The immunoglobulin class can be further divided into subclasses (isotypes) such as IgG1, IgG2, IgG3, IgG4, IgA1 or IgA2. The light chains of antibodies can be divided into kappa and lambda chains based on their constant region. In the present disclosure, antibodies and antibody fragments can belong to any immunoglobulin class and subclass and can have either κ and λ chains. In one embodiment, the immunoglobulin class of antibodies and antibody fragments is IgG.

As used herein, an anti-prion protein antibody or antibody fragment means an antibody or antibody fragment that binds to prion protein with sufficient affinity to exert the desired effect. The species of prion protein is not particularly limited, and includes prion proteins of humans, mice, bovine, sheep, and the like.

An anti-prion protein antibody can be obtained by a general method using a prion protein or a peptide consisting of a portion of its amino acid sequence as an immunogen. Although the length of the peptide is not particularly limited, it is 5-30 amino acids, preferably 5-20 amino acids, more preferably 8-15 amino acids. Such immunogens can be produced by known genetic engineering techniques based on the sequence of the gene encoding the prion protein or the amino acid sequence of the prion protein. In one embodiment, the anti-prion protein antibody is an antibody produced using a prion protein as an immunogen. In one embodiment, the anti-prion protein antibody is an antibody produced using a peptide consisting of a portion of the amino acid sequence of the prion protein and including the amino acid sequence of SEQ ID NO:9 as an immunogen.

Polyclonal antibodies can be produced by general methods described in "Antibodies: A Laboratory Manual, Lane, H. D. et al. eds., Cold Spring Harbor Laboratory Press, New York, 1989" and the like. Specifically, it can be produced by immunizing mammals such as rats, mice, rabbits, goats, and horses with a prion protein or a peptide consisting of a portion of its amino acid sequence.

Monoclonal antibodies can be obtained by known methods, such as a method of preparing antibody-producing hybridomas, a method of preparing an expression vector containing an antibody gene using genetic engineering techniques, and expressing it in cells.

Hybridomas secreting 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, bovine serum albumin, etc.) and, if necessary, an immunostimulator (Freund's complete or incomplete adjuvant, etc.). and immunize non-human mammals such as rats, mice, rabbits, goats and horses. Immunized animals are immunized multiple times at intervals of 3-10 days. The immunogen is generally administered at 1-100 μg. Next, immunocompetent cells (cells having antibody-producing ability in the immunized animal) are collected from the immunized animal that has undergone multiple immunizations, and myeloma cells that have no autoantibody-producing ability (e.g., mice, rats, guinea pigs, hamsters, rabbits, or cells derived from mammals such as humans). A polyethylene glycol method, an electrofusion method, or the like is used for cell fusion. Furthermore, cells that have successfully fused cells are selected based on the selectable marker possessed by the fused cells, and the reactivity of the antibody produced by the selected cells to the immunogen is confirmed by ELISA, radioimmunoassay, fluorescent antibody method, or the like. A hybridoma that produces the desired monoclonal antibody is obtained. Monoclonal antibodies can be isolated from the culture supernatant of in vitro culture of the resulting hybridomas. It can also be cultured in vivo such as ascites of mice, rats, guinea pigs, hamsters or rabbits, and isolated from the ascites.

Monoclonal antibodies can also be obtained by cloning antibody genes from the obtained hybridomas, inserting them into appropriate expression vectors and expressing them in host cells, as described later (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, transgenic animal production techniques are used to produce transgenic animals (e.g., cows, goats, sheep or pigs) in which the gene for the antibody of interest is integrated into the endogenous gene, and It is also possible to obtain a large amount of monoclonal antibodies derived from the antibody gene from .

The obtained monoclonal antibody is purified by appropriately combining methods well known in the art, such as chromatography using a protein A column, ion exchange chromatography, hydrophobic chromatography, ammonium sulfate salting-out method, gel filtration, affinity chromatography, and the like. can do.

A chimeric antibody is an antibody in which a variable region and a constant region derived from different origins are linked. For example, a chimeric antibody can be composed of a non-human animal-derived antibody variable region and a human antibody-derived constant region. For chimeric antibodies, for example, a polynucleotide encoding a variable region of an antibody derived from a mammal other than human and a polynucleotide encoding a constant region of a human antibody are linked, incorporated into an expression vector, and the expression vector is transferred to a host. It can be obtained by introducing and expressing.

The variable region of an antibody is usually composed of three complementarity determining regions (also referred to as CDRs) flanked by four framework regions (also referred to as FRs). FRs and CDRs are generally in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 for both light and heavy chains. Multiple methods for defining antibody variable regions and CDRs have been reported, for example, the Kabat definition (Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD. 1991), Chothia definition (Chothia et al., J. Mol. Biol., 1987; 196: 901-917), AbM definition (Martin et al., Proc.Natl.Acad.Sci.USA, 1989; 86: 9268- 9272), the definition of Contact (MacCallum et al., J. Mol. Biol. 1996; 262: 732-745), and the definition of IMGT (Lefranc et al., Dev Comp Immunol. 2003; 27(1): 55- 77) and others. The Kabat definitions are used herein unless otherwise stated.

CDRs are regions that substantially determine the binding specificity of an antibody, and their amino acid sequences are highly diverse. On the other hand, amino acid sequences that constitute FRs show high homology even among antibodies with different binding specificities. CDR grafting therefore allows the binding specificity of one antibody to be grafted onto another antibody.

