Anti-human TLR7 antibodies

Abstract

To provide a humanized anti-human TLR7 monoclonal antibody having human TLR7 function-inhibiting activity-suppressing action and capable of becoming a therapeutic or prophylactic agent for immunoinflammation-related diseases, etc.SOLUTION: Antibodies or antigen-binding fragments of antibodies are provided that (a) specifically bind to human TLR7 or monkey TLR7 and do not bind to mouse TLR7 or rat TLR7; and (b) inhibit the function of human TLR7 or monkey TLR7.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to anti-human Toll-like receptor (TLR) 7 antibodies for the treatment of disorders and diseases. [Background technology]

[0002] Ten types of TLRs, pattern recognition molecules, are known to exist in humans and form a single family. Among these, TLR7 (variant 1) is a single-pass transmembrane protein consisting of 1049 amino acids. It senses single-stranded RNA and mediates host defense responses through the production of type I interferons and cytokines. It has also been suggested that it may play an important role in the pathogenesis of various inflammatory and autoimmune diseases (Non-Patent Document 1). A variant of TLR7 (variant 2), a single-pass transmembrane protein consisting of 1049 amino acids, is also known and is known to have the same function as TLR7 (variant 1) (Non-Patent Document 2). Many reports suggest the involvement of TLR7 in diseases such as psoriasis and systemic lupus erythematosus, and inhibiting TLR7 function is expected to be a promising treatment for these diseases (Non-Patent Document 3).

[0003] Furthermore, while TLR7 deficiency has been shown to alleviate symptoms in disease models, reports suggest that TLR9 deficiency is involved in the exacerbation of the disease. Therefore, drugs that can specifically inhibit TLR7 are considered promising for the treatment of inflammatory diseases and other conditions (Non-patent Document 4).

[0004] Although there have been attempts to develop nucleic acids and small molecular weight compounds aimed at inhibiting human TLR7, it has been difficult to achieve specific inhibition of human TLR7 using nucleic acids and small molecular weight compounds. Furthermore, although mouse anti-mouse TLR7 antibodies are known as TLR7 antibodies (Patent Document 1), there is a need for research and development of new human TLR7-specific inhibitors. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] WO2014 / 174704 [Non-patent literature]

[0006] [Non-Patent Document 1] Takeuchi O, Akira S. Cell. 2010 Mar 19;140(6):805-2 [Non-patent document 2] Chuang TH, Ulevitch RJ, Eur Cytokine Netw. 2000 Sep;11(3):372-8 [Non-patent document 3] Lyn-Cook BD, Xie C, Oates J, Treadwell E, Word B, Hammons G, Wiley K. Mol Immunol. 2014 Sep;61(1):38-43. [Non-patent document 4] Christensen SR, Shupe J, Nickerson K, Kashgarian M, Flavell RA, Shlomchik MJ Immunity. 2006 Sep;25(3):417-28. Summary of the Invention [Problem to be solved by the invention]

[0007] An object of the present invention is to provide an agent for treating and / or preventing immune inflammation-related diseases, allergic diseases, infectious diseases, and cancer. [Means for solving the problem]

[0008] To achieve the above object, the present inventors conducted a search for substances effective in treating and / or preventing immune-inflammatory diseases, allergic diseases, infectious diseases, and cancer, and obtained a mouse anti-human TLR7 antibody. The obtained mouse anti-human TLR7 antibody was humanized, and the CDRs, framework, and variable regions of the humanized antibody were further modified to complete the present invention.

[0009] That is, the present invention includes the following inventions. (1) An antibody or an antigen-binding fragment of the antibody, characterized by having the following properties: (a) specifically binds to human TLR7 or monkey TLR7 and does not bind to mouse TLR7 or rat TLR7; and (b) Inhibiting the function of human TLR7 or monkey TLR7. (2) The antibody or antigen-binding fragment thereof according to (1) above, wherein human TLR7 is a molecule consisting of the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 4, monkey TLR7 is a molecule consisting of the amino acid sequence set forth in SEQ ID NO: 85, mouse TLR7 is a molecule consisting of the amino acid sequence set forth in SEQ ID NO: 83, or rat TLR7 is a molecule consisting of the amino acid sequence set forth in SEQ ID NO: 84. (3) In binding to human TLR7, an antibody having a heavy chain having a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 5 and a light chain having a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 11; an antibody having a heavy chain having a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 7 and a light chain having a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 13; or A heavy chain having a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 9 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 15 Variable region an antibody having The antibody or antigen-binding fragment thereof according to (1) or (2) above, which has competitive inhibitory activity against (4) (a) a heavy chain complementarity determining region (CDRH1) comprising the amino acid sequence set forth in SEQ ID NO: 17, a CDRH2 comprising the amino acid sequence set forth in SEQ ID NO: 18, and a CDRH3 comprising the amino acid sequence set forth in SEQ ID NO: 19; and as light chain complementarity determining regions, CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 20, CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 21, and CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 22; (b) a heavy chain complementarity-determining region comprising CDRH1 having the amino acid sequence set forth in SEQ ID NO: 23, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 24, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 25; and a light chain complementarity determining region comprising CDRL1 having the amino acid sequence set forth in SEQ ID NO: 26, CDRL2 having the amino acid sequence set forth in SEQ ID NO: 27, and CDRL3 having the amino acid sequence set forth in SEQ ID NO: 28; or (c) a heavy chain complementarity-determining region comprising CDRH1 having the amino acid sequence set forth in SEQ ID NO: 29, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 30, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 31; and as light chain complementarity determining regions, CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 32, CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 33, and CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 34; The antibody or antigen-binding fragment thereof according to any one of (1) to (3) above, comprising: (5) a heavy chain complementarity determining region (CDRH1) comprising the amino acid sequence set forth in SEQ ID NO: 17, a CDRH2 comprising the amino acid sequence set forth in SEQ ID NO: 18, and a CDRH3 comprising the amino acid sequence set forth in SEQ ID NO: 19; and as light chain complementarity determining regions, CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 20, CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 21, and CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 22; The antibody or antigen-binding fragment of the antibody according to (4) above, which has the following structure: (6) (a) a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 5 and a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 11; (b) a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 7 and a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 13, or (c) a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 9 and a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 15; The antibody or antigen-binding fragment thereof according to any one of (1) to (5) above, comprising: (7) The antibody or antigen-binding fragment thereof according to any one of (1) to (6) above, wherein the constant region is a human-derived constant region. (8) (a) a heavy chain consisting of the amino acid sequence from positions 20 to 465 in the amino acid sequence set forth in SEQ ID NO: 35 and a light chain consisting of the amino acid sequence from positions 21 to 233 in the amino acid sequence set forth in SEQ ID NO: 36; (b) a heavy chain consisting of the amino acid sequence from positions 20 to 471 in the amino acid sequence set forth in SEQ ID NO: 37 and a light chain consisting of the amino acid sequence from positions 21 to 233 in the amino acid sequence set forth in SEQ ID NO: 38; or (c) a heavy chain consisting of the amino acid sequence from positions 20 to 470 in the amino acid sequence set forth in SEQ ID NO: 39 and a light chain consisting of the amino acid sequence from positions 21 to 234 in the amino acid sequence set forth in SEQ ID NO: 40; The antibody or antigen-binding fragment of the antibody according to (7) above, which has the following structure: (9) The antibody or antigen-binding fragment thereof according to any one of (1) to (8) above, which has been humanized. (10) (I) (a) the amino acid sequence set forth in SEQ ID NO: 41; (b) the amino acid sequence set forth in SEQ ID NO: 42; (c) an amino acid sequence having at least 95% or more homology to the sequence of (a) or (b); and (d) 1 to 10 amino acids are deleted, substituted, or added in the sequence of (a) or (b). The amino acid sequence a heavy chain variable region consisting of an amino acid sequence selected from the group consisting of: (e) the amino acid sequence set forth in SEQ ID NO: 43; (f) the amino acid sequence set forth in SEQ ID NO: 44; (g) an amino acid sequence having at least 95% homology to the sequence of (e) or (f); and (h) 1 to 10 amino acids are deleted, substituted, or added in the sequence of (e) or (f). The amino acid sequence a light chain variable region consisting of an amino acid sequence selected from the group consisting of: (II)(a) the amino acid sequence set forth in SEQ ID NO: 50; (b) an amino acid sequence having at least 95% homology to the sequence of (a); and (c) an amino acid sequence in which 1 to 10 amino acids are deleted, substituted, or added in the sequence of (a); a heavy chain variable region consisting of an amino acid sequence selected from the group consisting of: (d) the amino acid sequence set forth in SEQ ID NO: 51; (e) an amino acid sequence having at least 95% homology to the sequence of (d); and (f) an amino acid sequence in which 1 to 10 amino acids are deleted, substituted, or added in the sequence of (d); Acid sequence a light chain variable region consisting of an amino acid sequence selected from the group consisting of: The antibody or antigen-binding fragment of the antibody according to (9) above, which has the following structure: (11) (a) a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 41 and a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 43; (b) a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 41 and a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 44; (c) a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 42 and a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 43; (d) a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 42 and a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 44, or (e) a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 50 and a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 51; The antibody or antigen-binding fragment thereof according to (10) above, comprising: (12) A heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 42 and a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 44 The antibody or antigen-binding fragment thereof according to (11) above, comprising: (13) (a) a heavy chain consisting of the amino acid sequence from positions 20 to 465 in the amino acid sequence set forth in SEQ ID NO: 45 and a light chain consisting of the amino acid sequence from positions 21 to 233 in the amino acid sequence set forth in SEQ ID NO: 48; (b) a heavy chain consisting of the amino acid sequence from positions 20 to 465 in the amino acid sequence set forth in SEQ ID NO: 45 and a light chain consisting of the amino acid sequence from positions 21 to 233 in the amino acid sequence set forth in SEQ ID NO: 49; (c) a heavy chain consisting of the amino acid sequence from positions 20 to 465 in the amino acid sequence set forth in SEQ ID NO: 46 and a light chain consisting of the amino acid sequence from positions 21 to 233 in the amino acid sequence set forth in SEQ ID NO: 48; (d) a heavy chain consisting of the amino acid sequence from positions 20 to 465 in the amino acid sequence set forth in SEQ ID NO: 46 and a light chain consisting of the amino acid sequence from positions 21 to 233 in the amino acid sequence set forth in SEQ ID NO: 49; (e) a heavy chain consisting of the amino acid sequence from positions 20 to 462 in the amino acid sequence set forth in SEQ ID NO: 47 and a light chain consisting of the amino acid sequence from positions 21 to 233 in the amino acid sequence set forth in SEQ ID NO: 48; (f) a heavy chain consisting of the amino acid sequence from positions 20 to 462 in the amino acid sequence set forth in SEQ ID NO: 47 and a light chain consisting of the amino acid sequence from positions 21 to 233 in the amino acid sequence set forth in SEQ ID NO: 49; (g) a heavy chain consisting of the amino acid sequence from positions 20 to 471 in the amino acid sequence set forth in SEQ ID NO: 52 and a light chain consisting of the amino acid sequence from positions 21 to 233 in the amino acid sequence set forth in SEQ ID NO: 53, or (h) a heavy chain consisting of the amino acid sequence from positions 20 to 468 in the amino acid sequence set forth in SEQ ID NO: 54 and a light chain consisting of the amino acid sequence from positions 21 to 233 in the amino acid sequence set forth in SEQ ID NO: 53; The antibody or antigen-binding fragment of the antibody according to (11) above, which has the following structure: (14) The antibody or antigen-binding fragment of the antibody according to (13) above, which has a heavy chain consisting of the amino acid sequence from the 20th to the 465th positions in the amino acid sequence set forth in SEQ ID NO: 46 and a light chain consisting of the amino acid sequence from the 21st to the 233rd positions in the amino acid sequence set forth in SEQ ID NO: 49. (15) An antibody or an antigen-binding fragment of the antibody, characterized by having the following properties: (a) specifically binds to human TLR7; (b) inhibiting the function of human TLR7; and (c)(1) a heavy chain complementarity-determining region (CDRH1) consisting of the amino acid sequence set forth in SEQ ID NO: 17, a CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 18, and a CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 19; and a light chain complementarity-determining region (CDRL1) consisting of the amino acid sequence set forth in SEQ ID NO: 20 and the amino acid sequence set forth in SEQ ID NO: 21. (2) a heavy chain complementarity determining region comprising a CDRH1 comprising the amino acid sequence set forth in SEQ ID NO: 23, a CDRH2 comprising the amino acid sequence set forth in SEQ ID NO: 24, and a CDRH3 comprising the amino acid sequence set forth in SEQ ID NO: 25, and a light chain complementarity determining region comprising a CDRL1 comprising the amino acid sequence set forth in SEQ ID NO: 26, a CDRL2 comprising the amino acid sequence set forth in SEQ ID NO: 27, and a CDRL3 comprising the amino acid sequence set forth in SEQ ID NO: 28, or (3) Heavy chain complementarity-determining regions: CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 29, CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 30, and CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 31; and light chain complementarity-determining regions: CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 32, CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 33, and CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 34. It has. (16) A heavy chain complementarity determining region (CDRH1) comprising the amino acid sequence set forth in SEQ ID NO: 17, a CDRH2 comprising the amino acid sequence set forth in SEQ ID NO: 18, and a CDRH3 comprising the amino acid sequence set forth in SEQ ID NO: 19; and The light chain complementarity determining regions are CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 20, CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 21, and CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 22. The antibody or antigen-binding fragment of the antibody according to (15) above, comprising: (17) (a) a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 5 and a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 11; (b) a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 7 and a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 13, or (c) a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 9 and a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 15 The antibody or antigen-binding fragment of the antibody according to (15) or (16) above, comprising: (18) The antibody or antigen-binding fragment thereof according to any one of (15) to (17) above, wherein the constant region is a human-derived constant region. (19) (a) a heavy chain consisting of the amino acid sequence from positions 20 to 465 in the amino acid sequence set forth in SEQ ID NO: 35 and a light chain consisting of the amino acid sequence from positions 21 to 233 in the amino acid sequence set forth in SEQ ID NO: 36; (b) a heavy chain consisting of the amino acid sequence from positions 20 to 471 in the amino acid sequence set forth in SEQ ID NO: 37 and a light chain consisting of the amino acid sequence from positions 21 to 233 in the amino acid sequence set forth in SEQ ID NO: 38; or (c) a heavy chain consisting of the amino acid sequence from positions 20 to 470 in the amino acid sequence set forth in SEQ ID NO: 39 and a light chain consisting of the amino acid sequence from positions 21 to 234 in the amino acid sequence set forth in SEQ ID NO: 40 The antibody or antigen-binding fragment of the antibody according to (18) above, which has the following structure: (20) The antibody or antigen-binding fragment thereof according to any one of (15) to (19) above, which has been humanized. (21)(I) (a) the amino acid sequence set forth in SEQ ID NO: 41; (b) the amino acid sequence set forth in SEQ ID NO: 42; (c) an amino acid sequence having at least 95% or more homology to the sequence of (a) or (b); and (d) an amino acid sequence in which 1 to 10 amino acids are deleted, substituted, or added in the sequence of (a) or (b); a heavy chain variable region consisting of an amino acid sequence selected from the group consisting of: and (e) the amino acid sequence set forth in SEQ ID NO: 43; (f) the amino acid sequence set forth in SEQ ID NO: 44; (g) an amino acid sequence having at least 95% homology to the sequence of (e) or (f); and (h) A sequence of (e) or (f) in which 1 to 10 amino acids are deleted, substituted, or added. The amino acid sequence a light chain variable region consisting of an amino acid sequence selected from the group consisting of: (II) (a) the amino acid sequence set forth in SEQ ID NO: 50; (b) an amino acid sequence having at least 95% or more homology to the sequence of (a), and (c) an amino acid sequence in which 1 to 10 amino acids are deleted, substituted, or added in the sequence of (a). a heavy chain variable region consisting of an amino acid sequence selected from the group consisting of: and (d) the amino acid sequence set forth in SEQ ID NO: 51; (e) an amino acid sequence having at least 95% or more homology to the sequence of (d), and (f) an amino acid sequence in which 1 to 10 amino acids are deleted, substituted or added in the sequence of (d). Acid sequence a light chain variable region consisting of an amino acid sequence selected from the group consisting of: The antibody or antigen-binding fragment of the antibody according to (20) above, comprising: (22) (a) a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 41 and a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 43; (b) a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 41 and a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 44; (c) a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 42 and a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 43; (d) a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 42 and a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 44, or (e) a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 50 and a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 51 The antibody or antigen-binding fragment thereof according to (21) above, comprising: (23) The antibody or antigen-binding fragment of the antibody according to (22) above, having a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 42 and a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 44. (24) (a) a heavy chain consisting of the amino acid sequence from positions 20 to 465 in the amino acid sequence set forth in SEQ ID NO: 45 and a light chain consisting of the amino acid sequence from positions 21 to 233 in the amino acid sequence set forth in SEQ ID NO: 48; (b) a heavy chain consisting of the amino acid sequence from positions 20 to 465 in the amino acid sequence set forth in SEQ ID NO: 45 and a light chain consisting of the amino acid sequence from positions 21 to 233 in the amino acid sequence set forth in SEQ ID NO: 49; (c) a heavy chain consisting of the amino acid sequence from positions 20 to 465 in the amino acid sequence set forth in SEQ ID NO: 46 and a light chain consisting of the amino acid sequence from positions 21 to 233 in the amino acid sequence set forth in SEQ ID NO: 48; (d) a heavy chain consisting of the amino acid sequence from positions 20 to 465 in the amino acid sequence set forth in SEQ ID NO: 46 and a light chain consisting of the amino acid sequence from positions 21 to 233 in the amino acid sequence set forth in SEQ ID NO: 49; (e) a heavy chain consisting of the amino acid sequence from positions 20 to 462 in the amino acid sequence set forth in SEQ ID NO: 47 and a light chain consisting of the amino acid sequence from positions 21 to 233 in the amino acid sequence set forth in SEQ ID NO: 48; (f) a heavy chain consisting of the amino acid sequence from positions 20 to 462 in the amino acid sequence set forth in SEQ ID NO: 47 and the amino acid sequence from positions 21 to 233 in the amino acid sequence set forth in SEQ ID NO: 49 a light chain consisting of the amino acid sequence (g) a heavy chain consisting of the amino acid sequence from positions 20 to 471 in the amino acid sequence set forth in SEQ ID NO: 52 and a light chain consisting of the amino acid sequence from positions 21 to 233 in the amino acid sequence set forth in SEQ ID NO: 53, or (h) a heavy chain consisting of the amino acid sequence from positions 20 to 468 in the amino acid sequence set forth in SEQ ID NO: 54 and a light chain consisting of the amino acid sequence from positions 21 to 233 in the amino acid sequence set forth in SEQ ID NO: 53 The antibody or antigen-binding fragment of the antibody according to (22) above, comprising: (25) The antibody or antigen-binding fragment of the antibody according to (24) above, having a heavy chain consisting of the amino acid sequence from the 20th to the 465th positions in the amino acid sequence set forth in SEQ ID NO: 46 and a light chain consisting of the amino acid sequence from the 21st to the 233rd positions in the amino acid sequence set forth in SEQ ID NO: 49. (26) An antibody or an antigen-binding fragment of the antibody having a heavy chain consisting of the amino acid sequence from positions 20 to 465 in the amino acid sequence set forth in SEQ ID NO: 45 and a light chain consisting of the amino acid sequence from positions 21 to 233 in the amino acid sequence set forth in SEQ ID NO: 48. (27) An antibody or an antigen-binding fragment of the antibody, having a heavy chain consisting of the amino acid sequence from positions 20 to 465 in the amino acid sequence set forth in SEQ ID NO: 46 and a light chain consisting of the amino acid sequence from positions 21 to 233 in the amino acid sequence set forth in SEQ ID NO: 48. (28) An antibody or an antigen-binding fragment of the antibody having a heavy chain consisting of the amino acid sequence from positions 20 to 465 in the amino acid sequence set forth in SEQ ID NO: 46 and a light chain consisting of the amino acid sequence from positions 21 to 233 in the amino acid sequence set forth in SEQ ID NO: 49. (29) An antibody or an antigen-binding fragment of the antibody, having a heavy chain consisting of the amino acid sequence from positions 20 to 462 in the amino acid sequence set forth in SEQ ID NO: 47 and a light chain consisting of the amino acid sequence from positions 21 to 233 in the amino acid sequence set forth in SEQ ID NO: 49. (30) An antibody or an antigen-binding fragment of the antibody having a heavy chain consisting of the amino acid sequence from positions 20 to 471 in the amino acid sequence set forth in SEQ ID NO: 52 and a light chain consisting of the amino acid sequence from positions 21 to 233 in the amino acid sequence set forth in SEQ ID NO: 53. (31) An antibody or an antigen-binding fragment of the antibody having a heavy chain consisting of the amino acid sequence from positions 20 to 468 in the amino acid sequence set forth in SEQ ID NO: 54 and a light chain consisting of the amino acid sequence from positions 21 to 233 in the amino acid sequence set forth in SEQ ID NO: 53. (32) The antibody or antigen-binding fragment thereof according to any one of (26) to (31) above, which has the properties of (a) specifically binding to human TLR7 and (b) inhibiting the function of human TLR7. (33) An antigen-binding fragment of the antibody according to any one of (1) to (32) above, wherein the antigen-binding fragment is selected from the group consisting of Fab, F(ab)2, Fab', and Fv. (34) The antibody according to any one of (1) to (33) above, or the antigen-binding ability of the antibody A polynucleotide having a polynucleotide sequence encoding the fragment. (35) (1) a polynucleotide sequence encoding CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 17, a polynucleotide sequence encoding CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 18, and a polynucleotide sequence encoding CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 19; a polynucleotide sequence encoding CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 20, a polynucleotide sequence encoding CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 21, and a polynucleotide sequence encoding CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 22; (2) a polynucleotide sequence encoding CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 23, a polynucleotide sequence encoding CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 24, and a polynucleotide sequence encoding CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 25; and A polynucleotide sequence encoding CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 26 a polynucleotide sequence encoding a CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO:27 and a polynucleotide sequence encoding a CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO:28; or (3) a polynucleotide sequence encoding CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 29, a polynucleotide sequence encoding CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 30, and a polynucleotide sequence encoding CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 31; and a polynucleotide sequence encoding CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 32, a polynucleotide sequence encoding CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 33, and a polynucleotide sequence encoding CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 34; The polynucleotide according to (34) above, having the following structure: (36)(I) (a) a polynucleotide sequence encoding the heavy chain variable region set forth in SEQ ID NO: 77; (b) a polynucleotide sequence encoding the heavy chain variable region set forth in SEQ ID NO: 78; and (c) a polynucleotide sequence that hybridizes under stringent conditions to a polynucleotide consisting of a polynucleotide sequence complementary to the polynucleotide sequence of (a) or (b); a polynucleotide sequence selected from the group consisting of: (d) a polynucleotide sequence encoding the light chain variable region set forth in SEQ ID NO: 79; (e) a polynucleotide sequence encoding the light chain variable region set forth in SEQ ID NO: 80; and (f) a polynucleotide sequence that hybridizes under stringent conditions to a polynucleotide consisting of a polynucleotide sequence complementary to the polynucleotide sequence of (d) or (e); a polynucleotide sequence selected from the group consisting of: (II) (a) a polynucleotide sequence encoding the heavy chain variable region set forth in SEQ ID NO: 81; and (b) a polynucleotide sequence that hybridizes under stringent conditions to a polynucleotide having a polynucleotide sequence complementary to the polynucleotide sequence of (a); a polynucleotide sequence selected from the group consisting of: (c) a polynucleotide sequence encoding the light chain variable region set forth in SEQ ID NO: 82; and (d) a polynucleotide sequence that hybridizes under stringent conditions to a polynucleotide having a polynucleotide sequence complementary to the polynucleotide sequence of (c); a polynucleotide sequence selected from the group consisting of: The polynucleotide according to (35) above, having the following structure: (37) (a) a polynucleotide sequence encoding a heavy chain variable region set forth in SEQ ID NO: 77 and a polynucleotide sequence encoding a light chain variable region set forth in SEQ ID NO: 79; (b) a polynucleotide sequence encoding a heavy chain variable region set forth in SEQ ID NO: 77 and a polynucleotide sequence encoding a light chain variable region set forth in SEQ ID NO: 80; (c) a polynucleotide sequence encoding a heavy chain variable region set forth in SEQ ID NO: 78 and a polynucleotide sequence encoding a light chain variable region set forth in SEQ ID NO: 79; (d) a polynucleotide sequence encoding the heavy chain variable region set forth in SEQ ID NO: 78 and a polynucleotide sequence encoding the light chain variable region set forth in SEQ ID NO: 80; or (e) a polynucleotide sequence encoding a heavy chain variable region set forth in SEQ ID NO: 81 and a polynucleotide sequence encoding a light chain variable region set forth in SEQ ID NO: 82; The polynucleotide according to (36) above, having the following structure: (38) A method comprising the steps of: (1) preparing a polynucleotide according to any one of (34) to (37); Expression vector. (39) A host cell transformed with the expression vector according to (38) above. (40) The host cell according to (39) above, wherein the host cell is a eukaryotic cell. (41) A method for producing an antibody or an antigen-binding fragment thereof, comprising the steps of culturing the host cell according to (39) or (40) above, and recovering the antibody of interest or an antigen-binding fragment of the antibody from the culture obtained in the step. (42) An antibody or an antigen-binding fragment of the antibody, characterized by being obtained by the production method according to (41) above. (43) The antibody according to any one of (1) to (32) above, comprising one or more modifications selected from the group consisting of glycosylation at an N-linkage, glycosylation at an O-linkage, N-terminal processing, C-terminal processing, deamidation, isomerization of aspartic acid, oxidation of methionine, addition of a methionine residue to the N-terminus, amidation of a proline residue, and deletion of one or two amino acids at the carboxyl terminus of the heavy chain. (44) The antibody according to (43) above, wherein the two heavy chains are heavy chains consisting of any one or a combination of any two of the heavy chains selected from the group consisting of full-length heavy chains and deletion heavy chains in which one or two amino acids are deleted at the carboxyl terminus. (45) The antibody according to (44) above, in which one amino acid is deleted at the carboxyl terminus of each of the two heavy chains. (46) The antibody according to any one of (43) to (45) above, wherein the proline residue at the carboxyl terminus of the heavy chain is further amidated. (47) A pharmaceutical composition for treating and / or preventing a disease, characterized by containing as an active ingredient at least one of the antibodies or antigen-binding fragments of the antibodies described in (1) to (33) and (42) to (46) above, or the expression vector described in (38) above. (48) The pharmaceutical composition according to (47) above, which is for treating a disease caused by the function of human TLR7. (49) The pharmaceutical composition according to (48) above, wherein the disease caused by the function of human TLR7 is an immune inflammation-related disease, an allergic disease, an infectious disease, or cancer. (50) The pharmaceutical composition according to (49), wherein the immune inflammation-related disease is systemic lupus erythematosus, rheumatoid arthritis, juvenile idiopathic arthritis, adult Still's disease, ankylosing spondylitis, systemic sclerosis, polymyositis, dermatomyositis, psoriatic arthritis, osteoarthritis, mixed connective tissue disease, or muscular dystrophy. (51) The pharmaceutical composition according to (49) or (50) above, wherein the immune inflammation-related disease is systemic lupus erythematosus. (52) The pharmaceutical composition according to any one of (47) to (51) above, for use in combination with an immunosuppressant, an anti-inflammatory agent, an antiallergic agent, an anti-infective agent, or an anti-cancer agent. (53) A method for treating a disease caused by the function of human TLR7, comprising administering to an individual at least one of the antibodies or antigen-binding fragments of the antibodies according to (1) to (33) and (42) to (46) above. (54) A method for treating a disease caused by the function of human TLR7, comprising administering to an individual simultaneously, separately, or consecutively at least one of the antibodies or antigen-binding fragments of the antibodies according to (1) to (33) and (42) to (46) above, and an immunosuppressant, an anti-inflammatory agent, an anti-allergic agent, an anti-infective agent, or an anti-cancer agent. (55) The method for treating a disease caused by the function of human TLR7 according to (53) or (54) above, wherein the disease is an immune inflammation-related disease, an allergic disease, an infectious disease, or cancer. (56) The method for treating immune inflammation according to (55), wherein the immune inflammation-related disease is systemic lupus erythematosus, rheumatoid arthritis, juvenile idiopathic arthritis, adult Still's disease, ankylosing spondylitis, systemic sclerosis, polymyositis, dermatomyositis, psoriatic arthritis, osteoarthritis, mixed connective tissue disease, or muscular dystrophy. (57) The method for treating an immune inflammatory disease according to (55) or (56) above, wherein the immune inflammatory disease is systemic lupus erythematosus. (58) The method of any one of the above for use in treating or preventing a disease caused by the function of human TLR7. An antibody or an antigen-binding fragment of the antibody according to any one of (1) to (33) and (42) to (46), or an expression vector according to (38). (59) The antibody or antigen-binding fragment thereof, or expression vector according to (58) above, wherein the disease caused by the function of human TLR7 is an immune inflammation-related disease, an allergic disease, an infectious disease, or cancer. (60) The antibody or antigen-binding fragment thereof or expression vector according to (59), wherein the immune inflammation-related disease is systemic lupus erythematosus, rheumatoid arthritis, juvenile idiopathic arthritis, adult Still's disease, ankylosing spondylitis, systemic sclerosis, polymyositis, dermatomyositis, psoriatic arthritis, osteoarthritis, mixed connective tissue disease, or muscular dystrophy. (61) The antibody or antigen-binding fragment thereof, or expression vector according to (59) or (60) above, wherein the immunoinflammatory disease is systemic lupus erythematosus. (62) The antibody or antigen-binding fragment thereof or expression vector according to any one of (58) to (61) above, for use in combination with an immunosuppressant, anti-inflammatory agent, antiallergic agent, anti-infective agent, or anti-cancer agent. (63) Use of the antibody or antigen-binding fragment thereof according to any one of (1) to (33) and (42) to (46) above, or the expression vector according to (38) above, for the manufacture of a therapeutic or preventive agent for a disease caused by the function of human TLR7. (64) The use according to (63) above, wherein the disease caused by the function of human TLR7 is an immune inflammation-related disease, an allergic disease, an infectious disease, or cancer. (65) The use according to (64) above, wherein the immune inflammation-related disease is systemic lupus erythematosus, rheumatoid arthritis, juvenile idiopathic arthritis, adult Still's disease, ankylosing spondylitis, systemic sclerosis, polymyositis, dermatomyositis, psoriatic arthritis, osteoarthritis, mixed connective tissue disease, or muscular dystrophy. (66) The use according to (64) or (65) above, wherein the immune inflammation-related disease is systemic lupus erythematosus. (67) The use according to any one of (63) to (66) above, wherein the therapeutic or prophylactic agent is used in combination with an immunosuppressant, an anti-inflammatory agent, an antiallergic agent, an anti-infective agent, or an anti-cancer agent. [Effects of the Invention]

