CN110913894B - anti-CSF-1R antibodies, antigen binding fragments thereof and medical uses thereof - Google Patents

Abstract

The invention provides anti-CSF-1R antibodies, antigen-binding fragments thereof and pharmaceutical uses thereof; also provided are chimeric antibodies, humanized antibodies and medical uses thereof comprising CDR regions of the anti-CSF-1R antibodies.

Description

anti-CSF-1R antibodies, antigen binding fragments thereof and medical uses thereof

The present application claims priority from chinese patent application CN201711015488.5, filed on 26/10/2017. The present application refers to the above-mentioned chinese patent application in its entirety.

Technical Field

The present invention relates to an anti-CSF-1R antibody having immunoreactivity to a human CSF-1R receptor, and an antigen-binding fragment thereof, a chimeric antibody, a humanized antibody, and a pharmaceutical composition comprising the human anti-CSF-1R antibody and the antigen-binding fragment thereof, which comprise the CDR regions of the anti-CSF-1R antibody, and use thereof as an anti-cancer drug.

Background

The statements herein merely provide background information related to the present disclosure and may not necessarily constitute prior art.

Tumor immunotherapy is a long-standing hotspot in the field of tumor therapy, where T cell tumor immunotherapy is in turn at a central position. Tumor escape is a great obstacle for tumor immunotherapy, and most tumors express antigens that can be recognized by the host immune system to varying degrees, but in many cases, the tumor cells promote the wild growth of the tumor by virtue of their own suppressive effect on the immune system, due to inefficient activation of the immune cells, which triggers an inadequate immune response. The tumor immunotherapy is to fully utilize and mobilize various immune cells in the body of a tumor patient by various modes such as immune checkpoint inhibition, direct activation, tumor microenvironment activation and the like to kill the tumor.

Macrophages (macrophages) are an important cell population in the process of anti-tumor immune regulation and can directly kill tumor cells. However, in recent years, it has been proved that macrophages are classified into two types, M1 and M2, and macrophages in the tumor tissue interstitium, i.e., tumor-associated macrophages (TAMs), belong to the M2 type, and are not involved in the processes of tumor occurrence, growth, invasion and metastasis, as M1 type macrophages do. TAMs are derived from monocytes in the lymphatic circulation or macrophages remaining in the tissues and are the main type of leukocyte infiltrating many tumor stroma. Once activated, TAMs often become the primary source of cytokines, growth factors, and proteases in the tumor microenvironment, promoting tumor growth, proliferation, angiogenesis, invasion, metastasis, and chemotherapy resistance.

CSF-1R (colony stimulating factor-1 receptor, otherwise known as FMS, C-FMS, CD115, etc.) is a single-pass transmembrane receptor with an N-terminal extracellular domain (characterized by a repetitive Ig domain) and a C-terminal intracellular domain for tyrosine kinase activity. Binding of a CSF-1 or interleukin-34 (IL-34) ligand to CSF-1R results in receptor dimerization and up-regulation of tyrosine kinase activity, phosphorylation of CSF-1R tyrosine residues, and downstream signaling. Both CSF-1 and IL-34 ligands are capable of stimulating monocyte survival, proliferation and differentiation into macrophages. CSF-1R is expressed predominantly on cells of the monocytic lineage and in the female reproductive tract and placenta.

Many tumor cells secrete CSF-1.CSF-1 activates monocytes/M2 macrophages via CSF-1R. Intratumoral CSF-1 levels correlate with intratumoral TAM levels, while high levels of TAM correlate with poorer patient prognosis. CSF-1 has been found to promote tumor growth and progression to metastasis in human breast cancer xenografts in mice. On the other hand, CSF-1 and its receptor are also involved in various inflammatory and autoimmune diseases. CSF-1R stimulates proliferation, survival, activation and maturation of differentiated myeloid lineage cells and stimulates the ability of differentiated myeloid lineage cells to mediate disease pathology in a pathological setting. Accordingly, various classes of antagonists of CSF-1R receptor signaling, including monoclonal antibodies and the like, can be used to treat various CSF-1R mediated related diseases, such as cancer, inflammatory conditions and autoimmune diseases, by blocking monocyte activation to M2 macrophages.

At present, a plurality of pharmaceutical companies across countries develop monoclonal antibodies aiming at CSF-1R, and can achieve the purpose of killing tumor cells by antagonizing CSF1-R to inhibit TAM in a tumor microenvironment or be used for relevant macrophage-mediated autoimmune diseases and the like. Sheer, C.J. et al describe antibodies against CSF-1R that inhibit CSF-1 activity (Sherr, C.J. et al, 1989, blood 73. W02009/026303 discloses anti-CSF-1R antibodies that bind to human CSF-1R and in vivo mouse tumor models using anti-mouse CSF-1R antibodies. W02011/123381 discloses an anti-CSF-1R antibody that internalizes CSF-1R and has ADCC activity. W02011/123381 also discloses an in vivo mouse tumor model using anti-mouse CSF-1R antibodies. W02011/140249 discloses anti-CSF-1R antibodies that block the binding of CSF-1 to CSF-1R, which are believed to be useful in the treatment of cancer. W02009/112245 discloses anti-CSF-IR IgGl antibodies that inhibit CSF-1 binding to CSF-IR, which are believed to be useful in the treatment of cancer, inflammatory bowel disease and rheumatoid arthritis. W02011/131407 discloses anti-CSF-IR antibodies that inhibit CSF-l binding to CSF-IR, which are believed to be useful in the treatment of bone loss and cancer. W02011/107553 discloses anti-CSF-IR antibodies that inhibit CSF-l binding to CSF-IR, which are believed to be useful in the treatment of bone loss and cancer. W02011/070024 discloses anti-CSF-IR antibodies that bind to human CSF-IR fragment deID 4. Although therapeutic applications of anti-CSF-IR antibodies have been previously described for the treatment of certain cancers, there remains a need to develop new antibodies that are more suitable for clinical use.

Disclosure of Invention

Some embodiments of the invention provide an anti-CSF-1R antibody or antigen-binding fragment thereof, comprising:

an antibody light chain variable region comprising at least 1LCDR selected from the group consisting of seq id nos: the amino acid sequence of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16 or SEQ ID NO:96; and

an antibody heavy chain variable region comprising at least 1HCDR selected from the group consisting of seq id nos: SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:95,SEQ ID NO:97 or SEQ ID NO:98.

in other embodiments, the above anti-CSF-1R antibody or antigen-binding fragment thereof, comprising an antibody light chain variable region and an antibody heavy chain variable region, wherein:

a) The antibody light chain variable region comprises a sequence selected from the group consisting of SEQ ID NOs: 42. the amino acid sequence of SEQ ID NO:43 and SEQ ID NO: LCDR variants having 3, 2, 1 or 0 amino acid mutations based on LCDR1, LCDR2 and LCDR3, respectively, as shown in 44; the antibody heavy chain variable region comprises a sequence selected from the group consisting of SEQ ID NOs: 39. the amino acid sequence of SEQ ID NO:40 and SEQ ID NO: an HCDR variant having 3, 2, 1 or 0 amino acid mutations based on HCDR1, HCDR2 and HCDR3, respectively, as shown in 41;

b) The antibody light chain variable region comprises a sequence selected from the group consisting of SEQ ID NOs: 50. SEQ ID NO:51 and SEQ ID NO: LCDR variants having 3, 2, 1 or 0 amino acid mutations based on LCDR1, LCDR2 and LCDR3, respectively, as shown at 52; the antibody heavy chain variable region comprises a sequence selected from the group consisting of SEQ ID NOs: 47. SEQ ID NO:48 and SEQ ID NO: HCDR variants having 3, 2, 1 or 0 amino acid mutations based on HCDR1, HCDR2 and HCDR3, respectively, as shown in 49;

c) The antibody light chain variable region comprises a sequence selected from the group consisting of SEQ ID NOs: 58. SEQ ID NO:59 and SEQ ID NO: LCDR variants having 3, 2, 1 or 0 amino acid mutations based on LCDR1, LCDR2 and LCDR3, respectively, as shown at 60; the antibody heavy chain variable region is selected from the group consisting of SEQ ID NO: 55. SEQ ID NO:56 and SEQ ID NO: HCDR variants having 3, 2, 1 or 0 amino acid mutations based on HCDR1, HCDR2 and HCDR3 as shown in 57, respectively;

d) The antibody light chain variable region comprises a sequence selected from the group consisting of SEQ ID NOs: 66. SEQ ID NO:67 and SEQ ID NO: LCDR variants having 3, 2, 1 or 0 amino acid mutations based on LCDR1, LCDR2 and LCDR3, respectively, as shown in 68; the antibody heavy chain variable region comprises a sequence selected from the group consisting of SEQ ID NOs: 63. SEQ ID NO:64 and SEQ ID NO:65 HCDR1, HCDR2 and HCDR3 having 3, 2, 1 or 0 amino acid mutations, respectively;

e) The antibody light chain variable region comprises a sequence selected from the group consisting of SEQ ID NOs: 74. SEQ ID NO:75 and SEQ ID NO: LCDR variants having 3, 2, 1 or 0 amino acid mutations based on LCDR1, LCDR2 and LCDR3, respectively, as shown at 76; the antibody heavy chain variable region comprises a sequence selected from the group consisting of SEQ ID NOs: 71. the amino acid sequence of SEQ ID NO:72 and SEQ ID NO: an HCDR variant having 3, 2, 1 or 0 amino acid mutations based on HCDR1, HCDR2 and HCDR3, respectively, as shown in 73;

f) The antibody light chain variable region comprises a sequence selected from the group consisting of SEQ ID NOs: 82. SEQ ID NO:83 and SEQ ID NO: LCDR variants having 3, 2, 1 or 0 amino acid mutations based on LCDR1, LCDR2 and LCDR3, respectively, as shown at 84; the antibody heavy chain variable region comprises a sequence selected from the group consisting of SEQ ID NOs: 79. SEQ ID NO:80 and SEQ ID NO: HCDR variants having 3, 2, 1 or 0 amino acid mutations based on HCDR1, HCDR2 and HCDR3, respectively, as shown at 81;

g) The antibody light chain variable region comprises a sequence selected from the group consisting of SEQ ID NOs: 90. SEQ ID NO:91 and SEQ ID NO:92, and LCDR1, LCDR2 and LCDR3 having 3, 2, 1 or 0 amino acid mutations, respectively; the antibody heavy chain variable region comprises a sequence selected from the group consisting of SEQ ID NOs: 87. the amino acid sequence of SEQ ID NO:88 and SEQ ID NO:89 HCDR1, HCDR2 and HCDR3, each having 3, 2, 1 or 0 amino acid mutations;

h) The antibody light chain variable region comprises a sequence selected from the group consisting of SEQ ID NOs: 6. the amino acid sequence of SEQ ID NO:7 and SEQ ID NO: LCDR variants having 3, 2, 1 or 0 amino acid mutations based on LCDR1, LCDR2 and LCDR3, respectively, as shown in fig. 8; the variable region of the antibody heavy chain comprises a sequence selected from the group consisting of SEQ ID NOs: 3. the amino acid sequence of SEQ ID NO:4 and SEQ ID NO:5 HCDR1, HCDR2 and HCDR3 having 3, 2, 1 or 0 amino acid mutations, respectively;

i) The antibody light chain variable region comprises a sequence selected from the group consisting of SEQ ID NOs: 14. the amino acid sequence of SEQ ID NO:15 and SEQ ID NO: LCDR variants having 3, 2, 1 or 0 amino acid mutations based on LCDR1, LCDR2 and LCDR3, respectively, as shown in 16; the antibody heavy chain variable region comprises a sequence selected from the group consisting of SEQ ID NOs: 11. SEQ ID NO:12 and SEQ ID NO:13 HCDR1, HCDR2 and HCDR3 having 3, 2, 1 or 0 amino acid mutations, respectively;

j) The antibody light chain variable region comprises a sequence selected from the group consisting of SEQ ID NOs: 14. the amino acid sequence of SEQ ID NO:15 and SEQ ID NO:16 with 3, 2, 1 or 0 amino acid mutations based on LCDR1, LCDR2 and LCDR3, respectively; the antibody heavy chain variable region comprises a sequence selected from the group consisting of: SEQ ID NO:98. SEQ ID NO:12 and SEQ ID NO:13 HCDR1, HCDR2 and HCDR3 having 3, 2, 1 or 0 amino acid mutations, respectively;

k) The antibody light chain variable region comprises a sequence selected from the group consisting of SEQ ID NOs: 14. SEQ ID NO15 and SEQ ID NO: LCDR variants having 3, 2, 1 or 0 amino acid mutations based on LCDR1, LCDR2 and LCDR3, respectively, as shown in 16; the antibody heavy chain variable region comprises a sequence selected from the group consisting of: SEQ ID NO:98. SEQ ID NO:97 and SEQ ID NO:13 HCDR1, HCDR2 and HCDR3 having 3, 2, 1 or 0 amino acid mutations therein, respectively;

1) The antibody light chain variable region comprises a sequence selected from the group consisting of SEQ ID NOs: 96. the amino acid sequence of SEQ ID NO:7 and SEQ ID NO: LCDR variants having 3, 2, 1 or 0 amino acid mutations based on LCDR1, LCDR2 and LCDR3, respectively, as shown in fig. 8; the antibody heavy chain variable region comprises a sequence selected from the group consisting of: SEQ ID NO: 3. SEQ ID NO:4 and SEQ ID NO:5 HCDR1, HCDR2 and HCDR3 having 3, 2, 1 or 0 amino acid mutations, respectively;

m) comprises a light chain variable region selected from the group consisting of SEQ ID NOs: 6. SEQ ID NO:7 and SEQ ID NO: LCDR variants having 3, 2, 1 or 0 amino acid mutations based on LCDR1, LCDR2 and LCDR3, respectively, as shown in fig. 8; the antibody heavy chain variable region comprises a sequence selected from the group consisting of: SEQ ID NO: 3. the amino acid sequence of SEQ ID NO:95 and SEQ ID NO:5 HCDR1, HCDR2 and HCDR3 having 3, 2, 1 or 0 amino acid mutations, respectively;

n) comprises a light chain variable region selected from the group consisting of the amino acid sequences set forth in SEQ ID NO: 96. the amino acid sequence of SEQ ID NO:7 and SEQ ID NO: LCDR variants having 3, 2, 1 or 0 amino acid mutations based on LCDR1, LCDR2 and LCDR3, respectively, as shown in fig. 8; the antibody heavy chain variable region comprises a sequence selected from the group consisting of: SEQ ID NO: 3. the amino acid sequence of SEQ ID NO:95 and SEQ ID NO:5 HCDR1, HCDR2 and HCDR3 having 3, 2, 1 or 0 amino acid mutations, respectively; or

o) said antibody light chain variable region comprises a sequence selected from the group consisting of: the amino acid sequence of SEQ ID NO: 14. the amino acid sequence of SEQ ID NO:15 and SEQ ID NO: LCDR variants having 3, 2, 1 or 0 amino acid mutations based on LCDR1, LCDR2 and LCDR3, respectively, as shown in 16; the antibody heavy chain variable region comprises a sequence selected from the group consisting of: the amino acid sequence of SEQ ID NO: 11. SEQ ID NO:97 and SEQ ID NO:13 HCDR1, HCDR2 and HCDR3 are variants having 3, 2, 1 or 0 amino acid mutations, respectively.

