TW202206099A - Pharmaceutical composition and method for preventing or treating cancer with combined use of anti-human fn14 antibody and immune checkpoint inhibitor - Google Patents

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. The present invention provides: 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 composition and method for preventing or treating cancer by concomitant use of anti-human Fn14 antibody and immune checkpoint inhibitor

The present invention relates to the combined use of an anti-human Fn14 antibody and an immune checkpoint inhibitor for preventing or treating cancer, and an anti-human Fn14 antibody for preventing or treating steroid myopathy.

Fibroblast growth factor-inducible 14 (Fibroblast growth factor-inducible 14; Fn14) (also known as TNFRSF12A) is a member of the tumor necrosis factor receptor superfamily. In addition, Fn14 also binds to TNF (Tumor necrosis factor, tumor necrosis factor)-like weak inducer of apoptosis (TNF-like weak inducer of apoptosis; Tweak), and is known as a Tweak receptor. Regarding the activation of Tweak-dependent or independent Fn14, it is known to activate the NFkB signaling pathway, control cell proliferation, migration, differentiation, and apoptosis, as well as inflammation (non-patented) involved in angiogenesis, tissue damage, and regeneration. Reference 1).

As a correlation 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 3).

On the other hand, activation of Fn14 may lead to the production of inflammatory cytokines, which may worsen the inflammatory state. As an agonist antibody to human Fn14, enalatumab is reported to be in clinical development (Patent Document 1), but enalatumab has been reported to have hepatotoxicity in Phase I clinical trials (Non-Patent Document 4). ), suggesting that it may be related to inflammation caused by enatuzumab treatment (Non-Patent Document 5).

Mouse monoclonal antibody CRCBT-06-002 has been reported as an antibody that binds to human Fn14, has antagonist activity, and does not have agonist activity under specific conditions (Patent Document 2). CRCBT-06-002 was reported to have antagonist activity in inhibiting IL-8 production induced by Tweak stimulation in human malignant melanoma-derived cell line A375 cells, and was effective in a mouse model of cancer cachexia. However, the agonist activity to induce IL-8 production from A375 cells in the absence of Tweak still exists (Patent Document 2). There are no known anti-Fn14 antagonist antibodies that do not have agonist activity and avoid unwanted side effects.

Cancer immunotherapy is being researched and developed as a groundbreaking treatment for advanced cancers that are resistant to previous treatments. However, the vast majority of cancer patients do not respond to this treatment, and more powerful and innovative immunotherapies are expected to be available through single-agent or combination therapy. In addition to the increased expression of Fn14 in solid cancers, the expression of Fn14 is also increased in immune cells represented by dendritic cells and macrophages when stimulated by antigens. Further analysis of Tweak knockout mice suggests that Tweak-Fn14 signaling is inhibited It has a useful role in cancer immunotherapy (Non-Patent Document 6), but the anti-tumor effect of the anti-Fn14 antagonist antibody and the effect of combined use with existing cancer immunotherapy have not been clarified.

Excessive immune responses are known as adverse events in patients administered immune checkpoint inhibitors. In response to such adverse events, the administration of steroids is being studied (The Journal of experimental medicine, 2019, Vol.216, p.2701-2713).

Steroids are drugs that synthesize glucocorticoid components from hormones produced by the adrenal cortex, and are used for collagen diseases (rheumatoid arthritis, systemic lupus erythematosus, etc.), bronchial asthma, pneumonia, One of the standard treatments for various pathologies such as kidney disease, skin disease, allergy disease, etc. On the other hand, side effects (steroid myopathy (steroid-induced myopathy or steroid-induced myopathy)) associated with concomitant use of steroids are known to cause muscle wasting and muscle weakness, but other than discontinuing steroids or reducing the amount of steroids There is no other effective treatment. [Prior Art Literature] [Patent Literature]

[Patent Document 1] International Publication No. 2009/020933 [Patent Document 2] International Publication No. 2013/026099 [Non-patent literature]

[Non-Patent Document 1] Winkles JA, Nat Rev Drug Discov., 2008, Vol.7, p.411-425 [Non-Patent Document 2] Culp PA et al., Clin Cancer Res., 2010, Vol.16, p.497-508 [Non-Patent Document 3] Hu G et al., Tumor Biol., 2017, Vol.39 June, p.1-9 [Non-Patent Document 4] Lam ET et al., Mol Cancer Ther., 2018, Vol.17, p.215-221 [Non-Patent Document 5] Choi D et al., Am J Pharmacol Toxicol., 2017, Vol.12, p.18-38 [Non-Patent Document 6] Maeker et al., Cell, 2005, Vol.123, p.931-944

[Problems to be Solved by Invention]

The 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 a method for preventing or treating steroid myopathy by using an anti-human Fn14 antibody. [Technical means to solve problems]

When the present invention is completed, an anti-human Fn14 antibody comprising the following heavy chain variable region and light chain variable region is produced, wherein the heavy chain variable region includes: amino acids numbered 31 to 35 comprising amino acid number 31 of SEQ ID NO: 2 CDR1 of the sequence, CDR2 comprising the amino acid sequence of the amino acid number 50 to 65 of SEQ ID NO: 2, and CDR3 comprising the amino acid sequence of the amino acid number 98 to 114 of SEQ ID NO: 2, the above-mentioned light chain variable The region includes: CDR1 comprising the amino acid sequence of SEQ ID NO: 4 from 24 to 40, CDR2 comprising the amino acid sequence of SEQ ID NO: 4 from 56 to 62, and the amine comprising SEQ ID NO: 4 CDR3 (Examples 2 to 4) of the amino acid sequence of amino acid numbers 95 to 103. With regard to this antibody, it was found to bind to human Fn14 (Example 6), inhibit NFkB activation and IL-8 production by Tweak stimulation (Examples 7 and 8); and was not induced in the absence of Tweak IL-8 production (Example 8). The result provides an anti-human Fn14 antibody with antagonist activity and no agonist activity. Furthermore, the present inventors have repeatedly carried out a large number of creative studies, and as a result, they have found that the antibody obtained by murineization of the above-mentioned antibody (Example 4) and the anti-PD-1 antibody are used in a mouse cancer model to inhibit tumor growth (Example 4). Example 9), thereby completing the present invention related to the combined use of an anti-human Fn14 antibody and an immune checkpoint inhibitor. Furthermore, it was found that the above-mentioned anti-human Fn14 antibody inhibited the reduction of muscle strength and muscle weight in a mouse model of steroid myopathy (Example 10), thereby completing the present invention related to the application of anti-Fn14 antibody to steroid myopathy.

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, and used in combination with an immune checkpoint inhibitor. [2] The pharmaceutical composition according to [1], wherein the anti-Fn14 antibody or antigen-binding fragment thereof does not exhibit agonist activity for human Fn14 and inhibits the activation of human Fn14 by Tweak stimulation. [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] The pharmaceutical composition according to [3] or [4], wherein the anti-PD-1 antibody is nivolumab, pembrolizumab, pidilizumab, or simitrop cemiplimab, spartalizumab, MEDI0680, dostarlimab, cetrelimab, toripalimab, AMP-224, PF-06801591, tislelizumab, ABBV-181, BI 754091, or SHR-1210. [6] The pharmaceutical composition according to any one of [1] to [3], wherein the immune checkpoint inhibitor is an anti-PD-L1 antibody. [7] The pharmaceutical composition according to [3] or [6], wherein the anti-PD-L1 antibody is atezolizumab, durvalumab, BMS-936559, avelumab ), lodapolimab, CX-072, FAZ053, KN035, or MDX-1105. [8] The pharmaceutical composition according to any one of [1] to [7], wherein the anti-Fn14 antibody or antigen-binding fragment thereof is an anti-human Fn14 antibody or an antigen-binding fragment thereof comprising the following heavy chain variable region and light chain variable region Fragment, the above-mentioned heavy chain variable region comprises: CDR1 comprising the amino acid sequence of SEQ ID NO: 2 from 31 to 35, CDR2 comprising the amino acid sequence of SEQ ID NO: 2 from 50 to 65, and the CDR3 comprising the amino acid sequence of the amino acid number 98 of SEQ ID NO: 2 to 114, the above-mentioned light chain variable region comprises: the CDR1 comprising the amino acid sequence of the amino acid number 24 of SEQ ID NO: 4 to 40; The CDR2 of the amino acid sequence of SEQ ID NO: 4 from 56 to 62, and the CDR3 including the amino acid sequence of SEQ ID NO: 4 of the amino acid sequence of 95 to 103. [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): (1) An anti-human Fn14 antibody or an antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequences of amino acid numbers 1 to 125 of SEQ ID NO: 2 , the above-mentioned light chain variable region comprises the amino acid sequence of amino acid number 1 to 114 of SEQ ID NO: 4; and (2) An antibody or antigen-binding fragment thereof produced by post-translational modification of the anti-human Fn14 antibody or antigen-binding fragment thereof of (1) above. [10] The pharmaceutical composition according to any one of [1] to [9], wherein the anti-Fn14 antibody or its antigen-binding fragment is an anti-human Fn14 antibody or its antigen-binding agent comprising the following heavy chain variable region and light chain variable region Fragment, the variable region of the heavy chain comprises the amino acid sequence of amino acid number 1 to 125 of SEQ ID NO: 2, and the variable region of the light chain comprises the amino acid sequence of amino acid number 1 to 114 of SEQ ID NO: 4. [11] The pharmaceutical composition according to any one of [1] to [9], wherein the anti-Fn14 antibody or its antigen-binding fragment is an anti-human Fn14 antibody selected from the following (3) and (4): (3) an anti-human Fn14 antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 2, and the light chain comprises the amino acid sequence shown in SEQ ID NO: 4; and (4) An antibody produced by post-translational modification of the antibody of (3) above. [12] The pharmaceutical composition according to any one of [1] to [11], wherein the anti-Fn14 antibody or an antigen-binding fragment thereof is an anti-human Fn14 antibody comprising the following heavy chain and light chain, wherein the heavy chain comprises SEQ ID NO: 2 The amino acid sequence of the above-mentioned light chain comprises the amino acid sequence shown in SEQ ID NO: 4. [13] The pharmaceutical composition according to [9] or [11], wherein the post-translational modification is pyroglutamylation at the N-terminal of the heavy chain variable region and/or deletion of lysine at the C-terminal of the heavy chain.

Moreover, according to this invention, the invention of the following [14] to [21] is provided, for example. [14] A pharmaceutical composition comprising an anti-Fn14 antibody or antigen-binding fragment thereof and an excipient and for preventing or treating steroid myopathy. [15] The pharmaceutical composition as described in [14], wherein the anti-Fn14 antibody or antigen-binding fragment thereof does not exhibit agonist activity for human Fn14 and inhibits the activation of human Fn14 by Tweak stimulation. [16] The pharmaceutical composition according to [14] or [15], wherein the anti-Fn14 antibody or antigen-binding fragment thereof is an anti-human Fn14 antibody or antigen-binding fragment thereof comprising the following heavy chain variable region and light chain variable region, The chain variable region includes: CDR1 comprising the amino acid sequence of SEQ ID NO: 2 from 31 to 35, CDR2 comprising the amino acid sequence of SEQ ID NO: 2 from 50 to 65, and CDR2 comprising the amino acid sequence of SEQ ID NO: 2 The CDR3 of the amino acid sequence of the amino acid number 98 of 2 to 114, the above-mentioned light chain variable region comprises: the CDR1 of the amino acid sequence of the amino acid number 24 to 40 comprising the sequence number 4, the CDR1 comprising the amino acid sequence of the sequence number 4. CDR2 of the amino acid sequence of amino acid number 56 to 62, and CDR3 of amino acid sequence of amino acid number 95 to 103 of SEQ ID NO: 4 are included. [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): (1) An anti-human Fn14 antibody or an antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequences of amino acid numbers 1 to 125 of SEQ ID NO: 2 , the above-mentioned light chain variable region comprises the amino acid sequence of amino acid number 1 to 114 of SEQ ID NO: 4; and (2) An antibody or antigen-binding fragment thereof produced by post-translational modification of the anti-human Fn14 antibody or antigen-binding fragment thereof of (1) above. [18] The pharmaceutical composition according to any one of [14] to [17], wherein the anti-Fn14 antibody or antigen-binding fragment thereof is an anti-human Fn14 antibody or antigen-binding fragment thereof comprising the following heavy chain variable regions and light chain variable regions Fragment, the variable region of the heavy chain comprises the amino acid sequence of amino acid number 1 to 125 of SEQ ID NO: 2, and the variable region of the light chain comprises the amino acid sequence of amino acid number 1 to 114 of SEQ ID NO: 4. [19] The pharmaceutical composition according to any one of [14] to [17], wherein the anti-Fn14 antibody or antigen-binding fragment thereof is an anti-human Fn14 antibody selected from the following (3) and (4): (3) an anti-human Fn14 antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 2, and the light chain comprises the amino acid sequence shown in SEQ ID NO: 4; and (4) The antibody produced by post-translational modification of the antibody of (3) above is an anti-human Fn14 antibody. [20] The pharmaceutical composition according to any one of [14] to [19], wherein the anti-Fn14 antibody or an antigen-binding fragment thereof is an anti-human Fn14 antibody comprising the following heavy chain and light chain, wherein the heavy chain comprises SEQ ID NO: 2 The amino acid sequence of the above-mentioned light chain comprises the amino acid sequence shown in SEQ ID NO: 4. [twenty one] The pharmaceutical composition according to [17] or [19], wherein the post-translational modification is pyroglutamylation at the N-terminal of the heavy chain variable region and/or deletion of lysine at the C-terminal of the heavy chain.

Further, according to the present invention, for example, the following inventions [22] to [34] are provided. [twenty two] A method for preventing or treating cancer, comprising the steps 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. [twenty three] The method as described in [22], wherein the anti-Fn14 antibody or antigen-binding fragment thereof does not exhibit agonist activity for human Fn14 and inhibits the activation of human Fn14 by Tweak stimulation. [twenty four] 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] The method according to any one of [24] or [25], wherein the anti-PD-1 antibody is nivolumab, pembrolizumab, pidilizumab, cemiplimab, spartalizumab, MEDI0680, dostarlimab, cetrelimab, toripalimab, AMP- 224, PF-06801591, tislelizumab, ABBV-181, BI 754091, or SHR-1210. [27] The method according to any one of [22] to [24], wherein the immune checkpoint inhibitor is an anti-PD-L1 antibody. [28] The method according to [24] or [27], wherein the anti-PD-L1 antibody is atezolizumab, durvalumab, BMS-936559, avelumab, Lodapolimab, CX-072, FAZ053, KN035, or MDX-1105. [29] The method according to any one of [22] to [28], wherein the anti-Fn14 antibody or antigen-binding fragment thereof is an anti-human Fn14 antibody or antigen-binding fragment thereof comprising the following heavy chain variable region and light chain variable region, The above-mentioned heavy chain variable region includes: CDR1 comprising the amino acid sequence of SEQ ID NO: 2 from 31 to 35, CDR2 comprising the amino acid sequence of SEQ ID NO: 2 from 50 to 65, and a CDR2 comprising The CDR3 of the amino acid sequence of SEQ ID NO: 2 from 98 to 114, the above-mentioned light chain variable region includes: CDR1 comprising the amino acid sequence of SEQ ID NO: 4 from 24 to 40, including the CDR1 of SEQ ID NO: 4 CDR2 of the amino acid sequence of amino acid number 56 to 62 of SEQ ID NO: 4, and CDR3 of the amino acid sequence of amino acid number 95 to 103 of SEQ ID NO: 4 are included. [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 antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequences of amino acid numbers 1 to 125 of SEQ ID NO: 2 , the above-mentioned light chain variable region comprises the amino acid sequence of amino acid number 1 to 114 of SEQ ID NO: 4; and (2) An antibody or antigen-binding fragment thereof produced by post-translational modification of the anti-human Fn14 antibody or antigen-binding fragment thereof of (1) above. [31] The method according to any one of [22] to [30], wherein the anti-Fn14 antibody or antigen-binding fragment thereof is an anti-human Fn14 antibody or antigen-binding fragment thereof comprising the following heavy chain variable region and light chain variable region, The above-mentioned heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 2 of amino acid number 1 to 125, and the above-mentioned light chain variable region comprises the amino acid sequence of SEQ ID NO: 4 of amino acid number 1 to 114. [32] The method according to any one of [22] to [30], wherein the anti-Fn14 antibody or antigen-binding fragment thereof is an anti-human Fn14 antibody selected from the following (3) and (4): (3) an anti-human Fn14 antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 2, and the light chain comprises the amino acid sequence shown in SEQ ID NO: 4; and (4) An antibody produced by post-translational modification of the antibody of (3) above. [33] The method according to any one of [22] to [32], wherein the anti-Fn14 antibody or its antigen-binding fragment is an anti-human Fn14 antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises the amine shown in SEQ ID NO: 2 The amino acid sequence, the above-mentioned light chain comprises the amino acid sequence shown in SEQ ID NO: 4. [34] The method as described in [30] or [32], wherein the post-translational modification is pyroglutamylation at the N-terminus of the heavy chain variable region and/or deletion of lysine at the C-terminus of the heavy chain.

