TW201917136A - Anti-CSF-1R antibody, antigen-binding fragment thereof and pharmaceutical use thereof - Google Patents

Abstract

The present invention relates to an anti-CSF-1R antibody, an antigen-binding fragment thereof, and a pharmaceutical use thereof. Further, the present invention relates to a chimeric antibody, a humanized antibody comprising a CDR region of the anti-CSF-1R antibody, and a pharmaceutical composition comprising the human anti-CSF-1R antibody and the antigen-binding fragment thereof, and the use thereof as an anticancer drug. In particular, the invention relates to the use of a humanized anti-CSF-1R antibody for the manufacture of a medicament for the treatment of a CSF-1R mediated disease or disorder.

Description

Anti-CSF-1R antibody, its antigen-binding fragment and its medical use

This application claims priority from Chinese patent application CN201711015488.5 with an application date of October 26, 2017. This application cites the full text of the aforementioned Chinese patent application.

The present invention relates to an anti-CSF-1R antibody having immunoreactivity to the human CSF-1R receptor, and an antigen-binding fragment thereof, a chimeric antibody, a humanized antibody comprising the CDR region of the anti-CSF-1R antibody, and Pharmaceutical composition comprising human anti-CSF-1R antibody and antigen-binding fragment thereof, and its use as an anticancer drug.

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

Tumor immunotherapy is a long-term hot spot in the field of tumor therapy, of which T cell tumor immunotherapy is at the core. Tumor escape is a huge obstacle for tumor immunotherapy. Most tumors display antigens that can be recognized by the host's immune system to varying degrees. However, in many cases, inadequate immune response is triggered by inefficient activation of immune cells, so tumors Cells use their own suppressive effect on the immune system to promote the crazy growth of tumors. Tumor immunotherapy is to use various methods such as immune checkpoint inhibition, direct activation, and tumor microenvironment activation to fully utilize and mobilize various types of immune cells in tumor patients to kill tumors.

Macrophages are an important cell population in the anti-tumor immune regulation process, which can directly kill tumor cells. However, recent studies have confirmed that macrophages are divided into two types: M1 and M2. Macrophages in the tumor tissue, that is, tumor-associated macrophages (TAM) belong to the M2 category, and do not exert anti-tumor effects like M1 macrophages. Role, but participate in the process of tumorigenesis, growth, invasion and metastasis. TAM is derived from monocytes in the lymphatic circulation or macrophages remaining in tissues, and is the main type of white blood cells that infiltrate many types of tumor stroma. Once activated, TAM often becomes the main 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, also known as FMS, C-FMS, and CD115, etc.) is an N-terminal extracellular domain (characterized by repeated Ig domains) and tyrosine kinase activity One-way Transmembrane Receptor of the C-terminal Intracellular Domain. Binding of CSF-1 or interleukin-34 (IL-34) ligand to CSF-1R results in receptor dimerization and up-regulation of tyrosine kinase activity, CSF-1R tyrosine residue phosphorylation, and downstream signaling And other events. Both CSF-1 and IL-34 ligands can stimulate monocyte survival, proliferation, and differentiation into macrophages. CSF-1R is mainly expressed on cells of the monocyte lineage and in the female reproductive tract and placenta.

Many tumor cells secrete CSF-1. CSF-1 activates monocytes / M2 macrophages through CSF-1R. The degree of CSF-1 in the tumor is related to the degree of TAM in the tumor, while the high degree of TAM is related to the poor prognosis of patients. In human breast cancer xenografts in mice, CSF-1 has been found to promote tumor growth and development into metastasis. On the other hand, CSF-1 and its receptors are also involved in various inflammatory and autoimmune diseases. CSF-1R stimulates the proliferation, survival, activation, and maturation of differentiated myeloid cells, and stimulates the ability of the differentiated myeloid cells to mediate disease pathology in a pathological environment. Therefore, various antagonists related to CSF-1R receptor signal transmission, including monoclonal antibodies, can be used to treat various CSF-1R-mediated related diseases by blocking the activation of monocytes into M2 macrophages. Such as cancer, inflammatory conditions and autoimmune diseases.

