WO2021215469A1

WO2021215469A1 - PHARMACEUTICAL COMPOSITION AND METHOD FOR PREVENTING OR TREATING CANCER WITH COMBINED USE OF ANTI-HUMAN Fn14 ANTIBODY AND IMMUNE CHECKPOINT INHIBITOR

Info

Publication number: WO2021215469A1
Application number: PCT/JP2021/016166
Authority: WO
Inventors: 政勝川上; 亮永島
Original assignee: アステラス製薬株式会社
Priority date: 2020-04-22
Filing date: 2021-04-21
Publication date: 2021-10-28
Also published as: TW202206099A; WO2021214905A1

Abstract

The present invention addresses the problem of providing a pharmaceutical composition for preventing or treating cancer and a pharmaceutical composition for preventing or treating steroid myopathy. Provided are: a pharmaceutical composition for preventing or treating cancer that comprises an anti Fn14 antibody or an antigen-binding fragment thereof and an excipient, said pharmaceutical composition being to be used in combination with an immune checkpoint inhibitor; and a pharmaceutical composition for preventing or treating steroid myopathy that comprises an anti Fn14 antibody or an antigen-binding fragment thereof and an excipient.

Description

Pharmaceutical compositions and methods for preventing or treating cancer by using an anti-human Fn14 antibody in combination with an immune checkpoint inhibitor.

The present invention relates to a combination of an anti-human Fn14 antibody for the prevention or treatment of cancer and an immune checkpoint inhibitor, and an anti-human Fn14 antibody for the prevention or treatment of steroid myopathy.

Fibroblast growth factor-inducible 14 (Fn14) (also referred to as TNFRSF12A) is a member of the tumor necrosis factor receptor superfamily. In addition, Fn14 binds to a TNF-like weak apoptosis factor (TNF-like walk inducer of apoptosis; Tweek) and is also known as a Tweek receptor. Twake-dependent or independent activation of Fn14 activates the NFkB signaling pathway and controls cell proliferation, migration, differentiation, and apoptosis, as well as inflammation involved in angiogenesis, tissue damage, and regeneration. Is known (Non-Patent Document 1).

As for the association with cancer, it has been reported that Fn14 is overexpressed in various solid cancers (Non-Patent Document 2), and that Fn14 is involved in tumor progression and metastasis (non-patent document 2). Patent Document 3).

On the other hand, activation of Fn14 may induce the production of inflammatory cytokines and exacerbate the inflammatory state. As an agonist antibody against human Fn14, Enavatuzumab (Patent Document 1), which is being clinically developed, has been reported, but Enavatuzumab has been reported to be hepatotoxic in a phase I clinical trial (Non-Patent Document 4), and Enavatuzumab. It has been suggested that it may be due to the inflammation caused by the treatment (Non-Patent Document 5).

A mouse monoclonal antibody CRCBT-06-002 has been reported as an antibody that binds to human Fn14 and has antagonistic activity and does not have agonistic activity under specific conditions (Patent Document 2). CRCBT-06-002 has been reported to have antagonistic activity that inhibits Twake-stimulated IL-8 production in human malignant melanoma-derived cell line A375 cells, and efficacy in a mouse cancer cachexia model. However, the agonist activity that induces IL-8 production from A375 cells in the absence of Tweak remains (Patent Document 2). There are no known anti-Fn14 antagonist antibodies that have no agonist activity and can avoid unwanted side effects.

Cancer immunotherapy is being researched and developed as an epoch-making treatment method for advanced cancer that is resistant to conventional treatment. However, most cancer patients are refractory to this treatment, and there is a long-awaited, more powerful and innovative immunotherapy with single or combination therapies. In addition to solid cancer, Fn14 is upregulated in immune cells such as dendritic cells and macrophages when stimulated with an antigen, and analysis of Tweak knockout mice shows that Twake-Fn14 signal inhibition has a useful function in cancer immunotherapy. Although it has been suggested (Non-Patent Document 6), the antitumor effect of the anti-Fn14 antagonist antibody and the combined effect with existing cancer immunotherapy are unknown.

Excessive immune response is known as an adverse event that occurs in patients who receive immune checkpoint inhibitors. In order to deal with such adverse events, administration of steroids is being investigated (The Journal of experimental medicine, 2019, Vol. 216, p. 2701-2713).

Among the hormones produced in the adrenal cortex, steroids are drugs that synthesize glucocorticoid components, and have collagen diseases (rheumatoid arthritis, systemic lupus erythematosus, etc.), bronchial asthma, pneumonia, kidney disease through anti-inflammatory and anti-allergic effects. It is used as one of the standard therapies in various pathological conditions such as skin diseases and allergic diseases. On the other hand, steroids are known to have side effects (steroid myopathy or steroid-induced myopathy) that are accompanied by muscular atrophy and muscle weakness, but there are effective treatments other than discontinuation or dose reduction of steroids. Not done.

International Publication No. 2009/020933 International Publication No. 2013/026099

An object of the present invention is to provide a method for preventing or treating cancer by using an anti-human Fn14 antibody in combination with an immune checkpoint inhibitor, or to prevent or treat steroid myopathy with an anti-human Fn14 antibody. To provide a way to do it.

In completing the present invention, from CDR1 consisting of the amino acid sequences of amino acid numbers 31 to 35 of SEQ ID NO: 2, CDR2 consisting of the amino acid sequences of amino acid numbers 50 to 65 of SEQ ID NO: 2, and amino acid number 98 of SEQ ID NO: 2. A heavy chain variable region including CDR3 consisting of amino acid sequences up to 114, CDR1 consisting of amino acid numbers 24 to 40 of SEQ ID NO: 4, and CDR2 consisting of amino acid numbers 56 to 62 of SEQ ID NO: 4. , And an anti-human Fn14 antibody comprising a light chain variable region comprising CDR3 consisting of the amino acid sequences 95 to 103 of amino acid numbers 95 to 103 of SEQ ID NO: 4 (Examples 2-4). This antibody binds to human Fn14 (Example 6), inhibits NFkB activation and IL-8 production by Twake stimulation (Examples 7 and 8), and produces IL-8 in the absence of Twake. It was found that it did not induce (Example 8). As a result, an anti-human Fn14 antibody having antagonistic activity but not agonistic activity was provided. As a result of further considerable creative studies by the present inventors, the combined use of the antibody (Example 4) obtained by converting the above antibody into a mouse and an anti-PD-1 antibody suppresses tumor growth in a mouse cancer model. Finding that (Example 9), the present invention relating to the combined use of an anti-human Fn14 antibody and an immune checkpoint inhibitor was completed. Further, the anti-human Fn14 antibody was found to suppress a decrease in muscle strength and muscle mass in a mouse steroid myopathy model (Example 10), and the present invention relating to the application of the anti-Fn14 antibody to steroid myopathy was completed. I let you.

According to the present invention, for example, the following inventions [1] to [13] are provided.
[1]
A pharmaceutical composition comprising an anti-Fn14 antibody or an antigen-binding fragment thereof and an excipient for preventing or treating cancer, which is used in combination with an immune checkpoint inhibitor.
[2]
The pharmaceutical composition according to [1], wherein the anti-Fn14 antibody or an antigen-binding fragment thereof is an anti-Fn14 antibody or an antigen-binding fragment thereof that inhibits the activation of human Fn14 by Twake stimulation without exhibiting agonist activity against human Fn14. thing.
[3]
The pharmaceutical composition according to [1] or [2], wherein the immune checkpoint inhibitor is an anti-PD-1 antibody or an anti-PD-L1 antibody.
[4]
The pharmaceutical composition according to any one of [1] to [3], wherein the immune checkpoint inhibitor is an anti-PD-1 antibody.
[5]
Anti-PD-1 antibodies include nivolumab, pembrolizumab, pidirizumab, cemiplimab, spartarizumab, spartarizumab, MEDI0680, , AMP-224, PF-06801591, tivolumab, ABBV-181, BI 754091, or SHR-1210, the pharmaceutical composition according to [3] or [4].
[6]
The pharmaceutical composition according to any one of [1] to [3], wherein the immune checkpoint inhibitor is an anti-PD-L1 antibody.
[7]
Anti-PD-L1 antibodies are atezolizumab, durvalumab, BMS-936559, avelumab, rodapolimab, CX-072, FAZ053, KN035, or MD. Or the pharmaceutical composition according to [6].
[8]
The anti-Fn14 antibody or its antigen-binding fragment is CDR1 consisting of the amino acid sequences of amino acids 31 to 35 of SEQ ID NO: 2, CDR2 consisting of the amino acid sequences of amino acids 50 to 65 of SEQ ID NO: 2, and the amino acid of SEQ ID NO: 2. A heavy chain variable region containing CDR3 consisting of amino acid sequences of numbers 98 to 114, CDR1 consisting of amino acid numbers 24 to 40 of amino acid number 4 of SEQ ID NO: 4, and amino acids of amino acid numbers 56 to 62 of SEQ ID NO: 4. An anti-human Fn14 antibody or antigen-binding fragment thereof, comprising a light chain variable region comprising CDR2 consisting of a sequence and CDR3 consisting of amino acid numbers 95 to 103 of amino acid numbers 95 to 103 of SEQ ID NO: 4, [1] to [7]. ] The pharmaceutical composition according to any one of.
[9]
The pharmaceutical composition according to any one of [1] to [8], wherein the anti-Fn14 antibody or an antigen-binding fragment thereof is an anti-human Fn14 antibody or an antigen-binding fragment thereof selected from the following (1) and (2). Stuff:
(1) An anti-human Fn14 antibody or an anti-human Fn14 antibody containing a heavy chain variable region consisting of the amino acid sequences of amino acid numbers 1 to 125 of SEQ ID NO: 2 and a light chain variable region consisting of the amino acid sequences of amino acid numbers 1 to 114 of SEQ ID NO: 4. The antigen-binding fragment; and (2) the antibody or antigen-binding fragment thereof produced by post-translational modification of the anti-human Fn14 antibody of (1) above or the antigen-binding fragment thereof.
[10]
The anti-Fn14 antibody or its antigen-binding fragment comprises a heavy chain variable region consisting of the amino acid sequences of amino acids 1 to 125 of SEQ ID NO: 2 and a light chain variable region consisting of the amino acid sequences of amino acids 1 to 114 of SEQ ID NO: 4. The pharmaceutical composition according to any one of [1] to [9], which comprises an anti-human Fn14 antibody or an antigen-binding fragment thereof.
[11]
The pharmaceutical composition according to any one of [1] to [9], wherein the anti-Fn14 antibody or an antigen-binding fragment thereof is an anti-human Fn14 antibody selected from the following (3) and (4):
(3) Anti-human Fn14 antibody comprising a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 2 and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 4; and (4) post-translational modification of the antibody of (3) above. Anti-human Fn14 antibody, which is an antibody produced by.
[12]
The anti-Fn14 antibody or its antigen-binding fragment is an anti-human Fn14 antibody containing a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 2 and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 4 [1] to [11]. ] The pharmaceutical composition according to any one of.
[13]
The pharmaceutical composition according to [9] or [11], wherein the post-translational modification is pyroglutamylation at the N-terminal of the heavy chain and / or lysine deletion at the C-terminal of the heavy chain.

Further, according to the present invention, for example, the following inventions [14] to [21] are provided.
[14]
A pharmaceutical composition for the prevention or treatment of steroid myopathy, which comprises an anti-Fn14 antibody or an antigen-binding fragment thereof and an excipient.
[15]
The pharmaceutical composition according to [14], wherein the anti-Fn14 antibody or an antigen-binding fragment thereof is an anti-Fn14 antibody or an antigen-binding fragment thereof that inhibits the activation of human Fn14 by Twake stimulation without exhibiting agonistic activity against human Fn14. thing.
[16]
The anti-Fn14 antibody or its antigen-binding fragment is CDR1 consisting of the amino acid sequences of amino acids 31 to 35 of SEQ ID NO: 2, CDR2 consisting of the amino acid sequences of amino acids 50 to 65 of SEQ ID NO: 2, and the amino acid of SEQ ID NO: 2. A heavy chain variable region containing CDR3 consisting of amino acid sequences of numbers 98 to 114, CDR1 consisting of amino acid numbers 24 to 40 of amino acid number 4 of SEQ ID NO: 4, and amino acids of amino acid numbers 56 to 62 of SEQ ID NO: 4. An anti-human Fn14 antibody or antigen-binding fragment thereof, comprising a light chain variable region comprising CDR2 consisting of the sequence and CDR3 consisting of the amino acid sequences 95 to 103 of amino acid numbers 95 to 103 of SEQ ID NO: 4, [14] or [15]. ] The pharmaceutical composition according to.
[17]
The pharmaceutical composition according to any one of [14] to [16], wherein the anti-Fn14 antibody or an antigen-binding fragment thereof is an anti-human Fn14 antibody or an antigen-binding fragment thereof selected from the following (1) and (2). Stuff:
(1) An anti-human Fn14 antibody or an anti-human Fn14 antibody containing a heavy chain variable region consisting of the amino acid sequences of amino acid numbers 1 to 125 of SEQ ID NO: 2 and a light chain variable region consisting of the amino acid sequences of amino acid numbers 1 to 114 of SEQ ID NO: 4. The antigen-binding fragment; and (2) the antibody or antigen-binding fragment thereof produced by post-translational modification of the anti-human Fn14 antibody of (1) above or the antigen-binding fragment thereof.
[18]
The anti-Fn14 antibody or its antigen-binding fragment comprises a heavy chain variable region consisting of the amino acid sequences of amino acids 1 to 125 of SEQ ID NO: 2 and a light chain variable region consisting of the amino acid sequences of amino acids 1 to 114 of SEQ ID NO: 4. The pharmaceutical composition according to any one of [14] to [17], which comprises an anti-human Fn14 antibody or an antigen-binding fragment thereof.
[19]
The pharmaceutical composition according to any one of [14] to [17], wherein the anti-Fn14 antibody or an antigen-binding fragment thereof is an anti-human Fn14 antibody selected from the following (3) and (4):
(3) Anti-human Fn14 antibody comprising a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 2 and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 4; and (4) post-translational modification of the antibody of (3) above. Anti-human Fn14 antibody, which is an antibody produced by.
[20]
The anti-Fn14 antibody or its antigen-binding fragment is an anti-human Fn14 antibody containing a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 2 and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 4 [14] to [19]. ] The pharmaceutical composition according to any one of.
[21]
The pharmaceutical composition according to [17] or [19], wherein the post-translational modification is pyroglutamylation at the N-terminal of the heavy chain and / or lysine deletion at the C-terminal of the heavy chain.

Further, according to the present invention, for example, the following inventions [22] to [34] are provided.
[22]
A method for preventing or treating cancer, which comprises the step of administering to a patient a therapeutically effective amount of an anti-Fn14 antibody or an antigen-binding fragment thereof and an immune checkpoint inhibitor.
[23]
The method according to [22], wherein the anti-Fn14 antibody or an antigen-binding fragment thereof is an anti-Fn14 antibody or an antigen-binding fragment thereof that inhibits the activation of human Fn14 by Twake stimulation without exhibiting agonist activity against human Fn14.
[24]
The method according to [22] or [23], wherein the immune checkpoint inhibitor is an anti-PD-1 antibody or an anti-PD-L1 antibody.
[25]
The method according to any one of [22] to [24], wherein the immune checkpoint inhibitor is an anti-PD-1 antibody.
[26]
Anti-PD-1 antibodies include nivolumab, pembrolizumab, pidirizumab, cemiplimab, spartarizumab, spartarizumab, MEDI0680, , AMP-224, PF-06801591, tivolumab, ABBV-181, BI 754091, or SHR-1210, according to [24] or [25].
[27]
The method according to any one of [22] to [24], wherein the immune checkpoint inhibitor is an anti-PD-L1 antibody.
[28]
Anti-PD-L1 antibodies are atezolizumab, durvalumab, BMS-936559, avelumab, rodapolimab, CX-072, FAZ053, KN035, or MD. Or the method according to [27].
[29]
The anti-Fn14 antibody or its antigen-binding fragment is CDR1 consisting of the amino acid sequences of amino acids 31 to 35 of SEQ ID NO: 2, CDR2 consisting of the amino acid sequences of amino acids 50 to 65 of SEQ ID NO: 2, and the amino acid of SEQ ID NO: 2. A heavy chain variable region containing CDR3 consisting of amino acid sequences of numbers 98 to 114, CDR1 consisting of amino acid numbers 24 to 40 of amino acid number 4 of SEQ ID NO: 4, and amino acids of amino acid numbers 56 to 62 of SEQ ID NO: 4. An anti-human Fn14 antibody or antigen-binding fragment thereof comprising a light chain variable region comprising CDR2 consisting of a sequence and CDR3 consisting of amino acid numbers 95 to 103 of amino acid numbers 95 to 103 of SEQ ID NO: 4, [22] to [28]. ] The method described in any of.
[30]
The method according to any one of [22] to [29], wherein the anti-Fn14 antibody or an antigen-binding fragment thereof is an anti-human Fn14 antibody or an antigen-binding fragment thereof selected from the following (1) and (2):
(1) An anti-human Fn14 antibody or an anti-human Fn14 antibody containing a heavy chain variable region consisting of the amino acid sequences of amino acid numbers 1 to 125 of SEQ ID NO: 2 and a light chain variable region consisting of the amino acid sequences of amino acid numbers 1 to 114 of SEQ ID NO: 4. The antigen-binding fragment; and (2) the antibody or antigen-binding fragment thereof produced by post-translational modification of the anti-human Fn14 antibody of (1) above or the antigen-binding fragment thereof.
[31]
The anti-Fn14 antibody or its antigen-binding fragment comprises a heavy chain variable region consisting of the amino acid sequences of amino acids 1 to 125 of SEQ ID NO: 2 and a light chain variable region consisting of the amino acid sequences of amino acids 1 to 114 of SEQ ID NO: 4. The method according to any one of [22] to [30], which comprises an anti-human Fn14 antibody or an antigen-binding fragment thereof.
[32]
The method according to any one of [22] to [30], wherein the anti-Fn14 antibody or an antigen-binding fragment thereof is an anti-human Fn14 antibody selected from the following (3) and (4):
(3) Anti-human Fn14 antibody comprising a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 2 and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 4; and (4) post-translational modification of the antibody of (3) above. Anti-human Fn14 antibody, which is an antibody produced by.
[33]
The anti-Fn14 antibody or its antigen-binding fragment is an anti-human Fn14 antibody containing a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 2 and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 4, [22] to [32]. ] The method according to any one of the items.
[34]
The method according to [30] or [32], 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.