Humanized antibodies are composed of non-human animal-derived antibody CDRs, human antibody-derived FRs, and human antibody-derived constant regions. Humanized antibodies can be obtained by grafting the CDRs of non-human animal-derived antibodies into human antibodies. Overlap extension PCR is exemplified as a method for obtaining humanized antibodies. Specifically, PCR is performed using oligonucleotides as primers having overlapping portions at the ends of the CDRs of non-human animal-derived antibodies (e.g., mouse antibodies) and the FRs of human antibodies, and non-human animal-derived to synthesize a polynucleotide in which the CDRs of the antibody of the above are linked to the FRs of a human antibody. Next, the obtained polynucleotide is ligated to a polynucleotide encoding a constant region of a human antibody, incorporated into an expression vector, and the expression vector introduced into a host for expression, thereby obtaining a humanized antibody. (European Patent Application Publication No. 239400; WO 96/02576).

Human antibodies can be obtained, for example, by sensitizing human lymphocytes with a desired antigen in vitro and then fusing the sensitized lymphocytes with human myeloma cells (Japanese Patent Publication No. 1-59878). Human myeloma cells, which are fusion partners, can be used, for example, U266. Human antibodies can also be obtained by immunizing a transgenic animal having a full repertoire of human antibody genes with a desired antigen (WO 93/12227, WO 92/03918, WO 94/02602, WO 94/25585, WO 96/34096, WO 96/33735) (incorporated herein by reference). Furthermore, a technique for obtaining human antibodies by panning using a human antibody library is also known (International Publication No. 92/01047, International Publication No. 92/20791, International Publication No. 93/06213, International Publication 93/11236, WO 93/19172, WO 95/01438, WO 95/15388). For example, the variable region of a human antibody is expressed on the surface of a phage as a single-chain antibody (scFv) by a phage display method, phages that bind to an antigen are selected, and the genes of the selected phage are analyzed to determine the The DNA sequences encoding the human antibody V regions that bind can be determined. Next, this V region sequence is ligated in-frame with a human antibody C region sequence, inserted into an appropriate expression vector, and this expression vector is introduced into a host for expression, thereby obtaining a human antibody. can.

Multispecific antibodies are antibodies that bind at least two different sites. Multispecific antibodies include bispecific antibodies, trispecific antibodies, and the like. In one embodiment, the multispecific antibody binds a prion protein and one or more other antigens. Multispecific antibodies can be produced, for example, by genetic engineering techniques or by combining two or more antibodies that recognize different antigens.

Antibody fragments can be obtained, for example, by digesting antibodies with proteases such as papain and pepsin. Alternatively, it can be obtained by introducing an expression vector containing a polynucleotide encoding the antibody fragment into a host cell and expressing it (for example, Co, M. S. et al., J. Immunol. (1994) 152, 2968-2976; Better, M. and Horwitz, A. H., Methods Enzymol. (1989) 178, 476-496; Pluckthun, A. and Skerra, A., Methods Enzymol. (1989) 178, 497-515; (1986) 121, 652-663; Rousseaux, J. et al., Methods Enzymol. (1986) 121, 663-669; Bird, R. E. and Walker, B. W., Trends Biotechnol. ).

As described above, antibodies and antibody fragments can be obtained by introducing expression vectors containing polynucleotides encoding them into cells and expressing them. Specifically, an expression vector is constructed such that a sequence encoding an antibody or antibody fragment is expressed under an expression control region such as an enhancer or promoter, and a host cell is transformed with this expression vector to produce an antibody or antibody. Express the fragment.

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

That is, the present disclosure provides a polynucleotide encoding an antibody or antibody fragment of this disclosure, an expression vector comprising said polynucleotide, and a transformed cell comprising a polynucleotide capable of expressing an antibody or antibody fragment of this disclosure.

Whether antibodies and antibody fragments bind to the prion protein can be determined by enzyme immunoassay (EIA) (including ELISA), radioimmunoassay (RIA), chemiluminescence immunoassay (CIA), fluorescence immunoassay ( It can be confirmed by an immunoassay method such as FIA), BIACORE ^{(registered trademark)} surface plasmon resonance assay, or the like. Binding to prion protein can also be confirmed by a competition assay. For example, it can be confirmed by examining whether the antibody or antibody fragment competes with an antibody that has been confirmed to bind to the prion protein (referred to as a reference antibody).

As used herein, an antibody or antibody fragment that competes with a reference antibody means an antibody or antibody fragment that significantly reduces the binding of the reference antibody to prion protein. A competition assay can be performed, for example, as follows. The prion protein is bound to a solid surface and to this solid phase is added the antibody or antibody fragment to be evaluated along with a labeled reference antibody. After reacting for a certain period of time, the solid phase is washed and the signal from the label bound to the solid phase surface is measured. An antibody or antibody fragment that significantly reduces the signal compared to the absence of the antibody or antibody fragment being evaluated can be determined to compete with the reference antibody. In one embodiment, an antibody or antibody fragment of the disclosure reduces binding of a reference antibody to a prion protein by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, Or reduce by 95% or more. In one embodiment, the reference antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:7 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:8.

In one embodiment, the anti-prion protein antibody or antibody fragment is a monoclonal antibody or antibody fragment that recognizes the amino acid sequence QGSPG (SEQ ID NO: 9) as an epitope. This sequence is present in the prion proteins of almost all mammals (eg humans, gorillas, chimpanzees, mice, hamsters, rats, mink, sheep, goats, cows). Such a monoclonal antibody or antibody fragment can be obtained by using a prion protein or a peptide consisting of a part of its amino acid sequence and containing the amino acid sequence of SEQ ID NO:9 as an immunogen. Whether the antibody or antibody fragment recognizes the amino acid sequence of SEQ ID NO: 9 as an epitope is determined, for example, by the antibody or antibody fragment binding to the prion protein with the heavy chain variable region containing the amino acid sequence of SEQ ID NO: 7 and It can be confirmed by examining whether it competes with an anti-prion protein antibody containing a light chain variable region containing the amino acid sequence of SEQ ID NO:8.