Many multinational pharmaceutical companies are currently developing monoclonal antibodies against CSF-1R, which can be used to kill tumor cells by antagonizing CSF1-R to inhibit TAM in the tumor microenvironment, or for related macrophage-mediated Autoimmune diseases, etc. Sheer, C.J. et al. Described anti-CSF-1R antibodies that inhibit CSF-1 activity (Sherr, C.J. et al., 1989, Blood 73: 1786-1793). WO2009 / 026303 discloses an anti-CSF-1R antibody that binds human CSF-1R and an in vivo mouse tumor model using an anti-mouse CSF-1R antibody. WO2011 / 123381 discloses an anti-CSF-1R antibody that internalizes CSF-1R and has ADCC activity. WO2011 / 123381 also discloses an in vivo mouse tumor model using an anti-mouse CSF-1R antibody. WO2011 / 140249 discloses an anti-CSF-1R antibody that blocks the binding of CSF-1 to CSF-1R, which is believed to be useful in treating cancer. WO2009 / 112245 discloses an anti-CSF-IR IgGl antibody that inhibits CSF-1 binding to CSF-IR, which is believed to be useful in the treatment of cancer, inflammatory bowel disease and rheumatoid arthritis. WO2011 / 131407 discloses an anti-CSF-IR antibody that inhibits CSF-1 binding to CSF-IR, which is believed to be useful in treating bone loss and cancer. WO2011 / 107553 discloses an anti-CSF-IR antibody that inhibits CSF-1 binding to CSF-IR, which is believed to be useful in treating bone loss and cancer. WO2011 / 070024 discloses an anti-CSF-IR antibody that binds human CSF-IR fragment deID4. Although the therapeutic application of anti-CSF-IR antibodies in the treatment of certain cancers has been previously described, there remains a need to develop new types of antibodies that are more suitable for clinical applications.

Some embodiments of the present invention provide an anti-CSF-1R antibody or an antigen-binding fragment thereof, comprising: an antibody light chain variable region comprising at least one LCDR selected from the following sequence: SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 96; and an antibody heavy chain variable region, The variable region of the antibody heavy chain comprises at least one HCDR selected from the following sequences: SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 95, SEQ ID NO: 97 or SEQ ID NO: 98.