Moreover, according to this invention, the invention of the following [35] - [42] is provided, for example. [35] A method for preventing or treating steroid myopathy, comprising the steps of: administering a therapeutically effective amount of an anti-Fn14 antibody or an antigen-binding fragment thereof to a patient. [36] The method as described in [35], wherein the anti-Fn14 antibody or antigen-binding fragment thereof does not exhibit agonist activity for human Fn14 and inhibits the activation of human Fn14 by Tweak stimulation. [37] The method according to [35] or [36], wherein the anti-Fn14 antibody or its antigen-binding fragment is an anti-human Fn14 antibody or its antigen-binding fragment comprising the following heavy chain variable region and light chain variable region, and the above-mentioned heavy chain can be The variable region includes: CDR1 comprising the amino acid sequence of SEQ ID NO: 2 from 31 to 35, CDR2 comprising the amino acid sequence of SEQ ID NO: 2 from 50 to 65, and CDR2 comprising SEQ ID NO: 2. The CDR3 of the amino acid sequences of amino acid numbers 98 to 114, the light chain variable region includes: CDR1 comprising the amino acid sequences of amino acid numbers 24 to 40 of SEQ ID NO: 4, the amino group comprising SEQ ID NO: 4 CDR2 of the amino acid sequence of acid numbers 56 to 62, and CDR3 of the amino acid sequence of amino acid number 95 to 103 of SEQ ID NO: 4 are included. [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 antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequences of amino acid numbers 1 to 125 of SEQ ID NO: 2 , the above-mentioned light chain variable region comprises the amino acid sequence of amino acid number 1 to 114 of SEQ ID NO: 4; and (2) An antibody or antigen-binding fragment thereof produced by post-translational modification of the anti-human Fn14 antibody or antigen-binding fragment thereof of (1) above. [39] The method according to any one of [35] to [38], wherein the anti-Fn14 antibody or antigen-binding fragment thereof is an anti-human Fn14 antibody or antigen-binding fragment thereof comprising the following heavy chain variable regions and light chain variable regions, The above-mentioned heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 2 of amino acid number 1 to 125, and the above-mentioned light chain variable region comprises the amino acid sequence of SEQ ID NO: 4 of amino acid number 1 to 114. [40] The method according to any one of [35] to [38], wherein the anti-Fn14 antibody or antigen-binding fragment thereof is an anti-human Fn14 antibody selected from the following (3) and (4): (3) an anti-human Fn14 antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 2, and the light chain comprises the amino acid sequence shown in SEQ ID NO: 4; and (4) An antibody produced by post-translational modification of the antibody of (3) above. [41] The method according to any one of [35] to [40], wherein the anti-Fn14 antibody or its antigen-binding fragment is an anti-human Fn14 antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises the amine shown in SEQ ID NO: 2 The amino acid sequence, the above-mentioned light chain comprises the amino acid sequence shown in SEQ ID NO: 4. [42] The method as described in [38] or [40], wherein 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.

Moreover, according to this invention, the invention of the following [43] - [55] is provided, for example. [43] An anti-Fn14 antibody or antigen-binding fragment thereof used in combination with an immune checkpoint inhibitor and for preventing or treating cancer. [44] The anti-Fn14 antibody or antigen-binding fragment thereof as described in [43], wherein the anti-Fn14 antibody or antigen-binding fragment thereof does not exhibit agonist activity for human Fn14 and inhibits activation of human Fn14 by Tweak stimulation. [45] The anti-Fn14 antibody or the 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] The anti-Fn14 antibody or its antigen-binding fragment according to [45] or [46], wherein the anti-PD-1 antibody is nivolumab, pembrolizumab, or pidilizumab ), cemiplimab, spartalizumab, MEDI0680, dostarlimab, cetrelimab, toripalimab , AMP-224, PF-06801591, tislelizumab, 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] The anti-Fn14 antibody or its antigen-binding fragment as described in [45] or [48], wherein the anti-PD-L1 antibody is atezolizumab, durvalumab, BMS-936559, Avi avelumab, lodapolimab, CX-072, FAZ053, KN035, or MDX-1105. [50] The anti-Fn14 antibody or antigen-binding fragment thereof according to any one of [43] to [49], which is an anti-human Fn14 antibody or antigen-binding fragment thereof comprising the following heavy chain variable region and light chain variable region, wherein the above The heavy chain variable region includes: CDR1 comprising the amino acid sequence of amino acid number 31 to 35 of SEQ ID NO: 2, CDR2 comprising the amino acid sequence of amino acid number 50 to 65 of SEQ ID NO: 2, and a sequence comprising CDR3 of the amino acid sequence of amino acid number 98 to 114 of number 2, the above-mentioned light chain variable region includes: CDR1 comprising the amino acid sequence of amino acid number 24 to 40 of SEQ ID NO: 4, including SEQ ID NO: 4 The CDR2 of the amino acid sequence of SEQ ID NO: 56 to 62, and the CDR3 of the amino acid sequence of amino acid number 95 to 103 of SEQ ID NO: 4 are included. [51] The anti-Fn14 antibody or its antigen-binding fragment according to any one of [43] to [50], which is selected from the following (1) and (2) anti-human Fn14 antibodies or its antigen-binding fragment: (1) An anti-human Fn14 antibody or an antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequences of amino acid numbers 1 to 125 of SEQ ID NO: 2 , the above-mentioned light chain variable region comprises the amino acid sequence of amino acid number 1 to 114 of SEQ ID NO: 4; and (2) An antibody or antigen-binding fragment thereof produced by post-translational modification of the anti-human Fn14 antibody or antigen-binding fragment thereof of (1) above. [52] The anti-Fn14 antibody or antigen-binding fragment thereof according to any one of [43] to [51], which is an anti-human Fn14 antibody or an antigen-binding fragment thereof comprising the following heavy chain variable region and light chain variable region, wherein the above The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 2 from 1 to 125, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 4 from 1 to 114. [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) an anti-human Fn14 antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 2, and the light chain comprises the amino acid sequence shown in SEQ ID NO: 4; and (4) The antibody produced by post-translational modification of the antibody of (3) above is an anti-human Fn14 antibody. [54] The anti-Fn14 antibody or antigen-binding fragment thereof according to any one of [43] to [53], which is an anti-human Fn14 antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises the amine group shown in SEQ ID NO: 2 The acid sequence, the above-mentioned light chain comprises the amino acid sequence shown in SEQ ID NO: 4. [55] The anti-Fn14 antibody or antigen-binding fragment thereof according to [51] or [53], wherein the post-translational modification is pyroglutamylation at the N-terminal of the heavy chain variable region and/or lysine deletion at the C-terminal of the heavy chain.

Moreover, according to this invention, the invention of the following [56] - [63] is provided, for example. [56] An anti-Fn14 antibody or an antigen-binding fragment thereof for preventing or treating steroid myopathy. [57] The anti-Fn14 antibody or antigen-binding fragment thereof as described in [56], wherein the anti-Fn14 antibody or antigen-binding fragment thereof does not exhibit agonist activity for human Fn14 and inhibits activation of human Fn14 by Tweak stimulation. [58] The anti-Fn14 antibody or antigen-binding fragment thereof according to any one of [56] or [57], which is an anti-human Fn14 antibody or an antigen-binding fragment thereof comprising the following heavy chain variable region and light chain variable region, wherein the above The heavy chain variable region includes: CDR1 comprising the amino acid sequence of amino acid number 31 to 35 of SEQ ID NO: 2, CDR2 comprising the amino acid sequence of amino acid number 50 to 65 of SEQ ID NO: 2, and a sequence comprising CDR3 of the amino acid sequence of amino acid number 98 to 114 of number 2, the above-mentioned light chain variable region includes: CDR1 comprising the amino acid sequence of amino acid number 24 to 40 of SEQ ID NO: 4, including SEQ ID NO: 4 The CDR2 of the amino acid sequence of SEQ ID NO: 56 to 62, and the CDR3 of the amino acid sequence of amino acid number 95 to 103 of SEQ ID NO: 4 are included. [59] The anti-Fn14 antibody or its antigen-binding fragment according to any one of [56] to [58], which is selected from the following (1) and (2) anti-human Fn14 antibodies or its antigen-binding fragment: (1) An anti-human Fn14 antibody or an antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequences of amino acid numbers 1 to 125 of SEQ ID NO: 2 , the above-mentioned light chain variable region comprises the amino acid sequence of amino acid number 1 to 114 of SEQ ID NO: 4; and (2) An antibody or antigen-binding fragment thereof produced by post-translational modification of the anti-human Fn14 antibody or antigen-binding fragment thereof of (1) above. [60] The anti-Fn14 antibody or antigen-binding fragment thereof according to any one of [56] to [59], which is an anti-human Fn14 antibody or antigen-binding fragment thereof comprising the following heavy chain variable region and light chain variable region, wherein the above The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 2 from 1 to 125, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 4 from 1 to 114. [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) an anti-human Fn14 antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 2, and the light chain comprises the amino acid sequence shown in SEQ ID NO: 4; and (4) The antibody produced by post-translational modification of the antibody of (3) above is an anti-human Fn14 antibody. [62] The anti-Fn14 antibody or antigen-binding fragment thereof according to any one of [56] to [61], which is an anti-human Fn14 antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises the amine group shown in SEQ ID NO: 2 The acid sequence, the above-mentioned light chain comprises the amino acid sequence shown in SEQ ID NO: 4. [63] The anti-Fn14 antibody or antigen-binding fragment thereof according to [59] or [61], wherein the post-translational modification is pyroglutamylation at the N-terminal of the heavy chain variable region and/or lysine deletion at the C-terminal of the heavy chain.

Moreover, according to this invention, the invention of the following [64] - [76] is provided, for example. [64] Use of an anti-Fn14 antibody or an antigen-binding fragment thereof, which is to manufacture a pharmaceutical composition for preventing or treating cancer, and the pharmaceutical composition is used in combination with an immune checkpoint inhibitor. [65] Use as described in [64], wherein the anti-Fn14 antibody or antigen-binding fragment thereof does not exhibit agonist activity against human Fn14 and inhibits the activation of human Fn14 by Tweak stimulation. [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 one of [64] to [66], wherein the immune checkpoint inhibitor is an anti-PD-1 antibody. [68] The use as described in [66] or [67], wherein the anti-PD-1 antibody is nivolumab, pembrolizumab, pidilizumab, cimirizumab cemiplimab, spartalizumab, MEDI0680, dostarlimab, cetrelimab, toripalimab, AMP-224, PF- 06801591, tislelizumab, ABBV-181, BI 754091, or SHR-1210. [69] The use according to any one of [64] to [66], wherein the immune checkpoint inhibitor is an anti-PD-L1 antibody. [70] The use as described in [66] or [69], wherein the anti-PD-L1 antibody is atezolizumab, durvalumab, BMS-936559, avelumab, Lodapolimab, CX-072, FAZ053, KN035, or MDX-1105. [71] The use according to any one of [64] to [70], wherein the anti-Fn14 antibody or antigen-binding fragment thereof is an anti-human Fn14 antibody or antigen-binding fragment thereof comprising the following heavy chain variable region and light chain variable region, The above-mentioned heavy chain variable region includes: CDR1 comprising the amino acid sequence of SEQ ID NO: 2 from 31 to 35, CDR2 comprising the amino acid sequence of SEQ ID NO: 2 from 50 to 65, and a CDR2 comprising The CDR3 of the amino acid sequence of SEQ ID NO: 2 from 98 to 114, the above-mentioned light chain variable region includes: CDR1 comprising the amino acid sequence of SEQ ID NO: 4 from 24 to 40, including the CDR1 of SEQ ID NO: 4 CDR2 of the amino acid sequence of amino acid number 56 to 62 of SEQ ID NO: 4, and CDR3 of the amino acid sequence of amino acid number 95 to 103 of SEQ ID NO: 4 are included. [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 antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequences of amino acid numbers 1 to 125 of SEQ ID NO: 2 , the above-mentioned light chain variable region comprises the amino acid sequence of amino acid number 1 to 114 of SEQ ID NO: 4; and (2) An antibody or antigen-binding fragment thereof produced by post-translational modification of the anti-human Fn14 antibody or antigen-binding fragment thereof of (1) above. [73] The use according to any one of [64] to [72], wherein the anti-Fn14 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequences of amino acid numbers 1 to 125 of SEQ ID NO: 2 and an anti-human Fn14 antibody or an antigen-binding fragment thereof comprising the light chain variable region of the amino acid sequence of amino acid number 1 to 114 of SEQ ID NO: 4. [74] The use according to any one of [64] to [72], wherein the anti-Fn14 antibody or antigen-binding fragment thereof is an anti-human Fn14 antibody selected from the following (3) and (4): (3) an anti-human Fn14 antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 2, and the light chain comprises the amino acid sequence shown in SEQ ID NO: 4; and (4) The antibody produced by post-translational modification of the antibody of (3) above is an anti-human Fn14 antibody. [75] The use according to any one of [64] to [74], wherein the anti-Fn14 antibody or an antigen-binding fragment thereof is an anti-human Fn14 antibody comprising the following heavy chain and light chain, and the above-mentioned heavy chain comprises the amine shown in SEQ ID NO: 2 The amino acid sequence, the above-mentioned light chain comprises the amino acid sequence shown in SEQ ID NO: 4. [76] The use as described in [72] or [74], wherein 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.

Moreover, according to this invention, the invention of the following [77] - [84] is provided, for example. [77] Use of an anti-Fn14 antibody or an antigen-binding fragment thereof, which is used to manufacture a pharmaceutical composition for preventing or treating steroid myopathy. [78] Use as described in [77], wherein the anti-Fn14 antibody or antigen-binding fragment thereof does not exhibit agonist activity for human Fn14 and inhibits the activation of human Fn14 induced by Tweak stimulation. [79] The use as described in [77] or [78], wherein the anti-Fn14 antibody or its antigen-binding fragment is an anti-human Fn14 antibody or its antigen-binding fragment comprising the following heavy chain variable region and light chain variable region, wherein the heavy chain can be The variable region includes: CDR1 comprising the amino acid sequence of SEQ ID NO: 2 from 31 to 35, CDR2 comprising the amino acid sequence of SEQ ID NO: 2 from 50 to 65, and CDR2 comprising SEQ ID NO: 2. The CDR3 of the amino acid sequences of amino acid numbers 98 to 114, the light chain variable region includes: CDR1 comprising the amino acid sequences of amino acid numbers 24 to 40 of SEQ ID NO: 4, the amino group comprising SEQ ID NO: 4 CDR2 of the amino acid sequence of acid numbers 56 to 62, and CDR3 of the amino acid sequence of amino acid number 95 to 103 of SEQ ID NO: 4 are included. [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 antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequences of amino acid numbers 1 to 125 of SEQ ID NO: 2 , the above-mentioned light chain variable region comprises the amino acid sequence of amino acid number 1 to 114 of SEQ ID NO: 4; and (2) An antibody or antigen-binding fragment thereof produced by post-translational modification of the anti-human Fn14 antibody or antigen-binding fragment thereof of (1) above. [81] 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 or antigen-binding fragment thereof comprising the following heavy chain variable region and light chain variable region, The above-mentioned heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 2 of amino acid number 1 to 125, and the above-mentioned light chain variable region comprises the amino acid sequence of SEQ ID NO: 4 of amino acid number 1 to 114. [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) an anti-human Fn14 antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 2, and the light chain comprises the amino acid sequence shown in SEQ ID NO: 4; and (4) The antibody produced by post-translational modification of the antibody of (3) above is an anti-human Fn14 antibody. [83] The use according to any one of [77] to [82], wherein the anti-Fn14 antibody or its antigen-binding fragment is an anti-human Fn14 antibody comprising the following heavy chain and light chain, and the above-mentioned heavy chain comprises the amine shown in SEQ ID NO: 2 The amino acid sequence, the above-mentioned light chain comprises the amino acid sequence shown in SEQ ID NO: 4. [84] The use as described in [80] or [82], wherein the post-translational modification is pyroglutamylation at the N-terminus of the heavy chain variable region and/or deletion of lysine at the C-terminus of the heavy chain. [Effect of invention]

The anti-human Fn14 antibody used in the present invention does not exhibit agonist activity against human Fn14 but inhibits the activation of human Fn14 induced by Tweak stimulation, and thus has an inflammatory inhibitory effect. The pharmaceutical composition of the present invention may be used as a preventive or therapeutic agent for cancer or steroid myopathy. The prophylactic or therapeutic method of the present invention may possibly prevent or treat cancer or steroid myopathy. The anti-human Fn14 antibody of the present invention may be used to prevent or treat cancer or steroid myopathy. In addition, the anti-Fn14 antibody of the present invention may be used for the production of a pharmaceutical composition for the prevention or treatment of cancer or steroid myopathy.