In other embodiments, an anti-CSF-1R antibody or antigen-binding fragment thereof, as described above, comprises an antibody light chain variable region and an antibody heavy chain variable region, wherein:

p) comprises the variable regions of the antibody light chain as set forth in SEQ ID NOs: 42. SEQ ID NO:43 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 as shown at 44; the heavy chain variable region of the antibody comprises the amino acid sequences shown as SEQ ID NO: 39. the amino acid sequence of SEQ ID NO:40 and SEQ ID NO:41 HCDR1, HCDR2 and HCDR3;

q) comprises the amino acid sequences shown in SEQ ID NO: 50. the amino acid sequence of SEQ ID NO:51 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 shown at 52; the heavy chain variable region of the antibody comprises the amino acid sequences shown as SEQ ID NO: 47. SEQ ID NO:48 and SEQ ID NO: HCDR1, HCDR2 and HCDR3 shown at 49;

r) comprises the amino acid sequences shown in SEQ ID NO: 58. SEQ ID NO:59 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 as shown at 60; the heavy chain variable region of the antibody comprises the amino acid sequences shown as SEQ ID NO: 55. the amino acid sequence of SEQ ID NO:56 and SEQ ID NO: HCDR1, HCDR2 and HCDR3 shown at 57;

s) comprises the amino acid sequence as set forth in SEQ ID NO: 66. SEQ ID NO:67 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 as shown at 68; the heavy chain variable region of the antibody comprises the amino acid sequences shown as SEQ ID NO: 63. SEQ ID NO:64 and SEQ ID NO:65 HCDR1, HCDR2 and HCDR3;

t) the antibody light chain variable region comprises the amino acid sequences as set forth in SEQ ID NOs: 74. SEQ ID NO:75 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 shown at 76; the heavy chain variable region of the antibody comprises the amino acid sequences shown as SEQ ID NO: 71. SEQ ID NO:72 and SEQ ID NO: HCDR1, HCDR2 and HCDR3 shown at 73;

u) comprises the amino acid sequences shown in SEQ ID NO: 82. the amino acid sequence of SEQ ID NO:83 and SEQ ID NO: LCDR1, LCDR2, and LCDR3 as shown at 84; the heavy chain variable region of the antibody comprises the amino acid sequences shown as SEQ ID NO: 79. SEQ ID NO:80 and SEQ ID NO: HCDR1, HCDR2 and HCDR3 shown at 81;

v) the antibody light chain variable region comprises the amino acid sequences shown in SEQ ID NO: 90. SEQ ID NO:91 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 shown at 92; the heavy chain variable region of the antibody comprises the amino acid sequences shown as SEQ ID NO: 87. SEQ ID NO:88 and SEQ ID NO: HCDR1, HCDR2 and HCDR3 shown in 89;

w) the antibody light chain variable region comprises the amino acid sequences as set forth in SEQ ID NOs: 6. SEQ ID NO:7 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 as shown in 8; the heavy chain variable region of the antibody comprises the amino acid sequences shown as SEQ ID NO: 3. the amino acid sequence of SEQ ID NO:4 and SEQ ID NO:5 HCDR1, HCDR2 and HCDR3;

x) comprises the variable regions as set forth in SEQ ID NOs: 14. SEQ ID NO:15 and SEQ ID NO:16, LCDR1, LCDR2, and LCDR3; the heavy chain variable region of the antibody comprises the amino acid sequences shown as SEQ ID NO: 11. SEQ ID NO:12 and SEQ ID NO:13 HCDR1, HCDR2 and HCDR3;

y) comprises the amino acid sequences shown in SEQ ID NO: 14. SEQ ID NO:15 and SEQ ID NO:16, LCDR1, LCDR2, and LCDR3; the variable region of the antibody heavy chain comprises the following components: the amino acid sequence of SEQ ID NO:98. SEQ ID NO:12 and SEQ ID NO:13 HCDR1, HCDR2 and HCDR3;

z) comprises the amino acid sequences shown in SEQ ID NO: 14. SEQ ID NO15 and SEQ ID NO:16, LCDR1, LCDR2, and LCDR3; the variable region of the antibody heavy chain comprises the following components: SEQ ID NO:98. the amino acid sequence of SEQ ID NO:97 and SEQ ID NO:13 HCDR1, HCDR2 and HCDR3;

aa) comprises the variable region of the antibody light chain as set forth in SEQ ID NO: 96. SEQ ID NO:7 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 as shown in fig. 8; the variable region of the antibody heavy chain comprises the following components: SEQ ID NO: 3. SEQ ID NO:4 and SEQ ID NO:5 HCDR1, HCDR2 and HCDR3;

ab) comprises the amino acid sequence set forth in SEQ ID NO: 6. the amino acid sequence of SEQ ID NO:7 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 as shown in 8; the variable region of the antibody heavy chain comprises the following components: the amino acid sequence of SEQ ID NO: 3. SEQ ID NO:95 and SEQ ID NO:5 HCDR1, HCDR2 and HCDR3;

ac) comprises the variable regions as set forth in SEQ ID NOs: 96. SEQ ID NO:7 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 as shown in fig. 8; the variable region of the antibody heavy chain comprises the following components: SEQ ID NO: 3. SEQ ID NO:95 and SEQ ID NO:5 HCDR1, HCDR2 and HCDR3;

ad) the antibody light chain variable region comprises: SEQ ID NO: 14. SEQ ID NO:15 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 as shown at 16; the variable region of the antibody heavy chain comprises the following components: SEQ ID NO: 11. SEQ ID NO:97 and SEQ ID NO:13 HCDR1, HCDR2 and HCDR3.

In other embodiments, an anti-CSF-1R antibody or antigen-binding fragment thereof as described above, wherein said antibody or antigen-binding fragment thereof is a murine antibody or a chimeric antibody.

In other embodiments, the antibody or antigen-binding fragment as described above, wherein

a) The amino acid sequence of the light chain variable region is shown as SEQ ID NO:38, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO:37 is shown in the figure; or alternatively

b) The amino acid sequence of the light chain variable region is shown as SEQ ID NO:46, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO:45 is shown; or alternatively

c) The amino acid sequence of the light chain variable region is shown as SEQ ID NO:54, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO: shown at 53; or

d) The amino acid sequence of the light chain variable region is shown as SEQ ID NO:62, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO: 61; or

e) The amino acid sequence of the light chain variable region is shown as SEQ ID NO:70, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO: 69; or

f) The amino acid sequence of the light chain variable region is shown as SEQ ID NO:78, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO: 77; or

g) The amino acid sequence of the light chain variable region is shown as SEQ ID NO:86, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO:85 is shown; or alternatively

h) The amino acid sequence of the light chain variable region is shown as SEQ ID NO:2 or a variant thereof, preferably having 0-10 amino acid mutation in the light chain variable region, most preferably N37T amino acid mutation, and the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:1 or a variant thereof, said variant preferably having 0-10 amino acid mutations in the heavy chain variable region, preferably the amino acid mutation is N55T; or alternatively

i) The amino acid sequence of the light chain variable region is shown as SEQ ID NO:10, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO:9 or a variant thereof, said variant preferably having 0-10 amino acid mutations in the light chain variable region, preferably mutations being amino acid mutations of M34I, N55T or combinations thereof.

In other embodiments, an anti-CSF-1R antibody or antigen-binding fragment thereof as described above, wherein the antibody or antigen-binding fragment thereof is a humanized antibody or antigen-binding fragment thereof.

In other embodiments, the anti-CSF-1R antibody or antigen-binding fragment thereof, as described above, wherein the light chain FR region sequence on the humanized antibody light chain variable region is derived from a light chain FR region sequence as set forth in SEQ ID NO:22, a human germline light chain IGkV4-1 sequence; or from a nucleic acid sequence as set forth in SEQ ID NO:24, a human germline light chain IGkV1-13 sequence;

and/or, the humanized antibody heavy chain variable region further comprises a heavy chain FR region of human IgG1, igG2, igG3 or IgG4 or a variant thereof, preferably comprises a human IgG1, igG2 or IgG4 heavy chain FR region, more preferably comprises a human IgG1 or IgG4 heavy chain FR region; preferably, the heavy chain FR region sequence in the heavy chain variable region of the humanized antibody is derived from the amino acid sequence set forth in SEQ ID NO:21, human germline heavy chain IGHV1-46 sequence; or from a nucleic acid sequence as set forth in SEQ ID NO:23, and a human germline heavy chain IGHV1-2 sequence.

In other embodiments, the anti-CSF-1R antibody or antigen-binding fragment thereof as described above, wherein the humanized antibody light chain variable region sequence is as set forth in SEQ ID NO:30 or SEQ ID NO:34 or a variant thereof; the variant preferably has 0-10 amino acid mutations in the light chain variable region, wherein SEQ ID NO:30 most preferred amino acid mutations are S31N, T37N: SEQ ID NO:34 is preferably mutated to F87A, F91Y or a combination thereof;

and/or the sequence of the heavy chain variable region of the humanized antibody is shown as SEQ ID NO: 26. SEQ ID NO:100 or SEQ ID NO:33 or a variant thereof; the variant preferably has 0-10 amino acid mutations in the heavy chain variable region, wherein SEQ ID NO:26 preferred amino acid mutations are S30T, T55N, L70M or combinations thereof, most preferred amino acid mutation is T55N; SEQ ID NO:33 preferred amino acid mutations are Q1E, M34I, T55N, E89D or combinations thereof, most preferred amino acid mutations are M34I, T55N or combinations thereof;

preferably, the heavy chain variable region has the sequence of SEQ ID NO: 31. 32, 33 or 100 and the light chain variable region has the sequence of SEQ ID NO: 34. 35 or 36;

or, the sequence of the heavy chain variable region is SEQ ID NO: 25. 26, 27 or 28 and the light chain variable region has the sequence of SEQ ID NO:29 or 30.

In other embodiments, the anti-CSF-1R antibody or antigen-binding fragment thereof, as described above, wherein the chimeric or humanized antibody further comprises a light chain and/or a heavy chain constant region, the light chain constant region sequence being as set forth in SEQ ID NO:102, and/or the heavy chain constant region sequence is as set forth in SEQ ID NO: shown at 101.

In other embodiments, an anti-CSF-1R antibody or antigen-binding fragment thereof, as described above, wherein the humanized antibody:

comprises a sequence shown as SEQ ID NO:19, and a heavy chain having the sequence set forth in SEQ ID NO:20, a light chain;

comprises a sequence shown as SEQ ID NO:17, and a heavy chain having the sequence shown in SEQ ID NO:18, a light chain; or comprises a sequence shown as SEQ ID NO:99, and the heavy chain having the sequence shown in SEQ ID NO:20, or a light chain as shown.

In other embodiments, the murine antibody or fragment thereof as described above, wherein the antibody light chain variable region further comprises a light chain FR region of a murine kappa, lambda chain or variant thereof.

In other embodiments, the murine antibody or fragment thereof as described above further comprises a light chain constant region of a murine kappa, lambda chain or variant thereof.

In other embodiments, the murine antibody or fragment thereof as described above further comprises a heavy chain FR region of murine IgG1, igG2, igG3, igG4 or a variant thereof in the antibody heavy chain variable region.

In other embodiments, the murine antibody or fragment thereof as described above further comprises a heavy chain constant region of murine IgG1, igG2, igG3, igG4 or a variant thereof.

In other embodiments, an anti-CSF-1R antibody or antigen-binding fragment thereof as described above, wherein said antibody is a chimeric antibody or fragment thereof.

In other embodiments, a CSF-1R chimeric antibody or fragment thereof as described above, wherein the chimeric antibody light chain variable region sequence is: SEQ ID NO:2 or SEQ ID NO:10. the variant preferably has 0-10 amino acid changes in the light chain variable region, most preferably SEQ ID NO:2 to N37T.

In other embodiments, the CSF-1R chimeric antibody or fragment thereof as described above, wherein the chimeric antibody heavy chain variable region sequence is: SEQ ID NO:1 or SEQ ID NO:9, wherein SEQ ID NO:1 the preferred amino acid mutation is T55N; SEQ ID NO: preferred mutations of 9 are amino acid mutations of M34I, T55N or a combination thereof. In a preferred embodiment of the invention, a CSF-1R chimeric antibody or fragment thereof as described above further comprises a light chain constant region of a human kappa, lambda chain or variant thereof.

In other embodiments, the CSF-1R chimeric antibody or fragment thereof as described above further comprises a heavy chain constant region of human IgG1, igG2, igG3 or IgG4 or a variant thereof.

In other embodiments, an anti-CSF-1R antibody or antigen-binding fragment thereof as described above, wherein the antibody is a humanized antibody or antigen-binding fragment thereof.

In other embodiments, the CSF-1R humanized antibody or fragment thereof described above, the humanized antibody light chain variable region further comprises a light chain FR region of a human kappa, lambda chain or variant thereof.

In a preferred embodiment of the invention, a CSF-1R humanized antibody or fragment thereof as described above, wherein the humanized antibody light chain variable region sequence is as set forth in SEQ ID NO:30 or SEQ ID NO:34, or a variant thereof.

In other embodiments, a CSF-1R humanized antibody or fragment thereof as described above, wherein the humanized antibody light chain sequence is as set forth in SEQ ID NO:18 or SEQ ID NO:20, or a variant thereof; the humanized antibody variant preferably has 0-10 amino acid changes in the light chain variable region; wherein SEQ ID NO:18 to S31N, T37N or a combination thereof, most preferably to T37N; SEQ ID NO:20 preferred amino acid mutations are those of F87A, F91Y or a combination thereof. In a preferred embodiment of the invention, a CSF-1R humanized antibody or fragment thereof as described above further comprises a light chain constant region of a human kappa, lambda chain or variant thereof.

In other embodiments, the CSF-1R humanized antibody or fragment thereof as described above, the humanized antibody heavy chain variable region further comprises a heavy chain FR region of human IgG1, igG2, igG3, igG4 or a variant thereof.

In other embodiments, the CSF-1R humanized antibody or fragment thereof as described above further comprises a heavy chain constant region of human IgG1, igG2, igG3 or IgG4 or a variant thereof, preferably a human IgG1, igG2 or IgG4 heavy chain constant region. More preferably, igG 1is used which eliminates ADCC (antibody-dependent cell-mediated cytotoxicity) toxicity after IgG4 or amino acid mutation.

In another aspect, some embodiments of the invention also provide an isolated monoclonal antibody, or antigen-binding fragment thereof, that competes for binding to CSF-1R with an anti-CSF-1R antibody, or antigen-binding fragment thereof, as described above.

In a preferred embodiment of the present invention, an antibody as described above (the antibody described herein and below is the above-described anti-CSF-1R antibody or the above-described monoclonal antibody) or an antigen-binding fragment thereof, wherein the antigen-binding fragment is Fab, fv, sFv, F (ab') 2, linear antibody, single-chain antibody, nanobody, domain antibody, and multispecific antibody.

In another aspect, some embodiments of the invention also provide a multispecific antibody comprising a light chain variable region and/or a heavy chain variable region of an antibody or antigen-binding fragment thereof as described above.

In another aspect, some embodiments of the invention also provide a single chain antibody comprising the light chain variable region and/or the heavy chain variable region of the antibody or antigen binding fragment thereof as described above.

In another aspect, some embodiments of the present invention also provide an antibody-drug conjugate, wherein the antibody comprises a light chain variable region and/or a heavy chain variable region of the antibody or antigen binding fragment thereof as described above.

In another aspect, some embodiments of the invention also provide a nucleic acid molecule encoding an antibody or antigen-binding fragment thereof as described above, a multispecific antibody as described above, or a single chain antibody as described above.

In another aspect, some embodiments of the invention also provide a DNA sequence encoding an antibody or antigen-binding fragment thereof as described above.

In another aspect, some embodiments of the present invention also provide an expression vector comprising a DNA sequence as described above.

In some embodiments of the invention there is also provided a host cell transformed with an expression vector as described above.

In some embodiments, a host cell as described above, said host cell being a bacterium, preferably E.coli.

In other embodiments, one of the host cells described above is a yeast, preferably pichia pastoris.

In other embodiments, a host cell as described above is a mammalian cell, preferably a Chinese Hamster Ovary (CHO) cell, a Human Embryonic Kidney (HEK) 293 cell, or an NS0 cell.

In another aspect, some embodiments of the invention also provide an antibody-drug conjugate comprising a light chain variable region and a heavy chain variable region as described above. Such antibody-drug conjugates are well known in the art and are formed by antibody-linker-drug (toxin) interconnections, known linkers including cleavage linkers, such as linkers including but not limited to SMCC, SPDP, and the like; toxins are also well known in the art, such as DM1, DM4, MMAE, MMAF, and the like.

In another aspect, some embodiments of the invention also provide a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof as described above and a pharmaceutically acceptable buffer, excipient, diluent, or carrier.

