CN110913894A

CN110913894A - anti-CSF-1R antibodies, antigen binding fragments thereof and medical uses thereof

Info

Publication number: CN110913894A
Application number: CN201880044519.6A
Authority: CN
Inventors: 闫树德; 黄浩; 张连山; 曹国庆; 蒋家骅
Original assignee: Jiangsu Hengrui Medicine Co Ltd; Shanghai Hengrui Pharmaceutical Co Ltd
Current assignee: Jiangsu Hengrui Medicine Co Ltd; Shanghai Hengrui Pharmaceutical Co Ltd
Priority date: 2017-10-26
Filing date: 2018-10-25
Publication date: 2020-03-24
Anticipated expiration: 2038-10-25
Also published as: WO2019080889A1; TW201917136A; TWI714895B; CN110913894B

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 through various modes such as immune check point inhibition, direct activation, tumor microenvironment activation and the like, and has the effect of killing 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 interstitial tissue, i.e., tumor-associated macrophages (TAMs), belong to the M2 class, and do not play an anti-tumor role like the M1 class macrophages, but participate in the processes of tumor occurrence, growth, invasion and metastasis. 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 major 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 of 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 monocyte/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, autoimmune diseases, and the like, 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 which inhibit CSF-1 activity (Sherr, C.J. et al, 1989, Blood73: 1786-1793). W02009/026303 discloses anti-CSF-1R antibodies that bind to human CSF-1R and in vivo mouse tumor models using anti-murine 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-murine 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-l 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 1 LCDR selected from the group consisting of seq id nos: 6,7, 8, 14, 15, 16 or 96; and

an antibody heavy chain variable region comprising at least 1 HCDR selected from the group consisting of seq id nos: 3,4, 5, 11, 12, 13, 95, 97 or 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 LCDR variants selected from LCDR1, LCDR2 and LCDR3 having 3, 2, 1 or 0 amino acid mutations respectively on the basis of the amino acid sequences shown as SEQ ID NO 42, SEQ ID NO 43 and SEQ ID NO 44; the heavy chain variable region of the antibody comprises an HCDR variant selected from the group consisting of HCDR1, HCDR2, and HCDR3 having 3, 2, 1, or 0 amino acid mutations, respectively, as shown in SEQ ID NO:39, SEQ ID NO:40, and SEQ ID NO: 41;

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

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

d) the antibody light chain variable region comprises an LCDR variant selected from the group consisting of LCDR1, LCDR2 and LCDR3 having 3, 2, 1 or 0 amino acid mutations shown in SEQ ID NO 66, SEQ ID NO 67 and SEQ ID NO 68, respectively; the heavy chain variable region of the antibody comprises an HCDR variant selected from the group consisting of HCDR1, HCDR2, and HCDR3 having 3, 2, 1, or 0 amino acid mutations, respectively, as shown in SEQ ID NO 63, SEQ ID NO 64, and SEQ ID NO 65;

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

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

g) the antibody light chain variable region comprises an LCDR variant selected from the group consisting of LCDR1, LCDR2 and LCDR3 having 3, 2, 1 or 0 amino acid mutations based on SEQ ID NO 90, SEQ ID NO 91 and SEQ ID NO 92, respectively; the heavy chain variable region of the antibody comprises an HCDR variant selected from the group consisting of HCDR1, HCDR2, and HCDR3 having 3, 2, 1, or 0 amino acid mutations, respectively, as shown in SEQ ID NO 87, SEQ ID NO 88, and SEQ ID NO 89;

h) the antibody light chain variable region comprises LCDR variants selected from LCDR1, LCDR2 and LCDR3 shown as SEQ ID NO 6, SEQ ID NO 7 and SEQ ID NO 8 respectively having 3, 2, 1 or 0 amino acid mutations; the heavy chain variable region of the antibody comprises an HCDR variant selected from the group consisting of HCDR1, HCDR2, and HCDR3 having 3, 2, 1, or 0 amino acid mutations, respectively, as shown in SEQ ID NO 3, SEQ ID NO 4, and SEQ ID NO 5;