The present invention will be described in detail below.

Antibodies exist in five classes, namely IgG, IgM, IgA, IgD and IgE. The basic structure of antibody molecules is common to all types, including heavy chains with molecular weights of 50,000 to 70,000 and light chains of 20,000 to 30,000. The heavy chain includes a polypeptide chain usually comprising about 440 amino acids, and each class has a characteristic structure, corresponding to IgG, IgM, IgA, IgD, IgE, called Igγ, Igμ, Igα, Igδ, Igε. Furthermore, IgG has the following subtypes, namely, IgG1, IgG2, IgG3, and IgG4, and the heavy chains corresponding to each are called Igγ1, Igγ2, Igγ3, and Igγ4. The light chain includes a polypeptide chain usually containing about 220 amino acids, and two known L-type and K-type are known as Igλ and Igκ, respectively. In the peptide composition of the basic structure of an antibody molecule, two identical heavy chains and two light chains are bound together by disulfide bonds (S-S bonds) and non-covalent bonds, and have a molecular weight of 150,000 to 190,000. 2 light chains can be paired with any heavy chain. Each antibody molecule is always formed from 2 identical light chains and 2 identical heavy chains.

There are four intra-chain SS bonds in the heavy chain (five in Igμ, Igε) and two in the light chain, and every 100-110 amino acid residues form a loop, and the three-dimensional structure is in each loop. similar between them, called structural units or regions. The region located at the N-terminus of the heavy chain and light chain is called the variable region, that is, even if the sample comes from the same species (subtype) of the same animal, the amino acid sequence is not constant, and each region is called the heavy chain. Variable region (VH) and light chain variable region (VL). The amino acid sequence on the C-terminal side of the variable region is substantially constant in each class or subtype and is called a constant region (each region is represented by 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 the site. On the other hand, biological activities such as binding to complement or to cells expressing various Fc receptors reflect structural differences in the constant regions of each class of Ig. It can be seen that the variability of the variable regions of the light chain and the heavy chain is mainly limited to three smaller hypervariable regions that exist in any chain, and these regions are called complementarity determining regions (CDRs; respectively from the N-terminal side). are CDR1, CDR2, CDR3). The remainder of the variable region, called the framework region (FR), is relatively constant.

Various antigen-binding fragments including VH and VL of antibodies also have antigen-binding activity, and examples of such representative antigen-binding fragments include single-chain variable fragment (scFv), Fab, Fab', F(ab ') ₂ . scFvs include monovalent antibody fragments of VH and VL linked by a linker, and Fabs include monovalent antibody fragments of a light chain and a heavy chain fragment including a portion of the VH, CH1 region and hinge region. A Fab' is a monovalent antibody fragment comprising a light chain and a heavy chain fragment comprising a VH, CH1 region and a portion of a hinge region, in which a portion of the hinge region comprises cysteine residues that constitute an inter-heavy chain SS bond. F(ab') ₂ fragments are bivalent antibody fragments in which 2 Fab' fragments are bound by inter-heavy chain SS bonds 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 does not exhibit agonist activity against human Fn14 and inhibits activation of human Fn14 by Tweak stimulation. Whether a certain antibody does not exhibit agonist activity for human Fn14 and inhibits the activation of human Fn14 by Tweak stimulation can be confirmed, for example, by the methods described in Examples 7 and 8.

In one embodiment, the anti-human Fn14 antibody or antigen-binding fragment thereof used in the present invention has the following characteristics: An anti-human Fn14 antibody or an antigen-binding fragment thereof comprising the following heavy chain variable region and light chain variable region, the above-mentioned heavy chain variable region comprising: comprising the amino acid sequences of amino acid numbers 31 to 35 of SEQ ID NO: 2 CDR1, CDR2 comprising the amino acid sequences of amino acid numbers 50 to 65 of SEQ ID NO: 2, and CDR3 comprising the amino acid sequences of amino acid numbers 98 to 114 of SEQ ID NO: 2, the above-mentioned light chain variable region includes : CDR1 comprising the amino acid sequences of SEQ ID NO: 4 from 24 to 40, CDR2 comprising the amino acid sequences of SEQ ID NO: 4 from 56 to 62, and CDR2 comprising the amino acid sequences of SEQ ID NO: 4 CDR3 of the amino acid sequences numbered 95 to 103.

In one embodiment, the anti-human Fn14 antibody or antigen-binding fragment thereof used in the present invention comprises the following heavy chain variable region and light chain variable region, wherein the heavy chain variable region comprises amino acid number 1 of SEQ ID NO: 2 Up to the amino acid sequence of 125, the above-mentioned light chain variable region comprises the amino acid sequence of the amino acid number 1 of SEQ ID NO: 4 to the amino acid sequence of 114.

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

The numbering of residues related to the introduction of amino acid variation in the constant region of the antibody used in the present specification is based on the EU index (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Ed., United States Public Health Service, National Institute of Health, Bethesda). L234A and L235A are leucine at positions 234 and 235 of amino acids 234 and 235 according to the EU index of Kabat et al. in the human Igγ1 constant region replaced by alanine. Examples of the human Igγ1 constant region having amino acid variations of L234A and L235A include human Igγ1 constant regions comprising the amino acid sequences of amino acid numbers 126 to 455 of SEQ ID NO: 2.

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

In the context of expressing antibodies in cells, antibodies are known to undergo post-translational modifications. Examples of post-translational modifications include: deletion of lysine at the C-terminal of the heavy chain by carboxypeptidase; The modification of pyroglutamic acid; glycosylation; acidification; deamidation; .7, p.2426-2447).

The anti-human Fn14 antibodies or antigen-binding fragments thereof used in the present invention also include post-translationally modified anti-human Fn14 antibodies or antigen-binding fragments thereof. Examples of the post-translationally modified anti-human Fn14 antibody or antigen-binding fragment thereof used in the present invention include pyroglutamylation of the N-terminal of the variable region of the heavy chain and/or isolation of the C-terminal of the heavy chain. An amino acid-deleted anti-human Fn14 antibody or antigen-binding fragment thereof. It is known in the art that such post-translational modification of N-terminal pyroglutamate or C-terminal lysine deletion does not affect activity (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 is an antibody or antigen-binding fragment thereof produced by post-translational modification of the following anti-human Fn14 antibody or antigen-binding fragment thereof, the above-mentioned anti-human Fn14 antibody or its antigen The binding fragment includes: a heavy chain variable region comprising the amino acid sequence of amino acid number 1 to 125 of SEQ ID NO: 2 and a light chain variable region comprising the amino acid sequence of amino acid number 1 to 114 of SEQ ID NO: 4 Area. 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 an anti-human Fn14 antibody or an antigen-binding fragment thereof comprising the following heavy chain variable regions and light chain variable regions, wherein the heavy chain variable regions are as follows: The region comprises the amino acid sequence of amino acid number 1 to 125 of SEQ ID NO: 2, and the glutamine of amino acid number 1 of SEQ ID NO: 2 is modified by pyroglutamic acid, and the above-mentioned light chain variable region comprises SEQ ID NO: 4 The amino acid number 1 to the amino acid sequence of 114.

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

In one embodiment, the anti-human Fn14 antibody system used in the present invention comprises the following heavy chain and light chain anti-human Fn14 antibodies, the above-mentioned heavy chain comprises the amino acid sequence of amino acid number 1 to 454 of SEQ ID NO: 2, And the glutamine of the amino acid number 1 of SEQ ID NO: 2 is modified by pyroglutamic acid, and the above-mentioned light chain comprises 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 .

Manufacturers can also produce fusions of antibodies or antigen-binding fragments thereof with other peptides or proteins based on the present invention; and modified bodies formed by combining modifiers. Antibodies or antigen-binding fragments thereof in these forms can also be used in the present invention. Other peptides or proteins that can be used for fusion are not particularly limited as long as the fusion can bind to Fn14, and examples thereof include human serum albumin, various tag peptides, artificial helical motif peptides, maltose-binding protein, glutathione Glypeptide S-transferase, various toxins, other peptides or proteins that can promote multimerization, etc. The modifier used for modification is not particularly limited as long as the modifier binds to Fn14, and examples thereof include polyethylene glycol, sugar chains, phospholipids, liposomes, and low molecular weight compounds.

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

"Anti-human Fn14 antibody" in this specification means an antibody that binds to human Fn14. Whether or not it binds to human Fn14 can be confirmed using a known binding activity assay method. As a method for measuring the binding activity, for example, methods such as enzyme-binding immunosorbent assay (ELISA) can be mentioned. In the case of using ELISA, for example, human Fn14 protein is immobilized in an ELISA culture plate, and a test antibody is added and reacted, and two enzymes such as anti-IgG antibody labeled with an enzyme such as horseradish peroxidase (HRP) are added. secondary antibody reacts. After the reaction, washing is performed, and the binding to the secondary antibody is measured by the activity of a reagent for detecting its activity (for example, in the case of HRP labeling, TMB Microwell Peroxidase Substrate (Kirkegaard & Perry Laboratories, 50-76-03)) or the like Identification was performed by which it was possible to confirm whether or not the test antibody binds to human Fn14. As a specific evaluation method, the method described in the following Example 6 can be used.

The anti-human Fn14 antibody used in the present invention includes antibodies that bind to other animal-derived Fn14 (eg, mouse Fn14) in addition to human Fn14 as long as it is an antibody that binds to human Fn14.

The anti-human Fn14 antibody or antigen-binding fragment thereof used in the present invention can be easily prepared by the practitioner based on the sequence information of the heavy chain and light chain of the antibody used in the present invention disclosed in this specification, using methods well known in the art. The anti-human Fn14 antibody or its antigen-binding fragment used in the present invention is not particularly limited. For example, the following <Method for producing the anti-human Fn14 antibody used in the present invention and the anti-human Fn14 antibody produced by the method> can be used. Manufactured by the method described.

The anti-human Fn14 antibody or its antigen-binding fragment used in the present invention can be further purified if necessary, and then formulated according to the usual practice, and used for the prevention or treatment of inflammatory diseases such as excessive angiogenesis, weight loss, muscle atrophy, and Cachexia and other wasting diseases.

<Polynucleotide for production of anti-human Fn14 antibody used in the present invention> The polynucleotides used to produce the anti-human Fn14 antibodies used in the present invention include polynucleotides that include a heavy chain variable region encoding the anti-human Fn14 antibodies or antigen-binding fragments thereof used in the present invention The polynucleotide of the base sequence, and the polynucleotide comprising the 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 comprising 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 following polynucleotide, which includes the nucleotide sequence encoding the SEQ ID NO: The amino acid numbers of 2 are the base sequences of the heavy chain variable regions of the amino acid sequences of 1 to 125.

As a polynucleotide including the base sequence encoding the heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 1 to 125, for example: 1 to 375 of the base sequence of the polynucleotide.

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 is the following polynucleotide comprising the amine shown in SEQ ID NO: 2 The base sequence of the heavy chain of the base sequence.

As a polynucleotide comprising the nucleotide sequence encoding the heavy chain of the amino acid sequence shown in SEQ ID NO: 2, for example, a polynucleotide comprising the nucleotide sequence shown in SEQ ID NO: 1 may be mentioned.

In one embodiment, the polynucleotide comprising the base 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 following polynucleotide, which includes the nucleotide sequence number The base sequence of the light chain variable region of the amino acid number 1 of 4 to the amino acid sequence of 114.

As a polynucleotide including the base sequence encoding the light chain variable region including the amino acid sequence of SEQ ID NO: 4 from 1 to 114, for example, the base number 1 including SEQ ID NO: 3 can be exemplified. Polynucleotides up to the base sequence of 342.

In one embodiment, the polynucleotide comprising the base sequence encoding the light chain variable region of the anti-human Fn14 antibody used in the present invention is the following polynucleotide comprising the amine shown in SEQ ID NO: 4 The nucleotide sequence of the light chain of the nucleotide sequence.

As the polynucleotide including the nucleotide sequence encoding the light chain including the amino acid sequence shown in SEQ ID NO: 4, for example, a polynucleotide including the nucleotide sequence shown in SEQ ID NO: 3 can be exemplified.

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

<Expression vector for production of anti-human Fn14 antibody used in the present invention> The expression vector used to produce the anti-human Fn14 antibody used in the present invention includes the following expression vector, which comprises: including the base encoding the heavy chain variable region of the anti-human Fn14 antibody or antigen-binding fragment thereof used in the present invention A polynucleotide of sequence and/or a polynucleotide comprising 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, as an expression vector for producing the anti-human Fn14 antibody used in the present invention, there can be exemplified: a polynucleoside comprising a nucleotide sequence encoding the heavy chain of the anti-human Fn14 antibody used in the present invention Acid expression vectors, including expression vectors comprising polynucleotides encoding the base sequence of the light chain of the anti-human Fn14 antibody used in the present invention, or expression vectors including the heavy chain encoding the anti-human Fn14 antibody used in the present invention. A polynucleotide of the base sequence and an expression vector comprising the polynucleotide of the base sequence encoding the light chain of the antibody.

As an expression vector for expressing the polynucleotide for producing the anti-human Fn14 antibody used in the present invention, as long as it is in eukaryotic cells (eg, animal cells, insect cells, plant cells, yeast) and/or prokaryotic cells A polynucleotide comprising a base sequence encoding the heavy chain variable region of the anti-human Fn14 antibody or antigen-binding fragment thereof used in the present invention is expressed in various host cells (eg, Escherichia coli) and/or comprising a polynucleotide encoding the present invention The polynucleotide of the base sequence of the light chain variable region of the anti-human Fn14 antibody or the antigen-binding fragment thereof to be used is not particularly limited if it can produce a polypeptide encoded by the same. As such an expression vector, a plasmid vector, a viral vector (for example, adenovirus, retrovirus) etc. are mentioned, for example, For example, pEE6.4 or pEE12.4 can be used. Furthermore, antibody genes can also be expressed by introducing variable region gene fragments into expression vectors such as AG-γ1 or AG-κ (for example, refer to WO94/20632) having human Ig constant region genes in advance.

The expression vector used to produce the anti-human Fn14 antibody used in the present invention may comprise a promoter capable of being functionally linked to the polynucleotide used to produce the anti-human Fn14 antibody used in the present invention. As the promoter for expressing the polynucleotide for producing the anti-human Fn14 antibody used in the present invention in animal cells, for example, promoters derived from viruses such as CMV, RSV, and SV40, and actin Promoter, EF (elongation factor) 1α promoter, heat shock promoter, etc. Examples of promoters used for expression in bacteria (eg, Escherichia) include trp promoter, lac promoter, λPL promoter, tac promoter, and the like. Moreover, as a promoter for expression in yeast, GAL1 promoter, GAL10 promoter, PH05 promoter, PGK promoter, GAP promoter, ADH promoter etc. are mentioned, for example.

In the case of using animal cells, insect cells, or yeast as host cells, the expression vector used to produce the anti-human Fn14 antibody used in the present invention may include a start codon and a stop codon. In this case, the expression vector used to produce the anti-human Fn14 antibody used in the present invention may also include: enhancer sequences, 5 of the genes encoding the antibody of the present invention or its heavy chain variable region or light chain variable region. 'side' and 3'-side untranslated regions, secretion signal sequences, splice junctions, polyadenylation sites, or replicable units, and the like. In the case of using Escherichia coli as the host cell, the expression vector for producing the anti-human Fn14 antibody used in the present invention may comprise: a start codon, a stop codon, a terminator region, and a replicable unit. In this case, the expression vector used to produce the anti-human Fn14 antibody used in the present invention may also include selection markers commonly used depending on the purpose (eg, tetracycline resistance gene, ampicillin resistance gene, kanamycin resistance gene). , neomycin resistance gene, dihydrofolate reductase gene).

<Host cell for producing the anti-human Fn14 antibody used in the present invention> As a host cell for producing the anti-human Fn14 antibody used in the present invention, a polynucleotide comprising a nucleotide sequence encoding the heavy chain of the anti-human Fn14 antibody used in the present invention, and a A host cell transformed with an expression vector of a polynucleotide of the base sequence of the light chain of the antibody; and a polynucleoside comprising the base sequence encoding the heavy chain of the anti-human Fn14 antibody used in the present invention An expression vector for an acid, and a host cell transformed with an expression vector comprising a polynucleotide encoding the light chain nucleotide sequence of the antibody.