In other embodiments, an antibody or fragment thereof, multispecific antibody, single chain antibody, nucleic acid molecule, or pharmaceutical composition as described above is administered in the treatment of cancer, wherein the administration is before, during, substantially simultaneously with, or after the initiation of therapy with another anti-cancer treatment.

In other embodiments, a CSF-1R humanized antibody or fragment thereof as described above, wherein the anti-cancer treatment is selected from the group consisting of an anti-angiogenic agent, a chemotherapeutic agent, radiation, tumor immunotherapy, or a combination thereof.

In other embodiments, a CSF-1R humanized antibody or fragment thereof as described above, wherein the chemotherapeutic agent is selected from the group consisting of: paclitaxel (paclitaxel, torpedo, modified paclitaxel (Abraxane and Opaxio)), doxorubicin, modified doxorubicin (Caelyx or Doxil)), sunitinib, sorafenib and other multi-kinase inhibitors, oxaliplatin, cisplatin, carboplatin, etoposide, gemcitabine and vinblastine.

In other embodiments, a CSF-1R humanized antibody or fragment thereof as described above, wherein the tumor immunotherapy is selected from the group consisting of: t cell cement (engaging agent), targeted immunosuppression, cancer vaccine/enhanced dendritic cell function and adoptive cell transfer.

In another aspect, some embodiments of the invention further provide a use of an antibody or antigen-binding fragment thereof as described above in the manufacture of a medicament for treating a CSF-1R or CSF-1 mediated disease or disorder; wherein the disease is preferably cancer, an autoimmune disease, an inflammatory disease or osteolytic bone loss; most preferably, the cancer is breast cancer, endometrial cancer, squamous cell carcinoma, follicular lymphoma, renal cell carcinoma, uveal melanoma, cervical cancer, head and neck cancer, hodgkin's disease, astrocytic cancer, lung adenocarcinoma, mesothelioma, choriocarcinoma, melanoma, breast cancer, ovarian cancer, prostate cancer, pancreatic cancer, kidney cancer, lung cancer, liver cancer, brain cancer, stomach cancer, colorectal cancer, bladder cancer, esophageal cancer, cervical cancer, multiple myeloma, leukemia, lymphoma, and glioblastoma; said autoimmune disease is most preferably autoimmune encephalomyelitis, systemic lupus erythematosus, multiple sclerosis, arthromeningitis, psoriasis, and rheumatoid arthritis; the osteolytic bone loss is selected from the group consisting of osteoporosis, metastasis-induced osteolytic bone loss, and rheumatoid arthritis-induced bone loss. The present invention further provides a method of treating and preventing CSF-1R or CSF-1 mediated diseases or disorders, comprising administering to a patient in need thereof a therapeutically effective amount of an antibody or antigen-binding fragment thereof as described above, or a pharmaceutical composition comprising the same; wherein the disease is preferably cancer, an autoimmune disease, an inflammatory disease or osteolytic bone loss; most preferably, the cancer is breast cancer, endometrial cancer, squamous cell carcinoma, follicular lymphoma, renal cell carcinoma, uveal melanoma, cervical cancer, head and neck cancer, hodgkin's disease, astrocytic cancer, lung adenocarcinoma, mesothelioma, choriocarcinoma, melanoma, breast cancer, ovarian cancer, prostate cancer, pancreatic cancer, kidney cancer, lung cancer, liver cancer, brain cancer, stomach cancer, colorectal cancer, bladder cancer, esophageal cancer, cervical cancer, multiple myeloma, leukemia, lymphoma, and glioblastoma; said autoimmune disease is most preferably autoimmune encephalomyelitis, systemic lupus erythematosus, multiple sclerosis, arthromeningitis, psoriasis, and rheumatoid arthritis; the osteolytic bone loss is selected from the group consisting of osteoporosis, metastasis-induced osteolytic bone loss, and rheumatoid arthritis-induced bone loss.

Drawings

FIG. 1: ELISA (enzyme-Linked immuno sorbent assay) in-vitro binding experiments of nine CSF-1R chimeric antibodies show that except C27C, the other antibodies have strong cross binding with cynomolgus monkey CSF-1R protein.

FIG. 2 is a schematic diagram: humanized antibody and high CSF-1R expression CHO cell in vitro binding activity experiment. The humanized antibodies huC11 and huC19 had cell binding strengths comparable to their original chimeric antibodies, C11C and C19C, respectively.

FIG. 3: functional experiments of humanized antibody huC11 blocking monocyte proliferation. Antibodies block IL-34 stimulated monocyte proliferation. Antibodies block CSF-1-stimulated monocyte proliferation.

FIG. 4: functional experiment of humanized antibody huC19 for blocking monocyte proliferation. Antibodies block IL-34 stimulated monocyte proliferation. (B) the antibody blocks CSF-1-stimulated monocyte proliferation.

FIG. 5: in vivo efficacy experiments with humanized antibody huC 19I. The antibody inhibited the growth of MDA-MB-231 tumor cells.

FIG. 6: in vivo efficacy experiments for humanized antibody huC 19I. The anti-CSF 1R antibody had no significant effect on animal body weight compared to the control group.

Detailed Description

1. Term(s) for

In order that the invention may be more readily understood, certain technical and scientific terms are specifically defined below. Unless clearly defined otherwise herein, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The three letter codes and the one letter codes for amino acids used in the present invention are as described in j.biol.chem,243, p3558 (1968).

The term "antibody" as used herein refers to an immunoglobulin, which is a tetrapeptide chain structure of two identical heavy chains and two identical light chains linked by interchain disulfide bonds. The constant regions of immunoglobulin heavy chains differ in their amino acid composition and arrangement, and thus in their antigenicity. Accordingly, immunoglobulins can be classified into five classes, otherwise known as the isotype of immunoglobulins, i.e., igM, igD, igG, igA and IgE, with their corresponding heavy chains being the μ, δ, γ, α and ε chains, respectively. The same class of igs can be divided into different subclasses according to differences in amino acid composition of the hinge region and the number and position of disulfide bonds in the heavy chain, for example, igG can be divided into IgG1, igG2, igG3 and IgG4. Light chains are classified as either kappa or lambda chains by differences in the constant regions. The second of the five classes of Ig may have either a kappa chain or a lambda chain.

In the present invention, the antibody light chain variable region of the present invention may further comprise a light chain constant region comprising a human or murine kappa or lambda chain or a variant thereof.

In the present invention, the antibody heavy chain variable region of the present invention may further comprise a heavy chain constant region comprising human or murine IgG1,2,3,4 or variants thereof.

The sequences of the antibody heavy and light chains, near the N-terminus, are widely varied by about 110 amino acids, as are the variable regions (V-regions); the remaining amino acid sequence near the C-terminus is relatively stable and is a constant region (C region). The variable regions include 3 hypervariable regions (HVRs) and 4 Framework Regions (FRs) which are relatively sequence-conserved. The 3 hypervariable regions determine the specificity of the antibody, also known as Complementarity Determining Regions (CDRs). Each of the light chain variable region (VL) and the heavy chain variable region (VH) is composed of 3 CDR regions and 4 FR regions, and the sequence from the amino terminus to the carboxyl terminus is: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The 3 CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the 3 CDR regions of the heavy chain refer to HCDR1, HCDR2 and HCDR3. The CDR amino acid residues of the VL and VH regions of the antibodies or antigen binding fragments of the invention conform in number and position to known IMGT numbering rules.

The term "antigen presenting cell" or "APC" is a cell that displays foreign antigens complexed with MHC on its surface. T cells recognize this complex using the T Cell Receptor (TCR). Examples of APCs include, but are not limited to, dendritic Cells (DCs), peripheral Blood Mononuclear Cells (PBMCs), monocytes, B lymphoblastoid cells, and monocyte-derived Dendritic Cells (DCs). The term "antigen presentation" refers to the process by which APCs capture antigens and enable them to be recognized by T cells, for example as a component of an MHC-I/MHC-II conjugate.

The term "CSF-1R" refers to the colony stimulating factor-1 receptor protein, a single pass transmembrane receptor with an N-terminal extracellular domain and a C-terminal intracellular domain of tyrosine kinase activity. CSF-1R is a member of the RTK family that contains an immunoglobulin (Ig) motif, characterized by a repetitive Ig domain in the extracellular portion of the receptor. Activation of CSF-1R is mediated by its ligand M-CSF or interleukin IL-34, whose signaling plays important physiological roles in immune response, bone remodeling, and in the reproductive system.

The term "recombinant human antibody" includes human antibodies made, expressed, created or isolated by recombinant methods, involving techniques and methods well known in the art, such as (1) antibodies isolated from transgenes of human immunoglobulin genes, transchromosomal animals (e.g., mice), or hybridomas made therefrom; (2) Antibodies isolated from host cells transformed to express the antibodies, such as transfectomas; (3) antibodies isolated from a library of recombinant combinatorial human antibodies; and (4) antibodies prepared, expressed, created or isolated by splicing human immunoglobulin gene sequences to other DNA sequences, and the like. Such recombinant human antibodies comprise variable and constant regions that utilize specific human germline immunoglobulin sequences encoded by germline genes, but also include subsequent rearrangements and mutations such as occur during antibody maturation.

The term "murine antibody" is used in the present invention as a monoclonal antibody to human CSF-1R prepared according to the knowledge and skill in the art. Preparation is performed by injecting a subject with the CSF-1R antigen and then isolating hybridomas that express antibodies having the desired sequence or functional properties. In a preferred embodiment of the invention, the murine CSF-1R antibody or antigen binding fragment thereof may further comprise a light chain constant region of a murine kappa, lambda chain or variant thereof, or further comprise a heavy chain constant region of a murine IgG1, igG2, igG3 or IgG4 or variant thereof.

The term "human antibody" includes antibodies having variable and constant regions of human germline immunoglobulin sequences. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody" does not include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences (i.e., "humanized antibodies").

The term "humanized antibody", also known as CDR-grafted antibody (CDR), refers to an antibody produced by grafting a mouse CDR sequence into a human antibody variable region framework. Can overcome the strong immune response induced by the chimeric antibody because of carrying a large amount of mouse protein components. To avoid a decrease in activity associated with a decrease in immunogenicity, the human antibody variable regions may be subjected to minimal back-mutation to maintain activity.

The term "chimeric antibody" refers to an antibody obtained by fusing a variable region of a murine antibody to a constant region of a human antibody, and can reduce an immune response induced by the murine antibody. Establishing a chimeric antibody, selecting and establishing a hybridoma secreting a mouse-derived specific monoclonal antibody, cloning a variable region gene from a mouse hybridoma cell, cloning a constant region gene of a human antibody according to needs, connecting the mouse variable region gene and the human constant region gene into a chimeric gene, inserting the chimeric gene into a human vector, and finally expressing a chimeric antibody molecule in a eukaryotic industrial system or a prokaryotic industrial system. The constant region of the human antibody may be selected from the heavy chain constant region of human IgG1, igG2, igG3 or IgG4 or a variant thereof, preferably comprising human IgG2 or IgG4 heavy chain constant region, or IgG1 with no ADCC (antibody-dependent cell-mediated cytotoxicity) toxicity after amino acid mutation.

The term "antigen-binding fragment" refers to antigen-binding fragments and antibody analogs of antibodies, which typically include at least a portion of the antigen-binding or variable region (e.g., one or more CDRs) of a parent antibody. Antibody fragments retain at least some of the binding specificity of the parent antibody. Typically, an antibody fragment retains at least 10% of parent binding activity when expressed as activity on a molar basis. Preferably, the antibody fragment retains at least 20%, 50%, 70%, 80%, 90%, 95%, or 100% or more of the binding affinity of the parent antibody to the target. Examples of antigen-binding fragments include, but are not limited to: fab, fab ', F (ab') 2, fv fragments, linear antibodies, single chain antibodies, nanobodies, domain antibodies, and multispecific antibodies. Engineered antibody variants are reviewed in Holliger and Hudson (2005) nat biotechnol.23: 1126-1136.

A "Fab fragment" consists of one light chain and one heavy chain of CH1 and variable regions. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.

The "Fc" region contains two heavy chain fragments comprising the CH1 and CH2 domains of an antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by the hydrophobic interaction of the CH3 domains.

A "Fab ' fragment" contains a portion of one light chain and one heavy chain comprising the VH domain and the CH1 domain and the region between the CH1 and CH2 domains, whereby an interchain disulfide bond can be formed between the two heavy chains of the two Fab ' fragments to form a F (ab ') 2 molecule.

An "F (ab') 2 fragment" contains two light chains and two heavy chains comprising part of the constant region between the CH1 and CH2 domains, whereby an interchain disulfide bond is formed between the two heavy chains. Thus, a F (ab ') 2 fragment consists of two Fab' fragments held together by a disulfide bond between the two heavy chains.

The "Fv region" comprises variable regions from both the heavy and light chains, but lacks the constant region.

The term "multispecific antibody" is used in its broadest sense to encompass antibodies having polyepitopic specificity. These multispecific antibodies include, but are not limited to: an antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH-VL unit has polyepitopic specificity; an antibody having two or more VL and VH regions, each VH-VL unit binding to a different target or a different epitope of the same target; an antibody having two or more single variable regions, each single variable region binding to a different target or a different epitope of the same target; full length antibodies, antibody fragments, diabodies, bispecific diabodies and triabodies (triabodies), antibody fragments that have been linked together covalently or non-covalently, and the like.

The term "single-chain antibody" is a single-chain recombinant protein composed of a heavy chain variable region (VH) and a light chain variable region (VL) of an antibody linked by a linker peptide, which is the smallest antibody fragment having a complete antigen-binding site.

The term "domain antibody fragment" is an immunologically functional immunoglobulin fragment that contains only heavy chain variable regions or light chain variable regions. In certain instances, two or more VH regions are covalently linked to a peptide linker to form a bivalent domain antibody fragment. The two VH regions of the bivalent domain antibody fragment may target the same or different antigens.

The term "binds to CSF-1R" in the context of the invention means capable of interacting with human CSF-1R. The term "antigen binding site" of the present invention refers to a three-dimensional spatial site that is not antigenically contiguous and is recognized by an antibody or antigen binding fragment of the present invention.

"amino acid mutation" in the analogous "mutation of 3, 2, 1 or 0 amino acids" means that there is a mutation of an amino acid in a variant protein or polypeptide as compared to the original protein or polypeptide, including the insertion, deletion or substitution of a corresponding number of amino acids in the original protein or polypeptide.

In a similar manner as "in SEQ ID NO: x, SEQ ID NO: y and SEQ ID NO: in the description of CDR variants "having 3, 2, 1 or 0 amino acid mutations based on CDR1, CDR2 and CDR3, respectively, as indicated by Z, an exemplary explanation is that the mutation to the CDR may comprise a mutation of 3, 2, 1 or 0 amino acids, and that the same or different number of amino acid residues may optionally be selected for mutation among CDR1, CDR2 and CDR3, for example in the amino acid sequence as shown in SEQ ID NO: x, SEQ ID NO: y and SEQ ID NO: and on the basis of CDR1, CDR2 and CDR3 shown by Z, carrying out 1 amino acid mutation on CDR1 and carrying out 0 amino acid mutation on CDR2 and CDR3. When a CDR is subjected to 0 amino acid mutation, the CDR variant having 0 amino acid mutation is still the CDR itself.

The term "epitope" refers to a site on an antigen to which an immunoglobulin or antibody specifically binds. Epitopes can be formed from contiguous amino acids, or non-contiguous amino acids juxtaposed by tertiary folding of the protein. Epitopes formed by adjacent amino acids are typically retained after exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost after denaturing solvent treatment. Epitopes typically comprise at least 3-15 amino acids in a unique spatial conformation. Methods for determining what epitope is bound by a given antibody are well known in the art and include immunoblot and immunoprecipitation detection assays, and the like. Methods of determining the spatial conformation of an epitope include techniques in the art and those described herein, such as X-ray crystallography and two-dimensional nuclear magnetic resonance, among others.