[0010] According to the present invention, it is possible to obtain a therapeutic and / or prophylactic agent for immune inflammation-related diseases, allergic diseases, infectious diseases, and cancer, which has as its mechanism of action the inhibition of TLR7 function. [Brief explanation of the drawings]

[0011] [Figure 1] FIG. 1 shows the amino acid sequence of human TLR7 (variant 1) (SEQ ID NO: 2). [Figure 2] FIG. 2 shows the amino acid sequence of human TLR7 (variant 2) (SEQ ID NO: 4). [Figure 3] FIG. 3 shows the amino acid sequence of mouse TLR7 (SEQ ID NO: 83). [Figure 4] FIG. 4 shows the amino acid sequence of rat TLR7 (SEQ ID NO: 84). [Figure 5] FIG. 5 shows the amino acid sequence of monkey TLR7 (SEQ ID NO: 85). [Figure 6] FIG. 6 shows that the AT01 antibody, the NB7 antibody, and the FAN2 antibody inhibit IL-6 production from CL-264-treated human peripheral blood mononuclear cells (hereinafter, referred to as PBMC) in a concentration-dependent manner. [Figure 7]7 shows the amino acid sequence of the heavy chain containing a signal sequence (SEQ ID NO: 35) and the amino acid sequence of the light chain containing a signal sequence (SEQ ID NO: 36) of the cAT01 antibody. In the heavy chain amino acid sequence, the amino acid sequence at positions 1 to 19 is the signal sequence, the amino acid sequence at positions 20 to 135 is the heavy chain variable region, and the amino acid sequence at positions 136 to 465 is the heavy chain constant region. In the light chain amino acid sequence, the amino acid sequence at positions 1 to 20 is the signal sequence, the amino acid sequence at positions 21 to 126 is the light chain variable region, and the amino acid sequence at positions 127 to 233 is the light chain constant region. [Figure 8] 8 shows the amino acid sequence of the heavy chain containing a signal sequence (SEQ ID NO: 37) and the amino acid sequence of the light chain containing a signal sequence (SEQ ID NO: 38) of the cNB7 antibody. In the heavy chain amino acid sequence, the amino acid sequence at positions 1 to 19 is the signal sequence, the amino acid sequence at positions 20 to 141 is the heavy chain variable region, and the amino acid sequence at positions 142 to 471 is the heavy chain constant region. In the light chain amino acid sequence, the amino acid sequence at positions 1 to 20 is the signal sequence, the amino acid sequence at positions 21 to 126 is the light chain variable region, and the amino acid sequence at positions 127 to 233 is the light chain constant region. [Figure 9] 9 shows the amino acid sequence of the heavy chain containing a signal sequence (SEQ ID NO: 39) and the amino acid sequence of the light chain containing a signal sequence (SEQ ID NO: 40) of the cFAN2 antibody. In the heavy chain amino acid sequence, the amino acid sequence at positions 1 to 19 is the signal sequence, the amino acid sequence at positions 20 to 140 is the heavy chain variable region, and the amino acid sequence at positions 141 to 470 is the heavy chain constant region. In the light chain amino acid sequence, the amino acid sequence at positions 1 to 20 is the signal sequence, the amino acid sequence at positions 21 to 127 is the light chain variable region, and the amino acid sequence at positions 128 to 234 is the light chain constant region. [Figure 10] FIG. 10 shows that the chimeric anti-human TLR7 antibodies cAT01, cNB7, and cFAN2 inhibit IL-6 production from CL-264-treated human PBMCs in a concentration-dependent manner. [Figure 11]11 shows the amino acid sequence (SEQ ID NO: 45) of the heavy chain including the signal sequence of the humanized heavy chain huAT01_H1_IgG1LALA. In this sequence, the amino acid sequence from positions 1 to 19 is the signal sequence, the amino acid sequence from positions 20 to 135 is the variable region, and the amino acid sequence from positions 136 to 465 is the constant region. Furthermore, the amino acid sequence from positions 45 to 54 is CDRH1, the amino acid sequence from positions 69 to 78 is CDRH2, and the amino acid sequence from positions 118 to 124 is CDRH3. [Figure 12] 12 shows the amino acid sequence (SEQ ID NO: 46) of the heavy chain including the signal sequence of the humanized heavy chain huAT01_H3_IgG1LALA. In this sequence, the amino acid sequence from positions 1 to 19 is the signal sequence, the amino acid sequence from positions 20 to 135 is the variable region, and the amino acid sequence from positions 136 to 465 is the constant region. Furthermore, the amino acid sequence from positions 45 to 54 is CDRH1, the amino acid sequence from positions 69 to 78 is CDRH2, and the amino acid sequence from positions 118 to 124 is CDRH3. [Figure 13] 13 shows the amino acid sequence (SEQ ID NO: 47) of the heavy chain including the signal sequence of the humanized heavy chain huAT01_H3_IgG4Pro. In this sequence, the amino acid sequence from positions 1 to 19 is the signal sequence, the amino acid sequence from positions 20 to 135 is the variable region, and the amino acid sequence from positions 136 to 462 is the constant region. Furthermore, the amino acid sequence from positions 45 to 54 is CDRH1, the amino acid sequence from positions 69 to 78 is CDRH2, and the amino acid sequence from positions 118 to 124 is CDRH3. [Figure 14]Figure 14 shows a comparison of the amino acid sequence of the variable region of cAT01_H, the heavy chain of chimeric antibody cAT01 (SEQ ID NO: 5) (hereinafter referred to as cAT01_H), with the amino acid sequence of the variable region of humanized antibody heavy chain huAT01_H1_IgG1LALA (SEQ ID NO: 41) (hereinafter referred to as huAT01_H1) and the amino acid sequence of the variable region of huAT01_H3_IgG1LALA (SEQ ID NO: 42) (hereinafter referred to as huAT01_H3). In the sequences of huAT01_H1 and huAT01_H3, "·" indicates the same amino acid residue as in cAT01_H, and where an amino acid residue is listed, indicates a substituted amino acid residue. [Figure 15] 15 shows the amino acid sequence (SEQ ID NO: 48) of the humanized light chain huAT01_L1, including the signal sequence. In this sequence, the amino acid sequence from positions 1 to 20 is the signal sequence, the amino acid sequence from positions 21 to 126 is the variable region, and the amino acid sequence from positions 127 to 233 is the constant region. Furthermore, amino acid positions 44 to 54 are CDRL1, amino acid positions 70 to 76 are CDRL2, and amino acid positions 109 to 116 are CDRL3. [Figure 16] 16 shows the amino acid sequence (SEQ ID NO: 49) of the humanized light chain huAT01_L2, including the signal sequence. In this sequence, the amino acid sequence from positions 1 to 20 is the signal sequence, the amino acid sequence from positions 21 to 126 is the variable region, and the amino acid sequence from positions 127 to 233 is the constant region. Furthermore, amino acid positions 44 to 54 are CDRL1, amino acid positions 70 to 76 are CDRL2, and amino acid positions 109 to 116 are CDRL3. [Figure 17]17 shows a comparison of the amino acid sequence of the variable region of cAT01_L, the light chain of chimeric antibody cAT01 (SEQ ID NO: 11) (hereinafter referred to as cAT01_L), with the amino acid sequence of the variable region of humanized antibody light chain huAT01_L1 (SEQ ID NO: 43) (hereinafter referred to as huAT01_L1) and the amino acid sequence of the variable region of huAT01_L2 (SEQ ID NO: 44) (hereinafter referred to as huAT01_L2). In the sequences of huAT01_L1 and huAT01_L2, "·" indicates the same amino acid residue as in cAT01_L, and where an amino acid residue is listed, indicates a substituted amino acid residue. [Figure 18] 18 shows the amino acid sequence (SEQ ID NO: 52) of the humanized heavy chain huNB7_H3_IgG1LALA, including the signal sequence. In this sequence, the amino acid sequence from positions 1 to 19 is the signal sequence, the amino acid sequence from positions 20 to 141 is the variable region, and the amino acid sequence from positions 142 to 471 is the constant region. Furthermore, amino acid positions 45 to 55 are CDRH1, amino acid positions 70 to 78 are CDRH2, and amino acid positions 118 to 130 are CDRH3. [Figure 19] 19 shows the amino acid sequence (SEQ ID NO: 54) of the heavy chain including the signal sequence of the humanized heavy chain huNB7_H3_IgG4Pro. In this sequence, the amino acid sequence from positions 1 to 19 is the signal sequence, the amino acid sequence from positions 20 to 141 is the variable region, and the amino acid sequence from positions 142 to 468 is the constant region. Furthermore, amino acid positions 45 to 55 are CDRH1, amino acid positions 70 to 78 are CDRH2, and amino acid positions 118 to 130 are CDRH3. [Figure 20] 20 shows a comparison of the amino acid sequence of the variable region of cNB7_H, the heavy chain of chimeric antibody cNB7 (SEQ ID NO: 7) (hereinafter referred to as cNB7_H), and the amino acid sequence of the variable region of humanized antibody heavy chain huNB7_H3_IgG1LALA (SEQ ID NO: 50) (hereinafter referred to as huNB7_H3). In huNB7_H3, "·" indicates the same amino acid residue as in cNB7_H, and the positions where amino acid residues are listed indicate substituted amino acid residues. [Figure 21] 21 shows the amino acid sequence (SEQ ID NO: 53) of the humanized light chain huNB7_L3, including the signal sequence. In this sequence, the amino acid sequence from positions 1 to 20 is the signal sequence, the amino acid sequence from positions 21 to 126 is the variable region, and the amino acid sequence from positions 127 to 233 is the constant region. Furthermore, amino acid positions 44 to 54 are CDRL1, amino acid positions 70 to 76 are CDRL2, and amino acid positions 109 to 116 are CDRL3. [Figure 22] 22 shows a comparison of the amino acid sequence of the variable region of cNB7_L, the light chain of chimeric antibody cNB7 (SEQ ID NO: 13) (hereinafter referred to as cNB7_L), and the amino acid sequence of the variable region of the humanized antibody light chain huNB7_L3 (SEQ ID NO: 51) (hereinafter referred to as huNB7_L3). In huNB7_L3, "·" indicates the same amino acid residue as in cNB7_L, and the positions where amino acid residues are listed indicate substituted amino acid residues. [Figure 23] Figure 23 shows that humanized anti-human TLR7 antibodies (huAT01_H1L1_IgG1LALA, huAT01_H3L1_IgG1LALA, huAT01_H3L2_IgG1LALA, huAT01_H3L2_IgG4Pro, huNB7_H3L3_IgG1LALA, huNB7_H3L3_IgG4Pro) suppress IL-6 production from CL-264-treated human PBMCs in a concentration-dependent manner. [Figure 24]Figure 24-1 shows the amino acid sequence of CDRH1 (SEQ ID NO: 17), the amino acid sequence of CDRH2 (SEQ ID NO: 18), and the amino acid sequence of CDRH3 (SEQ ID NO: 19), as well as the amino acid sequence of CDRL1 (SEQ ID NO: 20), the amino acid sequence of CDRL2 (SEQ ID NO: 21), and the amino acid sequence of CDRL3 (SEQ ID NO: 22) of the AT01 antibody; Figure 24-2 shows the amino acid sequence of CDRH1 (SEQ ID NO: 23), the amino acid sequence of CDRH2 (SEQ ID NO: 24), and the amino acid sequence of CDRH3 (SEQ ID NO: 25), as well as the amino acid sequence of CDRL1 (SEQ ID NO: 26), the amino acid sequence of CDRL2 (SEQ ID NO: 27), and the amino acid sequence of CDRL3 (SEQ ID NO: 28) of the NB7 antibody; Figure 24-3 shows the amino acid sequence of CDRH1 (sequence number 29), CDRH2 (sequence number 30), and CDRH3 (sequence number 31) of the FAN2 antibody, as well as the amino acid sequence of CDRL1 (sequence number 32), CDRL2 (sequence number 33), and CDRL3 (sequence number 34). [Figure 25] FIG. 25 shows the results of flow cytometry analysis of the binding of the humanized anti-human TLR7 antibody (huAT01_H3L2_IgG1LALA) to an antigen (human TLR7: variant 1), demonstrating that the antibody specifically binds to the antigen. [Figure 26] FIG. 26 shows the results of flow cytometry analysis of the binding of the humanized anti-human TLR7 antibody (huAT01_H3L2_IgG1LALA) to an antigen (human TLR7: variant 2), demonstrating that the antibody specifically binds to the antigen. [Figure 27] FIG. 27 shows the results of flow cytometry analysis of the binding of humanized anti-human TLR7 antibody (huAT01_H3L2_IgG1LALA) to an antigen (monkey TLR7), demonstrating that the antibody specifically binds to the antigen. [Figure 28] FIG. 28 shows the results of flow cytometry analysis of the binding of humanized anti-human TLR7 antibody (huAT01_H3L2_IgG1LALA) to an antigen (mouse TLR7), demonstrating that the antibody does not bind to the antigen. [Figure 29]FIG. 29 shows the results of flow cytometry analysis of the binding of humanized anti-human TLR7 antibody (huAT01_H3L2_IgG1LALA) to an antigen (rat TLR7), demonstrating that the antibody does not bind to the antigen. DETAILED DESCRIPTION OF THE INVENTION

[0012] As used herein, the term "gene" includes not only DNA but also mRNA, cDNA, and cRNA.

