RU2787044C2 - Antibody against human fn14 - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention presents: an antibody against human Fn14 or its antigen-binding fragment and its use for the production of a pharmaceutical composition for the prevention or treatment of cancerous cachexia, polynucleotides encoding a variable region of heavy and light chains of the antibody against human Fn14, an expression vector, host cells, and methods for the production of an antibody against Fn14. In addition, pharmaceutical compositions for suppression of inflammation by inhibition of activation of human Fn14, a method for the prevention or treatment of cancerous cachexia, as well as pharmaceutical compositions for the prevention or treatment of cancer or cancerous cachexia are presented.

EFFECT: invention allows for the production of an antibody against human Fn14, which binds to human Fn14 for inhibition of its action, thereby preventing or treatment of cachexia of cancer patients.

30 cl, 7 dwg, 3 tbl, 10 ex

Description

FIELD OF TECHNOLOGY

[0001]

The present invention relates to an antibody against human Fn14.

BACKGROUND OF THE INVENTION

[0002]

Cachexia is a complex metabolic syndrome associated with an underlying disease characterized by muscle loss with or without fat loss. A prominent clinical sign of cachexia is weight loss in adults or undergrowth in children (Non-Patent Document 1). Approximately 80% of patients with advanced cancer have cachexia, which is called cancer cachexia. Cachexia worsens a patient's prognosis or quality of life (QOL) due to treatment resistance and is associated with increased mortality. However, there are no effective treatments for cachexia. Although the mechanism of occurrence of cachexia is unclear, in recent years, cachexia has been considered a whole-body inflammatory condition caused by various cytokines (Non-Patent Document 2).

[0003]

Fibroblast growth factor inducible protein 14 (Fn14) (also referred to as TNFRSF12A) is a member of the tumor necrosis factor receptor family. Fn14 is also known as the Tweak receptor, which binds to a TNF-like weak inducer of apoptosis (Tweak). Tweak-dependent or independent Fn14 activation is known to activate the NFkB signaling cascade and control cell proliferation, migration, differentiation and apoptosis, as well as inflammation involved in angiogenesis, tissue damage and regeneration (Non-Patent Document 3).

[0004]

Regarding the relationship with cancers, it has been described that Fn14 is overexpressed in various solid cancers (Non-Patent Document 4) and that Fn14 is involved in tumor progression and metastasis (Non-Patent Document 5). Furthermore, in a mouse model of cancer cachexia, an anti-Fn14 antibody has been described to be effective in improving the symptoms of cachexia, and its effect results from the inhibition of Fn14 in a tumor (Non-Patent Document 6).

[0005]

Meanwhile, Fn14 activation can induce the production of an inflammatory cytokine and worsen the inflammatory state. Enavatuzumab (Patent Document 1), which is under clinical development, has been described as a human Fn14 agonist antibody. In a phase I clinical trial of enavatuzumab, hepatotoxicity was described (Non-Patent Document 7), and it was suggested that treatment with enavatuzumab may cause inflammation (Non-Patent Document 8).

[0006]

The mouse monoclonal antibody CRCBT-06-002 has been described as an antibody that binds to human Fn14 under certain conditions and has an antagonist activity but no agonist activity (Patent Document 2). CRCBT-06-002 has been described to have antagonist activity that inhibits IL-8 production induced by Tweak stimulation in human malignant melanoma-derived cell line A375 and is effective in a mouse model of cancer patient cachexia. However, agonist activity that induces IL-8 production from A375 cells in the absence of Tweak is retained (Patent Document 2).

[0007]

An Fn14 antagonist antibody that does not have agonist activity and may be devoid of unwanted side effects is not known.

BACKGROUND OF THE INVENTION

PATENT DOCUMENTS

[0008]

[Patent document 1] WO 2009/020933

[Patent Document 2] WO 2013/026099

non-patent document

[0009]

[Non-Patent Document 1] Evans WJ et al., Clin Nutr. 2008 Vol. 27, p. 793-799

[Non-Patent Document 2] Baracos VE et al., Nat Rev Dis Primers. 2018 Vol. 4 Article number 17105, p. 1-18

[Non-Patent Document 3] Winkles JA, Nat Rev Drug Discov. 2008 Vol. 7, p. 411-425

[Non-Patent Document 4] Culp PA et al., Clin Cancer Res. 2010 Vol. 16, p. 497-508

[Non-Patent Document 5] HU G et al., Tumor Biol. 2017 Vol. 39 June, p. 1-9

[Non-Patent Document 6] Johnston AJ et al., Cell. 2015 Vol. 162, p. 1365-1378

[Non-Patent Document 7] Lam ET et al., Mol Cancer Ther. 2018 Vol. 17, p. 215-221

[Non-Patent Document 8] Choi D et al., Am J Pharmacol Toxicol. 2017 Vol. 12, p. 18-38.

Description of the invention

Problems solved by the invention

[0010]

It is an object of the present invention to provide an anti-human Fn14 antibody having excellent safety, which retains high antagonist activity and has reduced agonist activity compared to conventional antibodies.

Ways to solve problems

[0011]

The inventors have made extensive studies to obtain an anti-human Fn14 antibody and as a result have obtained an anti-human Fn14 antibody comprising a heavy chain variable region containing a CDR1 consisting of the amino acid sequence at amino acid positions 31-35 of SEQ ID NO: 2, a CDR2 consisting of the amino acid sequence from amino acid positions 50-65 of SEQ ID NO: 2, and a CDR3 consisting of an amino acid sequence from amino acid positions 98-114 of SEQ ID NO: 2, and a light chain variable region containing a CDR1 consisting of an amino acid sequence from amino acid positions 24- 40 of SEQ ID NO: 4, a CDR2 consisting of the amino acid sequence from amino acid positions 56-62 of SEQ ID NO: 4, and a CDR3 consisting of the amino acid sequence from amino acid positions 95-103 of SEQ ID NO: 4 (examples 2-4). The inventors found that the antibody binds to human Fn14 (Example 6), inhibits NFkB activation and IL-8 production induced by Tweak stimulation (Examples 7 and 8), and does not induce IL-8 production in the absence of Tweak (Example 8). As a result, an anti-human Fn14 antibody having an antagonist activity but not an agonist activity was provided, and the present invention was accomplished. Moreover, the inventors found that the antibody described above, which is murinized (Example 4), suppresses the loss of body weight and muscle mass in a cancer model of cachexia in mice (Example 9), and that the survival period is increased when the antibody and gemcitabine is used in combination as compared to administering the antibody alone or gemcitabine alone (Example 10).

[0012]

In accordance with the present invention, for example, the following invention is provided.

[1]

Anti-human Fn14 antibody, or antigen-binding fragment thereof, containing:

a heavy chain variable region comprising a CDR1 consisting of an amino acid sequence from amino acid positions 31-35 of SEQ ID NO: 2, a CDR2 consisting of an amino acid sequence from amino acid positions 50-65 of SEQ ID NO: 2, and a CDR3 consisting of an amino acid sequence from amino acid positions 98-114 SEQ ID NO: 2; and

a light chain variable region comprising a CDR1 consisting of an amino acid sequence from amino acid positions 24-40 of SEQ ID NO: 4, a CDR2 consisting of an amino acid sequence from amino acid positions 56-62 of SEQ ID NO: 4, and a CDR3 consisting of an amino acid sequence from amino acid positions 95-103 of SEQ ID NO: 4.

[2]

An anti-human Fn14 antibody or antigen-binding fragment thereof according to [1], selected from the following (1) and (2):

(1) an anti-human Fn14 antibody or antigen-binding fragment thereof, comprising a heavy chain variable region consisting of an amino acid sequence of amino acid positions 1-125 of SEQ ID NO: 2 and a light chain variable region consisting of an amino acid sequence of amino acid positions 1-114 SEQ ID NO: 4; and

(2) an antibody or antigen-binding fragment thereof generated by post-translational modification of an anti-human Fn14 antibody or antigen-binding fragment thereof according to (1).

[3]

An antibody against human Fn14 or its antigen-binding fragment according to [2], containing:

a heavy chain variable region consisting of an amino acid sequence from amino acid positions 1-125 of SEQ ID NO: 2; and

a light chain variable region consisting of an amino acid sequence from amino acid positions 1-114 of SEQ ID NO: 4.

[four]

An anti-human Fn14 antibody or antigen-binding fragment thereof according to [2], wherein the post-translational modification is pyroglutamylation at the N-terminus of the heavy chain variable region and/or a lysine deletion at the C-terminus of the heavy chain.

[five]

An antibody against human Fn14 or its antigen-binding fragment according to [2], containing:

a heavy chain variable region consisting of an amino acid sequence from amino acid positions 1-125 of SEQ ID NO: 2, wherein the glutamine at amino acid position 1 of SEQ ID NO: 2 is modified to pyroglutamic acid; and

a light chain variable region consisting of an amino acid sequence from amino acid positions 1-114 of SEQ ID NO: 4.

[6]

An anti-human Fn14 antibody according to [2] selected from the following (3) and (4):

(3) an anti-human Fn14 antibody comprising a heavy chain consisting of an amino acid sequence corresponding to SEQ ID NO: 2 and a light chain consisting of an amino acid sequence corresponding to SEQ ID NO: 4; and

(4) an anti-human Fn14 antibody generated by post-translational modification of the antibody according to (3).

[7]

Antibody against human Fn14 according to [6], containing:

a heavy chain consisting of the amino acid sequence corresponding to SEQ ID NO: 2; and

a light chain consisting of the amino acid sequence corresponding to SEQ ID NO: 4

[8]

An anti-human Fn14 antibody according to [6], where the post-translational modification is pyroglutamylation at the N-terminus of the heavy chain and/or a lysine deletion at the C-terminus of the heavy chain.

[nine]

Antibody against human Fn14 according to [8], containing:

a heavy chain consisting of an amino acid sequence from amino acid positions 1-454 of SEQ ID NO: 2, in which glutamine at amino acid position 1 of SEQ ID NO: 2 is modified to pyroglutamic acid; and

a light chain consisting of the amino acid sequence corresponding to SEQ ID NO: 4.

[10]

An antigen-binding fragment according to any one of [1]-[5], which is a single chain fragment of the variable region, Fab, Fab' or F(ab') ₂ .

[eleven]

Polynucleotide containing:

a base sequence encoding the heavy chain variable region of an anti-human Fn14 antibody or its antigen-binding fragment according to [3].

[12]

Polynucleotide containing:

a base sequence encoding the light chain variable region of an anti-human Fn14 antibody or its antigen-binding fragment according to [3].

[13]

An expression vector containing:

polynucleotide according to [11] and/or [12].

[fourteen]

Host cell selected from the group consisting of the following (a)-(d):

(a) a host cell transformed with an expression vector containing a polynucleotide containing a base sequence encoding the heavy chain variable region of an anti-human Fn14 antibody or an antigen-binding fragment thereof according to [3], and a polynucleotide containing a base sequence encoding a light chain variable region of the antibody, or its antigen-binding fragment;

(b) a host cell transformed with an expression vector containing a polynucleotide containing a base sequence encoding the heavy chain variable region of an anti-human Fn14 antibody or antigen-binding fragment thereof according to [3], and an expression vector containing a polynucleotide containing a base sequence encoding the variable region the light chain of an antibody or antigen-binding fragment thereof;

(c) a host cell transformed with an expression vector containing a polynucleotide containing a base sequence encoding the heavy chain variable region of an anti-human Fn14 antibody or antigen-binding fragment thereof according to [3]; and

(d) a host cell transformed with an expression vector containing a polynucleotide containing a base sequence encoding the light chain variable region of an anti-human Fn14 antibody or antigen-binding fragment thereof according to [3].

[15]

Host cell selected from the group consisting of the following (e)-(h):

(e) a host cell transformed with an expression vector containing a polynucleotide containing a base sequence encoding the heavy chain of an anti-human Fn14 antibody according to [7] and a polynucleotide containing a base sequence encoding a light chain of the antibody;

(f) a host cell transformed with an expression vector containing a polynucleotide containing a base sequence encoding the heavy chain of an anti-human Fn14 antibody according to [7] and an expression vector containing a polynucleotide containing a base sequence encoding an antibody light chain;

(g) a host cell transformed with an expression vector containing a polynucleotide containing a base sequence encoding the heavy chain of an anti-human Fn14 antibody according to [7]; and

(h) a host cell transformed with an expression vector containing a polynucleotide containing a base sequence encoding a light chain of an anti-human Fn14 antibody according to [7].

[sixteen]

A method for producing an anti-human Fn14 antibody or antigen-binding fragment thereof, the method comprising:

the step of cultivating a host cell selected from the group consisting of the following (A)-(C) to express an anti-human Fn14 antibody or an antigen-binding fragment thereof:

(A) a host cell transformed with an expression vector containing a polynucleotide containing a base sequence encoding the heavy chain variable region of an anti-human Fn14 antibody or an antigen-binding fragment thereof according to [3], and a polynucleotide containing a base sequence encoding a light chain variable region of an antibody, or its antigen-binding fragment;

(B) a host cell transformed with an expression vector containing a polynucleotide containing a base sequence encoding the heavy chain variable region of an anti-human Fn14 antibody or antigen-binding fragment thereof according to [3], and an expression vector containing a polynucleotide containing a base sequence encoding the variable region the light chain of an antibody or antigen-binding fragment thereof; and

(C) a host cell transformed with an expression vector containing a polynucleotide containing a base sequence encoding the heavy chain variable region of an anti-human Fn14 antibody or an antigen-binding fragment thereof according to [3], and a host cell transformed with an expression vector containing a polynucleotide containing the sequence bases encoding the variable region of the light chain of the antibody or its antigen-binding fragment.