Further, according to the present invention, for example, the following inventions [35] to [42] are provided.
[35]
A method for preventing or treating steroid myopathy, which comprises the step of administering to a patient a therapeutically effective amount of an anti-Fn14 antibody or an antigen-binding fragment thereof.
[36]
The method according to [35], wherein the anti-Fn14 antibody or an antigen-binding fragment thereof is an anti-Fn14 antibody or an antigen-binding fragment thereof that inhibits the activation of human Fn14 by Twake stimulation without exhibiting agonist activity against human Fn14.
[37]
The anti-Fn14 antibody or its antigen-binding fragment is CDR1 consisting of the amino acid sequences of amino acids 31 to 35 of SEQ ID NO: 2, CDR2 consisting of the amino acid sequences of amino acids 50 to 65 of SEQ ID NO: 2, and the amino acid of SEQ ID NO: 2. A heavy chain variable region containing CDR3 consisting of amino acid sequences of numbers 98 to 114, CDR1 consisting of amino acid numbers 24 to 40 of amino acid number 4 of SEQ ID NO: 4, and amino acids of amino acid numbers 56 to 62 of SEQ ID NO: 4. An anti-human Fn14 antibody or antigen-binding fragment thereof, comprising a light chain variable region comprising CDR2 consisting of the sequence and CDR3 consisting of the amino acid sequences 95 to 103 of amino acid numbers 95 to 103 of SEQ ID NO: 4, [35] or [36]. ] The method described in.
[38]
The method according to any one of [35] to [37], wherein the anti-Fn14 antibody or an antigen-binding fragment thereof is an anti-human Fn14 antibody or an antigen-binding fragment thereof selected from the following (1) and (2):
(1) An anti-human Fn14 antibody or an anti-human Fn14 antibody containing a heavy chain variable region consisting of the amino acid sequences of amino acid numbers 1 to 125 of SEQ ID NO: 2 and a light chain variable region consisting of the amino acid sequences of amino acid numbers 1 to 114 of SEQ ID NO: 4. The antigen-binding fragment; and (2) the antibody or antigen-binding fragment thereof produced by post-translational modification of the anti-human Fn14 antibody of (1) above or the antigen-binding fragment thereof.
[39]
The anti-Fn14 antibody or its antigen-binding fragment comprises a heavy chain variable region consisting of the amino acid sequences of amino acids 1 to 125 of SEQ ID NO: 2 and a light chain variable region consisting of the amino acid sequences of amino acids 1 to 114 of SEQ ID NO: 4. The method according to any of [35] to [38], which comprises an anti-human Fn14 antibody or an antigen-binding fragment thereof.
[40]
The method according to any one of [35] to [38], wherein the anti-Fn14 antibody or an antigen-binding fragment thereof is an anti-human Fn14 antibody selected from the following (3) and (4):
(3) Anti-human Fn14 antibody comprising a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 2 and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 4; and (4) post-translational modification of the antibody of (3) above. Anti-human Fn14 antibody, which is an antibody produced by.
[41]
The anti-Fn14 antibody or its antigen-binding fragment is an anti-human Fn14 antibody containing a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 2 and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 4, [35] to [40]. ] The method described in any of.
[42]
The method according to [38] or [40], 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.

Further, according to the present invention, for example, the following inventions [43] to [55] are provided.
[43]
An anti-Fn14 antibody or antigen-binding fragment thereof for use in the prevention or treatment of cancer, which is used in combination with an immune checkpoint inhibitor.
[44]
[43] The anti-Fn14 according to [43], wherein the anti-Fn14 antibody or an antigen-binding fragment thereof is an anti-Fn14 antibody or an antigen-binding fragment thereof that inhibits the activation of human Fn14 by Twake stimulation without exhibiting agonist activity against human Fn14. Antibodies or antigen-binding fragments thereof.
[45]
The anti-Fn14 antibody or antigen-binding fragment thereof according to [43] or [44], wherein the immune checkpoint inhibitor is an anti-PD-1 antibody or an anti-PD-L1 antibody.
[46]
The anti-Fn14 antibody or antigen-binding fragment thereof according to any one of [43] to [45], wherein the immune checkpoint inhibitor is an anti-PD-1 antibody.
[47]
Anti-PD-1 antibodies include nivolumab, pembrolizumab, pidirizumab, cemiplimab, spartarizumab, spartarizumab, MEDI0680, The anti-Fn14 antibody or antigen-binding fragment thereof according to [45] or [46], which is AMP-224, PF-06801591, tivolumab, ABBV-181, BI 754091, or SHR-1210.
[48]
The anti-Fn14 antibody or antigen-binding fragment thereof according to any one of [43] to [45], wherein the immune checkpoint inhibitor is an anti-PD-L1 antibody.
[49]
Anti-PD-L1 antibodies are atezolizumab, dulvalumab, BMS-936559, avelumab, rodapolimab, CX-072, FAZ053, KN035, or MD. Alternatively, the anti-Fn14 antibody or antigen-binding fragment thereof according to [48].
[50]
It consists of CDR1 consisting of the amino acid sequences of amino acid numbers 31 to 35 of SEQ ID NO: 2, CDR2 consisting of the amino acid sequences of amino acid numbers 50 to 65 of SEQ ID NO: 2, and the amino acid sequence of amino acid numbers 98 to 114 of SEQ ID NO: 2. The heavy chain variable region containing CDR3, CDR1 consisting of the amino acid sequences of amino acids 24 to 40 of SEQ ID NO: 4, CDR2 consisting of the amino acid sequences of amino acids 56 to 62 of SEQ ID NO: 4, and SEQ ID NO: 4 The anti-Fn14 antibody according to any one of [43] to [49], which is an anti-human Fn14 antibody or an antigen-binding fragment thereof, which comprises a light chain variable region containing CDR3 consisting of the amino acid sequences of amino acids 95 to 103. Or its antigen-binding fragment.
[51]
The anti-Fn14 antibody or antigen-binding fragment thereof according to any one of [43] to [50], which is an anti-human Fn14 antibody or an antigen-binding fragment thereof selected from the following (1) and (2):
(1) An anti-human Fn14 antibody or an anti-human Fn14 antibody containing a heavy chain variable region consisting of the amino acid sequences of amino acid numbers 1 to 125 of SEQ ID NO: 2 and a light chain variable region consisting of the amino acid sequences of amino acid numbers 1 to 114 of SEQ ID NO: 4. The antigen-binding fragment; and (2) the antibody or antigen-binding fragment thereof produced by post-translational modification of the anti-human Fn14 antibody of (1) above or the antigen-binding fragment thereof.
[52]
An anti-human Fn14 antibody or an antigen-binding fragment thereof containing a heavy chain variable region consisting of the amino acid sequences of amino acids 1 to 125 of SEQ ID NO: 2 and a light chain variable region consisting of the amino acid sequences of amino acids 1 to 114 of SEQ ID NO: 4. The anti-Fn14 antibody or antigen-binding fragment thereof according to any one of [43] to [51].
[53]
The anti-Fn14 antibody or antigen-binding fragment thereof according to any one of [43] to [51], which is an anti-human Fn14 antibody selected from the following (3) and (4):
(3) Anti-human Fn14 antibody comprising a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 2 and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 4; and (4) post-translational modification of the antibody of (3) above. Anti-human Fn14 antibody, which is an antibody produced by.
[54]
The anti-human Fn14 antibody according to any one of [43] to [53], which is an anti-human Fn14 antibody containing a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 2 and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 4. Fn14 antibody or an antigen-binding fragment thereof.
[55]
The anti-Fn14 antibody or antigen-binding fragment thereof according to [51] or [53], wherein the post-translational modification is polyglutamylation at the N-terminal of the heavy chain variable chain and / or lysine deletion at the C-terminal of the heavy chain.

Further, according to the present invention, for example, the following inventions [56] to [63] are provided.
[56]
An anti-Fn14 antibody or antigen-binding fragment thereof for use in the prevention or treatment of steroid myopathy.
[57]
[56] The anti-Fn14 according to [56], wherein the anti-Fn14 antibody or an antigen-binding fragment thereof is an anti-Fn14 antibody or an antigen-binding fragment thereof that inhibits the activation of human Fn14 by Twake stimulation without exhibiting agonist activity against human Fn14. Antibodies or antigen-binding fragments thereof.
[58]
It consists of CDR1 consisting of the amino acid sequences of amino acid numbers 31 to 35 of SEQ ID NO: 2, CDR2 consisting of the amino acid sequences of amino acid numbers 50 to 65 of SEQ ID NO: 2, and the amino acid sequence of amino acid numbers 98 to 114 of SEQ ID NO: 2. The heavy chain variable region containing CDR3, CDR1 consisting of the amino acid sequences of amino acids 24 to 40 of SEQ ID NO: 4, CDR2 consisting of the amino acid sequences of amino acids 56 to 62 of SEQ ID NO: 4, and SEQ ID NO: 4 The anti-Fn14 antibody according to any one of [56] or [57], which is an anti-human Fn14 antibody or an antigen-binding fragment thereof, which comprises a light chain variable region containing CDR3 consisting of the amino acid sequences of amino acids 95 to 103. Or its antigen-binding fragment.
[59]
The anti-Fn14 antibody or antigen-binding fragment thereof according to any one of [56] to [58], which is an anti-human Fn14 antibody or an antigen-binding fragment thereof selected from the following (1) and (2):
(1) An anti-human Fn14 antibody or an anti-human Fn14 antibody containing a heavy chain variable region consisting of the amino acid sequences of amino acid numbers 1 to 125 of SEQ ID NO: 2 and a light chain variable region consisting of the amino acid sequences of amino acid numbers 1 to 114 of SEQ ID NO: 4. The antigen-binding fragment; and (2) the antibody or antigen-binding fragment thereof produced by post-translational modification of the anti-human Fn14 antibody of (1) above or the antigen-binding fragment thereof.
[60]
An anti-human Fn14 antibody or an antigen-binding fragment thereof containing a heavy chain variable region consisting of the amino acid sequences of amino acids 1 to 125 of SEQ ID NO: 2 and a light chain variable region consisting of the amino acid sequences of amino acids 1 to 114 of SEQ ID NO: 4. The anti-Fn14 antibody or antigen-binding fragment thereof according to any one of [56] to [59].
[61]
The anti-Fn14 antibody or antigen-binding fragment thereof according to any one of [56] to [59], which is an anti-human Fn14 antibody selected from the following (3) and (4):
(3) Anti-human Fn14 antibody comprising a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 2 and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 4; and (4) post-translational modification of the antibody of (3) above. Anti-human Fn14 antibody, which is an antibody produced by.
[62]
The anti-Fn14 antibody according to any one of [56] to [61], which is an anti-human Fn14 antibody containing a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 2 and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 4. Or its antigen-binding fragment.
[63]
The anti-Fn14 antibody or antigen-binding fragment thereof according to [59] or [61], wherein the post-translational modification is polyglutamylation at the N-terminal of the heavy chain variable chain and / or lysine deletion at the C-terminal of the heavy chain.

Further, according to the present invention, for example, the following inventions [64] to [76] are provided.
[64]
Use of an anti-Fn14 antibody or antigen-binding fragment thereof in the manufacture of a pharmaceutical composition for the prevention or treatment of cancer, wherein the pharmaceutical composition is to be used in combination with an immune checkpoint inhibitor.
[65]
The use according to [64], wherein the anti-Fn14 antibody or antigen-binding fragment thereof is an anti-Fn14 antibody or antigen-binding fragment thereof that inhibits the activation of human Fn14 by Twake stimulation without exhibiting agonistic activity against human Fn14.
[66]
The use according to [64] or [65], wherein the immune checkpoint inhibitor is an anti-PD-1 antibody or an anti-PD-L1 antibody.
[67]
The use according to any of [64] to [66], wherein the immune checkpoint inhibitor is an anti-PD-1 antibody.
[68]
Anti-PD-1 antibodies include nivolumab, pembrolizumab, pidirizumab, cemiplimab, spartarizumab, spartarizumab, MEDI0680, , AMP-224, PF-06801591, tislelizumab, ABBV-181, BI 754091, or SHR-1210, the use according to [66] or [67].
[69]
The use according to any of [64] to [66], wherein the immune checkpoint inhibitor is an anti-PD-L1 antibody.
[70]
Anti-PD-L1 antibodies are atezolizumab, durvalumab, BMS-936559, avelumab, rodapolimab, CX-072, FAZ053, KN035, or MD. Or the use according to [69].
[71]
The anti-Fn14 antibody or its antigen-binding fragment is CDR1 consisting of the amino acid sequences of amino acids 31 to 35 of SEQ ID NO: 2, CDR2 consisting of the amino acid sequences of amino acids 50 to 65 of SEQ ID NO: 2, and the amino acid of SEQ ID NO: 2. A heavy chain variable region containing CDR3 consisting of amino acid sequences of numbers 98 to 114, CDR1 consisting of amino acid numbers 24 to 40 of amino acid number 4 of SEQ ID NO: 4, and amino acids of amino acid numbers 56 to 62 of SEQ ID NO: 4. An anti-human Fn14 antibody or antigen-binding fragment thereof comprising a light chain variable region comprising CDR2 consisting of a sequence and CDR3 consisting of amino acid numbers 95 to 103 of amino acid numbers 95 to 103 of SEQ ID NO: 4, [64] to [70]. ] Use described in any of.
[72]
The use according to any one of [64] to [71], wherein the anti-Fn14 antibody or an antigen-binding fragment thereof is an anti-human Fn14 antibody or an antigen-binding fragment thereof selected from the following (1) and (2):
(1) An anti-human Fn14 antibody or an anti-human Fn14 antibody containing a heavy chain variable region consisting of the amino acid sequences of amino acid numbers 1 to 125 of SEQ ID NO: 2 and a light chain variable region consisting of the amino acid sequences of amino acid numbers 1 to 114 of SEQ ID NO: 4. The antigen-binding fragment; and (2) the antibody or antigen-binding fragment thereof produced by post-translational modification of the anti-human Fn14 antibody of (1) above or the antigen-binding fragment thereof.
[73]
The anti-Fn14 antibody or its antigen-binding fragment comprises a heavy chain variable region consisting of the amino acid sequences of amino acids 1 to 125 of SEQ ID NO: 2 and a light chain variable region consisting of the amino acid sequences of amino acids 1 to 114 of SEQ ID NO: 4. The use according to any of [64] to [72], which comprises an anti-human Fn14 antibody or an antigen-binding fragment thereof.
[74]
The use according to any of [64] to [72], wherein the anti-Fn14 antibody or an antigen-binding fragment thereof is an anti-human Fn14 antibody selected from the following (3) and (4):
(3) Anti-human Fn14 antibody comprising a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 2 and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 4; and (4) post-translational modification of the antibody of (3) above. Anti-human Fn14 antibody, which is an antibody produced by.
[75]
The anti-Fn14 antibody or its antigen-binding fragment is an anti-human Fn14 antibody comprising a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 2 and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 4 [64] to [74]. ] Is used as described in any one of the items.
[76]
The use according to [72] or [74], 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.

Further, according to the present invention, for example, the following inventions [77] to [84] are provided.
[77]
Use of anti-Fn14 antibody or antigen-binding fragment thereof in the manufacture of a pharmaceutical composition for the prevention or treatment of steroid myopathy.
[78]
The use according to [77], wherein the anti-Fn14 antibody or antigen-binding fragment thereof is an anti-Fn14 antibody or antigen-binding fragment thereof that inhibits the activation of human Fn14 by Twake stimulation without exhibiting agonistic activity against human Fn14.
[79]
The anti-Fn14 antibody or its antigen-binding fragment is CDR1 consisting of the amino acid sequences of amino acids 31 to 35 of SEQ ID NO: 2, CDR2 consisting of the amino acid sequences of amino acids 50 to 65 of SEQ ID NO: 2, and the amino acid of SEQ ID NO: 2. A heavy chain variable region containing CDR3 consisting of amino acid sequences of numbers 98 to 114, CDR1 consisting of amino acid numbers 24 to 40 of amino acid number 4 of SEQ ID NO: 4, and amino acids of amino acid numbers 56 to 62 of SEQ ID NO: 4. An anti-human Fn14 antibody or antigen-binding fragment thereof, comprising a light chain variable region comprising CDR2 consisting of the sequence and CDR3 consisting of the amino acid sequences 95 to 103 of amino acid numbers 95 to 103 of SEQ ID NO: 4, [77] or [78]. ] Use described in.
[80]
The use according to any one of [77] to [79], wherein the anti-Fn14 antibody or an antigen-binding fragment thereof is an anti-human Fn14 antibody or an antigen-binding fragment thereof selected from the following (1) and (2):
(1) An anti-human Fn14 antibody or an anti-human Fn14 antibody containing a heavy chain variable region consisting of the amino acid sequences of amino acid numbers 1 to 125 of SEQ ID NO: 2 and a light chain variable region consisting of the amino acid sequences of amino acid numbers 1 to 114 of SEQ ID NO: 4. The antigen-binding fragment; and (2) the antibody or antigen-binding fragment thereof produced by post-translational modification of the anti-human Fn14 antibody of (1) above or the antigen-binding fragment thereof.
[81]
The anti-Fn14 antibody or its antigen-binding fragment comprises a heavy chain variable region consisting of the amino acid sequences of amino acids 1 to 125 of SEQ ID NO: 2 and a light chain variable region consisting of the amino acid sequences of amino acids 1 to 114 of SEQ ID NO: 4. The use according to any of [77] to [80], which comprises an anti-human Fn14 antibody or an antigen-binding fragment thereof.
[82]
The use according to any one of [77] to [80], wherein the anti-Fn14 antibody or antigen-binding fragment thereof is an anti-human Fn14 antibody selected from the following (3) and (4):
(3) Anti-human Fn14 antibody comprising a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 2 and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 4; and (4) post-translational modification of the antibody of (3) above. Anti-human Fn14 antibody, which is an antibody produced by.
[83]
The anti-Fn14 antibody or its antigen-binding fragment is an anti-human Fn14 antibody comprising a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 2 and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 4 [77] to [82]. ] Use described in any of.
[84]
The use according to [80] or [82], 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.

The anti-human Fn14 antibody used in the present invention has an anti-inflammatory effect by inhibiting the activation of human Fn14 by Twake stimulation without exhibiting agonist activity against human Fn14. The pharmaceutical composition of the present invention may be used as a prophylactic or therapeutic agent for cancer or steroid myopathy. The prophylactic or therapeutic method of the present invention may be able to prevent or treat cancer or steroid myopathy. The anti-human Fn14 antibody of the present invention may be used for the prevention or treatment of cancer or steroid myopathy. In addition, the anti-Fn14 antibody of the present invention may be used in the production of a pharmaceutical composition for the prevention or treatment of cancer or steroid myopathy.

It shows the inhibitory effect of anti-human Fn14 antibody on the production of inflammatory cytokine IL-8 by Twake stimulation in A375 cells. The vertical axis shows the inhibition rate (%), and the horizontal axis shows the antibody concentration (ng / mL). It shows the secretory action of IL-8 by inducing anti-human Fn14 antibody stimulation in A375 cells. The vertical axis shows the IL-8 concentration (pg / mL), and the horizontal axis shows the antibody concentration (ng / mL). Effect of 4-1h surrogate and anti-PD-1 antibody in combination in B16F10 mouse cancer-bearing model. The vertical axis shows the tumor volume (mm ³ ), and the horizontal axis shows the number of days after cancer-bearing (day). The change in mRNA expression of TOX in the tumor in the combination of 4-1h surrogate and anti-PD-1 antibody in the B16F10 mouse cancer-bearing model is shown. The vertical axis shows the mRNA expression level of each group when the Control group is 1. The effect (Grip strength) of 4-1h surrogate in a steroid myopathy model is shown. The effect of 4-1h surrogate on a steroid myopathy model (Quadriceps muscle weight) is shown.

The present invention will be described in detail below.