In one embodiment, the antibody or antibody fragment is
as CDR1, the sequence of SEQ ID NO: 1, or a sequence in which 1, 2 or 3 amino acids are modified in the sequence of SEQ ID NO: 1;
As CDR2, the sequence of SEQ ID NO: 2, or a sequence with 1, 2 or 3 amino acids modified in the sequence of SEQ ID NO: 2, and as CDR3, the sequence of SEQ ID NO: 3, or 1, 2 in the sequence of SEQ ID NO: 3 or a sequence in which 3 amino acids are modified,
and/or the sequence of SEQ ID NO: 4, or a sequence with 1, 2 or 3 amino acid alterations in the sequence of SEQ ID NO: 4 as CDR1,
As CDR2, the sequence of SEQ ID NO: 5, or a sequence with 1, 2 or 3 amino acids modified in the sequence of SEQ ID NO: 5, and as CDR3, the sequence of SEQ ID NO: 6, or 1, 2 in the sequence of SEQ ID NO: 6 or a sequence in which 3 amino acids are modified,
a light chain variable region comprising
including.

In one embodiment, the antibody or antibody fragment is
as CDR1 the sequence of SEQ ID NO: 1,
as CDR2 the sequence of SEQ ID NO: 2 and as CDR3 the sequence of SEQ ID NO: 3;
and/or the sequence of SEQ ID NO: 4 as CDR1,
as CDR2 the sequence of SEQ ID NO: 5 and as CDR3 the sequence of SEQ ID NO: 6;
a light chain variable region comprising
including.

In one embodiment, the antibody or antibody fragment is
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 amino acid sequence of SEQ ID NO:7;
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 amino acid sequence of SEQ ID NO:8.
The heavy chain variable region of this embodiment includes a heavy chain variable region having the same CDRs 1-3 as the CDRs 1-3 in the amino acid sequence of SEQ ID NO: 7, for example, SEQ ID NO: 1 as CDR1, SEQ ID NO: 2 as CDR2, and SEQ ID NO: 2 as CDR3. a heavy chain variable region comprising the sequence of SEQ ID NO:3. The light chain variable region of this embodiment includes a light chain variable region having the same CDRs 1-3 as the CDRs 1-3 in the amino acid sequence of SEQ ID NO: 8, such as SEQ ID NO: 4 as CDR1, SEQ ID NO: 5 as CDR2, and SEQ ID NO: 5 as CDR3. A light chain variable region comprising the sequence of SEQ ID NO:6 is included.

In one embodiment, the antibody or antibody fragment is
The amino acid sequence of SEQ ID NO: 7, or an amino acid sequence in which 1 to 20, 1 to 15, 1 to 10, 1 to 5, or 1 to 3 amino acids are modified in the amino acid sequence of SEQ ID NO: 7 a heavy chain variable region, and/or
The amino acid sequence of SEQ ID NO: 8, or an amino acid sequence in which 1 to 20, 1 to 15, 1 to 10, 1 to 5, or 1 to 3 amino acids are modified in the amino acid sequence of SEQ ID NO: 8 comprising a light chain variable region.
The heavy chain variable region of this embodiment includes a heavy chain variable region having the same CDRs 1-3 as the CDRs 1-3 in the amino acid sequence of SEQ ID NO: 7, for example, SEQ ID NO: 1 as CDR1, SEQ ID NO: 2 as CDR2, and SEQ ID NO: 2 as CDR3. a heavy chain variable region comprising the sequence of SEQ ID NO:3. The light chain variable region of this embodiment includes a light chain variable region having the same CDRs 1-3 as the CDRs 1-3 in the amino acid sequence of SEQ ID NO: 8, such as SEQ ID NO: 4 as CDR1, SEQ ID NO: 5 as CDR2, and SEQ ID NO: 5 as CDR3. A light chain variable region comprising the sequence of SEQ ID NO:6 is included.

In one embodiment, the antibody or antibody fragment is
a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7, and/or
A light chain variable region comprising the amino acid sequence of SEQ ID NO:8.

In one embodiment, the antibody or antibody fragment is
a heavy chain variable region having CDRs 1-3 identical to those in the amino acid sequence of SEQ ID NO:7, and/or a light chain variable region having CDRs 1-3 identical to those in the amino acid sequence of SEQ ID NO:8.
In this embodiment, the numbering system for determining CDRs is not particularly limited, but may be Kabat, Chothia, AbM, or IMGT, for example.

In one embodiment, the antibody or antibody fragment is an anti-prion protein antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:7 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:8 in binding to the prion protein. Competing antibodies or antibody fragments.

In one embodiment, the antibody or antibody fragment is an antibody or antibody fragment capable of binding to a prion protein expressed on the cell surface and activating a Src family kinase. In a further embodiment, the antibody or antibody fragment is an antibody or antibody fragment capable of binding to prion protein expressed on the surface of macrophages and activating a Src family kinase. In a further embodiment, the antibody or antibody fragment is an antibody or antibody fragment capable of binding to prion proteins expressed on the surface of macrophages, activating Src family kinases and inducing polarization into anti-inflammatory M2 macrophages. . The ability to activate Src family kinases and the ability to induce polarization to anti-inflammatory M2 macrophages can be confirmed by known methods, for example, Chida et al. PLoS Pathogen 2020: 16 ( 8): Can be confirmed according to the description of e1008823.

As used herein, amino acid alteration includes amino acid deletion, substitution, insertion and addition. Modifications may be deletions, substitutions, insertions, additions, or combinations of two or more of these. Amino acid modification can be performed by known methods such as site-directed mutagenesis. Thus, techniques for modifying the CDR or variable region sequences to obtain antibodies with maintained or improved affinity for antigens are well known in the art.