[0013] As used herein, the term "polynucleotide" is used interchangeably with nucleic acid and includes DNA, RNA, probes, oligonucleotides, and primers.

[0014] In this specification, the terms "polypeptide" and "protein" are used interchangeably.

[0015] As used herein, the term "RNA fraction" refers to a fraction containing RNA.

[0016] As used herein, the term "cells" includes cells within an animal body and cultured cells.

[0017] As used herein, "TLR7" is used synonymously with TLR7 protein.

[0018] As used herein, "antigen-binding fragment of an antibody" refers to a partial fragment of an antibody that has antigen-binding activity, and includes Fab, F(ab')2, Fv, scFv, diabody, linear antibody, and multispecific antibody formed from antibody fragments. Also included in the antigen-binding fragment of an antibody is Fab', a monovalent fragment of the variable region of an antibody obtained by treating F(ab')2 under reducing conditions. However, this is not limited to these molecules, as long as they have the ability to bind to an antigen. These antigen-binding fragments include not only those obtained by treating the full-length antibody protein molecule with an appropriate enzyme, but also those produced in appropriate host cells using genetically engineered antibody genes. This also includes proteins that have been extracted.

[0019] It is known that the heavy and light chains of an antibody molecule each contain three complementarity determining regions (CDRs). The complementarity determining regions, also known as hypervariable regions, are located within the variable regions of the heavy and light chains of an antibody and are regions with particularly high variability in their primary structure. They are separated into three regions in the primary structure of the heavy and light polypeptide chains. In this specification, the complementarity determining regions of an antibody are referred to as CDRH1, CDRH2, and CDRH3 in the order from the amino-terminus of the heavy chain amino acid sequence, and CDRL1, CDRL2, and CDRL3 in the order from the amino-terminus of the light chain amino acid sequence. These regions are adjacent to each other in the three-dimensional structure and determine the specificity for the antigen to which they bind. The amino acid sequence of each CDR is described based on the AbM definition (Martin, ACR, Cheetham, JC and Rees, AR (1989) Proc. Natl. Acad. Sci. USA, 86, 9268-9272).

[0020] In the present invention, "hybridizing under stringent conditions" means hybridizing at 68°C in a commercially available hybridization solution, ExpressHyb Hybridization Solution (CLONTECH), or hybridizing using a DNA-immobilized filter at 68°C in the presence of 0.7-1.0 M NaCl, followed by washing at 68°C using a 0.1-2x SSC solution (1x SSC consists of 150 mM NaCl and 15 mM sodium citrate), allowing identification, or hybridizing under equivalent conditions.

[0021] 1.TLR7 The human TLR7 used in the present invention can be directly purified from human B cells or dendritic cells, or prepared as a cell membrane fraction of the above cells. Human TLR7 can also be synthesized in vitro or produced in host cells by genetic engineering. Specifically, human TLR7 cDNA can be inserted into an expression vector, and then synthesized in a solution containing enzymes, substrates, and energy sources necessary for transcription and translation. Alternatively, human TLR7 can be expressed by transforming other prokaryotic or eukaryotic host cells to obtain the protein.

[0022] Similarly, monkey TLR7, mouse TLR7, and rat TLR7 can be directly purified from monkey, mouse, and rat TLR7-expressing cells, respectively, or prepared as cell membrane fractions of the above cells. Alternatively, monkey TLR7, mouse TLR7, and rat TLR7 can be synthesized in vitro or produced in host cells by genetic engineering.

[0023] The polynucleotide sequences encoding the amino acid sequences of human TLR7 (variant 1) and human TLR7 (variant 2) are set forth in SEQ ID NO: 1 or SEQ ID NO: 3, respectively, in the Sequence Listing, and the amino acid sequences of human TLR7 (variant 1) and human TLR7 (variant 2) are set forth in SEQ ID NO: 2 or SEQ ID NO: 4, respectively, in the Sequence Listing.

[0024] Human TLR7 cDNA can be obtained, for example, by the so-called PCR method, which involves performing a polymerase chain reaction (hereinafter referred to as "PCR") using a cDNA library from an organ expressing human TLR7 mRNA as a template and primers that specifically amplify human TLR7 cDNA (Saiki, R.K., et al., Science, (1988) 239, 487-49).

[0025] Human TLR7 cDNA also includes polynucleotides that hybridize under stringent conditions with polynucleotides consisting of nucleotide sequences complementary to the nucleotide sequence encoding human TLR7 and that encode proteins with biological activity equivalent to that of human TLR7. Furthermore, human TLR7 cDNA also includes splicing variants transcribed from the human TLR7 gene locus or polynucleotides that hybridize to these variants under stringent conditions and that encode proteins with biological activity equivalent to that of human TLR7.

[0026] Human TLR7 also includes proteins that have biological activity equivalent to that of human TLR7, consisting of an amino acid sequence in which one, two, three, or four or five amino acids have been substituted, deleted, or added in the amino acid sequence of human TLR7 or in the amino acid sequence in which the signal sequence has been removed from the amino acid sequence of human TLR7.Human TLR7 also includes proteins that have biological activity equivalent to that of human TLR7, consisting of an amino acid sequence encoded by a splicing variant transcribed from the human TLR7 gene locus or an amino acid sequence in which one, two, three, or four or five amino acids have been substituted, deleted, or added in the amino acid sequence.

[0027] The polynucleotide sequence encoding the amino acid sequence of monkey TLR7 is set forth in SEQ ID NO:58 of the Sequence Listing, and the amino acid sequence of monkey TLR7 is set forth in SEQ ID NO:85 of the Sequence Listing.

[0028] Monkey TLR7 cDNA can be obtained, for example, by PCR using a cDNA library from an organ expressing monkey TLR7 mRNA as a template and primers that specifically amplify monkey TLR7 cDNA.

[0029] Additionally, the cDNA of simian TLR7 also includes polynucleotides that hybridize under stringent conditions with polynucleotides consisting of nucleotide sequences complementary to the nucleotide sequence encoding simian TLR7 and that encode proteins with biological activity equivalent to that of simian TLR7. Furthermore, the cDNA of simian TLR7 also includes splicing variants transcribed from the simian TLR7 gene locus or polynucleotides that hybridize thereto under stringent conditions and that encode proteins with biological activity equivalent to that of simian TLR7.

[0030] Monkey TLR7 also includes proteins that have biological activity equivalent to that of simian TLR7, which consist of an amino acid sequence in which one, two, three, or four or five amino acids have been substituted, deleted, or added in the amino acid sequence of simian TLR7, or in the amino acid sequence in which the signal sequence has been removed from the amino acid sequence of simian TLR7. Furthermore, simian TLR7 also includes proteins that have biological activity equivalent to that of simian TLR7, which consist of an amino acid sequence encoded by a splicing variant transcribed from the simian TLR7 gene locus, or an amino acid sequence in which one, two, three, or four or five amino acids have been substituted, deleted, or added in the amino acid sequence.

[0031] The polynucleotide sequence encoding the amino acid sequence of monkey TLR7 is set forth in SEQ ID NO:58 of the Sequence Listing, and the amino acid sequence of monkey TLR7 is set forth in SEQ ID NO:85 of the Sequence Listing.

[0032] Monkey TLR7 cDNA can be obtained, for example, by PCR using a cDNA library from an organ expressing monkey TLR7 mRNA as a template and primers that specifically amplify monkey TLR7 cDNA.

[0033] The polynucleotide sequence encoding the amino acid sequence of mouse TLR7 is set forth in SEQ ID NO: 56 of the Sequence Listing, and the amino acid sequence of mouse TLR7 is set forth in SEQ ID NO: 83 of the Sequence Listing. It is being done.

[0034] Mouse TLR7 cDNA can be obtained, for example, by PCR using a cDNA library from an organ expressing mouse TLR7 mRNA as a template and primers that specifically amplify mouse TLR7 cDNA.

[0035] The polynucleotide sequence encoding the amino acid sequence of rat TLR7 is set forth in SEQ ID NO:57 in the Sequence Listing, and the amino acid sequence of rat TLR7 is set forth in SEQ ID NO:84 in the Sequence Listing.

[0036] Rat TLR7 cDNA can be obtained, for example, by PCR using a cDNA library from an organ expressing rat TLR7 mRNA as a template and primers that specifically amplify rat TLR7 cDNA.

[0037] 2. Anti-TLR7 antibodies and their production The present invention provides an antibody or an antigen-binding fragment thereof characterized by having the following properties: (a) specifically binds to human TLR7 or monkey TLR7 and does not bind to mouse TLR7 or rat TLR7; and (b) Inhibiting the function of human TLR7 or monkey TLR7.

[0038] The antibody or antigen-binding fragment thereof of the present invention specifically binds to human TLR7 or simian TLR7 and inhibits its function.

[0039] The binding activity of an antibody or an antigen-binding fragment thereof to TLR7 is evaluated by flow cytometry analysis according to standard methods.

[0040] Furthermore, as used herein, inhibiting the function of TLR7 refers to suppressing the production of IL-6 and / or type I interferon by TLR7-expressing cells. The activity of inhibiting the function of TLR7 is evaluated in vitro by incubating TLR7-expressing cells (e.g., PBMCs) in the presence of an antibody or an antigen-binding fragment thereof and measuring the concentration of IL-6 or type I interferon in the culture supernatant.

[0041] In one embodiment of the present invention, human TLR7 is a molecule consisting of the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 4, monkey TLR7 is a molecule consisting of the amino acid sequence set forth in SEQ ID NO: 85, mouse TLR7 is a molecule consisting of the amino acid sequence set forth in SEQ ID NO: 83, or rat TLR7 is a molecule consisting of the amino acid sequence set forth in SEQ ID NO: 84. For example, in the present invention, human TLR7 can be a molecule consisting of the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 4, monkey TLR7 can be a molecule consisting of the amino acid sequence set forth in SEQ ID NO: 85, mouse TLR7 can be a molecule consisting of the amino acid sequence set forth in SEQ ID NO: 83, and rat TLR7 can be a molecule consisting of the amino acid sequence set forth in SEQ ID NO: 84.

[0042] For example, the antibody or antigen-binding fragment thereof of the present invention may be an antibody or antigen-binding fragment thereof against human TLR7, and has the following characteristics: (a) specifically binds to human TLR7 and does not bind to mouse TLR7 and / or rat TLR7; and (b) inhibiting the function of human TLR7; wherein human TLR7 is a molecule consisting of the amino acid sequence set forth in SEQ ID NO:2 or SEQ ID NO:4, mouse TLR7 is a molecule consisting of the amino acid sequence set forth in SEQ ID NO:83, and rat TLR7 is a molecule consisting of the amino acid sequence set forth in SEQ ID NO:84.

[0043] For example, the antibody or antigen-binding fragment thereof of the present invention may be an antibody or antigen-binding fragment thereof against monkey TLR7, and has the following characteristics: (a) specifically binds to monkey TLR7 and does not bind to mouse TLR7 and / or rat TLR7; and (b) inhibiting the function of monkey TLR7; wherein monkey TLR7 is a molecule consisting of the amino acid sequence set forth in SEQ ID NO: 85, mouse TLR7 is a molecule consisting of the amino acid sequence set forth in SEQ ID NO: 83, and rat TLR7 is a molecule consisting of the amino acid sequence set forth in SEQ ID NO: 84.

[0044] The anti-TLR7 antibody of the present invention and the method for producing it will be described below using the anti-human TLR7 antibody as an example. Anti-monkey TLR7 antibodies can also be produced in the same manner as anti-human TLR7 antibodies.

[0045] The antibodies against human TLR7 of the present invention can be obtained by immunizing an animal with human TLR7 or any polypeptide selected from the amino acid sequence of human TLR7, and collecting and purifying the antibodies produced in the body using standard methods.

[0046] The human TLR7 antigen can be obtained by genetically engineering the human TLR7 gene in host cells. Specifically, a vector capable of expressing the TLR7 gene is prepared, introduced into host cells to express the gene, and the expressed TLR7 is then purified.

[0047] The antibodies against human TLR7 of the present invention can also be obtained using DNA immunization, a method in which an antigen-expressing plasmid is transfected into an animal such as a mouse or rat to express the antigen in the animal, thereby inducing immunity against the antigen.

[0048] Gene transfer methods include direct injection of plasmids into muscles, intravenous injection of complexes of plasmids with liposomes or polyethyleneimine, methods using viral vectors, injection of gold particles with plasmids attached using a gene gun, and hydrodynamic methods in which a large amount of plasmid solution is rapidly injected intravenously.

[0049] Regarding gene transfer by intramuscular injection of an expression plasmid, a technique called in vivo electroporation, in which the plasmid is injected intramuscularly and then electroporated at the same site, is known as a method for improving the expression level (Aihara H, Miyazaki J. Nat Biotechnol. 1998 Sep;16(9):867-70 or Mir LM, Bureau MF, Gehl J, Rangara R, Rouy D, Caillaud JM, Delaere P, Branellec D, Schwartz B, Scherman D. Proc Natl Acad Sci U S A. 1999 Apr 13;96(8):4262-7.) This method further improves expression levels by treating the muscle with hyaluronidase before intramuscular injection of the plasmid (McMahon JM1, Signori E, Wells KE, Fazio VM, Wells DJ. Gene Ther. 2001 Aug;8(16):1264-70).

[0050] Also, known methods (e.g., Kohler and Milstein, Nature (1975) 256, pp. 495-497; Kennet, R. ed., Monoclonal Antibodies, pp. 365-367, Plenum Press, NY (1980)), antibody-producing cells that produce antibodies against TLR7 are fused with myeloma cells to establish hybridomas and obtain monoclonal antibodies. Specific examples of such methods are described in International Publication Nos. WO09 / 48072 (published April 16, 2009) and WO10 / 117011 (published October 14, 2010).

[0051] Examples of mouse anti-human TLR7 antibodies established in this manner include the AT01 antibody, the NB7 antibody, and the FAN2 antibody.

[0052] The amino acid sequence of the heavy chain variable region of the AT01 antibody is shown in SEQ ID NO: 5 in the Sequence Listing, and the polynucleotide sequence encoding said sequence is shown in SEQ ID NO: 6 in the Sequence Listing. The amino acid sequence of the heavy chain variable region of the NB7 antibody is shown in SEQ ID NO: 7 in the Sequence Listing, and the polynucleotide sequence encoding said sequence is shown in SEQ ID NO: 8 in the Sequence Listing. The amino acid sequence of the heavy chain variable region of the FAN2 antibody is shown in SEQ ID NO: 9 in the Sequence Listing, and the polynucleotide sequence encoding said sequence is shown in SEQ ID NO: 10 in the Sequence Listing.

[0053] In addition, the amino acid sequence of the light chain variable region of the AT01 antibody is shown in SEQ ID NO: 11 in the Sequence Listing, and the polynucleotide sequence encoding said sequence is shown in SEQ ID NO: 12 in the Sequence Listing; the amino acid sequence of the light chain variable region of the NB7 antibody is shown in SEQ ID NO: 13 in the Sequence Listing, and the polynucleotide sequence encoding said sequence is shown in SEQ ID NO: 14 in the Sequence Listing; and the amino acid sequence of the light chain variable region of the FAN2 antibody is shown in SEQ ID NO: 15 in the Sequence Listing, and the polynucleotide sequence encoding said sequence is shown in SEQ ID NO: 16 in the Sequence Listing.

[0054] The heavy chain variable region of the AT01 antibody has a CDRH1 consisting of the amino acid sequence (GYTFTNNWLH) shown in SEQ ID NO: 17, a CDRH2 consisting of the amino acid sequence (DIYPSNGRTN) shown in SEQ ID NO: 18, and a CDRH3 consisting of the amino acid sequence (ERGYFDY) shown in SEQ ID NO: 19. The light chain variable region of this antibody has a CDRL1 consisting of the amino acid sequence (KASQDINKYIA) shown in SEQ ID NO: 20, a CDRL2 consisting of the amino acid sequence (YTSTLQP) shown in SEQ ID NO: 21, and a CDRL3 consisting of the amino acid sequence (LQYDYLLT) shown in SEQ ID NO: 22. The amino acid sequences of the above CDRs are also shown in Figure 24-1.

[0055] The heavy chain variable region of the NB7 antibody has a CDRH1 consisting of the amino acid sequence (GYSITSDYAWN) set forth in SEQ ID NO: 23, a CDRH2 consisting of the amino acid sequence (HISYRGNTN) set forth in SEQ ID NO: 24, and a CDRH3 consisting of the amino acid sequence (WNYYGYVDYAMDY) set forth in SEQ ID NO: 25. The light chain variable region of the antibody has a CDRL1 consisting of the amino acid sequence (RASQDISNYLN) set forth in SEQ ID NO: 26, a CDRL2 consisting of the amino acid sequence (YTSRLHS) set forth in SEQ ID NO: 27, and a CDRL3 consisting of the amino acid sequence (QQGDTFPT) set forth in SEQ ID NO: 28. The amino acid sequences of the above CDRs are also shown in Figure 24-2.

[0056] The heavy chain variable region of the FAN2 antibody has a CDRH1 consisting of the amino acid sequence (GFSLTGYGVN) set forth in SEQ ID NO: 29, a CDRH2 consisting of the amino acid sequence (MIWGDGSTD) set forth in SEQ ID NO: 30, and a CDRH3 consisting of the amino acid sequence (DKGYDGYYYAMDY) set forth in SEQ ID NO: 31. The light chain variable region of the antibody has a CDRL1 consisting of the amino acid sequence (RASENIYSYLA) set forth in SEQ ID NO: 32, a CDRL2 consisting of the amino acid sequence (DAKTLAE) set forth in SEQ ID NO: 33, and a CDRL3 consisting of the amino acid sequence (QHHYGIPYT) set forth in SEQ ID NO: 34. The amino acid sequences of the above CDRs are also shown in Figure 24-3.

[0057] In one embodiment, the antibody or antigen-binding fragment thereof of the present invention exhibits binding to human TLR7 similar to the heavy chain variable region and light chain variable region of the AT01 antibody, the NB7 antibody, or the FAN2 antibody. It may have competitive inhibitory activity with an antibody having the amino acid sequence of

[0058] In another embodiment, the antibody or antigen-binding fragment thereof may be an antibody or antigen-binding fragment thereof having CDRH1-3 of the AT01 antibody as the complementarity-determining region in the heavy chain and CDRL1-3 of the AT01 antibody as the complementarity-determining region in the light chain; an antibody or antigen-binding fragment thereof having CDRH1-3 of the NB7 antibody as the complementarity-determining region in the heavy chain and CDRL1-3 of the NB7 antibody as the complementarity-determining region in the light chain; or an antibody or antigen-binding fragment thereof having CDRH1-3 of the FAN2 antibody as the complementarity-determining region in the heavy chain and CDRL1-3 of the FAN2 antibody as the complementarity-determining region in the light chain. In a preferred embodiment, the antibody or antigen-binding fragment thereof may be an antibody or antigen-binding fragment thereof having CDRH1-3 of the AT01 antibody as the complementarity-determining region in the heavy chain and CDRL1-3 of the AT01 antibody as the complementarity-determining region in the light chain.

[0059] In yet another embodiment, the antibody or antigen-binding fragment thereof of the present invention may be an antibody having the heavy chain variable region and light chain variable region sequences of the AT01 antibody, NB7 antibody, or FAN2 antibody.

[0060] The antibodies of the present invention include not only the above-mentioned monoclonal antibodies against human TLR7, but also genetically engineered antibodies that have been artificially modified for the purpose of reducing heterologous antigenicity to humans, such as chimeric antibodies, humanized antibodies, and human antibodies. These antibodies can be produced using known methods.

[0061] Chimeric antibodies include antibodies in which the variable and constant regions are heterologous, such as chimeric antibodies in which the variable region of a mouse- or rat-derived antibody is fused to a human-derived constant region (see Proc. Natl. Acad. Sci. USA, 81, 6851-6855, (1984)). Chimeric antibodies derived from a mouse anti-human TLR7 antibody (AT01 antibody) include antibodies consisting of a heavy chain with a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 5 and a light chain with a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 11. An example of such a chimeric antibody derived from the AT01 antibody is an antibody consisting of a heavy chain consisting of the amino acid sequence of positions 20 to 465 in SEQ ID NO: 35 and a light chain consisting of the amino acid sequence of positions 21 to 233 in SEQ ID NO: 36. Herein, the above antibody is referred to as "cAT01" or "cAT01 antibody."

[0062] Furthermore, chimeric antibodies derived from mouse anti-human TLR7 antibodies (NB7 antibodies) include antibodies consisting of a heavy chain having a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 7 and a light chain having a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 13. An example of such a chimeric antibody derived from the NB7 antibody is an antibody consisting of a heavy chain consisting of the amino acid sequence from positions 20 to 471 of SEQ ID NO: 37 and a light chain consisting of the amino acid sequence from positions 21 to 233 of SEQ ID NO: 38. Herein, such antibodies are referred to as "cNB7" or "cNB7 antibodies."