In one embodiment, the host cell used to produce the anti-human Fn14 antibody used in the present invention comprises a host cell selected from the group consisting of the following (a) to (d) for producing the anti-human Fn14 used in the present invention. A host cell transformed from an antibody expression vector. (a) A host cell transformed from an expression vector comprising a polynucleotide comprising a heavy chain variable region encoding the anti-human Fn14 antibody or antigen-binding fragment thereof used in the present invention A polynucleotide of base sequence, and a polynucleotide comprising a base sequence encoding the light chain variable region of the antibody or antigen-binding fragment thereof; (b) A host cell transformed from an expression vector comprising a polynucleoside comprising a base sequence encoding the heavy chain variable region of the anti-human Fn14 antibody or antigen-binding fragment thereof used in the present invention An expression vector for an acid, and an expression vector comprising a polynucleotide comprising the base sequence encoding the light chain variable region of the antibody or antigen-binding fragment thereof; (c) A host cell transformed from an expression vector comprising a polynucleotide comprising a heavy chain variable region encoding the anti-human Fn14 antibody or antigen-binding fragment thereof used in the present invention base sequence polynucleotides; and (d) A host cell transformed from an expression vector comprising a polynucleotide comprising a light chain variable region encoding the anti-human Fn14 antibody or antigen-binding fragment thereof used in the present invention base sequence of polynucleotides.

In one embodiment, the host cell for producing the anti-human Fn14 antibody used in the present invention comprises a host cell selected from the group consisting of (e) to (h) below for producing the anti-human Fn14 antibody used in the present invention. A host cell transformed from an antibody expression vector. (e) A host cell transformed from an expression vector comprising a polynucleotide comprising a polynucleotide encoding the nucleotide sequence of the heavy chain of the anti-human Fn14 antibody used in the present invention , and a polynucleotide comprising the base sequence encoding the light chain of the antibody; (f) a host cell transformed from an expression vector comprising an expression vector comprising a polynucleotide encoding the nucleotide sequence of the heavy chain of the anti-human Fn14 antibody used in the present invention, and an expression vector comprising An expression vector of a polynucleotide encoding the base sequence of the light chain of the antibody; (g) A host cell transformed from an expression vector comprising a polynucleotide comprising a polynucleotide encoding the nucleotide sequence of the heavy chain of the anti-human Fn14 antibody used in the present invention ;as well as (h) A host cell transformed from an expression vector comprising a polynucleotide comprising a polynucleotide encoding the base sequence of the light chain of the anti-human Fn14 antibody used in the present invention .

The transformed host cell is not particularly limited as long as it is an expression vector suitable for use and can be transformed with the expression vector to express the antibody. Examples of the transformed host cells include various cells (for example, animal cells (for example, CHO-K1SV cells), insect cells (for example, Sf9), bacteria (Escherichia, etc.), yeast (Saccharomyces, Pichia, etc.), for example, CHO cells (CHO-K1SV cells, CHO-DG44 cells, etc.), 293 cells can be used , NS0 cells and other cultured cells.

The method for transformation into host cells is not particularly limited, and for example, calcium phosphate method, electroporation method, etc. can be used.

<The method for producing the anti-human Fn14 antibody used in the present invention and the anti-human Fn14 antibody produced by the method> The method for producing an anti-human Fn14 antibody or an antigen-binding fragment thereof used in the present invention includes the following method for producing an anti-human Fn14 antibody or an antigen-binding fragment thereof, comprising: culturing a method selected from the group consisting of (A) to (C) below The steps of expressing anti-human Fn14 antibodies or antigen-binding fragments thereof in host cells. (A) A host cell transformed from an expression vector comprising a polynucleotide comprising a heavy chain variable region encoding the anti-human Fn14 antibody or antigen-binding fragment thereof used in the present invention A polynucleotide of base sequence, and a polynucleotide comprising a base sequence encoding the light chain variable region of the antibody or antigen-binding fragment thereof; (B) a host cell transformed from an expression vector comprising a polynucleoside comprising a base sequence encoding the heavy chain variable region of the anti-human Fn14 antibody or antigen-binding fragment thereof used in the present invention An expression vector for an acid, and an expression vector comprising a polynucleotide comprising the base sequence encoding the light chain variable region of the antibody or antigen-binding fragment thereof; and (C) a host cell transformed from an expression vector comprising a polynucleotide comprising a base sequence encoding the heavy chain variable region of the anti-human Fn14 antibody or antigen-binding fragment thereof used in the present invention, and a host cell comprising A host cell transformed from an expression vector comprising a polynucleotide encoding the base sequence of the light chain variable region of the antibody or its antigen-binding fragment.

In one embodiment, the method for producing an anti-human Fn14 antibody used in the present invention includes the following method for producing an anti-human Fn14 antibody, comprising: culturing a host cell selected from the group consisting of (D) to (F) below , and the steps to express the anti-human Fn14 antibody. (D) A host cell transformed from an expression vector comprising a polynucleotide comprising a polynucleotide encoding the nucleotide sequence of the heavy chain of the anti-human Fn14 antibody used in the present invention , and a polynucleotide comprising the base sequence encoding the light chain of the antibody; (E) a host cell transformed from an expression vector comprising an expression vector comprising a polynucleotide encoding the nucleotide sequence of the heavy chain of the anti-human Fn14 antibody used in the present invention, and an expression vector comprising An expression vector of a polynucleotide encoding the base sequence of the light chain of the antibody; and (F) A host cell transformed from an expression vector comprising a polynucleotide comprising the nucleotide sequence encoding the heavy chain of the anti-human Fn14 antibody used in the present invention, and a host cell comprising a light chain comprising the light chain encoding the antibody A host cell transformed from an expression vector of a polynucleotide of base sequence.

The method for producing the anti-human Fn14 antibody or antigen-binding fragment thereof used in the present invention only includes the steps of culturing a host cell for producing the anti-human Fn14 antibody used in the present invention and expressing the anti-human Fn14 antibody or antigen-binding fragment thereof , there is no particular limitation. In one embodiment, as the host cell used in the method, the above-mentioned host cells (D) and (F) can be exemplified.

The culture of the transformed host cells can be carried out by well-known methods. Culture conditions such as temperature, pH value of the medium, and culture time can be appropriately selected. When the host cell is an animal cell, as the medium, for example, a MEM medium containing about 5 to 20% fetal bovine serum can be used (Eagle H, Science. 1959, Vol. 130 No. 3373, p. 432-437) , DMEM medium (Dulbecco R and Freeman G, Virology.1959, Vol.8, p.396-397), RPMI1640 medium (Moore GE et al., J Am Med Assoc.1967, Vol.199, p.519), 199 medium (Morgan JF et al., Proc Soc Exp Biol Med. 1950, Vol. 73, p. 1-8) and the like. The pH value of the medium is preferably about 6 to 8, and the culture is usually carried out at about 30 to 40° C. for about 15 to 336 hours while aeration or stirring is performed as necessary. When the host cells are insect cells, as the medium, for example, Grace's medium containing fetal bovine serum (Smith GE et al., Proc Natl Acad Sci USA. 1985, Vol. 82, p. 8404) and the like can be used. The pH value of the medium is preferably about 5 to 8, and the culture is usually carried out at about 20 to 40° C. for about 15 to 100 hours while aeration or stirring is performed as necessary. When the host cell is Escherichia coli or yeast, as the medium, for example, a liquid medium containing a nutrient source is suitable. The nutrient medium preferably contains the carbon source, inorganic nitrogen source or organic nitrogen source required for the growth of the transformed host cell. Examples of carbon sources include glucose, dextran, soluble starch, and sucrose, and examples of inorganic nitrogen sources or organic nitrogen sources include ammonium salts, nitrates, amino acids, corn steep liquor, Egg whites, casein, gravy, soybean meal, potato extract, etc. If necessary, other nutrients (for example, inorganic salts (for example, calcium chloride, sodium dihydrogen phosphate, magnesium chloride), vitamins, etc.), antibiotics (for example, tetracycline, neomycin, ampicillin, kanamycin, etc.) may also be contained ). The pH of the medium is preferably about 5-8. When the host cell is Escherichia coli, as a preferred medium, for example, LB medium, M9 medium (Mol. Clo., Cold Spring Harbor Laboratory, 2001, Vol.3, A2.2) can be used. During the culture, it is usually carried out at about 14 to 39° C. for about 3 to 24 hours while aeration or stirring is performed as necessary. When the host cell is yeast, as the medium, for example, Burkholder minimal medium (Bostian KA et al., Proc Natl Acad Sci USA. 1980, Vol. 77, p. 4504-4508) and the like can be used. During the culture, it is usually carried out at about 20 to 35° C. for about 14 to 144 hours while aeration or stirring is performed as necessary. By culturing as described above, the anti-human Fn14 antibody or antigen-binding fragment thereof used in the present invention can be expressed.

The method for producing the anti-human Fn14 antibody or antigen-binding fragment thereof used in the present invention includes the steps of culturing a host cell for producing the anti-human Fn14 antibody used in the present invention and expressing the anti-human Fn14 antibody or antigen-binding fragment thereof , and may further comprise the step of recovering the anti-human Fn14 antibody or antigen-binding fragment thereof from the transformed host cell, eg, performing isolation or purification. Examples of isolation or purification methods include: methods using solubility such as salting out and solvent precipitation; methods using differences in molecular weight such as dialysis, ultrafiltration, and gel filtration; ion exchange chromatography; hydroxyapatite Chromatography and other methods using charging; affinity chromatography and other methods using specific affinity; reverse-phase high performance liquid chromatography and other methods using the difference in hydrophobicity; isoelectric point electrophoresis and other methods using the difference in isoelectric points method etc. For example, antibodies stored in the culture supernatant can be purified by various chromatography methods, such as column chromatography using a protein A column or a protein G column.

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

<Immune checkpoint inhibitor used in the present invention> As used in this specification, "immune checkpoint inhibitor" or "checkpoint inhibitor" refers to a molecule that completely or partially reduces one or more checkpoint proteins, or inhibits more than one checkpoint inhibitor. Checkpoint protein, or interfere with more than one checkpoint protein, or negatively regulate more than one checkpoint protein; or completely or partially reduce the expression of more than one checkpoint protein, or inhibit the expression of more than one checkpoint protein Expression, or interfere with the expression of more than one checkpoint protein, or negatively regulate the expression of more than one checkpoint protein. In one embodiment, the immune checkpoint inhibitor binds to more than one checkpoint protein. In one embodiment, the immune checkpoint inhibitor binds to more than one molecule that modulates a checkpoint protein. In one embodiment, the immune checkpoint inhibitor binds to one or more checkpoint protein precursors, eg, at the DNA level or at the RNA level. In the present invention, any drug that functions as a checkpoint inhibitor can be used.

The term "part" used in the present specification means, for example, at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 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 inhibitory signaling (eg, with PD-1, PD-L1, CTLA-4, LAG-3, B7- Antibodies targeting H3, B7-H4, or TIM-3). Such ligands and receptors are outlined in Pardoll, D., Nature. 12:252-264, 2012. In addition, the present specification also describes immune checkpoint proteins that can be targeted.

In one embodiment, an immune checkpoint inhibitor prevents inhibitory signals associated with immune checkpoints. In one embodiment, an immune checkpoint inhibitor is an antibody or fragment thereof that disrupts immune checkpoint-related inhibitory signaling. In one embodiment, the immune checkpoint inhibitor is a low molecular weight inhibitor that disrupts inhibitory signaling. In one embodiment, the immune checkpoint inhibitor is an inhibitor that destroys the peptide matrix that inhibits signaling. In one embodiment, an immune checkpoint inhibitor is an inhibitory nucleic acid molecule that destroys inhibiting signaling. In one embodiment, an immune checkpoint inhibitor is one that interferes with the interaction of a checkpoint antagonist protein (eg, an antibody or fragment thereof that interferes with the interaction between PD-1 and PD-L1 or PD-L2). Antibodies, fragments thereof, or antibody mimetics. In one embodiment, the immune checkpoint inhibitor is an antibody, fragment thereof, 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 between TIM-3 and its ligand. In one embodiment, the immune checkpoint inhibitor interferes with inhibitory signaling mediated by CD39 and/or CD73, 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 and its receptor and/or B7-H4 and its receptor. In one embodiment, the immune checkpoint inhibitor interferes with the interaction of BTLA with its ligand HVEM. In one embodiment, the immune checkpoint inhibitor interferes 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 more than one of its ligands. In one embodiment, the immune checkpoint inhibitor interferes with the interaction of TIM-3 with more than one of its ligands Galectin-9, PtdSer, HMGB1 and CEACAM1. In one embodiment, the immune checkpoint inhibitor interferes with the interaction of TIGIT with more than one of its ligands PVR, PVRL2 and PVRL3. In one embodiment, the immune checkpoint inhibitor interferes with the interaction of CD94/NKG2A and HLA-E. In one embodiment, the immune checkpoint inhibitor interferes with the interaction of VISTA with more than one of its binding partners. In one embodiment, the immune checkpoint inhibitor interferes with the interaction of one or more sialic acid-binding Ig-like lectins (Siglec) with their respective ligands. In one embodiment, the immune checkpoint inhibitor interferes with CD20 signaling. In one embodiment, the immune checkpoint inhibitor interferes with the interaction of GARP with more than one 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, the immune checkpoint inhibitor interferes with NOX signaling. In one embodiment, the immune checkpoint inhibitor interferes with IDO and/or TDO signaling.

As described in this specification, inhibition or blockade of inhibitory immune checkpoint signaling prevents or reverses immunosuppression, establishing or enhancing T cell immunity to cancer cells. In one embodiment, inhibition of immune checkpoint signaling, as described herein, reduces or inhibits immune system insufficiency. In one embodiment, inhibition of immune checkpoint signaling does not render dysfunctional immune cells more dysfunctional, as described in this specification. In one embodiment, inhibition of immune checkpoint signaling further reduces the insufficiency of dysfunctional T cells, as described herein.

Regarding immune cells (eg, T cells), the term "insufficient" as used in the present specification refers to a state in which immune responsiveness to antigen stimulation is reduced. The term includes the common elements of both depletion and/or anergy that can cause antigen recognition, but the subsequent immune response has no effect on controlling infection or tumor proliferation. In addition, insufficiency includes a state in which antigen recognition is delayed due to insufficiency of immune cells. Insufficiency includes: unresponsiveness to antigen recognition; and impairment of the ability to translate antigen recognition into downstream T cell effector functions (eg, proliferation, cytokine production (eg, IL-2), and/or target cell killing).

With respect to immune cells (eg T cells), the term "anergy" as used in this specification refers to unresponsiveness to antigenic stimulation resulting from incomplete or insufficient signaling through the T cell receptor (TCR). state. Also, in the absence of co-stimulation, T-cell anergy may also occur when stimulated by antigen, with the result that, even in the presence of co-stimulation, cells become unresponsive to subsequent activation by antigen . The presence of IL-2 usually nullifies the state of unresponsiveness. Anergic T cells do not undergo clonal proliferation, and/or do not acquire effector function.

With regard to immune cells (eg T cells), the term "depletion" used in this specification refers to immune cell depletion such as T cell depletion as a state of T cell insufficiency, which is produced during various chronic infections and cancers The continuous TCR messaging results. Consumption differs from unresponsiveness in that it results from continuous message delivery rather than from incomplete or unfinished message delivery. Depletion is defined by insufficient effector function, persistent expression of inhibitory receptors, and a transcriptional state that differs from that of functional effector or memory T cells. Consumption interferes with optimal control of diseases such as infections and tumors. Depletion can result from both exogenous negative regulatory pathways (eg, immunomodulatory cytokines) as well as cellular endogenous negative regulatory pathways (such as the inhibitory immune checkpoint pathways described in this specification).

The meaning of "enhancement of T cell function" is as follows: induce, induce, or stimulate in such a way that T cells have sustained or enhanced biological function; or regenerate or reactivate depleted or inert T cells. Examples of enhancing T cell function include increased secretion of γ-interferon from CD8+ T cells, increased proliferation, and increased levels of antigen responsiveness (eg, tumor clearance rate) from those before intervention.

Immune checkpoint inhibitors can also be inhibitory nucleic acid molecules. The term "inhibitory nucleic acid" or "inhibitory nucleic acid molecule" as used herein refers to a nucleic acid molecule (eg, DNA or RNA) that completely or partially reduces, inhibits, interferes with, or negatively regulates one or more checkpoint proteins. As inhibitory nucleic acid molecules, oligonucleotides, siRNA, shRNA, antisense DNA or RNA molecules, and aptamers (eg, DNA or RNA aptamers) can be exemplified, but not limited thereto.