[17]

A method for producing an anti-human Fn14 antibody, comprising:

a step of culturing a host cell selected from the group consisting of the following (D) to (F) to express an anti-human Fn14 antibody:

(D) a host cell transformed with an expression vector containing a polynucleotide containing a base sequence encoding the heavy chain of an anti-human Fn14 antibody according to [7] and a polynucleotide containing a base sequence encoding a light chain of the antibody;

(E) a host cell transformed with an expression vector containing a polynucleotide containing a base sequence encoding the heavy chain of an anti-human Fn14 antibody according to [7] and an expression vector containing a polynucleotide containing a base sequence encoding an antibody light chain; and

(F) a host cell transformed with an expression vector containing a polynucleotide containing a base sequence encoding the heavy chain of an anti-human Fn14 antibody according to [7] and a host cell transformed with an expression vector containing a polynucleotide containing a base sequence encoding an antibody light chain against Fn14 man.

[eighteen]

An anti-human Fn14 antibody or antigen-binding fragment thereof, produced by the method according to [16].

[nineteen]

An anti-human Fn14 antibody produced by the method according to [17].

[20]

Pharmaceutical composition containing:

an anti-human Fn14 antibody or antigen-binding fragment thereof according to any one of [1]-[10], [18] and [19]; and

pharmaceutically acceptable excipient.

[21]

Pharmaceutical composition containing:

an antibody against human Fn14 or its antigen-binding fragment according to [3];

an antibody against human Fn14 or its antigen-binding fragment according to [5]; and

pharmaceutically acceptable excipient.

[22]

Pharmaceutical composition containing:

anti-human Fn14 antibody according to [7];

anti-human Fn14 antibody according to [9]; and

pharmaceutically acceptable excipient.

[23]

The pharmaceutical composition according to any one of [20]-[22], which is a pharmaceutical composition for preventing or treating cachexia in cancer patients.

[24]

A method for preventing or treating cachexia in cancer patients, the method comprising:

the step of administering a therapeutically effective amount of an anti-human Fn14 antibody or antigen-binding fragment thereof according to any one of [1]-[10], [18] and [19].

[25]

An anti-human Fn14 antibody or antigen-binding fragment thereof according to any one of [1]-[10], [18] and [19], which are intended to prevent or treat cachexia in cancer patients.

[26]

The use of an anti-human Fn14 antibody or antigen-binding fragment thereof according to any of [1]-[10], [18] and [19] for the production of a pharmaceutical composition for the prevention or treatment of cachexia in cancer patients.

[27]

Pharmaceutical composition for preventing or treating cancer or cachexia in cancer patients, comprising:

an anti-human Fn14 antibody or an antigen-binding fragment thereof according to any one of [1]-[10], [18] and [19] as an active ingredient,

wherein the pharmaceutical composition is used in combination with an anti-cancer agent.

[28]

Pharmaceutical composition for preventing or treating cancer or cachexia in cancer patients, comprising:

cancer remedy,

wherein the pharmaceutical composition is used in combination with an anti-human Fn14 antibody or antigen-binding fragment thereof according to any one of [1]-[10], [18] and [19].

Invention Effects

[0013]

The anti-human Fn14 antibody of the present invention acts to suppress inflammation by inhibiting human Fn14 activation induced by Tweak stimulation without exhibiting human Fn14 agonist activity, and can be used as an agent for preventing or treating cachexia in cancer patients.

Brief description of the drawings

[0014]

[Figure 1] Figure 1 shows the inhibitory effect of an anti-human Fn14 antibody on Tweak stimulation-induced production of the inflammatory cytokine IL-8 in A375 cells. The vertical axis represents the level of inhibition (%) and the horizontal axis represents the antibody concentration (ng/mL).

[Figure 2] Figure 2 shows IL-8 secretion induced by stimulation with anti-human Fn14 antibody in A375 cells. The vertical axis represents the concentration of IL-8 (pg/ml), and the horizontal axis represents the concentration of the antibody (ng/ml).

[Figure 3] Figure 3 shows the inhibitory effect of 4-1m murinized anti-human Fn14 antibody on weight loss induced by transplantation of C26 mouse colon cancer cell line into mice. The vertical axis represents body weight (g), and the horizontal axis represents the number of days since the injection of malignant cells.

[Figure 4] Figure 4 shows the inhibitory effect of murinized anti-human Fn14 antibody 4-1m on muscle mass reduction induced by transplantation of C26 mouse colon cancer cell line into mice. The vertical axis represents the mass (mg) of the tibialis anterior muscle.

[Fig. 5] Fig. 5 shows the inhibitory effect of murinized anti-human Fn14 antibody 4-1m on the decrease in muscle function induced by transplantation of C26 mouse colon cancer cell line into mice. The vertical axis represents the compression force (g).

[Fig. 6] Fig. 6 shows the effect of murinized anti-human Fn14 antibody 4-1m in combination with gemcitabine on the survival time of mice transplanted with C26 mouse colon cancer cell line. The vertical axis represents survival (%), and the horizontal axis represents the number of days elapsed since the injection of malignant cells.

[Figure 7] Figure 7 shows the effect of the combined use of murinized anti-human Fn14 antibody 4-1m with gemcitabine on tumor volume after transplantation of C26 mouse colon cancer cell line into mice. The vertical axis represents the volume of the tumor (mm ³ ), and the horizontal axis represents the number of days since the injection of malignant cells.

Embodiments of the invention

[0015]

Hereinafter, the present invention is described in detail.

[0016]

There are five classes of antibodies: IgG, IgM, IgA, IgD and IgE. The basic structure of an antibody molecule is organized into heavy chains having a molecular weight of 50,000 to 70,000 and light chains having a molecular weight of 20,000 to 30,000, in each of the classes in general. The heavy chain usually consists of a polypeptide chain containing approximately 440 amino acids, has a distinct structure for each of the classes and is designated Igγ, Igμ, Igα, Igδ and Igε, which correspond to IgG, IgM, IgA, IgD and IgE, respectively. In addition, four subclasses of IgG1, IgG2, IgG3, and IgG4 are present in IgG, and the heavy chains corresponding to them are respectively referred to as Igγ1, Igγ2, Igγ3, and Igγ4. The light chain usually consists of a 220 amino acid polypeptide chain of which two types are known, the L type and the K type, and are referred to as Igλ and Igκ. In the peptide configuration of the basic structure of an antibody molecule, two homologous heavy chains and two homologous light chains are linked by disulfide bonds (S-S bond) and non-covalent bonds, and their molecular weight ranges from 150,000 to 190,000. The two types of light chains can be paired with any heavy chain. The corresponding antibody molecules generally consist of two identical light chains and two identical heavy chains.

[0017]

With regard to interchain S-S bonds, four S-S bonds are present in the heavy chain (five in the Igμ and Igε chains) and two such bonds are present in the light chain; one loop is formed per 100-110 amino acid residues, and this spatial structure is similar among the loops, and they are called a building block or domain. The domain located at the N-terminal side of both the heavy chain and the light chain, whose amino acid sequence is not constant even in the case of a sample from the same class (subclass) of the same animal species, is called the variable region, and the corresponding domains are called the variable region of the heavy chain ( VH) and the light chain variable region (VL). The amino acid sequence of the C-terminal side of the variable region is substantially constant within each class or subclass and is referred to as the constant region (the respective domains are referred to as CH1, CH2, CH3 or CL, respectively).

[0018]

The antigenic binding site of an antibody is organized from VH and VL, and the binding specificity depends on the amino acid sequence of this site. On the other hand, biological activities such as binding to complement and various Fc receptor expressing cells reflect differences in constant region structures among Ig classes. It is understood that the variability of the light chain and heavy chain variable regions is limited for the most part to the three small hypervariable regions present on both chains, and these regions are referred to as complementarity determining regions (CDRs: CDR1, CDR2 and CDR3 on the N-terminal side). The remainder of the variable region is called the framework region (FR) and is relatively constant.

[0019]

Various antigen-binding fragments containing VH and VL antibodies also have antigen-binding activity, and representative examples of such antigen-binding fragment include single chain variable region fragment (scFv), Fab, Fab and F(ab') ₂ . scFv is a monovalent antibody fragment that consists of VH and VL connected to each other via a linker. A Fab is a monovalent antibody fragment that consists of a light chain and a heavy chain fragment containing a VH, a CH1 domain, and part of a hinge region. Fab' is a monovalent antibody fragment that consists of a light chain and a heavy chain fragment containing a VH, a CH1 domain and part of the hinge region, and part of the hinge region includes cysteine residues that form an SS bond between heavy chains. The F(ab') ₂ fragment is a bivalent antibody fragment in which two Fab' fragments are linked by an SS linkage between heavy chains in the hinge region.

[0020]

<Anti-human Fn14 antibody of the present invention>

The anti-human Fn14 antibody or antigen-binding fragment thereof of the present invention includes an anti-human Fn14 antibody or antigen-binding fragment thereof having the following characteristics.

An anti-human Fn14 antibody or antigen-binding fragment thereof, comprising a heavy chain variable region comprising a CDR1 consisting of an amino acid sequence from amino acid positions 31-35 of SEQ ID NO: 2, a CDR2 consisting of an amino acid sequence from amino acid positions 50-65 of SEQ ID NO: 2 and a CDR3 consisting of the amino acid sequence of amino acid positions 98-114 of SEQ ID NO: 2, and a light chain variable region containing CDR1 consisting of the amino acid sequence of amino acid positions 24-40 of SEQ ID NO: 4, CDR2 consisting of an amino acid sequence from amino acid positions 56-62 of SEQ ID NO: 4, and a CDR3 consisting of an amino acid sequence from amino acid positions 95-103 of SEQ ID NO: 4.

[0021]

In one embodiment, an anti-human Fn14 antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region consisting of the amino acid sequence of amino acid positions 1-125 of SEQ ID NO: 2 and a light chain variable region consisting of the amino acid sequence of amino acid positions 1-114 SEQ ID NO: 4.

[0022]

In one embodiment, an anti-human Fn14 antibody or antigen-binding fragment thereof of the present invention has the characteristics described above and further comprises a heavy chain constant region and a light chain constant region. Any subclass constant region can be selected as the constant region (eg, Igγ1, Igγ2, Igγ3 or Igγ4 as the heavy chain constant region, and Igλ or Igκ as the light chain constant region). In one embodiment, an anti-human Fn14 antibody or antigen-binding fragment thereof of the present invention comprises a human Igγ1 constant region as a heavy chain constant region and a human Igκ constant region as a light chain constant region.

[0023]

The residue number associated with the introduction of amino acid mutations in the constant region of the antibody used can be determined according to the EU index (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Ed., United States Public Health Service, National Institute of Health, Bethesda). L234A and L235A are substitutions of leucine at amino acid position 234 and at amino acid position 235 for alanine in the human Igγ1 constant region according to the EU index described by Kabat et al., respectively. Examples of a human Igγ1 constant region having amino acid L234A and L235A mutations include a human Igγ1 constant region consisting of an amino acid sequence from amino acid positions 126-455 of SEQ ID NO: 2.

[0024]

In one embodiment, an anti-human Fn14 antibody of the present invention comprises a heavy chain consisting of the amino acid sequence corresponding to SEQ ID NO: 2 and a light chain consisting of the amino acid sequence corresponding to SEQ ID NO: 4.

[0025]

It is known that when an antibody is expressed in cells, the antibody is modified after translation. Examples of post-translational modification include deletion of lysine at the C-terminus of the heavy chain by carboxypeptidase, modification of glutamine or glutamic acid at the N-terminus of the heavy chain and light chain to pyroglutamic acid by pyroglutamylation, glycosylation, oxidation, deamidation, and glycation, and such post-translational modifications are known to occur in various antibodies (Liu H et al., J Phar Sci. 2008, Vol. 97 No. 7, p. 2426-2447).

[0026]

The anti-human Fn14 antibody or antigen-binding fragment thereof of the present invention also includes an anti-human Fn14 antibody or antigen-binding fragment thereof modified after translation. Examples of an anti-human Fn14 antibody or an antigen-binding fragment thereof of the present invention modified after translation include anti-human Fn14 antibodies that have undergone pyroglutamylation at the N-terminus of the heavy chain variable region and/or a lysine deletion at the C-terminus of the heavy chain, or antigen-binding fragments thereof. . It is known in the art that such post-translational modification due to pyroglutamylation at the N-terminus or deletion of lysine at the C-terminus has no effect on antibody activity (Lyubarskaya Y et al., Anal Biochem. 2006, Vol. 348, p. 24-39 ).

[0027]

In one embodiment, an anti-human Fn14 antibody of the present invention comprises an antibody, or an antigen-binding fragment thereof, formed by post-translational modification of an anti-human Fn14 antibody, or an antigen-binding fragment thereof, comprising a heavy chain variable region consisting of the amino acid sequence of amino acid positions 1-125 of SEQ ID NO : 2, and a light chain variable region consisting of an amino acid sequence from amino acid positions 1-114 of SEQ ID NO: 4.