Sequence identity refers to the percentage of identical amino acid residues between two sequences that are optimally aligned (maximum match) over the entire region of the sequences being compared. There may be additions or deletions (eg gaps) in the optimal alignment of two sequences. Sequence identity can be calculated using programs such as FASTA, BLAST, and CLUSTAL W provided in public databases (eg, DDBJ (http://www.ddbj.nig.ac.jp)). Alternatively, it can be determined using commercially available sequence analysis software (eg, Vector NTI (registered trademark) software, GENETYX (registered trademark) ver. 12).

As used herein, an amino acid sequence "comprising" a given amino acid sequence includes an amino acid sequence in which one or more amino acid residues are added to the given amino acid sequence, and a sequence consisting of the given amino acid sequence. be.

The antibody or antibody fragment may have a modified constant region for regulation of antibody-dependent cellular cytotoxicity (ADCC) activity, complement-dependent cellular cytotoxicity (CDC) activity, pharmacokinetics, and the like. Modifications include, for example, amino acid sequence or sugar chain modifications and polymer conjugation. The antibody or antibody fragment may also be drug conjugated. Drugs can be small compounds, peptides, nucleic acids, and the like.

In examples, anti-prion protein antibodies improve serological test values in NASH model animals, suppress inflammation, fat accumulation, and fibrosis in liver tissue, and suppress anti-inflammatory M2 macrophages in liver tissue. and decreased inflammatory M1 macrophages, and these effects were observed with both prophylactic and therapeutic administration. Accordingly, the antibodies or antibody fragments of the disclosure can be used to treat or prevent NASH.

Nonalcoholic steatohepatitis (NASH) is a form of nonalcoholic fatty liver disease (NAFLD). NAFLD is generally defined as imaging or histological evidence of fatty accumulation (>5% of hepatocytes) in the liver, ruling out fatty liver secondary to alcohol, drugs, and genetic disease. NAFLD is classified histologically into non-alcoholic fatty liver (NAFL) and NASH. In general, NAFL is defined as having fat accumulation in 5% or more of the hepatocytes and no hepatocellular injury (balloon-like degeneration of hepatocytes), and NASH is histologically defined as fat in 5% or more of the Accumulation, defined as hepatocellular injury (balloon-like degeneration of hepatocytes) and inflammation. NAFL and NASH are known to reciprocate. The pathology of NAFL hardly progresses, while NASH is progressive, leading to liver fibrosis and liver cirrhosis and liver cancer. NASH findings include steatosis (also called fat accumulation), inflammation, inflammatory cell infiltration, hepatocellular injury (balloon-like degeneration of hepatocytes), and fibrosis.

Treatment of NASH includes inhibition of progression, remission, and cure of the disease, as well as inhibition of exacerbation, amelioration, and elimination of one or more features thereof. Prevention of NASH includes suppression of onset.

An anti-prion protein antibody or antibody fragment, or a pharmaceutical composition containing the same is administered to a subject in an amount capable of exhibiting a desired effect (herein referred to as an effective amount). In the present disclosure, subjects are mammals (eg, humans, mice, rats, hamsters, rabbits, cats, dogs, cows, sheep, monkeys). In one embodiment, the subject is human.

The subject can be a subject with NASH (eg, a NASH patient) or a subject at risk of developing NASH. Subjects at risk of developing NASH include, for example, subjects with NAFL (eg, NAFL patients) and subjects with one or more risk factors for NASH. Risk factors for NASH include obesity, insulin resistance, metabolic syndrome, and gene mutations that contribute to the onset and progression of NASH.

The dosage is appropriately selected depending on the age, body weight, health condition, etc. of the subject of administration. An anti-prion protein antibody or antibody fragment, or a pharmaceutical composition comprising the same, for example, the amount of the antibody or antibody fragment is 1 μg/kg to 100 mg/kg, 10 μg/kg to 10 mg/kg, or 100 μg/kg per adult per day. ~1 mg/kg may be administered, which may be administered in a single dose or divided into multiple doses (eg, 2, 3, or 4 doses). Dosing frequency can be a single dose, daily administration, or several days (e.g., 2, 3, or 4 days), one week, several weeks (e.g., 2, 3, or 4 weeks), one month. Or it may be administered once every few months (eg, every 2, 3, or 4 months).

Anti-prion protein antibodies or antibody fragments, or pharmaceutical compositions comprising same, may be administered systemically or locally. An anti-prion protein antibody or antibody fragment, or a pharmaceutical composition comprising the same, can be administered, for example, by intravenous, intramuscular, subcutaneous, transdermal, nasal, pulmonary, or oral administration. In one embodiment, the anti-prion protein antibody or antibody fragment, or pharmaceutical composition comprising same, is administered intravenously.

A pharmaceutical composition can be formulated by a conventional method. In addition to the anti-prion protein antibody or antibody fragment, the pharmaceutical composition contains sterile water, physiological saline, stabilizers, excipients, antioxidants, buffers, preservatives, surfactants, chelating agents, binders, and the like. can include Dosage forms include, for example, injections, transdermal formulations, inhalants, nasal drops, and oral formulations.

In one aspect, the present disclosure provides a method for treating or preventing non-alcoholic steatohepatitis (NASH) comprising administering an anti-prion protein antibody or antibody fragment to a subject in need thereof. offer.
In one aspect, the disclosure provides an anti-prion protein antibody or antibody fragment for the manufacture of a medicament for treating or preventing non-alcoholic steatohepatitis (NASH).
In one aspect, the present disclosure provides the use of anti-prion protein antibodies or antibody fragments for treating or preventing non-alcoholic steatohepatitis (NASH).

Illustrative embodiments of the disclosure are described below.

1. A pharmaceutical composition comprising an anti-prion protein antibody or antibody fragment for treating or preventing non-alcoholic steatohepatitis (NASH).