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

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

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

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

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

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

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

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

p) the antibody light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO 42, SEQ ID NO 43 and SEQ ID NO 44, respectively; the heavy chain variable region of the antibody comprises HCDR1, HCDR2 and HCDR3 shown as SEQ ID NO 39, SEQ ID NO 40 and SEQ ID NO 41 respectively;

q) the antibody light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO 50, SEQ ID NO 51 and SEQ ID NO 52, respectively; the heavy chain variable region of the antibody comprises HCDR1, HCDR2 and HCDR3 shown as SEQ ID NO 47, SEQ ID NO 48 and SEQ ID NO 49 respectively;

r) the antibody light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO 58, SEQ ID NO 59 and SEQ ID NO 60, respectively; the heavy chain variable region of the antibody comprises HCDR1, HCDR2 and HCDR3 shown as SEQ ID NO 55, SEQ ID NO 56 and SEQ ID NO 57 respectively;

s) the antibody light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO 66, SEQ ID NO 67 and SEQ ID NO 68, respectively; the heavy chain variable region of the antibody comprises HCDR1, HCDR2 and HCDR3 shown as SEQ ID NO 63, SEQ ID NO 64 and SEQ ID NO 65 respectively;

t) the antibody light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO 74, SEQ ID NO 75 and SEQ ID NO 76, respectively; the heavy chain variable region of the antibody comprises HCDR1, HCDR2 and HCDR3 shown as SEQ ID NO 71, SEQ ID NO 72 and SEQ ID NO 73 respectively;

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

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

w) the antibody light chain variable region comprises LCDR1, LCDR2 and LCDR3 shown as SEQ ID NO 6, SEQ ID NO 7 and SEQ ID NO 8, respectively; the heavy chain variable region of the antibody comprises HCDR1, HCDR2 and HCDR3 shown as SEQ ID NO 3, SEQ ID NO 4 and SEQ ID NO 5 respectively;

x) the antibody light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO 14, SEQ ID NO15 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;

y) the antibody light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO 14, SEQ ID NO15 and SEQ ID NO 16, respectively; the variable region of the antibody heavy chain comprises the following components: 98, 12 and 13 of HCDR1, 2 and 3;

z) comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:14, SEQ ID NO15 and SEQ ID NO:16, respectively; the variable region of the antibody heavy chain comprises the following components: 98, 97 and 13 as shown in SEQ ID NO, HCDR1, HCDR2 and HCDR 3;

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

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

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

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.

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

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

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

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

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, said variant preferably having 0-10 amino acid mutations in the light chain variable region, preferably mutations being M34I, N55T or a combination thereof amino acid mutations.

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 described above, wherein the light chain FR region sequence on the humanized antibody light chain variable region is derived from the human germline light chain IGkV4-1 sequence set forth as SEQ ID NO: 22; or from the human germline light chain IGkV1-13 sequence shown as SEQ ID NO. 24;

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 heavy chain FR region of human IgG1, IgG2 or IgG4, more preferably comprises a heavy chain FR region of human IgG1 or IgG 4; preferably, the heavy chain FR region sequence in the heavy chain variable region of the humanized antibody is derived from the human germline heavy chain IGHV1-46 sequence shown as SEQ ID NO: 21; or from the human germline heavy chain IGHV1-2 sequence shown in SEQ ID NO. 23.

In other embodiments, the anti-CSF-1R antibody or antigen-binding fragment thereof described above, wherein said 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 the most preferred amino acid mutations of SEQ ID NO:30 are S31N, T37N; 34 to F87A, F91Y or a combination thereof;

and/or, the humanized antibody heavy chain variable region sequence is shown as SEQ ID NO 26, SEQ ID NO 100 or SEQ ID NO 33 or the variant thereof; the variant preferably has 0-10 amino acid mutations in the heavy chain variable region, wherein the preferred amino acid mutation of SEQ ID No. 26 is S30T, T55N, L70M or a combination thereof, and the most preferred amino acid mutation is T55N; 33 preferred amino acid mutations of SEQ ID No. 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 heavy chain constant region having the sequence 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 heavy chain with a sequence shown as SEQ ID NO. 19 and a light chain with a sequence shown as SEQ ID NO. 20;

comprises a heavy chain with a sequence shown as SEQ ID NO. 17 and a light chain with a sequence shown as SEQ ID NO. 18; 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.