In one embodiment, the post-translational modification is pyroglutamilation at the N-terminus of the heavy chain variable region and/or a lysine deletion at the C-terminus of the heavy chain.

[0028]

In one embodiment, an anti-human Fn14 antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region consisting of an amino acid sequence from amino acid positions 1-125 of SEQ ID NO: 2, wherein the glutamine at amino acid position 1 of SEQ ID NO: 2 is modified to pyroglutamic acid, and a light chain variable region consisting of an amino acid sequence from amino acid positions 1-114 of SEQ ID NO: 4.

[0029]

In one embodiment, an anti-human Fn14 antibody of the present invention is formed by post-translational modification of an anti-human Fn14 antibody comprising a heavy chain consisting of the amino acid sequence corresponding to SEQ ID NO: 2 and a light chain consisting of the amino acid sequence corresponding to SEQ ID NO: 4 and the post-translational modification is pyroglutamilation at the N-terminus of the heavy chain and/or deletion of a lysine at the C-terminus of the heavy chain.

[0030]

In one embodiment, an anti-human Fn14 antibody of the present invention comprises a heavy chain consisting of an amino acid sequence from amino acid positions 1-454 of SEQ ID NO: 2, wherein the glutamine at amino acid position 1 of SEQ ID NO: 2 is modified to pyroglutamic acid, and a light a chain consisting of the amino acid sequence of SEQ ID NO: 4.

[0031]

In one embodiment, the antigen-binding fragment of the present invention is a scFv, Fab, Fab' or F(ab') ₂ .

[0032]

Any person skilled in the art can make a fusion form in which an antibody or an antigen-binding fragment thereof is fused to another peptide or protein, or can also make a modified form to which a modifying agent is bound, based on the present invention, and the antibody of the present invention also includes these forms of antibodies or their antigen-binding fragments. Other peptides or proteins used for fusion are not specifically limited provided that the fusion form binds to Fn14, and examples include human serum albumin, various tag peptides, artificial helix motif peptide, maltose binding proteins, glutathione-S-transferase, various toxins and other peptides or proteins capable of promoting multimerization. The modifying agent used for modification is not particularly limited as long as the modified form binds to Fn14, and examples thereof include polyethylene glycol, sugar chains, phospholipids, liposomes, and small molecular weight compounds.

[0033]

In one embodiment, the modifying agent used to modify the antibody or antigen-binding fragment of the present invention is polyethylene glycol.

[0034]

"Anti-human Fn14 antibody" as used herein means an antibody that binds to human Fn14. The presence of binding of an anti-Fn14 antibody to human Fn14 can be confirmed using a known method for measuring binding activity. Examples of a method for measuring binding activity include enzyme-linked immunosorbent assay (ELISA). In the case of ELISA, for example, the human Fn14 protein is immobilized on an ELISA plate and the test antibody to be reacted is added thereto. After the reaction, a secondary antibody reaction is carried out, such as an anti-IgG antibody labeled with an enzyme, such as horseradish peroxidase (HRP). Washing is carried out after the reaction, and then it can be confirmed whether the test antibody binds to human Fn14 by identifying the binding of the secondary antibody by measuring the activity using an activity detecting reagent (for example, in the case of HRP labeling, Microwell TMB peroxidase substrate (Kirkegaard & Perry Laboratories, Inc., 50-76-03)). As a specific measurement method, the method described in Example 6 below can be used.

[0035]

The anti-human Fn14 antibody of the present invention includes, in addition to binding to human Fn14, an antibody that binds to Fn14 derived from other animals (eg, mouse Fn14), provided that the antibody binds to human Fn14.

[0036]

An anti-human Fn14 antibody or antigen-binding fragment thereof of the present invention can be easily prepared by one skilled in the art using a method known in the art based on the heavy chain and light chain sequence information of the antibody of the present invention as disclosed herein. The anti-human Fn14 antibody or antigen-binding fragment thereof of the present invention is not particularly limited, and can be produced by the method described in <Method for producing an anti-human Fn14 antibody of the present invention, and an anti-human Fn14 antibody obtained by this method> described below.

[0037]

The anti-human Fn14 antibody or antigen-binding fragment thereof of the present invention is further purified as needed, formulated according to a conventional method, and can be used to prevent or treat inflammatory diseases such as excessive angiogenesis or wasting diseases such as weight loss, muscle wasting and cachexia.

[0038]

<Polynucleotide of the present invention>

The polynucleotide of the present invention comprises a polynucleotide comprising a base sequence encoding the heavy chain variable region of an anti-human Fn14 antibody or an antigen-binding fragment thereof of the present invention, and a polynucleotide comprising a base sequence encoding a light chain variable region of an anti-human Fn14 antibody or an antigen-binding fragment thereof of the present invention. invention.

[0039]

In one embodiment, the polynucleotide comprising a base sequence encoding the heavy chain variable region of an anti-human Fn14 antibody or antigen-binding fragment thereof of the present invention is a polynucleotide comprising a base sequence encoding a heavy chain variable region consisting of the amino acid sequence of amino acid positions 1- 125 SEQ ID NO: 2.

[0040]

Examples of a polynucleotide containing a base sequence encoding a heavy chain variable region shown by an amino acid sequence from amino acid positions 1-125 of SEQ ID NO: 2 include a polynucleotide containing a base sequence from base positions 1-375 of SEQ ID NO: 1.

[0041]

In one embodiment, a polynucleotide comprising a base sequence encoding the heavy chain variable region of an anti-human Fn14 antibody of the present invention contains a base sequence encoding a heavy chain consisting of the amino acid sequence corresponding to SEQ ID NO: 2.

[0042]

Examples of a polynucleotide containing a base sequence encoding a heavy chain consisting of an amino acid sequence corresponding to SEQ ID NO: 2 include a polynucleotide containing a base sequence corresponding to SEQ ID NO: 1.

[0043]

In one embodiment, a polynucleotide comprising a base sequence encoding the light chain variable region of an anti-human Fn14 antibody or antigen-binding fragment thereof of the present invention comprises a base sequence encoding a light chain variable region consisting of the amino acid sequence of amino acid positions 1-114 of SEQ ID NO : four.

[0044]

Examples of a polynucleotide containing a base sequence encoding a light chain variable region consisting of an amino acid sequence from amino acid positions 1-114 of SEQ ID NO: 4 include a polynucleotide containing a base sequence from base positions 1-342 of SEQ ID NO: 3.

[0045]

In one embodiment, a polynucleotide comprising a base sequence encoding the light chain variable region of an anti-human Fn14 antibody of the present invention comprises a light chain encoding base sequence consisting of the amino acid sequence corresponding to SEQ ID NO: 4.

[0046]

Examples of a polynucleotide containing a base sequence encoding a light chain consisting of an amino acid sequence corresponding to SEQ ID NO: 4 include a polynucleotide containing a base sequence corresponding to SEQ ID NO: 3.

[0047]

The polynucleotide of the present invention can be easily obtained by a person skilled in the art using a method known in the art based on the base sequence. For example, a polynucleotide of the present invention can be synthesized using a gene synthesis method known in the art. As a gene synthesis method, various methods can be used, such as the antibody gene synthesis method described in WO90/07861, known to a person skilled in the art.

[0048]

<Expression vector of the present invention>

The expression vector of the present invention comprises an expression vector comprising a polynucleotide comprising a base sequence encoding the heavy chain variable region of an anti-Fn14 antibody or an antigen-binding fragment thereof of the present invention, and/or a polynucleotide containing a base sequence encoding the light chain variable region of an anti-Fn14 antibody. human or its antigennegative fragment according to the present invention.

[0049]

In one embodiment, examples of an expression vector of the present invention include an expression vector comprising a polynucleotide comprising a base sequence encoding the heavy chain of an anti-human Fn14 antibody of the present invention, an expression vector comprising a polynucleotide comprising a base sequence encoding a light chain of an anti-human Fn14 antibody according to of the present invention, and an expression vector containing a polynucleotide containing a base sequence encoding the heavy chain of an anti-human Fn14 antibody of the present invention, and a polynucleotide containing a base sequence encoding a light chain of the antibody.

[0050]

Expression vectors used to express a polynucleotide of the present invention are not particularly limited, provided that the polynucleotide containing a base sequence encoding the heavy chain variable region of an anti-human Fn14 antibody or antigen-binding fragment thereof of the present invention, and/or the polynucleotide containing the base sequence, encoding the light chain variable region of an anti-human Fn14 antibody or antigen-binding fragment thereof of the present invention can be expressed in a variety of eukaryotic host cells (e.g., animal cells, insect cells, plant cells, and yeast cells) and/or prokaryotic host cells (e.g., , Escherichia coli ), and you can get the polypeptides encoded by them. Examples of an expression vector include plasmid vectors and viral vectors (eg adenovirus or retrovirus). For example, you can use pEE6.4 or pEE12.4. The antibody gene can also be expressed by introducing the variable region gene segment into an expression vector already carrying a human Ig constant region gene such as AG-γ1 and AG-κ (eg see WO94/20632).

[0051]

The expression vector of the present invention may contain a promoter that is operably linked to a polynucleotide of the present invention. Examples of a promoter for expressing a polynucleotide of the present invention in an animal cell include a virus-derived promoter such as CMV, RSV or SV40, an actin promoter, an EF 1α promoter (elongation factor), and a heat shock protein promoter. Examples of a promoter for expression of a polynucleotide of the present invention by bacteria (eg, Escherichia ) include the trp promoter, the lac promoter, the λPL promoter, and the tac promoter. In addition, examples of a promoter for expressing a polynucleotide of the present invention in yeast include the GAL1 promoter, the GAL10 promoter, the PH05 promoter, the PGK promoter, the GAP promoter, and the ADH promoter.

[0052]

When using an animal cell, insect cell or yeast cell as the host cell, the expression vector of the present invention may contain an initiation codon and a termination codon. In this case, the expression vector of the present invention may contain an enhancer sequence, an untranslated region on the 5' side and on the 3' side of the genes encoding the antibody of the present invention or its heavy chain variable region or light chain variable region, a secretion signal sequence, a dot splicing, polyadenylation site or replicating element. In the case where Escherichia coli is used as the host cell, the expression vector of the present invention may contain an initiation codon, a termination codon, a terminator region, and a replication element. In this case, the expression vector of the present invention may contain a selectable marker (for example, tetracycline resistance genes, ampicillin resistance genes, kanamycin resistance genes, neomycin resistance genes, or dihydrofolate reductase genes) that are commonly used depending on the intended use.

[0053]

<Transformed Host Cell of the Present Invention>

Examples of the transformed cell of the present invention include a host cell transformed with an expression vector containing a polynucleotide containing a base sequence encoding a heavy chain of an anti-human Fn14 antibody of the present invention and a polynucleotide containing a base sequence encoding an antibody light chain, and a host cell, transformed with an expression vector containing a polynucleotide containing a base sequence encoding the heavy chain of an anti-human Fn14 antibody of the present invention, and an expression vector containing a polynucleotide containing a base sequence encoding a light chain of an antibody.

[0054]

In one embodiment, the transformed host cell of the present invention includes a host cell transformed with an expression vector of the present invention, which is selected from the group consisting of the following (a)-(d):

(a) a host cell transformed with an expression vector comprising a polynucleotide comprising a base sequence encoding the heavy chain variable region of an anti-human Fn14 antibody or antigen-binding fragment thereof of the present invention, and a polynucleotide comprising a base sequence encoding the light chain variable region of the antibody or its an antigen-binding fragment;

(b) a host cell transformed with an expression vector containing a polynucleotide containing a base sequence encoding the heavy chain variable region of an anti-human Fn14 antibody or antigen-binding fragment thereof of the present invention, and an expression vector containing a polynucleotide containing a base sequence encoding a light variable region the chain of the antibody or its antigen-binding fragment;

(c) a host cell transformed with an expression vector containing a polynucleotide containing a base sequence encoding the heavy chain variable region of an anti-human Fn14 antibody or antigen-binding fragment thereof of the present invention; and

(d) a host cell transformed with an expression vector containing a polynucleotide containing a base sequence encoding a light chain variable region of an anti-human Fn14 antibody or antigen-binding fragment thereof of the present invention.

[0055]

In one embodiment, the transformed host cell of the present invention includes a host cell transformed with an expression vector of the present invention, which is selected from the group consisting of the following (e)-(h):

(e) a host cell transformed with an expression vector containing a polynucleotide containing a base sequence encoding the heavy chain of an anti-human Fn14 antibody of the present invention and a polynucleotide containing a base sequence encoding a light chain of the antibody;

(f) a host cell transformed with an expression vector containing a polynucleotide containing a base sequence encoding the heavy chain of an anti-human Fn14 antibody of the present invention and an expression vector containing a polynucleotide containing a base sequence encoding an antibody light chain;

(g) a host cell transformed with an expression vector containing a polynucleotide containing a base sequence encoding the heavy chain of an anti-human Fn14 antibody of the present invention; and

(h) a host cell transformed with an expression vector containing a polynucleotide containing a base sequence encoding a light chain of an anti-human Fn14 antibody of the present invention.