There are five classes of antibodies: IgG, IgM, IgA, IgD and IgE. The basic structure of an antibody molecule is common to each class and is composed of a heavy chain having a molecular weight of 50,000 to 70,000 and a light chain having a molecular weight of 20,000 to 30,000. A heavy chain usually consists of a polypeptide chain containing about 440 amino acids, has a characteristic structure for each class, and corresponds to IgG, IgM, IgA, IgD, IgE, Igγ, Igμ, Igα, Igδ, Igε. It is called. Further, IgG has subclasses of IgG1, IgG2, IgG3, and IgG4, and the heavy chains corresponding to each are called Igγ1, Igγ2, Igγ3, and Igγ4. The light chain usually consists of a polypeptide chain containing about 220 amino acids, and two types, L-type and K-type, are known and are called Igλ and Igκ, respectively. The peptide composition of the basic structure of an antibody molecule is that two heavy chains and two light chains that are homologous to each other are bonded by disulfide bonds (SS bonds) and non-covalent bonds, and have a molecular weight of 150,000 to 190,000. .. The two light chains can be paired with any heavy chain. Each antibody molecule is always made up of two identical light chains and two identical heavy chains.

There are four intrachain SS bonds in the heavy chain (five in Igμ and Igε) and two in the light chain, forming one loop for each amino acid residue 100-110, and this three-dimensional structure. Is similar between loops and is called a structural unit or domain. The domain located at the N-terminal of both heavy chain and light chain is called a variable region because its amino acid sequence is not constant even if it is a standard from the same class (subclass) of allogeneic animals, and each domain is called a variable region. , They are called heavy chain variable region (VH) and light chain variable region (VL), respectively. The amino acid sequence on the C-terminal side of the variable region is almost constant for each class or subclass and is called a constant region (each domain is represented as CH1, CH2, CH3 or CL, respectively).

The antigen binding site of an antibody is composed of VH and VL, and the specificity of binding 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 the difference in the structure of the constant region of each class Ig. The variability of the variable regions of the light and heavy chains has been found to be largely limited to the three small hypervariable regions present in both chains, which are the complementarity determining regions (CDRs; N-terminus, respectively). From the side, they are called CDR1, CDR2, CDR3). The rest of the variable region is called the framework region (FR) and is relatively constant.

Various antigen-binding fragments containing VH and VL of the antibody also have antigen-binding activity, and as such typical antigen-binding fragments, single-chain variable region fragments (scFv), Fab, Fab', F (ab') ₂ can be mentioned. scFv is a monovalent antibody fragment composed of linker-linked VH and VL, and Fab is composed of a light chain and a heavy chain fragment containing a VH, CH1 domain and part of the hinge region. It is a monovalent antibody fragment. Fab'is a monovalent antibody fragment composed of a light chain and a heavy chain fragment containing a VH, CH1 domain and a part of a hinge region, and the part of this hinge region is an inter-heavy chain SS. It contains the cysteine residues that made up the bond. The F (ab') ₂ fragment is a divalent antibody fragment in which two Fab'fragments are bound by an inter-heavy chain SS bond in the hinge region.

<Anti-human Fn14 antibody used in the present invention>
In one embodiment, the anti-human Fn14 antibody or antigen-binding fragment thereof used in the present invention is an anti-human Fn14 antibody that inhibits the activation of human Fn14 by Twake stimulation without exhibiting agonistic activity against human Fn14. .. Whether or not an antibody inhibits the activation of human Fn14 by Twake stimulation without exhibiting agonistic activity against human Fn14 can be confirmed, for example, by the method described in Examples 7 and 8.

In one embodiment, the anti-human Fn14 antibody or antigen-binding fragment thereof used in the present invention is an anti-human Fn14 antibody or antigen-binding fragment thereof having the following characteristics;
It consists of CDR1 consisting of the amino acid sequences of amino acid numbers 31 to 35 of SEQ ID NO: 2, CDR2 consisting of the amino acid sequences of amino acid numbers 50 to 65 of SEQ ID NO: 2, and the amino acid sequence of amino acid numbers 98 to 114 of SEQ ID NO: 2. The heavy chain variable region including CDR3, CDR1 consisting of the amino acid sequences of amino acids 24 to 40 of SEQ ID NO: 4, CDR2 consisting of the amino acid sequences of amino acids 56 to 62 of SEQ ID NO: 4, and the amino acid of SEQ ID NO: 4 An anti-human Fn14 antibody or antigen-binding fragment thereof comprising a light chain variable region containing CDR3 consisting of the amino acid sequences of numbers 95 to 103.

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

In one embodiment, the anti-human Fn14 antibody or antigen-binding fragment thereof used in the present invention has the above characteristics and further comprises a heavy chain constant region and a light chain constant region. As the constant region, any subclass constant region (for example, Igγ1, Igγ2, Igγ3 or Igγ4 as a heavy chain, and Igλ or Igκ as a light chain) can be selected. In one embodiment, the anti-human Fn14 antibody or antigen-binding fragment thereof used in 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.

Regarding the residue numbers related to the introduction of amino acid mutations in the constant region of the antibody used in the present specification, the EU index (Kabat et al., 1991, Sciences of Proteins of Immunological Information, 5th Ed., United States Public Health Service, United States Public Health Service). of Health, Bethesda). L234A and L235A are the substitutions of leucine at positions 234 and 235 with alanine according to the EU index of Kabat et al. In the human Igγ1 constant region. Examples of the human Igγ1 constant region having the amino acid mutations of L234A and L235A include the human Igγ1 constant region consisting of the amino acid sequences of amino acid numbers 126 to 455 of SEQ ID NO: 2.

In one embodiment, the anti-human Fn14 antibody used in the present invention comprises an anti-human Fn14 antibody comprising a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 2 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 4. Is.

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

The anti-human Fn14 antibody or its antigen-binding fragment used in the present invention also includes a post-translationally modified anti-human Fn14 antibody or its antigen-binding fragment. Examples of post-translationally modified anti-human Fn14 antibodies or antigen-binding fragments thereof used in the present invention have undergone heavy chain variable region N-terminal polyglutamylation and / or heavy chain C-terminal lysine deletion. Examples thereof include anti-human Fn14 antibody or an antigen-binding fragment thereof. It is known in the art that such post-translational modification by N-terminal pyroglutamylation or C-terminal lysine deletion does not affect the activity of the antibody (Lyubarskaya Y et al., Anal Biochem. 2006). , Vol. 348, p. 24-39).

In one embodiment, the anti-human Fn14 antibody used in the present invention comprises a heavy chain variable region consisting of the amino acid sequences of amino acids 1 to 125 of SEQ ID NO: 2 and amino acids of amino acids 1 to 114 of SEQ ID NO: 4. An antibody or an antigen-binding fragment thereof produced by post-translational modification of an anti-human Fn14 antibody containing a light chain variable region consisting of a sequence or an antigen-binding fragment thereof. Also, in one embodiment, the post-translational modification is pyroglutamylation at the N-terminus of the heavy chain variable region and / or lysine deletion at the C-terminus of the heavy chain.

In one embodiment, the anti-human Fn14 antibody or antigen-binding fragment thereof used in the present invention is a heavy chain variable region consisting of the amino acid sequences of amino acid numbers 1 to 125 of SEQ ID NO: 2, and is a heavy chain variable region of SEQ ID NO: 2. An anti-human Fn14 antibody or an antigen-binding fragment thereof, which comprises a heavy chain variable region in which glutamine of amino acid number 1 is modified with pyroglutamic acid, and a light chain variable region consisting of the amino acid sequences of amino acids 1 to 114 of SEQ ID NO: 4. be.

In one embodiment, the anti-human Fn14 antibody used in the present invention is an anti-human Fn14 antibody comprising a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 2 and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 4. An antibody produced by post-translational modification, which is an anti-human Fn14 antibody in which the post-translational modification is pyroglutamylation at the N-terminal of the heavy chain and / or lysine deletion at the C-terminal of the heavy chain.

In one embodiment, the anti-human Fn14 antibody used in the present invention is a heavy chain consisting of the amino acid sequences of amino acids 1 to 454 of SEQ ID NO: 2, and glutamine of amino acid number 1 of SEQ ID NO: 2 is pyro. An anti-human Fn14 antibody comprising a glutamic acid-modified heavy chain and a light chain consisting of the amino acid sequence of SEQ ID NO: 4.

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

Based on the present invention, a person skilled in the art can prepare a fusion of an antibody or an antigen-binding fragment thereof with another peptide or protein, or prepare a modified product to which a modifier is bound. , Antibodies in these forms or antigen-binding fragments thereof can also be used in the present invention. Other peptides and proteins used for fusion are not particularly limited as long as the fusion binds to Fn14, and for example, human serum albumin, various tag peptides, artificial helix motif peptides, maltose-binding proteins, glutathione S transferase, various toxins, etc. Other examples include peptides and proteins that can promote multimerization. The modifier used for the modification is not particularly limited as long as the modified product binds to Fn14, and examples thereof include polyethylene glycol, sugar chains, phospholipids, liposomes, and low molecular weight compounds.

In one embodiment, the modifier used to modify the antibody or antigen-binding fragment thereof used in the present invention is polyethylene glycol.

As used herein, the term "anti-human Fn14 antibody" means an antibody that binds to human Fn14. Whether or not it binds to human Fn14 can be confirmed by using a known method for measuring binding activity. Examples of the method for measuring the binding activity include a method such as Enzyme-Linked ImmunoSorbent Assay (ELISA). When ELISA is used, for example, a human Fn14 protein is immobilized on an ELISA plate, a test antibody is added thereto and reacted, and then an anti-IgG antibody labeled with an enzyme such as horseradish peroxidase (HRP) is used. React the secondary antibody of. After the reaction, after washing, binding of the secondary antibody by activity measurement using a reagent for detecting the activity (for example, in the case of HRP labeling, TMB Microwellell Peroxidase Substrate (Kirkegard & Perry Laboratories, 50-76-03)) or the like. By identifying, it is possible to confirm whether or not the test antibody binds to human Fn14. As a specific evaluation method, a method as described in Example 6 described later can be used.

The anti-human Fn14 antibody used in the present invention includes an antibody that binds to human Fn14 as long as it binds to human Fn14, as well as an antibody that also binds to Fn14 derived from other animals (for example, mouse Fn14). Is done.

The anti-human Fn14 antibody or antigen-binding fragment thereof used in the present invention is known in the art based on the heavy chain and light chain sequence information of the antibody used in the present invention disclosed herein. It can be readily made by one of ordinary skill in the art using the method. The anti-human Fn14 antibody or an antigen-binding fragment thereof used in the present invention is not particularly limited, but is, for example, <a method for producing an anti-human Fn14 antibody used in the present invention and a method for producing the same, which will be described later. It can be produced according to the method described in Anti-Human Fn14 Antibody>.

The anti-human Fn14 antibody or antigen-binding fragment thereof used in the present invention is further purified if necessary and then formulated according to a conventional method for inflammatory diseases such as excessive angiogenesis, weight loss, muscle wasting, and cachexia. It can be used for the prevention or treatment of debilitating diseases such as cachexia.

<Polynucleotide for producing the anti-human Fn14 antibody used in the present invention>
The polynucleotide for producing the anti-human Fn14 antibody used in the present invention includes a polynucleotide containing a base sequence encoding the heavy chain variable region of the anti-human Fn14 antibody used in the present invention or an antigen-binding fragment thereof. Also included are polynucleotides containing a base sequence encoding the light chain variable region of the anti-human Fn14 antibody or antigen-binding fragment thereof used in the present invention.

In one embodiment, the polynucleotide containing the nucleotide sequence encoding the heavy chain variable region of the anti-human Fn14 antibody or antigen-binding fragment thereof used in the present invention is the amino acid sequence of amino acid numbers 1 to 125 of SEQ ID NO: 2. It is a polynucleotide containing a base sequence encoding a heavy chain variable region consisting of.

Examples of the polynucleotide containing the base sequence encoding the heavy chain variable region shown in the amino acid sequences of amino acids 1 to 125 of SEQ ID NO: 2 include polys containing the base sequences of base numbers 1 to 375 of SEQ ID NO: 1. Nucleotides can be mentioned.

In one embodiment, the polynucleotide comprising the nucleotide sequence encoding the heavy chain variable region of the anti-human Fn14 antibody used in the present invention has a nucleotide sequence encoding the heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 2. It is a polynucleotide containing.

Examples of the polynucleotide containing the base sequence encoding the heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 2 include the polynucleotide containing the base sequence shown in SEQ ID NO: 1.

In one embodiment, the polynucleotide comprising the nucleotide sequence encoding the light chain variable region of the anti-human Fn14 antibody or antigen-binding fragment thereof used in the present invention is the amino acid sequence of amino acid numbers 1 to 114 of SEQ ID NO: 4. It is a polynucleotide containing a base sequence encoding a light chain variable region consisting of.

Examples of the polynucleotide containing the base sequence encoding the light chain variable region consisting of the amino acid sequences of amino acids 1 to 114 of SEQ ID NO: 4 include the polynucleotide containing the base sequences of base numbers 1 to 342 of SEQ ID NO: 3. Can be mentioned.

In one embodiment, the polynucleotide comprising the nucleotide sequence encoding the light chain variable region of the anti-human Fn14 antibody used in the present invention has a nucleotide sequence encoding the light chain consisting of the amino acid sequence shown in SEQ ID NO: 4. It is a polynucleotide containing.

Examples of the polynucleotide containing the base sequence encoding the light chain consisting of the amino acid sequence shown in SEQ ID NO: 4 include the polynucleotide containing the base sequence shown in SEQ ID NO: 3.

The polynucleotide for producing the anti-human Fn14 antibody used in the present invention can be easily prepared by a person skilled in the art based on its base sequence using a method known in the art. For example, the polynucleotide for producing the anti-human Fn14 antibody used in the present invention can be synthesized by using a gene synthesis method known in the art. As such a gene synthesis method, various methods known to those skilled in the art such as the antibody gene synthesis method described in WO90 / 07861 can be used.

<Expression vector for producing the anti-human Fn14 antibody used in the present invention>
The expression vector for producing the anti-human Fn14 antibody used in the present invention includes a polynucleotide containing a nucleotide sequence encoding the heavy chain variable region of the anti-human Fn14 antibody used in the present invention or an antigen-binding fragment thereof. / Or an expression vector containing a polynucleotide containing a nucleotide sequence encoding the light chain variable region of the anti-human Fn14 antibody or antigen-binding fragment thereof used in the present invention.

In one embodiment, the expression vector for producing the anti-human Fn14 antibody used in the present invention includes a polynucleotide containing a base sequence encoding the heavy chain of the anti-human Fn14 antibody used in the present invention. An expression vector, an expression vector containing a polynucleotide containing a nucleotide sequence encoding the light chain of the anti-human Fn14 antibody used in the present invention, or a nucleotide sequence encoding the heavy chain of the anti-human Fn14 antibody used in the present invention. Examples thereof include an expression vector containing a polynucleotide containing a polynucleotide and a polynucleotide containing a nucleotide sequence encoding the light chain of the antibody.

Expression vectors used to express the polynucleotide for producing the anti-human Fn14 antibody used in the present invention include eukaryotic cells (eg, animal cells, insect cells, plant cells, yeast) and / or prokaryotic cells. A polynucleotide containing a nucleotide sequence encoding the heavy chain variable region of the anti-human Fn14 antibody or antigen-binding fragment thereof used in the present invention in various host cells of (for example, Escherichia coli) and / or used in the present invention. It is not particularly limited as long as it can express a polynucleotide containing a nucleotide sequence encoding the light chain variable region of an anti-human Fn14 antibody or an antigen-binding fragment thereof and can produce a polypeptide encoded by these. Examples of such an expression vector include a plasmid vector and a viral vector (for example, adenovirus and retrovirus), and for example, pEE6.4 and pEE12.4 can be used. It is also possible to express an antibody gene by introducing a variable region gene fragment into an expression vector having a human Ig constant region gene in advance, such as AG-γ1 or AG-κ (see, for example, WO94 / 20632).

The expression vector for producing the anti-human Fn14 antibody used in the present invention may include a promoter operably linked to a polynucleotide for producing the anti-human Fn14 antibody used in the present invention. Examples of promoters for expressing the polynucleotide for producing the anti-human Fn14 antibody used in the present invention in animal cells include virus-derived promoters such as CMV, RSV, and SV40, actin promoters, and EF (elongation factor). Examples thereof include a 1α promoter and a heat shock promoter. Examples of promoters for expression in bacteria (for example, Escherichia spp.) include trp promoter, lac promoter, λPL promoter, tac promoter and the like. Examples of promoters for expression in yeast include GAL1 promoter, GAL10 promoter, PH05 promoter, PGK promoter, GAP promoter, ADH promoter and the like.

When animal cells, insect cells, or yeast are used as host cells, the expression vector for producing the anti-human Fn14 antibody used in the present invention may contain a start codon and a stop codon. In this case, the expression vector for producing the anti-human Fn14 antibody used in the present invention is the enhancer sequence, the 5'side of the enhancer sequence, the antibody of the present invention or the gene encoding the heavy chain variable region or the light chain variable region thereof, and 3 It may include untranslated regions on the'side, secretory signal sequences, splicing junctions, polyadenylation sites, or replicable units. When Escherichia coli is used as the host cell, the expression vector for producing the anti-human Fn14 antibody used in the present invention may contain a start codon, a stop codon, a terminator region, and a replicable unit. In this case, the expression vector for producing the anti-human Fn14 antibody used in the present invention is a selectable marker usually used depending on the purpose (for example, tetracycline resistance gene, ampicillin resistance gene, kanamycin resistance gene, neomycin resistance gene, etc. It may contain a dihydrofolate reductase gene).

<Host cell for producing the anti-human Fn14 antibody used in the present invention>
Host cells for producing the anti-human Fn14 antibody used in the present invention include a polynucleotide containing a base sequence encoding the heavy chain of the anti-human Fn14 antibody used in the present invention, and a light chain of the antibody. An expression vector containing a host cell transformed with an expression vector containing a nucleotide sequence encoding a nucleotide sequence, and a polynucleotide containing a nucleotide sequence encoding the heavy chain of the anti-human Fn14 antibody used in the present invention. , And an expression vector containing a polynucleotide containing a nucleotide sequence encoding the light chain of the antibody.

In one embodiment, the host cell for producing the anti-human Fn14 antibody used in the present invention is the anti-antibody used in the present invention selected from the group consisting of the following (a) to (d). Includes host cells transformed with an expression vector for producing human Fn14 antibody.
(A) Encodes a polynucleotide containing a nucleotide sequence encoding the heavy chain variable region of the anti-human Fn14 antibody or its antigen-binding fragment used in the present invention, and the light chain variable region of the antibody or its antigen-binding fragment. Host cells transformed with an expression vector containing a polynucleotide containing a base sequence;
(B) An expression vector containing a polynucleotide containing a nucleotide sequence encoding a heavy chain variable region of an anti-human Fn14 antibody or an antigen-binding fragment thereof used in the present invention, and a light chain variable of the antibody or its antigen-binding fragment. Host cells transformed with an expression vector, which contains a polynucleotide containing a nucleotide sequence encoding a region;
(C) Host cells transformed with an expression vector containing a nucleotide containing a nucleotide sequence encoding the heavy chain variable region of the anti-human Fn14 antibody or antigen-binding fragment thereof used in the present invention; and (d) the present invention. A host cell transformed with an expression vector containing a polynucleotide containing a nucleotide sequence encoding the light chain variable region of the anti-human Fn14 antibody or antigen-binding fragment thereof used in the above.