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 the amino acid mutation of 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: 1 or 9, wherein the preferred amino acid mutation of SEQ ID No. 1is T55N; preferred mutations of SEQ ID NO 9 are amino acid mutations of M34I, T55N or combinations 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 as 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 shown in SEQ ID NO 30 or SEQ ID NO 34, or a variant thereof.

In other embodiments, a humanized antibody of CSF-1R or a 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 the preferred amino acid mutation of SEQ ID NO. 18 is that of S31N, T37N or a combination thereof, and the most preferred amino acid mutation is T37N; 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 variants thereof, preferably comprising a human IgG1, IgG2 or IgG4 heavy chain constant region. More preferably, IgG4 or IgG1 which eliminates ADCC (antibody-dependent cell-mediated cytotoxicity) toxicity after amino acid mutation is used.

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 invention, an antibody as described above (herein and below the antibody is an anti-CSF-1R antibody as described above or a monoclonal antibody as described above) or an antigen-binding fragment thereof, wherein the antigen-binding fragment is Fab, Fv, sFv, F (ab') 2, a linear antibody, a single-chain antibody, a nanobody, a domain antibody, and a 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 a 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 cleavable 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 humanized antibody of CSF-1R or fragment thereof as described above, wherein the chemotherapeutic agent is selected from the group consisting of taxanes (paclitaxel, docetaxel, 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 humanized antibody or fragment thereof of CSF-1R as described above, wherein the tumor immunotherapy is selected from the group consisting of T cell cement (engaging agent), targeted immunosuppression, cancer vaccine/enhancement of dendritic cell function, and adoptive cell metastasis.

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 in vitro binding experiments of nine CSF-1R chimeric antibodies showed that except C27C, each antibody had strong cross-binding with cynomolgus monkey CSF-1R protein.

FIG. 2: 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 with humanized antibody huC11 blocked monocyte proliferation. (A) The antibody blocks IL-34 stimulated monocyte proliferation. (B) The antibody blocks CSF-1-stimulated monocyte proliferation.

FIG. 4: functional experiments with humanized antibody huC19 blocked monocyte proliferation. (A) The antibody blocks 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 with humanized antibody huC 19I. The anti-CSF 1R antibody had no significant effect on animal body weight compared to the control group.

Detailed Description

Term of

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 formed by two identical heavy chains and two identical light chains linked by interchain disulfide bonds, the constant regions of the heavy chains of the immunoglobulin have different amino acid compositions and sequences, and thus are different in antigenicity, and accordingly, the immunoglobulin can be classified into five classes, or isotypes called immunoglobulins, i.e., IgM, IgD, IgG, IgA, and IgE, and the corresponding heavy chains thereof are μ chain, δ chain, γ chain, α chain, and ε 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, being 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, FR 4. The 3 CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR 3; the 3 CDR regions of the heavy chain are referred to as HCDR1, HCDR2 and HCDR 3. 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 lymphoblasts, 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 variants thereof, preferably comprising human IgG2 or IgG4 heavy chain constant region, or IgG1 which has 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 the 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 and one heavy chain of CH1 and the variable domains. 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 the 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 (diabodies), bispecific diabodies and triabodies (triabodies), antibody fragments that have been covalently or non-covalently linked together, 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 accordance with the present 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 description similar to "CDR variants having 3, 2, 1 or 0 amino acid mutations on the basis of CDR1, CDR2 and CDR3 as shown in SEQ ID NO X, SEQ ID NO Y and SEQ ID NO Z, respectively", an exemplary explanation is that the mutations to the CDRs may comprise 3, 2, 1 or 0 amino acid mutations, and the same or different number of amino acid residues may optionally be selected for mutation between CDR1, CDR2 and CDR3, for example 1 amino acid mutation to CDR1 and 0 amino acid mutation to CDR2 and CDR3 on the basis of CDR1, CDR2 and CDR3 as shown in SEQ ID NO X, SEQ ID NO Y and SEQ ID NO Z. 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 terms "specific binding", "selectively binding" and "selective binding" as used herein refer to binding of an antibody to an epitope on a predetermined antigen. Typically, antibodies are less than about 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^-7M or even smaller equilibrium dissociation constant (K)_D) 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 (e.g., BSA, etc.). The term "antibody recognizing an antigen" is used interchangeably herein with the term "specifically binding antibody".