[0063] Furthermore, chimeric antibodies derived from mouse anti-human TLR7 antibodies (FAN2 antibodies) include antibodies consisting of a heavy chain having a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 9 and a light chain having a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 15. An example of such a chimeric antibody derived from the FAN2 antibody is an antibody consisting of a heavy chain consisting of the amino acid sequence from positions 20 to 470 of SEQ ID NO: 39 and a light chain consisting of the amino acid sequence from positions 21 to 234 of SEQ ID NO: 40. In this specification, such antibodies are referred to as "cFAN2" or "cFAN2 antibodies."

[0064] The sequence of the chimeric antibody against human TLR7 is used to reduce heterologous antigenicity to humans. Humanized antibodies, which are recombinant antibodies, can be prepared by artificially modifying the CDRs of the humanized antibodies, for example, to enhance their reactivity to the target protein. The antibodies of the present invention also include antibodies in which the CDRs of the humanized antibodies have been modified. These antibodies can be produced using known methods.

[0065] Examples of humanized antibodies include antibodies in which only the CDRs have been incorporated into a human-derived antibody (see Nature (1986) 321, pp. 522-525), and antibodies in which not only the CDR sequences but also some framework amino acid residues have been transplanted into a human antibody (International Publication No. WO90 / 07861).

[0066] For example, humanized antibodies derived from the cAT01 antibody and the cNB7 antibody retain all six CDR sequences derived from cAT01 or cNB7, and have the activity of inhibiting the function of human TLR7.

[0067] Suitable examples of the humanized antibody include a humanized antibody derived from the cAT01 antibody (huAT01 antibody): an antibody comprising a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 41 and a light chain comprising a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 43; an antibody comprising a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 41 and a light chain comprising a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 44; an antibody comprising a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 42 and a light chain comprising a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 43; an antibody comprising a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 42 and a light chain comprising a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 44; Examples include:

[0068] Furthermore, by combining sequences that show high homology with the amino acid sequences of the heavy chain variable regions and the light chain variable regions of the above-mentioned huAT01 antibody, it is possible to select an antibody with activity equivalent to that of the antibody. Such homology is generally 80% or more, preferably 90% or more, more preferably 95% or more, and most preferably 99% or more (however, each CDR is identical to the amino acid sequences of each of the above-mentioned antibodies). The amino acid sequence of the heavy chain variable region or the light chain variable region (wherein each C 1 to several (e.g., 2, 3, 4, 5, 6, 7, 8, It is also possible to select antibodies having activity equivalent to that of each of the above antibodies by combining amino acid sequences in which 9 or 10 amino acid residues have been substituted, deleted, or added.

[0069] A more suitable example is: an antibody comprising a heavy chain consisting of the amino acid sequence from positions 20 to 465 in the amino acid sequence set forth in SEQ ID NO: 45 and a light chain consisting of the amino acid sequence from positions 21 to 233 in the amino acid sequence set forth in SEQ ID NO: 48; an antibody comprising a heavy chain consisting of the amino acid sequence from positions 20 to 465 in the amino acid sequence set forth in SEQ ID NO: 45 and a light chain consisting of the amino acid sequence from positions 21 to 233 in the amino acid sequence set forth in SEQ ID NO: 49; an antibody comprising a heavy chain consisting of the amino acid sequence from positions 20 to 465 in the amino acid sequence set forth in SEQ ID NO: 46 and a light chain consisting of the amino acid sequence from positions 21 to 233 in the amino acid sequence set forth in SEQ ID NO: 48; an antibody comprising a heavy chain consisting of the amino acid sequence from positions 20 to 465 in the amino acid sequence set forth in SEQ ID NO: 46 and a light chain consisting of the amino acid sequence from positions 21 to 233 in the amino acid sequence set forth in SEQ ID NO: 49; An antibody comprising a heavy chain consisting of the amino acid sequence from positions 20 to 462 in the amino acid sequence set forth in SEQ ID NO: 47 and a light chain consisting of the amino acid sequence from positions 21 to 233 in the amino acid sequence set forth in SEQ ID NO: 48; or an antibody comprising a heavy chain consisting of the amino acid sequence from positions 20 to 462 in the amino acid sequence set forth in SEQ ID NO: 47 and a light chain consisting of the amino acid sequence from positions 21 to 233 in the amino acid sequence set forth in SEQ ID NO: 49; Examples include:

[0070] Furthermore, examples of humanized antibodies (huNB7 antibodies) derived from the cNB7 antibody include antibodies consisting of a heavy chain containing a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 50 and a light chain containing a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 51.

[0071] Furthermore, by combining sequences that show high homology with the amino acid sequences of the heavy chain variable region and the light chain variable region of the above-mentioned huNB7 antibody, it is possible to select an antibody having activity equivalent to that of the antibody. Such homology is generally 80% or more, preferably 90% or more, more preferably 95% or more, and most preferably 99% or more (however, each CDR is identical to each of the above-mentioned antibodies). In addition, the amino acid sequence of the heavy chain variable region or the light chain variable region (provided that the 1 to several (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or It is also possible to select antibodies having activity equivalent to that of the above antibodies by combining amino acid sequences in which amino acid residues (10 or 10) have been substituted, deleted, or added.

[0072] A more suitable example is: An antibody comprising a heavy chain consisting of the amino acid sequence from positions 20 to 471 in the amino acid sequence set forth in SEQ ID NO: 52 and a light chain consisting of the amino acid sequence from positions 21 to 233 in the amino acid sequence set forth in SEQ ID NO: 53; or an antibody comprising a heavy chain consisting of the amino acid sequence from positions 20 to 468 in the amino acid sequence set forth in SEQ ID NO: 54 and a light chain consisting of the amino acid sequence from positions 21 to 233 in the amino acid sequence set forth in SEQ ID NO: 53; Examples include:

[0073] The antibody of the present invention may be one in which the binding ability to human TLR7 is altered by further introducing mutations into the above-mentioned humanized antibody. This technique is called affinity maturation, and a specific example is ribosome display. Ribosome display is a method for isolating the gene sequence of a protein that binds to a target molecule using a ternary complex in which a protein and its genetic information, mRNA, are bound via ribosomes (Stafford R.L. et al., Protein Eng. Des. Sel. 2014(4):97-109).

[0074] To avoid cytotoxicity to normal human TLR7-expressing cells, it is desirable for the antibody to have low effector activity. It is known that effector activity differs depending on the antibody subclass. IgG4 has low ADCC and CDC activity, while IgG2 has CDC activity but low ADCC activity. Based on this characteristic, it is possible to prepare antibodies with reduced ADCC and CDC activity by substituting the IgG1 constant region with the IgG2 or IgG4 constant region. Furthermore, by substituting a portion of the sequence of the IgG1 constant region with that of IgG2 or IgG4, it is possible to prepare IgG1 antibodies with reduced ADCC and CDC activity. For example, according to Marjan Hezareh et al., Journal of Virology, 75(24):12161-12168 (2001), substituting the leucine residues at positions 234 and 235 of IgG1 (numbers are EU index according to Kabat et al.) with alanine residues, respectively, results in a decrease in ADCC and CDC activity. It has been shown that DCC and CDC activity are reduced.

[0075] The present invention also encompasses modified antibodies or antigen-binding fragments thereof. The term "modified" refers to antibodies or antigen-binding fragments thereof that have been chemically or biologically modified. Chemical modifications include attachment of chemical moieties to the amino acid backbone, chemical modifications of N- or O-linked carbohydrate chains, and the like. Biological modifications include post-translational modifications (e.g., N- or O-linked glycosylation, N- or C-terminal processing, deamidation, aspartic acid isomerization, and methionine oxidation), and those in which a methionine residue has been added to the N-terminus by expression in a prokaryotic host cell. Also encompassed within the meaning of such modifications are those labeled to enable detection or isolation of the antibodies or antigens of the present invention, such as enzyme-labeled, fluorescent-labeled, and affinity-labeled antibodies. Such modified antibodies or antigen-binding fragments of the present invention are useful for improving the stability and blood retention of the original antibodies of the present invention, reducing antigenicity, and detecting or isolating such antibodies or antigens.

[0076] It is known that the lysine residue at the carboxyl terminus of the heavy chain of an antibody produced in cultured mammalian cells is deleted (Journal of Chromatography A, 705: 129-134 (1995)), and it is also known that two amino acid residues, glycine and lysine, are deleted at the carboxyl terminus of the heavy chain, and a proline residue newly positioned at the carboxyl terminus is amidated (Analytical Biochemistry, 360: 75-83 (2007)).

[0077] However, these deletions and modifications of the heavy chain sequence do not affect the antigen-binding ability and effector functions (such as complement activation and antibody-dependent cellular cytotoxicity) of the antibody.

[0078] Therefore, modified antibodies and antigen-binding fragments of the antibodies of the present invention include those in which one or two amino acids are deleted at the carboxyl terminus of the heavy chain, and those in which the deletion is amidated (for example, a heavy chain in which the proline residue at the carboxyl terminus is amidated), etc. However, as long as the antigen-binding ability and effector function are maintained, the deletions at the carboxyl terminus of the heavy chain of the antibody of the present invention are not limited to the above types.

[0079] The two heavy chains constituting the antibody of the present invention may be a combination of any one heavy chain selected from the group consisting of full-length and the above-mentioned deletion variants, or a combination of any two heavy chains. The quantitative ratio of each deletion variant may be affected by the type of cultured mammalian cells producing the antibody of the present invention and the culture conditions, but the main component of the antibody of the present invention can be an antibody in which one amino acid residue is deleted at the carboxyl terminus in both of the two heavy chains. Specifically, heavy chains consisting of amino acid sequences in which one or two amino acids are deleted at the carboxyl terminus of the heavy chain sequences shown in the amino acid sequence of positions 20 to 465 in SEQ ID NO: 35, the amino acid sequence of positions 20 to 471 in SEQ ID NO: 37, the amino acid sequence of positions 20 to 470 in SEQ ID NO: 39, the amino acid sequence of positions 20 to 465 in SEQ ID NO: 45, the amino acid sequence of positions 20 to 465 in SEQ ID NO: 46, the amino acid sequence of positions 20 to 462 in SEQ ID NO: 47, the amino acid sequence of positions 20 to 471 in SEQ ID NO: 52, and the amino acid sequence of positions 20 to 468 in SEQ ID NO: 54 can also be used in the antibodies of the present invention.

[0080] Antibodies obtained by the above methods can be evaluated for their binding to antigens, and suitable antibodies can be selected. Another example of an index for comparing antibody properties is antibody stability. Differential scanning calorimetry (DSC) is a method that can quickly and accurately measure the thermal denaturation midpoint (Tm), which is a good index of the relative structural stability of proteins. Differences in thermal stability can be compared by measuring Tm values ​​using DSC and comparing the values. It is known that the storage stability of an antibody correlates to a certain extent with its thermal stability (Lori Burton, et. al., Pharmaceutical Development and Technology (2007) 12, pp. 265-273), and suitable antibodies can be selected using thermal stability as an indicator. Other indicators for antibody selection include high yield in appropriate host cells and low aggregation in aqueous solution. For example, an antibody with the highest yield does not necessarily exhibit the highest thermal stability, so it is necessary to comprehensively assess the above-mentioned indicators to select an antibody that is most suitable for administration to humans.

[0081] Also known is a method for obtaining single-chain immunoglobulin by linking the full-length sequences of the heavy and light chains of an antibody using an appropriate linker (Lee, H.S., et al., Molecular Immunology (1999) 36, pp. 61-71; Shirrmann, T., et al., mAbs (2010), 2, (1) pp. 1-4). By dimerizing, such single-chain immunoglobulins can retain a structure and activity similar to that of antibodies, which are originally tetrameric. Furthermore, the antibodies of the present invention may have a single heavy-chain variable region and no light-chain sequence. Such antibodies are called single domain antibodies (sdAb) or nanobodies, and have been observed in camels and llamas, where they have been reported to retain their antigen-binding ability (Muyldemans S. et al., Protein Eng. (1994) 7(9), 1129-35, Hamers-Casterman C. et al., Nature (1993) 363(6428)446-8). The above-mentioned antibodies can also be considered as a type of antigen-binding fragment of the antibody of the present invention.

[0082] Furthermore, antibody-dependent cellular cytotoxicity can be enhanced by modulating the glycosylation of the antibody of the present invention. Techniques for modulating the glycosylation of antibodies are known, including, but not limited to, those disclosed in WO99 / 54342, WO00 / 61739, WO02 / 31140, WO2007 / 133855, and WO2013 / 120066.

[0083] When the antibody gene is isolated and then introduced into an appropriate host to produce the antibody, a combination of an appropriate host and an expression vector can be used.

[0084] Specific examples of antibody genes include combinations of genes encoding the heavy chain and light chain sequences of the antibodies described herein. More specifically, antibody genes include, for example, a combination of a polynucleotide having a polynucleotide sequence encoding the antibody or antigen-binding fragment thereof of the present invention, a polynucleotide having a polynucleotide sequence encoding the amino acid sequence of the heavy chain variable region of the antibody or antigen-binding fragment thereof of the present invention, and a polynucleotide having a polynucleotide sequence encoding the amino acid sequence of the light chain variable region. The heavy chain sequence gene constituting the antibody gene also includes a polynucleotide having a polynucleotide sequence contained in a polynucleotide that hybridizes under stringent conditions with a polynucleotide consisting of a polynucleotide sequence complementary to the polynucleotide sequence encoding the heavy chain variable region. Furthermore, the light chain sequence gene constituting the antibody gene also includes a polynucleotide having a polynucleotide sequence contained in a polynucleotide that hybridizes under stringent conditions with a polynucleotide consisting of a polynucleotide sequence complementary to the polynucleotide sequence encoding the light chain variable region.

[0085] When transforming host cells, the heavy and light chain sequence genes are expressed in the same expression vector. The gene may be inserted into a vector or into a separate expression vector. When eukaryotic cells are used as hosts, animal cells, plant cells, and eukaryotic microorganisms can be used. Examples of animal cells include (1) mammalian cells, such as monkey COS cells (Gluzman, Y. Cell (1981) 23, pp. 175-182, ATCC CRL-1650), mouse fibroblast NIH3T3 (ATCC No. CRL-1658), and dihydrofolate reductase-deficient strains of Chinese hamster ovary cells (CHO cells, ATCC CCL-61) (Urlaub, G. and Chasin, LA Proc. Natl. Acad. Sci. USA (1980) 77, pp. 4126-4220). Prokaryotic cells include, for example, Escherichia coli and Bacillus subtilis. Antibodies can be obtained by transforming these cells with the desired antibody gene and culturing the transformed cells in vitro. In the above culture methods, the yield may vary depending on the antibody sequence, and it is possible to select antibodies with equivalent binding activity that can be easily produced as a pharmaceutical using the yield as an indicator.

[0086] The isotype of the antibodies of the present invention is not limited, and examples include IgG (IgG1, IgG2, IgG3, IgG4), IgM, IgA (IgA1, IgA2), IgD, and IgE, but IgG or IgM is preferred, and IgG1 or IgG4 is more preferred.

[0087] The antibody of the present invention may also be an antigen-binding fragment of the antibody having the antigen-binding site thereof, or a modified version thereof. The antibody fragment can be obtained by treating the antibody with protease such as papain or pepsin, or by modifying the antibody gene by genetic engineering techniques and expressing it in appropriate cultured cells. Among such antibody fragments, a fragment that retains all or part of the functions of the full-length antibody molecule can be called an antigen-binding fragment of the antibody.

[0088] Antibody functions generally include antigen-binding activity, activity to neutralize antigen activity, activity to enhance antigen activity, antibody-dependent cellular cytotoxicity, complement-dependent cytotoxicity, and complement-dependent cellular cytotoxicity. The function retained by the antigen-binding fragment of the antibody of the present invention is binding activity to human TLR7, preferably activity to inhibit the function of human TLR7, and more preferably activity to suppress the production of IL-6 and / or type I interferon by TLR7-expressing cells.

[0089] For example, antibody fragments include Fab, F(ab'), Fv, single-chain Fv (scFv) in which heavy and light chain Fvs are linked via an appropriate linker, diabodies, linear antibodies, and multispecific antibodies formed from antibody fragments. Also included in antibody fragments is Fab', which is a monovalent fragment of the variable region of an antibody obtained by treating F(ab') under reducing conditions.

[0090] Furthermore, the antibodies of the present invention may be multispecific antibodies having specificity for at least two different antigens. Although such molecules typically bind to two antigens (i.e., bispecific antibodies), the term "multispecific antibodies" as used herein encompasses antibodies having specificity for more than two (e.g., three) antigens.

[0091] The multispecific antibodies of the present invention may be full-length antibodies or fragments of such antibodies (for example, F(ab')2 bispecific antibodies). Bispecific antibodies can be prepared by combining the heavy and light chains (HL pairs) of two types of antibodies, or by fusing hybridomas producing different monoclonal antibodies to prepare bispecific antibody-producing fusion cells. It can be produced (Millstein et al., Nature (1983) 305, pp. 537-539).

[0092] The antibody of the present invention may be a single-chain antibody (also referred to as scFv). Single-chain antibodies can be obtained by linking the heavy chain variable region and light chain variable region of an antibody with a polypeptide linker (Pluckthun, The Pharmacology of Monoclonal Antibodies, 113 (Rosenberg and Moore, eds., Springer Verlag, New York, pp. 269-315 (1994)); Nature Biotechnology (2005), 23, pp. 1126-1136). Alternatively, a BiscFv fragment prepared by linking two scFvs with a polypeptide linker can be used as a bispecific antibody.

[0093] Methods for producing single-chain antibodies are well known in the art (see, for example, U.S. Pat. Nos. 4,946,778, 5,260,203, 5,091,513, and 5,455,030). In this scFv, the heavy chain variable region and the light chain variable region are linked via a linker that does not form a conjugate, preferably a polypeptide linker (Huston, J. et al., Proc. Natl. Acad. Sci. USA (1988), 85, pp. 5879-5883). The heavy chain variable region and the light chain variable region in the scFv may be derived from the same antibody or from different antibodies. The polypeptide linker linking the variable regions may be, for example, any single-chain peptide consisting of 12 to 19 residues.

[0094] DNA encoding an scFv can be obtained by using the DNA encoding the heavy chain or heavy chain variable region of the antibody and the DNA encoding the light chain or light chain variable region, respectively, with the entire sequence or a DNA portion encoding a desired amino acid sequence as a template, amplifying the DNA by PCR using a primer pair that specifies both ends of the template, and then further amplifying DNA encoding a polypeptide linker portion and a primer pair that specifies the ends of the template so that the DNA can be linked to the heavy chain and light chain, respectively.

[0095] Furthermore, once DNA encoding scFv is prepared, expression vectors containing the DNA and hosts transformed with the expression vectors can be obtained according to standard methods, and scFv can be obtained using the hosts according to standard methods. These antibody fragments can be produced by obtaining and expressing the genes in the same manner as described above.

[0096] The antibodies of the present invention may be multimerized to increase their affinity for an antigen. The antibody to be multimerized may be a single type of antibody, or multiple antibodies that recognize multiple epitopes of the same antigen. Methods for multimerizing antibodies include binding of an IgG CH3 domain to two scFvs, binding to streptavidin, and introduction of a helix-turn-helix motif.

[0097] The antibody of the present invention may be a polyclonal antibody, which is a mixture of multiple anti-human TLR7 antibodies with different amino acid sequences. An example of a polyclonal antibody is a mixture of multiple antibodies with different CDRs. Such polyclonal antibodies can be obtained by culturing a mixture of cells producing different antibodies and purifying the culture (see WO2004 / 061104).

[0098] As a modified antibody, an antibody conjugated with various molecules such as polyethylene glycol (PEG) can also be used.

[0099] The antibodies of the present invention may also be conjugated with other drugs (immunoconjugates). Examples of such antibodies include antibodies conjugated with radioactive substances or pharmacological compounds (Nature Biotechnology (2005) 23, pp. 1137-1146).

[0100] The obtained antibody can be purified to homogeneity. Antibodies can be separated and purified using methods commonly used for proteins. For example, antibodies can be separated and purified by appropriately selecting and combining methods such as column chromatography, filter filtration, ultrafiltration, salting out, dialysis, preparative polyacrylamide gel electrophoresis, and isoelectric focusing (Strategies for Protein Purification and Characterization: A Laboratory Course Manual, Daniel R. Marshak et al. eds., Cold Spring, 1999). Antibodies: A Laboratory Manual. Ed Harlow and David Lane, Cold Spring Harbor Laboratory (1988)).

[0101] Examples of chromatography include affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration chromatography, reversed-phase chromatography, and adsorption chromatography. These chromatographies can be performed using liquid chromatography such as HPLC or FPLC. Examples of columns used in affinity chromatography include Protein A columns and Protein G columns. For example, columns using Protein A columns include Hyper D, POROS, and Sepharose FF (Pharmacia). It is also possible to purify antibodies by utilizing their binding to antigens using a carrier on which an antigen is immobilized.

[0102] 3. Medicine containing anti-human TLR7 antibody Antibodies that inhibit the function of human TLR7 can be obtained from the anti-human TLR7 antibodies obtained by the method described above in Section "2. Anti-TLR7 antibodies and their production." These antibodies that inhibit the function of human TLR7 can inhibit the biological activity of human TLR7 in vivo, i.e., the activation of human TLR7-expressing cells, such as blood cells, by human TLR7 ligands, and can therefore be used as pharmaceuticals to treat and / or prevent diseases caused by the function of human TLR7.

[0103] Diseases and conditions caused by the function of human TLR7 include immune-inflammatory diseases, allergic diseases, infectious diseases, and cancer.