2. an anti-prion protein antibody or antibody fragment,
as CDR1, the sequence of SEQ ID NO: 1, or a sequence in which 1, 2 or 3 amino acids are modified in the sequence of SEQ ID NO: 1;
As CDR2, the sequence of SEQ ID NO: 2, or a sequence with 1, 2 or 3 amino acids modified in the sequence of SEQ ID NO: 2, and as CDR3, the sequence of SEQ ID NO: 3, or 1, 2 in the sequence of SEQ ID NO: 3 or a sequence in which 3 amino acids are modified,
a heavy chain variable region comprising
as CDR1, the sequence of SEQ ID NO: 4, or a sequence in which 1, 2 or 3 amino acids are modified in the sequence of SEQ ID NO: 4;
As CDR2, the sequence of SEQ ID NO: 5, or a sequence with 1, 2 or 3 amino acids modified in the sequence of SEQ ID NO: 5, and as CDR3, the sequence of SEQ ID NO: 6, or 1, 2 in the sequence of SEQ ID NO: 6 or a sequence in which 3 amino acids are modified,
a light chain variable region comprising
2. The pharmaceutical composition according to 1 above, comprising:

3. an anti-prion protein antibody or antibody fragment,
as CDR1 the sequence of SEQ ID NO: 1,
as CDR2 the sequence of SEQ ID NO: 2 and as CDR3 the sequence of SEQ ID NO: 3;
a heavy chain variable region comprising, and as CDR1, the sequence of SEQ ID NO:4;
as CDR2 the sequence of SEQ ID NO: 5 and as CDR3 the sequence of SEQ ID NO: 6;
a light chain variable region comprising
3. The pharmaceutical composition according to 1 or 2 above, comprising:

4. an anti-prion protein antibody or antibody fragment,
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 amino acid sequence of SEQ ID NO:7; and
4. The light chain variable region according to any one of 1 to 3 above, comprising a sequence having 80% or more, 85% or more, 90% or more, or 95% or more sequence identity with the amino acid sequence of SEQ ID NO:8. pharmaceutical composition.

5. an anti-prion protein antibody or antibody fragment,
The amino acid sequence of SEQ ID NO: 7, or an amino acid sequence in which 1 to 20, 1 to 15, 1 to 10, 1 to 5, or 1 to 3 amino acids are modified in the amino acid sequence of SEQ ID NO: 7 a heavy chain variable region, and
The amino acid sequence of SEQ ID NO: 8, or an amino acid sequence in which 1 to 20, 1 to 15, 1 to 10, 1 to 5, or 1 to 3 amino acids are modified in the amino acid sequence of SEQ ID NO: 8 5. The pharmaceutical composition according to any one of 1 to 4 above, which comprises a light chain variable region.

6. an anti-prion protein antibody or antibody fragment,
a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:7; and a light chain variable region comprising the amino acid sequence of SEQ ID NO:8;
6. The pharmaceutical composition according to any one of 1 to 5 above, comprising

7. an anti-prion protein antibody or antibody fragment,
1 above, comprising a heavy chain variable region having the same CDRs 1 to 3 as the CDRs 1 to 3 in the amino acid sequence of SEQ ID NO: 7, and a light chain variable region having the same CDRs 1 to 3 as the CDRs 1 to 3 in the amino acid sequence of SEQ ID NO: 8. Pharmaceutical composition as described.

8. The anti-prion protein antibody or antibody fragment competes for binding to the prion protein with an anti-prion protein antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:7 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:8. 2. The pharmaceutical composition according to 1 above, which is an antibody or an antibody fragment.

9. 9. The pharmaceutical composition according to any one of 1 to 8 above, wherein the anti-prion protein antibody or antibody fragment is an antibody or antibody fragment capable of binding to prion protein expressed on the cell surface and activating Src family kinase. .

10. A method for treating or preventing non-alcoholic steatohepatitis (NASH) comprising administering an anti-prion protein antibody or antibody fragment to a subject in need thereof.

11. Use of an anti-prion protein antibody or antibody fragment for the manufacture of a medicament for treating or preventing non-alcoholic steatohepatitis (NASH).

12. Use of an anti-prion protein antibody or antibody fragment for treating or preventing non-alcoholic steatohepatitis (NASH).

The invention is further illustrated by the following examples, which are not intended to limit the invention in any way.

In this test, an anti-prion protein antibody (38-2 antibody) disclosed in WO 2016/163354 was used. The 38-2 antibody was obtained by immunizing prion protein-deficient mice with a mouse recombinant prion protein having an amino acid sequence corresponding to amino acids 23 to 231 of mouse prion protein (GenBank accession No. M13685) (SEQ ID NO: 10). It is a monoclonal antibody. The sequences of the heavy and light chain variable regions of the 38-2 antibody are shown below. CDRs 1-3 of the heavy chain variable region (SEQ ID NOs: 1-3) and CDRs 1-3 of the light chain variable region (SEQ ID NOs: 4-6) as determined by Kabat numbering are underlined. WO 2016/163354 shows that the 38-2 antibody recognizes the 41st to 45th amino acid sequence QGSPG (SEQ ID NO: 9) of the mouse prion protein as an epitope.