The term "compete" 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 antigen binding proteins, as determined by the following assay: in such assays, the antigen binding protein to be detected (e.g., an antibody or antigen binding fragment thereof) prevents or inhibits (e.g., reduces) specific binding of a reference antigen binding protein (e.g., a ligand or a 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: 242-; solid phase direct biotin-avidin EIA (see, e.g., Kirkland et al, 1986, J.Immunol.137: 3614-), 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: 7-15); solid phase direct biotin-avidin EIA (see, e.g., Cheung, et al, 1990, Virology 176: 546-552); and directly labeled RIA (Moldenhauer et al, 1990, Scand. J. Immunol.32: 77-82). 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., decrease) the 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 to 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 enterobacteriaceae family (enterobacteriaceae), such as strains of Escherichia coli (Escherichia coli) or Salmonella (Salmonella); bacillaceae (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. Human FR germline sequences can be obtained from the website http:// imgt. cities.fr of ImmunoGeneTiCs (IMGT) or from the 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 stably transfect CHO cells. As a more recommended prior art, mammalian expression systems lead to 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, for example, hybridoma technology, recombinant technology, phage display technology, synthetic techniques (e.g., CDR-grafting), or other known techniques.

"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, for example, a cell in vitro and ex vivo by a reagent, a diagnostic, a binding composition, or by another cell. "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 any particular disease symptom (also referred to as a "therapeutically effective amount") can 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 disease symptom has been reduced can be assessed by any clinical test commonly used by physicians or other health professional 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 … …" or variants thereof as used throughout the specification and claims is meant to encompass all such elements or groups of elements, and optionally other elements of similar or different nature than the elements, which other 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 test cell and cultures derived therefrom, regardless of the number of transfers. 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 clearly visible 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 in admixture with other chemical components, as well as other components such as physiologically/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 conventional reagents are purchased in the market.

Example 1 immunizing antigen, screening sequence and preparation of antigen

The sequence encoding His-Tag-tagged human CSF-1R and the sequence encoding huFc-tagged human CSF-1R were synthesized by Integrated DNA Technology (IDT) and cloned into pTT5 vector (Biovector), respectively. Recombinant CSF-1R protein in 293T cells (ATCC, CRL-3216)^TM) Purification was performed after expression. 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 procedure 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 5 mM. 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 a 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. The target protein eluted by the imidazole in PBS 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 Protein A 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 10⁶And/ml. Mu.g of the vector encoding huCSF-1R or gene was mixed with 50ul of LF-LTX (Life Technologies, # A12621) in 1ml of 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 are generated by immunizing mice. Experiments were performed with Swiss Webster 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 regulated for 12/12 hours at the temperature of 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 + 15 paw pad (footpad) per emulsified antigen, 3 days prior to splenocyte fusion boosting, and Intraperitoneally (IP) injection of 15. mu.g + 15 paw pad (footpad) per physiological saline 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 cells 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 at 30,000-50,000 cells/well for 7-14 days, taking the culture medium supernatant, performing antibody screening on the hybridoma supernatant by using CSF-1R recombinant protein and ELISA experiment as described in example 4, further using CHO-S cells stably expressing CSF-1R as a positive antibody strain, comparing the blank CHO-S cells to eliminate non-specifically bound antibody hybridoma strains, performing screening by a flow sorting method,thus, about 400 strains were identified that bound the recombinant protein and also bound the hybridomas that the cells expressed the antigen. Subsequently, as described in example 5, about 50 hybridomas having a blocking function were identified and subcloned using experimental screening for a CSF-1 ligand blocking function based on CSF-1R stably transfected cells, and then purified antibodies were collected and subjected to experimental screening for an IL-34 ligand blocking function based on CSF-1R stably transfected cells, to identify 11 antibody subclones having a double blocking function of CSF-1 and IL-34. Hybridoma subcloned cells in logarithmic growth phase were harvested, RNA extracted using Trizol (Invitrogen,15596-^TMReverse Transcriptase, Takara # 2680A). The cDNA obtained by reverse transcription is subjected to PCR amplification by using mouse Ig-Primer Set (Novagen, TB326 Rev.B 0503) and then sequenced, and after the repetitive sequence of the CDR region is eliminated, the sequences of nine murine antibodies, namely C11, C19, C6, C8, C16, C18, C23, C27 and C30 are finally obtained.