The term "specific binding" as used herein"," selectively binds "refers to binding of an antibody to an epitope on a predetermined antigen. Typically, antibodies are present at about less than 10 when measured in an instrument by Surface Plasmon Resonance (SPR) techniques using recombinant human CSF-1R as the analyte and an antibody as the ligand ^-7 M or an even smaller equilibrium dissociation constant (KD) binds to a predetermined antigen and binds to the predetermined antigen with at least twice the affinity as it binds to a non-specific antigen other than the predetermined antigen or closely related antigens, such as BSA and the like. The term "antigen-recognizing antibody" is used interchangeably herein with the term "specifically binding antibody".

The term "competition" when used in the context of antigen binding proteins that compete for the same epitope (e.g., neutralizing antigen binding proteins or neutralizing antibodies) means competition between the antigen binding proteins, as determined by the following assay: in such assays, the antigen binding protein (e.g., antibody or antigen binding fragment thereof) to be detected prevents or inhibits (e.g., reduces) specific binding of a reference antigen binding protein (e.g., ligand or reference antibody) to a common antigen. Numerous types of competitive binding assays are available for determining whether an antigen binding protein competes with another, such as: solid phase direct or indirect Radioimmunoassays (RIA), solid phase direct or indirect Enzyme Immunoassays (EIA), sandwich competition assays (see, e.g., stahli et al, 1983, methods in Enzymology 9; solid phase direct biotin-avidin EIA (see, e.g., kirkland et al, 1986, J.Immunol.137, 3614-3619), solid phase direct labeling assay, solid phase direct labeling sandwich assay (see, e.g., harlow and Lane,1988, antibodies, A Laboratory Manual, cold Spring Harbor Press); direct labeling of RIA with a solid phase of I-125 label (see, e.g., morel et al, 1988, mol. Immunol.25; solid phase direct biotin-avidin EIA (see, e.g., cheung, et al, 1990, virology 176; and directly labeled RIA (Moldenhauer et al, 1990, scand. J. Immunol.32. Typically, the assay involves the use of a purified antigen (either on a solid surface or on a cell surface) that binds to a test antigen binding protein with an unlabeled label and a labeled reference antigen binding protein. Competitive inhibition is measured by measuring the amount of label bound to a solid surface or cells in the presence of the antigen binding protein to be detected. Typically, the antigen binding protein to be tested is present in excess. Antigen binding proteins identified by competitive assays (competing antigen binding proteins) include: an antigen binding protein that binds to the same epitope as a reference antigen binding protein; and an antigen binding protein that binds a contiguous epitope sufficiently close to the binding epitope of the reference antigen binding protein that the two epitopes sterically hinder binding from occurring. Additional details regarding methods for determining competitive binding are provided in the disclosed embodiments. Typically, when a competing antigen binding protein is present in excess, it will inhibit (e.g., reduce) specific binding of at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, or 75% or more of a reference antigen binding protein to a common antigen. In certain instances, binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97%, or 97% or more.

The term "cross-reactive" refers to the ability of an antibody of the invention to bind CSF-1R from a different species. For example, an antibody of the invention that binds human CSF-1R may also bind CSF-1R of another species. Cross-reactivity is measured by detecting specific reactivity with purified antigens in binding assays (e.g., SPR and ELISA), or binding or functional interactions with cells that physiologically express CSF-1R. Methods of determining cross-reactivity include standard binding assays as described herein, such as Surface Plasmon Resonance (SPR) analysis, or flow cytometry.

The term "host cell" refers to a cell into which an expression vector has been introduced. Host cells may include bacterial, microbial, plant or animal cells. Bacteria susceptible to transformation include members of the family Enterobacteriaceae (Enterobacteriaceae), such as strains of Escherichia coli (Escherichia coli) or Salmonella (Salmonella); bacillus family (Bacillus) such as Bacillus subtilis; pneumococcus (Pneumococcus); streptococcus (Streptococcus) and Haemophilus influenzae (Haemophilus influenzae). Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris. Suitable animal host cell lines include CHO (chinese hamster ovary cell line), HEK293 cells (shown without limitation as HEK293E cells), and NS0 cells.

The terms "inhibit" or "block" are used interchangeably and encompass both partial and complete inhibition/blocking. Inhibition/blocking of a ligand preferably reduces or alters the normal level or type of activity that occurs in the absence of inhibition or blocking when ligand binding occurs. Inhibition and blocking are also intended to include any measurable decrease in ligand binding affinity when contacted with an anti-CSF-1R antibody, as compared to a ligand not contacted with an anti-CSF-1R antibody.

The term "inhibit growth" (e.g., in relation to a cell) is intended to include any measurable decrease in cell growth.

The terms "induce an immune response" and "enhance an immune response" are used interchangeably and refer to stimulation (i.e., passive or adaptive) of an immune response to a particular antigen. The term "induction" with respect to induction of CDC or ADCC refers to stimulation of a specific direct cell killing mechanism.

The term "ADCC", i.e., antibody-dependent cell-mediated cytotoxicity, as used herein refers to the direct killing of antibody-coated target cells by Fc fragments of cells expressing Fc receptors through recognition of the antibody. The ADCC effector function of an antibody may be enhanced or reduced or eliminated by modification of the Fc-fragment of the IgG. The modification refers to mutation in the heavy chain constant region of the antibody.

Methods for producing and purifying antibodies and antigen-binding fragments are well known and can be found in the prior art, such as the antibody test technical guide of cold spring harbor, chapters 5-8 and 15. For example, a mouse may be immunized with human CSF-1R or a fragment thereof, and the resulting antibody may be renatured, purified, and subjected to amino acid sequencing by conventional methods. Antigen-binding fragments can likewise be prepared by conventional methods. The antibodies or antigen-binding fragments of the invention are genetically engineered to incorporate one or more human FR regions in a CDR region of non-human origin. The human FR germline sequence can be obtained from the website http of Imminogenetics (IMGT): i/imgt. Cines. Fr or from immunoglobulin journal, 2001ISBN 012441351.

The engineered antibodies or antigen binding fragments of the invention can be prepared and purified using conventional methods. The cDNA sequence of the corresponding antibody can be cloned and recombined into the GS expression vector. Recombinant immunoglobulin expression vectors can be stably transfected into CHO cells. As a more recommended prior art, mammalian expression systems result in glycosylation of antibodies, particularly at the highly conserved N-terminus of the Fc region. Stable clones were obtained by expressing antibodies that specifically bind to antigens of human origin. Positive clones were expanded in bioreactor serum-free medium to produce antibodies. The antibody-secreting culture medium can be purified and collected by conventional techniques. The antibody can be concentrated by filtration by a conventional method. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves, ion exchange. The resulting product is either immediately frozen, e.g., -70 ℃, or lyophilized.

The antibody of the present invention refers to a monoclonal antibody. The monoclonal antibodies (mAbs) of the present invention refer to antibodies derived from a single clonal cell line, which is not limited to eukaryotic, prokaryotic, or phage clonal cell lines. Monoclonal antibodies or antigen-binding fragments can be obtained by recombination using techniques such as hybridoma technology, recombinant technology, phage display technology, synthetic techniques (e.g., CDR-grafting), or other techniques known in the art.

"administration" and "treatment," when applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, refers to contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. "administration" and "treatment" may refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. The treatment of the cells comprises contacting the reagent with the cells and contacting the reagent with a fluid, wherein the fluid is in contact with the cells. "administering" and "treating" also mean treating, e.g., a cell, by an agent, diagnosis, binding composition, or by another cell in vitro and ex vivo. "treatment" when applied to a human, veterinary or research subject refers to therapeutic treatment, prophylactic or preventative measures, research and diagnostic applications.

By "treating" is meant administering a therapeutic agent, such as a composition comprising any of the binding compounds of the invention, either internally or externally to a patient who has one or more symptoms of a disease for which the therapeutic agent is known to have a therapeutic effect. Typically, the therapeutic agent is administered in the subject patient or population in an amount effective to alleviate one or more symptoms of the disease, whether by inducing regression of such symptoms or inhibiting the development of such symptoms to any clinically useful degree. The amount of therapeutic agent effective to alleviate the symptoms of any particular disease (also referred to as a "therapeutically effective amount") may vary depending on a variety of factors, such as the disease state, age, and weight of the patient, and the ability of the drug to produce a desired therapeutic effect in the patient. Whether a symptom of a disease has been alleviated can be assessed by any clinical test commonly used by physicians or other health care professionals to assess the severity or progression of the symptom. Although embodiments of the invention (e.g., methods of treatment or articles of manufacture) may be ineffective in alleviating the symptoms of the target disease in every patient, they should alleviate the symptoms of the target disease in a statistically significant number of patients as determined by any statistical test known in the art, such as Student's t-test, chi-square test, U-test by Mann and Whitney, kruskal-Wallis test (H-test), jonckhere-Terpsra test, and Wilcoxon test.

The term "consisting essentially of 8230 \\8230%, \8230composition" or variations thereof, as used throughout the specification and claims, is meant to encompass all such elements or groups of elements, and optionally additional elements of similar or different nature to those described, which additional elements do not materially alter the basic or novel characteristics of a given dosing regimen, method or composition. As a non-limiting example, a binding compound consisting essentially of the amino acid sequence mentioned may also comprise one or more amino acids, which do not significantly affect the properties of the binding compound.

The term "naturally occurring" as applied to an object in accordance with the present invention refers to the fact that the object may be found in nature. For example, a polypeptide sequence or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and that has not been intentionally modified by man in the laboratory is naturally occurring.

An "effective amount" includes an amount sufficient to ameliorate or prevent a symptom or condition of a medical condition. An effective amount also means an amount sufficient to allow or facilitate diagnosis. The effective amount for a particular patient or veterinary subject may vary depending on the following factors: such as the condition to be treated, the general health of the patient, the method and dosage of administration, and the severity of side effects. An effective amount may be the maximum dose or dosage regimen that avoids significant side effects or toxic effects.

"exogenous" refers to a substance that is to be produced outside an organism, cell, or human body by context. "endogenous" refers to a substance produced in a cell, organism, or human body by background.

"homology" refers to sequence similarity between two polynucleotide sequences or between two polypeptides. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if each position of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared x 100%. For example, two sequences are 60% homologous if there are 6 matches or homologies at 10 positions in the two sequences when the sequences are optimally aligned. In general, comparisons are made when aligning two sequences to obtain the greatest percentage of homology.

As used herein, the expressions "cell," "cell line," and "cell culture" are used interchangeably, and all such designations include progeny thereof. Thus, the words "transformant" and "transformed cell" include the primary subject cell and cultures derived therefrom, regardless of the number of metastases. It is also understood that all progeny may not be precisely identical in DNA content due to deliberate or inadvertent mutations. Mutant progeny that have the same function or biological activity as screened for in the originally transformed cell are included. Where different names are intended, they are clear from the context.

"optional" or "optionally" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs or does not. For example, "optionally comprising 1-3 antibody heavy chain variable regions" means that antibody heavy chain variable regions of a particular sequence may, but need not, be present.

"pharmaceutical composition" means a mixture containing one or more compounds described herein, or a physiologically/pharmaceutically acceptable salt or prodrug thereof, and other chemical components, as well as other components such as physiological/pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to facilitate administration to an organism, facilitate absorption of the active ingredient, and exert biological activity.

The invention provides an anti-CSF-1R antibody with high affinity, high selectivity and high biological activity, a monoclonal antibody immunotherapy for tumors or autoimmune diseases and related applications thereof, and other related medicaments, compositions and methods for treating CSF-1R positive tumors or autoimmune diseases.

Detailed Description

The present invention will be further described with reference to the following examples, which are not intended to limit the scope of the present invention. The experimental method of the present invention, in which the specific conditions are not specified, is usually performed according to conventional conditions, such as the antibody technical laboratory manual of cold spring harbor, molecular cloning manual; or according to the conditions recommended by the manufacturer of the raw material or the goods. Reagents of specific sources are not indicated, and are conventional reagents purchased in the market.

Example 1 immunizing antigen, screening sequence of antigen and preparation

The sequence encoding His-Tag-tagged human CSF-1R and huFc-tagged human CSF-1R were synthesized by Integrated DNA Technology (IDT) and cloned into pTT5 vector (Biovector), respectively. The recombinant CSF-1R protein was purified after expression in 293T cells (ATCC, CRL-3216. TM.). The purified protein was used in the experiments described in the following examples.

Sequence of Human CSF-1R ECD-His:

sequence of Human CSF-1R-huFc:

EXAMPLE 2 CSF-1R recombinant protein preparation

1. Purification of His-tagged CSF-1R recombinant protein:

the supernatant samples expressed by HEK293 cells were centrifuged at high speed to remove impurities and the buffer was replaced with Phosphate Buffer (PBS) and imidazole was added to a final concentration of 5mM. The nickel column was equilibrated with a PBS solution containing 5mM imidazole, and washed 2-5 column volumes. The displaced supernatant sample was applied to the column. The column was washed with PBS solution containing 5mM imidazole until the a280 reading dropped to baseline. The column was then washed with PBS +10mM imidazole to remove non-specifically bound contaminating proteins and the effluent was collected. The target protein was eluted with 300mM imidazole in PBS and the peak was collected.

The collected eluate was further purified by ion exchange (SP column). Preparing a solution A: 0.01M PB, pH8.0. Preparing a solution B: solution A +1M NaCl. Firstly, the target protein eluted by the PBS solution of imidazole is replaced to solution A, the solution A is used for balancing an SP column, the sample is loaded, the concentration gradient of the solution B is 0-100%, the elution is carried out in 10 times of the column volume, and each elution peak is collected. And carrying out electrophoresis and peptide mapping on the obtained protein, identifying by liquid chromatography-mass spectrometry (LC-MS), and subpackaging for later use.

2. Purification procedure of Fc-tagged CSF-1R recombinant protein (h-CSF-1R-Fc):

the supernatant samples expressed by HEK293 cells were centrifuged at high speed to remove impurities and the buffer was changed to PBS. The ProteinA affinity column was equilibrated with 10mM phosphate buffer and washed 2-5 column volumes. The displaced supernatant sample was applied to the column. The column was washed with buffer containing 25 column volumes until the a280 reading dropped to baseline. The target protein was eluted with 0.8% acetic acid buffer solution at pH 3.5, and the peak was collected, neutralized with 1M Tris-Cl pH8.0 buffer solution immediately after dispensing, and then replaced with PBS pH6.9 using Millipore's Amico-15 filter column. The obtained protein is identified by electrophoresis, peptide diagram and LC-MS and then is subpackaged for standby.

3. Preparation of CHO stably transfected cell line expressing human CSF-1R antigen:

the full-length sequence encoding the human CSF-1R protein (huCSF-1R) was synthesized by Integrated DNA Technology (IDT) and cloned into the pcDNA3.1 vector, pcDNA3.1/puro (Invitrogen # V79020), respectively. CHO-S (ATCC) cells were cultured in CD-CHO medium (Life Technologies, # 10743029) to 0.5X 10 ⁶ And (4) the concentration is/ml. Mu.g of the vector encoding huCSF-1R or gene was mixed with 50ul LF-LTX (Life Technologies, # A12621) in 1ml Opti-MEM medium (Life Technologies, # 31985088), incubated at room temperature for 20 minutes, added to CHO cell culture broth and placed in a carbon dioxide incubator for culture. After 24 hours the medium was replaced with fresh medium and puromycin at 10. Mu.g/ml was added. Then replacing a new culture solution every 2-3 days, and screening for 10-12 days to obtain the stable CHO-S cell pool.