[0056]

The transformed host cell is not particularly limited as long as the host cell is suitable for the expression vector used to transform the cell and can express the antibody. Examples of the transformed host cell include various cells such as natural cells or artificially produced cells that are commonly used in the field of the present invention (for example, animal cells (for example, CHO-K1SV cells), insect cells (for example, Sf9), bacteria (for example , Escherichia ), yeast (for example, Saccharomyces or Pichia ), etc.). For example, cultured cells such as CHO cells (CHO-K1SV cells and CHO-DG44 cells), 293 cells, and NS0 cells can be used.

[0057]

The method for transforming the host cell is not particularly limited, and for example, the calcium phosphate method or the electroporation method can be used.

[0058]

<Method for producing an anti-human Fn14 antibody of the present invention, and an anti-human Fn14 antibody obtained by this method>

The method for producing an anti-human Fn14 antibody or antigen-binding fragment thereof of the present invention includes a method for producing an anti-human Fn14 antibody or antigen-binding fragment thereof, comprising the step of culturing a host cell selected from the group consisting of the following (A) to (C) to express the antibody against human Fn14 or its antigen-binding fragment:

(A) a host cell transformed with an expression vector containing a polynucleotide containing a base sequence encoding the heavy chain variable region of an anti-human Fn14 antibody or antigen-binding fragment thereof of the present invention, and a polynucleotide containing a base sequence encoding the light chain variable region of the antibody or its an antigen-binding fragment;

(B) a host cell transformed with an expression vector containing a polynucleotide containing a base sequence encoding the heavy chain variable region of an anti-human Fn14 antibody or an antigen-binding fragment thereof of the present invention, and an expression vector containing a polynucleotide containing a base sequence encoding a light variable region. the chain of the antibody or its antigen-binding fragment; and

(C) a host cell transformed with an expression vector containing a polynucleotide containing a base sequence encoding the heavy chain variable region of an anti-human Fn14 antibody or antigen-binding fragment thereof of the present invention, and a host cell transformed with an expression vector containing a polynucleotide containing the base sequence encoding the variable region of the light chain of the antibody or its antigen-binding fragment.

[0059]

In one embodiment, the method for producing an anti-human Fn14 antibody of the present invention comprises a method for producing an anti-human Fn14 antibody comprising the step of culturing a host cell selected from the group consisting of the following (D)-(F) to express the anti-human Fn14 antibody:

(D) a host cell transformed with an expression vector containing a polynucleotide containing a base sequence encoding the heavy chain of an anti-human Fn14 antibody of the present invention and a polynucleotide containing a base sequence encoding an antibody light chain;

(E) a host cell transformed with an expression vector containing a polynucleotide containing a base sequence encoding the heavy chain of an anti-human Fn14 antibody of the present invention and an expression vector containing a polynucleotide containing a base sequence encoding an antibody light chain; and

(F) a host cell transformed with an expression vector containing a polynucleotide containing a base sequence encoding the heavy chain of an anti-human Fn14 antibody of the present invention, and a host cell transformed with an expression vector containing a polynucleotide containing a base sequence encoding an antibody light chain.

[0060]

The method for producing an anti-human Fn14 antibody or an antigen-binding fragment thereof of the present invention is not particularly limited as long as the method includes the step of culturing the transformed host cell of the present invention to express the anti-human Fn14 antibody or antigen-binding fragment thereof. In one embodiment, examples of a host cell used in the method include host cells according to (A)-(D) described above.

[0061]

The transformed host cell can be cultured in a known manner. Culture conditions such as temperature, culture medium pH, and culture time are selected appropriately. In the case where the host cell is an animal cell, examples of the culture medium include MEM culture medium supplemented with approximately 5%-20% fetal bovine serum (Eagle H, Science. 1959, Vol. 130, No. 3373, p. 432-437 ), DMEM culture medium (Dulbecco R and Freeman G, Virology. 1959, Vol. 8, p. 396-397), RPMI1640 culture medium (Moore GE et al., J Am Med Assoc. 1967, Vol. 199, p. 519) and culture medium 199 (Morgan JF et al., Proc Soc Exp Biol Med. 1950, Vol. 73, p. 1-8). Preferably, the pH of the culture medium is about 6-8, and cultivation is usually carried out at about 30°C to 40°C for about 15-336 hours with optimal ventilation and agitation. In the case where the host cell is an insect cell, Grace's culture medium (Smith GE et al., Proc Natl Acad Sci USA., 1985, Vol. 82, p. 8404) supplemented with fetal calf serum can be used as the culture medium. Preferably, the pH of the culture medium is about 5-8, and cultivation is usually carried out at about 20°C - 40°C for about 15 hours to 100 hours with optional ventilation and agitation. In the case where the host cell is Escherichia coli or yeast, the culture medium is suitable, for example, liquid culture medium supplemented with a nutrient source. The nutrient culture medium preferably includes a carbon source, an inorganic nitrogen source, or an organic nitrogen source necessary for the growth of the transformed host cell. Examples of the carbon source include glucose, dextran, soluble starch and sucrose, and examples of the inorganic nitrogen source include ammonium salts, nitrates, amino acids, liquid corn extract, peptone, casein, meat extract, soy flour and potato extract. Other nutrients (eg, inorganic salts (eg, calcium chloride, sodium dihydrogen phosphate, and magnesium chloride), vitamins) and antibiotics (eg, tetracycline, neomycin, ampicillin, and kanamycin) may be included if desired. Preferably the pH of the culture medium is about 5-8. In the case where the host cell is Escherichia coli , for example, LB culture medium or M9 culture medium (Mol. Clo., Cold Spring Harbor Laboratory, 2001, Vol. 3, A2.2) can be preferably used as the culture medium. Cultivation is usually carried out at about 14°C to 39°C for about 3 to 24 hours with optional ventilation and agitation. In the case where the host cell is a yeast cell, for example Burkholder Minimal Medium (Bostian KA et al., Proc Natl Acad Sci USA. 1980, Vol. 77, p. 4504-4508) can be used as a culture medium. Cultivation is usually carried out at about 20°C to 35°C for about 14 to 144 hours with optional ventilation or agitation. By carrying out cultivation in the manner described above, it is possible to express an anti-human Fn14 antibody or an antigen-binding fragment thereof of the present invention.

[0062]

The method for producing an anti-human Fn14 antibody or antigen-binding fragment thereof of the present invention may further include, in addition to the step of culturing the transformed host cell of the present invention to express the anti-human Fn14 antibody or antigen-binding fragment thereof, the step of extracting, for example, isolating or purifying the anti-human Fn14 antibody or antigen-binding fragment thereof from the transformed host cell and/or culture supernatant. Examples of the isolation or purification method include methods using solubility such as salting out and solvent precipitation method, methods using differences in molecular weight such as dialysis, ultrafiltration and gel filtration, methods using electric charge such as ion exchange chromatography and chromatography. with hydroxyapatite, methods using specific affinity such as affinity chromatography, methods using differences in hydrophobicity such as reverse phase high performance liquid chromatography, and methods using differences in isoelectric point such as isoelectric focusing electrophoresis. For example, the antibody accumulated in the culture supernatant can be purified by various chromatography methods, such as column chromatography using a protein A column or a protein G column.

[0063]

The anti-human Fn14 antibody or antigen-binding fragment thereof of the present invention also includes an anti-human Fn14 antibody or antigen-binding fragment thereof obtained by the method for producing an anti-human Fn14 antibody or antigen-binding fragment thereof of the present invention.

[0064]

<Pharmaceutical composition of the present invention>

The pharmaceutical composition of the present invention includes a pharmaceutical composition comprising an anti-human Fn14 antibody or an antigen-binding fragment thereof of the present invention and a pharmaceutically acceptable excipient. The pharmaceutical composition of the present invention can be obtained in a manner commonly used with excipients, i.e. medical excipients or medical carriers commonly used in the art. Examples of dosage forms of the pharmaceutical compositions include a parenteral drug such as an injection drug and a drip drug, and they can be administered by intravenous administration, subcutaneous administration, intramuscular administration, and the like. When preparing a medicinal product, excipients, carriers and additives in accordance with dosage forms can be used in a pharmaceutically acceptable range.

[0065]

The pharmaceutical composition of the present invention may contain a plurality of types of anti-human Fn14 antibodies or antigen-binding fragments thereof of the present invention. For example, the present invention also includes a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof that is not post-translationally modified, and an antibody or antigen-binding fragment thereof formed by post-translational modification of the antibody or antigen-binding fragment thereof.

[0066]

In one embodiment, a pharmaceutical composition of the present invention comprising an anti-human Fn14 antibody, or antigen-binding fragment thereof, also comprises the following pharmaceutical composition.

A pharmaceutical composition containing an anti-human Fn14 antibody or antigen-binding fragment thereof, comprising a heavy chain variable region consisting of an amino acid sequence of amino acid positions 1-125 of SEQ ID NO: 2, and a light chain variable region consisting of an amino acid sequence of amino acid positions 1- 114 SEQ ID NO: 4, and an anti-human Fn14 antibody or antigen-binding fragment thereof, formed by post-translational modification of the antibody or antigen-binding fragment thereof.

[0067]

The pharmaceutical composition of the present invention also includes a pharmaceutical composition comprising an antibody in which the lysine at the C-terminus of the heavy chain is removed, an antibody or antigen-binding fragment thereof modified after translation at the N-terminus, an antibody in which the lysine at the C-terminus of the heavy chain is removed and which is modified after translation at the N-terminus, and/or an antibody which has a lysine at the C-terminus of the heavy chain and is not modified after translation at the N-terminus.

[0068]

In one embodiment, a pharmaceutical composition of the present invention comprising an anti-human Fn14 antibody also includes a pharmaceutical composition comprising two or more types of anti-human Fn14 antibodies, among the following (5)-(8):

(5) an anti-human Fn14 antibody comprising a heavy chain consisting of an amino acid sequence of amino acid positions 1-454 of SEQ ID NO: 2 and a light chain consisting of an amino acid sequence of SEQ ID NO: 4.

(6) an anti-human Fn14 antibody comprising a heavy chain consisting of an amino acid sequence corresponding to SEQ ID NO: 2, in which glutamine at amino acid position 1 of SEQ ID NO: 2 is modified to pyroglutamic acid, and a light chain consisting of an amino acid sequence, corresponding to SEQ ID NO: 4.

(7) an anti-human Fn14 antibody comprising a heavy chain consisting of an amino acid sequence of amino acid positions 1-454 of SEQ ID NO: 2, in which the glutamine at amino acid position 1 of SEQ ID NO: 2 is modified to pyroglutamic acid, and a light chain consisting of from the amino acid sequence corresponding to SEQ ID NO: 4.

(8) an anti-human Fn14 antibody comprising a heavy chain consisting of an amino acid sequence corresponding to SEQ ID NO: 2 and a light chain consisting of an amino acid sequence corresponding to SEQ ID NO: 4.

[0069]

In one embodiment, a pharmaceutical composition of the present invention comprising an anti-human Fn14 antibody, or antigen-binding fragment thereof, also comprises the following pharmaceutical composition. Pharmaceutical composition containing an anti-human Fn14 antibody containing a heavy chain consisting of an amino acid sequence corresponding to SEQ ID NO: 2 and a light chain consisting of an amino acid sequence corresponding to SEQ ID NO: 4, an anti-human Fn14 antibody containing a heavy chain, consisting of an amino acid sequence from amino acid positions 1-454 of SEQ ID NO: 2, in which glutamine at amino acid position 1 of SEQ ID NO: 2 is modified to pyroglutamic acid, and a light chain consisting of an amino acid sequence corresponding to SEQ ID NO: 4, and pharmaceutically acceptable excipient.

[0070]

The amount of the anti-human Fn14 antibody or antigen-binding fragment thereof of the present invention to be added to the formulation varies depending on the degree of the patient's symptoms, the age of the patient, the dosage form of the drug used, the binding titer of the antibody, and the like, and, for example, the antibody or its antigen-binding fragment can be used in an amount from about 0.001 mg/kg to 100 mg/kg during administration.

[0071]

The pharmaceutical composition of the present invention can be used as an agent for the prevention or treatment of diseases such as cachexia in cancer patients in which active human Fn14 is involved in the pathogenesis.

[0072]

The present invention relates to a pharmaceutical composition for preventing or treating cachexia in cancer patients, comprising an anti-human Fn14 antibody or an antigen-binding fragment thereof of the present invention. In addition, the present invention relates to a method for treating or preventing cachexia in cancer patients, comprising the step of administering a therapeutically effective amount of an anti-human Fn14 antibody or antigen-binding fragment of the present invention. In addition, the present invention provides an anti-human Fn14 antibody or antigen-binding fragment thereof of the present invention for use in the prevention or treatment of cachexia in cancer patients. In addition, the present invention relates to the use of an anti-human Fn14 antibody or an antigen-binding fragment thereof of the present invention for the manufacture of a pharmaceutical composition for the prevention or treatment of cachexia in cancer patients.