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 (R)	Sequence of	Numbering
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 chain and the light chain of the murine monoclonal antibody C6 have the following sequences:

C6 HCVR

C6 LCVR

it contains the following CDR sequences:

name (R)	Sequence of	Numbering
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 regions of the heavy chain and the light chain of the murine monoclonal antibody C8 have the following sequences:

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 chain and the light chain of the murine monoclonal antibody C16 have the following sequences:

C16 HCVR

C16 LCVR

it contains the following CDR sequences:

name (R)	Sequence of	Numbering
HCDR1	GYTFTTYDIN	SEQID NO：55

HCDR2	WVYPRDGSTKYNEKFKG	SEQID NO：56
HCDR3	SGLTGSPFAY	SEQID NO：57
LCDR1	RASESFDSYGNTFMH	SEQID NO：58
LCDR2	RASNLES	SEQID NO：59
LCDR3	QQNNEYPLT	SEQID NO：60

The variable regions of the heavy chain and the light chain of the murine monoclonal antibody C18 have the following sequences:

C18 HCVR

C18 LCVR

it contains the following CDR sequences:

name (R)	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 chain and the light chain of the murine monoclonal antibody C23 have the following sequences:

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 chain and the light chain of the murine monoclonal antibody C27 have the following sequences:

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 chain and the light chain of the murine monoclonal antibody C30 have the following sequences:

C30 HCVR

C30 LCVR

it contains the following CDR sequences:

name (R)	Sequence of	Numbering
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, respectively, and then transiently transfected into HEK293 cells to obtain individual chimeric antibodies against CSF-1R (C11C, C19C, C6C, C8C, C16C, C18C, C23C, C27C and C30C), purified, identified, and tested for relevant binding and functional activity as described in example 2 (Fc-tagged protein purification).

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 20 h. After washing the plate 1 time with PBST (PH7.4PBS containing 0.05% Tween-20) buffer, 120. mu.l/well of PBST/1% skim milk was added and incubated for 1h at room temperature for blocking. After washing the plate 1 time with PBST buffer, 1. mu.g/ml of biotin-labeled h-CSF-1R-his (or monkey and murine CSF-1R proteins to be assayed) diluted with PBST/1% skim milk was added and incubated at room temperature for 1 h. 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% mikk was added and incubated at room temperature for 1.5 h. 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 1 h. After PBST washing 3 times, 100. mu.l/well TMB was added and incubated at room temperature for 5-10 min. Add 100. mu.l/well 1M H₂SO₄The reaction was stopped, absorbance read at 450nm and ELISA binding EC calculated₅₀The 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 × 10⁷Cells/ml. After incubation at 4 ℃ for 30 minutes, 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. Add 100. mu.l of test antibody to each well with a concentration gradient of 0.085nM to 670nM, cell resuspension, 4 ℃ light protection incubation for 60 minutes. The supernatant was discarded by centrifugation and 100. mu.l of l of a 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, and 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 (0.01nM to 670nM) was incubated with CSF-1R/CHO cells and combined with additional incubation 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 PBS washing, 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 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, the 9-strain antibody 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 the reference antibody FPA 008.

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 standard amino coupling 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. Antigen-antibody binding kinetics were followed for 3 minutes and dissociation kinetics were followed for 10 minutes. The data obtained were analyzed using BIAevaluation software of GE in 1:1(Langmuir) binding model, in such a way that k of each chimeric antibody was determined_a(k_on)、kd(k_off) And K_DThe 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) constant domain, and 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 preferably being a human germline antibody template as shown below.

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

c11 preferably 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 were grafted onto selected corresponding humanized templates, the humanized variable regions were replaced, 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 back-mutated and the CDR regions were optimized for mutation of the chemically unstable asparagine residues, wherein the HCDR2 of murine antibody C11: EMNPNNGDSSYNQKFKG (SEQ ID NO: 4) is optimized as: EMNPNTGDSSYNQKFKG (SEQ ID NO: 95), LCDR 1: KSSQSLLNSGNQKNSLT (SEQ ID NO: 6) is 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). Expression vectors 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 CO₂Incubate 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 HCDR 1: AITFTDYYMN (SEQ ID NO:11) to: AITFTDYYIN (SEQ ID NO:98) to remove the chemically labile methionine residue, resulting in the humanized antibody huC19I, with the huC19I heavy chain variable region sequence shown in SEQ ID NO: 100. The full-length sequences of huC11, huC19, and huC19I, respectively, are shown in SEQ ID NOS: 17-20 and SEQ ID NO: 99.