In one embodiment, the host cell for producing the anti-human Fn14 antibody used in the present invention is selected from the group consisting of the following (e) to (h), and the anti-human used in the present invention. Includes host cells transformed with an expression vector for producing Fn14 antibodies.
(E) Transformation with an expression vector containing a polynucleotide containing a nucleotide sequence encoding the heavy chain of the anti-human Fn14 antibody used in the present invention and a polynucleotide containing a nucleotide sequence encoding the light chain of the antibody. Host cell;
(F) An expression vector containing a polynucleotide containing a nucleotide sequence encoding the heavy chain of the anti-human Fn14 antibody used in the present invention, and an expression vector containing a polynucleotide containing a nucleotide sequence encoding the light chain of the antibody. , Host cells transformed with;
(G) Host cells transformed with an expression vector containing a polynucleotide containing a nucleotide sequence encoding the heavy chain of the anti-human Fn14 antibody used in the present invention; and (h) the anti-human Fn14 used in the present invention. A host cell transformed with an expression vector containing a polynucleotide containing a nucleotide sequence encoding the light chain of an antibody.

The host cell to be transformed is not particularly limited as long as it is compatible with the expression vector to be used and can be transformed with the expression vector to express an antibody. Examples of the host cell to be transformed include various cells (for example, animal cells (for example, CHO-K1SV cells) and insect cells) such as natural cells or artificially established cells usually used in the technical field of the present invention. (For example, Sf9), bacteria (Escherichia spp., Etc.), yeast (Saccharomyces spp., Pikia spp., Etc.), etc.), for example, CHO cells (CHO-K1SV cells, CHO-DG44 cells, etc.), 293 cells, NS0. Cultured cells such as cells can be used.

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

<Method for producing the anti-human Fn14 antibody used in the present invention and the anti-human Fn14 antibody produced by the method>
In the method for producing the anti-human Fn14 antibody or its antigen-binding fragment used in the present invention, a host cell selected from the group consisting of the following (A) to (C) is cultured, and the anti-human Fn14 antibody or its antigen-binding fragment thereof is cultured. A method for producing an anti-human Fn14 antibody or an antigen-binding fragment thereof, which comprises the step of expressing an antigen-binding fragment.
(A) Encodes a polynucleotide containing a nucleotide sequence encoding the heavy chain variable region of the anti-human Fn14 antibody or its antigen-binding fragment used in the present invention, and the light chain variable region of the antibody or its antigen-binding fragment. Host cells transformed with an expression vector containing a polynucleotide containing a base sequence;
(B) An expression vector containing a nucleotide containing a nucleotide sequence encoding the heavy chain variable region of the anti-human Fn14 antibody or its antigen-binding fragment used in the present invention, and a light chain variable of the antibody or its antigen-binding fragment. Host cells transformed with an expression vector containing a polynucleotide containing a nucleotide sequence encoding a region; and (C) encode the heavy chain variable region of the anti-human Fn14 antibody or antigen-binding fragment thereof used in the present invention. A host cell transformed with an expression vector containing a polynucleotide containing a base sequence, and an expression vector containing a polynucleotide containing a base sequence encoding the light chain variable region of the antibody or its antigen-binding fragment. Host cell.

In one embodiment, in the method for producing the anti-human Fn14 antibody used in the present invention, a host cell selected from the group consisting of the following (D) to (F) is cultured to obtain an anti-human Fn14 antibody. Included is a method of producing an anti-human Fn14 antibody that comprises the steps of expression.
(D) Transformation with an expression vector containing a polynucleotide containing a nucleotide sequence encoding the heavy chain of the anti-human Fn14 antibody used in the present invention and a polynucleotide containing a nucleotide sequence encoding the light chain of the antibody. Host cell;
(E) An expression vector containing a nucleotide containing a base sequence encoding the heavy chain of the anti-human Fn14 antibody used in the present invention, and an expression vector containing a polynucleotide containing a base sequence encoding the light chain of the antibody. Host cells transformed with, and (F) host cells transformed with an expression vector containing a nucleotide containing a nucleotide sequence encoding the heavy chain of the anti-human Fn14 antibody used in the present invention, and said. Host cells transformed with an expression vector containing a polynucleotide containing a nucleotide sequence encoding the light chain of an antibody.

The method for producing the anti-human Fn14 antibody or its antigen-binding fragment used in the present invention is to culture a host cell for producing the anti-human Fn14 antibody used in the present invention and to produce the anti-human Fn14 antibody or its antigen-binding fragment. As long as it includes the step of expressing the fragment, it is not particularly limited. In one embodiment, the host cell used in the method includes the above-mentioned host cells (D) and (F).

The transformed host cell can be cultured by a known method. Culturing conditions, such as temperature, medium pH, and culturing time, are appropriately selected. When the host cell is an animal cell, the medium is, for example, MEM medium (Eagle H, Science. 1959, Vol. 130 No. 3373, p. 432-437) containing about 5 to 20% fetal bovine serum, DMEM. Medium (Dulvecco R and Freeman G, Virology. 1959, Vol. 8, p. 396-397), RPMI 1640 medium (Moore GE et al., J Am Med Assoc. 1967, Vol. 199, p. 319), 199 medium. (Morgan JF et al., Proc Soc Exp Biol Med. 1950, Vol. 73, p. 1-8) and the like can be used. The pH of the medium is preferably about 6-8, and the culture is usually carried out at about 30-40 ° C. for about 15-336 hours with aeration and stirring as needed. When the host cell is an insect cell, for example, a Grace's medium containing fetal bovine serum (Mith GE et al., Proc Natl Acad Sci USA. 1985, Vol. 82, p. 8404) or the like is used. Can be done. The pH of the medium is preferably about 5-8, and the culture is usually carried out at about 20-40 ° C. for about 15-100 hours with aeration and stirring as needed. When the host cell is Escherichia coli or yeast, the medium is, for example, a liquid medium containing a nutrient source. The nutrient medium preferably contains a carbon source, an inorganic nitrogen source or an organic nitrogen source necessary for the growth of the transformed host cell. Examples of carbon sources include glucose, dextran, soluble starch, sucrose, etc., and examples of inorganic nitrogen sources or organic nitrogen sources include ammonium salts, nitrates, amino acids, corn steep liquor, peptone, casein, and meat extract. , Soybean starch, potato extract, etc. If desired, it may contain other nutrients (eg, calcium chloride, sodium dihydrogen phosphate, magnesium chloride, etc.), antibiotics (eg, tetracycline, neomycin, ampicillin, kanamycin, etc.). good. The pH of the medium is preferably about 5-8. When the host cell is Escherichia coli, as a preferable medium, for example, LB medium, M9 medium (Mol. Clo., Cold Spring Harbor Laboratory, 2001, Vol. 3, A2.2) and the like can be used. Culturing is usually carried out at about 14-39 ° C. for about 3-24 hours with aeration and stirring as needed. When the host cell is yeast, for example, Burkholder's minimum medium (Bostian KA et al., Proc Natl Acad Sci USA. 1980, Vol. 77, p. 4504-4508) can be used as the medium. Culturing is usually carried out at about 20-35 ° C. for about 14-144 hours with aeration and stirring as needed. By culturing as described above, the anti-human Fn14 antibody used in the present invention or an antigen-binding fragment thereof can be expressed.

The method for producing an anti-human Fn14 antibody or an antigen-binding fragment thereof used in the present invention involves culturing a host cell for producing the anti-human Fn14 antibody used in the present invention and binding the anti-human Fn14 antibody or an antigen-binding fragment thereof. In addition to the step of expressing the fragment, it can further include the step of recovering, for example, isolating or purifying the anti-human Fn14 antibody or its antigen-binding fragment from the transformed host cell. Examples of the isolation or purification method include a method using solubility such as salting out and solvent precipitation, a method using difference in molecular weight such as dialysis, ultrafiltration, and gel filtration, ion exchange chromatography, and hydroxylapatite chromatography. A method using charge such as imaging, a method using specific affinity such as affinity chromatography, a method using difference in hydrophobicity such as reverse phase high performance liquid chromatography, isoelectric focusing such as isoelectric focusing. There is a method of utilizing the difference between the two. For example, the antibody accumulated in the culture supernatant can be purified by various types of chromatography, for example, column chromatography using a protein A column or a protein G column.

The anti-human Fn14 antibody or its antigen-binding fragment used in the present invention includes the anti-human Fn14 antibody or its antigen-binding fragment produced by the method for producing the anti-human Fn14 antibody or its antigen-binding fragment used in the present invention. Is also included.

<Immune checkpoint inhibitor used in the present invention>
As used herein, an "immune checkpoint inhibitor" or "checkpoint inhibitor" completely or partially reduces, inhibits, or interferes with one or more checkpoint proteins. A molecule that, or negatively regulates, or completely or partially reduces, inhibits, interferes with, or negatively regulates the expression of one or more checkpoint proteins. In one embodiment, the immune checkpoint inhibitor binds to one or more checkpoint proteins. In one embodiment, the immune checkpoint inhibitor binds to one or more molecules that regulate the checkpoint protein. In one embodiment, the immune checkpoint inhibitor binds to a precursor of one or more checkpoint proteins, eg, at the DNA or RNA level. Any agent that functions as a checkpoint inhibitor can be used in the present invention.

As used herein, the term "partially" refers to, for example, at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, at the level of inhibition of a checkpoint protein. It means 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%.

In one embodiment, immune checkpoint inhibitors suitable for use in the methods disclosed herein are antagonists of the inhibitory signal (eg, PD-1, PD-L1, CTLA-4, LAG-3, B7). -An antibody that targets H3, B7-H4, or TIM-3). These ligands and receptors are described in Pardol, D. et al. , Nature. It is outlined at 12: 252-264, 2012. Further immune checkpoint proteins that can be targeted are also described herein.

In one embodiment, an immune checkpoint inhibitor prevents an inhibitory signal associated with an immune checkpoint. In one embodiment, an immune checkpoint inhibitor is an antibody or fragment thereof that disrupts the inhibitory signaling associated with an immune checkpoint. In one embodiment, the immune checkpoint inhibitor is a small molecule inhibitor that disrupts inhibitory signaling. In one embodiment, the immune checkpoint inhibitor is a peptide-based inhibitor that disrupts inhibitory signaling. In one embodiment, an immune checkpoint inhibitor is an inhibitory nucleic acid molecule that disrupts inhibitory signaling. In one embodiment, the immune checkpoint inhibitor is an interaction between a checkpoint antagonist protein (eg, an antibody or fragment thereof that interferes with the interaction between PD-1 and PD-L1 or PD-L2). An antibody, a fragment thereof, or an antibody imitator that interferes with. In one embodiment, the immune checkpoint inhibitor is an antibody, fragment, or antibody mimetic that interferes with the interaction between CTLA-4 and CD80 or CD86. In one embodiment, the immune checkpoint inhibitor is an antibody, fragment thereof, or antibody mimetic that interferes with the interaction between LAG-3 and its ligand, or TIM-3 and its ligand. In one embodiment, immune checkpoint inhibitors interfere with CD39 and / or CD73-mediated inhibitory signaling and / or the interaction of A2AR and / or A2BR with adenosine. In one embodiment, the immune checkpoint inhibitor interferes with the interaction of B7-H3 with its receptor and / or B7-H4 with its receptor. In one embodiment, the immune checkpoint inhibitor interferes with the interaction of BTLA with its ligand HVEM. In one embodiment, immune checkpoint inhibitors interfere with the interaction of one or more KIRs with their respective ligands. In one embodiment, the immune checkpoint inhibitor interferes with the interaction of LAG-3 with one or more of its ligands. In one embodiment, the immune checkpoint inhibitor interferes with the interaction of TIM-3 with one or more of its ligands, Galectin-9, PtdSer, HMGB1 and CEACAM1. In one embodiment, the immune checkpoint inhibitor interferes with the interaction of TIGIT with one or more of its ligands, PVR, PVRL2 and PVRL3. In one embodiment, the immune checkpoint inhibitor interferes with the interaction of CD94 / NKG2A with HLA-E. In one embodiment, the immune checkpoint inhibitor interferes with the interaction of VISTA with one or more of its binding partners. In one embodiment, the immune checkpoint inhibitor interferes with the interaction of one or more Siglec with their respective ligands. In one embodiment, immune checkpoint inhibitors interfere with CD20 signaling. In one embodiment, the immune checkpoint inhibitor interferes with the interaction of GARP with one or more of its ligands. In one embodiment, the immune checkpoint inhibitor interferes with the interaction of CD47 with SIRPα. In one embodiment, the immune checkpoint inhibitor interferes with the interaction of PVRIG with PVRL2. In one embodiment, the immune checkpoint inhibitor interferes with the interaction of CSF1R with CSF1. In one embodiment, immune checkpoint inhibitors interfere with NOX signaling. In one embodiment, immune checkpoint inhibitors interfere with IDO and / or TDO signaling.

As described herein, inhibition or blocking of inhibitory immune checkpoint signaling prevents or reverses immunosuppression and establishes or enhances T cell immunity to cancer cells. In one embodiment, inhibition of immune checkpoint signaling reduces or inhibits immune system dysfunction, as described herein. In one embodiment, inhibition of immune checkpoint signaling, as described herein, prevents dysfunctional immune cells from becoming more dysfunctional. In one embodiment, inhibition of immune checkpoint signaling reduces dysfunction of dysfunctional T cells, as described herein.

For immune cells (eg, T cells) The term "dysfunction" as used herein refers to a condition in which the immune response to antigen stimulation is reduced. Although the term includes common elements of both depletion and / or anergy in which antigen recognition can occur, subsequent immune responses are not effective in controlling infection or tumor growth. Dysfunction also includes a condition in which antigen recognition is delayed due to dysfunctional immune cells. Dysfunction is impaired in non-responsiveness to antigen recognition and the ability to convert antigen recognition to downstream T cell effector function (eg, proliferation, cytokine production (eg, IL-2) and / or target cell killing). included.

As used herein for immune cells (eg, T cells), the term "anergy" refers to a state of non-responsiveness to antigenic stimuli resulting from incomplete or inadequate signals delivered through the T cell receptor (TCR). To say. T cell anergy can also occur during stimulation with the antigen in the absence of co-stimulation, so that the cell becomes refractory to subsequent activation by the antigen, even in the context of co-stimulation. .. Non-responsive conditions can often be overridden by the presence of IL-2. Anergic T cells do not undergo clonal proliferation and / or acquire effector function.

As used herein for immune cells (eg, T cells), the term "depletion" refers to T cell depletion as a condition of T cell dysfunction resulting from persistent TCR signaling that occurs during many chronic infections and cancers. Immune cell depletion such as. Anergy is distinguished from anergy in that it results from continuous signaling rather than through incomplete or incomplete signaling. Depletion is defined by poor effector function, sustained expression of inhibitory receptors, and transcriptional states that differ from those of functional effector or memory T cells. Depletion interferes with optimal control of diseases (eg, infections and tumors). Depletion can result from both exogenous negative regulatory pathways (eg, immunomodulatory cytokines) as well as intracellular negative regulatory pathways (inhibitory immune checkpoint pathways as described herein).

"Enhancement of T cell function" induces, induces, or stimulates T cells to have sustained or amplified biological function, or regenerates or reactivates depleted or inactive T cells. Means that. Examples of enhanced T cell function include increased secretion of γ-interferon from CD8 + T cells, increased proliferation, and increased antigen responsiveness compared to such levels prior to intervention (eg, tumor clearance). Be done.

The immune checkpoint inhibitor may be an inhibitory nucleic acid molecule. As used herein, the term "inhibiting nucleic acid" or "inhibiting nucleic acid molecule" is a nucleic acid molecule that completely or partially reduces, inhibits, interferes with, or negatively regulates one or more checkpoint proteins. (For example, DNA or RNA). Inhibitor nucleic acid molecules include, but are not limited to, oligonucleotides, siRNA, shRNA, antisense DNA or RNA molecules, and aptamers (eg, DNA or RNA aptamers).

The term "oligonucleotide" as used herein refers to a nucleic acid molecule capable of reducing protein expression, in particular the expression of checkpoint proteins such as the checkpoint proteins described herein. Oligonucleotides are short DNA or RNA molecules, typically containing 2-50 nucleotides. Oligonucleotides may be single-stranded or double-strand. The checkpoint inhibitor oligonucleotide can be an antisense oligonucleotide. An antisense oligonucleotide is a single-stranded DNA or RNA molecule that is complementary to the sequence of another sequence, particularly the nucleic acid sequence (or fragment thereof) of a checkpoint protein. Antisense RNA is typically used to prevent protein translation of mRNA, eg, mRNA encoding a checkpoint protein, by binding to said mRNA. Antisense DNA is typically used to target specific complementary (coding or non-coding) RNA. Upon binding, such DNA / RNA hybrids are degraded by the enzyme RNase H, and morpholino antisense oligonucleotides can be used for gene knockdown in vertebrates (eg, JExp Med. , 2006, 203: 871-81).

The term "siRNA" or "small interfering RNA" or "small interfering RNA" is used interchangeably herein and has a complementary nucleotide sequence, such as a gene encoding a checkpoint protein. A double-stranded RNA molecule having a typical length of 20 to 25 base pairs that interferes with the expression of the gene. In one embodiment, siRNA interferes with mRNA and thus blocks translation (eg, translation of immune checkpoint proteins). Transfection of exogenous siRNA can be used for gene knockdown, but this effect can only be transient, especially in rapidly dividing cells. Stable transfection can be achieved, for example, by RNA modification or by using expression vectors. Useful modifications and vectors for stable transfection of cells with siRNA are known in the art. The siRNA sequence can also be modified to introduce a short loop between the two strands to give rise to a "small hairpin RNA" or "SHRNA". The shRNA can be processed into a functional siRNA by the Dicer. shRNA has a relatively low degradation rate and turnover. Therefore, the immune checkpoint inhibitor can be shRNA.

As used herein, the term "aptamer" typically refers to a single-stranded nucleic acid molecule such as DNA or RNA 25-70 nucleotides in length that can bind to a target molecule such as a polypeptide. In one embodiment, the aptamer binds to an immune checkpoint protein, such as the immune checkpoint protein described herein. For example, the aptamers according to the present disclosure may specifically bind to a molecule in an immune checkpoint protein or polypeptide, or a signaling pathway that regulates the expression of an immune checkpoint protein or polypeptide. The production and therapeutic use of aptamers is well known in the art (eg, US5,475,096).

The terms "small molecule inhibitor" or "small molecule" are used interchangeably herein and, as described above, reduce or inhibit one or more checkpoint proteins in whole or in part. A low molecular weight organic compound (usually up to 1000 daltons) that acts, interferes, or negatively regulates. Such small molecule inhibitors are usually synthesized by organic chemistry, but can also be isolated from natural sources such as plants, fungi, and microorganisms. The low molecular weight allows the small molecule inhibitor to diffuse rapidly across the cell membrane. For example, various A2AR antagonists known in the art are organic compounds having a molecular weight of less than 500 daltons.