4. Cynomolgus monkey CSF-1R-His (cynoCSF-1R-His) and mouse CSF-1R-His proteins were purchased from Acro biosciences.

EXAMPLE 3 preparation of antibodies

Anti-human CSF-1R monoclonal antibodies were generated by immunizing mice. Experiments were performed with SwissWebster white mice, female, 6 weeks old (Charles River Corp.). A breeding environment: SPF grade. After the mice are purchased, the mice are raised in a laboratory environment for 1 week, and the light/dark period is adjusted at 12/12 hours, and the temperature is 20-25 ℃; the humidity is 40-60%. The immune antigen is human CSF-1R recombinant protein (huCSF-1R-Fc) with Fc tag. Titermax (sigma Lot Num: T2684) was used as adjuvant. The ratio of antigen to adjuvant (titermax) was 1: 1, and vaccination was performed after emulsification for days 0, 21, 35, 49, 63. Day 0 was injected Intraperitoneally (IP) with 15 μ g + paw pad (footpad) 25/individual post-emulsification antigen. 21 35, 49, 63 days Intraperitoneally (IP) injection of 15. Mu.g + paw pad (footpad) 15/only of post-emulsification antigen, boosting 3 days before splenocyte fusion, intraperitoneally (IP) injection of 15. Mu.g + paw pad (footpad) 15/only of normal salineAntigen solution. Blood was examined at day 42, 56 and 70, and the antibody titer in the mouse serum was determined by detecting the mouse serum using enzyme linked immunosorbent assay (ELISA) and Fluorescence Activated Cell Sorter (FACS). After the 5 th immunization, mice with high antibody titers in serum and titers tending to plateau were selected for splenocyte fusion, and splenic lymphocytes were fused with myeloma Sp2/0 cells using an optimized electrofusion procedure (

CRL-8287 ^TM ) And carrying out fusion to obtain hybridoma cells.

After culturing the fused hybridoma cells for 30,000-50,000 cells/well for 7-14 days, taking culture medium supernatant, performing antibody screening on the hybridoma supernatant by using CSF-1R recombinant protein and ELISA experiment as described in example 4, and obtaining positive antibody strain, further using CHO-S cells stably expressing CSF-1R, comparing blank CHO-S cells to eliminate non-specific binding antibody hybridoma strain, and performing screening by using flow sorting method, thereby determining about 400 hybridomas which bind to the recombinant protein and also bind to cell expression antigen. Subsequently, as described in example 5, about 50 hybridomas having a blocking agent function were identified and subcloned using CSF-1 ligand blocking function experimental screening based on CSF-1R stably transfected cells, and then purified antibodies were collected and subjected to IL-34 ligand blocking function experimental screening based on CSF-1R stably transfected cells, and 11 antibody subclones having CSF-1 and IL-34 double blocking functions were identified. Hybridoma subcloned cells in logarithmic growth phase were harvested, RNA extracted using Trizol (Invitrogen, 15596-018) and reverse transcribed (PrimeScript) ^TM Reverse Transcriptase, takara # 2680A). And (3) carrying out PCR amplification on the cDNA obtained by reverse transcription by using mouse Ig-Primer Set (Novagen, TB326 Rev.B 0503), sequencing, and removing the repetitive sequence of the CDR region to finally obtain the sequences C11, C19, C6, C8, C16, C18, C23, C27 and C30 of the nine murine antibodies.

The variable regions of the heavy chain and the light chain of the murine monoclonal antibody C11 have the following sequences:

C11 HCVR

C11 LCVR

it contains the following CDR sequences:

name(s)	Sequence of	Number of
			HCDR1	GYTFSNYYMN	SEQ ID NO：3
HCDR2	EMNPNNGDSSYNQKFKG	SEQ ID NO：4
			HCDR3	RSPWWFFDV	SEQ ID NO：5
LCDR1	KSSQSLLNSGNQKNSLT	SEQ ID NO：6
			LCDR2	WASTRES	SEQ ID NO：7
LCDR3	QNDYTYPFT	SEQ ID NO：8

The heavy and light chain variable region sequences of C19 are as follows:

C19 HCVR

C19 LCVR

it contains the following CDR sequences:

name (R)	Sequence of	Numbering
			HCDR1	AITFTDYYMN	SEQ ID NO：11
HCDR2	DIYPNNGGTTYNQKFKG	SEQ ID NO：12
			HCDR3	EKITMEYYYAMDY	SEQ ID NO：13
LCDR1	RASESVSSHDIHLIH	SEQ ID NO：14
			LCDR2	AASSLES	SEQ ID NO：15
LCDR3	QQSIEDPPT	SEQ ID NO：16

The variable regions of the heavy and light chains of murine mab C6 were as follows:

C6 HCVR

C6 LCVR

it contains the following CDR sequences:

name(s)	Sequence of	Number of
			HCDR1	GYTFTGYYMH	SEQID NO：39
HCDR2	EINPNTGSCTYNQKFTG	SEQID NO：40
			HCDR3	LNYYWYFDV	SEQID NO：41
LCDR1	KSSQTLLNSNDQKNYLA	SEQID NO：42
			LCDR2	FASTRDS	SEQID NO：43
LCDR3	QQDYSTPFT	SEQID NO：44

The variable sequences of the mouse monoclonal antibody C8 heavy and light chains were as follows: c8 HCVR

C8 LCVR

It contains the following CDR sequences:

name (R)	Sequence of	Numbering
			HCDR1	GYTFTNYWIA	SEQID NO：47
HCDR2	EMYPGGGGDNHHEKFKN	SEQID NO：48
			HCDR3	RDYGNPCFDY	SEQID NO：49
LCDR1	RASQGVTTSSHSYMH	SEQID NO：50
			LCDR2	YASNLES	SEQID NO：51
LCDR3	QHSWEIPYT	SEQID NO：52

The variable regions of the heavy and light chains of murine mab C16 were as follows:

C16 HCVR

C16 LCVR

it contains the following CDR sequences:

the variable regions of the heavy and light chains of murine mab C18 were as follows:

C18 HCVR

C18 LCVR

it contains the following CDR sequences:

name(s)	Sequence of	Numbering
			HCDR1	AITFTDYYMN	SEQID NO：63
HCDR2	DINPNNGGTTYNQKFKG	SEQID NO：64
			HCDR3	EKISMEYYYAMDY	SEQID NO：65
LCDR1	RASESVSSHDIHLMH	SEQID NO：66
			LCDR2	AASNLES	SEQID NO：67
LCDR3	QQSIEDPPT	SEQID NO：68

The variable regions of the heavy and light chains of murine mab C23 were as follows:

C23 HCVR

C23 LCVR

it contains the following CDR sequences:

name (R)	Sequence of	Numbering
			HCDR1	GYTFTVYYMN	SEQID NO：71
HCDR2	DIDPNTGDSTYNQKFRG	SEQID NO：72
			HCDR3	YDGYIDY	SEQID NO：73
LCDR1	RSSQSIVHSNRHTYLE	SEQID NO：74
			LCDR2	GVSNRFS	SEQID NO：75
LCDR3	FQGTHVPLT	SEQID NO：76

The variable regions of the heavy and light chains of murine mab C27 were as follows:

C27 HCVR

C27 LCVR

it contains the following CDR sequences:

name (R)	Sequence of	Numbering
			HCDR1	GFNIKDYYMH	SEQID NO：79
HCDR2	RFDPENGDTIYDWKFQD	SEQID NO：80
			HCDR3	SGDYMFDY	SEQID NO：81
LCDR1	ITSTGVDDDFN	SEQID NO：82
			LCDR2	EGNTLRP	SEQID NO：83
LCDR3	LQSDHLPFT	SEQID NO：84

The variable regions of the heavy and light chains of murine mab C30 were as follows:

C30 HCVR

C30 LCVR

it contains the following CDR sequences:

name (R)	Sequence of	Number of
			HCDR1	GYTFSSYWIE	SEQID NO：87
HCDR2	EILPGSGSTNYNEKFKG	SEQID NO：88
			HCDR3	NYDGSLYPMDY	SEQID NO：89
LCDR1	RTSESVSIHGTHLMH	SEQID NO：90
			LCDR2	AASNLES	SEQID NO：91
LCDR3	QQSIEDPPT	SEQID NO：92

The heavy and light chain variable regions of each mouse antibody were cloned into pTT vector plasmid (Biovector) containing human IgG4 heavy chain constant region and kappa light chain constant region, and transiently transfected into HEK293 cells to obtain individual chimeric antibodies (C11C, C19C, C6C, C8C, C16C, C18C, C23C, C27C and C30C) against CSF-1R, purified, identified, and tested for relevant binding and functional activity as described in example 2 (purification of Fc-tagged proteins).

EXAMPLE 4 in vitro binding Activity assay of antibodies

Neutralizing avidin for binding to biotin was diluted to 1. Mu.g/ml with PBS pH7.4 buffer, added to a 96-well plate in a volume of 100. Mu.l/well, and left at 4 ℃ for 16h to 20h. After washing the plate 1 time with PBST (0.05% Tween-20 in PH7.4PBS), 120. Mu.l/well of PBST/1% skim milk was added and the blocking was performed by incubation at room temperature for 1h. After washing the plate 1 time with PBST buffer, adding 1. Mu.g/ml biotin-labeled h-CSF-1R-his (or monkey, mouse CSF-1R protein waiting for testing), incubated for 1h at room temperature. After washing the plate 3 times with PBST buffer, the CSF-1R antibody to be tested (murine antibody/chimeric antibody/humanized antibody) diluted to the appropriate concentration with PBST/1% mil was added and incubated at room temperature for 1.5h. The reaction system was removed and after washing The plate 3 times with PBST, a secondary anti-mouse antibody or a secondary anti-human antibody (The Jackson Laboratory) labeled with Horseradish Peroxidase (HRP) diluted with PBST/1% skim milk was added at 100. Mu.l/well and incubated at room temperature for 1h. After PBST washing 3 times, 100. Mu.l/well TMB was added and incubated at room temperature for 5-10min. Add 100. Mu.l/well of 1M H ₂ SO ₄ The reaction was stopped, absorbance read at 450nm and ELISA binding EC calculated ₅₀ The value is obtained.

CHO-S cells highly expressing huCSF-1R were centrifuged at 1000rpm for 5 minutes, the pellet was collected and suspended in 10-15ml of pre-cooled flow buffer and the cells counted. Centrifuging at 1000rpm for 5 min in a 50ml centrifuge tube, collecting cells, discarding supernatant, resuspending the precipitate with precooled blocking buffer solution at density of 0.5-1.0 × 10 ⁷ Cells/ml. After incubation at 4 ℃ for 30 minutes, the resuspension was added to 96-well plates at 100. Mu.l per well. After centrifugation of the 96-well plate at 1500rpm for 5 minutes, the supernatant was discarded. Mu.l of the antibody to be tested was added to each well in a concentration gradient of 0.085nM to 670nM, the cells were resuspended and incubated at 4 ℃ for 60 min in the absence of light. The supernatant was discarded by centrifugation, and 100. Mu.l of a 1: 400 diluted FITC-labeled secondary antibody (BD Biosciences) was added. The cells were resuspended and incubated at 4 ℃ for 60 minutes in the dark. The cells were washed twice with flow buffer and fixed by resuspending the cells in 1% paraformaldehyde for flow detection.

In the same ELISA method, cross-binding of each antibody to cynomolgus monkey CSF-1R was detected using CSF-1R-His protein, and the data are shown in fig. 1, except that C27C did not bind to monkey antigen protein, the remaining antibodies and FPA008 reference all had strong cross-binding activity to monkey CSF-1R.

EXAMPLE 5 in vitro blocking function assay of antibodies

1. Blockade of CSF-1R binding by anti-CSF-1R antibodies on CSF-1R/CHO cells

A concentration gradient of anti-CSF-1R antibody (0.01 nM-670 nM) was incubated with CSF-1R/CHO cells and combined by addition of biotinylated CSF-1 protein or APC-tagged IL-34 protein. In which the CSF-1 assay was further combined with a streptavidin-PE tag. After washing with PBS, the cells were subjected to flow cytometry to detect the fluorescence signal intensity corresponding to CSF-1 or IL-34.

2. Blocking experiment of CSF-1 or IL-34 induced proliferation of human monocyte by anti-CSF-1R antibody

Peripheral Blood Mononuclear Cells (PBMC) in healthy human blood were first isolated and extracted using Ficoll-Paque gradient separation kit (GE Healthcare Bio-Sciences), and then mononuclear cells in PBMC were purified and extracted using Percoll kit (GE Healthcare Bio-Sciences). The isolated monocytes were then incubated in a blank antibody-free or concentration-gradient anti-CSF-1R antibody for 3 days at 37 ℃ in a carbon dioxide incubator with human CSF-1 or IL-34 (R & D Systems) stimulation. And (3) measuring the ATP content in each culture sample to be detected by using a CellTiter-Glo kit (Promega), and finally measuring the corresponding proliferation level of the mononuclear cells according to the linear positive correlation between the ATP content and the number of the activated mononuclear cells.

The results of the binding and primary functional screening experiments for 30 subcloned murine antibodies and the reference antibody FPA008 from the company FivePrime are summarized in the following table. Selected 9 murine antibodies after sequencing are shown in bold.

The blocking function data of the chimeric antibody are shown in the table below, 9 strains of antibodies can effectively block CSF-1 or IL-34 from stimulating monocyte proliferation, wherein the chimeric antibodies such as C11C, C19C, C23C and C27C have very low IC50, and the blocking effect is equivalent to or better than that of a reference antibody FPA008.

Example 6 in vitro binding affinity and kinetics experiments

The experiment was measured using the Surface Plasmon Resonance (SPR) method. An anti-human IgG polyclonal antibody was covalently linked to a CM5 (GE) chip by a standard amino coupling method using a kit supplied by Biacore, and then the chimeric antibody or humanized antibody to be tested according to the present invention was captured to a stationary phase using this antibody. The h-CSF-1R-His protein (example 1) diluted in the same buffer with a concentration gradient of 25-800nM was injected in each cycle and regenerated with the regeneration reagent in the kit after injection. The antigen-antibody binding kinetics were followed for 3 min and the dissociation kinetics for 10min. The data obtained were analyzed using the BIAevaluation software of GE in a 1: 1 (Langmuir) binding model, in such a way that the k of each chimeric antibody was determined _a (k _on )、kd(k _off ) And K _D The values are shown in the following table.

Example 7 mouse antibody humanization experiments

Humanization of murine anti-human CSF-1R monoclonal antibodies was performed as described in many publications in the art. Briefly, the present invention humanizes two optimal candidate molecules, C11 and C19, determined by evaluating the overall functional activity, sequence specificity, druggability, etc., using a human constant domain in place of the parent (murine antibody) constant domain, selecting a human antibody sequence based on the homology between the murine and human antibodies.

Based on the obtained typical structure of VH/VL CDR of the murine antibody, the variable region sequences of the heavy and light chains are compared with the germline database of the human antibody to obtain a human germline template with high homology. Wherein the human germline light chain framework region is from a human kappa light chain gene and the human germline heavy chain framework region is from a human heavy chain, the antibody of the invention is preferably a human germline antibody template as shown below.

C11 preferably human germline heavy chain template IGHV1-46 (SEQ ID NO: 21):

c11 preferred human germline light chain template IGkV4-1 (SEQ ID NO: 22):

c19 preferably human germline heavy chain template IGHV1-2 (SEQ ID NO: 23):

c19 preferably human germline light chain template IGkV1-13 (SEQ ID NO: 24):

the CDR regions of murine antibodies C11 and C19 are grafted onto a selected corresponding humanized template, replacing the humanized variable regions, and recombined with IgG constant regions (preferably IgG4 for the heavy chain and kappa for the light chain). Then, based on the three-dimensional structure of the murine antibody, the embedded residues, residues that interact directly with the CDR regions, and residues that have an important influence on the conformation of VL and VH were subjected to back mutation, and the CDR regions were optimized for mutation of the chemically unstable asparagine residues, wherein the HCDR2: EMNPNNGDSSYNQKFKG (SEQ ID NO: 4) was optimized as: EMNPNTGDSSYNQKFKG (SEQ ID NO: 95), LCDR1: KSSQSLLNSGNQKNSLT (SEQ ID NO: 6) was optimized as: KSSQSLLNSGNQKTSLT (SEQ ID NO: 96); HCDR2 of murine antibody C19: DIYPNNGGTTYNQKFKG (SEQ ID NO: 12) was optimized as: DIYPNTGGTTYNQKFKG (SEQ ID NO: 97). Thus, an antibody comprising a combination of the following humanized light and heavy chain variable region sequences was designed and tested.

huC11-VH-a(SEQ ID NO：25)：

huC11-VH-b(SEQ ID NO：26)：

huC11-VH-c(SEQ ID NO：27)：

huC11-VH-d(SEQ ID NO：28)：

huC11-VL-a(SEQ ID NO：29)：

huC11-VL-b(SEQ ID NO：30)：

huC19-VH-a(SEQ ID NO：31)：

huC19-VH-b(SEQ ID NO：32)：

huC19-VH-c(SEQ ID NO：33)：

huC19-VL-a(SEQ ID NO：34)：

huC19-VL-b(SEQ ID NO：35)：

huC19-VL-c(SEQ ID NO：36)：

cDNA fragments were synthesized based on the amino acid sequences of the light and heavy chains of each of the humanized antibodies above and inserted into pcDNA3.1 expression vector (Life Technologies Cat. No. V790-20). The expression vector and transfection reagent PEI (Polysciences, inc. Cat. No. 23966) were transfected into HEK293 cells (Life Technologies Cat. No. 11625019) at a 1: 2 ratio and placed in CO ₂ Incubate in incubator for 4-5 days. The expressed antibody was recovered by centrifugation, and then purified by the method of example 2 (purification of Fc-tagged protein) to obtain a humanized antibody protein of the present invention.