[0073]

The present invention relates to a pharmaceutical composition for the prevention or treatment of cancer or cachexia in cancer patients, which contains an anti-human Fn14 antibody or antigen-binding fragment of the present invention and is used in combination with an anti-cancer agent. The present invention relates to a method for preventing or treating cancer or cachexia in cancer patients, which comprises the step of administering effective amounts of an anti-human Fn14 antibody or antigen-binding fragment thereof of the present invention and an anti-cancer agent. The present invention provides an anti-human Fn14 antibody or antigen-binding fragment thereof of the present invention, which is used in combination with an anti-cancer agent for use in the prevention or treatment of cancer or cachexia in cancer patients. The present invention relates to the use of an anti-human Fn14 antibody or antigen-binding fragment thereof of the present invention for the manufacture of a pharmaceutical composition for the prevention or treatment of cancer or cachexia in cancer patients, and to the use of the pharmaceutical composition in combination with an anti-cancer agent.

[0074]

The present invention relates to a pharmaceutical composition for the prevention or treatment of cancer or cachexia in cancer patients, which contains an anti-cancer agent and is used in combination with an anti-human Fn14 antibody or antigen-binding fragment thereof of the present invention. The present invention relates to an anti-cancer agent which is used in combination with an anti-human Fn14 antibody or antigen-binding fragment thereof of the present invention for use in the prevention or treatment of cancer or cachexia in cancer patients. The present invention relates to the use of an anti-cancer agent for the manufacture of a pharmaceutical composition for the prevention or treatment of cancer or cachexia in cancer patients, and to the use of the pharmaceutical composition in combination with an anti-human Fn14 antibody or antigen-binding fragment thereof of the present invention.

[0075]

In one embodiment, the anti-cancer agent used in the present invention is gemcitabine.

[0076]

For a better understanding of the present invention, specific examples are provided to which reference is made, however, they are only examples and the present invention is not limited to them.

Examples

[0077]

For parts using commercially available kits or reagents, experiments were performed according to the attached protocol, unless otherwise indicated.

[0078]

(Example 1: Preparation of human and mouse Fn14-Fc fusion proteins)

The inventors have prepared a human Fn14-human Fc fusion protein and a mouse Fn14-mouse Fc fusion protein for use as an antigen for producing an anti-Fn14 antibody and a material for use in a screening test. The human Fn14-human Fc fusion protein is a fusion protein in which the C-terminus of the human Fn14 extracellular partial sequence (amino acids 1 to 79 of NCBI accession number: NP_057223.1) and the N-terminus of the human Fc region (amino acids 106 to 330 of NCBI accession number: P01857.1) are linked by a peptide linker (SEQ ID NO: 9). The mouse Fn14-mouse Fc fusion protein is a fusion protein in which the C-terminus of the extracellular mouse Fn14 partial sequence (amino acids 1 to 75 of the NCBI sequence accession number: AAF07882.1) and the N-terminus of the mouse Fc region are linked, and in particular a fusion protein in which genes encoding the mouse extracellular partial Fn14 sequence are included in a multiple cloning site between the hEF1-HTLV promoter region and the mIgG2B-Fc region of pFUSE-mIgG2B-Fc1 (InvivoGen, pfuse-mg2bfc1) with using restriction enzymes EcoRI and EcoRV, and expressed. Expression vectors were prepared in which the genes encoding the fusion proteins described above were inserted into the pEE12.4 GS vector (Lonza) and introduced into CHO-K1SV cells (Lonza), respectively. The human Fn14-human Fc fusion protein and the mouse Fn14-mouse Fc fusion protein were each purified from the culture supernatant of CHO-K1SV cells according to a conventional method.

[0079]

(Example 2: Preparation of anti-human Fn14 antibody)

The antibody was obtained using "VelocImmune" human monoclonal antibody technology with mice (VelocImmune antibody technology: Regeneron Pharmaceuticals, Inc. (US Pat. No. 6,596,541)). An antibody produced by VelocImmune technology is an antibody (also referred to as a chimeric antibody) having a human antibody variable region and a mouse antibody constant region. VelocImmune mice were immunized with an immune response adjuvant along with human Fn14 protein in which the human Fc region was excised and removed from the human Fn14-human Fc fusion protein prepared according to Example 1 using FabRICATOR (Sigma, 77661). Lymphocytes derived from the lymph node of the immunized mouse were fused with mouse-derived myeloma cells SP2/0-Ag14 (ATCC: CRL-1581) according to the conventional method to obtain hybridomas, and the hybridomas were subjected to monocloning. An antibody-producing hybridoma that binds to human Fn14 and suppresses NFkB activation induced by Tweak stimulation (hereinafter referred to as "Tweak-induced NFkB activation" in the examples below) was selected, and the antibody was purified.

[0080]

(Example 3: NFkB Activation Assay)

To evaluate the effect of the antibody of Example 2 as a human Fn14 agonist, the effect of the antibody on NFkB activation in the absence of Tweak was evaluated by reporter analysis. Specifically, HEK293 cells (ATCC, CRL-1573) were obtained, which were stably transduced with pGL4.32 luciferase reporter vector (Promega K.K., E8491) having an NFkB transcriptional response element incorporated therein (hereinafter referred to as NFkB/HEK293 cells), and used for evaluation.

[0081]

NFkB/HEK293 cells were suspended in DMEM containing 10% fetal calf serum (Sigma, D6429) at 1.25×10 ⁵ cells/ml and plated at 80 μl/well in a white 96-well clear bottom plate (Corning Incorporated, 3610). After culturing for 2 hours in a CO ₂ incubator set at 37°C with an atmosphere of 5% CO ₂ , a 12-step dilution series was prepared for the purified antibody prepared according to Example 2 in the culture medium described above with approximately 3-fold denominator progression from a final concentration of 1 ng/ml to 300 μg/ml, and then added in an amount of 20 μl/well. After culturing overnight at 37° C. in 5% CO ₂ , luciferase expression was measured using the ONE-Glo Luciferase Assay System (Promega Corporation) to quantify NFkB activation.

[0082]

As a result, an anti-human Fn14 antibody was selected that did not induce NFkB activation; did not show agonist activity and was named 4-1.

[0083]

(Example 4: Preparation of fully humanized antibody and murinized antibody)

The genes encoding the heavy chain and light chain of the antibody were cloned from hybridomas producing the antibody selected in Example 3, and the sequence was determined. Once the antibody sequence has been determined, genes encoding signal sequences (Wittle N et al., Protein Engineering. 1987, Vol. 1, No. 6, p. 499-505) and human Igγ1 constant region genes (consisting of the base sequence at base positions 376 -1365 SEQ ID NO: 1) having the amino acid mutations L234A and L235A, respectively, were ligated at the 5' side and 3' side of the heavy chain variable region genes, and the heavy chain genes were inserted into the pEE6.4 GS vector (Lonza). In addition, genes encoding signal sequences (Wittle N et al., Protein Engineering. 1987, Vol. 1, No. 6, p. 499-505) and constant region genes (consisting of a sequence of bases at base positions 343 to 660 SEQ ID NO: 3) Human κ chains were respectively ligated to the 5' side and 3' side of the light chain variable region genes, and the light chain genes were inserted into the pEE12.4 GS vector. These GS vectors were fragmented with NotI-HF and PvuI-HF restriction enzymes and ligated using <Mighty Mix> DNA Ligation Kit (Takara Bio Inc., 6023) to obtain a GS vector in which both heavy chain genes and and light chain genes. The antibody was purified in a conventional manner from the culture supernatant of CHO-K1SV cells obtained by vector transfection, and thus a fully humanized 4-1 antibody was obtained and was named 4-1h.

[0084]

The base sequence of the resulting heavy chain 4-1h corresponds to SEQ ID NO: 1, the amino acid sequence encoded by it corresponds to SEQ ID NO: 2, the base sequence of the light chain corresponds to SEQ ID NO: 3, and the amino acid sequence encoded by it corresponds to SEQ ID NO : 4. The heavy chain variable region corresponding to SEQ ID NO: 2 consists of the amino acid sequence from amino acid positions 1-125 of SEQ ID NO: 2, and the light chain variable region corresponding to SEQ ID NO: 4 consists of the amino acid sequence from positions amino acids 1-114 of SEQ ID NO: 4. Heavy chain variable region CDR1, CDR2 and CDR3 4-1h consist of the amino acid sequences of amino acid positions 31-35, 50-65 and 98-114 of SEQ ID NO: 2, respectively. Light chain variable region CDR1, CDR2 and CDR3 4-1h consist of amino acid sequences at amino acid positions 24-40, 56-62 and 95-103 of SEQ ID NO: 4, respectively.

[0085]

Murinized antibody 4-1h (hereinafter referred to as 4-1m) was obtained to reduce the immunogenic risk in the evaluation of antibodies against human Fn14 by in vivo test in mice. The base sequence encoding the 4-1m variable region was generated by partially replacing the 4-1h light chain and heavy chain framework (FR) with the FRs of other mouse antibodies.

[0086]

4-1m was obtained using the same method as the method for obtaining a vector, expression of antibodies and purification for 4-1h, described above. For the heavy chain constant region, genes encoding a mouse Igγ2a constant region (consisting of the sequence of bases at base positions 376-1365 of SEQ ID NO: 5) having the D265A amino acid mutation were used, and for the light chain constant region, constant region genes (consisting of the sequence bases at base positions 343-660 of SEQ ID NO: 7) mouse κ chain.

[0087]

The base sequence of the resulting heavy chain 4-1m corresponds to SEQ ID NO: 5, the amino acid sequence encoded by it corresponds to SEQ ID NO: 6, the light chain sequence corresponds to SEQ ID NO: 7, and the amino acid sequence encoded by it corresponds to SEQ ID NO: 8. The heavy chain variable region corresponding to SEQ ID NO: 6 consists of the amino acid sequence of amino acid positions 1-125 of SEQ ID NO: 6, and the light chain variable region corresponding to SEQ ID NO: 8 consists of the amino acid sequence of amino acid positions 1-114 SEQ ID NO: 8. CDR1, CDR2 and CDR3 of the heavy chain variable region 4-1m consists of amino acid sequences at amino acid positions 31-35, 50-65 and 98-114 of SEQ ID NO: 6, respectively. Light chain variable region CDR1, CDR2 and CDR3 4-1m consists of amino acid sequences at amino acid positions 24-40, 56-62 and 95-103 of SEQ ID NO: 8, respectively.

[0088]

(Example 5: Amino acid modification analysis of a fully humanized antibody)

Purified 4-1h was subjected to amino acid modification analysis and as a result most of the purified antibodies had pyroglutamylation at the N-terminus of the heavy chain and a lysine deletion at the C-terminus.

[0089]

(Example 6: Human and mouse Fn14 binding ELISA for fully humanized antibody and murinized antibody)

Conducted an assessment of the activity of binding 4-1h and 4-1m, obtained according to example 4, with the protein Fn14. Fn14 human-human Fc fusion protein and mouse Fn14-mouse Fc fusion protein prepared according to Example 1 were diluted with phosphate buffered saline (PBS) at 1 μg/ml and added to a MaxiSorp 384-well transparent plate (Nunc, 464718) in amount of 15 µl/well. Incubation was carried out overnight at 4°C for immobilization. The next day, the solid phase liquid was removed and washed with 0.05% Tween-20 Tris-buffered saline (TBS-T) was performed. To this was added PBS containing 20% Blocking One (Nacalai tesque, Inc., 03953-95) at 50 μl/well. The resulting material was left to stand at room temperature for 1 hour and then washed with TBS-T. Received a series of dilutions 4-1h or 4-1m, obtained according to example 4, by dilution of 12 steps with a 4-fold denominator progression from a maximum concentration of 30 μg/ml using TBS-T containing 5% Blocking One (hereinafter referred to as diluent) , and added in an amount of 20 μl/well. After incubation for 1 hour at room temperature, washing with TBS-T was performed. Horseradish peroxidase-labeled anti-human kappa light chain (SouthernBiotech, 2060-05) as a secondary antibody, diluted 5000-fold with diluent, at 20 µl/well for 4-1h detection, and horseradish peroxidase-labelled anti-mouse kappa light chain were added. (SouthernBiotech, 1050-05) as a secondary antibody, diluted 4000 times with diluent at 20 µl/well for 4-1m detection. After incubation for 1 hour at room temperature, washing with TBS-T was performed. Microwell TMB peroxidase substrate (Kirkegaard & Perry Laboratories, Inc., 50-76-03) was added and the resulting mixture was allowed to stand for 3 minutes for a 4-1h score or 5 minutes for a 4-1m score. The reaction was then stopped by adding 2 M sulfuric acid and absorbance was measured at 450 nm using a SpectraMax Paradigm (Molecular Devices, LLC). The test was performed in duplicate and EC ₅₀ values were calculated by 4-parameter logistic curve fitting.

[0090]

As a result, 4-1h and 4-1m obtained according to Example 4 were confirmed to have binding activity for human Fn14 and mouse Fn14 (Table 1).

Table 1: 4-1h and 4-1m binding activity to human Fn14 and mouse Fn14

[Table 1]

Name of the antibody EC ₅₀ (ng/ml) Person Mouse 4-1h 30.4 23.2 4-1m 18.2 16.5

[0091]

(Example 7: Tweak-Inducible NFkB Activation Inhibition Assay for a Fully Humanized Antibody)

To assess the activity of 4-1h, obtained according to example 4, as an Fn14 antagonist, the inhibitory effect induced by Tweak on NFkB activation was evaluated by reporter analysis.