The immune checkpoint inhibitor can be a fusion protein comprising an antibody, an antigen-binding fragment thereof, an antibody mimic, or an antibody moiety having an antigen-binding fragment of the required specificity. Antibodies or antigen-binding fragments thereof are as described herein. Antibodies or antigen-binding fragments thereof that are immune checkpoint inhibitors specifically include antibodies that bind to immune checkpoint proteins such as immune checkpoint receptors or immune checkpoint receptor ligands or antigen-binding fragments thereof. The antibody or antigen binding fragment can be fused to a peptide or protein, or can be bound to a modifier. In particular, the antibody or antigen-binding fragment thereof is a chimeric, humanized or human antibody. In one embodiment, an immune checkpoint inhibitor antibody or antigen-binding fragment thereof is an antagonist of an immune checkpoint receptor or an immune checkpoint receptor ligand. Also, in another embodiment, the antibody that is an immune checkpoint inhibitor is an isolated antibody. An antibody that is an immune checkpoint inhibitor or an antigen-binding fragment thereof according to the present disclosure can be an antibody that cross-competites for antigen binding with any known immune checkpoint inhibitor antibody. In one embodiment, the immune checkpoint inhibitor antibody cross-competes with one or more of the immune checkpoint inhibitor antibodies described herein. The ability of antibodies to cross-compete for binding to an antigen is the ability of these antibodies to bind to the same epitope region of the antigen, or to inhibit known immune checkpoints to that particular epitope region if they bind to another epitope. It is shown that the binding of the drug antibody can be sterically disturbed. Since these cross-competitive antibodies are expected to block the binding of immune checkpoints to their ligands by binding to the same epitope or by sterically interfering with the binding of the ligand, they are cross-competitive. Can have functional characteristics very similar to those of Cross-competitive antibodies are readily identified based on their ability to cross-competition with one or more known antibodies in standard binding assays (eg, Surface Plasmon Response analysis, ELISA assay or flow cytometry (WO2013 / 173223)). obtain. In one embodiment, an antibody or antigen-binding fragment thereof that cross-competites for binding to a given antigen or binds to the same epitope region of a given antigen is a monoclonal antibody. For administration to human patients, these cross-competitive antibodies can be chimeric antibodies, or humanized or human antibodies. Such chimeric, humanized or human monoclonal antibodies can be prepared and isolated by methods well known in the art.

The checkpoint inhibitor may also be in the soluble form of the molecule (or variant thereof) itself, eg, in the form of soluble PD-L1 or PD-L1 fusion.

In one embodiment, two or more checkpoint inhibitors can be used. Here, the two or more checkpoint inhibitors target different checkpoint pathways or the same checkpoint pathway. In one embodiment, the two or more checkpoint inhibitors are separate checkpoint inhibitors. If two or more separate checkpoint inhibitors are used, at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 separate checkpoint inhibitors are used and another one practice. In embodiments, 2, 3, 4 or 5 separate checkpoint inhibitors are used, and in another embodiment, 2, 3 or 4 separate checkpoint inhibitors are used, in yet another embodiment. Two or three separate checkpoint inhibitors are used, and in yet another embodiment, two separate checkpoint inhibitors are used.

In one embodiment, the immune checkpoint inhibitor is an inhibitor of the PD-1 / PD-L1 or PD-1 / PD-L2 signaling pathway. Therefore, one embodiment provides a subject to administer a checkpoint inhibitor of the PD-1 signaling pathway. In one embodiment, the checkpoint inhibitor of the PD-1 signaling pathway is a PD-1 inhibitor. In one embodiment, the checkpoint inhibitor of the PD-1 signaling pathway is a PD-1 ligand inhibitor such as a PD-L1 inhibitor or PD-L2 inhibitor. In one embodiment, the checkpoint inhibitor of the PD-1 signaling pathway is an antibody or antibody that disrupts the interaction between PD-1 and one or more of its ligands, PD-L1 and / or PD-L2. It is the antigen-binding fragment. Antibodies that bind to PD-1 and disrupt the interaction between PD-1 and one or more of its ligands are known in the art. In one embodiment, the antibody or antigen-binding fragment thereof specifically binds to PD-1. That is, in one embodiment, the checkpoint inhibitor is an anti-PD-1 antibody. In one embodiment, the antibody or antigen-binding fragment thereof specifically binds to PD-L1 and inhibits its interaction with PD-1, thereby increasing immune activity. That is, in one embodiment, the checkpoint inhibitor is an anti-PD-L1 antibody. In one embodiment, the antibody or antigen-binding fragment thereof specifically binds to PD-L2 and inhibits its interaction with PD-1, thereby increasing immune activity. That is, in one embodiment, the checkpoint inhibitor is an anti-PD-L2 antibody.

In one embodiment, immune checkpoint inhibitors are nivolumab (nivolumab, WO2006 / 121168), pembrolizumab (WO2008 / 156712), pidilizumab, WO2009 / 101611, semiprimab, cemiplimab, cemiplimab, cemiplimab, cemiplimab, cemiplimab, cemiplimab. Mab (spartarizumab), MEDI0680 (WO2012 / 145493), dostallimab, setrelimab, triparimab, AMP-224 (WO2010 / 027827, WO2011 / 066827, WO2011 / 06622) -181, BI 754991, or SHR-1210 (WO2015 / 085847).

In one embodiment, the immune checkpoint inhibitors are atezolizumab (US9,724,413), dulvalumab (WO2011 / 06638), BMS-936559 (WO2013 / 173223), avelumab (US2014 / 03). , Rodapolimab, CX-072 (WO2016 / 149201), FAZ053, KN035 (WO2017 / 02801, WO2017 / 02802), or MDX-1105 (US2015 / 0320859).

In one embodiment, the immune checkpoint inhibitor is an inhibitor of the CTLA-4 signaling pathway. Therefore, one embodiment provides a subject to administer a checkpoint inhibitor of the CTLA-4 signaling pathway. In one embodiment, the checkpoint inhibitor of the CTLA-4 signaling pathway is a CTLA-4 inhibitor. In one embodiment, the checkpoint inhibitor of the CTLA-4 signaling pathway is a CTLA-4 ligand inhibitor. In one embodiment, the checkpoint inhibitor of the CTLA-4 signaling pathway is an antibody or antigen-binding fragment thereof that disrupts the interaction between CTLA-4 and its ligand.

In one embodiment, the immune checkpoint inhibitor is an inhibitor of the TIGIT signaling pathway. Therefore, one embodiment provides a subject to administer a checkpoint inhibitor of the TIGIT signaling pathway. In one embodiment, the checkpoint inhibitor of the TIGIT signaling pathway is a TIGIT inhibitor. In one embodiment, the checkpoint inhibitor of the TIGIT signaling pathway is a TIGIT ligand inhibitor. In one embodiment, the checkpoint inhibitor of the TIGIT signaling pathway is an antibody or antigen-binding fragment thereof that disrupts the interaction between TIGIT and its ligand.

In one embodiment, the immune checkpoint inhibitor is an inhibitor of the B7 family signaling pathway. In one embodiment, the B7 family members are B7-H3 and B7-H4. One embodiment provides to administer a checkpoint inhibitor of B7-H3 and / or B7-H4 to a subject. Therefore, one embodiment provides that an antibody targeting B7-H3 or B7-H4 or an antigen-binding fragment thereof is administered to a subject. In one embodiment, the checkpoint inhibitor of the B7 family signaling pathway is an antibody or antigen-binding fragment thereof that disrupts the interaction between the B7 family and the receptor.

In one embodiment, the immune checkpoint inhibitor is an inhibitor of the BTLA signaling pathway. Therefore, one embodiment provides a subject to administer a checkpoint inhibitor of the BTLA signaling pathway. In one embodiment, the checkpoint inhibitor of the BTLA signaling pathway is a BTLA inhibitor. In one embodiment, the checkpoint inhibitor of the BTLA signaling pathway is an HVEM inhibitor. In one embodiment, the checkpoint inhibitor of the BTLA signaling pathway is an antibody or antigen-binding fragment thereof that disrupts the interaction between BTLA and HVEM.

In one embodiment, the immune checkpoint inhibitor is an inhibitor of one or more KIR signaling pathways. Therefore, one embodiment provides for administering to a subject a checkpoint inhibitor of one or more KIR signaling pathways. In one embodiment, the checkpoint inhibitor of one or more KIR signaling pathways is a KIR inhibitor. In one embodiment, the checkpoint inhibitor of one or more KIR signaling pathways is a KIR ligand inhibitor. In one embodiment, the checkpoint inhibitor of the KIR signaling pathway is an antibody or antigen-binding fragment thereof that disrupts the interaction between KIR and one or more of its ligands, KIR2DL1, KIR2DL2, and KIR2DL3.

In one embodiment, the immune checkpoint inhibitor is an inhibitor of the LAG-3 signaling pathway. Therefore, one embodiment provides a subject to administer a checkpoint inhibitor of the LAG-3 signaling pathway. In one embodiment, the checkpoint inhibitor of the LAG-3 signaling pathway is a LAG-3 inhibitor. In one embodiment, the checkpoint inhibitor of the LAG-3 signaling pathway is a LAG-3 ligand inhibitor. In one embodiment, the checkpoint inhibitor of the LAG-3 signaling pathway is an antibody or antigen-binding fragment thereof that disrupts the interaction between LAG-3 and its ligand.

In one embodiment, the immune checkpoint inhibitor is an inhibitor of the TIM-3 signaling pathway. Therefore, one embodiment provides a subject to administer a checkpoint inhibitor of the TIM-3 signaling pathway. In one embodiment, the checkpoint inhibitor of the TIM-3 signaling pathway is a TIM-3 inhibitor. In one embodiment, the checkpoint inhibitor of the TIM-3 signaling pathway is a TIM-3 ligand inhibitor. In one embodiment, the checkpoint inhibitor of the TIM-3 signaling pathway is an antibody or antigen-binding fragment thereof that disrupts the interaction between TIM-3 and its ligand.

In one embodiment, the immune checkpoint inhibitor is an inhibitor of the CD94 / NKG2A signaling pathway. Therefore, one embodiment provides that a checkpoint inhibitor of the CD94 / NKG2A signaling pathway is administered to a subject. In one embodiment, the checkpoint inhibitor of the CD94 / NKG2A signaling pathway is a CD94 / NKG2A inhibitor. In one embodiment, the checkpoint inhibitor of the CD94 / NKG2A signaling pathway is a CD94 / NKG2A ligand inhibitor. In one embodiment, the checkpoint inhibitor of the CD94 / NKG2A signaling pathway is an antibody or antigen-binding fragment thereof that disrupts the interaction between CD94 / NKG2A and its ligand.

In one embodiment, the immune checkpoint inhibitor is an inhibitor of the IDO signaling pathway. Therefore, one embodiment provides a subject to administer a checkpoint inhibitor of the IDO signaling pathway. In one embodiment, the checkpoint inhibitor of the IDO signaling pathway is an IDO inhibitor. In one embodiment, the checkpoint inhibitor of the IDO signaling pathway is an antibody or antigen-binding fragment thereof that disrupts the interaction between IDO and its ligand.

In one embodiment, the immune checkpoint inhibitor is an inhibitor of the adenosine signaling pathway. Therefore, one embodiment provides a subject to administer a checkpoint inhibitor of the adenosine signaling pathway. In one embodiment, the checkpoint inhibitor of the adenosine signaling pathway is a CD39 inhibitor. In one embodiment, the checkpoint inhibitor of the adenosine signaling pathway is a CD73 inhibitor. In one embodiment, the checkpoint inhibitor of the adenosine signaling pathway is an A2AR inhibitor. In one embodiment, the checkpoint inhibitor of the adenosine signaling pathway is an A2BR inhibitor.

In one embodiment, the immune checkpoint inhibitor is an inhibitor of the VISTA signaling pathway. Therefore, one embodiment provides a subject to administer a checkpoint inhibitor of the VISTA signaling pathway. In one embodiment, the checkpoint inhibitor of the VISTA signaling pathway is a VISTA inhibitor. In one embodiment, the checkpoint inhibitor of the VISTA signaling pathway is an antibody or antigen-binding fragment thereof that disrupts the interaction between VISTA and its binding partner.

In one embodiment, the immune checkpoint inhibitor is an inhibitor of one or more Siglec signaling pathways. Therefore, one embodiment provides for administering to a subject a checkpoint inhibitor of one or more Siglec signaling pathways. In one embodiment, the checkpoint inhibitor of one or more Siglec signaling pathways is a Siglec inhibitor. In one embodiment, the checkpoint inhibitor of one or more Siglec signaling pathways is a Siglec ligand inhibitor. In one embodiment, the checkpoint inhibitor of the Siglec signaling pathway is an antibody or antigen-binding fragment thereof that disrupts the interaction between Siglec and its ligand.

In one embodiment, the immune checkpoint inhibitor is an inhibitor of the CD20 signaling pathway. Therefore, one embodiment provides a subject to administer a checkpoint inhibitor of the CD20 signaling pathway. In one embodiment, the checkpoint inhibitor of the CD20 signaling pathway is a CD20 inhibitor.

In one embodiment, the immune checkpoint inhibitor is an inhibitor of the GARP signaling pathway. Therefore, one embodiment provides a subject to administer a checkpoint inhibitor of the GARP signaling pathway. In one embodiment, the checkpoint inhibitor of the GARP signaling pathway is a GARP inhibitor. In one embodiment, the checkpoint inhibitor of the GARP signaling pathway is an antibody or antigen-binding fragment thereof that disrupts the interaction between GARP and its ligand.

In one embodiment, the immune checkpoint inhibitor is a component of the CD47 signaling pathway. Therefore, one embodiment provides a subject to administer a checkpoint inhibitor of the CD47 signaling pathway. In one embodiment, the checkpoint inhibitor of the CD47 signaling pathway is a CD47 inhibitor. In one embodiment, the checkpoint inhibitor of the CD47 signaling pathway is a SIRPα inhibitor. In one embodiment, the checkpoint inhibitor of the CD47 signaling pathway is an antibody or antigen-binding fragment thereof that disrupts the interaction between CD47 and SIRPα.

In one embodiment, the immune checkpoint inhibitor is an inhibitor of the PVRIG signaling pathway. Therefore, one embodiment provides a subject to administer a checkpoint inhibitor of the PVRIG signaling pathway. In one embodiment, the checkpoint inhibitor of the PVRIG signaling pathway is a PVRIG inhibitor. In one embodiment, the checkpoint inhibitor of the PVRIG signaling pathway is a PVRIG ligand inhibitor. In one embodiment, the checkpoint inhibitor of the PVRIG signaling pathway is an antibody or antigen-binding fragment thereof that disrupts the interaction between PVRIG and its ligand.

In one embodiment, the immune checkpoint inhibitor is an inhibitor of the CSF1R signaling pathway. Therefore, one embodiment provides a subject to administer a checkpoint inhibitor of the CSF1R signaling pathway. In one embodiment, the checkpoint inhibitor of the CSF1R signaling pathway is a CSF1R inhibitor. In one embodiment, the checkpoint inhibitor of the CSF1R signaling pathway is a CSF1 inhibitor. In one embodiment, the checkpoint inhibitor of the CSF1R signaling pathway is an antibody or antigen-binding fragment thereof that disrupts the interaction between CSF1R and CSF1.

In one embodiment, the immune checkpoint inhibitor is an inhibitor of the NOX signaling pathway. Therefore, one embodiment provides a subject to administer a checkpoint inhibitor of the NOX signaling pathway. In one embodiment, the checkpoint inhibitor of the NOX signaling pathway is a NOX inhibitor.

In one embodiment, the immune checkpoint inhibitor is an inhibitor of the TDO signaling pathway. Therefore, one embodiment provides a subject to administer a checkpoint inhibitor of the TDO signaling pathway. In one embodiment, the checkpoint inhibitor of the TDO signaling pathway is a TDO inhibitor.

PD-1, PD-L1, CTLA-4, TIGIT, B7-H3, B7-H4, BTLA, KIR, LAG-3, TIM-3, CD94 / NKG2A, IDO, A2AR, A2BR, VISTA, Sigma, CD20, Inhibitors of CD39, CD73, GARP, CD47, PVRIG, CSF1R, NOX and TDO and their respective ligands are known, and some of them are already in clinical trials or approved. Based on these known immune checkpoint inhibitors, alternative immune checkpoint inhibitors can be used. Known inhibitors of immune checkpoint proteins can be used as is, or analogs thereof can be used, and in some cases, antibodies that cross-competition with any of the antibodies described herein, chimeric antibodies. Humanized or human antibodies can be used. Others where targeting results in increased T cell proliferation, enhanced T cell activation, increased cytokine (eg, IFN-γ, IL2) production, and / or stimulation of an immune response such as an antitumor immune response. Immune checkpoints can also be targeted by antagonists or antibodies.

Checkpoint inhibitors can be administered in any form and by any route known in the art. The form and route of administration depends on the form of the checkpoint inhibitor used. The checkpoint inhibitor can be administered in the form of any suitable pharmaceutical composition. Checkpoint inhibitors can be administered in the form of immune checkpoint inhibitors, eg, nucleic acids encoding inhibitory nucleic acid molecules or antibodies or fragments thereof (eg, DNA or RNA molecules). For example, the antibody can be delivered encoded in an expression vector. Nucleic acid molecules can be delivered, for example, in the form of plasmids or mRNA molecules, or complexed with delivery vehicles (eg, liposomes, lipoplexes or nucleic acid lipid particles). Checkpoint inhibitors can also be administered via an oncolytic virus that contains an expression cassette that encodes a checkpoint inhibitor. Checkpoint inhibitors can also be administered by administration of endogenous or allogeneic cells capable of expressing the checkpoint inhibitor (eg, in the form of cell-based therapy).

The term "cell-based therapy" refers to cells expressing immune checkpoint inhibitors (eg, T lymphocytes, dendritic cells, or stem cells) for the purpose of treating a disease or disorder (eg, cancer disease). Refers to porting to. In one embodiment, cell-based therapy comprises genetically engineered cells. In one embodiment, genetically engineered cells express immune checkpoint inhibitors. In one embodiment, an immune check in which the genetically engineered cell is an inhibitory nucleic acid molecule such as siRNA, shRNA, oligonucleotide, antisense DNA or RNA, aptamer, antibody or fragment thereof, or soluble immune checkpoint protein or fusion. Express point inhibitors. Genetically engineered cells can also express additional agents that enhance T cell function. Such agents are known in the art. Cell-based therapies for use in inhibiting immune checkpoint signaling are disclosed, for example, in WO 2018/222711.

As used herein, the term "oncolytic virus" is selected in cancerous or hypergrowth cells either in vitro or in vivo, while not affecting or minimally affecting normal cells. Refers to a virus that is capable of replicating, slowing its growth, or inducing the death of its cells. Tumor-lytic viruses for the delivery of immune checkpoint inhibitors are inhibitor nucleic acid molecules such as siRNA, shRNA, oligonucleotides, antisense DNA or RNA, aptamers, antibodies or fragments thereof, or soluble immune checkpoint proteins or fusions. Includes an expression cassette that can encode an immune checkpoint inhibitor. Oncolytic viruses are preferably replication competents, and expression cassettes are under the control of viral promoters (eg, early / late synthetic poxvirus promoters). Examples of tumor-dissolving viruses include bullous stomatitis virus (VSV), rabdovirus (eg, Seneca Valley virus; picornavirus such as SVV-001), coxsackie virus, parvovirus, Newcastle disease virus (NDV), simple herpes. Illustratively described in virus (HSV; Oncovex GMCSF), retrovirus (eg, influenza virus), measles virus, leovirus, Sindbis virus, vaccinia virus (WO2017 / 209053 (including Copenhagen, Western Reserve, Wyeth strain)). And adenoviruses (eg, Delta-24, Delta-24-RGD, ICOVIR-5, ICOVIR-7, ICOVIR-7, Anyx-015, ColorAd1, H101, AD5 / 3-D24-GMCSF). Can be mentioned. The production of recombinant oncolytic viruses, including soluble forms of immune checkpoint inhibitors, and methods of their use are disclosed in WO 2018/022831. As the oncolytic virus, an attenuated virus can be used.