In other embodiments, the above-mentioned anti-CSF-1R antibody or antigen-binding fragment thereof comprises an antibody light chain variable region and an antibody heavy chain variable region, wherein: a) the antibody light chain variable region comprises a selective region LCDR variants having 3, 2, 1, or 0 amino acid mutations, respectively, based on LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 42, SEQ ID NO: 43, and SEQ ID NO: 44; The variable region of the antibody heavy chain comprises 3, 2, 1 or 0 amine groups selected from HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO: 39, SEQ ID NO: 40 and SEQ ID NO: 41, respectively. Acid-mutated HCDR variants; b) the variable region of the antibody light chain comprises selected from LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 50, SEQ ID NO: 51, and SEQ ID NO: 52, respectively LCDR variants with 3, 2, 1 or 0 amino acid mutations; the antibody heavy chain variable region comprises a selected from the group consisting of SEQ ID NO: 47, SEQ ID NO: 48 and SEQ ID NO: 49 HCDR1, HCDR2, and HCDR3 based on HCDR variants having 3, 2, 1, or 0 amino acid mutations, respectively; c) the variable region of the antibody light chain comprises a region selected from SEQ ID NO: 58, SEQ ID NO: NO: 59 and LCDR variants having 3, 2, 1 or 0 amino acid mutations on the basis of LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 60; the variable region of the heavy chain of the antibody is selected from the group consisting of SEQ ID NO: 55. HCDR variants having 3, 2, 1 or 0 amino acid mutations on the basis of HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO: 56 and SEQ ID NO: 57; d) the antibody light chain The variable region comprises an LCDR selected from the group consisting of LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 66, SEQ ID NO: 67, and SEQ ID NO: 68 with 3, 2, 1, or 0 amino acid mutations, respectively. Variant; the variable region of the antibody heavy chain comprises 3, 2, 1 selected from HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 63, SEQ ID NO: 64, and SEQ ID NO: 65, respectively Or 0 amino acid mutant HCDR variants; e) the variable region of the antibody light chain comprises LCDR1, LCDR2 selected from the group consisting of SEQ ID NO: 74, SEQ ID NO: 75, and SEQ ID NO: 76 LCDR variants having 3, 2, 1 or 0 amino acid mutations on the basis of LCDR3 respectively; the variable region of the antibody heavy chain comprises HCDR1, HCDR2 and HCDR3 shown in NO: 73 HCDR variants having 3, 2, 1 or 0 amino acid mutations, respectively; f) the variable region of the antibody light chain comprises a group selected from SEQ ID NO: 82, SEQ ID NO: 83 and SEQ ID LCDR1, LCDR2, and LCDR3 shown in NO: 84 each have 3, 2, 1, or 0 amino acid mutations in the LCDR variants; the variable region of the antibody heavy chain comprises a region selected from SEQ ID NO: 79 HCDR variants having 3, 2, 1 or 0 amino acid mutations on the basis of HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO: 80 and SEQ ID NO: 81, respectively; g) the light chain of the antibody may be The variable region comprises an LCDR variant selected from the group consisting of LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 90, SEQ ID NO: 91, and SEQ ID NO: 92 with 3, 2, 1, or 0 amino acid mutations, respectively. The antibody heavy chain variable region comprises a member selected from the group consisting of HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 87, SEQ ID NO: 88, and SEQ ID NO: 89, respectively, having 3, 2, 1, or 0 amino acid mutant HCDR variants; h) the variable region of the antibody light chain comprises LCDR1, LCDR2 and LCDR1 selected from SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8; LCDR3 has 3, 2, 1, or 0 respectively Amino acid-mutated LCDR variants; the antibody heavy chain variable regions comprise selected from HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, respectively HCDR variants having 3, 2, 1 or 0 amino acid mutations; i) the variable region of the antibody light chain comprising a member selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16 The LCDR1, LCDR2, and LCDR3 shown have an LCDR variant having 3, 2, 1, or 0 amino acid mutations, respectively; the variable region of the antibody heavy chain comprises a region selected from SEQ ID NO: 11, SEQ ID HCDR variants having 3, 2, 1 or 0 amino acid mutations on the basis of HCDR1, HCDR2 and HCDR3 shown in NO: 12 and SEQ ID NO: 13 respectively; j) the variable region of the light chain of the antibody comprises Selected from LCDR variants having 3, 2, 1 or 0 amino acid mutations on the basis of LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, respectively; The variable region of the weight chain comprises 3, 2, 1 or 0 amino acids selected from HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 98, SEQ ID NO: 12, and SEQ ID NO: 13, respectively. Mutated HCDR variants; k) The body light chain variable region comprises 3, 2, 1, or 0 amino acids 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. A mutated LCDR variant; the antibody heavy chain variable region comprises a member selected from the group consisting of HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 98, SEQ ID NO: 97, and SEQ ID NO: 13, respectively, having 3, 2, 1 or 0 amino acid mutant HCDR variants; l) the variable region of the antibody light chain comprises an LCDR1 selected from the group consisting of SEQR NO: 96, SEQ ID NO: 7 and SEQ ID NO: 8; LCDR2 and LCDR3 have LCDR variants with 3, 2, 1 or 0 amino acid mutations respectively; the antibody heavy chain variable region contains a selected from: SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO : HCDR variants having 3, 2, 1 or 0 amino acid mutations on the basis of HCDR1, HCDR2 and HCDR3 shown in 5 respectively; m) the variable region of the light chain of the antibody comprises a member selected from the group consisting of SEQ ID NO: 6. LCDR variants having 3, 2, 1 or 0 amino acid mutations on the basis of LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 7 and SEQ ID NO: 8, respectively; the variable region of the antibody heavy chain contains a selection Freedom: SEQ ID NO: 3, SEQ ID NO: 95 and SEQ ID NO: 5 based on HCDR1, HCDR2 and HCDR3, respectively, HCDR variants having 3, 2, 1 or 0 amino acid mutations; n) the variable region of the antibody light chain comprises LCDR variants having 3, 2, 1 or 0 amino acid mutations on the basis of LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 96, SEQ ID NO: 7 and SEQ ID NO: 8, respectively; antibody heavy chain The variable region comprises one selected from HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 3, SEQ ID NO: 95, and SEQ ID NO: 5 with 3, 2, 1, or 0 amino acid mutations, respectively. HCDR variants; or o) the variable region of the antibody light chain comprises a member selected from the group consisting of LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, respectively LCDR variants with 3, 2, 1 or 0 amino acid mutations; the antibody heavy chain variable region comprises a HCDR1 selected from SEQ ID NO: 11, SEQ ID NO: 97, and SEQ ID NO: 13 HCDR2 and HCDR3 are based on HCDR variants with 3, 2, 1 or 0 amino acid mutations, respectively.