The term "oligonucleotide" used in the present specification refers to a nucleic acid molecule capable of reducing the expression of proteins, particularly the expression of checkpoint proteins such as the checkpoint proteins described in the present specification. Oligonucleotides are shorter DNA or RNA molecules, typically containing 2 to 50 nucleotides. Oligonucleotides are either single-stranded or double-stranded. The checkpoint inhibitor oligonucleotide can be an antisense oligonucleotide. Antisense oligonucleotides are single-stranded DNA or RNA molecules that are complementary to other sequences, particularly the nucleic acid sequences (or fragments thereof) of checkpoint proteins. Typically, antisense RNAs are used to prevent protein translation of mRNAs, such as mRNAs encoding checkpoint proteins, by binding to such mRNAs. Typically, antisense DNA is used to target a specific complementary (coding or non-coding) RNA. If bound, this DNA/RNA hybrid is cleaved by the enzyme RNase H, and N-morpholino antisense oligonucleotides can be used for gene knockdown in vertebrates (e.g., J Exp Med., 2006, 203:871-81).

The terms "siRNA" or "low molecular weight interfering RNA" or "low molecular weight inhibitory RNA" are used interchangeably in this specification and refer to a double-stranded RNA of a typical length of 20-25 base pairs that interferes with the expression of a particular gene Molecules, the above-mentioned specific gene is, for example, a gene encoding a checkpoint protein with a complementary nucleotide sequence. In one embodiment, siRNA interferes with mRNA, thereby preventing translation (eg, of immune checkpoint proteins). Transfection of exogenous siRNA can be used for gene knockdown, but the effect may be temporary, especially in rapidly dividing cells. Stable transfection can be achieved, for example, by RNA modification or by using expression vectors. Modifications and vectors for stably transfecting cells with siRNA are known in the art. The siRNA sequence can also be modified to introduce a shorter loop between the two strands to generate "small hairpin RNA" or "shRNA". shRNA can be processed by Dicer into functional siRNA. shRNA has a relatively low rate of decomposition and metabolic turnover. Thus, the immune checkpoint inhibitor can be a shRNA.

The term "aptamer" used in this specification typically refers to a single-stranded nucleic acid molecule such as DNA or RNA of 25 to 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 in this specification. For example, the aptamers of the present invention can specifically bind to immune checkpoint proteins or polypeptides, or molecules in message transduction pathways that modulate the expression of immune checkpoint proteins or polypeptides. The generation and use of aptamers in therapy is well known in the art (eg, US 5,475,096).

The terms "low-molecular-weight inhibitor" or "low-molecular-weight" are used interchangeably in this specification and, as described above, refer to the reduction of one or more checkpoint proteins, in whole or in part, or the inhibition of one or more checkpoint proteins, Low molecular weight organic compounds (usually up to 1000 Daltons) that interfere with, or negatively regulate, one or more checkpoint proteins. Such low molecular weight inhibitors are usually synthesized by organic chemistry, but can also be isolated from natural sources such as plants, fungi, and microorganisms. Low molecular weight enables rapid diffusion of low molecular weight inhibitors across cell membranes. For example, various A2AR antagonists known in the art are organic compounds having molecular weights of less than 500 Daltons.

The immune checkpoint inhibitor can be an antibody, an antigen-binding fragment thereof, an antibody mimetic, or a fusion protein comprising an antibody portion with the desired specific antigen-binding fragment. Antibodies or antigen-binding fragments thereof are as described in this specification. Antibodies or antigen-binding fragments thereof that are immune checkpoint inhibitors especially comprise antibodies or antigen-binding fragments thereof that bind to immune checkpoint proteins such as immune checkpoint receptors or immune checkpoint receptor ligands. Antibodies or antigen-binding fragments can be fused to peptides or proteins, or can be conjugated to modifying agents. In particular the antibody or antigen-binding fragment thereof is a chimeric, humanized or human antibody. In one embodiment, the antibody or antigen-binding fragment thereof that is an immune checkpoint inhibitor is an antagonist of an immune checkpoint receptor or an immune checkpoint receptor ligand. In another embodiment, the antibody is isolated from the antibody system as an immune checkpoint inhibitor. The antibody used as the immune checkpoint inhibitor or antigen-binding fragment thereof of the present invention may be an antibody that binds to the antigen cross-competitively with any known immune checkpoint inhibitor antibody. In one embodiment, the immune checkpoint inhibitor antibody system cross-competes with one or more immune checkpoint inhibitor antibodies described in this specification. Regarding the ability of antibodies to cross-competitively bind to antigens, it means that these antibodies can bind to the same epitope region of the antigen, or when binding to different epitopes, sterically interfere with known immune checkpoint inhibitor antibodies to specific Epitope region binding. These cross-competing antibodies are predicted to prevent the binding of immune checkpoints to their ligands by binding to the same epitope, or by sterically interfering with ligand binding, and thus may function very similarly to cross-competitors characteristic. Cross-competing antibodies can be readily identified in standard binding assays (eg, Surface Plasmon Resoncance assays, ELISA assays or flow cytometry assays (WO2013/173223)) based on their ability to cross-compete with more than one known antibody. In one embodiment, the antibody or antigen-binding fragment thereof that cross-competesly binds 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-competing antibodies can be chimeric, or humanized or human antibodies. Such chimeric, humanized or human monoclonal antibodies can be prepared and isolated by methods well known in the art.

Also, the checkpoint inhibitor may be a soluble form of the molecule (or a variant thereof) itself, such as a soluble PD-L1 or a PD-L1 fusion form.

In one embodiment, two or more checkpoint inhibitors may be used. Here, two or more checkpoint inhibitors target different checkpoint pathways or the same checkpoint pathway. In one embodiment, the two or more checkpoint inhibitors are different checkpoint inhibitors. Where more than 2 different checkpoint inhibitors are used, at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 different checkpoint inhibitors are used, in another embodiment, 2, 3, 4 or 5 different checkpoint inhibitors are used, in yet another embodiment 2, 3 or 4 different checkpoint inhibitors are used, in yet another embodiment 2 or 3 different checkpoint inhibitors are used The checkpoint inhibitor, in yet another embodiment, uses two different checkpoint inhibitors.

In one embodiment, the immune checkpoint inhibitor is an inhibitor of the PD-1/PD-L1 or PD-1/PD-L2 signaling pathway. Accordingly, one embodiment provides for administering to a subject 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 a PD-L2 inhibitor. In one embodiment, the checkpoint inhibitor of the PD-1 signaling pathway is an antibody or its antigen binding that disrupts the interaction between PD-1 and one or more of its ligands, PD-L1 and/or PD-L2 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, inhibiting 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, inhibiting 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, the immune checkpoint inhibitor is nivolumab (nivolumab, WO2006/121168), pembrolizumab (pembrolizumab, WO2008/156712), pidilizumab (WO2009/101611), Mipilimab (cemiplimab, WO2015/112800), spartalizumab, MEDI0680 (WO2012/145493), dostarlimab, cetrelimab, toripal toripalimab, AMP-224 (WO2010/027827, WO2011/066342), PF-06801591, tislelizumab, ABBV-181, BI 754091, or SHR-1210 (WO2015/085847) .

In one embodiment, the immune checkpoint inhibitor is atezolizumab (atezolizumab, US9,724,413), durvalumab (durvalumab, WO2011/066389), BMS-936559 (WO2013/173223), avelumab Monoclonal antibody (avelumab, US2014/0341917), lodapolimab, CX-072 (WO2016/149201), FAZ053, KN035 (WO2017/020801, WO2017/020802), or MDX-1105 (US2015/0320859).

In one embodiment, the immune checkpoint inhibitor is an inhibitor of the CTLA-4 signaling pathway. Accordingly, one embodiment provides for administering to a subject 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. Accordingly, one embodiment provides for administering to a subject 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 of signaling pathways. In one embodiment, the B7 family members are B7-H3 and B7-H4. One embodiment provides for administering to a subject a checkpoint inhibitor of B7-H3 and/or B7-H4. Accordingly, one embodiment provides for administering to a subject an antibody or antigen-binding fragment thereof that targets B7-H3 or B7-H4. In one embodiment, the checkpoint inhibitor of the B7 family messaging pathway is an antibody or antigen-binding fragment thereof that disrupts the interaction between the B7 family and receptors.

In one embodiment, the immune checkpoint inhibitor is an inhibitor of the BTLA signaling pathway. Accordingly, one embodiment provides for administering to a subject 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 more than one KIR signaling pathway. Accordingly, one embodiment provides for administration of a checkpoint inhibitor of more than one KIR signaling pathway to a subject. In one embodiment, the checkpoint inhibitor of more than one KIR signaling pathway is a KIR inhibitor. In one embodiment, the checkpoint inhibitor of more than one KIR signaling pathway 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. Accordingly, one embodiment provides for administering to a subject 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. Accordingly, one embodiment provides for administering to a subject 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. Accordingly, one embodiment provides for administering to a subject a checkpoint inhibitor of the CD94/NKG2A signaling pathway. 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. Accordingly, one embodiment provides for administration of a checkpoint inhibitor of the IDO signaling pathway to a subject. 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. Accordingly, one embodiment provides for administering to a subject 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. Accordingly, one embodiment provides for administering to a subject 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 sialic acid-binding immunoglobulin-like lectin signaling pathways. Accordingly, one embodiment provides a checkpoint inhibitor for administering to a subject one or more sialic acid-binding immunoglobulin-like lectin signaling pathways. In one embodiment, one or more checkpoint inhibitors of the sialic acid-binding immunoglobulin-like lectin signaling pathway are sialic acid-binding immunoglobulin-like lectin inhibitors. In one embodiment, one or more checkpoint inhibitors of the sialic acid-binding immunoglobulin-like lectin signaling pathway are sialic acid-binding immunoglobulin-like lectin ligand inhibitors. In one embodiment, the checkpoint inhibitor of the sialic acid-binding immunoglobulin-like lectin signaling pathway is an antibody or antigen-binding thereof that disrupts the interaction between the sialic acid-binding immunoglobulin-like lectin and its ligand Fragment.

In one embodiment, the immune checkpoint inhibitor is an inhibitor of the CD20 signaling pathway. Accordingly, one embodiment provides for administering to a subject 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. Accordingly, one embodiment provides for administering to a subject 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. Accordingly, one embodiment provides for administering to a subject 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. Accordingly, one embodiment provides for administering to a subject 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. Accordingly, one embodiment provides for administering to a subject 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. Accordingly, one embodiment provides for administering to a subject 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. Accordingly, one embodiment provides for administering to a subject 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, sialic acid binding Inhibitors of immunoglobulin-like lectins, CD20, CD39, CD73, GARP, CD47, PVRIG, CSF1R, NOX and TDO inhibitors and their respective ligands are known, and several of them have been used in clinical trials or have been Approved. Based on these well-known immune checkpoint inhibitors, alternative immune checkpoint inhibitors can be used. Well-known inhibitors of immune checkpoint proteins can be used directly, or their analogs can be used, and as the case may be, an antibody that cross-competes with any of the antibodies described in this specification, chimerization, humanization, or humanization of the antibody can be used antibody. Targeting results in increased T cell proliferation, enhanced T cell activation, increased production of cytokines (eg, IFN-γ, IL2), and/or stimulation of immune responses such as anti-tumor immune responses, in this case Other 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 mode and route of administration is dependent on the mode of checkpoint inhibitor used. The checkpoint inhibitor can be administered in the form of any appropriate pharmaceutical composition. Checkpoint inhibitors can be administered in the form of immune checkpoint inhibitors, eg, inhibitory nucleic acid molecules or nucleic acids (eg, DNA or RNA molecules) encoding antibodies or fragments thereof. For example, antibodies can be encoded by an expression vector and delivered. Nucleic acid molecules can be delivered, for example, in the form of plastids or mRNA molecules, or complexed with delivery vehicles such as liposomes, lipid complexes, or nucleic acid lipid particles. Also, the administration of the checkpoint inhibitor can be mediated by an oncolytic virus comprising an expression cassette encoding the checkpoint inhibitor. Also, checkpoint inhibitors can be administered by administering endogenous cells or allogeneic cells (eg, in the form of cell-matrix therapy) that express the checkpoint inhibitor.

The term "cell matrix therapy" refers to the transplantation of cells expressing immune checkpoint inhibitors (eg, T lymphocytes, dendritic cells, or stem cells) into a subject to treat a disease or disorder (eg, cancer). In one embodiment, the treatment of the cell matrix comprises genetically manipulated cells. In one embodiment, the genetically manipulated cells express an immune checkpoint inhibitor. In one embodiment, the genetically manipulated cells express an immune checkpoint inhibitor, i.e., siRNA, shRNA, oligonucleotide, antisense DNA or RNA, aptamer, antibody or fragment thereof, or a soluble immune checkpoint protein or fusions and other inhibitory nucleic acid molecules. Also, genetically manipulated cells may express further agents that enhance T cell function. Such agents are known in the art. Cell-based treatments for inhibiting immune checkpoint signaling are disclosed, for example, in WO2018/222711.

The term "oncolytic virus" as used in this specification refers to a virus that, although it does not or minimally affect normal cells, can cause cancer in either in vitro or in vivo. Either overgrown cells replicate selectively, delaying their growth, or inducing cell death. Oncolytic viruses used to deliver immune checkpoint inhibitors comprise expression cassettes encoding immune checkpoint inhibitors, i.e. siRNA, shRNA, oligonucleotides, antisense DNA or RNA, aptamers, antibodies or Fragments thereof, or inhibitory nucleic acid molecules such as soluble immune checkpoint proteins or fusions, and the like. The oncolytic virus is preferably replication competent, and the expression cassette is under the control of a viral promoter (eg, an early/late synthetic poxvirus promoter). Examples of oncolytic viruses include vesicular stomatitis virus (VSV), baculovirus (for example, Seneca Valley virus; picornaviruses such as SVV-001), coxsackie virus, parvovirus , Newcastle virus (NDV), herpes simplex virus (HSV; granulocyte macrophage colony stimulating factor GMCSF), retroviruses (eg, influenza virus), measles virus, Leo virus, Sindby virus, vaccinia virus ( exemplarily described in WO2017/209053 (including Copenhagen, Western Reserve, Wyeth strains), and adenoviruses (eg, Delta-24, Delta-24-RGD, ICOVIR-5, ICOVIR-7, ICOVIR-7, Onyx -015, ColoAd1, H101, AD5/3-D24-GMCSF). The generation of recombinant oncolytic viruses comprising soluble forms of immune checkpoint inhibitors and methods of use thereof are disclosed in WO2018/022831. Attenuated viruses can be used for oncolytic viruses.

<Pharmaceutical composition of the present invention, method of prevention or treatment of the present invention, antibody for prevention or treatment of the present invention, use in production of the present invention> The pharmaceutical composition of the present invention includes a pharmaceutical composition comprising 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 commonly used in the field, ie, pharmaceutical excipients or pharmaceutical carriers, and the like. Examples of the dosage form of these pharmaceutical compositions include parenteral preparations such as injections and drips, and can be administered by intravenous administration, subcutaneous administration, intramuscular administration, and the like. During formulation, excipients, carriers, additives, etc., which are compatible with these dosage forms, can be used within the scope of pharmaceutically acceptable.

The pharmaceutical composition of the present invention may comprise a plurality of anti-human Fn14 antibodies or antigen-binding fragments thereof used in the present invention. For example, the present invention also includes pharmaceutical compositions comprising an antibody or antigen-binding fragment thereof that has not undergone post-translational modification, and an antibody or antigen-binding fragment thereof produced by post-translational modification of the antibody or antigen-binding fragment thereof.

In one embodiment, the pharmaceutical composition of the present invention containing the anti-human Fn14 antibody or antigen-binding fragment thereof also includes the pharmaceutical composition described below. A pharmaceutical composition comprising: an anti-human Fn14 antibody or an antigen-binding fragment thereof comprising the following heavy chain variable regions and light chain variable regions; and an antibody produced by post-translational modification of the antibody or its antigen-binding fragment or an antigen-binding fragment thereof, wherein the variable region of the heavy chain comprises the amino acid sequences of the amino acid numbers 1 to 125 of SEQ ID NO: 2, and the variable region of the light chain comprises the amines of the amino acid numbers 1 to 114 of SEQ ID NO: 4 base acid sequence.