<Pharmaceutical composition of the present invention, prophylactic or therapeutic method of the present invention, antibody for prophylactic or therapeutic of the present invention, use in the production of the present invention>
The pharmaceutical composition of the present invention includes a pharmaceutical composition containing the anti-human Fn14 antibody or antigen-binding fragment thereof used in the present invention and a pharmaceutically acceptable excipient. The pharmaceutical composition of the present invention can be prepared by a commonly used method using excipients usually used in the art, that is, pharmaceutical excipients, pharmaceutical carriers and the like. Examples of dosage forms of these pharmaceutical compositions include parenteral preparations such as injections and infusions, which can be administered by intravenous administration, subcutaneous administration, intramuscular administration, or the like. In the formulation, excipients, carriers, additives and the like corresponding to these dosage forms can be used within a pharmaceutically acceptable range.

The pharmaceutical composition of the present invention may contain a plurality of anti-human Fn14 antibodies or antigen-binding fragments thereof used in the present invention. For example, a pharmaceutical composition containing an antibody or an antigen-binding fragment thereof which has not undergone post-translational modification, and an antibody or an antigen-binding fragment thereof produced by post-translational modification of the antibody or its antigen-binding fragment is also included in the present invention. ..

In one embodiment, the pharmaceutical composition of the present invention containing an anti-human Fn14 antibody or an antigen-binding fragment thereof also includes the pharmaceutical compositions described below.
An anti-human Fn14 antibody or an antigen-binding fragment thereof containing a heavy chain variable region consisting of the amino acid sequences of amino acid numbers 1 to 125 of SEQ ID NO: 2 and a light chain variable region consisting of the amino acid sequences of amino acid numbers 1 to 114 of SEQ ID NO: 4. , And an anti-human Fn14 antibody or an antigen-binding fragment thereof, which is an antibody or an antigen-binding fragment thereof produced by post-translational modification of the antibody or its antigen-binding fragment.

The pharmaceutical composition of the present invention lacks a heavy chain C-terminal lysine-deficient antibody, an N-terminal post-translational modified antibody or an antigen-binding fragment thereof, and a heavy chain C-terminal lysine deleted and undergoes N-terminal post-translational modification. Also included are pharmaceutical compositions containing the antibody and / or the antibody having a heavy chain C-terminal lysine and not undergoing N-terminal post-translational modification.

In one embodiment, the pharmaceutical composition of the present invention containing an anti-human Fn14 antibody also includes a pharmaceutical composition containing two or more of the following (5) to (8) anti-human Fn14 antibodies.
(5) An anti-human Fn14 antibody comprising a heavy chain consisting of the amino acid sequences 1 to 454 of SEQ ID NO: 2 and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 4.
(6) A heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 2, in which glutamine of amino acid number 1 of SEQ ID NO: 2 is modified to pyroglutamic acid and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 4. Anti-human Fn14 antibody, including.
(7) A heavy chain consisting of the amino acid sequences of amino acids 1 to 454 of SEQ ID NO: 2, in which glutamine of amino acid number 1 of SEQ ID NO: 2 is modified with pyroglutamic acid and the amino acid shown in SEQ ID NO: 4. An anti-human Fn14 antibody comprising a light chain consisting of a sequence.
(8) An anti-human Fn14 antibody comprising a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 2 and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 4.

In one embodiment, the pharmaceutical composition of the present invention containing an anti-human Fn14 antibody or an antigen-binding fragment thereof also includes the pharmaceutical composition described below. An anti-human Fn14 antibody containing a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 2 and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 4, and a heavy chain consisting of the amino acid sequences 1 to 454 of SEQ ID NO: 2. An anti-human Fn14 antibody comprising a heavy chain in which the glutamine of amino acid No. 1 of SEQ ID NO: 2 is modified with pyroglutamic acid and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 4 and a pharmaceutically acceptable addition. A pharmaceutical composition comprising a shaping agent.

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

The pharmaceutical composition of the present invention can be used as a prophylactic or therapeutic agent for cancer by being used in combination with an immune checkpoint inhibitor. In one embodiment, the pharmaceutical composition of the present invention can be used as a prophylactic or therapeutic agent for cancer by being used in combination with an anti-PD-1 antibody. In one embodiment, the pharmaceutical composition of the present invention can be used as a prophylactic or therapeutic agent for cancer by being used in combination with an anti-PD-L1 antibody.

As used herein, the term "cancer" refers to brain tumor, head and neck cancer, salivary adenocarcinoma, thyroid cancer, lung cancer, small cell lung cancer, breast cancer, mesenteric tumor, pancreatic cancer, liver cancer, biliary tract cancer. , Esophageal cancer, gastric cancer, gastrointestinal stromal tumor, small intestine cancer, colon cancer, kidney cancer, renal pelvis cancer, urinary tract cancer, bladder cancer, prostate cancer, cervical cancer, ovarian cancer , Uterine sarcoma, testicular tumor, malignant lymphoma, leukemia, chronic lymphocytic leukemia, multiple myeloma, skin cancer, melanoma and sarcoma.

In one embodiment, the cancer prevented or treated by the present invention is a cancer that expresses Fn14. In one embodiment, the cancer prevented or treated by the present invention is a cancer that is sensitive to anti-Fn14 antibodies.

The present invention is a pharmaceutical composition for preventing or treating cancer, which comprises an anti-human Fn14 antibody or an antigen-binding fragment thereof used in the present invention, and is used in combination with an immune checkpoint inhibitor. The composition is provided. The present invention provides a method for preventing or treating cancer, comprising the step of administering a therapeutically effective amount of an anti-human Fn14 antibody or antigen-binding fragment thereof and an immune checkpoint inhibitor used in the present invention. The present invention provides an anti-human Fn14 antibody or antigen-binding fragment thereof used in the present invention, which is used in combination with an immune checkpoint inhibitor for use in the prevention or treatment of cancer. The present invention is the use of the anti-human Fn14 antibody or antigen-binding fragment thereof used in the present invention in the production of a pharmaceutical composition for the prevention or treatment of cancer, wherein the pharmaceutical composition is used in combination with an immune checkpoint inhibitor. Provide use, which is to be done.

The present invention is a pharmaceutical composition for preventing or treating cancer, which comprises an immune checkpoint inhibitor, and is used in combination with the anti-human Fn14 antibody used in the present invention or an antigen-binding fragment thereof. Provide a pharmaceutical composition. The present invention provides immune checkpoint inhibitors that are used in combination with the anti-human Fn14 antibody or antigen-binding fragment thereof used in the present invention for use in the prevention or treatment of cancer. The present invention is the use of an immune checkpoint inhibitor in the production of a pharmaceutical composition for the prevention or treatment of cancer, wherein the pharmaceutical composition is used in combination with the anti-human Fn14 antibody used in the present invention or an antigen-binding fragment thereof. Provide use, which is to be done.

The pharmaceutical composition of the present invention includes a pharmaceutical composition containing an immune checkpoint inhibitor used in the present invention and a pharmaceutically acceptable excipient. The pharmaceutical composition of the present invention can be prepared by a commonly used method using excipients usually used in the art, that is, pharmaceutical excipients, pharmaceutical carriers and the like. Examples of dosage forms of these pharmaceutical compositions include parenteral preparations such as injections and infusions, which can be administered by intravenous administration, subcutaneous administration, intramuscular administration, or the like. In the formulation, excipients, carriers, additives and the like corresponding to these dosage forms can be used within a pharmaceutically acceptable range.

The amount of the immune checkpoint inhibitor used in the present invention for formulation varies depending on the degree and age of the patient's symptoms, the dosage form of the formulation used, the form of the inhibitor, etc., but is, for example, 0.001 mg. About / kg to 100 mg / kg can be used.

In formulation, the amount of anti-PD-1 antibody or anti-PD-L1 antibody used in the present invention or an antigen-binding fragment thereof added depends on the degree and age of the patient's symptoms, the dosage form of the formulation used, or the antibody. Although it depends on the binding titer and the like, for example, about 0.001 mg / kg to 100 mg / kg can be used.

In one embodiment, the anti-human Fn14 antibody is administered (ie, co-administered) to a subject (eg, patient) with an immune checkpoint inhibitor. In one embodiment, the immune checkpoint inhibitor and anti-human Fn14 antibody are administered to the subject as a single composition. In one embodiment, the immune checkpoint inhibitor and anti-human Fn14 antibody are administered to the subject simultaneously (at the same time as separate compositions). In one embodiment, the immune checkpoint inhibitor and anti-human Fn14 antibody are administered to the subject separately. In one embodiment, the immune checkpoint inhibitor is administered to the subject prior to the anti-human Fn14 antibody. In one embodiment, the immune checkpoint inhibitor is administered to the subject after the anti-human Fn14 antibody. In one embodiment, the immune checkpoint inhibitor and anti-human Fn14 antibody are administered to the subject on the same day. In one embodiment, the immune checkpoint inhibitor and anti-human Fn14 antibody are administered to the subject on different days.

In one embodiment, the present invention is a pharmaceutical composition for the prevention or treatment of cancer, comprising the following anti-Fn14 antibody or antigen-binding fragment thereof and excipient, which is used in combination with an anti-PD-1 antibody. Pharmaceutical composition:
It consists of CDR1 consisting of the amino acid sequences of amino acid numbers 31 to 35 of SEQ ID NO: 2, CDR2 consisting of the amino acid sequences of amino acid numbers 50 to 65 of SEQ ID NO: 2, and the amino acid sequence of amino acid numbers 98 to 114 of SEQ ID NO: 2. The heavy chain variable region containing CDR3, CDR1 consisting of the amino acid sequences of amino acids 24 to 40 of SEQ ID NO: 4, CDR2 consisting of the amino acid sequences of amino acids 56 to 62 of SEQ ID NO: 4, and SEQ ID NO: 4 An anti-human Fn14 antibody or an antigen-binding fragment thereof, which comprises a light chain variable region containing CDR3 consisting of the amino acid sequences of amino acids 95 to 103.

In one embodiment, the present invention is a pharmaceutical composition for the prevention or treatment of cancer, which comprises an anti-Fn14 antibody or an antigen-binding fragment thereof and an excipient selected from the following (1) and (2). It is a pharmaceutical composition used in combination with an anti-PD-1 antibody:
(1) An anti-human Fn14 antibody or an anti-human Fn14 antibody containing a heavy chain variable region consisting of the amino acid sequences of amino acid numbers 1 to 125 of SEQ ID NO: 2 and a light chain variable region consisting of the amino acid sequences of amino acid numbers 1 to 114 of SEQ ID NO: 4. The antigen-binding fragment; and (2) the antibody or antigen-binding fragment thereof produced by post-translational modification of the anti-human Fn14 antibody of (1) above or the antigen-binding fragment thereof.

In one embodiment, the present invention is a pharmaceutical composition for the prevention or treatment of cancer, comprising an anti-Fn14 antibody and an excipient selected from the following (3) and (4), wherein the anti-PD -1 A pharmaceutical composition used in combination with an antibody:
(3) Anti-human Fn14 antibody comprising a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 2 and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 4; and (4) Post-translation modification of the antibody of (3) above. Anti-human Fn14 antibody, which is an antibody produced by.

In one embodiment, the invention is a method of preventing or treating cancer that comprises administering to a patient a therapeutically effective amount of the following anti-Fn14 antibody or antigen-binding fragment thereof and an anti-PD-1 antibody:
It consists of CDR1 consisting of the amino acid sequences of amino acid numbers 31 to 35 of SEQ ID NO: 2, CDR2 consisting of the amino acid sequences of amino acid numbers 50 to 65 of SEQ ID NO: 2, and the amino acid sequence of amino acid numbers 98 to 114 of SEQ ID NO: 2. The heavy chain variable region containing CDR3, CDR1 consisting of the amino acid sequences of amino acids 24 to 40 of SEQ ID NO: 4, CDR2 consisting of the amino acid sequences of amino acids 56 to 62 of SEQ ID NO: 4, and SEQ ID NO: 4 An anti-human Fn14 antibody or an antigen-binding fragment thereof, which comprises a light chain variable region containing CDR3 consisting of the amino acid sequences of amino acids 95 to 103.

In one embodiment, the invention comprises administering to a patient a therapeutically effective amount of an anti-Fn14 antibody or antigen-binding fragment thereof selected from the following (1) and (2) and an anti-PD-1 antibody. , Cancer prevention or treatment methods:
(1) An anti-human Fn14 antibody or an anti-human Fn14 antibody containing a heavy chain variable region consisting of the amino acid sequences of amino acid numbers 1 to 125 of SEQ ID NO: 2 and a light chain variable region consisting of the amino acid sequences of amino acid numbers 1 to 114 of SEQ ID NO: 4. The antigen-binding fragment; and (2) the antibody or antigen-binding fragment thereof produced by post-translational modification of the anti-human Fn14 antibody of (1) above or the antigen-binding fragment thereof.

In one embodiment, the invention comprises the step of administering to a patient a therapeutically effective amount of an anti-Fn14 antibody and an anti-PD-1 antibody selected from the following (3) and (4) for cancer prevention. Or a treatment method:
(3) Anti-human Fn14 antibody comprising a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 2 and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 4; and (4) Post-translation modification of the antibody of (3) above. Anti-human Fn14 antibody, which is an antibody produced by.

In one embodiment, the invention is the following anti-Fn14 antibody or antigen-binding fragment thereof for use in the prevention or treatment of cancer in combination with an anti-PD-1 antibody:
It consists of CDR1 consisting of the amino acid sequences of amino acid numbers 31 to 35 of SEQ ID NO: 2, CDR2 consisting of the amino acid sequences of amino acid numbers 50 to 65 of SEQ ID NO: 2, and the amino acid sequence of amino acid numbers 98 to 114 of SEQ ID NO: 2. The heavy chain variable region containing CDR3, CDR1 consisting of the amino acid sequences of amino acids 24 to 40 of SEQ ID NO: 4, CDR2 consisting of the amino acid sequences of amino acids 56 to 62 of SEQ ID NO: 4, and SEQ ID NO: 4 An anti-human Fn14 antibody or an antigen-binding fragment thereof, which comprises a light chain variable region containing CDR3 consisting of the amino acid sequences of amino acids 95 to 103.

In one embodiment, the present invention is an anti-Fn14 antibody selected from the following (1) and (2) for use in the prevention or treatment of cancer, which is used in combination with an anti-PD-1 antibody, or an anti-Fn14 antibody thereof. Antigen binding fragment:
(1) An anti-human Fn14 antibody or an anti-human Fn14 antibody containing a heavy chain variable region consisting of the amino acid sequences of amino acid numbers 1 to 125 of SEQ ID NO: 2 and a light chain variable region consisting of the amino acid sequences of amino acid numbers 1 to 114 of SEQ ID NO: 4. The antigen-binding fragment; and (2) the antibody or antigen-binding fragment thereof produced by post-translational modification of the anti-human Fn14 antibody of (1) above or the antigen-binding fragment thereof.

In one embodiment, the present invention is an anti-Fn14 antibody selected from the following (3) and (4) for use in the prevention or treatment of cancer, which is used in combination with an anti-PD-1 antibody. :
(3) Anti-human Fn14 antibody comprising a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 2 and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 4; and (4) Post-translation modification of the antibody of (3) above. Anti-human Fn14 antibody, which is an antibody produced by.

In one embodiment, the present invention is the use of the following anti-Fn14 antibody or antigen-binding fragment thereof in the production of a pharmaceutical composition for the prevention or treatment of cancer, wherein the pharmaceutical composition is anti-PD-1. Used in combination with antibodies:
It consists of CDR1 consisting of the amino acid sequences of amino acid numbers 31 to 35 of SEQ ID NO: 2, CDR2 consisting of the amino acid sequences of amino acid numbers 50 to 65 of SEQ ID NO: 2, and the amino acid sequence of amino acid numbers 98 to 114 of SEQ ID NO: 2. The heavy chain variable region containing CDR3, CDR1 consisting of the amino acid sequences of amino acids 24 to 40 of SEQ ID NO: 4, CDR2 consisting of the amino acid sequences of amino acids 56 to 62 of SEQ ID NO: 4, and SEQ ID NO: 4 An anti-human Fn14 antibody or an antigen-binding fragment thereof, which comprises a light chain variable region containing CDR3 consisting of the amino acid sequences of amino acids 95 to 103.

In one embodiment, the present invention is the use of an anti-Fn14 antibody or antigen-binding fragment thereof selected from the following (1) and (2) in the production of a pharmaceutical composition for the prevention or treatment of cancer. , The pharmaceutical composition is to be used in combination with an anti-PD-1 antibody, use:
(1) An anti-human Fn14 antibody or an anti-human Fn14 antibody containing a heavy chain variable region consisting of the amino acid sequences of amino acid numbers 1 to 125 of SEQ ID NO: 2 and a light chain variable region consisting of the amino acid sequences of amino acid numbers 1 to 114 of SEQ ID NO: 4. The antigen-binding fragment; and (2) the antibody or antigen-binding fragment thereof produced by post-translational modification of the anti-human Fn14 antibody of (1) above or the antigen-binding fragment thereof.

In one embodiment, the present invention is the use of an anti-Fn14 antibody selected from the following (3) and (4) in the production of a pharmaceutical composition for the prevention or treatment of cancer, wherein the pharmaceutical composition. Is used in combination with anti-PD-1 antibody:
(3) Anti-human Fn14 antibody comprising a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 2 and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 4; and (4) Post-translation modification of the antibody of (3) above. Anti-human Fn14 antibody, which is an antibody produced by.

In one embodiment, the present invention is a pharmaceutical composition for the prevention or treatment of cancer, which comprises the following anti-Fn14 antibody or antigen-binding fragment thereof and excipient, and is used in combination with the anti-PD-L1 antibody. Pharmaceutical composition:
It consists of CDR1 consisting of the amino acid sequences of amino acid numbers 31 to 35 of SEQ ID NO: 2, CDR2 consisting of the amino acid sequences of amino acid numbers 50 to 65 of SEQ ID NO: 2, and the amino acid sequence of amino acid numbers 98 to 114 of SEQ ID NO: 2. The heavy chain variable region containing CDR3, CDR1 consisting of the amino acid sequences of amino acids 24 to 40 of SEQ ID NO: 4, CDR2 consisting of the amino acid sequences of amino acids 56 to 62 of SEQ ID NO: 4, and SEQ ID NO: 4 An anti-human Fn14 antibody or an antigen-binding fragment thereof, which comprises a light chain variable region containing CDR3 consisting of the amino acid sequences of amino acids 95 to 103.

In one embodiment, the present invention is a pharmaceutical composition for the prevention or treatment of cancer, which comprises an anti-Fn14 antibody or an antigen-binding fragment thereof and an excipient selected from the following (1) and (2). It is a pharmaceutical composition used in combination with an anti-PD-L1 antibody:
(1) An anti-human Fn14 antibody or an anti-human Fn14 antibody containing a heavy chain variable region consisting of the amino acid sequences of amino acid numbers 1 to 125 of SEQ ID NO: 2 and a light chain variable region consisting of the amino acid sequences of amino acid numbers 1 to 114 of SEQ ID NO: 4. The antigen-binding fragment; and (2) the antibody or antigen-binding fragment thereof produced by post-translational modification of the anti-human Fn14 antibody of (1) above or the antigen-binding fragment thereof.