Heavy chain variable region (SEQ ID NO:7)
MGWLWNLLFLMAAAQSAQAQIQLIQSGPELKKPGETVKISCKASGYTFT TFGMS WVKQALGKGLKWMG WINTYSGVPTYADDFKG RFAFSLEASASTAYLQINNLNNEDTATYFCSR YGSYAMDY WGQGTSVTVSS

CDR1 (SEQ ID NO: 1): TFGMS
CDR2 (SEQ ID NO:2): WINTYSGVPTYADDFKG
CDR3 (SEQ ID NO:3): YGSYAMDY

Light chain variable region (SEQ ID NO: 8)
MRCSLQFLGVLMFWISGVSGDIVITHDELSNPVTSGESVSISC RSSKSLLYKDGKTYLN WFLQRPGQSPQLLIY LMSTRAS GVSDRFSGSGSGTDFTLEISRVKAEDVGVYYC QQLVEFPLT FGAGTKLELKR

CDR1 (SEQ ID NO:4): RSSKSLLYKDGKTYLN
CDR2 (SEQ ID NO:5): LMSTRAS
CDR3 (SEQ ID NO: 6): QQLVEFPLT

In order to investigate whether anti-prion protein antibody has a protective effect against NASH, 38-2 anti-prion protein antibody was intraperitoneally administered to NASH model mice (Fig. 1). Mouse IgG (SIGMA, I5381-10MG) was similarly administered as a control. The NASH model mouse used in this study is a model mouse that causes NASH pathology such as fat deposition, inflammation, and fibrosis in liver tissue by feeding TSNO mice a high-calorie, high-fat diet (International Publication No. 2019/070024). Six-week-old TSNO mice were fed (custom-made diet: 69.5% MF diet containing 28.75% palm oil, 1.25% cholesterol, and 0.5% cholic acid, Oriental Yeast Co., Ltd.) one day before the start The first antibody administration (1 mg/animal) was performed on . Two weeks after starting the diet, a second antibody administration (1 mg/animal) was performed. Serological tests and pathological analyzes were performed three times, 4, 12 and 24 weeks after starting the diet.

Serological tests and pathological analyzes were performed as follows.

Serological Test After attaching a 25-gauge injection needle (TERMO) to a 1.0-mL tuberculin syringe (TERMO), 1.0 mL of blood was collected from the heart of a TSNO mouse. The collected blood was mixed with 5 units of heparin sodium (Ajinomoto Pharmaceuticals Co., Ltd.) and then centrifuged (1,700 G, 15 min, room temperature) to recover 0.4 mL of plasma. The recovered plasma was shipped to Oriental Yeast Co., Ltd. (Nagahama Plant, Nagahama Life Science Laboratory) in a frozen state, and then subjected to contract analysis (liver/biliary disease set). This analysis quantified total protein, albumin, AST, ALT, ALP, LDH, total cholesterol (T-CHO), free cholesterol (F-CHO), and triglycerides (TG) in plasma.

Pathological analysis After removing the liver of the TSNO mouse, the tissue was fixed in a mildform 10N solution (Wako). After embedding a tissue piece cut out from the fixed tissue in paraffin, a 5 μm-thick section cut with a sliding microtome was used for hematoxylin-eosin (HE) staining and immunostaining. For Sirius Red staining, 10 μm-thick slices were used.
HE staining was performed as follows. Deparaffinization (xylene, 3 times, 10 min each) → Dexylene (100% ethanol, 2 times, 2 min each) → Immersion (90% ethanol, 70% ethanol, 2 min each) → Running water (tap water, 2 min) → Washing ( Distilled water, 1 min) → staining (hematoxylin staining solution, 5 min) → coloring (tap water, 10 min) → staining (eosin staining solution, 30 sec) → washing with water (distilled water, 2 sec) → separation (70% ethanol, 30 sec) → Dehydration (100% ethanol, 3 times, 3 minutes each) → Clearing (xylene, 3 times, 3 minutes each) → Encapsulation. HE-stained images were observed with BIOREBO BZ-9000 (Keyence).
Immunostaining was performed as follows. Deparaffinization (xylene, 3 times, 10 min each) → Dexylene (100% ethanol, 2 times, 2 min each) → Water immersion (90% ethanol, 70% ethanol, 2 min each) → Running water (tap water, 2 min) → Antigen retrieval quenching (10 mM sodium citrate, 0.05% Tween-20, pH 6.0, 20 min, 105° C.) → fixation (4% PFA, 10 min) → washing (PBS, 3 times, 5 min each) → permeabilization (0.3 % Triton X-100, 15 min) → washing (PBS, 3 times, 5 min each) → blocking (10% FCS, overnight, 4°C) → primary antibody reaction (anti-CD68 antibody diluted 2,000 times with 10% FCS (abcam, ab125212) and anti-CD163 antibody (abcam, ab182422), overnight, 4° C.)→Washing (PBS, 3 times, 5 min each)→Secondary antibody reaction (Alexa Fluor diluted 2,000-fold with 10% FCS) 488 goat anti-rabbit IgG (Invitrogen, A11008), overnight, 4°C) → washing (PBS, 3 times, 5 min each) → mounting. Fluorescent images were observed with BIOREBO BZ-9000 (Keyence).
Sirius Red staining was performed as follows. Deparaffinization (xylene, 3 times, 10 min each) → dexylene (100% ethanol, 2 times, 2 min each) → immersion (90% ethanol, 70% ethanol, 2 min each) → running water (tap water, 10 min) → staining ( Sirius Red, 60 min)→Washing (Acidified water: solution of 5 ml of glacial acetic acid added to 1,000 ml of water, twice, 5 min each)→Dehydration (100% ethanol, 3 times, 5 min each)→Clearing (xylene, 3 times , 3 min each) → enclosed. Images of collagen fibers were observed with BIOREBO BZ-9000 (Keyence).
The tissue pieces fixed with mildform were immersed in a 10% sucrose solution overnight, 20% sucrose solution overnight, and 30% sucrose solution overnight. Subsequently, excess water was wiped off the tissue pieces, and the pieces were embedded with an embedding agent (OCT compound). Sections cut in a cryostat at a thickness of 10 μm were used for Oil Red staining.
Oil Red staining was performed as follows. Washing (60% isopropyl alcohol aqueous solution, 1 min)→staining (Oil Red O staining solution, 15 min, 37° C.)→washing (60% isopropyl alcohol aqueous solution, 10 sec)→encapsulation. Images of lipid droplets were observed with BIOREBO BZ-9000 (Keyence).