The final humanized huC11 (using VH-b heavy chain and VL-b light chain) and huC19 antibody molecules (using VH-c heavy chain and VL-a light chain) were selected by expression testing and back-mutation number comparison.

Further, for the huC19 humanized antibody, its HCDR1: AITFTDYYMN (SEQ ID NO: 11) was mutated to: AITFTDYIN (SEQ ID NO: 98) thus removed the chemically unstable methionine residue site, resulting in the humanized antibody huC19I, the huC19I heavy chain variable region sequence being as shown in SEQ ID NO: shown at 100. The full-length sequences of huC11, huC19, and huC19I are set forth in SEQ ID NO:17-20 and SEQ ID NO:99, respectively.

huC19-VH-d

huC11 HC

huC11 LC

huC19 HC

huC19 LC/huC19I LC

huC19I HC

Wherein, the heavy chain constant region sequence of the humanized antibody (SEQ ID NO: 101)

Light chain constant region sequence of humanized antibody (SEQ ID NO: 102)

Example 8 assay of humanized antibody Activity

The following experimental assays were performed in vitro on huC11 and huC19 antibodies:

1. in the cell binding experiment (same procedure as in example 4), the results are shown in FIG. 2, and the humanized antibodies huC11 and huC19 positively bind to CSF-1R-highly expressing CHO cells with binding capacity comparable to that of the corresponding chimeric antibodies C11C and C19C, while the chimeric antibody and the humanized antibody versions of C19 are stronger than the corresponding versions of C11.

2. Affinity kinetics experiments (same procedure as in example 6) resulted in the following table, which shows that the final preferred humanized antibodies huC11 and huC19 both retain strong affinity for human CSF-1R antigenic protein, with huC19 having stronger affinity than the reference antibody FPA008.

3. In vitro test for blocking functional Activity (the procedure was as in example 6), the results are shown in FIG. 3, FIG. 4 and the following tables, and the humanized antibodies huC11 and huC19 and their mutant huC19I all showed strong blocking function against CSF-1 and IL-34 induced monocyte proliferation, and the blocking functional activity of huC19 was comparable or slightly superior to that of the FPA008 reference antibody, while its mutant huC19I was slightly decreased to be similar to that of FPA008.

In vitro blocking functional Activity assay for huC11

In vitro blocking functional activity experiment of huC19 and mutant huC19I thereof

Example 9 efficacy of anti-CSF 1R antibodies in humanized tumor models

The peripheral blood of normal human is taken, and PBMC of healthy human is separated by density gradient centrifugation. Using CD14 ⁺ Sorting the mononuclear cells by the microbeads kit, and carrying out CD14 according to the protocol provided by the kit ⁺ Isolation of monocytes, i.e., 20ul Anti-CD14 microbeads per 107 cells were added and incubated for 15 minutes at 4 ℃. Then, the cells were loaded on a magnetic column, washed three times, and the cells in the magnetic column, i.e., CD14, were collected ⁺ A monocyte. CD14 ⁺ The monocytes were cultured in RPMI 1640 medium containing 100ng/ml M-CSF for 6 days, induced culture of macrohag cells was performed, the remaining cells were added to Mitomycin C-treated MDA-MB-231 cells, and PBMC and MDA-MB-231 were co-cultured for 6 days in the presence of IL-2 and 10% FBS in RPMI 1640 medium.

After 6 days, macrohage cells were collected and 1X 10 cells were collected ⁵ Individual cells, CD68, CD115, CD163, CD206 stained and FACS analyzed for success in inducing macrophage. PBMC cells, macrocage and freshly digested MDA-MB-231 cells (ATCC) were mixed at a ratio of 6.25: 1: 50, and inoculated subcutaneously into each NCG mouse (university of Nanjing-Nanjing Biomedicine institute, adapted for 5 days). The experimental animals are all raised in independent ventilation boxes with constant temperature and humidity, the raising room temperature is 18.0-26.0 ℃, the humidity is 40-70%,10-20 times/hour ventilation is carried out, and the light and shade alternation time is 12h/12h day and night.

The experiment was divided into a human IgG1 antibody control group, the reference antibody FPA8, and huC19I, each administered at a dose of 20mg/kg. Each group of 6 mice was administered once every two days by intraperitoneal injection 10 times (see Table 1). Daily performance of the animals was monitored daily for 24 days after dosing. Throughout the experiment, the length and width of the tumor, and the tumor volume (mm) were measured 2 times per week with a vernier caliper ³ ) =0.5 × (tumor major axis × tumor minor axis) ² ) And (4) calculating. Relative tumor inhibition TGI (%): TGI% = (1-T/C) × 100%. T/C% is the relative tumor proliferation rate, i.e., the percentage value of the relative tumor volume or tumor weight of the treated and control groups at a certain time point. T and C are the Tumor Volume (TV) or Tumor Weight (TW) at a particular time point for the treated group and IgG1 control group, respectively. All data are expressed by Mean + -SEM, and student's t test is used for comparing the tumor volume and tumor weight of the treatment group with the tumor volume and tumor weight of the control group, and p < 0.05 is used as the significant difference.

As shown in fig. 5 and fig. 6, CSF1R antibody huC19I exhibited anti-tumor effect in the Macrophage-PBMC-MDA-MB-231 tumor model with the final mean tumor volumes of each group: 294.32mm ³ 、230.46mm ³ 、131.19mm ³ Starting the process; the tumor inhibition rates (TGI) of the FPA8 group and the huC19I group were 22.62% and 55.95%, respectively. No significant weight loss of NCG mice occurred in any of the administration groups, indicating that NCG mice were well-tolerated by CSF1R antibody at this dose.

Experimental groups and dosing regimens are given in the table below:

sequence listing

<110> Hengrui pharmaceutical Co., ltd, jiangsu; shanghai Hengrui pharmaceuticals, inc

<120> anti-CSF-1R antibody, antigen-binding fragment thereof, and medical use thereof

<130> P19115415CP

<150> CN201711015488.5

<151> 2017-10-26

<160> 102

<170> PatentIn version 3.5

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Lys Leu Leu Ile Tyr Ala Ala Ser Ser Leu Glu Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Ala Thr Asp Phe Thr Leu Asn Ile His

65 70 75 80

Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Phe Cys Gln Gln Ser Ile

85 90 95

Glu Asp Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 11

<211> 10

<212> PRT

<213> mouse (Mus musculus)

<400> 11

Ala Ile Thr Phe Thr Asp Tyr Tyr Met Asn

1 5 10

<210> 12

<211> 17

<212> PRT

<213> mouse (Mus musculus)

<400> 12

Asp Ile Tyr Pro Asn Asn Gly Gly Thr Thr Tyr Asn Gln Lys Phe Lys

1 5 10 15

Gly

<210> 13

<211> 13

<212> PRT

<213> mouse (Mus musculus)

<400> 13

Glu Lys Ile Thr Met Glu Tyr Tyr Tyr Ala Met Asp Tyr

1 5 10

<210> 14

<211> 15

<212> PRT

<213> mouse (Mus musculus)

<400> 14

Arg Ala Ser Glu Ser Val Ser Ser His Asp Ile His Leu Ile His

1 5 10 15

<210> 15

<211> 7

<212> PRT

<213> mouse (Mus musculus)

<400> 15

Ala Ala Ser Ser Leu Glu Ser

1 5

<210> 16

<211> 9

<212> PRT

<213> mouse (Mus musculus)

<400> 16

Gln Gln Ser Ile Glu Asp Pro Pro Thr

1 5

<210> 17

<211> 448

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> PEPTIDE

<223> huC11 humanized antibody heavy chain

<400> 17

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Asn Tyr

20 25 30

Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Glu Met Asn Pro Asn Thr Gly Asp Ser Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Arg Val Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Arg Ser Pro Trp Trp Phe Phe Asp Val Trp Gly Gln Gly Thr

100 105 110

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

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

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

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

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

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

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

340 345 350

Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

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

405 410 415

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

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

435 440 445

<210> 18

<211> 220

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> PEPTIDE

<223> huC11 humanized antibody light chain

<400> 18

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

<210> 19

<211> 452

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> PEPTIDE

<223> huC19 humanized antibody heavy chain

<400> 19

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Ala Ile Thr Phe Thr Asp Tyr

20 25 30

Tyr Met Asn Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Asp Ile Tyr Pro Asn Thr Gly Gly Thr Thr Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Arg Val Thr Leu Thr Val Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Glu Lys Ile Thr Met Glu Tyr Tyr Tyr Ala Met Asp Tyr Trp

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

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

385 390 395 400

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

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

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

435 440 445

Ser Pro Gly Lys

450

<210> 20

<211> 218

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> PEPTIDE

<223> C19/C19I humanized antibody light chain

<400> 20

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Ser Ser His

20 25 30

Asp Ile His Leu Ile His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro

35 40 45

Lys Leu Leu Ile Tyr Ala Ala Ser Ser Leu Glu Ser Gly Val Pro Ser

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Ser Ile

85 90 95

Glu Asp Pro Pro Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

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

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 21

<211> 98

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 21

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

1 5 10 15

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

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

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

85 90 95

Ala Arg

<210> 22

<211> 101

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 22

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Tyr Tyr Ser Thr Pro

100

<210> 23

<211> 98

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 23

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

1 5 10 15

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

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg

<210> 24

<211> 95

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 24

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Ala

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Ser Tyr Pro

85 90 95

<210> 25

<211> 118

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> PEPTIDE

<223> C11 humanized antibody heavy chain a

<400> 25

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr

20 25 30

Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Glu Met Asn Pro Asn Thr Gly Asp Ser Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Arg Ser Pro Trp Trp Phe Phe Asp Val Trp Gly Gln Gly Thr

100 105 110

Met Val Thr Val Ser Ser

115

<210> 26

<211> 118

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> PEPTIDE

<223> C11 humanized antibody heavy chain b

<400> 26

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Asn Tyr

20 25 30

Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Glu Met Asn Pro Asn Thr Gly Asp Ser Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Arg Val Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Arg Ser Pro Trp Trp Phe Phe Asp Val Trp Gly Gln Gly Thr

100 105 110

Met Val Thr Val Ser Ser

115

<210> 27

<211> 118

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> PEPTIDE

<223> C11 humanized antibody heavy chain C

<400> 27

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Asn Tyr

20 25 30

Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Glu Met Asn Pro Asn Thr Gly Asp Ser Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Arg Val Thr Met Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Arg Ser Pro Trp Trp Phe Phe Asp Val Trp Gly Gln Gly Thr

100 105 110

Met Val Thr Val Ser Ser

115

<210> 28

<211> 118

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> PEPTIDE

<223> C11 humanized antibody heavy chain d

<400> 28

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Asn Tyr

20 25 30

Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Glu Met Asn Pro Asn Thr Gly Asp Ser Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Arg Val Thr Met Thr Arg Asp Lys Ser Thr Ser Thr Val Tyr

65 70 75 80

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

85 90 95

Ala Arg Arg Ser Pro Trp Trp Phe Phe Asp Val Trp Gly Gln Gly Thr

100 105 110

Met Val Thr Val Ser Ser

115

<210> 29

<211> 113

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> PEPTIDE

<223> C11 humanized antibody light chain a

<400> 29

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Lys

<210> 30

<211> 113

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> PEPTIDE

<223> C11 humanized antibody light chain b

<400> 30

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Lys

<210> 31

<211> 122

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> PEPTIDE

<223> C19 humanized antibody heavy chain a

<400> 31

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Ala Ile Thr Phe Thr Asp Tyr

20 25 30

Tyr Met Asn Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Asp Ile Tyr Pro Asn Thr Gly Gly Thr Thr Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Arg Val Thr Leu Thr Val Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Glu Lys Ile Thr Met Glu Tyr Tyr Tyr Ala Met Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 32

<211> 122

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> PEPTIDE

<223> C19 humanized antibody heavy chain b

<400> 32

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Ala Ile Thr Phe Thr Asp Tyr

20 25 30

Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Asp Ile Tyr Pro Asn Thr Gly Gly Thr Thr Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Arg Ala Thr Leu Thr Val Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Glu Lys Ile Thr Met Glu Tyr Tyr Tyr Ala Met Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 33

<211> 122

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> PEPTIDE

<223> C19 humanized antibody heavy chain C

<400> 33

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Ala Ile Thr Phe Thr Asp Tyr

20 25 30

Tyr Met Asn Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Asp Ile Tyr Pro Asn Thr Gly Gly Thr Thr Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Arg Val Thr Leu Thr Val Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Glu Lys Ile Thr Met Glu Tyr Tyr Tyr Ala Met Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 34

<211> 111

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> PEPTIDE

<223> C19 humanized antibody light chain a

<400> 34

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Ser Ser His

20 25 30

Asp Ile His Leu Ile His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro

35 40 45

Lys Leu Leu Ile Tyr Ala Ala Ser Ser Leu Glu Ser Gly Val Pro Ser

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Ser Ile

85 90 95

Glu Asp Pro Pro Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 35

<211> 111

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> PEPTIDE

<223> C19 humanized antibody light chain b

<400> 35

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Ser Ser His

20 25 30

Asp Ile His Leu Ile His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro

35 40 45

Lys Leu Leu Ile Tyr Ala Ala Ser Ser Leu Glu Ser Gly Val Pro Ser

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Gln Pro Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Ser Ile

85 90 95

Glu Asp Pro Pro Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 36

<211> 111

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> PEPTIDE

<223> C19 humanized antibody light chain C

<400> 36

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

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Ser Ser His

20 25 30

Asp Ile His Leu Ile His Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro

35 40 45

Lys Leu Leu Ile Tyr Ala Ala Ser Ser Leu Glu Ser Gly Val Pro Ser

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Ser Ile

85 90 95

Glu Asp Pro Pro Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 37

<211> 118

<212> PRT

<213> mouse (Mus musculus)

<400> 37

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

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr

20 25 30

Tyr Met His Trp Val Lys Gln Ser Pro Glu Lys Ser Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asn Pro Asn Thr Gly Ser Cys Thr Tyr Asn Gln Lys Phe

50 55 60

Thr Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Leu Asn Tyr Tyr Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr

100 105 110

Thr Val Thr Val Ser Ser

115

<210> 38

<211> 113

<212> PRT

<213> mouse (Mus musculus)

<400> 38

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

1 5 10 15

Gln Lys Val Thr Met Ser Cys Lys Ser Ser Gln Thr Leu Leu Asn Ser

20 25 30

Asn Asp Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Ser Pro Lys Leu Leu Val Tyr Phe Ala Ser Thr Arg Asp Ser Gly Val

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Gln

<210> 39

<211> 10

<212> PRT

<213> mouse (Mus musculus)

<400> 39

Gly Tyr Thr Phe Thr Gly Tyr Tyr Met His

1 5 10

<210> 40

<211> 17

<212> PRT

<213> mouse (Mus musculus)

<400> 40

Glu Ile Asn Pro Asn Thr Gly Ser Cys Thr Tyr Asn Gln Lys Phe Thr

1 5 10 15

Gly

<210> 41

<211> 9

<212> PRT

<213> mouse (Mus musculus)

<400> 41

Leu Asn Tyr Tyr Trp Tyr Phe Asp Val

1 5

<210> 42

<211> 17

<212> PRT

<213> mouse (Mus musculus)