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 for huC11 and huC19 antibodies:

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

2. Affinity kinetics experiments (same procedure as in example 6) resulted in the following table, and the final preferred humanized antibodies huC11 and huC19 both retained strong affinity for human CSF-1R antigenic protein, with huC19 having stronger affinity than the reference antibody FPA 008.

3. In vitro functional activity blocking assay (procedure same as example 6), results are shown in fig. 3, fig. 4 and table below, and humanized antibodies huC11, huC19 and its mutant huC19I all showed strong blocking function on CSF-1 and IL-34 induced monocyte proliferation function, and wherein the blocking functional activity of huC19 was comparable or slightly superior to that of FPA008 reference antibody, while its mutant huC19I was slightly reduced to be similar to that of FPA 008.

In vitro functional activity blocking assay for huC11

In vitro functional activity blocking assay for huC19 and mutant huC19I thereof

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

Taking the peripheral blood of normal peopleHealthy human PBMC were isolated 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. every 10⁷20ul of Anti-CD14 microbeads were added to each cell 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⁺Adding RPMI 1640 culture solution containing 100ng/ml M-CSF into the mononuclear cells, culturing for 6 days, performing macrophagg cell induction culture, adding the rest cells into MDA-MB-231 cells treated by Mitomycin C, and culturing the PBMC and the MDA-MB-231 for 6 days in a total way, wherein the culture solution is the RPMI 1640 culture solution containing IL-2 and 10% FBS.

After 6 days, macrohage cells were collected and 1X 10 cells were collected⁵Individual cells, CD68, CD115, CD163, CD206 stained and FACS analyzed for success in inducing macrophage. PBMC cells, macrophage 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 temperature of the raising room is 18.0-26.0 ℃, the humidity is 40-70%, the air exchange is carried out for 10-20 times/hour, and the light and shade alternation time is 12h/12h day and night.

The experiment was divided into a human IgG1 antibody control group, a reference antibody FPA8, and huC19I, each administered at a dose of 20 mg/kg. 6 mice per group were administered by intraperitoneal injection 10 times every two days (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 caliper³) 0.5 × (tumor major diameter × tumor minor diameter)²) And (4) calculating. Relative tumor inhibition ratio 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 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 as Mean + -SEM, and student's t test is used to compare tumor volume and tumor weight in the treated group with those in the control groupPresence or absence of significant difference, p<0.05 was significantly different.

As shown in fig. 5 and fig. 6, CSF1R antibody huC19I showed anti-tumor effect in the Macrophage-PBMC-MDA-MB-231 tumor model, with the final mean tumor volumes of each group being: 294.32mm³、230.46mm³、131.19mm³Starting 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 the CSF1R antibody at this dose.

The experimental groups and dosing regimens are shown in the following table:

Claims

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:

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

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

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

d) the antibody light chain variable region comprises an LCDR variant selected from the group consisting of LCDR1, LCDR2 and LCDR3 having 3, 2, 1 or 0 amino acid mutations shown in SEQ ID NO 66, SEQ ID NO 67 and SEQ ID NO 68, respectively; the heavy chain variable region of the antibody comprises an HCDR variant selected from the group consisting of HCDR1, HCDR2, and HCDR3 having 3, 2, 1, or 0 amino acid mutations, respectively, as shown in SEQ ID NO 63, SEQ ID NO 64, and SEQ ID NO 65;

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

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

g) the antibody light chain variable region comprises an LCDR variant selected from the group consisting of LCDR1, LCDR2 and LCDR3 having 3, 2, 1 or 0 amino acid mutations based on SEQ ID NO 90, SEQ ID NO 91 and SEQ ID NO 92, respectively; the heavy chain variable region of the antibody comprises an HCDR variant selected from the group consisting of HCDR1, HCDR2, and HCDR3 having 3, 2, 1, or 0 amino acid mutations, respectively, as shown in SEQ ID NO 87, SEQ ID NO 88, and SEQ ID NO 89;

h) the antibody light chain variable region comprises LCDR variants selected from LCDR1, LCDR2 and LCDR3 shown as SEQ ID NO 6, SEQ ID NO 7 and SEQ ID NO 8 respectively having 3, 2, 1 or 0 amino acid mutations; the heavy chain variable region of the antibody comprises an HCDR variant selected from the group consisting of HCDR1, HCDR2, and HCDR3 having 3, 2, 1, or 0 amino acid mutations, respectively, as shown in SEQ ID NO 3, SEQ ID NO 4, and SEQ ID NO 5;