[0092]

NFkB/HEK293 cells prepared according to Example 3 were suspended in DMEM containing 10% fetal calf serum at 1.25×10 ⁵ cells/ml and plated at 80 μl/well in a white 96-well clear bottom plate. . After culturing overnight at 37° C. with 5% CO ₂ a dilution series of 11 steps was obtained for 4-1h prepared according to example 4 in the above culture medium with approximately 3 times the progression denominator from the final concentration of 0.1 ng/ ml to 10 µg/ml and then added at 10 µl/well. After culturing for 30 minutes at 37° C. with 5% CO ₂ 10 μl of Tweak (PeproTech, Inc., 310-06) was added to a final concentration of 100 ng/ml. After cultivation for 5 hours at 37°C with 5% CO ₂ , NFkB activation was quantified according to the method of Example 3. The group in which only antibody-free culture medium was added was selected as the control group, the group with the addition of Tweak was determined as 0% inhibition, and the group without Tweak addition was defined as 100% inhibition to calculate the 50% inhibition concentration (IC ₅₀ value) of the antibody by 4-parameter logistic curve fitting. Antibodies were tested in duplicate and the IC ₅₀ values of the three trials were geometrically averaged to calculate a 95% confidence interval (Table 2). In this test, mouse anti-human Fn14 antibody CRCBT-06-002 (Patent Document 2) was used as a comparison antibody.

[0093]

As a result, 4-1h was found to have higher agonist activity than CRCBT-06-002.

Table 2: 4-1h inhibitory activity relative to Tweak-induced NFkB activation

[Table 2]

Name of the antibody IC ₅₀ (ng/ml)
(95% confidence interval) 4-1h 41.5
(27.2-63.4) CRCBT-06-002 115
(72.7-183)

[0094]

(Example 8: IL-8 Production Assay for Fully Humanized Antibody)

The functional activity of 4-1h, obtained according to example 4, was evaluated by analyzing the production of IL-8 in vitro . A375 cells (ATCC, CRL-1619) were suspended in DMEM containing 10% fetal calf serum at 5×10 ⁴ cells/ml and plated at 100 μl/well in a 96 well flat bottom plate (IWAKI CO. , ltd., 3860-096). After culturing overnight at 37°C with 5% CO ₂ was washed with PBS. Received a series of 10-fold dilutions 4-1h, obtained according to example 4, at a final concentration of 1 ng/ml to 100 μg/ml in the culture medium described above and added to the cells. After culturing overnight at 37° C. with 5% CO ₂ in the presence of Tweak at a final concentration of 5 ng/ml (assessment of antagonist activity) or in the absence of Tweak (assessment of agonist activity), the culture supernatant was collected and the concentration of IL-8 was measured in culture supernatant using the Quantikine human IL-8/CXCL8 ELISA kit (R&D Systems, D8000C). The final volumes in the evaluation of antagonist activity and the evaluation of agonist activity in the experiments were 65 μl/well and 100 μl/well, respectively. In this test, antibodies were tested in duplicate using CRCBT-06-002 as the reference antibody.

[0095]

In evaluating antagonist activity, the group in which only antibody-free culture medium was added was selected as the control group, the Tweak-supplemented group was defined as 0% inhibition, and the Tweak-free group was defined as 100% inhibition to calculate the level of inhibition. The arithmetic mean of the maximum levels of inhibition ± standard error in each of the four experiments are presented in table 3 (table 3). Antagonist activity is plotted in FIG. 1 and agonist activity is plotted in FIG. 2.

[0096]

As shown in Table 3 and Figure 1, 4-1h was found to show almost complete inhibitory activity on Tweak stimulation-induced IL-8 production, while CRCBT-06-002 only showed partial inhibitory activity. In addition, as shown in Figure 2, CRCBT-06-002 induced IL-8 production by itself in the absence of Tweak, while 4-1h of the present invention hardly induced IL-8 production.

[0097]

From the above, it was found that CRCBT-06-002 is a partial antagonist antibody having an agonist action, while 4-1h is a full antagonist antibody having a small agonist action against human Fn14.

Table 3: Inhibitory activity of 4-1h and CRCBT-06-002 on IL-8 production induced by Tweak stimulation

[Table 3]

Name of the antibody Maximum inhibition level (%)
Mean ± standard error 4-1h 96.4±0.4 GRCBT-06-002 53.0±6.8

[0098]

(Example 9: Action in a mouse model of cachexia of cancer patients)

Mice harboring C26 mouse colon cancer cell line (COLON 26) are one representative example of cancer patient cachexia models that exhibit weight loss and muscle mass reduction that are characteristics of cachexia (Aulino P et al., BCM Cancer. 2010, Vol. 10 363, p. 1-15) (Bonetto A et al., J Vis Exp. 2016, Nov. 30 117, p. 1-21).

[0099]

C26 cells (National Cancer Institute) suspended at 10 ⁶ cells/100 µl using a phenol red free BD basement membrane based on matrigel matrix (BD, 356237) were injected subcutaneously at 100 µl to male CD2F1 mice (Charles River Laboratories Japan Inc.) to produce a mouse that had a C26 tumor. After confirming that the body weight of the C26 tumor-bearing mice began to decrease, administration of the antibody was started. Specifically, 4-1m diluted with PBS was subcutaneously administered to mice bearing a C26 tumor at a dose of 3 mg/kg (dosage: 10 ml/kg) on the 25th, 28th, 30th, 32nd, and 35th day after cell injection. In the control group, an isotype control antibody was administered in a similar manner. Mouse anti-keyhole limpet hemocyanin (KLH) murine IgG2a, which is an antigen that does not exist in a biological body, was purchased and used according to a conventional method as an isotype control antibody. Administration started with 15 mice in each group. Two mice in the 4-1m administration group and seven mice in the control group died by day 37, which was the day the experiment ended.

[0100]

One day before the end of the experiment (day 36), the compression force was measured using a small animal compression force measuring device (MELQUEST Co., Ltd., GPM-100B). Body weight was measured at day 37, and the weight of the isolated tibialis anterior was measured as muscle mass. With regard to the scores determined on day 36 and day 37, a significant difference was determined between the 4-1m administration group and the control group using Student's t-test. In all cases, a p<0.05 value was considered significant and in the case where there was a significant difference, the symbol (*) is present on the graph (the symbol ** on the graph indicates that there was a significant difference with p <0.01).

[0101]

The change in body weight, consistently measured up to the 37th day, is shown in Fig.3. The weight loss progressed even after the start of administration in the control group. However, the decrease in body weight was suppressed in the 4-1m administration group and a significant increase in body weight was observed in the 4-1m administration group compared to the control group on day 37 (P=0.0019). In addition, an increase in muscle mass was observed in the 4-1m administration group (FIG. 4, p=0.0009). Moreover, the contraction force was also significantly increased (Fig. 5, p=0.0003) in the 4-1m injection group, and not only an increase in muscle mass was shown, but also an improvement in muscle function.

[0102]

The results described above showed that 4-1m is effective against cachexia and can be effective even when administered therapeutically after observing weight loss.

[0103]

(Example 10: Effect on Survival in a Mouse Model of Cancer Cachexia)

C26 tumor-bearing mice were prepared in the same manner as in Example 9. Administration was started to 5 mice in each group at the point in time when the body weight of C26 tumor-bearing mice began to decline. Specifically, 4-1m diluted in PBS was administered subcutaneously at a dose of 0.3 mg/kg (dosage: 10 ml/kg) three times a week from days 22 to 70 after cell injection, and then on days 71- e, 76th, 78th, 83rd and 85th days. In the control group, the same isotype control antibody was administered as in Example 9, in the same way as 4-1m. Gemcitabine (trade name: GEMZAR, Eli Lilly and Company) diluted in PBS was administered subcutaneously in the gemcitabine administration group at a dose of 10 mg/kg (dosage: 10 ml/kg). Both 4-1m and gemcitabine were administered in the gemcitabine-4-1m combination group on the same day. The long diameter and short diameter were successively measured using a thickness gauge (ASONE, VC01-150), and the tumor volume was calculated by the following formula.

Tumor volume=long diameter (mm) × short diameter (mm) × short diameter (mm)/2

Mice survival is presented as Kaplan-Meier survival curves in FIG. When differences in survival curves between groups were compared, testing was performed using a logarithmic rank test and p<0.05 was considered significant. The median survival times in the control group, the 4-1m administration group, and the gemcitabine administration group were 41 days, 57 days, and 55 days, respectively. Median survival in the gemcitabine-4-1m combination group was 83 days, and a significant difference was observed compared to the survival curve for the 4-1m alone or gemcitabine alone administration group.

Both when the antibody was administered alone and when the antibody was administered in combination with gemcitabine, there was no effect of 4-1m administration on tumor volume (FIG. 7).

The results described above indicated that 4-1m can prolong survival without affecting tumor size and may be more effective in improving survival when used in combination with anti-cancer agents such as gemcitabine.

Industrial Applicability

[0104]

The anti-human Fn14 antibody of the present invention is useful in the prevention or treatment of various diseases in which human Fn14 is involved. In addition, the method for producing a polynucleotide, an expression vector, a transformed host cell, or an antibody of the present invention is expected to be suitable for the production of an anti-human Fn14 antibody.

Free text sequence listing

[0105]

The numeric heading <223> of the sequence listing below provides a description of the "artificial sequence". In particular, the base sequences corresponding to SEQ ID NOs: 1, 3, 5 and 7 of the sequence listing are, respectively, the base sequences of the heavy chain and light chain of the anti-human Fn14 antibody, and the amino acid sequences corresponding to SEQ ID NOs: 2, 4 , 6 and 8 are, respectively, the amino acid sequences of the heavy chain and light chain encoded by SEQ ID NO: 1, 3, 5 and 7. The amino acid sequence corresponding to SEQ ID NO: 9 is the sequence of a peptide linker that binds the human Fn14 protein and a human Fc region protein.

--->

SEQUENCE LIST

<110> Astellas Pharma Inc.

<120> ANTIBODY AGAINST HUMAN FN14

<130> A19011A00

<150> JP 2018-205995

<151> 2018-10-31

<160> 9

<170>PatentIn version 3.5

<210> 1

<211> 1365

<212> DNA

<213> Artificial sequence

<220>

<223> H chain antibody 4-1h

<220>

<221> CDS

<222> (1)..(1365)

<400> 1

cag gtg cag cta cag cag tgg ggc gca gga ctg ttg aag cct tcg gag 48

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

acc ctg tcc ctc acc tgc gct gtc tat ggt ggg tcc ttc agt ggt tac 96

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

tac tgg agc tgg atc cgc cag ccc cca ggg aag ggg ctg gag tgg att 144

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

ggg gaa atc aat cat cgt gga agc acc aac tcc aac ccg tcc ctc aag 192

Gly Glu Ile Asn His Arg Gly Ser Thr Asn Ser Asn Pro Ser Leu Lys

50 55 60

agt cga gac acc ata tca gta gac acg tcc aag aac cag ttc tcc ctg 240

Ser Arg Asp Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

agg ctg agg tct gtg acc gcc gcg gac acg gct gtg tat tac tgt gcg 288

Arg Leu Arg Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

cgc gag ggt ata agt gga agt atg ggg atc tac gac tac agc gga atg 336

Arg Glu Gly Ile Ser Gly Ser Met Gly Ile Tyr Asp Tyr Ser Gly Met

100 105 110

gac gtc tgg ggc caa ggg acc acg gtc acc gtc tcc tca gcc tcc acc 384

Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr

115 120 125

aag ggc cca tcg gtc ttc ccc ctg gca ccc tcc tcc aag agc acc tct 432

Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser

130 135 140

ggg ggc aca gcg gcc ctg ggc tgc ctg gtc aag gac tac ttc ccc gaa 480

Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu

145 150 155 160

ccg gtg acg gtg tcg tgg aac tca ggc gcc ctg acc agc ggc gtg cac 528

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

165 170 175

acc ttc ccg gct gtc cta cag tcc tca gga ctc tac tcc ctt agt agc 576

Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

180 185 190

gtg gtg acc gtg ccc tcc agc agc ttg ggc acc cag acc tac atc tgc 624

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys

195 200 205

aac gtg aat cac aag ccc agc aac acc aag gtg gac aag aaa gtt gag 672

Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu

210 215 220

ccc aaa tct tgt gac aaa act cac aca tgc cca ccg tgc cca gca cct 720

Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

225 230 235 240

gaa gcc gct ggg gga ccg tca gtc ttc ctc ttc ccc cca aaa ccc aag 768

Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

245 250 255

gac acc ctc atg atc tcc cgg acc cct gag gtc aca tgc gtg gtg gtg 816

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

260 265 270

gac gtg agc cac gaa gac cct gag gtc aag ttc aac tgg tac gtg gac 864

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

275 280 285

ggc gtg gag gtg cat aat gcc aag aca aag ccg cgg gag gag cag tac 912

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

290 295 300

aac agc acg tac cgt gtg gtc agc gtc ctc acc gtc ctg cac cag gac 960

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

305 310 315 320

tgg ctg aat ggc aag gag tac aag tgc aag gtc tcc aac aaa gcc ctc 1008

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

325 330 335

cca gcc ccc atc gag aaa acc atc tcc aaa gcc aaa ggg cag ccc cga 1056

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

340 345 350

gaa cca cag gtg tac acc ctg ccc cca tcc cgg gat gag ctg acc aag 1104

Glu Pro Gln Val Tyr Thr Leu Pro Ser Arg Asp Glu Leu Thr Lys

355 360 365

aac cag gtc agc ctg acc tgc ctg gtc aaa ggc ttc tat ccc agc gac 1152

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

370 375 380

atc gcc gtg gag tgg gag agc aat ggg cag ccg gag aac aac tac aag 1200

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

385 390 395 400

acc acg cct ccc gtg ctg gac tcc gac ggc tcc ttc ttc ctc tac agc 1248

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

405 410 415

aag ctc acc gtg gac aag agc agg tgg cag cag ggg aac gtc ttc tca 1296

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

420 425 430

tgc tcc gtg atg cat gag gct ctg cac aac cac tac acg cag aag agc 1344

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

435 440 445

ctc tcc ctg tct ccg ggt aaa 1365

Leu Ser Leu Ser Pro Gly Lys

450 455

<210> 2

<211> 455

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 2

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asn His Arg Gly Ser Thr Asn Ser Asn Pro Ser Leu Lys