Statistical Analysis All statistical analyzes were performed with Student's t-test (Microsoft EXCEL software, version 16.37). ^** P<0.01, ^* P<0.05.

We investigated whether intraperitoneal antibody administration of 38-2 antibody affected food intake and body weight gain in TSNO mice (Fig. 2). For this purpose, food intake and body weight changes of TSNO mice administered with 38-2 antibody or control IgG were measured up to 11 weeks of age. As a result, there was no significant difference in food intake and body weight change between TSNO mice administered 38-2 antibody and TSNO mice administered control IgG antibody, and 38-2 antibody administration affected food intake and body weight change. found to not give

A serological test was performed to investigate whether the 38-2 antibody can suppress liver injury in NASH model mice (Fig. 3). The high-calorie/high-fat diet increased the serum levels of AST, ALT, ALP, and LDH from 4 weeks after the start of the diet compared to the normal diet. This result indicated that liver injury had already started 4 weeks after the start of the diet. However, those values were significantly lower in mice treated with 38-2 antibody compared to mice treated with control IgG. Similar results were also observed 22 weeks after the second antibody administration. The above results not only show that the 38-2 antibody can suppress liver injury in NASH model mice, but also that the effect can be sustained for at least 22 weeks.

In order to pathologically analyze the effects of the 38-2 antibody, HE staining of liver tissue was performed (Fig. 4). In the control IgG-administered mice, hepatocyte staining was highly transparent 4 weeks after the start of the high-calorie/high-fat diet, and accumulation (empty follicles) and infiltration of inflammatory cells were observed in the liver tissue after 12 weeks. rice field. However, compared to these mice, the 38-2 antibody-administered mice had less transparent staining of hepatocytes even 4 weeks after the start of the high-calorie, high-fat diet, and accumulation in the liver tissue after 12 weeks ( Empty follicles) and infiltration of inflammatory cells were also low. These results indicated that the 38-2 antibody suppressed fat accumulation and inflammatory cell infiltration in liver tissue of NASH model mice.

Oil Red staining was performed to further examine fat accumulation in the liver tissue of each mouse. Oil Red-stained photographs of liver tissue (FIG. 5), fat accumulation in liver tissue (FIG. 6), and the number of lipid droplets (20 μm or more in diameter) (FIG. 7) (n=3 for each group) are shown. Compared with the Oil Red staining of the liver tissue of the mice on the normal diet, the liver tissue of the mice on the high-calorie/high-fat diet was strongly positive for Oil Red staining already 4 weeks after the start of the diet. Compared to control IgG-treated mice, 38-2 antibody-treated mice had significantly reduced Oil Red staining and lipid droplet counts in all weeks examined. These results indicated that the 38-2 antibody suppressed fat accumulation in the liver tissue of NASH model mice, and the effect could last for at least 22 weeks.

Sirius Red staining was performed to examine the fibrosis of the liver tissue of each mouse. FIG. 8 shows Sirius Red-stained photographs of liver tissue (FIG. 8) and the extent of fibrosis in liver tissue (FIG. 9) (n=3 for each group). Sirius Red staining of normal-fed mice showed that only the perivascular fibrous tissue was stained. However, Sirius Red staining was positive in the liver tissue of mice on a high-calorie/high-fat diet. Compared to control IgG-treated mice, 38-2 antibody-treated mice had significantly less Sirius Red staining in all weeks examined. These results indicated that the 38-2 antibody suppressed fibrosis in NASH model mice, and the effect could be sustained for at least 22 weeks.

It was investigated whether the 38-2 antibody induces anti-inflammatory M2 macrophages in liver tissue of NASH model mice (Fig. 10). Inflammatory M1 macrophages were immunostained with CD68 antibody and anti-inflammatory M2 macrophages with CD163 antibody. There was an increase in the number of inflammatory M1 macrophages in the liver tissue of 38-2 antibody-treated mice compared to control antibody-treated mice at 4, 12, and 24 weeks after diet initiation. Suppressed and conversely increased anti-inflammatory M2 macrophages. These results indicated that the 38-2 antibody induced anti-inflammatory M2 macrophages in the liver tissue of NASH model mice and conversely suppressed inflammatory M1 macrophages.

In order to verify the therapeutic effect of the 38-2 antibody on NASH, the 38-2 antibody was intraperitoneally administered 4 weeks after the start of the diet (first dose) (1 mg/animal), and further intraperitoneally administered 2 weeks later ( 2nd time) (1 mg/animal) (Fig. 11). As a control, mouse IgG was similarly administered. Two weeks later, serology and pathological analysis were performed.

A serological test was performed to investigate whether the 38-2 antibody can treat liver damage in NASH model mice (Fig. 12). Serological tests after 4 weeks of treatment showed elevated AST and ALP in mice receiving control IgG compared to before treatment. Based on these results, it was considered that the high-calorie, high-fat diet progressed the liver injury. However, AST, ALT, and LDH were all significantly lower in mice treated with 38-2 antibody than in mice treated with control IgG, and were at levels similar to those prior to initiation of treatment. Among them, ALP was improved compared to before treatment. The above results indicated that the 38-2 antibody had a therapeutic effect on liver injury in NASH model mice.

The inhibitory effect of 38-2 antibody on liver injury in NASH model mice was pathologically analyzed. The liver tissue of each mouse was HE-stained (Fig. 13). In the liver tissue 4 weeks after the start of treatment, a large number of fat accumulations (empty follicles) and infiltration of inflammatory cells were observed in the control IgG-administered mice compared to before treatment. This result indicates that the high-calorie/high-fat diet progressed liver tissue damage. However, fat accumulation (empty follicles) and inflammatory cell infiltration were suppressed in mice administered 38-2 antibody compared to mice administered control IgG. The above results showed that the 38-2 antibody also pathologically suppressed liver tissue damage in NASH model mice.