<400> 42

Lys Ser Ser Gln Thr Leu Leu Asn Ser Asn Asp Gln Lys Asn Tyr Leu

1 5 10 15

Ala

<210> 43

<211> 7

<212> PRT

<213> mouse (Mus musculus)

<400> 43

Phe Ala Ser Thr Arg Asp Ser

1 5

<210> 44

<211> 9

<212> PRT

<213> mouse (Mus musculus)

<400> 44

Gln Gln Asp Tyr Ser Thr Pro Phe Thr

1 5

<210> 45

<211> 119

<212> PRT

<213> mouse (Mus musculus)

<400> 45

Gln Val Gln Leu Gln Gln Ser Gly Thr Glu Leu Val Gln Pro Gly Ala

1 5 10 15

Ser Val Lys Met Thr Cys Lys Thr Ser Gly Tyr Thr Phe Thr Asn Tyr

20 25 30

Trp Ile Ala Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Glu Met Tyr Pro Gly Gly Gly Gly Asp Asn His His Glu Lys Phe

50 55 60

Lys Asn Lys Ala Ser Leu Thr Val Asp Met Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Arg Leu Thr Phe Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Asp Tyr Gly Asn Pro Cys Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Ser Leu Thr Val Ser Ser

115

<210> 46

<211> 111

<212> PRT

<213> mouse (Mus musculus)

<400> 46

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

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Gln Gly Val Thr Thr Ser

20 25 30

Ser His Ser Tyr Met His Trp Tyr Gln Gln Lys Leu Gly Gln Ser Pro

35 40 45

Lys Leu Leu Ile Lys Tyr Ala Ser Asn Leu Glu Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His

65 70 75 80

Pro Val Glu Glu Glu Asp Thr Ala Thr Tyr Tyr Cys Gln His Ser Trp

85 90 95

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

100 105 110

<210> 47

<211> 10

<212> PRT

<213> mouse (Mus musculus)

<400> 47

Gly Tyr Thr Phe Thr Asn Tyr Trp Ile Ala

1 5 10

<210> 48

<211> 17

<212> PRT

<213> mouse (Mus musculus)

<400> 48

Glu Met Tyr Pro Gly Gly Gly Gly Asp Asn His His Glu Lys Phe Lys

1 5 10 15

Asn

<210> 49

<211> 10

<212> PRT

<213> mouse (Mus musculus)

<400> 49

Arg Asp Tyr Gly Asn Pro Cys Phe Asp Tyr

1 5 10

<210> 50

<211> 15

<212> PRT

<213> mouse (Mus musculus)

<400> 50

Arg Ala Ser Gln Gly Val Thr Thr Ser Ser His Ser Tyr Met His

1 5 10 15

<210> 51

<211> 7

<212> PRT

<213> mouse (Mus musculus)

<400> 51

Tyr Ala Ser Asn Leu Glu Ser

1 5

<210> 52

<211> 9

<212> PRT

<213> mouse (Mus musculus)

<400> 52

Gln His Ser Trp Glu Ile Pro Tyr Thr

1 5

<210> 53

<211> 119

<212> PRT

<213> mouse (Mus musculus)

<400> 53

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

1 5 10 15

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

20 25 30

Asp Ile Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Trp Val Tyr Pro Arg Asp Gly Ser Thr Lys Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Val Asp Thr Ser Ser Arg Thr Val Tyr

65 70 75 80

Met Glu Phe His Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys

85 90 95

Ala Arg Ser Gly Leu Thr Gly Ser Pro Phe Ala Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ala

115

<210> 54

<211> 111

<212> PRT

<213> mouse (Mus musculus)

<400> 54

Lys Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Arg

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Phe Asp Ser Tyr

20 25 30

Gly Asn Thr Phe Met His Trp Phe Gln Gln Lys Ser Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Asp

65 70 75 80

Pro Val Glu Ala Asp Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Asn Asn

85 90 95

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

100 105 110

<210> 55

<211> 10

<212> PRT

<213> mouse (Mus musculus)

<400> 55

Gly Tyr Thr Phe Thr Thr Tyr Asp Ile Asn

1 5 10

<210> 56

<211> 17

<212> PRT

<213> mouse (Mus musculus)

<400> 56

Trp Val Tyr Pro Arg Asp Gly Ser Thr Lys Tyr Asn Glu Lys Phe Lys

1 5 10 15

Gly

<210> 57

<211> 10

<212> PRT

<213> mouse (Mus musculus)

<400> 57

Ser Gly Leu Thr Gly Ser Pro Phe Ala Tyr

1 5 10

<210> 58

<211> 15

<212> PRT

<213> mouse (Mus musculus)

<400> 58

Arg Ala Ser Glu Ser Phe Asp Ser Tyr Gly Asn Thr Phe Met His

1 5 10 15

<210> 59

<211> 7

<212> PRT

<213> mouse (Mus musculus)

<400> 59

Arg Ala Ser Asn Leu Glu Ser

1 5

<210> 60

<211> 9

<212> PRT

<213> mouse (Mus musculus)

<400> 60

Gln Gln Asn Asn Glu Tyr Pro Leu Thr

1 5

<210> 61

<211> 122

<212> PRT

<213> mouse (Mus musculus)

<400> 61

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

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Ser Ala Ile Thr Phe Thr Asp Tyr

20 25 30

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

35 40 45

Gly Asp Ile Asn Pro Asn Asn Gly Gly Thr Thr Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Thr Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Glu Lys Ile Ser Met Glu Tyr Tyr Tyr Ala Met Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Ser Val Thr Val Ser Ser

115 120

<210> 62

<211> 111

<212> PRT

<213> mouse (Mus musculus)

<400> 62

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

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Ser Ser His

20 25 30

Asp Ile His Leu Met His Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro

35 40 45

Lys Leu Leu Ile Tyr Ala Ala Ser Asn Leu Glu Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Ala Thr Asp Phe Thr Leu Asn Ile His

65 70 75 80

Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Phe Cys Gln Gln Ser Ile

85 90 95

Glu Asp Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 63

<211> 10

<212> PRT

<213> mouse (Mus musculus)

<400> 63

Ala Ile Thr Phe Thr Asp Tyr Tyr Met Asn

1 5 10

<210> 64

<211> 17

<212> PRT

<213> mouse (Mus musculus)

<400> 64

Asp Ile Asn Pro Asn Asn Gly Gly Thr Thr Tyr Asn Gln Lys Phe Lys

1 5 10 15

Gly

<210> 65

<211> 13

<212> PRT

<213> mouse (Mus musculus)

<400> 65

Glu Lys Ile Ser Met Glu Tyr Tyr Tyr Ala Met Asp Tyr

1 5 10

<210> 66

<211> 15

<212> PRT

<213> mouse (Mus musculus)

<400> 66

Arg Ala Ser Glu Ser Val Ser Ser His Asp Ile His Leu Met His

1 5 10 15

<210> 67

<211> 7

<212> PRT

<213> mouse (Mus musculus)

<400> 67

Ala Ala Ser Asn Leu Glu Ser

1 5

<210> 68

<211> 9

<212> PRT

<213> mouse (Mus musculus)

<400> 68

Gln Gln Ser Ile Glu Asp Pro Pro Thr

1 5

<210> 69

<211> 116

<212> PRT

<213> mouse (Mus musculus)

<400> 69

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

1 5 10 15

Ser Val Lys Ile Ser Cys Gln Ala Ser Gly Tyr Thr Phe Thr Val Tyr

20 25 30

Tyr Met Asn Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile

35 40 45

Gly Asp Ile Asp Pro Asn Thr Gly Asp Ser Thr Tyr Asn Gln Lys Phe

50 55 60

Arg Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Tyr Asp Gly Tyr Ile Asp Tyr Trp Gly Gln Gly Thr Thr Leu

100 105 110

Thr Val Ser Ser

115

<210> 70

<211> 112

<212> PRT

<213> mouse (Mus musculus)

<400> 70

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

1 5 10 15

Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser

20 25 30

Asn Arg His Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

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

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Met Gly Ile Tyr Tyr Cys Phe Gln Gly

85 90 95

Thr His Val Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105 110

<210> 71

<211> 10

<212> PRT

<213> mouse (Mus musculus)

<400> 71

Gly Tyr Thr Phe Thr Val Tyr Tyr Met Asn

1 5 10

<210> 72

<211> 17

<212> PRT

<213> mouse (Mus musculus)

<400> 72

Asp Ile Asp Pro Asn Thr Gly Asp Ser Thr Tyr Asn Gln Lys Phe Arg

1 5 10 15

Gly

<210> 73

<211> 7

<212> PRT

<213> mouse (Mus musculus)

<400> 73

Tyr Asp Gly Tyr Ile Asp Tyr

1 5

<210> 74

<211> 16

<212> PRT

<213> mouse (Mus musculus)

<400> 74

Arg Ser Ser Gln Ser Ile Val His Ser Asn Arg His Thr Tyr Leu Glu

1 5 10 15

<210> 75

<211> 7

<212> PRT

<213> mouse (Mus musculus)

<400> 75

Gly Val Ser Asn Arg Phe Ser

1 5

<210> 76

<211> 9

<212> PRT

<213> mouse (Mus musculus)

<400> 76

Phe Gln Gly Thr His Val Pro Leu Thr

1 5

<210> 77

<211> 117

<212> PRT

<213> mouse (Mus musculus)

<400> 77

Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ala

1 5 10 15

Leu Val Lys Leu Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Tyr

20 25 30

Tyr Met His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile

35 40 45

Gly Arg Phe Asp Pro Glu Asn Gly Asp Thr Ile Tyr Asp Trp Lys Phe

50 55 60

Gln Asp Lys Ala Ile Ile Thr Ser Asp Thr Ser Ser Asn Thr Ala Tyr

65 70 75 80

Leu His Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Gly Asp Tyr Met Phe Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ala

115

<210> 78

<211> 107

<212> PRT

<213> mouse (Mus musculus)

<400> 78

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

1 5 10 15

Glu Lys Val Thr Ile Arg Cys Ile Thr Ser Thr Gly Val Asp Asp Asp

20 25 30

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

35 40 45

Ser Glu Gly Asn Thr Leu Arg Pro Gly Val Pro Ser Arg Phe Ser Ser

50 55 60

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

65 70 75 80

Glu Asp Val Ala Asp Tyr Phe Cys Leu Gln Ser Asp His Leu Pro Phe

85 90 95

Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 79

<211> 10

<212> PRT

<213> mouse (Mus musculus)

<400> 79

Gly Phe Asn Ile Lys Asp Tyr Tyr Met His

1 5 10

<210> 80

<211> 17

<212> PRT

<213> mouse (Mus musculus)

<400> 80

Arg Phe Asp Pro Glu Asn Gly Asp Thr Ile Tyr Asp Trp Lys Phe Gln

1 5 10 15

Asp

<210> 81

<211> 8

<212> PRT

<213> mouse (Mus musculus)

<400> 81

Ser Gly Asp Tyr Met Phe Asp Tyr

1 5

<210> 82

<211> 11

<212> PRT

<213> mouse (Mus musculus)

<400> 82

Ile Thr Ser Thr Gly Val Asp Asp Asp Phe Asn

1 5 10

<210> 83

<211> 7

<212> PRT

<213> mouse (Mus musculus)

<400> 83

Glu Gly Asn Thr Leu Arg Pro

1 5

<210> 84

<211> 9

<212> PRT

<213> mouse (Mus musculus)

<400> 84

Leu Gln Ser Asp His Leu Pro Phe Thr

1 5

<210> 85

<211> 120

<212> PRT

<213> mouse (Mus musculus)

<400> 85

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

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Thr Gly Tyr Thr Phe Ser Ser Tyr

20 25 30

Trp Ile Glu Trp Val Lys Gln Arg Pro Gly His Gly Leu Glu Trp Ile

35 40 45

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

50 55 60

Lys Gly Lys Ala Thr Phe Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Asn Tyr Asp Gly Ser Leu Tyr Pro Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Ser Val Thr Val Ser Ser

115 120

<210> 86

<211> 111

<212> PRT

<213> mouse (Mus musculus)

<400> 86

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

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Arg Thr Ser Glu Ser Val Ser Ile His

20 25 30

Gly Thr His Leu Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Ala Ala Ser Asn Leu Glu Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Glu Thr Asp Phe Thr Leu Asn Ile His

65 70 75 80

Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Phe Cys Gln Gln Ser Ile

85 90 95

Glu Asp Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 87

<211> 10

<212> PRT

<213> mouse (Mus musculus)

<400> 87

Gly Tyr Thr Phe Ser Ser Tyr Trp Ile Glu

1 5 10

<210> 88

<211> 17

<212> PRT

<213> mouse (Mus musculus)

<400> 88

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

1 5 10 15

Gly

<210> 89

<211> 11

<212> PRT

<213> mouse (Mus musculus)

<400> 89

Asn Tyr Asp Gly Ser Leu Tyr Pro Met Asp Tyr

1 5 10

<210> 90

<211> 15

<212> PRT

<213> mouse (Mus musculus)

<400> 90

Arg Thr Ser Glu Ser Val Ser Ile His Gly Thr His Leu Met His

1 5 10 15

<210> 91

<211> 7

<212> PRT

<213> mouse (Mus musculus)

<400> 91

Ala Ala Ser Asn Leu Glu Ser

1 5

<210> 92

<211> 9

<212> PRT

<213> mouse (Mus musculus)