The pharmaceutical compositions of the present invention also include the following pharmaceutical compositions comprising: an antibody with a C-terminal lysine deletion of the heavy chain, an antibody or an antigen-binding fragment thereof subjected to post-translational modification at the N-terminal, a heavy chain C-terminal lysine deletion, and An antibody that has undergone N-terminal post-translational modification, and/or an antibody that has a heavy chain C-terminal lysine and has not undergone 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 anti-human Fn14 antibodies of the following (5) to (8). (5) An anti-human Fn14 antibody comprising the following heavy chain and light chain, wherein the heavy chain comprises the amino acid sequences of SEQ ID NO: 2 to 454, and the light chain comprises the amino acid shown in SEQ ID NO: 4 sequence. (6) An anti-human Fn14 antibody comprising the following heavy chain and light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 2 and the glutamine of amino acid number 1 in SEQ ID NO: 2 is modified to pyroglutamine acid, the above-mentioned light chain comprises the amino acid sequence shown in SEQ ID NO: 4. (7) An anti-human Fn14 antibody comprising a heavy chain and a light chain, the heavy chain comprising the amino acid sequences of amino acid number 1 to 454 of SEQ ID NO: 2 and glutamine of amino acid number 1 of SEQ ID NO: 2 It is modified into pyroglutamic acid, and the above-mentioned light chain contains the amino acid sequence shown in SEQ ID NO: 4. (8) An anti-human Fn14 antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 2, and the light chain comprises 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. A pharmaceutical composition comprising: an anti-human Fn14 antibody comprising a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 2 and a light chain comprising the amino acid sequence shown in SEQ ID NO: 4; The amino acid sequence of amino acid number 1 to 454, and the glutamine of amino acid number 1 of SEQ ID NO: 2 is modified into a heavy chain of pyroglutamic acid and a light chain comprising the amino acid sequence shown in SEQ ID NO: 4 chain human Fn14 antibody; and a pharmaceutically acceptable excipient.

During formulation, the added amount of the anti-human Fn14 antibody or its antigen-binding fragment used in the present invention varies depending on the degree of symptoms of the patient, age, the dosage form of the preparation used, or the binding titer of the antibody, etc. Use about 0.001 mg/kg to 100 mg/kg.

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

In this specification, the term "cancer" includes brain tumor, head and neck cancer, salivary gland cancer, thyroid cancer, lung cancer, small cell lung cancer, breast cancer, mesothelioma, pancreatic cancer, liver cancer, biliary tract cancer, esophageal cancer, stomach cancer, gastrointestinal cancer Interstitial tumor, small bowel cancer, colorectal 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 for prevention or treatment by the present invention is a cancer expressing Fn14. In one embodiment, the cancer for prevention or treatment by the present invention is a cancer that is sensitive to anti-Fn14 antibodies.

The present invention provides a pharmaceutical composition comprising the anti-human Fn14 antibody or its antigen-binding fragment used in the present invention for preventing or treating cancer, and used in combination with an immune checkpoint inhibitor. The present invention provides a method for preventing or treating cancer, comprising the steps of: administering a therapeutically effective amount of the anti-human Fn14 antibody or antigen-binding fragment thereof used in the present invention and an immune checkpoint inhibitor. The present invention provides an anti-human Fn14 antibody or an antigen-binding fragment thereof used in the present invention, which is used in combination with an immune checkpoint inhibitor for preventing or treating cancer. The present invention provides a use of the anti-human Fn14 antibody or its antigen-binding fragment used in the present invention, which is to manufacture a pharmaceutical composition for preventing or treating cancer, and the pharmaceutical composition is used in combination with an immune checkpoint inhibitor.

In addition, the present invention provides a pharmaceutical composition comprising an immune checkpoint inhibitor for preventing or treating cancer, and used in combination with the anti-human Fn14 antibody or antigen-binding fragment thereof used in the present invention. The present invention provides an immune checkpoint inhibitor, which is used in combination with the anti-human Fn14 antibody or its antigen-binding fragment used in the present invention to prevent or treat cancer. The present invention provides the use of an immune checkpoint inhibitor for the manufacture of a pharmaceutical composition for preventing or treating cancer, and the pharmaceutical composition is used in combination with the anti-human Fn14 antibody or its antigen-binding fragment used in the present invention.

The pharmaceutical composition of the present invention includes a pharmaceutical composition comprising the 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 commonly used in the field, ie, pharmaceutical excipients or pharmaceutical carriers, and the like. Examples of the dosage form of these pharmaceutical compositions include parenteral preparations such as injections and drips, and can be administered by intravenous administration, subcutaneous administration, intramuscular administration, and the like. During formulation, excipients, carriers, additives, etc. that are compatible with these dosage forms can be used within the pharmaceutically acceptable range.

During formulation, the dosage of the immune checkpoint inhibitor used in the present invention varies depending on the degree of symptoms of the patient, age, the dosage form of the formulation used, or the form of the inhibitor, for example, 0.001 mg/kg can be used ~100 mg/kg or so.

During formulation, the amount of the anti-PD-1 antibody or anti-PD-L1 antibody or antigen-binding fragment thereof used in the present invention depends on the patient's degree of symptoms, age, the dosage form of the preparation used, or the binding effect of the antibody. Although the value varies, for example, about 0.001 mg/kg to 100 mg/kg can be used.

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

In one embodiment, the present invention is a pharmaceutical composition comprising the following anti-Fn14 antibody or an antigen-binding fragment thereof and an excipient for the prevention or treatment of cancer, and an anti-PD-1 antibody and use: An anti-human Fn14 antibody or an antigen-binding fragment thereof comprising the following heavy chain variable region and light chain variable region, the above-mentioned heavy chain variable region comprising: comprising the amino acid sequences of amino acid numbers 31 to 35 of SEQ ID NO: 2 CDR1, CDR2 comprising the amino acid sequences of amino acid numbers 50 to 65 of SEQ ID NO: 2, and CDR3 comprising the amino acid sequences of amino acid numbers 98 to 114 of SEQ ID NO: 2, the above-mentioned light chain variable region includes : CDR1 comprising the amino acid sequences of SEQ ID NO: 4 from 24 to 40, CDR2 comprising the amino acid sequences of SEQ ID NO: 4 from 56 to 62, and CDR2 comprising the amino acid sequences of SEQ ID NO: 4 CDR3 of the amino acid sequences numbered 95 to 103.

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

In one embodiment, the present invention is a pharmaceutical composition comprising an anti-Fn14 antibody selected from the following (3) and (4) and an excipient for preventing or treating cancer, which is combined with Concomitant use of anti-PD-1 antibodies: (3) an anti-human Fn14 antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 2, and the light chain comprises the amino acid sequence shown in SEQ ID NO: 4; and (4) The antibody produced by post-translational modification of the antibody of (3) above is an anti-human Fn14 antibody.

In one embodiment, the present invention is a method for preventing or treating cancer, comprising the steps of: administering to a patient a therapeutically effective amount of the following anti-Fn14 antibodies or antigen-binding fragments thereof and anti-PD-1 antibodies: An anti-human Fn14 antibody or an antigen-binding fragment thereof comprising the following heavy chain variable region and light chain variable region, the above-mentioned heavy chain variable region comprising: comprising the amino acid sequences of amino acid numbers 31 to 35 of SEQ ID NO: 2 CDR1, CDR2 comprising the amino acid sequences of amino acid numbers 50 to 65 of SEQ ID NO: 2, and CDR3 comprising the amino acid sequences of amino acid numbers 98 to 114 of SEQ ID NO: 2, the above-mentioned light chain variable region includes : CDR1 comprising the amino acid sequences of SEQ ID NO: 4 from 24 to 40, CDR2 comprising the amino acid sequences of SEQ ID NO: 4 from 56 to 62, and CDR2 comprising the amino acid sequences of SEQ ID NO: 4 CDR3 of the amino acid sequences numbered 95 to 103.

In one embodiment, the present invention is a method of preventing or treating cancer, comprising the steps of: administering to a patient a therapeutically effective amount of an anti-Fn14 antibody or an antigen-binding fragment thereof selected from the following (1) and (2) and anti-PD-1 antibodies: (1) An anti-human Fn14 antibody or an antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequences of amino acid numbers 1 to 125 of SEQ ID NO: 2 , the above-mentioned light chain variable region comprises the amino acid sequence of amino acid number 1 to 114 of SEQ ID NO: 4; and (2) An antibody or antigen-binding fragment thereof produced by post-translational modification of the anti-human Fn14 antibody or antigen-binding fragment thereof of (1) above.

In one embodiment, the present invention is a method for preventing or treating cancer, comprising the steps of: administering to a patient a therapeutically effective amount of an anti-Fn14 antibody and an anti-PD-1 selected from the following (3) and (4) antibody: (3) an anti-human Fn14 antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 2, and the light chain comprises the amino acid sequence shown in SEQ ID NO: 4; and (4) The antibody produced by post-translational modification of the antibody of (3) above is an anti-human Fn14 antibody.

In one embodiment, the present invention is the following anti-Fn14 antibody or antigen-binding fragment thereof, which is used in combination with an anti-PD-1 antibody and for preventing or treating cancer: An anti-human Fn14 antibody or an antigen-binding fragment thereof comprising the following heavy chain variable region and light chain variable region, the above-mentioned heavy chain variable region comprising: comprising the amino acid sequences of amino acid numbers 31 to 35 of SEQ ID NO: 2 CDR1, CDR2 comprising the amino acid sequences of amino acid numbers 50 to 65 of SEQ ID NO: 2, and CDR3 comprising the amino acid sequences of amino acid numbers 98 to 114 of SEQ ID NO: 2, the above-mentioned light chain variable region includes : CDR1 comprising the amino acid sequences of SEQ ID NO: 4 from 24 to 40, CDR2 comprising the amino acid sequences of SEQ ID NO: 4 from 56 to 62, and CDR2 comprising the amino acid sequences of SEQ ID NO: 4 CDR3 of the amino acid sequences numbered 95 to 103.

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

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

In one embodiment, the present invention is the use of the following anti-Fn14 antibodies or antigen-binding fragments thereof for the manufacture of a pharmaceutical composition for preventing or treating cancer, and the pharmaceutical composition is used in combination with an anti-PD-1 antibody: An anti-human Fn14 antibody or an antigen-binding fragment thereof comprising the following heavy chain variable region and light chain variable region, the above-mentioned heavy chain variable region comprising: comprising the amino acid sequences of amino acid numbers 31 to 35 of SEQ ID NO: 2 CDR1, CDR2 comprising the amino acid sequences of amino acid numbers 50 to 65 of SEQ ID NO: 2, and CDR3 comprising the amino acid sequences of amino acid numbers 98 to 114 of SEQ ID NO: 2, the above-mentioned light chain variable region includes : CDR1 comprising the amino acid sequences of SEQ ID NO: 4 from 24 to 40, CDR2 comprising the amino acid sequences of SEQ ID NO: 4 from 56 to 62, and CDR2 comprising the amino acid sequences of SEQ ID NO: 4 CDR3 of the amino acid sequences numbered 95 to 103.

In one embodiment, the present invention is the use of an anti-Fn14 antibody or an antigen-binding fragment thereof selected from the following (1) and (2) for the manufacture of a pharmaceutical composition for preventing or treating cancer, and the medicine Composition in combination with anti-PD-1 antibody: (1) An anti-human Fn14 antibody or an antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequences of amino acid numbers 1 to 125 of SEQ ID NO: 2 , the above-mentioned light chain variable region comprises the amino acid sequence of amino acid number 1 to 114 of SEQ ID NO: 4; and (2) An antibody or antigen-binding fragment thereof produced by post-translational modification of the anti-human Fn14 antibody or antigen-binding fragment thereof of (1) above.

In one embodiment, the present invention is the use of an anti-Fn14 antibody selected from the following (3) and (4) for the manufacture of a pharmaceutical composition for preventing or treating cancer, and the pharmaceutical composition is combined with anti-PD Concomitant use of -1 antibody: (3) an anti-human Fn14 antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 2, and the light chain comprises the amino acid sequence shown in SEQ ID NO: 4; and (4) The antibody produced by post-translational modification of the antibody of (3) above is an anti-human Fn14 antibody.

In one embodiment, the present invention is a pharmaceutical composition comprising the following anti-Fn14 antibody or an antigen-binding fragment thereof and an excipient for the prevention or treatment of cancer, and an anti-PD-L1 antibody and use: An anti-human Fn14 antibody or an antigen-binding fragment thereof comprising the following heavy chain variable region and light chain variable region, the above-mentioned heavy chain variable region comprising: comprising the amino acid sequences of amino acid numbers 31 to 35 of SEQ ID NO: 2 CDR1, CDR2 comprising the amino acid sequences of amino acid numbers 50 to 65 of SEQ ID NO: 2, and CDR3 comprising the amino acid sequences of amino acid numbers 98 to 114 of SEQ ID NO: 2, the above-mentioned light chain variable region includes : CDR1 comprising the amino acid sequences of SEQ ID NO: 4 from 24 to 40, CDR2 comprising the amino acid sequences of SEQ ID NO: 4 from 56 to 62, and CDR2 comprising the amino acid sequences of SEQ ID NO: 4 CDR3 of the amino acid sequences numbered 95 to 103.

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

In one embodiment, the present invention is a pharmaceutical composition comprising an anti-Fn14 antibody selected from the following (3) and (4) and an excipient for preventing or treating cancer, and with Concomitant use of anti-PD-L1 antibodies: (3) an anti-human Fn14 antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 2, and the light chain comprises the amino acid sequence shown in SEQ ID NO: 4; and (4) An antibody produced by post-translational modification of the antibody of (3) above.

In one embodiment, the present invention is a method for preventing or treating cancer, comprising the steps of: administering to a patient a therapeutically effective amount of the following anti-Fn14 antibodies or antigen-binding fragments thereof and anti-PD-L1 antibodies: An anti-human Fn14 antibody or an antigen-binding fragment thereof comprising the following heavy chain variable region and light chain variable region, the above-mentioned heavy chain variable region comprising: comprising the amino acid sequences of amino acid numbers 31 to 35 of SEQ ID NO: 2 CDR1, CDR2 comprising the amino acid sequences of amino acid numbers 50 to 65 of SEQ ID NO: 2, and CDR3 comprising the amino acid sequences of amino acid numbers 98 to 114 of SEQ ID NO: 2, the above-mentioned light chain variable region includes : CDR1 comprising the amino acid sequences of SEQ ID NO: 4 from 24 to 40, CDR2 comprising the amino acid sequences of SEQ ID NO: 4 from 56 to 62, and CDR2 comprising the amino acid sequences of SEQ ID NO: 4 CDR3 of the amino acid sequences numbered 95 to 103.

In one embodiment, the present invention is a method of preventing or treating cancer, comprising the steps of: administering to a patient a therapeutically effective amount of an anti-Fn14 antibody or an antigen-binding fragment thereof selected from the following (1) and (2) and anti-PD-L1 antibodies: (1) An anti-human Fn14 antibody or an antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequences of amino acid numbers 1 to 125 of SEQ ID NO: 2 , the above-mentioned light chain variable region comprises the amino acid sequence of amino acid number 1 to 114 of SEQ ID NO: 4; and (2) An antibody or antigen-binding fragment thereof produced by post-translational modification of the anti-human Fn14 antibody or antigen-binding fragment thereof of (1) above.

In one embodiment, the present invention is a method for preventing or treating cancer, comprising the steps of: administering to a patient a therapeutically effective amount of an anti-Fn14 antibody and an anti-PD-L1 selected from the following (3) and (4) antibody: (3) an anti-human Fn14 antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 2, and the light chain comprises the amino acid sequence shown in SEQ ID NO: 4; and (4) The antibody produced by post-translational modification of the antibody of (3) above is an anti-human Fn14 antibody.

In one embodiment, the present invention is the following anti-Fn14 antibody or antigen-binding fragment thereof, which is used in combination with an anti-PD-L1 antibody and for the prevention or treatment of cancer: An anti-human Fn14 antibody or an antigen-binding fragment thereof comprising the following heavy chain variable region and light chain variable region, the above-mentioned heavy chain variable region comprising: comprising the amino acid sequences of amino acid numbers 31 to 35 of SEQ ID NO: 2 CDR1, CDR2 comprising the amino acid sequences of amino acid numbers 50 to 65 of SEQ ID NO: 2, and CDR3 comprising the amino acid sequences of amino acid numbers 98 to 114 of SEQ ID NO: 2, the above-mentioned light chain variable region includes : CDR1 comprising the amino acid sequences of SEQ ID NO: 4 from 24 to 40, CDR2 comprising the amino acid sequences of SEQ ID NO: 4 from 56 to 62, and CDR2 comprising the amino acid sequences of SEQ ID NO: 4 CDR3 of the amino acid sequences numbered 95 to 103.