[0104] Examples of immune-inflammatory diseases include connective tissue and musculoskeletal systems (systemic lupus erythematosus, rheumatoid arthritis, juvenile idiopathic arthritis, adult Still's disease, ankylosing spondylitis, systemic sclerosis, polymyositis, dermatomyositis, psoriatic arthritis, osteoarthritis, mixed connective tissue disease, muscular dystrophy, etc.), blood systems (autoimmune hemolytic anemia, aplastic anemia, idiopathic thrombocytopenic purpura, etc.), gastrointestinal systems (Crohn's disease, ulcerative colitis, ileitis, etc.), hepatobiliary pancreatic and endocrine systems (autoimmune hepatitis, viral hepatitis, alcoholic hepatitis, non-alcoholic steatohepatitis, primary sclerosing bile duct disease, etc.). inflammation, primary biliary cirrhosis, Sjögren's syndrome, type 1 diabetes, autoimmune thyroiditis, Graves' disease, Hashimoto's disease, etc.), respiratory system (chronic obstructive pulmonary disease, cystic fibrosis, interstitial pneumonia, etc.), cranial nervous system (multiple sclerosis, myasthenia gravis, meningitis, encephalomyelitis, autoimmune encephalitis, etc.), visual system (uveitis, trachoma, endophthalmitis, etc.), cardiovascular system (vasculitis syndrome, granulomatosis with polyangiitis, Wegener's granulomatosis, myocarditis, ischemic heart disease, atherosclerosis, etc.), skin and epidermal system (psoriasis, pemphigus, vitiligo, contact dermatitis, eczema, etc.), renal system (glomerulonephritis, diabetes mellitus) These include diseases related to the endocrine system (type 1 diabetes, autoimmune thyroiditis, Graves' disease, Hashimoto's disease, etc.), and systemic inflammation (Behçet's disease, antiphospholipid syndrome, IgG4-related disease, sepsis, bleeding, hypersensitivity reactions, transplant rejection, shock symptoms caused by cancer chemotherapy, etc.).

[0105] Examples of allergic diseases include atopic dermatitis, asthma, anaphylaxis, anaphylactoid reactions, food allergies, rhinitis, otitis media, drug reactions, insect sting reactions, plant reactions, latex allergies, conjunctivitis, and hives.

[0106] Examples of infectious diseases include diseases caused by infection with viruses (single-stranded RNA viruses, double-stranded RNA viruses, single-stranded DNA viruses, double-stranded DNA viruses, etc.), bacteria (Gram-negative bacteria, Gram-positive bacteria, acid-fast bacteria, actinomycetes, spirochetes, spiral bacteria, rickettsia, chlamydia, mycoplasma, etc.), fungi (tinea fungus, Candida, Cryptococcus, Aspergillus, Pneumocystis, Malassezia, etc.), parasites (filaria, flukes, tapeworms, flukes, echinococcus, entamoeba histolytica, fleas, lice, mites, roundworms, pinworms, etc.), etc.

[0107] Examples of cancer treatments include lymphoma, leukemia, breast cancer, lung cancer, and skin cancer.

[0108] Examples of the anti-human TLR7 antibody as a pharmaceutical include chimeric antibodies, humanized antibodies, and CDR-modified antibodies thereof prepared from the AT01 antibody and / or the NB7 antibody.

[0109] The in vitro inhibitory activity of anti-human TLR7 antibodies against human TLR7 function can be measured by their ability to suppress the activation of blood cells expressing human TLR7. For example, by adding various concentrations of anti-human TLR7 antibodies to human PBMCs, the inhibitory activity against CL264-stimulated IL-6 release from human PBMCs can be measured.

[0110] It is widely recognized and experimentally proven that human TLR7 ligand levels are elevated, human TLR7 expression is enhanced, or human TLR7 stimulation is involved in various immune-inflammatory diseases, including systemic lupus erythematosus. Human TLR7 ligands act on human TLR7-expressing cells to produce inflammatory cytokines, such as interleukin 6 (IL-6), leading to inflammatory responses and enhanced antibody production. This activation leads to the release of type I interferons, which in turn trigger a systemic immune-inflammatory response, resulting in the onset and exacerbation of autoimmune diseases, including systemic lupus erythematosus. Therefore, inhibiting the production of these cytokines, which are dependent on human TLR7 stimulation, could lead to the prevention and treatment of systemic lupus erythematosus and other conditions. Furthermore, the inhibitory activity of human TLR7 antibodies could be used as a marker to assess their therapeutic utility.

[0111] Preferred antibodies of the present invention include antibodies or antigen-binding fragments of the antibodies that specifically bind to human TLR7 having the amino acid sequence set forth in SEQ ID NO: 2 or 4 and inhibit the amount of IL-6 released by CL264-treated human PBMCs in a concentration-dependent manner.

[0112] Furthermore, the therapeutic or preventive effects of anti-human TLR7 antibodies against various diseases can be confirmed in an in vivo or ex vivo evaluation system in which the antibody is administered to monkeys that are cross-reactive with the anti-human TLR7 antibody, or in the following in vivo evaluation system in which the anti-human TLR7 antibody is administered to human TLR7 transgenic and mouse TLR7 knockout mice.

[0113] The effect on inflammatory cytokine production will be evaluated by comparing the cytokine production capacity in peripheral blood between the anti-TLR7 antibody group and the non-administered group in inflammation induced by intraperitoneal or intravenous administration of TLR7 ligand to mice.

[0114] The effect on atopic dermatitis will be evaluated by applying mite antigen cream to the ear or back 3 to 6 times every 3 days to 2 weeks, applying haptens to the ear, abdomen, or back daily to once a week, administering pruritus-inducing substances into the ear skin or subcutaneously into the back or subarachnoid space, or using NC / Nga mice, which are naturally atopic mice.The behaviors caused by atopic dermatitis will be induced or developed naturally by these methods, and will be quantified by measuring the number of scratches using a magnet attached to the soles of both mice and an itch-scratching behavior measuring device, and by examining the pathological characteristics of the skin, peripheral blood, and spinal cord tissue, and comparing them between an anti-human TLR7 antibody group and a non-administered group.

[0115] The effect on psoriasis will be evaluated by applying imiquimod or R848 to the ears and shaved backs, or by administering cytokines such as IL-23 into the ear skin of mice, inducing psoriasis-like dermatitis. The effects will be quantified by measuring the weight and thickness of the inflamed area, the myeloperoxidase activity of neutrophils infiltrating into the area, flow cytometry analysis of infiltrated cells, genetic analysis, cytokine concentration, etc., and comparing the results between the anti-human TLR7 antibody group and the non-treated group.

[0116] The effect on arthritis will be evaluated by injecting an emulsified mixture of bovine type II collagen solution and Freund's complete adjuvant intradermally into the base of the tail of mice, and then 2-3 weeks later administering an emulsified mixture of bovine type II collagen solution and Freund's incomplete adjuvant, or by administering an anti-bovine type II collagen antibody and a TLR ligand, and then scoring joint swelling, measuring footpad thickness, and measuring antibody, cytokine, and blood biomarker concentrations in blood and tissues, as well as the proliferation activity, cytokine production ability, and surface antigens of cells obtained from peripheral blood, spleen, lymph nodes, bone marrow, and joints, between an anti-human TLR7 antibody group and a non-administered group.

[0117] The effects on colitis will be evaluated by inducing colitis in mice by administering trinitrobenzenesulfonic acid into the intestines of mice fasted for 24 hours, allowing them to drink 1-10% dextran sodium sulfate aqueous solution ad libitum from a water bottle for 4 days to 2 weeks, or by transferring purified CD4+CD25-CD45RBhi T cells collected from the lymph nodes and spleen of human TLR7 transgenic mice into the abdominal cavity of Rag2-deficient mice.The effects on colitis will be evaluated by comparing body weight during the observation period, intestinal thickening, number and size of polyps, and histopathological characteristics at necropsy after the study, antibody, cytokine, and blood biomarker concentrations in blood and tissues, and the proliferative activity, cytokine production ability, and surface antigens of cells obtained from the intestine, peripheral blood, thymus, spleen, lymph nodes, bone marrow, and Peyer's patches between the anti-human TLR7 antibody group and the control group.

[0118] The effect on systemic lupus erythematosus will be evaluated by comparing the antibody, cytokine, and blood biomarker concentrations in blood and tissues collected over time from spontaneously developing NZB / WF1 mice, MRL / lpr mice, and BXSB mice, as well as antibody titers and biomarker concentrations in urine, and symptoms induced by transferring cells prepared from organs such as spleen and lymph nodes collected after the test into untreated mice between the anti-human TLR7 antibody group and the untreated group.

[0119] The effect on hepatitis will be evaluated by inducing hepatitis in mice by administering D-galactosamine alone or in combination with lipopolysaccharide intraperitoneally, or by administering concanavalin A alone into the tail vein, and comparing the AST and ALT concentrations, cytokine concentrations, and histopathological state of liver lesions in blood collected 1 hour to 1 week after administration of the inflammatory substance between the anti-human TLR7 antibody group and the non-administration group.

[0120] The antibodies thus obtained that inhibit the biological activity of human TLR7 are useful as medicines, particularly for the treatment of immune inflammation including systemic lupus erythematosus. connection The antibodies are useful as antibodies for preventing or treating diseases, allergic diseases, infectious diseases, cancer, and the like.

[0121] For example, anti-human TLR7 antibodies can be administered alone or in combination with at least one other therapeutic agent for the treatment or prevention of immune inflammation-related diseases, allergic diseases, infectious diseases, or cancer. Other therapeutic agents that can be administered in combination with anti-human TLR7 antibodies include, but are not limited to, corticosteroids, nonsteroidal anti-inflammatory drugs, nucleic acid antimetabolites, nucleic acid synthesis inhibitors, antifolates, calcineurin inhibitors, antimalarials, antithymocyte globulins, biologics targeting cell surface antigens, or biologics targeting cytokine interferons or cytokine interferon receptors.

[0122] Specific examples of the above therapeutic agents include methylprednisolone as an adrenocortical steroid, loxoprofen sodium hydrate, diclofenac sodium, indomethacin, and acetyl salicylic acid as nonsteroidal anti-inflammatory drugs, and mycophthamic acid as a nucleic acid antimetabolite. Examples include enolate mofetil, a nucleic acid synthesis inhibitor such as cyclophosphamide, a calcineurin inhibitor such as cyclosporin and taclorims, an antimalarial drug such as hydroxychloroquine, a folate metabolic antagonist such as methotrexate, antithymocyte globulins such as zetbulin, lymphoglobuline, and thymoglobulin, biological agents that target cell surface antigens such as alemtuzumab, rituximab, and abatacept, and biological agents that target cytokines / interferons or cytokine / interferon receptors such as infliximab, etanercept, adalimumab, tocilizumab, belimumab, and anifrolumab.

[0123] Depending on the state of the immune inflammatory disease, allergic disease, infectious disease, or cancer and the degree of treatment and / or prevention desired, two, three, or more types of other therapeutic agents can be administered, or these other therapeutic agents can be administered simultaneously by being encapsulated in the same formulation. The other therapeutic agent and the anti-human TLR7 antibody can also be administered simultaneously by being encapsulated in the same formulation. Alternatively, the anti-human TLR7 antibody and the other therapeutic agent can be administered simultaneously by being encapsulated in separate formulations. Furthermore, the other therapeutic agent and the anti-human TLR7 antibody can be administered separately, one after the other. That is, the other therapeutic agent can be administered first, followed by a therapeutic agent containing an anti-human TLR7 antibody or an antigen-binding fragment thereof as an active ingredient, or a therapeutic agent containing an anti-human TLR7 antibody or an antigen-binding fragment thereof as an active ingredient, followed by the other therapeutic agent. When administered in gene therapy, the gene for the proteinaceous therapeutic agent and the gene for the anti-human TLR7 antibody can be inserted downstream of separate or the same promoter regions and can be introduced into separate or the same vectors.

[0124] By binding a therapeutic agent to an anti-human TLR7 antibody or a fragment thereof, a targeted drug conjugate can be produced as described in M.C. Garnet, "Targeted drug conjugates: principles and progress," Advanced Drug Delivery Reviews, (2001) 53, 171-216. For this purpose, in addition to antibody molecules, any antibody fragment can be used as long as it does not completely lose T cell recognition ability. Examples of such fragments include Fab, F(ab')2, and Fv. Similarly, in the present invention, The binding mode of the anti-human TLR7 antibody or a fragment thereof to the therapeutic agent can be determined by the method described in M.C. Garnet, "Targeted Drug Various forms are possible, such as those described in "Conjugates: Principles and Progress," Advanced Drug Delivery Reviews, (2001) 53, 171-216; G.T. Hermanson, "Bioconjugate Techniques," Academic Press, California (1996); and Putnam and J. Kopecek, "Polymer Conjugates with Anticancer Activity," Advances in Polymer Science (1995) 122, 55-123. Examples of such forms include those in which the anti-human TLR7 antibody and the therapeutic agent are chemically linked directly or via a spacer such as an oligopeptide, or those in which they are linked via a suitable drug carrier. Examples of drug carriers include liposomes and water-soluble polymers. More specifically, examples of the manner in which these drug carriers are mediated include a manner in which an antibody and a therapeutic agent are encapsulated in a liposome, and the liposome and the antibody are conjugated, and a manner in which a therapeutic agent is chemically conjugated to a water-soluble polymer (a compound with a molecular weight of about 1,000 to 100,000) directly or via a spacer such as an oligopeptide, and the antibody is conjugated to the water-soluble polymer. The conjugation of an antibody (or a fragment thereof) to a drug carrier such as a therapeutic agent, liposome, or water-soluble polymer is described in G.T. Hermanson, "Bioconjugate Techniques," Academic Press, California (1996), Putnam and J. Kopecek, "Polymer Conjugates with Anticancer Activity," Advances in In Polymer Science (1995) 122, 55-123, etc. The incorporation of therapeutic agents into liposomes can be carried out by methods well known to those skilled in the art, such as the method described in DD Lasic, "Liposomes: From Physics to Applications," Elsevier Science Publishers BV, Amsterdam (1993), etc. The binding of therapeutic agents to water-soluble polymers can be carried out by methods well known to those skilled in the art, such as the method described in D. Putnam and This can be carried out by methods known to those skilled in the art, such as the method described in J. Kopecek, "Polymer Conjugates with Anticancer Activity," Advances in Polymer Science (1995) 122, 55-123. In addition to the above methods, conjugates of antibodies (or fragments thereof) and proteinaceous therapeutic agents (or fragments thereof) can also be prepared by genetic engineering methods known to those skilled in the art.

[0125] The present invention also provides pharmaceutical compositions containing, as an active ingredient, an anti-human TLR7 antibody or at least one antigen-binding fragment of the antibody, or an expression vector containing a polynucleotide having a polynucleotide sequence encoding the same, in an amount effective for treating and / or preventing a disease, as well as pharmaceutically acceptable diluents, carriers, solubilizers, emulsifiers, preservatives, and / or adjuvants.

[0126] The present invention also provides pharmaceutical compositions comprising an expression vector containing at least one anti-human TLR7 antibody or antigen-binding fragment of the antibody, or a polynucleotide having a polynucleotide sequence encoding the same, in an amount effective for treating and / or preventing a disease, at least one therapeutic agent in an amount effective for treating and / or preventing a disease, and a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative, and / or adjuvant.

[0127] Examples of therapeutic agents include, but are not limited to, the aforementioned folate antagonists, calcineurin inhibitors, corticosteroids, antithymocyte globulins, nucleic acid antimetabolites, nucleic acid synthesis inhibitors, biological preparations that target cell surface antigens, or biological preparations that target cytokines or cytokine receptors.

[0128] The substances used in the formulations acceptable for the pharmaceutical compositions of the present invention are preferably non-toxic to the recipients of the pharmaceutical compositions at the dosages and concentrations.

[0129] The pharmaceutical compositions of the present invention may contain formulatory agents to modify or maintain pH, osmolality, viscosity, clarity, color, isotonicity, sterility, stability, dissolution rate, sustained-release rate, absorption rate, or permeability, including, but not limited to, amino acids such as glycine, alanine, glutamine, asparagine, arginine, or lysine, antibacterial agents, antioxidants such as ascorbic acid, sodium sulfate, or sodium bisulfite, buffers such as phosphoric acid, citric acid, borate buffer, sodium bicarbonate, and Tris-HCl solution, bulking agents such as mannitol and glycine, chelating agents such as ethylenediaminetetraacetic acid (EDTA), caffeine, polyvinylpyrrolidine, complexing agents such as β-cyclodextrin and hydroxypropyl-β-cyclodextrin, bulking agents such as glucose, mannose, or dextrin, other carbohydrates such as monosaccharides and disaccharides, colorants, flavoring agents, diluents, and emulsifiers. 、Hydrophilic polymers such as polyvinylpyrrolidine, low-molecular-weight polypeptides, salt-forming counterions, preservatives such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorexidine, sorbic acid, or hydrogen peroxide, solvents such as glycerin, propylene glycol, or polyethylene glycol, sugar alcohols such as mannitol or sorbitol, suspending agents, sorbitan esters, polysorbates such as polysorbate 20 and polysorbate 80, surfactants such as triton, tromethamine, lecithin, or cholesterol, stabilizing agents such as sucrose or sorbitol, elasticity enhancers such as sodium chloride, potassium chloride, or mannitol / sorbitol, transport agents, excipients, and / or pharmaceutical adjuvants. The amount of these formulation substances added is preferably 0.01 to 100 times, and particularly 0.1 to 10 times, the weight of the anti-human TLR7 antibody. The composition of the pharmaceutical composition in the formulation can be appropriately determined by those skilled in the art depending on the applicable disease, applicable administration route, etc.

[0130] The excipients and carriers in pharmaceutical compositions can be liquid or solid. Suitable excipients and carriers include water for injection, physiological saline, artificial cerebrospinal fluid, and other substances commonly used for parenteral administration. Neutral physiological saline or physiological saline containing serum albumin can also be used as a carrier. Pharmaceutical compositions can include Tris buffer (pH 7.0-8.5), acetate buffer (pH 4.0-5.5), and citrate buffer (pH 3.0-6.2). These buffers can also contain sorbitol or other compounds. Pharmaceutical compositions of the present invention include those containing anti-human TLR7 antibodies and those containing an anti-human TLR7 antibody and at least one therapeutic agent. The pharmaceutical compositions of the present invention are prepared as lyophilized or liquid preparations with a selected composition and required purity. Pharmaceutical compositions containing anti-human TLR7 antibodies and those containing an anti-human TLR7 antibody and at least one agent for treating bone metabolic disorders can also be formulated as lyophilized preparations using a suitable excipient, such as sucrose.

[0131] The pharmaceutical compositions of the present invention can be prepared for parenteral administration or oral absorption through the gastrointestinal tract. The composition and concentration of the formulation can be determined depending on the administration method. The higher the affinity of the anti-human TLR7 antibody contained in the pharmaceutical composition of the present invention for human TLR7, i.e., the lower the dissociation constant (Kd value) for human TLR7, the lower the therapeutic effect can be achieved even at a lower dose administered to humans. Therefore, the dosage of the pharmaceutical composition of the present invention for humans can be determined based on this result. When administering an anti-human TLR7 antibody to humans in the present invention, the dosage is approximately 0.1 to 100 mg / kg, administered once every 1 to 180 days.

[0132] The pharmaceutical composition of the present invention may be in the form of an injection including intravenous drip, a suppository, a nasal agent, a sublingual agent, a transdermal agent, or the like.

[0133] While most approved antibody formulations are administered intravenously, subcutaneous administration is preferred in many clinical settings. Because the volume is limited to 1.0–1.5 mL, a highly concentrated antibody solution is required depending on the dose. However, as the concentration increases, the viscosity of the solution increases, making it impossible to inject using commonly used needles. Therefore, when selecting an injectable pharmaceutical composition, low viscosity is an important priority, and viscosity can be used as an indicator to select an appropriate antibody. [Example]

[0134] The present invention will be described in more detail below with reference to examples, but is not limited to these. Unless otherwise specified, genetic manipulation procedures in the following examples were carried out according to the methods described in "Molecular Cloning" (Sambrook, J., Fritsch, EF, and Maniatis, T., published by Cold Spring Harbor Laboratory Press in 1989), or, when commercially available reagents or kits were used, they were used according to the instructions provided with the commercially available products.

[0135] [Example 1] Preparation of mouse anti-human TLR7 antibody 1)-1 Immunity 1)-1-1 Establishment of the Ba / F3 cell line overexpressing human TLR7-Flag-His×6 / human Unc93B1-HA×2 The human TLR7 (variant 2) gene (SEQ ID NO: 3) was inserted into the retroviral vector pMXs (Cell Biolabs, Inc.), which adds Flag-His×6 to the C-terminus of the gene, using In Fusion® enzyme (Takara). The retroviral vector was then transfected into the HEK293-derived packaging cell line Plat-E (Cell Biolabs, Inc.) using the transfection reagent Fugene® 6 (Roche). After 24 hours, the culture supernatant was collected and used as a virus suspension. The virus suspension was mixed with the transfection reagent DOTAP (Roche) and added to the Ba / F3 cell line (RIKEN, BRC). The cells were then centrifuged at 2000 rpm for 1 hour to establish the human TLR7-Flag-His×6-expressing Ba / F3 cell line.

[0136] Human TLR7 encoded by the human TLR7 (variant 2) gene (SEQ ID NO: 3) has the amino acid sequence of SEQ ID NO: 4. This sequence is shown in FIG.