50 55 60

Ser Arg Asp Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Arg Leu Arg Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Glu Gly Ile Ser Gly Ser Met Gly Ile Tyr Asp Tyr Ser Gly Met

100 105 110

Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr

115 120 125

Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser

130 135 140

Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu

145 150 155 160

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

165 170 175

Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

180 185 190

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys

195 200 205

Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu

210 215 220

Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

225 230 235 240

Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

245 250 255

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

260 265 270

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

275 280 285

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

290 295 300

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

305 310 315 320

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

325 330 335

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

340 345 350

Glu Pro Gln Val Tyr Thr Leu Pro Ser Arg Asp Glu Leu Thr Lys

355 360 365

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

370 375 380

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

385 390 395 400

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

405 410 415

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

420 425 430

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

435 440 445

Leu Ser Leu Ser Pro Gly Lys

450 455

<210> 3

<211> 660

<212> DNA

<213> Artificial sequence

<220>

<223> Antibody L chain 4-1h

<220>

<221> CDS

<222> (1)..(660)

<400> 3

gac atc gtg atg acc cag tct cca gac tcc ctg gct gtg tct ctg ggc 48

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

gag agg gcc acc atc aac tgc aag tcc agg cag agt att tta tat agt 96

Glu Arg Ala Thr Ile Asn Cys Lys Ser Arg Gln Ser Ile Leu Tyr Ser

20 25 30

tcc aac aat aag aac tac tta act tgg tac cag cag aaa cca gga cag 144

Ser Asn Asn Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

cct cct aag ttg ctc att tac tgg gca tct acc cgg gaa tcc ggg gtc 192

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

cct gac cga ttc agt ggc agc ggg tct ggg aca gat ttc act ctc acc 240

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

atc agc agc ctg cag gct gaa gat gtg gca gtt tat tac tgt cag caa 288

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln

85 90 95

tat tat agt act cct tac act ttt ggc cag ggg acc aag ctg gag atc 336

Tyr Tyr Ser Thr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile

100 105 110

aaa cgg act gtg gct gca cca tct gtc ttc atc ttc ccg cca tct gat 384

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

115 120 125

gag cag ttg aaa tct gga act gcc tct gtt gtg tgc ctg ctg aat aac 432

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

130 135 140

ttc tat ccc aga gag gcc aaa gta cag tgg aag gtg gat aac gcc ctc 480

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

145 150 155 160

caa tcg ggt aac tcc cag gag agt gtc aca gag cag gac agc aag gac 528

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

165 170 175

agc acc tac agc ctc agc agc acc ctg acg ctg agc aaa gca gac tac 576

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

180 185 190

gag aaa cac aaa gtc tac gcc tgc gaa gtc acc cat cag ggc ctg agc 624

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

195 200 205

tcg ccc gtc aca aag agc ttc aac agg gga gag tgt 660

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215 220

<210> 4

<211> 220

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 4

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Arg Gln Ser Ile Leu Tyr Ser

20 25 30

Ser Asn Asn Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln

85 90 95

Tyr Tyr Ser Thr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile

100 105 110

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

115 120 125

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

130 135 140

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

145 150 155 160

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

165 170 175

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

180 185 190

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

195 200 205

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215 220

<210> 5

<211> 1368

<212> DNA

<213> Artificial sequence

<220>

<223> Antibody H chain 4-1m

<220>

<221> CDS

<222> (1)..(1368)

<400> 5

gat gtt cag ctg caa gag tct ggc cct ggc ctg gtc aag cct tct cag 48

Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

tct ctg tct ctg acc tgc gct gtg tac ggc ggc tct ttc tct ggc tac 96

Ser Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

tac tgg tcc tgg atc cgg cag ttc cct ggc aac aag ctg gaa tgg atg 144

Tyr Trp Ser Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp Met

35 40 45

ggc gag atc aac cac cgg ggc tcc acc aac tct aac ccc agc ctg aag 192

Gly Glu Ile Asn His Arg Gly Ser Thr Asn Ser Asn Pro Ser Leu Lys

50 55 60

tcc cgg atc tcc atc acc gtg gac acc tcc aag aac cag ttc ttt ctg 240

Ser Arg Ile Ser Ile Thr Val Asp Thr Ser Lys Asn Gln Phe Phe Leu

65 70 75 80

cag ctc aac tcc gtg aca acc gag gac acc gcc acc tac tac tgt gcc 288

Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys Ala

85 90 95

aga gag ggc atc tct ggc tcc atg ggc atc tac gac tac tcc ggc atg 336

Arg Glu Gly Ile Ser Gly Ser Met Gly Ile Tyr Asp Tyr Ser Gly Met

100 105 110

gat gtg tgg ggc cag ggc aca ctg gtt acc gtg tct gcc gct aag acc 384

Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala Ala Lys Thr

115 120 125

acc gct cct tcc gtg tat cct ctg gca cct gtg tgt ggc gac acc acc 432

Thr Ala Pro Ser Val Tyr Pro Leu Ala Pro Val Cys Gly Asp Thr Thr

130 135 140

gga agt tct gtg acc ctg gga tgt ctg gtc aag ggc tac ttc ccc gag 480

Gly Ser Ser Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu

145 150 155 160

cct gtg aca ctg acc tgg aac tct ggc tct ctg tcc tct ggc gtg cac 528

Pro Val Thr Leu Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His

165 170 175

acc ttt cca gcc gtg ctg cag tct gac ctg tac acc ctg tct agc tcc 576

Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser

180 185 190

gtg acc gtg acc tcc tct acc tgg cct agc cag tcc atc acc tgt aac 624

Val Thr Val Thr Ser Ser Thr Trp Pro Ser Gln Ser Ile Thr Cys Asn

195 200 205

gtg gcc cat cct gcc tcc agc acc aag gtg gac aag aag atc gag cct 672

Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Glu Pro

210 215 220

cgg ggc cct acc atc aag cct tgt cct cca tgc aag tgc ccc gct cct 720

Arg Gly Pro Thr Ile Lys Pro Cys Pro Pro Cys Lys Cys Pro Ala Pro

225 230 235 240

aat ctg ctc gga ggc ccc tcc gtg ttc atc ttc cca cct aag atc aag 768

Asn Leu Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Ile Lys

245 250 255

gac gtg ctg atg atc tcc ctg tct cct atc gtg acc tgc gtg gtg gtg 816

Asp Val Leu Met Ile Ser Leu Ser Pro Ile Val Thr Cys Val Val Val

260 265 270

gcc gtg tcc gag gat gat cct gac gtg cag atc agt tgg ttc gtg aac 864

Ala Val Ser Glu Asp Asp Pro Asp Val Gln Ile Ser Trp Phe Val Asn

275 280 285

aac gtg gaa gtg cac acc gct cag acc cag aca cac aga gag gac tac 912

Asn Val Glu Val His Thr Ala Gln Thr Gln Thr His Arg Glu Asp Tyr

290 295 300

aac agc acc ctg aga gtg gtg tct gcc ctg cct atc cag cat cag gat 960

Asn Ser Thr Leu Arg Val Val Ser Ala Leu Pro Ile Gln His Gln Asp

305 310 315 320

tgg atg tcc ggc aaa gag ttc aag tgc aaa gtg aac aac aag gac ctg 1008

Trp Met Ser Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Lys Asp Leu

325 330 335

cct gct cca atc gag cgg acc atc tct aag cct aag ggc tct gtc agg 1056

Pro Ala Pro Ile Glu Arg Thr Ile Ser Lys Pro Lys Gly Ser Val Arg

340 345 350

gcc cct cag gtg tac gtt ttg cct cca cct gag gaa gag atg acc aag 1104

Ala Pro Gln Val Tyr Val Leu Pro Pro Glu Glu Glu Met Thr Lys

355 360 365

aaa caa gtg acc ctg aca tgc atg gtc acc gac ttc atg ccc gag gac 1152

Lys Gln Val Thr Leu Thr Cys Met Val Thr Asp Phe Met Pro Glu Asp

370 375 380

atc tac gtg gaa tgg acc aac aac ggc aag acc gag ctg aac tac aag 1200

Ile Tyr Val Glu Trp Thr Asn Asn Gly Lys Thr Glu Leu Asn Tyr Lys

385 390 395 400

aac acc gag cca gtg ctg gac tcc gac ggc tcc tac ttc atg tac tcc 1248

Asn Thr Glu Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe Met Tyr Ser

405 410 415

aag ctg cgc gtc gag aag aag aac tgg gtc gag aga aac tcc tac tcc 1296

Lys Leu Arg Val Glu Lys Lys Asn Trp Val Glu Arg Asn Ser Tyr Ser

420 425 430

tgc tcc gtg gtg cac gag ggc ctg cac aat cac cac acc acc aag tcc 1344

Cys Ser Val Val His Glu Gly Leu His Asn His His Thr Thr Lys Ser

435 440 445

ttc tct cgg acc cct ggc aag tga 1368

Phe Ser Arg Thr Pro Gly Lys

450 455

<210> 6

<211> 455

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 6

Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Ser Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp Met

35 40 45

Gly Glu Ile Asn His Arg Gly Ser Thr Asn Ser Asn Pro Ser Leu Lys

50 55 60

Ser Arg Ile Ser Ile Thr Val Asp Thr Ser Lys Asn Gln Phe Phe Leu

65 70 75 80

Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys Ala

85 90 95

Arg Glu Gly Ile Ser Gly Ser Met Gly Ile Tyr Asp Tyr Ser Gly Met

100 105 110

Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala Ala Lys Thr

115 120 125

Thr Ala Pro Ser Val Tyr Pro Leu Ala Pro Val Cys Gly Asp Thr Thr

130 135 140

Gly Ser Ser Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu

145 150 155 160

Pro Val Thr Leu Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His

165 170 175

Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser

180 185 190

Val Thr Val Thr Ser Ser Thr Trp Pro Ser Gln Ser Ile Thr Cys Asn

195 200 205

Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Glu Pro

210 215 220

Arg Gly Pro Thr Ile Lys Pro Cys Pro Pro Cys Lys Cys Pro Ala Pro

225 230 235 240

Asn Leu Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Ile Lys

245 250 255

Asp Val Leu Met Ile Ser Leu Ser Pro Ile Val Thr Cys Val Val Val

260 265 270

Ala Val Ser Glu Asp Asp Pro Asp Val Gln Ile Ser Trp Phe Val Asn

275 280 285

Asn Val Glu Val His Thr Ala Gln Thr Gln Thr His Arg Glu Asp Tyr

290 295 300

Asn Ser Thr Leu Arg Val Val Ser Ala Leu Pro Ile Gln His Gln Asp

305 310 315 320

Trp Met Ser Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Lys Asp Leu

325 330 335

Pro Ala Pro Ile Glu Arg Thr Ile Ser Lys Pro Lys Gly Ser Val Arg

340 345 350

Ala Pro Gln Val Tyr Val Leu Pro Pro Glu Glu Glu Met Thr Lys

355 360 365

Lys Gln Val Thr Leu Thr Cys Met Val Thr Asp Phe Met Pro Glu Asp

370 375 380

Ile Tyr Val Glu Trp Thr Asn Asn Gly Lys Thr Glu Leu Asn Tyr Lys

385 390 395 400

Asn Thr Glu Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe Met Tyr Ser

405 410 415

Lys Leu Arg Val Glu Lys Lys Asn Trp Val Glu Arg Asn Ser Tyr Ser

420 425 430

Cys Ser Val Val His Glu Gly Leu His Asn His His Thr Thr Lys Ser

435 440 445

Phe Ser Arg Thr Pro Gly Lys

450 455

<210> 7

<211> 663

<212> DNA

<213> Artificial sequence

<220>

<223> Antibody L chain 4-1m

<220>

<221> CDS

<222> (1)..(663)