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

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

In one embodiment, the present invention is the use of the following anti-Fn14 antibodies or antigen-binding fragments thereof to manufacture a pharmaceutical composition for preventing or treating cancer, and the pharmaceutical composition is used in combination with an anti-PD-L1 antibody: An anti-human Fn14 antibody or an antigen-binding fragment thereof comprising the following heavy chain variable region and light chain variable region, the above-mentioned heavy chain variable region comprising: comprising the amino acid sequences of amino acid numbers 31 to 35 of SEQ ID NO: 2 CDR1, CDR2 comprising the amino acid sequences of amino acid numbers 50 to 65 of SEQ ID NO: 2, and CDR3 comprising the amino acid sequences of amino acid numbers 98 to 114 of SEQ ID NO: 2, the above-mentioned light chain variable region includes : CDR1 comprising the amino acid sequences of SEQ ID NO: 4 from 24 to 40, CDR2 comprising the amino acid sequences of SEQ ID NO: 4 from 56 to 62, and CDR2 comprising the amino acid sequences of SEQ ID NO: 4 CDR3 of the amino acid sequences numbered 95 to 103.

In one embodiment, the present invention is the use of an anti-Fn14 antibody or an antigen-binding fragment thereof selected from the following (1) and (2) for the manufacture of a pharmaceutical composition for preventing or treating cancer, and the medicine Composition in combination with anti-PD-L1 antibody: (1) An anti-human Fn14 antibody or an antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequences of amino acid numbers 1 to 125 of SEQ ID NO: 2 , the above-mentioned light chain variable region comprises the amino acid sequence of amino acid number 1 to 114 of SEQ ID NO: 4; and (2) An antibody or antigen-binding fragment thereof produced by post-translational modification of the anti-human Fn14 antibody or antigen-binding fragment thereof of (1) above.

In one embodiment, the present invention is the use of an anti-Fn14 antibody selected from the following (3) and (4) for the manufacture of a pharmaceutical composition for preventing or treating cancer, and the pharmaceutical composition is combined with anti-PD Concomitant use of -L1 antibody: (3) an anti-human Fn14 antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 2, and the light chain comprises the amino acid sequence shown in SEQ ID NO: 4; and (4) The antibody produced by post-translational modification of the antibody of (3) above is an anti-human Fn14 antibody.

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

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

In one embodiment, the steroid myopathy that is the subject of the present invention is a steroid myopathy resulting from the administration of steroids for the prevention or treatment of excessive immune responses caused by immune checkpoint inhibitors. In one embodiment, the steroid myopathy that is the subject of the present invention is a steroid myopathy resulting from administration of a steroid to a patient at the same time as, or before and after administration of the immune checkpoint inhibitor.

The present invention includes a pharmaceutical composition for preventing or treating steroid myopathy comprising the anti-human Fn14 antibody or antigen-binding fragment thereof used in the present invention. Furthermore, the present invention includes a method of treating or preventing steroid myopathy comprising the step of administering a therapeutically effective amount of the anti-human Fn14 antibody or antigen-binding fragment thereof used in the present invention. Furthermore, the present invention includes the anti-human Fn14 antibody or antigen-binding fragment thereof used in the present invention for preventing or treating steroid myopathy. Furthermore, the present invention includes the use of the anti-human Fn14 antibody or the antigen-binding fragment thereof used in the present invention to manufacture a pharmaceutical composition for preventing or treating steroid myopathy.

The specific embodiments referred to for further understanding of the present invention are provided here, but these are provided for illustrative purposes only and do not limit the present invention. [Example]

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

(Example 1: Acquisition of human and mouse Fn14-Fc fusion proteins) The present inventors prepared human Fn14-human Fc fusion proteins and mouse Fn14-mouse Fc fusion proteins, which were used as antigens for obtaining anti-Fn14 antibodies and materials for screening tests. The human Fn14-human Fc fusion protein is the C-terminus of the extracellular part of the sequence of human Fn14 (NCBI accession number: amino acids No. 1 to 79 of NP_057723.1), and the human Fc region (NCBI accession number: A fusion protein in which the N-terminus of amino acids No. 106 to No. 330 of P01857.1 was linked by a peptide linker (SEQ ID NO: 9). In addition, the mouse Fn14-mouse Fc fusion protein is the C-terminus of the extracellular partial sequence of the mouse Fn14 sequence (NCBI accession number: amino acids No. 1 to No. 75 of AAF07882.1), and mouse Fc The fusion protein formed by linking the N-terminus of the region, specifically, the genome encoding the extracellular partial sequence of the mouse Fn14 sequence was incorporated into pFUSE-mIgG2B-Fc1 (InvivoGen, pfuse-mg2bfc1) using restriction enzymes EcoRI and EcoRV. ) between the hEF1-HTLV promoter region and the mIgG2B-Fc region, and the fusion protein expressed. Expression vectors containing the gene encoding the above fusion protein were prepared in GS vector pEE12.4 (Lonza Corporation), respectively, and introduced into CHO-K1SV cells (Lonza Corporation). Conventionally, human Fn14-human Fc fusion protein and mouse Fn14-mouse Fc fusion protein were purified from the culture supernatant of the above-mentioned CHO-K1SV cells, respectively.

(Example 2: Acquisition of anti-human Fn14 antibody) Antibodies were produced using human monoclonal antibody development technology "VelocImmune" (VelocImmune antibody technology: Regeneron Corporation (US Pat. No. 6,596,541)) mice. Antibodies obtained by VelocImmune technology are antibodies (also called chimeric antibodies) having variable regions of human antibodies and constant regions of mouse antibodies. For VelocImmune mice, immunization with an adjuvant that elicits an immune response, and a human Fn14 protein obtained by cleavage of the human Fn14-human Fc fusion protein prepared in Example 1 and removal of the human Fc region using FabRICATOR (Sigma, 77661) . Conventionally, lymphocytes collected from the lymph nodes of immunized mice were fused with mouse-derived myeloma cells SP2/0-Ag14 (ATCC: CRL-1581) to prepare fusion tumors and line them up alone. . Fusionomas producing antibodies that bind to human Fn14 and inhibit NFkB activation by Tweak stimulation (hereinafter, referred to as "Tweak-induced NFkB activation" in the following Examples) were screened, and the antibodies were purified from the culture supernatant.

(Example 3: NFkB activation assay) To evaluate the agonist effect of the antibody found in Example 2 on human Fn14, the effect of the antibody on NFkB activation in the absence of Twea was evaluated by reporter gene analysis. Specifically, HEK293 cells (ATCC, CRL-1573) (hereinafter referred to as NFkB/HEK293) into which the luciferase reporter vector pGL4.32 (Promega, E8491) incorporating the NFkB transcription response sequence was stably introduced were prepared. cells) and used for evaluation.

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

As a result, an anti-human Fn14 antibody that does not induce NFkB activation, that is, does not exhibit agonist activity, was screened and named 4-1.

(Example 4: Production of fully human antibody and murine antibody) The genes encoding the heavy and light chains of the antibody were cloned from fusionomas producing the antibodies screened in Example 3 and sequenced. After the sequence determination of the antibody, the gene encoding the signal sequence (Wittle N et al., Protein Engineering. 1987, Vol. 1 No. 6, p. 499-505) is linked to the 5' side of the heavy chain variable region gene, and A human Igγ1 constant region gene with amino acid variations of L234A and L235A (including the nucleotide sequence of nucleotide number 376 to 1365 of SEQ ID NO: 1) was linked to the 3' side, and the heavy chain gene was inserted into GS Vector pEE6.4 (Lonza Corporation). In addition, a gene encoding a signal sequence (Wittle N et al., Protein Engineering. 1987, Vol.1 No.6, p.499-505) was linked to the 5' side of the light chain variable region gene, and the 3' side was The constant region gene of human kappa chain (including the nucleotide sequence of nucleotide number 343 to 660 of SEQ ID NO: 3) was linked, and the light chain gene was inserted into the GS vector pEE12.4. These GS vectors were cleaved with restriction enzymes using NotI-HF and PvuI-HF, and ligated using a DNA ligation kit <Mighty Mix> (TaKaRa, 6023) to construct two genes inserted into the heavy chain and light chain. GS carrier. According to convention, the fully human type antibody of 4-1 was obtained by purifying the antibody from the culture supernatant of CHO-K1SV cells transfected with the vector, and named 4-1h.

The base sequence of the produced 4-1h heavy chain is shown in SEQ ID NO: 1, the amino acid sequence encoded by it is shown in SEQ ID NO: 2, and the base sequence of the light chain is shown in SEQ ID NO: 3. The encoded amino acid sequence is shown in SEQ ID NO:4. The variable region of the heavy chain shown in SEQ ID NO: 2 includes the amino acid sequence of amino acid number 1 to 125 of SEQ ID NO: 2, and the variable region of the light chain shown in SEQ ID NO: 4 includes SEQ ID NO: 4 The amino acid numbering No. 1 to the amino acid sequence No. 114. The CDR1, CDR2, and CDR3 of the heavy chain variable region of 4-1h respectively comprise the amino acids of No. 31 to No. 35, No. 50 to No. 65, and No. 98 to No. 114 of SEQ ID NO: 2 sequence. The CDR1, CDR2, and CDR3 of the light chain variable region of 4-1h respectively comprise the amino acids No. 24 to No. 40, No. 56 to No. 62, and No. 95 to No. 103 of SEQ ID NO: 4 sequence.

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

4-1m was obtained using the same method as described above for vector construction, antibody expression and purification for 4-1h. Among them, the constant region of the heavy chain uses the gene encoding the mouse Igγ2a constant region gene with D265A amino acid variation (including the nucleotide sequences of nucleotides 376 to 1365 in SEQ ID NO: 5), and the constant region of the light chain is The constant region gene of mouse kappa chain (including the nucleotide sequence of nucleotide number 343 to 660 of SEQ ID NO: 7) was used for the region.

The base sequence of the produced 4-1m heavy chain is shown in SEQ ID NO: 5, the amino acid sequence encoded by it is shown in SEQ ID NO: 6, and the base sequence of the light chain is shown in SEQ ID NO: 7. The encoded amino acid sequence is shown in SEQ ID NO:8. The variable region of the heavy chain shown in SEQ ID NO: 6 includes the amino acid sequence of amino acid number 1 to 125 of SEQ ID NO: 6, and the variable region of the light chain shown in SEQ ID NO: 8 includes SEQ ID NO: 8 The amino acid numbering No. 1 to the amino acid sequence No. 114. The CDR1, CDR2, and CDR3 of the heavy chain variable region of 4-1m comprise amino acids No. 31 to No. 35, No. 50 to No. 65, and No. 98 to No. 114 of SEQ ID NO: 6, respectively sequence. The CDR1, CDR2, and CDR3 of the light chain variable region of 4-1m comprise amino acids No. 24 to No. 40, No. 56 to No. 62, and No. 95 to No. 103 of SEQ ID NO: 8, respectively sequence.

(Example 5: Amino Acid Modification Analysis of Fully Human Type Antibody) As a result of amino acid modification analysis of the purified 4-1 h, in the majority of the purified antibody, pyroglutamylation at the N-terminus of the heavy chain and deletion of lysine at the C-terminus were produced.

(Example 6: Human and mouse Fn14 binding ELISA of fully human type antibody and murine antibody) The binding activity of 4-1h and 4-1m obtained in Example 4 to Fn14 protein was evaluated. The human Fn14-human Fc fusion protein and mouse Fn14-mouse Fc fusion protein obtained in Example 1 were prepared at 1 μg/mL with phosphate buffered saline (PBS), and added to 15 μL per well. MaxiSorp 384-well transparent culture dish (Nunc, 464718), and cultured overnight at 4°C to solidify. The solid phase liquid was removed on the second day, and washed with tris buffered saline (TBS-T) containing 0.05% Tween-20. PBS containing 20% Blocking One (Nacalai Tesque, 03953-95) was added in 50 μL per well, and after standing at room temperature for 1 hour, washed with TBS-T. For 4-1h or 4-1m obtained in Example 4, use TBS-T containing 5% Blocking One (hereinafter referred to as diluent), from the highest concentration of 30 μg/mL by 4 times the common ratio of 12 stages Dilute to make dilution series and add 20 μL per well. After culturing at room temperature for 1 hour, it was washed with TBS-T. As a secondary antibody, 20 μL per well of 4-1h was added with horseradish peroxidase-labeled anti-human kappa light chain antibody (Southern Biotech, 2060-05) diluted 5000 times with the dilution solution, and added to 4-1m The horseradish peroxidase-labeled anti-mouse kappa light chain antibody (Southern Biotech, 1050-05) diluted 4000-fold with the diluent was added in 20 μL per well. After culturing at room temperature for 1 hour, wash with TBS-T, add TMB Microwell Peroxidase Substrate (Kirkegaard & Perry Laboratories, 50-76-03), stand for 3 minutes at 4-1h evaluation, and 4-1m evaluation After standing for 5 minutes, the reaction was terminated by the addition of 2M sulfuric acid, and the absorbance at 450 nm was measured by a multi-plate reader (Molecular Devices). The experiments were repeated and _EC50 values were calculated from 4-parameter logarithmic curve comparisons.

As a result, 4-1h and 4-1m prepared in Example 4 were confirmed to have binding activities to human Fn14 and mouse Fn14 (Table 1). Table 1: Binding activities of 4-1h and 4-1m to human and mouse Fn14 [Table 1] Antibody name _EC50 (ng/mL) Humanity mouse 4-1h 30.4 23.2 4-1m 18.2 16.5

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

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 were inoculated into a white 96-well culture plate with a transparent bottom middle. After culturing overnight at 37°C and 5% CO ₂ , for the 4-1 h obtained in Example 4, the above-mentioned medium was used to prepare 11 stages at a final concentration of 0.1 ng/mL up to about 3 times the common ratio of 10 μg/mL. After the dilution series, 10 μL per well was added. After incubation at 37°C and 5% CO ₂ for 30 minutes, 10 μL of Tweak (Peprotech, 310-06) was added so that the final concentration would be 100 ng/mL. After 5 hours of incubation at 37°C, 5% _CO2 , NFkB activation was quantified according to the method of Example 3. The group with only the medium added without the antibody was set as the control group, the Tweak-added group was regarded as 0% inhibition, and the Tweak non-added group was regarded as 100% inhibition, and the antibody with 50% inhibition was calculated by 4-parameter logarithmic curve comparison. concentration (IC ₅₀ value). For each antibody, the experiment was repeated, and the geometric mean of the _IC50 values of the three repetitions was performed to calculate a 95% confidence interval (Table 2). In this test, as a comparative antibody, mouse anti-human Fn14 antibody CRCBT-06-002 (Patent Document 2) was used.

As a result, it was confirmed that 4-1h has a stronger antagonist activity than CRCBT-06-002. Table 2: Inhibitory activity of 4-1h on Tweak-induced NFkB activation [Table 2] Antibody name IC ₅₀ (ng/mL) (95% confidence interval) 4-1h 41.5 (27.2-63.4) CRCBT-06-002 115 (72.7-183)

(Example 8: IL-8 production assay of fully human type antibody) The functional activity of 4-1 h obtained in Example 4 was evaluated by in vitro IL-8 production assay. A375 cells (ATCC, CRL-1619) were suspended at 5×10 ⁴ cells/mL in DMEM containing 10% fetal bovine serum, and 100 μL per well were seeded into 96-well flat-bottom culture dishes (IWAKI, 3860 -096). After culturing overnight at 37°C and 5% CO ₂ , the cells were washed with PBS. For the 4-1 h obtained in Example 4, a 10-fold dilution series with a final concentration of 1 ng/mL up to 100 μg/mL was prepared using the above-mentioned medium, and added to the cells. In the presence of Tweak at a final concentration of 5 ng/mL (evaluation of antagonist activity) or in the absence of Tweak (evaluation of agonist activity), after overnight incubation at 37°C, 5% CO ₂ , the culture medium was recovered. The IL-8 concentration in the culture supernatant was measured using the human IL-8/CXCL8 Quantikine ELISA kit (R&D Company, D8000C). In the evaluation of antagonist activity and agonist activity, experiments were performed with final volumes of 65 μL/well and 100 μL/well, respectively. In this test, CRCBT-06-002 was used as a comparative antibody, and the experiment was repeated for each antibody.

In the evaluation of antagonist activity, the group to which only the medium containing no antibody was added was set as the control group, the Tweak-added group was regarded as 0% inhibition, and the group not added with Tweak was regarded as 100% inhibition, and the inhibition rate was calculated. The arithmetic mean ± standard error of the maximum inhibition rate in each of the four experiments is shown in Table 3 (Table 3). A graph of antagonist activity is shown in FIG. 1 and a graph of agonist activity is shown in FIG. 2 .