Oil Red staining was performed to examine fat accumulation in the liver tissue of each mouse (Fig. 14). Even in the liver tissue before treatment, lipid droplets were observed due to the accumulation of fat due to the high-calorie/high-fat diet, and the entire liver tissue was positive for Oil Red staining. However, in the control antibody-administered mice, the number of lipid droplets was increased, and Oil Red staining of the liver tissue was more strongly stained. This result indicates that the high-calorie/high-fat diet promoted fat accumulation. However, the number of lipid droplets and the degree of Oil Red staining of liver tissue were significantly reduced in mice administered 38-2 antibody. These results indicated that the 38-2 antibody suppressed fat accumulation in liver tissue of NASH model mice.

Sirius Red staining was performed to examine the fibrosis of the liver tissue of each mouse (Fig. 15). Sirius Red staining of the liver tissue was strongly stained in the control antibody-administered mice compared with the liver tissue before treatment. This result indicates that the high-calorie/high-fat diet promoted fibrosis of the liver tissue. However, mice receiving the 38-2 antibody had a significantly reduced degree of Sirius Red staining. These results indicated that the 38-2 antibody suppressed fibrosis in liver tissue of NASH model mice.

The 38-2 antibody used in this example, together with different anti-prion protein antibodies (3S9 and 2H9), stimulates prion protein expressed on the cell surface of macrophages, activates Src family kinases, and converts macrophages to anti-inflammatory M2. It has been reported to polarize macrophages (Chida et al. PLoS Pathogen 2020: 16(8):e1008823). 3S9 and 2H9 are monoclonal antibodies that recognize different epitopes than the 38-2 antibody, 3S9 recognizes amino acids 141-161 of mouse prion protein and 2H9 recognizes a non-contiguous epitope contained in amino acids 151-221. As described above, the 38-2 antibody induces anti-inflammatory M2 macrophages in liver tissue, so the effect shown in the present application is not an effect specific to the 38-2 antibody, but an effect common to anti-prion protein antibodies. It was suggested that there is From the above, it was shown that the anti-prion protein antibody is effective in the treatment and prevention of NASH.

Claims (9)

A pharmaceutical composition comprising an anti-prion protein antibody or antibody fragment for treating or preventing non-alcoholic steatohepatitis (NASH). an anti-prion protein antibody or antibody fragment,
as CDR1, the sequence of SEQ ID NO: 1, or a sequence in which 1, 2 or 3 amino acids are modified in the sequence of SEQ ID NO: 1;
As CDR2, the sequence of SEQ ID NO: 2, or a sequence with 1, 2 or 3 amino acids modified in the sequence of SEQ ID NO: 2, and as CDR3, the sequence of SEQ ID NO: 3, or 1, 2 in the sequence of SEQ ID NO: 3 or a sequence in which 3 amino acids are modified,
a heavy chain variable region comprising
as CDR1, the sequence of SEQ ID NO: 4, or a sequence in which 1, 2 or 3 amino acids are modified in the sequence of SEQ ID NO: 4;
As CDR2, the sequence of SEQ ID NO: 5, or a sequence with 1, 2 or 3 amino acids modified in the sequence of SEQ ID NO: 5, and as CDR3, the sequence of SEQ ID NO: 6, or 1, 2 in the sequence of SEQ ID NO: 6 or a sequence in which 3 amino acids are modified,
a light chain variable region comprising
2. The pharmaceutical composition of claim 1, comprising an anti-prion protein antibody or antibody fragment,
as CDR1 the sequence of SEQ ID NO: 1,
as CDR2 the sequence of SEQ ID NO: 2 and as CDR3 the sequence of SEQ ID NO: 3;
a heavy chain variable region comprising, and as CDR1, the sequence of SEQ ID NO:4;
as CDR2 the sequence of SEQ ID NO: 5 and as CDR3 the sequence of SEQ ID NO: 6;
a light chain variable region comprising
3. The pharmaceutical composition of claim 1 or 2, comprising an anti-prion protein antibody or antibody fragment,
a heavy chain variable region comprising a sequence having 80% or more sequence identity with the amino acid sequence of SEQ ID NO:7; and
4. The pharmaceutical composition according to any one of claims 1 to 3, comprising a light chain variable region comprising a sequence having 80% or more sequence identity with the amino acid sequence of SEQ ID NO:8. an anti-prion protein antibody or antibody fragment,
a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence in which 1 to 20 amino acids are altered in the amino acid sequence of SEQ ID NO: 7; and
5. The medicament according to any one of claims 1 to 4, comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 8, or an amino acid sequence in which 1 to 20 amino acids are modified in the amino acid sequence of SEQ ID NO: 8. Composition. an anti-prion protein antibody or antibody fragment,
a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:7; and a light chain variable region comprising the amino acid sequence of SEQ ID NO:8;
The pharmaceutical composition according to any one of claims 1 to 5, comprising an anti-prion protein antibody or antibody fragment,
Claim 1, comprising a heavy chain variable region having the same CDRs 1 to 3 as the CDRs 1 to 3 in the amino acid sequence of SEQ ID NO: 7, and a light chain variable region having the same CDRs 1 to 3 as the CDRs 1 to 3 in the amino acid sequence of SEQ ID NO: 8. The pharmaceutical composition according to . The anti-prion protein antibody or antibody fragment competes for binding to the prion protein with an anti-prion protein antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:7 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:8. 2. The pharmaceutical composition of claim 1, which is an antibody or antibody fragment. The pharmaceutical composition according to any one of claims 1 to 8, wherein the anti-prion protein antibody or antibody fragment is an antibody or antibody fragment capable of binding to prion protein expressed on the cell surface and activating Src family kinase. thing.

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