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

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

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

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

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

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

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

p) the antibody light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO 42, SEQ ID NO 43 and SEQ ID NO 44, respectively; the heavy chain variable region of the antibody comprises HCDR1, HCDR2 and HCDR3 shown as SEQ ID NO 39, SEQ ID NO 40 and SEQ ID NO 41 respectively;

q) the antibody light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO 50, SEQ ID NO 51 and SEQ ID NO 52, respectively; the heavy chain variable region of the antibody comprises HCDR1, HCDR2 and HCDR3 shown as SEQ ID NO 47, SEQ ID NO 48 and SEQ ID NO 49 respectively;

r) the antibody light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO 58, SEQ ID NO 59 and SEQ ID NO 60, respectively; the heavy chain variable region of the antibody comprises HCDR1, HCDR2 and HCDR3 shown as SEQ ID NO 55, SEQ ID NO 56 and SEQ ID NO 57 respectively;

s) the antibody light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO 66, SEQ ID NO 67 and SEQ ID NO 68, respectively; the heavy chain variable region of the antibody comprises HCDR1, HCDR2 and HCDR3 shown as SEQ ID NO 63, SEQ ID NO 64 and SEQ ID NO 65 respectively;

t) the antibody light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO 74, SEQ ID NO 75 and SEQ ID NO 76, respectively; the heavy chain variable region of the antibody comprises HCDR1, HCDR2 and HCDR3 shown as SEQ ID NO 71, SEQ ID NO 72 and SEQ ID NO 73 respectively;

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

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

w) the antibody light chain variable region comprises LCDR1, LCDR2 and LCDR3 shown as SEQ ID NO 6, SEQ ID NO 7 and SEQ ID NO 8, respectively; the heavy chain variable region of the antibody comprises HCDR1, HCDR2 and HCDR3 shown as SEQ ID NO 3, SEQ ID NO 4 and SEQ ID NO 5 respectively;

x) the antibody light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO 14, SEQ ID NO15 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;

y) the antibody light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO 14, SEQ ID NO15 and SEQ ID NO 16, respectively; the variable region of the antibody heavy chain comprises the following components: 98, 12 and 13 of HCDR1, 2 and 3;

z) comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:14, SEQ ID NO15 and SEQ ID NO:16, respectively; the variable region of the antibody heavy chain comprises the following components: 98, 97 and 13 as shown in SEQ ID NO, HCDR1, HCDR2 and HCDR 3;

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

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

ac) the antibody light chain variable region comprises LCDR1, LCDR2 and LCDR3 shown as SEQ ID NO 96, SEQ ID NO 7 and SEQ ID NO 8, respectively; the variable region of the antibody heavy chain comprises the following components: HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO. 3, SEQ ID NO. 95 and SEQ ID NO. 5; 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.
The anti-CSF-1R antibody or antigen-binding fragment thereof of claim 1 or 2, wherein the antibody or antigen-binding fragment thereof is a murine antibody or a chimeric antibody.
The antibody or antigen-binding fragment of claim 3, 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

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

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

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

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, said variant preferably having 0-10 amino acid mutations in the light chain variable region, preferably mutations being M34I, N55T or a combination thereof amino acid mutations.
The anti-CSF-1R antibody or antigen-binding fragment thereof of claim 1 or 2, wherein the antibody or antigen-binding fragment thereof is a humanized antibody or antigen-binding fragment thereof.
The anti-CSF-1R antibody or antigen-binding fragment thereof of claim 5, wherein the light chain FR region sequence on the humanized antibody light chain variable region is derived from the human germline light chain IGkV4-1 sequence set forth in SEQ ID NO: 22; or from the human germline light chain IGkV1-13 sequence shown as SEQ ID NO. 24;