In one embodiment, the present invention is a pharmaceutical composition for the prevention or treatment of cancer, comprising an anti-Fn14 antibody and an excipient selected from the following (3) and (4), wherein the anti-PD -A pharmaceutical composition used in combination with an L1 antibody:
(3) Anti-human Fn14 antibody comprising a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 2 and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 4; and (4) Post-translation modification of the antibody of (3) above. Anti-human Fn14 antibody, which is an antibody produced by.

In one embodiment, the invention is a method of preventing or treating cancer that comprises administering to a patient a therapeutically effective amount of the following anti-Fn14 antibody or antigen-binding fragment thereof and an anti-PD-L1 antibody:
It consists of CDR1 consisting of the amino acid sequences of amino acid numbers 31 to 35 of SEQ ID NO: 2, CDR2 consisting of the amino acid sequences of amino acid numbers 50 to 65 of SEQ ID NO: 2, and the amino acid sequence of amino acid numbers 98 to 114 of SEQ ID NO: 2. The heavy chain variable region containing CDR3, CDR1 consisting of the amino acid sequences of amino acids 24 to 40 of SEQ ID NO: 4, CDR2 consisting of the amino acid sequences of amino acids 56 to 62 of SEQ ID NO: 4, and SEQ ID NO: 4 An anti-human Fn14 antibody or an antigen-binding fragment thereof, which comprises a light chain variable region containing CDR3 consisting of the amino acid sequences of amino acids 95 to 103.

In one embodiment, the present invention comprises administering to a patient a therapeutically effective amount of an anti-Fn14 antibody or antigen-binding fragment thereof selected from the following (1) and (2) and an anti-PD-L1 antibody. , Cancer prevention or treatment methods:
(1) An anti-human Fn14 antibody or an anti-human Fn14 antibody containing a heavy chain variable region consisting of the amino acid sequences of amino acid numbers 1 to 125 of SEQ ID NO: 2 and a light chain variable region consisting of the amino acid sequences of amino acid numbers 1 to 114 of SEQ ID NO: 4. The antigen-binding fragment; and (2) the antibody or antigen-binding fragment thereof produced by post-translational modification of the anti-human Fn14 antibody of (1) above or the antigen-binding fragment thereof.

In one embodiment, the invention comprises the step of administering to a patient a therapeutically effective amount of an anti-Fn14 antibody and an anti-PD-L1 antibody selected from the following (3) and (4) for cancer prevention. Or a treatment method:
(3) Anti-human Fn14 antibody comprising a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 2 and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 4; and (4) Post-translation modification of the antibody of (3) above. Anti-human Fn14 antibody, which is an antibody produced by.

In one embodiment, the invention is the following anti-Fn14 antibody or antigen-binding fragment thereof for use in the prevention or treatment of cancer in combination with an anti-PD-L1 antibody:
It consists of CDR1 consisting of the amino acid sequences of amino acid numbers 31 to 35 of SEQ ID NO: 2, CDR2 consisting of the amino acid sequences of amino acid numbers 50 to 65 of SEQ ID NO: 2, and the amino acid sequence of amino acid numbers 98 to 114 of SEQ ID NO: 2. The heavy chain variable region containing CDR3, CDR1 consisting of the amino acid sequences of amino acids 24 to 40 of SEQ ID NO: 4, CDR2 consisting of the amino acid sequences of amino acids 56 to 62 of SEQ ID NO: 4, and SEQ ID NO: 4 An anti-human Fn14 antibody or an antigen-binding fragment thereof, which comprises a light chain variable region containing CDR3 consisting of the amino acid sequences of amino acids 95 to 103.

In one embodiment, the present invention is an anti-Fn14 antibody selected from the following (1) and (2) for use in the prevention or treatment of cancer, which is used in combination with an anti-PD-L1 antibody, or an anti-Fn14 antibody thereof. Antigen binding fragment:
(1) An anti-human Fn14 antibody or an anti-human Fn14 antibody containing a heavy chain variable region consisting of the amino acid sequences of amino acid numbers 1 to 125 of SEQ ID NO: 2 and a light chain variable region consisting of the amino acid sequences of amino acid numbers 1 to 114 of SEQ ID NO: 4. The antigen-binding fragment; and (2) the antibody or antigen-binding fragment thereof produced by post-translational modification of the anti-human Fn14 antibody of (1) above or the antigen-binding fragment thereof.

In one embodiment, the present invention is an anti-Fn14 antibody selected from the following (3) and (4) for use in the prevention or treatment of cancer, which is used in combination with an anti-PD-L1 antibody. :
(3) Anti-human Fn14 antibody comprising a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 2 and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 4; and (4) Post-translation modification of the antibody of (3) above. Anti-human Fn14 antibody, which is an antibody produced by.

In one embodiment, the present invention is the use of the following anti-Fn14 antibody or antigen-binding fragment thereof in the production of a pharmaceutical composition for the prevention or treatment of cancer, wherein the pharmaceutical composition is anti-PD-L1. Used in combination with antibodies:
It consists of CDR1 consisting of the amino acid sequences of amino acid numbers 31 to 35 of SEQ ID NO: 2, CDR2 consisting of the amino acid sequences of amino acid numbers 50 to 65 of SEQ ID NO: 2, and the amino acid sequence of amino acid numbers 98 to 114 of SEQ ID NO: 2. The heavy chain variable region containing CDR3, CDR1 consisting of the amino acid sequences of amino acids 24 to 40 of SEQ ID NO: 4, CDR2 consisting of the amino acid sequences of amino acids 56 to 62 of SEQ ID NO: 4, and SEQ ID NO: 4 An anti-human Fn14 antibody or an antigen-binding fragment thereof, which comprises a light chain variable region containing CDR3 consisting of the amino acid sequences of amino acids 95 to 103.

In one embodiment, the present invention is the use of an anti-Fn14 antibody or antigen-binding fragment thereof selected from the following (1) and (2) in the production of a pharmaceutical composition for the prevention or treatment of cancer. , The pharmaceutical composition is to be used in combination with an anti-PD-L1 antibody, use:
(1) An anti-human Fn14 antibody or an anti-human Fn14 antibody containing a heavy chain variable region consisting of the amino acid sequences of amino acid numbers 1 to 125 of SEQ ID NO: 2 and a light chain variable region consisting of the amino acid sequences of amino acid numbers 1 to 114 of SEQ ID NO: 4. The antigen-binding fragment; and (2) the antibody or antigen-binding fragment thereof produced by post-translational modification of the anti-human Fn14 antibody of (1) above or the antigen-binding fragment thereof.

In one embodiment, the present invention is the use of an anti-Fn14 antibody selected from the following (3) and (4) in the production of a pharmaceutical composition for the prevention or treatment of cancer, wherein the pharmaceutical composition. Is used in combination with anti-PD-L1 antibody:
(3) Anti-human Fn14 antibody comprising a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 2 and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 4; and (4) Post-translation modification of the antibody of (3) above. Anti-human Fn14 antibody, which is an antibody produced by.

The pharmaceutical composition of the present invention can be used as a prophylactic or therapeutic agent for steroid myopathy or steroid-induced myopathy.

In one embodiment, the steroid myopathy that is the subject of the present invention is steroid myopathy caused by glucocorticoids. In one embodiment, the subject steroid myopathy of the invention is cortisol (including hydrocortisone and hydrocortisone succinate), prednisolone (including methylprednisolone and methylprednisolone succinate), triamcinolone (including triamcinolone acetonide), Steroid myopathy caused by dexamethasone or betametasone.

In one embodiment, the subject steroid myopathy of the present invention is a steroid myopathy caused by a steroid administered to prevent or treat an excessive immune response by an immune checkpoint inhibitor. In one embodiment, the subject steroid myopathy of the present invention is a steroid myopathy that occurs in a patient who has been administered a steroid at the same time as, or before or after the administration of an immune checkpoint inhibitor.

The present invention includes a pharmaceutical composition for the prevention or treatment of steroid myopathy, which comprises the anti-human Fn14 antibody used in the present invention or an antigen-binding fragment thereof. The present invention also includes a method for treating or preventing steroid myopathy, which comprises the step of administering a therapeutically effective amount of the anti-human Fn14 antibody or antigen-binding fragment thereof used in the present invention. The present invention also includes the anti-human Fn14 antibody or antigen-binding fragment thereof used in the present invention for use in the prevention or treatment of steroid myopathy. Furthermore, the present invention includes the use of the anti-human Fn14 antibody or antigen-binding fragment thereof used in the present invention in the production of a pharmaceutical composition for the prevention or treatment of steroid myopathy.

Specific examples referred to herein for further understanding of the invention are provided, but these are for illustrative purposes only and are not intended to limit the invention.

In some cases, unless otherwise specified, commercially available kits or reagents were used and experiments were performed according to the respective protocols.

(Example 1: Acquisition of human and mouse Fn14-Fc fusion protein)
The present inventors prepared a human Fn14-human Fc fusion protein and a mouse Fn14-mouse Fc fusion protein for use as an antigen for obtaining an anti-Fn14 antibody and a material used in a screening test. The human Fn14-human Fc fusion protein contains the C-terminus of the extracellular partial sequence of the human Fn14 sequence (NCBI accession number: NP_057723.1 amino acids 1 to 79) and the human Fc region (NCBI accession number: P01857). It is a fusion protein in which the N-terminal of the 106th to 330th amino acids of .1 is linked with a peptide linker (SEQ ID NO: 9). In addition, the mouse Fn14-mouse Fc fusion protein connects the C-terminal of the extracellular partial sequence of the mouse Fn14 sequence (NCBI accession number: AAF07882.1 1st to 75th amino acids) with the N-terminal of the mouse Fc region. The fusion protein, specifically, the gene encoding the extracellular partial sequence of the mouse Fn14 sequence, is located between the hEF1-HTLV promoter region and the mIgG2B-Fc region of pFUSE-mIgG2B-Fc1 (InvivoGen, pfuse-mg2bfc1). It is a fusion protein that has been incorporated and expressed at a multicloning site using the restriction enzymes EcoRI and EcoRV. Expression vectors in which the gene encoding the above-mentioned fusion protein was incorporated into the GS vector pEE12.4 (Lonza) were prepared and introduced into CHO-K1SV cells (Lonza). From the culture supernatant of the CHO-K1SV cells, a human Fn14-human Fc fusion protein and a mouse Fn14-mouse Fc fusion protein were purified according to a conventional method.

(Example 2: Acquisition of anti-human Fn14 antibody)
Antibodies were prepared using human monoclonal antibody development technology "Velosimune" (VelocImmune antibody technology: Regeneron (US Pat. No. 6,596,541)) mice. The antibody obtained by the belosimune technique is an antibody (also referred to as a chimeric antibody) having a variable region of a human antibody and a constant region of a mouse antibody. A human Fn14 protein obtained by cleaving and removing a human Fc region from the human Fn14-human Fc fusion protein prepared in Example 1 using FabRICATOR (Sigma, 77661), together with an adjuvant that induces an immune response, was added to a belosimune mouse. I was immunized. According to a conventional method, lymphocytes collected from the lymph nodes of immunized mice were fused with mouse-derived myeloma cells SP2 / 0-Ag14 (ATCC: CRL-1581) to prepare hybridomas and monocloned. Hybridomas that bind to human Fn14 and produce an antibody that suppresses NFkB activation by Twake stimulation (hereinafter referred to as "Twake-induced NFkB activation" in the examples below) are selected, and the antibody is selected from the culture supernatant. Was purified.

(Example 3: NFkB activation assay)
In order to evaluate the agonistic effect of the antibody found in Example 2 on human Fn14, the effect of the antibody on NFkB activation in the absence of Tweak was evaluated by a reporter assay. Specifically, HEK293 cells (ATCC, CRL-1573) (hereinafter referred to as NFkB / HEK293 cells) into which the luciferase reporter vector pGL4.32 (Promega, E8941) incorporating the NFkB transcription response sequence has been stably introduced are introduced. Was prepared and used for evaluation.

NFkB / HEK293 cells were suspended in DMEM (Sigma, D6429) containing 10% fetal bovine serum at 1.25x10 ⁵ cells / mL, and 80 μL per well was seeded on a clear bottom white 96-well plate (Corning, 3610). After culturing in a CO ₂ incubator set at 37 ° C. and 5% CO ₂ for 2 hours, the purified antibody obtained in Example 2 was diluted in 12 steps at a final concentration of about 3 times from 1 ng / mL to 300 μg / mL. After the series was prepared in the above medium, 20 μL was added per well. After culturing overnight at 37 ° C. and 5% CO ₂ , NFkB activation was quantified by measuring the expression level of luciferase using the luciferase measuring reagent ONE-Glo Luciferase Assay System (Promega).

As a result, anti-human Fn14 antibodies that did not induce NFkB activation, that is, did not show agonist activity, were selected and named 4-1.

(Example 4: Preparation of fully human antibody and mouse antibody)
The genes encoding the heavy and light chains of the antibody were cloned and sequenced from the hybridomas that produced the antibody selected in Example 3. After sequencing the antibody, the gene encoding the signal sequence (Wittle Net al., Protein Engineering. 1987, Vol. 1 No. 6, p. 499-505) is placed on the 5'side of the heavy chain variable region gene, and A human Igγ1 constant region gene having amino acid mutations of L234A and L235A on the 3'side (consisting of the base sequences 376 to 1365 of SEQ ID NO: 1) is linked, and this heavy chain gene is linked to the GS vector pEE6.4. It was inserted into (Lonza). A gene encoding a signal sequence (Wittle Net al., Protein Engineering. 1987, Vol. 1 No. 6, p. 499-505) is placed on the 5'side of the light chain variable region gene, and a gene encoding the signal sequence (P. 499-505) is placed on the 3'side. The constant region gene of the human kappa chain (consisting of the nucleotide sequences 343 to 660 of SEQ ID NO: 3) was linked, and this light chain gene was inserted into the GS vector pEE12.4. These GS vectors were digested with NotI-HF and PvuI-HF, ligated using DNA Ligation Kit <Mighty Mix> (TaKaRa, 6023), and both heavy and light chain genes were inserted. A GS vector was constructed. From the culture supernatant of CHO-K1SV cells transfected with this vector, the antibody was purified according to a conventional method to obtain a fully human antibody of 4-1 and named 4-1h.

The nucleotide sequence of the prepared 4-1h heavy chain is sequenced in SEQ ID NO: 1, the amino acid sequence encoded thereto is sequenced in SEQ ID NO: 2, the nucleotide sequence of the light chain is sequenced in SEQ ID NO: 3, and the amino acid sequence encoded thereto is sequenced. Each is shown in number 4. The variable region of the heavy chain shown in SEQ ID NO: 2 consists of the amino acid sequences of amino acid numbers 1 to 125 of SEQ ID NO: 2, and the variable region of the light chain shown in SEQ ID NO: 4 is the amino acid number of SEQ ID NO: 4. It consists of the amino acid sequences from the 1st to the 114th. CDR1, CDR2, and CDR3 of the heavy chain variable region of 4-1h consist of the amino acid sequences of positions 31 to 35, 50 to 65, and 98 to 114 of SEQ ID NO: 2, respectively. CDR1, CDR2, and CDR3 of the light chain variable region of 4-1h consist of the amino acid sequences of positions 24 to 40, 56 to 62, and 95 to 103 of SEQ ID NO: 4, respectively.

When evaluating an anti-human Fn14 antibody in a mouse in vivo test, a 4-1h mouse antibody (hereinafter referred to as 4-1m) was prepared in order to reduce the risk of immunogenicity. The framework regions (FRs) of the light and heavy chains of 4-1h were partially replaced with the FRs of other mouse antibodies to create a nucleotide sequence encoding a variable region of 4-1m.

4-1m was obtained using the same method as the above-mentioned vector construction of 4-1h, expression and purification of antibody. However, in the constant region of the heavy chain, a gene encoding a mouse Igγ2a constant region gene having a D265A amino acid mutation (consisting of the base sequences 376 to 1365 of SEQ ID NO: 5) is placed in the constant region of the light chain. Used the constant region gene of the mouse kappa chain (consisting of the nucleotide sequences from the 343rd to 660th nucleotide sequences of SEQ ID NO: 7).

The nucleotide sequence of the prepared 4-1 m heavy chain is sequenced in SEQ ID NO: 5, the amino acid sequence encoded by the sequence is sequenced in SEQ ID NO: 6, the nucleotide sequence of the light chain is sequenced in SEQ ID NO: 7, and the amino acid sequence encoded by the sequence is sequenced. Each is shown in number 8. The variable region of the heavy chain shown in SEQ ID NO: 6 consists of the amino acid sequences of amino acid numbers 1 to 125 of SEQ ID NO: 6, and the variable region of the light chain shown in SEQ ID NO: 8 is the amino acid number of SEQ ID NO: 8. It consists of the amino acid sequences from the 1st to the 114th. CDR1, CDR2, and CDR3 of the 4-1 m heavy chain variable region consist of the amino acid sequences of positions 31 to 35, 50 to 65, and 98 to 114 of SEQ ID NO: 6, respectively. CDR1, CDR2, and CDR3 of the light chain variable region of 4-1 m consist of the amino acid sequences of positions 24 to 40, 56 to 62, and 95 to 103 of SEQ ID NO: 8, respectively.

(Example 5: Amino acid modification analysis of fully human antibody)
As a result of amino acid modification analysis of the purified 4-1h, in most of the purified antibodies, pyroglutamylation at the N-terminal of the heavy chain and lysine deletion at the C-terminal occurred.

(Example 6: Human and mouse Fn14 binding ELISA of fully human antibody and mouse antibody)
The binding activity of 4-1h and 4-1m obtained in Example 4 to the Fn14 protein was evaluated. The human Fn14-human Fc fusion protein and the mouse Fn14-mouse Fc fusion protein obtained in Example 1 were prepared in 1 μg / mL with phosphate buffer saline (PBS) and maxi-soap 384-well transparent plate (Nunc, Inc., Inc.). 464718) was added with 15 μL per well and incubated overnight at 4 ° C. to solidify. The next day, the solid phase solution was removed, and the cells were washed with Tris-buffered saline (TBS-T) containing 0.05% Tween-20. 50 μL of PBS containing 20% Blocking One (Nacalai Tesque, 03953-95) was added per well, allowed to stand at room temperature for 1 hour, and then washed with TBS-T. Using TBS-T (hereinafter referred to as a diluent) containing 5% Blocking One, 4-1h or 4-1m obtained in Example 4 was used at a maximum concentration of 30 μg / mL in 12 steps and a 4-fold common ratio. By dilution, a dilution series was prepared and 20 μL was added per well. After incubating for 1 hour at room temperature, the cells were washed with TBS-T. As a secondary antibody, 4-1h is a hose radish peroxidase-labeled anti-human copper light chain antibody (Southern Biotech, 2060-05) diluted 5000 times with a diluent, and 4-1m is a hose diluted 4000 times with a diluent. Radish peroxidase-labeled anti-mouse copper light chain antibody (Southern Biotech, 1050-05) was added in an amount of 20 μL per well. After incubating at room temperature for 1 hour, the cells were washed with TBS-T, and TMB Microwellell Peroxidase Substrate (Kirkegard & Perry Laboratories, 50-76-03) was added for 3 minutes at the time of 4-1h evaluation and 5 minutes at the time of 4-1m evaluation. After allowing to stand for a minute, the reaction was stopped by adding 2M sulfuric acid, and the absorbance at 450 nm was measured with SpectraMax Paradigm (Molecular Devices). Were tested in duplicate was calculated by EC ₅₀ values of 4-parameter logistic curve fitting.