[0137] The human Unc93B1 gene (SEQ ID NO: 55) was integrated into the retroviral vector pMXs (Cell Biolabs, Inc.), which adds HAx2 to the C-terminus of the gene, using In Fusion (registered trademark) enzyme (Takara). The retroviral vector was transfected into the HEK293 cell-derived packaging cell line Plat-E (Cell Biolabs, Inc.). The cells were transfected into a 100-well plate (Biolabs, Inc.) using the transfection reagent Fugene (registered trademark) 6 (Roche). After 24 hours, the culture supernatant was collected and This virus suspension was mixed with the transfection reagent DOTAP (Roche) and added to the human TLR7-Flag-Hisx6-expressing Ba / F3 cell line established above. The mixture was then centrifuged at 2000 rpm for 1 hour to establish the human TLR7-Flag-Hisx6 / human Unc93B1-HAx2-expressing Ba / F3 cell line.

[0138] 1)-1-2 Mouse immunization The human TLR7-Flag-Hisx6 / human Unc93B1-HAx2-expressing Ba / F3 cell line established in 1)-1-1 was mixed with the adjuvant TiterMAX® Gold (TiterMax USA) and administered as an antigen to TLR9-deficient mice on a BALB / c background, generated at the Institute of Medical Science, The University of Tokyo, at weekly intervals three times in total, into the soles of the feet, the base of the tail, and intraperitoneally.

[0139] On the fourth occasion, the Ba / F3 cell line forcedly expressing human TLR7-Flag-Hisx6 / human Unc93B1-HAx2 suspended in 1x PBS was intraperitoneally administered.

[0140] Five days after the final immunization, the spleens were excised and used to prepare hybridomas.

[0141] 1)-2 Hybridoma production Immunized spleen cells were mixed with the Sp2 / o cell line (ATCC) and fused using an HVJ-E cell fusion kit (Ishihara Sangyo Kaisha). From the day after the fusion procedure, hybridomas were selected by culturing them in RPMI1640 (Life Technologies) containing HAT (Thermo Fisher Scientific) medium (10% FBS, 50 μM 2-mercaptoethanol (Life Technologies), 50 U / mL penicillin, and 50 μg / mL streptomycin (Life Technologies)). Monoclonal hybridomas were generated by collecting the hybridoma colonies that emerged.

[0142] 1)-3 Screening of human TLR7-binding antibodies by flow cytometry analysis Culture supernatants were collected from hybridomas that formed colonies under a microscope and used for screening. Ba / F3 cells expressing hTLR7-Flag-His×6 / hUnc93B1-HA×2 were permeabilized with 0.1% Saponin (Sigma-Aldrich), and non-expressing Ba / F3 cells were stained with the culture supernatants. Hybridomas producing anti-hTLR7 antibodies were selected by flow cytometry (LSRFortessa™ MX-20, Becton Dickinson Japan).

[0143] 1)-4 Screening of human TLR7 function-blocking antibodies by flow cytometry The human TLR7 (variant 2) gene (SEQ ID NO: 3) and the human Unc93B1 D34A mutant-HAx2 gene (SEQ ID NO: 86) were retrovirally transfected into the Ba / F3 cell line (RIKEN, BRC). Furthermore, an NF-κB-green fluorescent protein (GFP) reporter plasmid (STRATAGENE) was electroporated to establish a Ba / F3 cell line overexpressing hTLR7 / hUnc93B1 D34A mutant-HAx2, allowing analysis of NF-κB activation. The hybridoma culture supernatant was added to the cell line. After 4 hours, the cells were stimulated with 25 ng / ml of the TLR7 ligand R848 (InvivoGen). After 24 hours, GFP fluorescence was measured by flow cytometry to analyze the inhibitory effect of human TLR7 responses. Three hybridomas producing mouse anti-human TLR7 antibodies were selected and named AT01, NB7, and FAN2, respectively.

[0144] In the present specification, the antibodies produced by the hybridomas AT01, NB7, and FAN2 are referred to as the AT01 antibody, the NB7 antibody, and the FAN2 antibody, respectively.

[0145] 1)-5 Determining antibody isotype 1) The isotypes of the antibodies produced by each of the mouse anti-human TLR7 antibody-producing hybridomas (AT01, NB7, and FAN2) obtained in 4) were determined using IsoStrip Mouse The isotypes of the AT01, NB7, and FAN2 antibodies were determined using a Monoclonal Antibody Isotyping Kit (Merck). The results showed that the isotypes of the AT01, NB7, and FAN2 antibodies were all IgG1 / κ chain.

[0146] 1)-6 Preparation of mouse anti-human TLR7 antibody The mouse anti-human TLR7 monoclonal antibody was purified from ascites collected approximately 10 days after intraperitoneal injection of the hybridoma into ICR-CD1-Foxn1 nude mice (ICR-nu, Charles River Japan) pretreated with pristane (Sigma-Aldrich, now Merck).

[0147] First, pristane was intraperitoneally administered to ICR-nu mice and they were raised for 7 days or more. Next, AT01, NB7, and FAN2-producing hybridomas were cultured in 10% FBS, 50 μM 2-mercaptoethanol (Life Technologies), 50 U / mL The cells were cultured in RPMI1640 (Life Technologies) containing penicillin and 50 μg / mL streptomycin (Life Technologies) and grown to a sufficient amount, then suspended in 1×PBS and administered intraperitoneally to ICR-nu.

[0148] After 10 days, the ascites was collected, the blood cells were sedimented using a high-speed centrifuge, and the supernatant was collected. The ascites was diluted 10-fold with 1× PBS and then passed through a 0.45 μm filter (Millipore).

[0149] The antibody was purified from the ascites fluid using AKTAprime plus (GE Healthcare Japan) on a protein G column (GE Healthcare Japan).

[0150] Subsequently, the buffer was replaced with saline using PD-10 (GE Healthcare Japan) and the solution was concentrated to an antibody concentration of 1.0 mg / mL or higher.

[0151] Finally, the mixture was sterilized with Millex-GV 0.22 μm (Millipore) to prepare a purified sample.

[0152] [Example 2] In vitro evaluation of mouse anti-human TLR7 antibodies 2)-1 Evaluation of binding selectivity of mouse anti-human TLR7 antibodies 2)-1-1 Establishment of the Ba / F3 cell line overexpressing mouse TLR7-Flag-His×6 The mouse TLR7 gene (SEQ ID NO: 56) was integrated into the retroviral vector pMXs (Cell Biolabs, Inc.), which adds Flag-Hisx6 to the C-terminus of the gene, using In Fusion® enzyme (Takara). The retroviral vector was then transfected into the HEK293-derived packaging cell line Plat-E (Cell Biolabs, Inc.) using the transfection reagent Fugene® 6 (Roche). After 24 hours, the culture supernatant was collected and used as a virus suspension. The virus suspension was mixed with the transfection reagent DOTAP (Roche) and added to the Ba / F3 cell line (RIKEN, BRC). The cells were then centrifuged at 2000 rpm for 1 hour to establish the mouse TLR7-Flag-Hisx6-expressing Ba / F3 cell line.

[0153] Mouse TLR7 encoded by the mouse TLR7 gene (SEQ ID NO: 56) has the amino acid sequence of SEQ ID NO: 83. This sequence is shown in Figure 3.

[0154] 2)-1-2 Development of rat TLR7-Flag-His×6-expressing Ba / F3 cell line standing The rat TLR7 gene (SEQ ID NO: 57) was integrated into the retroviral vector pMXs (Cell Biolabs, Inc.), which adds Flag-Hisx6 to the C-terminus of the gene, using In Fusion® enzyme (Takara). The retroviral vector was then transfected into the HEK293-derived packaging cell line Plat-E (Cell Biolabs, Inc.) using the transfection reagent Fugene® 6 (Roche). After 24 hours, the culture supernatant was collected and used as a virus suspension. The virus suspension was mixed with the transfection reagent DOTAP (Roche) and added to the Ba / F3 cell line (RIKEN, BRC). The cells were centrifuged at 2000 rpm for 1 hour to establish the rat TLR7-Flag-Hisx6-expressing Ba / F3 cell line.

[0155] Rat TLR7 encoded by the rat TLR7 gene (SEQ ID NO: 57) has the amino acid sequence of SEQ ID NO: 84. This sequence is shown in FIG.

[0156] 2)-1-3 Establishment of monkey TLR7-Flag-His×6-expressing Ba / F3 cell line The simian TLR7 gene (SEQ ID NO: 58) was integrated into the retroviral vector pMXs (Cell Biolabs, Inc.), which adds Flag-Hisx6 to the C-terminus of the gene, using In Fusion® enzyme (Takara). The retroviral vector was then transfected into the HEK293-derived packaging cell line Plat-E (Cell Biolabs, Inc.) using the transfection reagent Fugene® 6 (Roche). After 24 hours, the culture supernatant was collected and used as a virus suspension. The virus suspension was mixed with the transfection reagent DOTAP (Roche) and added to the Ba / F3 cell line (RIKEN, BRC). The cells were centrifuged at 2000 rpm for 1 hour to construct a simian TLR7-Flag-Hisx6-expressing Ba / F3 cell line.

[0157] The simian TLR7 encoded by the simian TLR7 gene (SEQ ID NO: 58) has the amino acid sequence of SEQ ID NO: 85. This sequence is shown in FIG.

[0158] 2)-1-4 Evaluation of binding selectivity of mouse anti-human TLR7 antibodies by flow cytometry The Ba / F3 cell line established in 1)-1-1 expressing human TLR7-Flag-His×6 / human Unc93B1-HA×2, the Ba / F3 cell line established in 2)-1-1 expressing mouse TLR7-Flag-His×6, the Ba / F3 cell line established in 2)-1-2 expressing rat TLR7-Flag-His×6×2, and the Ba / F3 cell line established in 2)-1-3 expressing monkey TLR7-Flag-His×6 were permeabilized with 0.1% Saponin and incubated with AT01 antibody, NB antibody, and IgG. 7 antibodies The cells were stained with FAN2 and FAN2 antibodies and a secondary antibody, Goat anti-mouse IgG (H+L)-PE (eBioscience). The binding selectivity of each antibody was evaluated by flow cytometry and mean fluorescence intensity (MFI) was compared.

[0159] As a result, the AT01 antibody, NB7 antibody, and FAN2 antibody specifically bound to human TLR7 or monkey TLR7, but did not bind to mouse TLR7 or rat TLR7.

[0160] 2)-2 Evaluation of binding ability of mouse anti-human TLR7 antibodies The Ba / F3 cell line, established in 1)-1-1, expressing human TLR7-Flag-His×6 / human Unc93B1-HA×2, was permeabilized with 0.1% Saponin and incubated with AT01 antibody and NB 7 antibodies A dilution series of FAN2 antibody and the secondary antibody Goat anti-mouse IgG (H+L)-PE (eBioscience) were used. The binding activity of each antibody was evaluated by flow cytometry and the mean fluorescence intensity (MFI) was compared.

[0161] As a result, the binding activity was ranked in descending order of AT01 antibody, NB7 antibody, and FAN2 antibody.

[0162] 2)-3 Cytokine production suppression effect of mouse anti-human TLR7 antibody Human PBMCs were purchased as frozen products from Cellular Technology, and were thawed and used according to the manufacturer's instructions.

[0163] 10% FBS, 1mM Sodium Pyruvate (Life Techn. RPMI 1640 (Life Technologies) containing 0.1 mM MEM Non-Essential Amino Acids (Life Technologies), 50 μM 2-Mercaptoethanol (Life Technologies), 50 U / mL Penicillin, and 50 μg / mL Streptomycin (Life Technologies) at a concentration of 2.5 x 10 6 PBMCs prepared at a concentration of 100 cells / mL were seeded into 96-well cell culture plates at 100 μL per well, and mouse anti-human TLR7 antibodies AT01, NB7, or FAN2, or a mouse IgG control antibody (Biolegend) were added at 80 μL / well and pretreated in a 37°C incubator for 4 hours.

[0164] Then, 20 μL / well of 1 mg / mL CL-264 (InvivoGen) was added, and the plates were thoroughly mixed and then cultured at 37°C under 5% CO for approximately 20 hours. After thorough mixing, the mixture was centrifuged at 1500 rpm for 5 minutes, and the concentration of interleukin-6 (IL-6) in the supernatant was measured by the FRET method (Cisbio).

[0165] The results are shown in Figure 6. The mouse anti-human TLR7 antibody inhibited IL-6 production from CL-264-treated human PBMC in a concentration-dependent manner. The AT01, NB7, and FAN2 antibodies inhibited IL-6 production from human PBMC in a concentration-dependent manner. The half inhibitory concentrations (IC 50 ) were calculated from the concentrations between the two points on either side of 50% inhibitory activity and the inhibitory activity when those concentrations were added, and were 192 ng / mL, 771 ng / mL, and 8389 ng / mL, respectively.

[0166] On the other hand, no inhibition was observed with the mouse IgG control antibody even at a concentration of 10 μg / mL.

[0167] [Example 3] Determination of the nucleotide and amino acid sequences of cDNA encoding the variable region of mouse anti-human TLR7 antibody 3)-1 cDNA synthesis Total RNA was extracted from the AT01, NB7, and FAN2 hybridomas using TRIzol Reagent (Ambion), followed by cDNA synthesis using the PrimeScript 1st Strand cDNA Synthesis Kit (TAKARA).

[0168] 3)-2 Amplification and sequencing of mouse immunoglobulin heavy and light chain variable region gene fragments The nucleotide sequence encoding the variable region of the antibody contained in the synthesized cDNA was decoded by Genescript.

[0169] 3)-2-1 Mouse anti-human TLR7 antibody (AT01) The determined amino acid sequence encoded by the nucleotide sequence of the cDNA encoding the heavy chain variable region of the AT01 antibody is shown in SEQ ID NO:5 in the sequence listing.

[0170] The determined amino acid sequence encoded by the nucleotide sequence of the cDNA encoding the light chain variable region of the AT01 antibody is shown in SEQ ID NO: 11 in the sequence listing.

[0171] 3)-2-2 Mouse anti-human TLR7 antibody (NB7) The determined amino acid sequence encoded by the nucleotide sequence of the cDNA encoding the heavy chain variable region of the NB7 antibody is shown in SEQ ID NO: 7 in the sequence listing.

[0172] The determined amino acid sequence encoded by the nucleotide sequence of the cDNA encoding the light chain variable region of the NB7 antibody is shown in SEQ ID NO: 13 in the sequence listing.

[0173] 3)-2-3 Mouse anti-human TLR7 antibody (FAN2) The determined amino acid sequence encoded by the nucleotide sequence of the cDNA encoding the heavy chain variable region of the FAN2 antibody is shown in SEQ ID NO:9 in the sequence listing.

[0174] The determined amino acid sequence encoded by the nucleotide sequence of the cDNA encoding the light chain variable region of the FAN2 antibody is shown in SEQ ID NO: 15 in the sequence listing.

[0175] [Example 4] Preparation of chimeric anti-human TLR7 antibody 4)-1 Construction of expression vector for chimeric anti-TLR7 antibody 4)-1-1 Construction of chimeric light chain expression vector pCMA-LK Plasmid pcDNA3.3-TOPO / LacZ (Invitrogen) was digested with restriction enzymes XbaI and PmeI to obtain a 5.4-kb fragment, which was then ligated with a DNA fragment (SEQ ID NO: 59) encoding the human light chain signal sequence and the human kappa chain constant region using the In-Fusion HD PCR Cloning Kit (Clontech) to produce pcDNA3.3 / LK. pCMA-LK was constructed by removing the neomycin expression unit from pcDNA3.3 / LK.

[0176] 4)-1-2 Construction of chimeric IgG1-type heavy chain expression vector pCMA-G1 pCMA-LK was digested with XbaI and PmeI to remove the light chain signal sequence and human kappa chain constant region, and a DNA fragment (SEQ ID NO: 60) containing a DNA sequence encoding the human heavy chain signal sequence and human IgG1 constant region was ligated using an In-Fusion HD PCR cloning kit (Clontech) to construct pCMA-G1.

[0177] 4)-1-3 Construction of AT01 chimeric anti-human TLR7 antibody expression vector A DNA fragment containing a nucleotide sequence encoding the variable region of the heavy chain of the cAT01 antibody, represented by nucleotides 36 to 422 of the nucleotide sequence shown in SEQ ID NO: 61, was synthesized (GENEART). Using the In-Fusion HD PCR Cloning Kit (Clontech), pCMA-G1 was cleaved with the restriction enzyme BlpI and the synthesized DNA fragment was inserted into the site, thereby constructing an expression vector for the heavy chain of the cAT01 antibody. The heavy chain of the cAT01 antibody, including the signal sequence, has the amino acid sequence of SEQ ID NO: 35. The sequence is shown in Figure 7.

[0178] A DNA fragment (SEQ ID NO: 62) containing the DNA sequence encoding the cAT01 antibody light chain was synthesized (GENEART). Using pCMA-LK prepared in Example 4)-1-1, an expression vector for the cAT01 antibody light chain was constructed in the same manner as above. The light chain of the cAT01 antibody has the amino acid sequence of SEQ ID NO: 36, including the signal sequence, and this sequence is shown in Figure 7. 4)-1-4 Construction of NB7 chimeric anti-human TLR7 antibody expression vector A DNA fragment containing a nucleotide sequence encoding the variable region of the heavy chain of the cNB7 antibody, represented by nucleotides 36 to 440 of the nucleotide sequence shown in SEQ ID NO: 63, was synthesized (GENEART). A cNB7 heavy chain expression vector was constructed in the same manner as in Example 4)-1-3. The heavy chain of the cNB7 antibody has the amino acid sequence of SEQ ID NO: 37, including the signal sequence. The sequence is shown in Figure 8.

[0179] A DNA fragment (SEQ ID NO: 64) containing a DNA sequence encoding the cNB7 light chain was synthesized (GENEART). Using pCMA-LK prepared in Example 4)-1-1, a cNB7 light chain expression vector was constructed in the same manner as in Example 4)-1-3. The light chain of the cNB7 antibody was It has the amino acid sequence of SEQ ID NO: 38, including the signal sequence, which is shown in Figure 8.

[0180] 4)-1-5 Construction of FAN2 chimeric anti-human TLR7 antibody expression vector The nucleotide sequence shown in SEQ ID NO: 65 is represented by nucleotide numbers 36 to 437. A DNA fragment containing a nucleotide sequence encoding the variable region of the cFAN2 antibody heavy chain was synthesized (GENEART). A cFAN2 heavy chain expression vector was constructed in the same manner as in Example 4)-1-3. The heavy chain of the cFAN2 antibody has the amino acid sequence of SEQ ID NO: 39, including the signal sequence. This sequence is shown in Figure 9.

[0181] A DNA fragment (SEQ ID NO: 66) containing a DNA sequence encoding the cFAN2 light chain was synthesized (GENEART). Using pCMA-LK prepared in Example 4)-1-1, a cFAN2 light chain expression vector was constructed in the same manner as in Example 4)-1-3. The light chain has the amino acid sequence of SEQ ID NO: 40, including the signal sequence, which is shown in Figure 9.

[0182] 4)-2 Production of chimeric anti-human TLR7 antibodies 4)-2-1 Production of chimeric AT01 antibody FreeStyle 293F cells (Invitrogen) were subcultured and cultured according to the manufacturer's instructions. 1.2 × 10 cells were cultured in the logarithmic growth phase. 9 FreeStyle 293 F cells (Invitrogen) were seeded in a 3L Fernbach Erlenmeyer Flask (Corning) and diluted with FreeStyle293 expression medium (Invitrogen) to 2.0 × 10 6 cells / mL 0.24 mg of the heavy chain expression vector constructed in 4)-1-3 above, 0.36 mg of the light chain expression vector constructed in 4)-1-3 above, and 1.8 mg of Polyethyleneimine (Polyscience #24765) were added to 40 mL of Opti-Pro SFM medium (Invitrogen), and the mixture was gently stirred and left for another 5 minutes before being added to the FreeStyle 293F cells. Incubated at 37°C and 8% CO2. After culturing in a shaker at 90 rpm for 4 hours, 600 ml of EX-CELL VPRO medium (SAFC Biosciences), 18 ml of GlutaMAX I (GIBCO), and 30 ml of Yeastolate Ultrafiltrate (GIBCO) were added, and the cells were shaken at 90 rpm in an incubator at 37°C and 8% CO2 for 7 days. The culture supernatant obtained by culturing was filtered through a Disposable Capsule Filter (Advantec #CCS-045-E1H) to produce chimeric AT01 antibody.

[0183] 4)-2-2 Production of chimeric NB7 antibody Using the heavy chain expression vector and light chain expression vector constructed in 4)-1-4 above, Chimeric NB7 antibody was produced by the method described in )-2-1.

[0184] 4)-2-3 Production of chimeric FAN2 antibody The heavy chain expression vector and the light chain expression vector constructed in 4)-1-5 above were used to A chimeric FAN2 antibody was produced by the method described in 2-2-1.

[0185] 4)-3 Purification of chimeric anti-human TLR7 antibodies The target antibodies were purified from each culture supernatant obtained in Example 4)-2 using a single-step process of rProtein A affinity chromatography. The culture supernatant was applied to a column packed with MabSelectSuRe (GE Healthcare Bioscience) equilibrated with PBS, and the column was washed with at least two column volumes of PBS. The antibody-containing fractions were then eluted with 2 M arginine hydrochloride solution (pH 4.0), and the fractions containing the antibody were collected. The fractions were then buffer-exchanged to PBS(-) by dialysis (Thermo Scientific, Slide-A-Lyzer Dialysis Cassette). The antibody was concentrated using a Centrifugal UF Filter Device VIVASPIN20 (molecular weight cutoff UF10K, Sartorius) to adjust the IgG concentration to 10 mg / mL or higher. Finally, the purified sample was filtered through a Minisart-Plus filter (Sartorius).