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 heavy chain FR region of human IgG1, IgG2 or IgG4, more preferably comprises a heavy chain FR region of human IgG1 or IgG 4; preferably, the heavy chain FR region sequence in the heavy chain variable region of the humanized antibody is derived from the human germline heavy chain IGHV1-46 sequence shown as SEQ ID NO: 21; or from the human germline heavy chain IGHV1-2 sequence shown in SEQ ID NO. 23.
The anti-CSF-1R antibody or antigen-binding fragment thereof of claim 6, 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 the most preferred amino acid mutations of SEQ ID NO:30 are S31N, T37N; 34 to F87A, F91Y or a combination thereof;

and/or, the humanized antibody heavy chain variable region sequence is shown as SEQ ID NO 26, SEQ ID NO 100 or SEQ ID NO 33 or the variant thereof; the variant preferably has 0-10 amino acid mutations in the heavy chain variable region, wherein the preferred amino acid mutation of SEQ ID No. 26 is S30T, T55N, L70M or a combination thereof, and the most preferred amino acid mutation is T55N; 33 preferred amino acid mutations of SEQ ID No. 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.
The anti-CSF-1R antibody or antigen-binding fragment thereof of any one of claims 3-7, wherein the chimeric or humanized antibody further comprises a light chain and/or heavy chain constant region having the sequence 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.
The anti-CSF-1R antibody or antigen-binding fragment thereof of claim 8, 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;

comprises a heavy chain with a sequence shown as SEQ ID NO. 17 and a light chain with a sequence shown as SEQ ID NO. 18; 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.
An isolated monoclonal antibody or antigen-binding fragment thereof that competes for binding to CSF-1R with the anti-CSF-1R antibody or antigen-binding fragment thereof of any one of claims 1 to 9.
A multispecific antibody comprising a light chain variable region and/or a heavy chain variable region of the body or antigen-binding fragment thereof according to any one of claims 1 to 10.
A single chain antibody comprising the light chain variable region and/or the heavy chain variable region of the antibody or the antigen-binding fragment thereof according to any one of claims 1 to 10.
An antibody-drug conjugate, wherein the antibody comprises the light chain variable region and/or the heavy chain variable region of the antibody or the antigen-binding fragment thereof according to any one of claims 1 to 10.
A nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of any one of claims 1 to 10, the multispecific antibody of claim 11, or the single chain antibody of claim 12.
An expression vector comprising the nucleic acid of claim 14.
A host cell transformed with the expression vector of claim 15.
The host cell of claim 16, wherein the host cell is a bacterium, preferably e.coli;

or the host cell is saccharomycete, preferably pichia pastoris;

alternatively, the host cell is an animal cell, preferably a Chinese Hamster Ovary (CHO) cell, a Human Embryonic Kidney (HEK)293 cell, or an NS0 cell.
A method for producing the antibody or antigen-binding fragment thereof of any one of claims 1 to 10, the multispecific antibody or single-chain antibody of claim 11 or claim 12, comprising expressing the antibody or antigen-binding fragment thereof, the multispecific antibody or the single-chain antibody in the host cell of claim 16 or 17, and isolating the antibody or antigen-binding fragment thereof, the multispecific antibody or the single-chain antibody from the host cell.
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.
The antibody or antigen-binding fragment thereof of any one of claims 1 to 10, the multispecific antibody of claim 11, the single-chain antibody of claim 12, the antibody-drug conjugate of claim 13, the nucleic acid molecule of claim 14, or the pharmaceutical composition of claim 19 for use in 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 initiation of therapy with another anticancer therapy.
The use of claim 20, 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.
The use of claim 21, wherein the chemotherapeutic agent is selected from one or more of taxanes (paclitaxel injection, docetaxel, paclitaxel albumin, etc.), doxorubicin, modified doxorubicin (Caelyx or Doxil)), 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.
Use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, a multispecific antibody according to claim 11, a single-chain antibody according to claim 12, an antibody-drug conjugate according to claim 13, a nucleic acid molecule according to claim 14, or a pharmaceutical composition according to claim 19, 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.
A method of preventing a CSF-1R or CSF-1 mediated disease or disorder, which comprises administering to a patient in need thereof a therapeutically effective amount of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, a multispecific antibody according to claim 11, a single chain antibody according to claim 12, an antibody-drug conjugate according to claim 13, a nucleic acid molecule according to claim 14 or a pharmaceutical composition according to claim 19, 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.