As a result, it was confirmed that 4-1h and 4-1m prepared in Example 4 had binding activity to human Fn14 and mouse Fn14 (Table 1).
Table 1: Binding activity of 4-1h and 4-1m to human and mouse Fn14

(Example 7: Tweak-induced NFkB activation inhibition assay for fully human antibody)
In order to evaluate the antagonist activity of 4-1h obtained in Example 4 against Fn14, the Twake-induced NFkB activation inhibitory effect was evaluated by a reporter assay.

The NFkB / HEK293 cells prepared in Example 3 were suspended in DMEM containing 10% fetal bovine serum at 1.25 × 10 ⁵ cells / mL, and 80 μL per well was seeded on a clear bottom white 96-well plate. After culturing overnight at 37 ° C. and 5% CO ₂ , the 11-step dilution series was prepared for 4-1h obtained in Example 4 at a final concentration of about 3 times from 0.1 ng / mL to 10 μg / mL. After preparation in the medium, 10 μL was added per well. After culturing at 37 ° C. and 5% CO ₂ for 30 minutes, 10 μL of Twake (Peprotech, 310-06) was added to a final concentration of 100 ng / mL. After culturing at 37 ° C. and 5% CO ₂ for 5 hours, NFkB activation was quantified according to the method of Example 3. The group to which only the medium containing no antibody was added was set as the control group, the group to which Tweak was added was 0% inhibitory, the group to which Tweak was not added was 100% inhibitory, and the concentration of antibody which was 50% inhibited by 4-parameter logistic curve fitting ( IC ₅₀ value) was calculated. Experiment in duplicate for each antibody, and geometric mean IC ₅₀ values of 3 trials was calculated 95% confidence intervals (Table 2). In this test, a mouse anti-human Fn14 antibody CRCBT-06-002 (Patent Document 2) was used as a comparative antibody.

As a result, it was revealed that 4-1h has a stronger antagonist activity than CRCBT-06-002.
Table 2: Inhibitory activity against Twake-induced NFkB activation in 4-1h

(Example 8: IL-8 production assay for fully human antibody)
The functional activity of 4-1h obtained in Example 4 was evaluated in an in vitro IL-8 production assay. A375 cells (ATCC, CRL-1619) were ^{suspended in 5x10 4} cells / mL in DMEM containing 10% fetal bovine serum and 100 μL per well was seeded on a 96-well flat bottom plate (IWAKI, 3860-096). After culturing overnight at 37 ° C. and 5% CO ₂ , the cells were washed with PBS. For 4-1h obtained in Example 4, a 10-fold dilution series from a final concentration of 1 ng / mL to 100 μg / mL was prepared in the medium and added to cells. _{After culturing overnight at 37 ° C. and 5% CO 2} in the presence or absence of Twake (antagonist activity evaluation) or non-existence (agonist activity evaluation) at a final concentration of 5 ng / mL, the culture supernatant was collected and IL in the culture supernatant was collected. -8 concentrations were measured using a Human IL-8 / CXCL8 Quantikine ELISA kit (R & D, D8000C). The final volumes in the antagonist and agonist activity assessments were tested at 65 μL / well and 100 μL / well, respectively. In this test, CRCBT-06-002 was used as a comparative antibody, and each antibody was duplicated.

In the antagonist activity evaluation, the inhibition rate was calculated by setting the group to which only the medium containing no antibody was added as the control group, and assuming that the Twake-added group was 0% inhibitory and the Tweak-free group was 100% inhibitory. Table 3 shows the arithmetic mean ± standard error of the maximum inhibition rate in each of the four experiments (Table 3). A graph of antagonist activity is shown in FIG. 1 and a graph of agonist activity is shown in FIG.

As shown in Table 3 and FIG. 1, 4-1h showed almost complete inhibitory activity against Twake-stimulated IL-8 production, whereas CRCBT-06-002 showed only partial inhibitory activity. Became clear. Further, as shown in FIG. 2, CRCBT-06-002 induced IL-8 production by the antibody alone in the absence of Tweak, whereas 4-1h of the present invention hardly induced IL-8 production. rice field.

From the above, it was clarified that CRCBT-06-002 is a partial antagonist antibody having an agonistic action, whereas 4-1h is a complete antagonist antibody having an extremely weak agonistic action on human Fn14.
Table 3: Inhibitory activity of 4-1h and CRCBT-06-002 against IL-8 production by Tweak stimulation

(Example 9: Combined effect of 4-1h surrogate antibody and anti-PD-1 antibody in mouse cancer-bearing model)
The functional activity of the 4-1h surrogate antibody (4-1h surrogate) obtained in Example 4 was evaluated using a B16F10 mouse cancer-bearing model. Anti-PD-1 antibodies have been used clinically as a breakthrough treatment for advanced cancers that are refractory to conventional treatments. However, most cancer patients are refractory to this treatment. B16F10 cancer-bearing mice, which are mouse malignant melanoma cells, are partially effective against anti-PD-1 antibody, but most of them are refractory or have a limited effect, and drug responsiveness similar to clinical one can be obtained. Known as a model (British Journal of Cancer, 2019, 120, 346-355).

B16F10 (ATCC, CRL-6475) was cultured in DMEM containing 10% fetal bovine serum ^{, suspended in CORNING to 0.5x10 7} cells / mL, and 0.5x10 ⁶ cells / in C57BL / 6 mice. It was administered subcutaneously to the left back in 100 μL (day 0). When the average tumor volume reaches 100-200 mm ³ (average tumor volume 116.3 mm ³ , day 1), grouping is performed based on the tumor volume (n = 10x4), and drug administration is started for each group over time. The tumor diameter was measured and the tumor volume was calculated (Fig. 3). The tumor volume (mm ³ ) was calculated by the formula of Long axis (mm) x Short axis (mm) x Short axis (mm) / 2, and expressed as an average ± standard error. The composition of each group and the date of administration are as follows.

(1) Control group; rat IgG2a isotype control (Bio X cell Clone 2A3) 100 μg / head intraperitoneal administration; day1, day6, day8 and anti-Keyhole Limpet Hemocyanin (KLH) antibody (KLH) antibody (KLH) antibody (KLH) ) 0.3 mg / kg subcutaneous administration; day1, day3, day6, day8
(2) Anti-PD-1 administration group; InVivoMAb anti-mouse PD-1 (Bio X cell RMP1-14) 100 μg / head intraperitoneal administration; day1, day6, day8 and anti-KLH antibody 0.3 mg / kg subcutaneously Administration; day1, day3, day6, day8
(3) 4-1h surrogate administration group; rat IgG2a isotype control 100 μg / head intraperitoneal administration; day1, day6, day8 and 4-1h surrogate 0.3 mg / kg subcutaneous administration; day1, day3, day6, day8
(4) Combination group; InVivoMAb anti-mouse PD-1 100 μg / head intraperitoneal administration; day1, day6, day8 and 4-1h surrogate 0.3 mg / kg subcutaneous administration; day1, day3, day6, day8

When the tumor volume was measured on the final day of evaluation (day 10), the 4-1h surrogate group and the combination group significantly suppressed the tumor volume. Similarly, the 4-1h surrogate group and the combination group significantly suppressed the tumor volume as compared with the anti-PD-1 antibody administration group. In addition, the combination group significantly suppressed the tumor volume as compared with the 4-1h surrogate group (p <0.05). On the other hand, the anti-PD-1 antibody-administered group showed a tendency to suppress as compared with the control group (Fig. 3).

The tumor was resected on Day 10 under isoflurane (Pfizer) anesthesia and stored at -80 ° C. RNA was extracted from the stored tumor sample to prepare cDNA. The change in mRNA expression of each group of mouse TOX (TOX) in the tumor was comparatively evaluated (with the mRNA expression level of each molecule of the control group as 1) by quantitative PCR (qPCR) using the prepared cDNA (mean ± standard error). Notation) (Fig. 4). 18S rRNA was used as the endogenous control gene.

TOX is a molecule known as a transcription factor that induces T cell exhaustion. Suppression of TOX expression is expected as one of the drug discovery targets for avoiding T cell exhaustion (Nature Immunology, 2019, 20, 1092-194). In the qPCR analysis of TOX, the expression of the combination group was significantly suppressed (p <0.05) as compared with the control group. The 4-1h surrogate group and the anti-PD-1 antibody group showed a tendency to suppress expression as compared with the control group.

(Example 10: Effect of 4-1h surrogate on mouse steroid-induced muscular atrophy model)
The functional activity of 4-1h surrogate was evaluated in a steroid-induced myopathy (SIM) model. After grouping by body weight (n = 10x6), free drinking was started (day 0) after adjusting the concentration of corticosterone (Fujifilm Wako Pure Chemical Industries, Ltd.) to 100 μg / mL, and continued until day 22. Drug administration was performed on

days

0, 2, 5, 7, 9, 12, 14, 16, 19, and 21. The group composition is as follows.

(1) Sham group; sham with PBS (10 mL / kg subcutaneous administration)
(2) SIM control group; SIM with PBS (10 mL / kg subcutaneous administration)
(3) KLH group; SIM with anti-KLH antibody (manufactured by Astellas Pharma Inc.) (3 mg / kg subcutaneous administration)
(4) 4-1h surrogate (0.03 mg / kg subcutaneous administration) group; SIM with 4-1h surrogate (0.03 mg / kg subcutaneous administration)
(5) 4-1h surrogate (0.3 mg / kg subcutaneous administration) group; SIM with 4-1h surrogate (0.3 mg / kg subcutaneous administration)
(6) 4-1h surrogate (3 mg / kg subcutaneous administration) group; SIM with 4-1h surrogate (3 mg / kg subcutaneous administration)

Grip strength measurement (GPM-100; Melquest) was performed on Day 21, and the value divided by the weight (g) of each individual (Grip strength (kg / kg BW) = mean grip strength (kg) / BW (g) × As a result of comparison in 1000), the SIM control group had a significant decrease in muscle strength as compared with the Sham group (p <0.01). On the other hand, the 4-1h surrogate 0.3 mg / kg group and the 3 mg / kg group had significantly improved muscle strength compared to the SIM control group (p <0.01) (expressed as mean ± standard error) ( FIG. 5).

The weight of the quadriceps femoris (AG104; METTTLER TOLEDO) was measured on Day 22, and the value was divided by the weight (g) of each individual (Quadrieps muscle weight (mg / g BW) = quadriceps muscle weight (g) / BW (g)). When compared with × 1000), the SIM control group had a significant decrease in muscle mass as compared with the Sham group (p <0.01). On the other hand, the muscle mass of the 4-1h surrogate 0.03 mg / kg and 0.3 mg / kg groups and the 3 mg / kg group was significantly improved as compared with the SIM control group (p <0.05 and p). <0.01) (expressed as mean ± standard error) (Fig. 6).

The pharmaceutical composition of the present invention is expected to be useful for the prevention or treatment of cancer or steroid myopathy. The prophylactic or therapeutic method of the present invention is expected to be useful for the prevention or treatment of cancer or steroid myopathy. The anti-human Fn14 antibody of the present invention is expected to be useful as an anti-human Fn14 antibody for use in the prevention or treatment of cancer or steroid myopathy. In addition, the anti-Fn14 antibody of the present invention is expected to be useful for use in the production of a pharmaceutical composition for the prevention or treatment of cancer or steroid myopathy.

In the number heading <223> of the following sequence listing, the description of "Artificial Sequence" is described. Specifically, the nucleotide sequences represented by SEQ ID NOs: 1, 5 and 3, 7 in the sequence listing are the nucleotide sequences of the heavy chain and light chain of the anti-human Fn14 antibody, respectively, and SEQ ID NOs: 2, 4, 6 and The amino acid sequence represented by 8 is the amino acid sequence of the heavy chain and the light chain encoded by SEQ ID NOs: 1, 3, 5 and 7, respectively. The amino acid sequence represented by SEQ ID NO: 9 is a peptide linker sequence linking a human Fn14 protein and a human Fc region protein.

Claims

A pharmaceutical composition containing an anti-Fn14 antibody or an antigen-binding fragment thereof and an excipient for preventing or treating cancer, which is used in combination with an immune checkpoint inhibitor.
The pharmaceutical composition according to claim 1, wherein the anti-Fn14 antibody or an antigen-binding fragment thereof is an anti-Fn14 antibody or an antigen-binding fragment thereof that inhibits the activation of human Fn14 by Twake stimulation without exhibiting agonist activity against human Fn14. thing.
The pharmaceutical composition according to claim 1 or 2, wherein the immune checkpoint inhibitor is an anti-PD-1 antibody or an anti-PD-L1 antibody.
The pharmaceutical composition according to any one of claims 1 to 3, wherein the immune checkpoint inhibitor is an anti-PD-1 antibody.
Anti-PD-1 antibodies include nivolumab, pembrolizumab, pidirizumab, cemiplimab, spartarizumab, spartarizumab, MEDI0680, , AMP-224, PF-06801591, tivolumab, ABBV-181, BI 754991, or SHR-1210, the pharmaceutical composition according to claim 3 or 4.
The pharmaceutical composition according to any one of claims 1 to 3, wherein the immune checkpoint inhibitor is an anti-PD-L1 antibody.
The anti-PD-L1 antibody is atezolizumab, durvalumab, BMS-936559, avelumab, rodapolimab, CX-072, FAZ053, KN035, or MD. Or the pharmaceutical composition according to 6.
The anti-Fn14 antibody or its antigen-binding fragment is CDR1 consisting of the amino acid sequences of amino acids 31 to 35 of SEQ ID NO: 2, CDR2 consisting of the amino acid sequences of amino acids 50 to 65 of SEQ ID NO: 2, and the amino acid of SEQ ID NO: 2. Heavy chain variable region including CDR3 consisting of amino acid sequences of Nos. 98 to 114, CDR1 consisting of amino acid sequences of amino acids Nos. 24 to 40 of SEQ ID NO: 4, amino acids of amino acids Nos. 56 to 62 of SEQ ID NO: 4. Claims 1 to 7, which are anti-human Fn14 antibodies or antigen-binding fragments thereof, comprising a light chain variable region comprising CDR2 consisting of a sequence and CDR3 consisting of amino acid sequences 95 to 103 of SEQ ID NO: 4. The pharmaceutical composition according to any one of the following items.
The pharmaceutical composition according to any one of claims 1 to 8, wherein the anti-Fn14 antibody or an antigen-binding fragment thereof is an anti-human Fn14 antibody or an antigen-binding fragment thereof selected from the following (1) and (2). Stuff:
(1) An anti-human Fn14 antibody or an anti-human Fn14 antibody containing a heavy chain variable region consisting of the amino acid sequences of amino acid numbers 1 to 125 of SEQ ID NO: 2 and a light chain variable region consisting of the amino acid sequences of amino acid numbers 1 to 114 of SEQ ID NO: 4. The antigen-binding fragment; and (2) the antibody or antigen-binding fragment thereof produced by post-translational modification of the anti-human Fn14 antibody of (1) above or the antigen-binding fragment thereof.
The anti-Fn14 antibody or its antigen-binding fragment comprises a heavy chain variable region consisting of the amino acid sequences of amino acids 1 to 125 of SEQ ID NO: 2 and a light chain variable region consisting of the amino acid sequences of amino acids 1 to 114 of SEQ ID NO: 4. The pharmaceutical composition according to any one of claims 1 to 9, which is an anti-human Fn14 antibody or an antigen-binding fragment thereof.
The pharmaceutical composition according to any one of claims 1 to 9, wherein the anti-Fn14 antibody or an antigen-binding fragment thereof is an anti-human Fn14 antibody selected from the following (3) and (4):
(3) Anti-human Fn14 antibody comprising a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 2 and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 4; and (4) post-translational modification of the antibody of (3) above. Anti-human Fn14 antibody, which is an antibody produced by.
15. The pharmaceutical composition according to any one of the following items.
The pharmaceutical composition according to claim 9 or 11, wherein the post-translational modification is pyroglutamylation at the N-terminal of the heavy chain and / or lysine deletion at the C-terminal of the heavy chain.
A pharmaceutical composition for the prevention or treatment of steroid myopathy, which comprises an anti-Fn14 antibody or an antigen-binding fragment thereof and an excipient.
The pharmaceutical composition according to claim 14, wherein the anti-Fn14 antibody or an antigen-binding fragment thereof is an anti-Fn14 antibody or an antigen-binding fragment thereof that inhibits the activation of human Fn14 by Twake stimulation without exhibiting agonist activity against human Fn14. thing.
The anti-Fn14 antibody or its antigen-binding fragment is CDR1 consisting of the amino acid sequences of amino acids 31 to 35 of SEQ ID NO: 2, CDR2 consisting of the amino acid sequences of amino acids 50 to 65 of SEQ ID NO: 2, and the amino acid of SEQ ID NO: 2. A heavy chain variable region containing CDR3 consisting of amino acid sequences of numbers 98 to 114, CDR1 consisting of amino acid numbers 24 to 40 of amino acid number 4 of SEQ ID NO: 4, and amino acids of amino acid numbers 56 to 62 of SEQ ID NO: 4. 13. A claim 14 or 15, which is an anti-human Fn14 antibody or an antigen-binding fragment thereof, comprising a light chain variable region comprising CDR2 consisting of a sequence and CDR3 consisting of amino acid numbers 95 to 103 of amino acid number 4 of SEQ ID NO: 4. The pharmaceutical composition described.
The pharmaceutical composition according to any one of claims 14 to 16, wherein the anti-Fn14 antibody or an antigen-binding fragment thereof is an anti-human Fn14 antibody or an antigen-binding fragment thereof selected from the following (1) and (2):
(1) An anti-human Fn14 antibody or an anti-human Fn14 antibody containing a heavy chain variable region consisting of the amino acid sequences of amino acid numbers 1 to 125 of SEQ ID NO: 2 and a light chain variable region consisting of the amino acid sequences of amino acid numbers 1 to 114 of SEQ ID NO: 4. The antigen-binding fragment; and (2) the antibody or antigen-binding fragment thereof produced by post-translational modification of the anti-human Fn14 antibody of (1) above or the antigen-binding fragment thereof.
The anti-Fn14 antibody or its antigen-binding fragment comprises a heavy chain variable region consisting of the amino acid sequences of amino acids 1 to 125 of SEQ ID NO: 2 and a light chain variable region consisting of the amino acid sequences of amino acids 1 to 114 of SEQ ID NO: 4. The pharmaceutical composition according to any one of claims 14 to 17, which is an anti-human Fn14 antibody or an antigen-binding fragment thereof.
The pharmaceutical composition according to any one of claims 14 to 17, wherein the anti-Fn14 antibody or an antigen-binding fragment thereof is an anti-human Fn14 antibody selected from the following (3) and (4):
(3) Anti-human Fn14 antibody comprising a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 2 and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 4; and (4) post-translational modification of the antibody of (3) above. Anti-human Fn14 antibody, which is an antibody produced by.
Claims 14 to 19, wherein the anti-Fn14 antibody or an antigen-binding fragment thereof is an anti-human Fn14 antibody containing a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 2 and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 4. The pharmaceutical composition according to any one of the following items.
The pharmaceutical composition according to claim 17 or 19, wherein the post-translational modification is pyroglutamylation at the N-terminal of the heavy chain and / or lysine deletion at the C-terminal of the heavy chain.