[0186] [Example 5] In vitro activity of chimeric anti-human TLR7 antibodies 5)-1 Antigen-binding activity of chimeric anti-human TLR7 antibodies by flow cytometry Ba / F3 cells expressing human TLR7-Flag-Hisx6 / hUnc93B1-HAx2 were permeabilized with 0.1% Saponin and stained with a dilution series of the chimeric AT01, NB7, and FAN2 antibodies obtained in Example 4 and a secondary antibody, Goat anti-human IgG Fc-PE (eBioscience). The binding activity of the antibodies was evaluated by flow cytometry, and MFI was compared. The results showed that the binding activity was strongest in the following order: chimeric AT01 antibody (cAT01), chimeric NB7 antibody (cNB7), and chimeric FAN2 antibody (cFAN2).

[0187] 5)-2 Cytokine production suppression effect of chimeric anti-human TLR7 antibody Human PBMCs were purchased frozen from Cellular Technology and thawed according to the manufacturer's instructions. The cells were cultured at a concentration of 2.5x10 in RPMI 1640 (Life Technologies) containing 0.1 mM MEM Non-Essential Amino Acids (Life Technologies), 50 μM 2-Mercaptoethanol (Life Technologies), 50 U / mL Penicillin, and 50 μg / mL Streptomycin (Life Technologies). 6 PBMCs were prepared at a concentration of 100 cells / mL and seeded in a 96-well cell culture plate at 100 μL per well. Chimeric anti-human TLR7 antibodies cAT01, cNB7, cFAN2, or a human IgG control antibody (EUREKA Therapeutics) were added at 80 μL / well. The cells were pretreated for 4 hours in a 37°C incubator. Then, 20 μL / well of 1 mg / mL CL-264 (InvivoGen) was added, and the cells were cultured for approximately 20 hours at 37°C under 5% CO2. After stirring the plate well, the cells were cultured at 1500 rpm for 5 hours. The mixture was centrifuged for 1 minute, and the IL-6 concentration in the supernatant was measured using the FRET method (Cisbio).

[0188] Figure 10 shows that chimeric anti-human TLR7 antibodies inhibit IL-6 production from CL-264-treated human PBMCs in a concentration-dependent manner. cAT01, cNB7, and cFAN2 inhibited IL-6 production from human PBMCs in a concentration-dependent manner. The half inhibitory concentrations (IC 50 ) were calculated from the concentrations between the two points that sandwiched the 50% inhibitory activity and the inhibitory activity when those concentrations were added, and were 35 ng / mL, 196 ng / mL, and over 10,000 ng / mL, respectively.

[0189] On the other hand, no inhibition was observed with the human IgG control antibody even at a concentration of 10 μg / mL.

[0190] [Example 6] Production of humanized anti-human TLR7 antibody 6)-1 Design of humanized anti-human TLR7 antibodies 6)-1-1 Molecular modeling of antibody variable regions Molecular modeling of the variable regions was performed using a known method known as homology modeling (Methods in Enzymology, 203, 121-153, (1991)) and the commercially available protein three-dimensional structure analysis program BioLuminate (Schrodinger). The template was a sequence registered in the Protein Data Bank (Nuc. Acid Res. 35, D301-D303 (2007)) that has high sequence identity to the heavy and light chain variable regions. The structure used was

[0191] 6)-1-2 Design of humanized amino acid sequence Humanization was performed using a technique known as CDR grafting (Proc. Natl. Acad. Sci. USA 86, 10029-10033 (1989)). The consensus sequences defined in Kabat et al. (Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service National Institutes of Health, Bethesda, MD. (1991)) and the human sequences registered in IMGT (THE INTERNATIONAL IMMUNOGENETICS INFORMATION SYSTEM (registered trademark), http: / / www.imgt.org) were used. An acceptor with high identity was selected from the germline sequence, and the donor residue to be transferred onto the acceptor was determined according to the method described by Queen et al. (Proc. Natl. Acad. Sci. USA 86, 10029-10033 (1989)). The selection was made by analyzing the three-dimensional model based on the criteria given by the

[0192] 6)-1-3 Design of humanized amino acid sequence of AT01 antibody The consensus sequences of human gamma chain subgroup 1 and kappa chain subgroup 1 were chosen as acceptors for cAT01 because they share high identity with the framework regions of cAT01. Molecular modeling of the variable regions was performed using known structures (PDB ID :1 E4W) was used as the template.

[0193] 6)-1-3-1 Humanization of the AT01 antibody heavy chain (1) The designed heavy chain was named huAT01_H1_IgG1LALA. huAT01_H1_IgG1LALA has the amino acid sequence shown in SEQ ID NO: 45 as the full-length heavy chain amino acid sequence, including the signal sequence. This sequence is shown in Figure 11. In this sequence, the amino acid sequence from positions 1 to 19 is the signal sequence, the amino acid sequence from positions 20 to 135 is the variable region, and the amino acid sequence from positions 136 to 465 is the constant region. In addition, the amino acid sequence from positions 45 to 54 is CDRH1, and the amino acid sequence from positions 69 to 78 is the constant region. The amino acid sequence at positions 118 to 124 is CDRH3.

[0194] The nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 45 has the sequence set forth in SEQ ID NO: 67. The nucleotide sequence encoding the heavy chain variable region has the sequence set forth in SEQ ID NO: 7 to 7 It has the sequence described.

[0195] (2) The designed heavy chain was named huAT01_H3_IgG1LALA. huAT01_H3_IgG1LALA has the amino acid sequence shown in SEQ ID NO: 46 as the full-length heavy chain amino acid sequence, including the signal sequence. This sequence is shown in Figure 12. In this sequence, the amino acid sequence from positions 1 to 19 is the signal sequence, the amino acid sequence from positions 20 to 135 is the variable region, and the amino acid sequence from positions 136 to 465 is the constant region. In addition, the amino acid sequence from positions 45 to 54 is CDRH1, and the amino acid sequence from positions 69 to 78 is the constant region. The amino acid sequence at position 118 is CDRH2, and the amino acid sequence at positions 118 to 124 is C It is DRH3.

[0196] The nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 46 has the sequence set forth in SEQ ID NO: 68. Furthermore, the nucleotide sequence encoding the heavy chain variable region has the sequence set forth in SEQ ID NO: 78.

[0197] (3) The designed heavy chain was named huAT01_H3_IgG4Pro. huAT01_H3_IgG4Pro has the amino acid sequence shown in SEQ ID NO: 47 as the full-length heavy chain amino acid sequence, including the signal sequence. This sequence is shown in Figure 13. In this sequence, the amino acid sequence from positions 1 to 19 is the signal sequence, the amino acid sequence from positions 20 to 135 is the variable region, and the amino acid sequence from positions 136 to 462 is the constant region. In addition, the amino acid sequence from positions 45 to 54 is CDRH1, and the amino acid sequence from positions 69 to 78 is the constant region. The amino acid sequence of positions 118 to 124 is CDRH3.

[0198] The nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 47 has the sequence set forth in SEQ ID NO: 69. The nucleotide sequence encoding the heavy chain variable region has the sequence set forth in SEQ ID NO: 78.

[0199] A comparison of the amino acid sequence of the variable region of cAT01_H, the heavy chain of chimeric antibody cAT01 (SEQ ID NO: 5) (hereinafter referred to as cAT01_H), and the amino acid sequence of the variable region of humanized antibody heavy chain huAT01_H1_IgG1LALA (SEQ ID NO: 41) (hereinafter referred to as huAT01_H1) and the amino acid sequence of the variable region of huAT01_H3_IgG1LALA (SEQ ID NO: 42) (hereinafter referred to as huAT01_H3) is shown in Figure 14. In the sequences of huAT01_H1 and huAT01_H3, "·" indicates the same amino acid residue as in cAT01_H, and where an amino acid residue is listed, indicates a substituted amino acid residue.

[0200] 6)-1-3-2 Humanization of the AT01 antibody light chain The designed light chain was designated huAT01_L1. huAT01_L1 has the amino acid sequence shown in SEQ ID NO: 48, including the signal sequence, as the full-length amino acid sequence of the light chain. This sequence is shown in Figure 15. In this sequence, the amino acid sequence from positions 1 to 20 is the signal sequence, the amino acid sequence from positions 21 to 126 is the variable region, and the amino acid sequence from positions 127 to 233 is the constant region. Furthermore, amino acid positions 44 to 54 are CDRL1, amino acid positions 70 to 76 are CDRL2, and amino acid position 10 is the constant region. Numbers 9 to 116 are CDRL3.

[0201] The nucleotide sequence encoding the amino acid sequence of SEQ ID NO:48 has the sequence set forth in SEQ ID NO:70, and the nucleotide sequence encoding the light chain variable region has the sequence set forth in SEQ ID NO:79.

[0202] The designed light chain was designated huAT01_L2. huAT01_L2 has the full-length light chain amino acid sequence shown in SEQ ID NO: 49, including the signal sequence. This sequence is shown in Figure 16. In this sequence, the amino acid sequence from positions 1 to 20 is the signal sequence, the amino acid sequence from positions 21 to 126 is the variable region, and the amino acid sequence from positions 127 to 233 is the constant region. Furthermore, amino acid positions 44 to 54 are CDRL1, amino acid positions 70 to 76 are CDRL2, and amino acid position 10 is the constant region. Numbers 9 to 116 are CDRL3.

[0203] The nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 49 is set forth in SEQ ID NO: 71. The nucleotide sequence encoding the light chain variable region has the sequence set forth in SEQ ID NO: 80. do.

[0204] A comparison of the amino acid sequence of the variable region of cAT01_L, the light chain of chimeric antibody cAT01 (SEQ ID NO: 11) (hereinafter referred to as cAT01_L), with the amino acid sequence of the variable region of humanized antibody light chain huAT01_L1 (SEQ ID NO: 43) (hereinafter referred to as huAT01_L1) and the amino acid sequence of the variable region of huAT01_L2 (SEQ ID NO: 44) (hereinafter referred to as huAT01_L2) is shown in Figure 17. In the sequences of huAT01_L1 and huAT01_L2, "·" indicates the same amino acid residue as in cAT01_L, and where an amino acid residue is listed, indicates a substituted amino acid residue.

[0205] 6)-1-4 Design of humanized amino acid sequence of NB7 The human germline sequences IGHV4-30-4*02 and IGHJ6*01 were selected as acceptors for cNB7 because they share high identity with the framework regions of cNB7. Molecular modeling of the variable regions was performed using a known structure (PDB ID: 3CDF).

[0206] 6)-1-4-1 Humanization of the NB7 antibody heavy chain (1) The designed heavy chain was named huNB7_H3_IgG1LALA. huNB7_H3_IgG1LALA has the amino acid sequence shown in SEQ ID NO: 52, including the signal sequence, as the full-length heavy chain amino acid sequence. This sequence is shown in Figure 18. In this sequence, the amino acid sequence from positions 1 to 19 is the signal sequence, the amino acid sequence from positions 20 to 141 is the variable region, and the amino acid sequence from positions 142 to 471 is the constant region. Furthermore, amino acid positions 45 to 55 are CDRH1, and amino acid positions 70 to 78 are the constant region. The amino acid sequence from position 118 to position 130 corresponds to CDRH3.

[0207] The nucleotide sequence encoding the amino acid sequence of SEQ ID NO:52 is set forth in SEQ ID NO:72, and the nucleotide sequence encoding the heavy chain variable region has the sequence set forth in SEQ ID NO:81.

[0208] (2) The designed heavy chain was named huNB7_H3_IgG4Pro. huNB7_H3_IgG4Pro has the amino acid sequence shown in SEQ ID NO: 54, including the signal sequence, as the full-length heavy chain amino acid sequence. This sequence is shown in Figure 19. In this sequence, the amino acid sequence from positions 1 to 19 is the signal sequence, the amino acid sequence from positions 20 to 141 is the variable region, and the amino acid sequence from positions 142 to 468 is the constant region. Furthermore, amino acid positions 45 to 55 are CDRH1, and amino acid positions 70 to 78 are C DRH2, and amino acids 118 to 130 are CDRH3.

[0209] The nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 54 is set forth in SEQ ID NO: 73. The nucleotide sequence encoding the heavy chain variable region has the sequence set forth in SEQ ID NO: 81.

[0210] A comparison of the amino acid sequence of the variable region of cNB7_H, the heavy chain of chimeric antibody cNB7 (SEQ ID NO: 7) (hereinafter referred to as cNB7_H), and the amino acid sequence of the variable region of humanized antibody heavy chain huNB7_H3_IgG1LALA (SEQ ID NO: 50) (hereinafter referred to as huNB7_H3) is shown in Figure 20. In huNB7_H3, "·" indicates the same amino acid residue as in cNB7_H, and where an amino acid residue is listed, indicates a substituted amino acid residue.

[0211] 6)-1-4-2 Humanization of the NB7 antibody light chain The designed light chain was named huNB7_L3. huNB7_L3 has the amino acid sequence shown in SEQ ID NO: 53, including the signal sequence, as the full-length amino acid sequence of the light chain. The sequence is shown in Figure 21. In this sequence, the amino acid sequence from positions 1 to 20 is a signal sequence, the amino acid sequence from positions 21 to 126 is a variable region, and the amino acid sequence from positions 127 to 233 is a constant region. Furthermore, amino acid positions 44 to 54 are CDRL1, amino acid positions 70 to 76 are CDRL2, and amino acid position 109 is a constant region. The 116th position is CDRL3.

[0212] The nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 53 is set forth in SEQ ID NO: 74. The nucleotide sequence encoding the light chain variable region has the sequence set forth in SEQ ID NO: 82.

[0213] A comparison of the amino acid sequence of the variable region of cNB7_L, the light chain of chimeric antibody cNB7 (SEQ ID NO: 13) (hereinafter referred to as cNB7_L), and the amino acid sequence of the variable region of humanized antibody light chain huNB7_L3 (SEQ ID NO: 51) (hereinafter referred to as huNB7_L3) is shown in Figure 22. In huNB7_L3, "·" indicates the same amino acid residue as in cNB7_L, and where an amino acid residue is listed, indicates the substituted amino acid residue.

[0214] 6)-2 Design of humanized antibodies by combining heavy and light chains 6)-2-1 Design of humanized AT01 anti-TLR7 antibody (1) An antibody having huAT01_H1_IgG1LALA consisting of the amino acid sequence from positions 20 to 465 in SEQ ID NO: 45 as a heavy chain and huAT01_L1 consisting of the amino acid sequence from positions 21 to 233 in SEQ ID NO: 48 as a light chain was named "huAT01_H1L1_IgG1LALA antibody" or "huAT01_H1L1_IgG1LALA."

[0215] (2) An antibody having huAT01_H3_IgG1LALA consisting of the amino acid sequence from positions 20 to 465 in SEQ ID NO: 46 as a heavy chain and huAT01_L1 consisting of the amino acid sequence from positions 21 to 233 in SEQ ID NO: 48 as a light chain was named "huAT01_H3L1_IgG1LALA antibody" or "huAT01_H3L1_IgG1LALA."

[0216] (3) An antibody having huAT01_H3_IgG1LALA consisting of the amino acid sequence from positions 20 to 465 in SEQ ID NO: 46 as a heavy chain and huAT01_L2 consisting of the amino acid sequence from positions 21 to 233 in SEQ ID NO: 49 as a light chain was named "huAT01_H3L2_IgG1LALA antibody" or "huAT01_H3L2_IgG1LALA."

[0217] (4) An antibody having huAT01_H3_IgG4Pro consisting of the amino acid sequence of positions 20 to 462 in SEQ ID NO: 47 as a heavy chain and huAT01_L2 consisting of the amino acid sequence of positions 21 to 233 in SEQ ID NO: 49 as a light chain was designated "huAT01_H3L2_IgG4Pro antibody" or "huAT01_H3L2_IgG4Pro."

[0218] 6)-2-2 Design of humanized NB7 anti-TLR7 antibody (1) An antibody consisting of huNB7_H3_IgG1LALA, which consists of the amino acid sequence from positions 20 to 471 in SEQ ID NO: 52, as a heavy chain, and huNB7_L3, which consists of the amino acid sequence from positions 21 to 233 in SEQ ID NO: 53, as a light chain, was named "huNB7_H3L3_IgG1LALA antibody" or "huNB7_H3L3_IgG1LALA."

[0219] (2) a heavy chain consisting of the amino acid sequence from positions 20 to 468 in SEQ ID NO: 54 An antibody consisting of huNB7_H3_IgG4Pro having the amino acid sequence of positions 21 to 233 in SEQ ID NO: 53 as its light chain and huNB7_L3 having the amino acid sequence of positions 21 to 233 in SEQ ID NO: 53 as its light chain was designated "huNB7_H3L3_IgG4Pro antibody" or "huNB7_H3L3_IgG4Pro."

[0220] 6)-3 Generation of humanized anti-human TLR7 antibodies 6)-3-1 Humanized IgG1LALA type heavy chain expression vector pCMA-G1LAL Construction of A pCMA-G1LALA was constructed in the same manner as in Example 4)-1-2 using a DNA fragment (SEQ ID NO: 75) containing a nucleotide sequence encoding the human heavy chain signal sequence and the amino acid sequence of the human IgG1LALA constant region.

[0221] 6)-3-2 Humanized IgG4Pro type heavy chain expression vector pCMA-G4Pro construction pCMA-G4Pro was constructed in the same manner as in Example 4)-1-2 using a DNA fragment (SEQ ID NO: 76) containing a nucleotide sequence encoding a human heavy chain signal sequence and the amino acid sequence of a human IgG4Pro constant region.

[0222] 6)-3-3 Construction of AT01 humanized heavy chain expression vector 6)-3-3-1 Construction of huAT01_H1_IgG1LALA expression vector A DNA fragment represented by nucleotide numbers 36 to 422 of the nucleotide sequence of huAT01_H1_IgG1LALA shown in SEQ ID NO: 46 was synthesized (GENEART). Using an In-Fusion HD PCR cloning kit (Clontech), pCMA-G1LALA was cleaved with the restriction enzyme BlpI and the synthesized DNA fragment was inserted into the site, thereby constructing a huAT01_H1_IgG1LALA expression vector.

[0223] 6)-3-3-2 Construction of huAT01_H3_IgG1LALA expression vector A DNA fragment represented by nucleotide numbers 36 to 422 of the nucleotide sequence of huAT01_H3_IgG1LALA shown in SEQ ID NO: 68 was synthesized (GENEART). A huAT01_H3_IgG1LALA expression vector was constructed in the same manner as in Example 6)-3-3-1.

[0224] 6)-3-3-3 Construction of huAT01_H3_IgG4Pro expression vector A DNA fragment represented by nucleotide numbers 36 to 422 of the nucleotide sequence of huAT01_H3_IgG4Pro shown in SEQ ID NO: 69 was synthesized (GENEART). Using an In-Fusion HD PCR cloning kit (Clontech), pCMA-G4Pro was cleaved with the restriction enzyme BlpI and the synthesized DNA fragment was inserted into the site, thereby constructing a huAT01_H3_IgG4Pro expression vector.

[0225] 6)-3-4 Construction of AT01 humanized light chain expression vector 6)-3-4-1 Construction of huAT01_L1 expression vector A DNA fragment represented by nucleotide numbers 37 to 399 of the nucleotide sequence of huAT01_L1 shown in SEQ ID NO: 70 was synthesized (GENEART). Using an In-Fusion HD PCR cloning kit (Clontech), pCMA-LK was cleaved with the restriction enzyme BsiWI and the synthesized DNA fragment was inserted into the site, thereby constructing a huAT01_L1 expression vector.

[0226] 6)-3-4-2 Construction of huAT01_L2 expression vector Nucleotide number 37 of the nucleotide sequence of huAT01_L2 shown in SEQ ID NO: 71 A DNA fragment shown at positions 100 to 109 was synthesized (GENEART). Using an In-Fusion HD PCR cloning kit (Clontech), a huAT01_L1 expression vector was constructed in the same manner as in Example 6)-3-4-1.

[0227] 6)-3-5 Construction of NB7 humanized heavy chain expression vector 6)-3-5-1 Construction of huNB7_H3_IgG1LALA expression vector A DNA fragment represented by nucleotide numbers 36 to 440 of the nucleotide sequence of huNB7_H3_IgG1LALA shown in SEQ ID NO: 72 was synthesized (GENEART). A huNB7_H3_IgG1LALA expression vector was constructed in the same manner as in Example 6)-3-3-1.

[0228] 6)-3-5-2 Construction of huNB7_H3_IgG4Pro expression vector A DNA fragment represented by nucleotide numbers 36 to 440 of the nucleotide sequence of huNB7_H3_IgG4Pro shown in SEQ ID NO: 73 was synthesized (GENEART). A huNB7_H3_IgG4Pro expression vector was constructed in the same manner as in Example 6)-3-3-3.

[0229] 6)-3-6 Construction of NB7 humanized light chain expression vector 6)-3-6-1 Construction of huNB7_L3 expression vector A DNA fragment represented by nucleotide numbers 37 to 399 of the nucleotide sequence of huNB7_L3 shown in SEQ ID NO: 74 was synthesized (GENEART). A huNB7_L3 expression vector was constructed using an In-Fusion HD PCR cloning kit (Clontech) in the same manner as in Example 6)-3-4-1.

[0230] 6)-3-7 Preparation of humanized antibodies 6)-3-7-1 Production of humanized antibodies By combining the heavy chains and light chains designed in Example 6)-2, humanized antibodies huAT01_H1L1_IgG1LALA, huAT01_H3L1_IgG1LALA, huAT01_H3L2_IgG1LALA, huAT01_H3L2_IgG4Pro, huNB7_H3L3_IgG1LALA, and huNB7_H3L3_IgG4Pro were produced using the expression vectors constructed in Examples 6)-3-1 to 6)-3-6 in the same manner as in Example 4)-2.

[0231] 6)-3-7-2 Purification of humanized antibodies The culture supernatant obtained in Example 6)-3-7-1 was purified in a two-step process using rProtein A affinity chromatography and ceramic hydroxyapatite.