<400> 92

Gln Gln Ser Ile Glu Asp Pro Pro Thr

1 5

<210> 93

<211> 522

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> PEPTIDE

<223> sequence of Human CSF-1R ECD-His

<400> 93

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

1 5 10 15

Gly Arg Ala Ile Pro Val Ile Glu Pro Ser Val Pro Glu Leu Val Val

20 25 30

Lys Pro Gly Ala Thr Val Thr Leu Arg Cys Val Gly Asn Gly Ser Val

35 40 45

Glu Trp Asp Gly Pro Pro Ser Pro His Trp Thr Leu Tyr Ser Asp Gly

50 55 60

Ser Ser Ser Ile Leu Ser Thr Asn Asn Ala Thr Phe Gln Asn Thr Gly

65 70 75 80

Thr Tyr Arg Cys Thr Glu Pro Gly Asp Pro Leu Gly Gly Ser Ala Ala

85 90 95

Ile His Leu Tyr Val Lys Asp Pro Ala Arg Pro Trp Asn Val Leu Ala

100 105 110

Gln Glu Val Val Val Phe Glu Asp Gln Asp Ala Leu Leu Pro Cys Leu

115 120 125

Leu Thr Asp Pro Val Leu Glu Ala Gly Val Ser Leu Val Arg Val Arg

130 135 140

Gly Arg Pro Leu Met Arg His Thr Asn Tyr Ser Phe Ser Pro Trp His

145 150 155 160

Gly Phe Thr Ile His Arg Ala Lys Phe Ile Gln Ser Gln Asp Tyr Gln

165 170 175

Cys Ser Ala Leu Met Gly Gly Arg Lys Val Met Ser Ile Ser Ile Arg

180 185 190

Leu Lys Val Gln Lys Val Ile Pro Gly Pro Pro Ala Leu Thr Leu Val

195 200 205

Pro Ala Glu Leu Val Arg Ile Arg Gly Glu Ala Ala Gln Ile Val Cys

210 215 220

Ser Ala Ser Ser Val Asp Val Asn Phe Asp Val Phe Leu Gln His Asn

225 230 235 240

Asn Thr Lys Leu Ala Ile Pro Gln Gln Ser Asp Phe His Asn Asn Arg

245 250 255

Tyr Gln Lys Val Leu Thr Leu Asn Leu Asp Gln Val Asp Phe Gln His

260 265 270

Ala Gly Asn Tyr Ser Cys Val Ala Ser Asn Val Gln Gly Lys His Ser

275 280 285

Thr Ser Met Phe Phe Arg Val Val Glu Ser Ala Tyr Leu Asn Leu Ser

290 295 300

Ser Glu Gln Asn Leu Ile Gln Glu Val Thr Val Gly Glu Gly Leu Asn

305 310 315 320

Leu Lys Val Met Val Glu Ala Tyr Pro Gly Leu Gln Gly Phe Asn Trp

325 330 335

Thr Tyr Leu Gly Pro Phe Ser Asp His Gln Pro Glu Pro Lys Leu Ala

340 345 350

Asn Ala Thr Thr Lys Asp Thr Tyr Arg His Thr Phe Thr Leu Ser Leu

355 360 365

Pro Arg Leu Lys Pro Ser Glu Ala Gly Arg Tyr Ser Phe Leu Ala Arg

370 375 380

Asn Pro Gly Gly Trp Arg Ala Leu Thr Phe Glu Leu Thr Leu Arg Tyr

385 390 395 400

Pro Pro Glu Val Ser Val Ile Trp Thr Phe Ile Asn Gly Ser Gly Thr

405 410 415

Leu Leu Cys Ala Ala Ser Gly Tyr Pro Gln Pro Asn Val Thr Trp Leu

420 425 430

Gln Cys Ser Gly His Thr Asp Arg Cys Asp Glu Ala Gln Val Leu Gln

435 440 445

Val Trp Asp Asp Pro Tyr Pro Glu Val Leu Ser Gln Glu Pro Phe His

450 455 460

Lys Val Thr Val Gln Ser Leu Leu Thr Val Glu Thr Leu Glu His Asn

465 470 475 480

Gln Thr Tyr Glu Cys Arg Ala His Asn Ser Val Gly Ser Gly Ser Trp

485 490 495

Ala Phe Ile Pro Ile Ser Ala Gly Ala His Thr His Pro Pro Asp Glu

500 505 510

Gly Ser His His His His His His His His

515 520

<210> 94

<211> 745

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> PEPTIDE

<223> sequence of Human CSF-1R-huFc

<400> 94

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

1 5 10 15

Gly Arg Ala Ile Pro Val Ile Glu Pro Ser Val Pro Glu Leu Val Val

20 25 30

Lys Pro Gly Ala Thr Val Thr Leu Arg Cys Val Gly Asn Gly Ser Val

35 40 45

Glu Trp Asp Gly Pro Pro Ser Pro His Trp Thr Leu Tyr Ser Asp Gly

50 55 60

Ser Ser Ser Ile Leu Ser Thr Asn Asn Ala Thr Phe Gln Asn Thr Gly

65 70 75 80

Thr Tyr Arg Cys Thr Glu Pro Gly Asp Pro Leu Gly Gly Ser Ala Ala

85 90 95

Ile His Leu Tyr Val Lys Asp Pro Ala Arg Pro Trp Asn Val Leu Ala

100 105 110

Gln Glu Val Val Val Phe Glu Asp Gln Asp Ala Leu Leu Pro Cys Leu

115 120 125

Leu Thr Asp Pro Val Leu Glu Ala Gly Val Ser Leu Val Arg Val Arg

130 135 140

Gly Arg Pro Leu Met Arg His Thr Asn Tyr Ser Phe Ser Pro Trp His

145 150 155 160

Gly Phe Thr Ile His Arg Ala Lys Phe Ile Gln Ser Gln Asp Tyr Gln

165 170 175

Cys Ser Ala Leu Met Gly Gly Arg Lys Val Met Ser Ile Ser Ile Arg

180 185 190

Leu Lys Val Gln Lys Val Ile Pro Gly Pro Pro Ala Leu Thr Leu Val

195 200 205

Pro Ala Glu Leu Val Arg Ile Arg Gly Glu Ala Ala Gln Ile Val Cys

210 215 220

Ser Ala Ser Ser Val Asp Val Asn Phe Asp Val Phe Leu Gln His Asn

225 230 235 240

Asn Thr Lys Leu Ala Ile Pro Gln Gln Ser Asp Phe His Asn Asn Arg

245 250 255

Tyr Gln Lys Val Leu Thr Leu Asn Leu Asp Gln Val Asp Phe Gln His

260 265 270

Ala Gly Asn Tyr Ser Cys Val Ala Ser Asn Val Gln Gly Lys His Ser

275 280 285

Thr Ser Met Phe Phe Arg Val Val Glu Ser Ala Tyr Leu Asn Leu Ser

290 295 300

Ser Glu Gln Asn Leu Ile Gln Glu Val Thr Val Gly Glu Gly Leu Asn

305 310 315 320

Leu Lys Val Met Val Glu Ala Tyr Pro Gly Leu Gln Gly Phe Asn Trp

325 330 335

Thr Tyr Leu Gly Pro Phe Ser Asp His Gln Pro Glu Pro Lys Leu Ala

340 345 350

Asn Ala Thr Thr Lys Asp Thr Tyr Arg His Thr Phe Thr Leu Ser Leu

355 360 365

Pro Arg Leu Lys Pro Ser Glu Ala Gly Arg Tyr Ser Phe Leu Ala Arg

370 375 380

Asn Pro Gly Gly Trp Arg Ala Leu Thr Phe Glu Leu Thr Leu Arg Tyr

385 390 395 400

Pro Pro Glu Val Ser Val Ile Trp Thr Phe Ile Asn Gly Ser Gly Thr

405 410 415

Leu Leu Cys Ala Ala Ser Gly Tyr Pro Gln Pro Asn Val Thr Trp Leu

420 425 430

Gln Cys Ser Gly His Thr Asp Arg Cys Asp Glu Ala Gln Val Leu Gln

435 440 445

Val Trp Asp Asp Pro Tyr Pro Glu Val Leu Ser Gln Glu Pro Phe His

450 455 460

Lys Val Thr Val Gln Ser Leu Leu Thr Val Glu Thr Leu Glu His Asn

465 470 475 480

Gln Thr Tyr Glu Cys Arg Ala His Asn Ser Val Gly Ser Gly Ser Trp

485 490 495

Ala Phe Ile Pro Ile Ser Ala Gly Ala His Thr His Pro Pro Asp Glu

500 505 510

Gly Ser Gly Gly Gly Gly Asp Lys Thr His Thr Cys Pro Pro Cys Pro

515 520 525

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

530 535 540

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

545 550 555 560

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

565 570 575

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

580 585 590

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

595 600 605

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

610 615 620

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

625 630 635 640

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

645 650 655

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

660 665 670

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

675 680 685

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

690 695 700

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

705 710 715 720

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

725 730 735

Lys Ser Leu Ser Leu Ser Pro Gly Lys

740 745

<210> 95

<211> 17

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> after optimization of C11HCDR2

<400> 95

Glu Met Asn Pro Asn Thr Gly Asp Ser Ser Tyr Asn Gln Lys Phe Lys

1 5 10 15

Gly

<210> 96

<211> 17

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

After <223> C11LCDR1 optimization

<400> 96

Lys Ser Ser Gln Ser Leu Leu Asn Ser Gly Asn Gln Lys Thr Ser Leu

1 5 10 15

Thr

<210> 97

<211> 17

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> after optimization of C19HCDR2

<400> 97

Asp Ile Tyr Pro Asn Thr Gly Gly Thr Thr Tyr Asn Gln Lys Phe Lys

1 5 10 15

Gly

<210> 98

<211> 10

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> huC19I HCDR1

<400> 98

Ala Ile Thr Phe Thr Asp Tyr Tyr Ile Asn

1 5 10

<210> 99

<211> 452

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> PEPTIDE

<223> huC19I HC

<400> 99

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Ala Ile Thr Phe Thr Asp Tyr

20 25 30

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

35 40 45

Gly Asp Ile Tyr Pro Asn Thr Gly Gly Thr Thr Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Arg Val Thr Leu Thr Val Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Glu Lys Ile Thr Met Glu Tyr Tyr Tyr Ala Met Asp Tyr Trp

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

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

385 390 395 400

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

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

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

435 440 445

Ser Pro Gly Lys

450

<210> 100

<211> 122

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> PEPTIDE

<223> C19 humanized antibody light chain d

<400> 100

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Ala Ile Thr Phe Thr Asp Tyr

20 25 30

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

35 40 45

Gly Asp Ile Tyr Pro Asn Thr Gly Gly Thr Thr Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Arg Val Thr Leu Thr Val Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Glu Lys Ile Thr Met Glu Tyr Tyr Tyr Ala Met Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 101

<211> 330

<212> PRT

<213> Intelligent (Homo sapiens)

<220>

<221> PEPTIDE

<223> heavy chain constant region of humanized antibody

<400> 101

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

1 5 10 15

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

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

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

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210> 102

<211> 107

<212> PRT

<213> Intelligent (Homo sapiens)

<220>

<221> PEPTIDE

<223> light chain constant region of humanized antibody

<400> 102

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

Claims (26)

1. An anti-CSF-1R antibody or antigen-binding fragment thereof, comprising an antibody light chain variable region and an antibody heavy chain variable region, wherein:

x) the antibody light chain variable region comprises LCDR1, LCDR2 and LCDR3 shown as SEQ ID NO. 14, SEQ ID NO. 15 and SEQ ID NO. 16 respectively; the heavy chain variable region of the antibody comprises HCDR1, HCDR2 and HCDR3 shown as SEQ ID NO.11, SEQ ID NO. 12 and SEQ ID NO. 13 respectively;

z) the antibody light chain variable region comprises LCDR1, LCDR2 and LCDR3 shown as SEQ ID NO. 14, SEQ ID NO. 15 and SEQ ID NO. 16, respectively; the variable region of the antibody heavy chain comprises the following components: HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO. 98, SEQ ID NO. 97 and SEQ ID NO. 13;

or ad) the antibody light chain variable region comprises: LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO. 14, SEQ ID NO. 15 and SEQ ID NO. 16; the variable region of the antibody heavy chain comprises the following components: HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO.11, SEQ ID NO. 97 and SEQ ID NO. 13.

2. The anti-CSF-1R antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof is a murine antibody, a chimeric antibody, a humanized antibody, or an antigen-binding fragment thereof.

3. The anti-CSF-1R antibody or antigen-binding fragment thereof of claim 2, wherein

i) The amino acid sequence of the light chain variable region is shown as SEQ ID NO:10, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO:9 or a variant thereof having an amino acid mutation to M34I, N55T or a combination thereof.

4. The anti-CSF-1R antibody or antigen-binding fragment thereof of claim 2, wherein the light chain FR region sequence on the humanized antibody light chain variable region is derived from the human germline light chain IGkV1-13 sequence set forth in SEQ ID NO: 24;

and/or, the humanized antibody heavy chain variable region further comprises a heavy chain FR region of a human IgG1, igG2, igG3 or IgG4 or variant thereof.

5. The anti-CSF-1R antibody or antigen-binding fragment thereof of claim 4, wherein the heavy chain FR region sequence on the humanized antibody heavy chain variable region is derived from the human germline heavy chain IGHV1-2 sequence set forth in SEQ ID NO. 23.

6. The anti-CSF-1R antibody or antigen-binding fragment thereof of claim 4, wherein the humanized antibody light chain variable region sequence is the sequence set forth in SEQ ID No. 34 or a variant thereof; the variant has amino acid mutations of F87A, F91Y or combinations thereof;

and/or, the humanized antibody heavy chain variable region sequence is shown as SEQ ID NO:100 or SEQ ID NO:33 or the variant thereof; wherein, the amino acid mutation of the variant of the sequence shown in SEQ ID NO. 33 is Q1E, M34I, T55N, E89D or the combination thereof.

7. The anti-CSF-1R antibody or antigen-binding fragment thereof according to claim 6, wherein the variant of the sequence shown in SEQ ID NO. 33 has an amino acid mutation to M34I, T55N or a combination thereof.

8. The anti-CSF-1R antibody or antigen-binding fragment thereof of claim 6, wherein the sequence of the heavy chain variable region is SEQ ID NO: 31. 32, 33 or 100 and the light chain variable region has the sequence of SEQ ID NO: 34. 35 or 36.

9. The anti-CSF-1R antibody or antigen-binding fragment thereof of any one of claims 2-8, wherein the chimeric or humanized antibody further comprises a light chain and/or a heavy chain constant region, the light chain constant region sequence being as set forth in SEQ ID NO:102, and/or the heavy chain constant region sequence is as set forth in SEQ ID NO:101, respectively.

10. The anti-CSF-1R antibody or antigen-binding fragment thereof of claim 9, wherein the humanized antibody:

comprises a heavy chain with a sequence shown as SEQ ID NO. 19 and a light chain with a sequence shown as SEQ ID NO. 20;

or comprises a heavy chain with a sequence shown as SEQ ID NO. 99 and a light chain with a sequence shown as SEQ ID NO. 20.

11. A multispecific antibody comprising a light chain variable region and a heavy chain variable region of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10.

12. A single-chain antibody comprising the light chain variable region and the heavy chain variable region of the antibody or the antigen-binding fragment thereof according to any one of claims 1 to 10.

13. An antibody-drug conjugate, wherein the antibody comprises the light chain variable region and the heavy chain variable region of the antibody or the antigen-binding fragment thereof according to any one of claims 1 to 10.

14. A nucleic acid molecule encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, the multispecific antibody according to claim 11, or the single-chain antibody according to claim 12.

15. An expression vector comprising the nucleic acid of claim 14.

16. A host cell transformed with the expression vector of claim 15.

17. The host cell of claim 16, wherein the host cell is a bacterium; or the host cell is saccharomycete; alternatively, the host cell is an animal cell.

18. The host cell of claim 17, wherein the bacterium is escherichia coli; the yeast is pichia pastoris; the animal cell is a Chinese Hamster Ovary (CHO) cell, a Human Embryonic Kidney (HEK) 293 cell or an NS0 cell.

19. A method for producing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, the multispecific antibody according to claim 11, or the single-chain antibody according to claim 12, comprising expressing the antibody or antigen-binding fragment thereof, the multispecific antibody, or the single-chain antibody in the host cell according to any one of claims 16 to 18, and isolating the antibody or antigen-binding fragment thereof, the multispecific antibody, or the single-chain antibody from the host cell.

20. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, a multispecific antibody according to claim 11, or a single-chain antibody according to claim 12, and a pharmaceutically acceptable buffer, excipient, diluent, or carrier.

21. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, the multispecific antibody according to claim 11, the single-chain antibody according to claim 12, the antibody-drug conjugate according to claim 13, the nucleic acid molecule according to claim 14 or the pharmaceutical composition according to claim 20 for the manufacture of a medicament for anticancer therapy, wherein the antibody or antigen-binding fragment thereof, the multispecific antibody, the single-chain antibody, the antibody-drug conjugate, the nucleic acid molecule or the pharmaceutical composition is administered before, during, substantially simultaneously with or after the initiation of therapy with another anticancer therapy.

22. The use of claim 21, wherein the other therapy for the anti-cancer treatment is selected from the group consisting of an anti-angiogenic agent, a chemotherapeutic agent, radiation, tumor immunotherapy, or a combination thereof.

23. The use of claim 22, wherein the chemotherapeutic agent is selected from one or more of taxanes, doxorubicin, modified doxorubicin, sunitinib, sorafenib and other multi-kinase inhibitors, oxaliplatin, cisplatin, carboplatin, etoposide, gemcitabine and vinblastine; and/or the presence of a gas in the gas,

the tumor immunotherapy is selected from the group consisting of T cell cement (engaging agent), targeted immunosuppression, cancer vaccine/enhanced dendritic cell function, and adoptive cell transfer.

24. The use of claim 22, wherein the taxane is selected from the group consisting of paclitaxel injection, docetaxel, paclitaxel albumin; the modified doxorubicin is selected from Caelyx or Doxil.

25. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, the multispecific antibody according to claim 11, the single-chain antibody according to claim 12, the antibody-drug conjugate according to claim 13, the nucleic acid molecule according to claim 14 or the pharmaceutical composition according to claim 20 for the preparation of a medicament for the treatment of cancer.

26. The use of claim 25, wherein the cancer is selected from the group consisting of breast cancer, endometrial cancer, squamous cell carcinoma, follicular lymphoma, renal cell carcinoma, uveal melanoma, cervical cancer, head and neck cancer, hodgkin's disease, astrocytic cancer, lung adenocarcinoma, mesothelioma, choriocarcinoma, melanoma, breast cancer, ovarian cancer, prostate cancer, pancreatic cancer, renal cancer, lung cancer, liver cancer, brain cancer, gastric cancer, colorectal cancer, bladder cancer, esophageal cancer, cervical cancer, multiple myeloma, leukemia, lymphoma, and glioblastoma.

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