As shown in Table 3 and Fig. 1 , it was clear that 4-1h showed almost complete inhibitory activity against IL-8 production by Tweak stimulation, whereas CRCBT-06-002 showed only partial inhibitory activity. Also, as shown in FIG. 2 , CRCBT-06-002 induced IL-8 production alone in the absence of Tweak, whereas 4-1 h of the present invention hardly induced IL-8 production.

From the above, it is clear that CRCBT-06-002 is a partial antagonist antibody with agonist effect, while 4-1h is a complete antagonist antibody with weak agonist effect on human Fn14. Table 3: Inhibitory activity of 4-1h and CRCBT-06-002 on IL-8 production induced by Tweak stimulation [Table 3] Antibody name Maximum inhibition rate (%) mean ± standard error 4-1h 96.4±0.4 CRCBT-06-002 53.0±6.8

(Example 9: Combination 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 by the B16F10 mouse cancer-bearing model. Anti-PD-1 antibodies are used clinically as a breakthrough therapy for advanced cancers that are resistant to previous treatments. However, most cancer patients do not respond to this treatment. Mouse malignant melanoma cells, namely B16F10 cancer-bearing mice, are partially effective against anti-PD-1 antibodies, but most of them do not respond or have limited effects, and it is known as a model that can obtain the same drug responsiveness as clinical (British Journal of Cancer). , 2019, 120, 346-355).

B16F10 (ATCC, CRL-6475) was cultured in DMEM containing 10% fetal bovine serum, and then suspended in Matrigel (CORNING) in a manner of 0.5×10 ⁷ cells/mL, and 0.5×10 ⁶ cells /100 μL was administered subcutaneously to the left back of C57BL/6 mice (day 0). At the point when the mean tumor volume became 100-200 mm ³ (mean tumor volume was 116.3 mm ³ , day 1), grouping based on tumor volume (n=10×4) was performed, and drug administration was started on a group-by-group basis over time. Tumor diameter was measured and tumor volume was calculated (Figure 3). The tumor volume (mm ³ ) was calculated by the calculation formula of long axis (mm)×short axis (mm)×short axis (mm)/2, and described as mean±standard error. The composition of each group and the Japanese system of investment are as follows. (1) Control group: rat IgG2a isotype control (Bio X cell company pure line 2A3) 100 μg/horse intraperitoneal administration; Day 1, Day 6, Day 8 and anti-Keyhole Hemocyanin (KLH) Antibody (KLH_173A1_ma0, manufactured by Astellas Pharma Co., Ltd.) 0.3 mg/kg subcutaneously administered; Day 1, Day 3, Day 6, Day 8 (2) Anti-PD-1 administration group: InVivoMAb anti-mouse PD-1 (Bio X cell company RMP1-14) 100 μg/horse intraperitoneal administration; day 1, day 6, day 8 and subcutaneous administration of anti-KLH antibody 0.3 mg/kg; day 1, day 3 Day, Day 6, Day 8 (3) 4-1h Substitute administration group: rat IgG2a isotype control 100 μg/horse intraperitoneal administration; Day 1, Day 6, Day 8 and 4-1h Substitute 0.3 mg/kg subcutaneous administration; Day 1, Day 3, Day 6, Day 8 (4) Concomitant group: InVivoMAb anti-mouse PD-1 100 μg/horse intraperitoneal administration; Day 1 , 6 days, 8 days and 4-1h substitutes were administered subcutaneously at 0.3 mg/kg; tumor volumes were measured on the last day of evaluation (day 10) on days 1, 3, 6, and 8, As a result, the tumor volume of the 4-1h surrogate group and the combination group was significantly suppressed. Similarly, compared with the anti-PD-1 antibody-administered group, the tumor volume of the 4-1 h surrogate group and the combination group was significantly suppressed. Also, compared with the 4-1h surrogate group, the tumor volume of the combined group was significantly inhibited (p<0.05). On the other hand, the anti-PD-1 antibody-administered group showed a tendency of inhibition compared with the control group ( FIG. 3 ).

Tumors were excised on day 10 under isoflurane (Pfizer) anesthesia and stored at minus 80 degrees. RNA was extracted from deposited tumor samples to prepare cDNA. By quantitative PCR (qPCR) using the prepared cDNA, the mRNA expression changes of each group of mouse TOX (TOX) in the tumor (the mRNA expression level of each molecule in the control group was taken as 1) were compared and evaluated (averaged). ± standard error to record) (Figure 4). The endogenous control gene line used 18S rRNA.

TOX is a molecule known as a transcription factor that induces T cell fatigue. Inhibition of TOX expression is expected as a drug development goal to avoid T cell fatigue (Nature Immunology, 2019, 20, 1092-1094). In the qPCR analysis of TOX, the expression of the concomitant group was significantly inhibited (p<0.05) compared with the control group. The 4-1h surrogate group and the anti-PD-1 antibody group showed an inhibitory tendency compared with the control group.

(Example 10: Effect of 4-1h Substitute in Murine Steroid-Induced Muscle Atrophy Model) The functional activity of the 4-1 h surrogates was assessed by a Steroid-Induced Myopathy (SIM) model. After grouping by body weight (n=10×6), the concentration of corticosterone (Fujifilm Wako Pure Chemical Industries, Ltd.) was adjusted to 100 μg/mL, and the mice were allowed to drink water freely (day 0) and continued to the 22nd day until days. Drug administration was performed on day 0, day 2, day 5, day 7, day 9, day 12, day 14, day 16, day 19, and day 21. The group composition system is shown below. (1) Sham treatment group; Sham treatment and PBS (10 mL/kg subcutaneous administration) (2) SIM control group; SIM and PBS (10 mL/kg subcutaneous administration) (3) KLH population; SIM and anti-KLH antibody (manufactured by Astellas Pharma Co., Ltd.) (3 mg/kg subcutaneously) (4) 4-1h substitute (0.03 mg/kg subcutaneous administration) population; SIM and 4-1h substitute (0.03 mg/kg subcutaneous administration) (5) 4-1h substitute (0.3 mg/kg subcutaneous administration) population; SIM and 4-1h substitute (0.3 mg/kg subcutaneous administration) (6) 4-1h substitute (3 mg/kg subcutaneous administration) population; SIM and 4-1h substitute (3 mg/kg subcutaneous administration) Muscle strength (Grip strength) was measured on the 21st day (GPM-100; Melquest), and divided by the body weight (g) of each individual, using the obtained value (Grip strength (kg/kg BW)=average The grip force (kg)/BW (g)×1000) was compared, and the result was that the muscle strength of the SIM control group was significantly reduced (p<0.01) compared with the sham treatment group. On the other hand, the 4-1 h replacement 0.3 mg/kg and 3 mg/kg groups had significantly improved muscle strength (p<0.01) compared to the SIM control group (reported as mean ± SE) (Figure 5).

The weight of the quadriceps femoris was measured on the 22nd day (AG104; METTLER TOLEDO), and divided by the body weight (g) of each individual, using the obtained value (quadriceps weight (mg/g BW) = quadriceps femoris Muscle weight (g)/BW (g)×1000) was compared, and the result showed that the muscle weight of the SIM control group was significantly reduced compared with the sham treatment group (p<0.01). On the other hand, the 4-1h substitutes 0.03 mg/kg and 0.3 mg/kg group, 3 mg/kg group, compared with the SIM control group, the muscle weight was significantly improved (p < 0.05 and p < 0.01) (mean ± 0.01). Standard error records) (Figure 6). [Industrial Availability]

It is expected that the pharmaceutical composition of the present invention can be used for the prevention or treatment of cancer or steroid myopathy. It is expected that the prophylactic or therapeutic method of the present invention can be used to prevent or treat cancer or steroid myopathy. It is expected that the anti-human Fn14 antibody of the present invention can be used as an anti-human Fn14 antibody for preventing or treating cancer or steroid myopathy. In addition, it is expected that the anti-Fn14 antibody of the present invention can be used for the production of a pharmaceutical composition for preventing or treating cancer or steroid myopathy. [Sequence Listing Non-Key Text]

The description of "artificial sequence" is described in the numerical title <223> of the sequence listing below. Specifically, the nucleotide sequences represented by SEQ ID NOs: 1, 5, 3, and 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 8 The amino acid sequences shown are those of the heavy and light chains encoded by SEQ ID NOs: 1, 3, 5 and 7, respectively. The amino acid sequence shown in SEQ ID NO: 9 is a peptide linker sequence that connects the human Fn14 protein and the human Fc region protein.

Figure 1 shows the inhibitory effect of anti-human Fn14 antibody on the production of the inflammatory cytokine IL-8 induced by Tweak stimulation in A375 cells. The vertical axis represents the inhibition rate (%), and the horizontal axis represents the antibody concentration (ng/mL). Figure 2 shows the secretion of IL-8 induced by anti-human Fn14 antibody stimulation in A375 cells. The vertical axis represents the IL-8 concentration (pg/mL), and the horizontal axis represents the antibody concentration (ng/mL). Figure 3 shows the combined effect of 4-1h substitute and anti-PD-1 antibody in B16F10 mouse cancer-bearing model. The vertical axis represents tumor volume (mm ³ ), and the horizontal axis represents days after cancer bearing (day). Figure 4 shows the changes of TOX mRNA expression in the tumor when the 4-1h substitute and the anti-PD-1 antibody were used in combination in the B16F10 mouse cancer-bearing model. The vertical axis represents the mRNA expression level of each group when the control group is set to 1. Figure 5 shows the effect of 4-1h replacement in a steroid myopathy model (grip force). Figure 6 shows the effect (quadriceps weight) of a 4-1h surrogate in a steroid myopathy model.

Claims (21)

A pharmaceutical composition comprising an anti-Fn14 antibody or an antigen-binding fragment thereof and an excipient for preventing or treating cancer, and used in combination with an immune checkpoint inhibitor. The pharmaceutical composition of claim 1, wherein the anti-Fn14 antibody or antigen-binding fragment thereof does not exhibit agonist activity for human Fn14 and inhibits activation of human Fn14 by Tweak stimulation. The pharmaceutical composition of claim 1 or 2, wherein the immune checkpoint inhibitor is an anti-PD-1 antibody or an anti-PD-L1 antibody. The pharmaceutical composition of any one of claims 1 to 3, wherein the immune checkpoint inhibitor is an anti-PD-1 antibody. The pharmaceutical composition according to claim 3 or 4, wherein the anti-PD-1 antibody is nivolumab, pembrolizumab, pidilizumab, and cimirizumab (cemiplimab), spartalizumab, MEDI0680, dostarlimab, cetrelimab, toripalimab, AMP-224, PF-06801591 , tislelizumab, ABBV-181, BI 754091, or SHR-1210. The pharmaceutical composition of any one of claims 1 to 3, wherein the immune checkpoint inhibitor is an anti-PD-L1 antibody. The pharmaceutical composition of claim 3 or 6, wherein the anti-PD-L1 antibody is atezolizumab, durvalumab, BMS-936559, avelumab, Dalimumab (lodapolimab), CX-072, FAZ053, KN035, or MDX-1105. The pharmaceutical composition according to any one of claims 1 to 7, wherein the anti-Fn14 antibody or antigen-binding fragment thereof is an anti-human Fn14 antibody or antigen-binding fragment thereof comprising the following heavy chain variable region and light chain variable region, the above The heavy chain variable region includes: CDR1 comprising the amino acid sequence of amino acid number 31 to 35 of SEQ ID NO: 2, CDR2 comprising the amino acid sequence of amino acid number 50 to 65 of SEQ ID NO: 2, and a sequence comprising CDR3 of the amino acid sequence of amino acid number 98 to 114 of number 2, the above-mentioned light chain variable region includes: CDR1 comprising the amino acid sequence of amino acid number 24 to 40 of SEQ ID NO: 4, including SEQ ID NO: 4 The CDR2 of the amino acid sequence of SEQ ID NO: 56 to 62, and the CDR3 of the amino acid sequence of amino acid number 95 to 103 of SEQ ID NO: 4 are included. The pharmaceutical composition of any one of claims 1 to 8, wherein the anti-Fn14 antibody or antigen-binding fragment thereof is selected from the following (1) and (2) anti-human Fn14 antibodies or antigen-binding fragments thereof: (1) An anti-human Fn14 antibody or an antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequences of amino acid numbers 1 to 125 of SEQ ID NO: 2 , the above-mentioned light chain variable region comprises the amino acid sequence of amino acid number 1 to 114 of SEQ ID NO: 4; and (2) An antibody or antigen-binding fragment thereof produced by post-translational modification of the anti-human Fn14 antibody or antigen-binding fragment thereof of (1) above. The pharmaceutical composition according to any one of claims 1 to 9, wherein the anti-Fn14 antibody or antigen-binding fragment thereof is an anti-human Fn14 antibody or antigen-binding fragment thereof comprising the following heavy chain variable regions and light chain variable regions, the above The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 2 from 1 to 125, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 4 from 1 to 114. The pharmaceutical composition of any one of claims 1 to 9, wherein the anti-Fn14 antibody or antigen-binding fragment thereof is selected from the following anti-human Fn14 antibodies in (3) and (4): (3) an anti-human Fn14 antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 2, and the light chain comprises the amino acid sequence shown in SEQ ID NO: 4; and (4) The antibody produced by post-translational modification of the antibody of (3) above is an anti-human Fn14 antibody. The pharmaceutical composition according to any one of claims 1 to 11, wherein the anti-Fn14 antibody or antigen-binding fragment thereof is an anti-human Fn14 antibody comprising the following heavy chain and light chain, and the above-mentioned heavy chain comprises the amine group shown in SEQ ID NO: 2 The acid sequence, the above-mentioned light chain comprises the amino acid sequence shown in SEQ ID NO: 4. The pharmaceutical composition of claim 9 or 11, wherein 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. A pharmaceutical composition comprising an anti-Fn14 antibody or antigen-binding fragment thereof and an excipient and for preventing or treating steroid myopathy. The pharmaceutical composition of claim 14, wherein the anti-Fn14 antibody or antigen-binding fragment thereof does not exhibit agonist activity for human Fn14 and inhibits activation of human Fn14 by Tweak stimulation. The pharmaceutical composition according to claim 14 or 15, wherein the anti-Fn14 antibody or antigen-binding fragment thereof is an anti-human Fn14 antibody or antigen-binding fragment thereof comprising the following heavy chain variable region and light chain variable region, and the above-mentioned heavy chain variable region The region includes: CDR1 comprising the amino acid sequence of SEQ ID NO: 2 from 31 to 35, CDR2 comprising the amino acid sequence of SEQ ID NO: 2 from 50 to 65, and the amine comprising SEQ ID NO: 2 The CDR3 of the amino acid sequence of amino acid number 98 to 114, the above-mentioned light chain variable region includes: CDR1 comprising the amino acid sequence of amino acid number 24 to 40 of SEQ ID NO: 4, including the amino acid of SEQ ID NO: 4 CDR2 of the amino acid sequence of No. 56 to 62, and CDR3 of the amino acid sequence of amino acid No. 95 to 103 of SEQ ID NO: 4 are included. The pharmaceutical composition of any one of claims 14 to 16, wherein the anti-Fn14 antibody or antigen-binding fragment thereof is an anti-human Fn14 antibody or antigen-binding fragment thereof selected from the following (1) and (2): (1) An anti-human Fn14 antibody or an antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequences of amino acid numbers 1 to 125 of SEQ ID NO: 2 , the above-mentioned light chain variable region comprises the amino acid sequence of amino acid number 1 to 114 of SEQ ID NO: 4; and (2) An antibody or antigen-binding fragment thereof produced by post-translational modification of the anti-human Fn14 antibody or antigen-binding fragment thereof of (1) above. The pharmaceutical composition according to any one of claims 14 to 17, wherein the anti-Fn14 antibody or antigen-binding fragment thereof is an anti-human Fn14 antibody or antigen-binding fragment thereof comprising the following heavy chain variable regions and light chain variable regions, the above The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 2 from 1 to 125, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 4 from 1 to 114. The pharmaceutical composition of any one of claims 14 to 17, wherein the anti-Fn14 antibody or antigen-binding fragment thereof is an anti-human Fn14 antibody selected from the following (3) and (4): (3) an anti-human Fn14 antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 2, and the light chain comprises the amino acid sequence shown in SEQ ID NO: 4; and (4) The antibody produced by post-translational modification of the antibody of (3) above is an anti-human Fn14 antibody. The pharmaceutical composition according to any one of claims 14 to 19, wherein the anti-Fn14 antibody or antigen-binding fragment thereof is an anti-human Fn14 antibody comprising the following heavy chain and light chain, and the above-mentioned heavy chain comprises the amine group shown in SEQ ID NO: 2 The acid sequence, the above-mentioned light chain comprises the amino acid sequence shown in SEQ ID NO: 4. The pharmaceutical composition of claim 17 or 19, wherein 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.

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