[0232] The culture supernatant was loaded onto a column (GE After application to a column (manufactured by Healthcare Biosciences), the column was washed with at least two column volumes of PBS, and the antibody was then eluted with 2 M arginine hydrochloride solution (pH 4.0).

[0233] The antibody-containing fraction was subjected to buffer exchange with PBS by dialysis (Thermo Scientific, Slide-A-Lyzer Dialysis Cassette), diluted five-fold with 5 mM sodium phosphate / 50 mM MES / pH 7.0 buffer, and then applied to a ceramic hydroxyapatite column (Nippon Bio-Rad, Bio-Scale CHT Type-1 Hydroxyapatite Column) equilibrated with 5 mM NaPi / 50 mM MES / 30 mM NaCl / pH 7.0 buffer.

[0234] A linear gradient elution with sodium chloride was performed, and the antibody-containing fractions were collected. The fractions were then dialyzed (Thermo Scientific, Slide-A-Lyzer Dialysis Cassette) against HBS (25 mM histidine / 5% The buffer was replaced with a buffer containing sorbitol (pH 6.0). The antibody was concentrated using a Centrifugal UF Filter Device VIVASPIN20 (molecular weight cutoff UF10K, Sartorius) to adjust the IgG concentration to 50 mg / mL. Finally, the antibody was filtered through a Minisart-Plus filter (Sartorius) to obtain a purified sample.

[0235] [Example 7] In vitro activity of humanized anti-human TLR7 antibodies 7)-1 Cytokine production suppression effect of humanized anti-human TLR7 antibody Human PBMCs were purchased as frozen products from Cellular Technology, and were thawed and used according to the manufacturer's instructions.

[0236] 10% FBS, 1mM Sodium Pyruvate (Life Techn. RPMI 1640 (Life Technologies) containing 0.1 mM MEM Non-Essential Amino Acids (Life Technologies), 50 μM 2-Mercaptoethanol (Life Technologies), 50 U / mL Penicillin, and 50 μg / mL Streptomycin (Life Technologies) at a concentration of 2.5 x 10 6PBMCs adjusted to a concentration of 100 cells / mL were seeded into a 96-well cell culture plate at 100 μL per well, and humanized anti-human TLR7 antibodies huAT01_H1L1_IgG1LALA, huAT01_H3L1_IgG1LALA, huAT01_H3L2_IgG1LALA, huAT01_H3L2_IgG4Pro, huNB7_H3L3_IgG1LALA, huNB7_H3L3_IgG4Pro, or a human IgG control antibody (EUREKA Therapeutics) were added at 80 μL / well. ell was added and the cells were pretreated in an incubator at 37°C for 4 hours.

[0237] Then, 20 μL / well of 1 mg / mL CL-264 (InvivoGen) was added, and after thorough mixing, the cells were cultured at 37° C. under 5% CO 2 for approximately 20 hours.

[0238] After thoroughly stirring the plate, it was centrifuged at 1500 rpm for 5 minutes, and the IL-6 concentration in the supernatant was measured by the FRET method (Cisbio).

[0239] FIG. 23 shows that humanized anti-human TLR7 antibodies inhibit IL-6 production from CL-264-treated human PBMCs in a concentration-dependent manner.

[0240] huAT01_H1L1_IgG1LALA, huAT01_H3L1_IgG1LALA, huAT01_H3L2_IgG1LALA, huAT01_H3L2_IgG4Pro, huNB7_H3L3_IgG1LALA, and huNB7_H3L3_IgG4Pro inhibited IL-6 production from human PBMCs in a concentration-dependent manner.

[0241] Half-maximal inhibitory concentrations (IC 50) were calculated from the concentrations between the two points that sandwiched 50% inhibitory activity and the inhibitory activity when those concentrations were added, and were 74 ng / mL, 138 ng / mL, 38 ng / mL, 24 ng / mL, 307 ng / mL, and 31 ng / mL, respectively.

[0242] [Example 8] Confirmation of antigen binding of humanized anti-human TLR7 antibodies 8)-1 Preparation of human TLR7 (variant 1)-Flag-expressing HEK293 cells Lenti - X® HEK293T cells (Clontech) were cultured at 4.5 × 10 6 At a concentration of 75cm 2 The cells were seeded in a flask (Sumitomo Bakelite Co., Ltd.) and cultured overnight at 37°C in 5% CO2 in DMEM medium (Fujifilm Wako Pure Chemical Industries, Ltd.) containing 10% FBS, 50 U / mL penicillin, and 50 μg / mL streptomycin (Life Technologies).

[0243] The next day, pcDNA3.1 prepared by Genscript - Lentivirus expression of human TLR7 (variant 1)-Flag plasmid - X® HEK293T cells (Clontech) were transfected with the transfection reagent Lipofectamine ( Gene transfer was performed using a 2000 (Life Technologies) The cells were then further cultured overnight at 37°C in 5% CO2.

[0244] The next day, the expression plasmid-transfected cells were treated with TrypLE Express (Life Technologies), and the treated cells were washed with Flow Cytometry Staining Buffer (Life Technologies). Flow Cytometry Staining Buffer (Life Tech The cells were suspended in a PBS (manufactured by Nologies) and the resulting cell suspension was used for flow cytometry analysis.

[0245] pcDNA3.1 - Expression of human TLR7 in cells transfected with the human TLR7 (variant 1)-Flag expression plasmid was confirmed by flow cytometry (Miltenyi Biotec) using an anti-Flag-PE antibody (Biolegend) or an isotype control antibody (Biolegend) as a negative control.

[0246] The amino acid sequence of human TLR7 (variant 1) and the polynucleotide sequence encoding it are set forth in SEQ ID NOs: 2 and 1, respectively, in the Sequence Listing.

[0247] 8)-2 Preparation of human TLR7 (variant 2)-Flag-expressing HEK293 cells Lenti - X® HEK293T cells (Clontech) were cultured at 4.5 × 10 6 At a concentration of 75cm 2 The cells were seeded in a flask (Sumitomo Bakelite Co., Ltd.) and cultured overnight at 37°C in 5% CO2 in DMEM medium (Fujifilm Wako Pure Chemical Industries, Ltd.) containing 10% FBS, 50 U / mL penicillin, and 50 μg / mL streptomycin (Life Technologies).

[0248] The next day, pcDNA3.1 prepared by Genscript - Lentivirus expression of human TLR7 (variant 2)-Flag plasmid - X® HEK293T cells (Clontech) were transfected with the transfection reagent Lipofectamine ( Gene transfer was performed using a 2000 (Life Technologies) The cells were then further cultured overnight at 37°C in 5% CO2.

[0249] The next day, the expression vector-transfected cells were treated with TrypLE Express (Life Technologies), and the treated cells were washed with Flow Cytometry Staining Buffer (Life Technologies). low Cytometry Staining Buffer(Life Techn The cells were suspended in a PBS (manufactured by Biologics) and the resulting cell suspension was used for flow cytometry analysis.

[0250] pcDNA3.1 - Expression of human TLR7 in cells transfected with the human TLR7 (variant 2)-Flag expression plasmid was confirmed by flow cytometry (Miltenyi Biotec) using anti-Flag-PE antibody (Biolegend) or an isotype control antibody (Biolegend) as a negative control.

[0251] 8)-3 Preparation of monkey TLR7-Flag-expressing HEK293 cells Lenti - X® HEK293T cells (Clontech) were cultured at 4.5 × 10 6 At a concentration of 75cm 2 The cells were seeded in a flask (Sumitomo Bakelite Co., Ltd.) and cultured overnight at 37°C in 5% CO2 in DMEM medium (Fujifilm Wako Pure Chemical Industries, Ltd.) containing 10% FBS, 50 U / mL penicillin, and 50 μg / mL streptomycin (Life Technologies).

[0252] The next day, pcDNA3.1 prepared by Genscript - Lentivirus-mediated ischemia-reactive protein (TLR7-Flag) expression plasmid. -X (registered trademark) HEK293T cells (Clontech) were transfected with the transfection reagent Lipofectamine (registered trademark) 2000 (Life Technologies), and further cultured overnight at 37°C and 5% CO 2 .

[0253] The next day, the expression vector-transfected cells were treated with TrypLE Express (Life Technologies), and the treated cells were washed with Flow Cytometry Staining Buffer (Life Technologies). low Cytometry Staining Buffer(Life Techn The cells were suspended in a PBS (manufactured by Biologics) and the resulting cell suspension was used for flow cytometry analysis.

[0254] pcDNA3.1 - Expression of monkey TLR7 in the monkey TLR7-Flag expression plasmid-transfected cells was confirmed by flow cytometry (Miltenyi Biotec) using anti-Flag-PE antibody (Biolegend) or an isotype control antibody (Biolegend) as a negative control.

[0255] 8)-4 Preparation of HEK293 cells overexpressing mouse TLR7-Flag Lenti - X® HEK293T cells (Clontech) were cultured at 4.5 × 10 6 At a concentration of 75cm 2 The cells were seeded in a flask (Sumitomo Bakelite Co., Ltd.) and cultured overnight at 37°C in 5% CO2 in DMEM medium (Fujifilm Wako Pure Chemical Industries, Ltd.) containing 10% FBS, 50 U / mL penicillin, and 50 μg / mL streptomycin (Life Technologies).

[0256] The next day, pcDNA3.1 prepared by Genscript -Lentivirus-mediated ischemia-reactive protein expression (TLR7-Flag) was detected in the mouse TLR7-Flag expression plasmid. - X (registered trademark) HEK293T cells (Clontech) were transfected with the transfection reagent Lipofectamine (registered trademark) 2000 (Life Technologies), and further cultured overnight at 37°C and 5% CO 2 .

[0257] The next day, the expression vector-transfected cells were treated with TrypLE Express (Life Technologies), and the treated cells were washed with Flow Cytometry Staining Buffer (Life Technologies). low Cytometry Staining Buffer(Life Techn The cells were suspended in a PBS (manufactured by Biologics) and the resulting cell suspension was used for flow cytometry analysis.

[0258] pcDNA3.1 - Mouse TLR7-Flag expression plasmid-transfected cells mouse The expression of TLR7 was confirmed by detection by flow cytometry (Miltenyi Biotec) using an anti-Flag-PE antibody (Biolegend) or, as a negative control, an isotype control antibody (Biolegend).

[0259] 8)-5 Preparation of rat TLR7-Flag-expressing HEK293 cells Lenti - X® HEK293T cells (Clontech) were cultured at 4.5 × 10 6 At a concentration of 75cm 2 The cells were seeded in a flask (Sumitomo Bakelite Co., Ltd.) and cultured overnight at 37°C in 5% CO2 in DMEM medium (Fujifilm Wako Pure Chemical Industries, Ltd.) containing 10% FBS, 50 U / mL penicillin, and 50 μg / mL streptomycin (Life Technologies).

[0260] The next day, pcDNA3.1 prepared by Genscript - Lentivirus-mediated ischemia-reactive protein expression (TLR7-Flag) was transfected with a rat TLR7 - X (registered trademark) HEK293T cells (Clontech) were transfected with the transfection reagent Lipofectamine (registered trademark) 2000 (Life Technologies), and further cultured overnight at 37°C and 5% CO 2 .

[0261] The next day, the expression vector-transfected cells were treated with TrypLE Express (Life Technologies), and the treated cells were washed with Flow Cytometry Staining Buffer (Life Technologies). low Cytometry Staining Buffer(Life Techn The cells were suspended in a PBS (manufactured by Biologics) and the resulting cell suspension was used for flow cytometry analysis.

[0262] pcDNA3.1 - Rat TLR7-Flag expression plasmid transfected cells rat The expression of TLR7 was confirmed by detection by flow cytometry (Miltenyi Biotec) using an anti-Flag-PE antibody (Biolegend) or, as a negative control, an isotype control antibody (Biolegend).

[0263] 8)-6 Evaluation of binding selectivity of humanized anti-human TLR7 antibodies by flow cytometry HEK293 cells overexpressing human TLR7 (variant 1)-Flag prepared in 8)-1, HEK293 cells overexpressing human TLR7 (variant 2)-Flag prepared in 8)-2, HEK293 cells overexpressing monkey TLR7-Flag prepared in 8)-3, HEK293 cells overexpressing mouse TLR7-Flag prepared in 8)-4, and HEK293 cells overexpressing rat TLR7-Flag prepared in 8)-5 were fixed / permeabilized with Fixation and Permeabilization Solution (Becton Dickinson), and then incubated with huAT01_H3L2_IgG1LALA, a humanized anti-human TLR7 antibody, and Alexa Fluor (registered trademark) as a secondary antibody. Staining was performed using 647 AffiniPure Goat anti-human IgG + IgM (H+L) (Jackson ImmunoResearch Inc.).

[0264] The binding of the humanized anti-human TLR7 antibody to human TLR7 was detected by flow cytometry (Miltenyi Biotec), and the flow cytometry data was analyzed using software. Data were analyzed using Flowjo, a software program.

[0265] As a result, huAT01_H3L2_IgG1LALA, one of the humanized anti-human TLR7 antibodies, specifically bound to human TLR7 (variant 1), human TLR7 (variant 2), or monkey TLR7, but did not bind to mouse TLR7 or rat TLR7. The results are shown in Figures 25 to 29. [Industrial Applicability]

[0266] The humanized anti-TLR7 antibodies of the present invention have the effect of suppressing the inhibitory activity of human TLR7 function, and can be used as therapeutic or preventive agents for immune-inflammatory diseases and the like. [Sequence List Free Text]

[0267] SEQ ID NO: 1: Nucleotide sequence encoding human TLR7 (variant 1) SEQ ID NO: 2: Amino acid sequence of human TLR7 (variant 1) SEQ ID NO: 3: Nucleotide sequence encoding human TLR7 (variant 2) SEQ ID NO: 4: Amino acid sequence of human TLR7 (variant 2) SEQ ID NO: 5: Amino acid sequence of the heavy chain variable region of the AT01 antibody SEQ ID NO: 6: Nucleotide sequence encoding the heavy chain variable region of the cAT01 antibody SEQ ID NO: 7: Amino acid sequence of the heavy chain variable region of the NB7 antibody SEQ ID NO: 8: Nucleotide sequence encoding the heavy chain variable region of the cNB7 antibody SEQ ID NO: 9: Amino acid sequence of the heavy chain variable region of the FAN2 antibody SEQ ID NO: 10: Nucleotide sequence encoding the heavy chain variable region of the cFAN2 antibody SEQ ID NO: 11: Amino acid sequence of the light chain variable region of the AT01 antibody SEQ ID NO: 12: Nucleotide sequence encoding the light chain variable region of the cAT01 antibody SEQ ID NO: 13: Amino acid sequence of the light chain variable region of the NB7 antibody SEQ ID NO: 14: Nucleotide sequence encoding the light chain variable region of the cNB7 antibody SEQ ID NO: 15: Amino acid sequence of the light chain variable region of the FAN2 antibody SEQ ID NO: 16: Nucleotide sequence encoding the light chain variable region of the cFAN2 antibody SEQ ID NO: 17: Amino acid sequence of CDRH1 of AT01 antibody SEQ ID NO: 18: Amino acid sequence of CDRH2 of AT01 antibody SEQ ID NO: 19: Amino acid sequence of CDRH3 of AT01 antibody SEQ ID NO: 20: Amino acid sequence of CDRL1 of AT01 antibody SEQ ID NO: 21: Amino acid sequence of CDRL2 of AT01 antibody SEQ ID NO: 22: Amino acid sequence of CDRL3 of AT01 antibody SEQ ID NO: 23: Amino acid sequence of CDRH1 of NB7 antibody SEQ ID NO: 24: Amino acid sequence of CDRH2 of NB7 antibody SEQ ID NO: 25: Amino acid sequence of CDRH3 of NB7 antibody SEQ ID NO: 26: Amino acid sequence of CDRL1 of NB7 antibody SEQ ID NO: 27: Amino acid sequence of CDRL2 of NB7 antibody SEQ ID NO: 28: Amino acid sequence of CDRL3 of NB7 antibody SEQ ID NO: 29: Amino acid sequence of CDRH1 of FAN2 antibody SEQ ID NO: 30: Amino acid sequence of CDRH2 of FAN2 antibody SEQ ID NO: 31: Amino acid sequence of CDRH3 of FAN2 antibody SEQ ID NO: 32: Amino acid sequence of CDRL1 of FAN2 antibody SEQ ID NO: 33: Amino acid sequence of CDRL2 of FAN2 antibody SEQ ID NO: 34: Amino acid sequence of CDRL3 of FAN2 antibody SEQ ID NO: 35: Amino acid sequence of the heavy chain of AT01 chimeric anti-human TLR7 antibody (cAT01) SEQ ID NO: 36: Amino acid sequence of the light chain of AT01 chimeric anti-human TLR7 antibody (cAT01) SEQ ID NO: 37: Amino acid sequence of the heavy chain of NB7 chimeric anti-human TLR7 antibody (cNB7) SEQ ID NO: 38: Amino acid sequence of the light chain of NB7 chimeric anti-human TLR7 antibody (cNB7) SEQ ID NO: 39: Amino acid sequence of the heavy chain of FAN2 chimeric anti-human TLR7 antibody (cFAN2) SEQ ID NO: 40: Amino acid sequence of the light chain of FAN2 chimeric anti-human TLR7 antibody (cFAN2) SEQ ID NO: 41: Amino acid sequence of the heavy chain variable region of huAT01_H1_IgG1LALA SEQ ID NO: 42: Amino acid sequence of the heavy chain variable region of huAT01_H3_IgG1LALA SEQ ID NO: 43: Amino acid sequence of the light chain variable region of huAT01_L1 SEQ ID NO: 44: Amino acid sequence of the light chain variable region of huAT01_L2 SEQ ID NO: 45: Amino acid sequence of huAT01_H1_IgG1LALA SEQ ID NO: 46: Amino acid sequence of huAT01_H3_IgG1LALA SEQ ID NO: 47: Amino acid sequence of huAT01_H3_IgG4Pro SEQ ID NO: 48: Amino acid sequence of huAT01_L1 SEQ ID NO: 49: Amino acid sequence of huAT01_L2 SEQ ID NO: 50: Amino acid sequence of the heavy chain variable region of huNB7_H3_IgG1LALA SEQ ID NO: 51: Amino acid sequence of the light chain variable region of huNB7_L3 SEQ ID NO: 52: Amino acid sequence of huNB7_H3_IgG1LALA SEQ ID NO: 53: Amino acid sequence of huNB7_L3 SEQ ID NO: 54: Amino acid sequence of huNB7_H3_IgG4Pro SEQ ID NO: 55: Nucleotide sequence of the human Unc93B1 gene SEQ ID NO: 56: Nucleotide sequence of the mouse TLR7 gene SEQ ID NO: 57: Nucleotide sequence of the rat TLR7 gene SEQ ID NO: 58: Nucleotide sequence of the monkey TLR7 gene SEQ ID NO: 59: Nucleotide sequence of a DNA fragment containing a nucleotide sequence encoding a human light chain signal sequence and a human kappa chain constant region SEQ ID NO: 60: Nucleotide sequence of a DNA fragment containing a nucleotide sequence encoding a human heavy chain signal sequence and a human IgG1 constant region SEQ ID NO: 61: Nucleotide sequence encoding the heavy chain of the cAT01 antibody SEQ ID NO: 62: Nucleotide sequence of a DNA fragment containing the nucleotide sequence encoding the light chain of the cAT01 antibody SEQ ID NO: 63: Nucleotide sequence encoding the heavy chain of the cNB7 antibody SEQ ID NO: 64: Nucleotide sequence of a DNA fragment containing the nucleotide sequence encoding the light chain of the cNB7 antibody SEQ ID NO: 65: Nucleotide sequence encoding the heavy chain of the cFAN2 antibody SEQ ID NO: 66: Nucleotide sequence of a DNA fragment containing the nucleotide sequence encoding the light chain of the cFAN2 antibody SEQ ID NO: 67: Nucleotide sequence encoding huAT01_H1_IgG1LALA SEQ ID NO: 68: Nucleotide sequence encoding huAT01_H3_IgG1LALA SEQ ID NO: 69: Nucleotide sequence encoding huAT01_H3_IgG4Pro SEQ ID NO: 70: Nucleotide sequence encoding huAT01_L1 SEQ ID NO: 71: Nucleotide sequence encoding huAT01_L2 SEQ ID NO: 72: Nucleotide sequence encoding huNB7_H3_IgG1LALA SEQ ID NO: 73: Nucleotide sequence encoding huNB7_H3_IgG4Pro SEQ ID NO: 74: Nucleotide sequence encoding huNB7_L3 SEQ ID NO: 75: Nucleotide sequence of a DNA fragment containing a nucleotide sequence encoding a human heavy chain signal sequence and a human IgG1 LALA constant region SEQ ID NO: 76: Nucleotide sequence of a DNA fragment containing a nucleotide sequence encoding a human heavy chain signal sequence and a human IgG4Pro constant region SEQ ID NO: 77: Nucleotide sequence encoding the heavy chain variable region of huAT01_H1 SEQ ID NO: 78: Nucleotide sequence encoding the heavy chain variable region of huAT01_H3 SEQ ID NO: 79: Nucleotide sequence encoding the light chain variable region of huAT01_L1 SEQ ID NO: 80: Nucleotide sequence encoding the light chain variable region of huAT01_L2 SEQ ID NO: 81: Nucleotide sequence encoding the heavy chain variable region of huNB7_H3 SEQ ID NO: 82: Nucleotide sequence encoding the light chain variable region of huNB7_L3 SEQ ID NO: 83: Amino acid sequence of mouse TLR7 SEQ ID NO: 84: Amino acid sequence of rat TLR7 SEQ ID NO: 85: Amino acid sequence of monkey TLR7 SEQ ID NO: 86: Nucleotide sequence of human Unc93B1 D34A mutant-HAx2 gene

Claims

[Claim 1] The invention described herein.

Images