<400> 7

gac atc gtg atg tct cag agc cct tcc tct ctg gcc gtg tcc gtg gga 48

Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala Val Ser Val Gly

1 5 10 15

gag aaa gtg acc atg tcc tgc aag tcc cgg cag tcc atc ctg tac tcc 96

Glu Lys Val Thr Met Ser Cys Lys Ser Arg Gln Ser Ile Leu Tyr Ser

20 25 30

tcc aac aac aag aac tac ctg acc tgg tat cag cag aag ccc ggc cag 144

Ser Asn Asn Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

tct cct aag ctg ctg atc tac tgg gcc tcc acc aga gaa tct ggc gtg 192

Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

ccc gat aga ttc acc ggc tct ggc tct ggc acc gac ttt acc ctg acc 240

Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

atc tcc tcc gtg aag gcc gag gat ctg gct gtg tac tac tgc cag cag 288

Ile Ser Ser Val Lys Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln

85 90 95

tac tac agc acc cct tac acc ttt ggc tcc ggc acc aag ctg gaa atc 336

Tyr Tyr Ser Thr Pro Tyr Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile

100 105 110

aag aga gct gac gcc gct cct acc gtg tct atc ttc cca cct agc tcc 384

Lys Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser

115 120 125

gag cag ctg acc tct ggc gga gct tct gtc gtg tgc ttc ctg aac aac 432

Glu Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn

130 135 140

ttc tac ccc aag gac atc aac gtg aag tgg aag atc gac ggc tcc gag 480

Phe Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu

145 150 155 160

aga cag aac ggc gtg ctg aac tct tgg acc gac cag gac tcc aag gac 528

Arg Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp

165 170 175

agc acc tac tcc atg tcc tcc aca ctg acc ctg aca aag gac gag tac 576

Ser Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr

180 185 190

gag cgg cac aac tcc tat acc tgc gag gct acc cac aag acc tcc acc 624

Glu Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr

195 200 205

tct cca atc gtg aag tcc ttc aac cgg aac gag tgc tga 663

Ser Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys

210 215 220

<210> 8

<211> 220

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 8

Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala Val Ser Val Gly

1 5 10 15

Glu Lys Val Thr Met Ser Cys Lys Ser Arg Gln Ser Ile Leu Tyr Ser

20 25 30

Ser Asn Asn Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Val Lys Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln

85 90 95

Tyr Tyr Ser Thr Pro Tyr Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile

100 105 110

Lys Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser

115 120 125

Glu Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn

130 135 140

Phe Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu

145 150 155 160

Arg Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp

165 170 175

Ser Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr

180 185 190

Glu Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr

195 200 205

Ser Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys

210 215 220

<210> 9

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 9

Ile Glu Gly Arg Met Asp Gly Gly Gly

15

<---

Claims (80)

1. An antibody against human Fn14 (fibroblast growth factor inducible protein 14) or an antigen-binding fragment thereof, containing: heavy chain variable region containing CDR1 consisting of the amino acid sequence from amino acid positions 31-35 of SEQ ID NO:2, a CDR2 consisting of the amino acid sequence from amino acid positions 50-65 of SEQ ID NO:2, and CDR3 consisting of the amino acid sequence from amino acid positions 98-114 of SEQ ID NO:2; and light chain variable region containing CDR1 consisting of the amino acid sequence from amino acid positions 24-40 of SEQ ID NO:4, a CDR2 consisting of the amino acid sequence from amino acid positions 56-62 of SEQ ID NO:4, and CDR3 consisting of the amino acid sequence from amino acid positions 95-103 of SEQ ID NO:4. 2. An anti-human Fn14 antibody or antigen-binding fragment thereof according to claim 1, selected from the following (1) and (2): (1) an anti-human Fn14 antibody or antigen-binding fragment thereof, comprising a heavy chain variable region consisting of an amino acid sequence from amino acid positions 1-125 of SEQ ID NO:2, and a light chain variable region consisting of an amino acid sequence from amino acid positions 1-114 of SEQ ID NO:4; and (2) an antibody or antigen-binding fragment thereof generated by post-translational modification of an anti-human Fn14 antibody or antigen-binding fragment thereof according to (1). 3. An anti-human Fn14 antibody or antigen-binding fragment thereof according to claim 2, containing: a heavy chain variable region consisting of an amino acid sequence from amino acid positions 1-125 of SEQ ID NO:2; and a light chain variable region consisting of an amino acid sequence from amino acid positions 1-114 of SEQ ID NO:4. 4. An anti-human Fn14 antibody or antigen-binding fragment thereof according to claim 2, wherein the post-translational modification is pyroglutamylation at the N-terminus of the heavy chain variable region and/or a lysine deletion at the C-terminus of the heavy chain. 5. An anti-human Fn14 antibody or antigen-binding fragment thereof according to claim 2, containing: a heavy chain variable region consisting of an amino acid sequence from amino acid positions 1-125 of SEQ ID NO:2, in which glutamine at amino acid position 1 of SEQ ID NO:2 is modified to pyroglutamic acid; and a light chain variable region consisting of an amino acid sequence from amino acid positions 1-114 of SEQ ID NO:4. 6. An anti-human Fn14 antibody according to claim 2, selected from the following (3) and (4): (3) an anti-human Fn14 antibody containing a heavy chain consisting of the amino acid sequence corresponding to SEQ ID NO:2, and a light chain consisting of the amino acid sequence corresponding to SEQ ID NO:4; and (4) an anti-human Fn14 antibody generated by post-translational modification of the antibody according to (3). 7. Antibody against human Fn14 according to claim 6, containing: a heavy chain consisting of the amino acid sequence corresponding to SEQ ID NO:2; and a light chain consisting of the amino acid sequence corresponding to SEQ ID NO:4. 8. The anti-human Fn14 antibody of claim 6, wherein the post-translational modification is pyroglutamylation at the N-terminus of the heavy chain and/or deletion of a lysine at the C-terminus of the heavy chain. 9. An anti-human Fn14 antibody according to claim 8, comprising: a heavy chain consisting of an amino acid sequence from amino acid positions 1-454 of SEQ ID NO:2, in which glutamine at amino acid position 1 of SEQ ID NO:2 is modified to pyroglutamic acid; and a light chain consisting of the amino acid sequence corresponding to SEQ ID NO:4. 10. An anti-human Fn14 antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein the antigen-binding fragment is a single chain variable region fragment, Fab, Fab' or F(ab') ₂ . 11. A polynucleotide encoding the heavy chain variable region of an anti-human Fn14 antibody or antigen-binding fragment thereof according to claim 3. 12. A polynucleotide encoding the light chain variable region of an anti-human Fn14 antibody or antigen-binding fragment thereof according to claim 3. 13. An expression vector containing: polynucleotide according to claims 11 and/or 12. 14. A host cell for producing an anti-human Fn14 antibody or antigen-binding fragment thereof according to claim 3, wherein the cell is selected from the group consisting of: (a) a host cell transformed with an expression vector comprising a polynucleotide containing a base sequence encoding the heavy chain variable region of an anti-human Fn14 antibody or an antigen-binding fragment thereof according to claim 3, and a polynucleotide containing a base sequence encoding a light chain variable region of an anti-human Fn14 antibody. Fn14 human or its antigennegative fragment according to claim 3; (b) a host cell transformed with an expression vector containing a polynucleotide containing a base sequence encoding the heavy chain variable region of an anti-human Fn14 antibody or antigen-binding fragment thereof according to claim 3, and an expression vector containing a polynucleotide containing a base sequence encoding the variable region light chain of an anti-human Fn14 antibody or antigen-binding fragment thereof according to claim 3. 15. A host cell for obtaining the heavy chain variable region of an anti-human Fn14 antibody or antigen-binding fragment thereof according to claim 3, transformed with an expression vector containing a polynucleotide containing a base sequence encoding the heavy chain variable region of an anti-human Fn14 antibody or antigen-binding fragment thereof according to claim .3. 16. A host cell for obtaining a light chain variable region of an anti-human Fn14 antibody or an antigen-binding fragment thereof according to claim 3, transformed with an expression vector containing a polynucleotide containing a base sequence encoding a light chain variable region of an anti-human Fn14 antibody or an antigen-binding fragment thereof according to claim 16. .3. 17. A host cell for producing an anti-human Fn14 antibody according to claim 7, wherein the cell is selected from the group consisting of the following (a)-(b): (a) a host cell transformed with an expression vector comprising a polynucleotide comprising a base sequence encoding the heavy chain of the anti-human Fn14 antibody of claim 7 and a polynucleotide containing a base sequence encoding the light chain of the anti-human Fn14 antibody of claim 7; (b) a host cell transformed with an expression vector containing a polynucleotide containing a base sequence encoding the heavy chain of an anti-human Fn14 antibody according to claim 7 and an expression vector containing a polynucleotide containing a base sequence encoding a light chain of an anti-human Fn14 antibody according to claim 7 .7. 18. A host cell for producing the heavy chain of the anti-human Fn14 antibody of claim 7, wherein the host cell is transformed with an expression vector containing a polynucleotide containing a base sequence encoding the heavy chain of the anti-human Fn14 antibody of claim 7. 19. A host cell for producing the light chain of an anti-human Fn14 antibody according to claim 7, wherein the host cell is transformed with an expression vector containing a polynucleotide containing a base sequence encoding the light chain of an anti-human Fn14 antibody according to claim 7. 20. A method for producing an anti-human Fn14 antibody or antigen-binding fragment thereof, wherein the method includes: the step of cultivating a host cell selected from the group consisting of the following (A)-(C) to express an anti-human Fn14 antibody or an antigen-binding fragment thereof: (A) a host cell transformed with an expression vector comprising a polynucleotide comprising a base sequence encoding the heavy chain variable region of an anti-human Fn14 antibody or an antigen-binding fragment thereof according to claim 3, and a polynucleotide comprising a base sequence encoding a light chain variable region of the antibody, or its antigen-binding fragment; (B) a host cell transformed with an expression vector containing a polynucleotide containing a base sequence encoding the heavy chain variable region of an anti-human Fn14 antibody or antigen-binding fragment thereof according to claim 3, and an expression vector containing a polynucleotide containing a base sequence encoding the variable region the light chain of an antibody or antigen-binding fragment thereof; and (C) a host cell transformed with an expression vector containing a polynucleotide containing a base sequence encoding the heavy chain variable region of an anti-human Fn14 antibody or an antigen-binding fragment thereof according to claim 3, and a host cell transformed with an expression vector containing a polynucleotide containing the sequence bases encoding the variable region of the light chain of the antibody or its antigen-binding fragment. 21. A method for producing antibodies against human Fn14, including: a step of culturing a host cell selected from the group consisting of the following (D) to (F) to express an anti-human Fn14 antibody: (D) a host cell transformed with an expression vector containing a polynucleotide containing a base sequence encoding the heavy chain of an anti-human Fn14 antibody according to claim 7 and a polynucleotide containing a base sequence encoding a light chain of an antibody; (E) a host cell transformed with an expression vector containing a polynucleotide containing a base sequence encoding the heavy chain of an anti-human Fn14 antibody according to claim 7 and an expression vector containing a polynucleotide containing a base sequence encoding an antibody light chain; and (F) a host cell transformed with an expression vector containing a polynucleotide containing a base sequence encoding the heavy chain of an anti-human Fn14 antibody according to claim 7, and a host cell transformed with an expression vector containing a polynucleotide containing a base sequence encoding an antibody light chain against Fn14 man. 22. A pharmaceutical composition for suppressing inflammation by inhibiting human Fn14 activation induced by Tweak stimulation, comprising: an anti-human Fn14 antibody or antigen-binding fragment thereof according to any one of claims 1-10; and pharmaceutically acceptable excipient. 23. A pharmaceutical composition for suppressing inflammation by inhibiting human Fn14 activation induced by Tweak stimulation, comprising: an anti-human Fn14 antibody or antigen-binding fragment thereof according to claim 3; an anti-human Fn14 antibody or antigen-binding fragment thereof according to claim 5; and pharmaceutically acceptable excipient. 24. A pharmaceutical composition for suppressing inflammation by inhibiting human Fn14 activation induced by Tweak stimulation, comprising: an anti-human Fn14 antibody according to claim 7; an anti-human Fn14 antibody according to claim 9; and pharmaceutically acceptable excipient. 25. A pharmaceutical composition according to any one of claims 22 to 24, which is a pharmaceutical composition for the prevention or treatment of cancer cachexia. 26. A method for preventing or treating cancer cachexia, wherein the method includes: the step of administering a therapeutically effective amount of an anti-human Fn14 antibody or antigen-binding fragment thereof according to any one of claims 1-10. 27. An anti-human Fn14 antibody, or antigen-binding fragment thereof, according to any one of claims 1 to 10, which is for use in the prevention or treatment of cancer cachexia. 28. The use of an anti-human Fn14 antibody or antigen-binding fragment thereof according to any one of claims 1 to 10 for the manufacture of a pharmaceutical composition for the prevention or treatment of cancer cachexia. 29. Pharmaceutical composition for the prevention or treatment of cancer or cancer cachexia, containing: an anti-human Fn14 antibody or an antigen-binding fragment thereof according to any one of claims 1 to 10, as an active ingredient, wherein the pharmaceutical composition is used in combination with an anti-cancer agent. 30. Pharmaceutical composition for the prevention or treatment of cancer or cancer cachexia, containing: cancer remedy, wherein the pharmaceutical composition is used in combination with an anti-human Fn14 antibody or antigen-binding fragment thereof according to any one of claims 1-10.

Images