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 is variable The region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 42, SEQ ID NO: 43 and SEQ ID NO: 44, respectively; the variable region of the antibody heavy chain comprises as shown in SEQ ID NO: 39, SEQ ID HCDR1, HCDR2 and HCDR3 shown in NO: 40 and SEQ ID NO: 41; q) the variable region of the antibody light chain comprises as shown in SEQ ID NO: 50, SEQ ID NO: 51 and SEQ ID NO: 52 respectively LCDR1, LCDR2 and LCDR3 shown; the antibody heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 47, SEQ ID NO: 48 and SEQ ID NO: 49, respectively; r) said The variable region of the antibody light chain comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 58, SEQ ID NO: 59, and SEQ ID NO: 60; NO: 55, HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO: 56 and SEQ ID NO: 57; s) The variable region of the light chain of the antibody comprises SEQ ID NO: 66, SEQ ID NO: 67 and SEQ ID NO: 68 LCDR1, LCDR2 and LCDR3 shown; the antibody heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 63, SEQ ID NO: 64 and SEQ ID NO: 65, respectively; t) said The variable region of the antibody light chain comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 74, SEQ ID NO: 75, and SEQ ID NO: 76; NO: 71, HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO: 72 and SEQ ID NO: 73; u) the variable region of the light chain of the antibody comprises SEQ ID NO: 82, SEQ ID NO: 83 and LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 84; the antibody heavy chain variable region comprises HCDR1, HCDR2 and HCDR1 shown in SEQ ID NO: 79, SEQ ID NO: 80 and SEQ ID NO: 81 respectively HCDR3; v) the variable region of the antibody light chain 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 of the antibody is variable The region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 87, SEQ ID NO: 88 and SEQ ID NO: 89, respectively; w) the variable region of the antibody light chain comprises as shown in SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8 LCDR1, LCDR2 and LCDR3; the antibody heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, respectively; x) The antibody light chain variable region includes LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, respectively; the antibody heavy chain variable region includes as shown in SEQ ID NO : 11. HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO: 12 and SEQ ID NO: 13; y) the variable region of the light chain of the antibody comprises SEQ ID NO: 14, SEQ ID NO: 15 and SEQ, respectively LCDR1, LCDR2, and LCDR3 shown in ID NO: 16; the variable region of the antibody heavy chain includes HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 98, SEQ ID NO: 12, and SEQ ID NO: 13, respectively; z The variable region of the antibody light chain includes 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 includes, respectively, SEQ ID NO : 98. HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 97 and SEQ ID NO: 13; aa) the antibody light chain variable region comprises SEQ ID NO: 96, SEQ ID NO: 7 and SEQ, respectively IDR: LCDR1, LCDR2 shown in 8 LCDR3; the antibody heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, respectively; ab) the antibody light chain variable region comprises LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8 respectively; the heavy chain variable region of the antibody includes: SEQ ID NO: 3, SEQ ID NO: 95, and HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 5; ac) the variable region of the light chain of the antibody comprises LCDR1, shown in SEQ ID NO: 96, SEQ ID NO: 7, and SEQ ID NO: 8, respectively LCDR2 and LCDR3; the antibody heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 3, SEQ ID NO: 95, and SEQ ID NO: 5, respectively; ad) the variable light chain of the antibody The regions include LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, respectively; the antibody heavy chain variable regions include as follows: SEQ ID NO: 11, SEQ ID NO : HCDR1, HCDR2 and HCDR3 shown in 97 and SEQ ID NO: 13.

In other embodiments, the anti-CSF-1R antibody or antigen-binding fragment thereof as described above, wherein the 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 as shown in SEQ ID NO: 38, and the amine of the heavy chain variable region is The amino acid sequence is shown in SEQ ID NO: 37; or b) the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 46, and the amino acid sequence of the heavy chain variable region is shown in SEQ. ID NO: 45; or c) the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 54, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 53 Or d) the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 62, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 61; or e) The amino acid sequence of the light chain variable region is shown in SEQ ID NO: 70, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 69; or f) the light chain may be The amino acid sequence of the variable region is shown in SEQ ID NO: 78, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 77; or g) the amino group of the light chain variable region The acid sequence is shown in SEQ ID NO: 86, and the heavy chain may be The amino acid sequence of the region is shown in SEQ ID NO: 85; or h) the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 2 or a variant thereof, and the variant is preferably in light The variable region of the chain has an amino acid mutation of 0-10. The most preferred amino acid mutation is N37T. The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 1 or a variant thereof. The variant preferably has an amino acid mutation of 0-10 in the heavy chain variable region, and the preferred amino acid mutation is N55T; or i) the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 10 As shown, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 9 or a variant thereof, and the variant preferably has a 0-10 amino acid mutation in the light chain variable region, preferably The mutation is an amino acid mutation of M34I, N55T, or a combination thereof.

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

In other embodiments, the anti-CSF-1R antibody or antigen-binding fragment thereof as described above, wherein the sequence of the light chain FR region on the variable region of the light chain of the humanized antibody is derived from SEQ ID NO: 22 The human germline light chain IGkV4-1 sequence shown; or derived from the human germline light chain IGkV1-13 sequence shown in SEQ ID NO: 24; and / or, the humanized antibody heavy chain variable region is further A heavy chain FR region comprising human IgG1, IgG2, IgG3 or IgG4 or a variant thereof, preferably a human IgG1, IgG2 or IgG4 heavy chain FR region, more preferably a human IgG1 or IgG4 heavy chain FR region; preferably The sequence of the heavy chain FR region on the variable region of the heavy chain of the humanized antibody is derived from the human germline heavy chain IGHV1-46 sequence shown in SEQ ID NO: 21; or is derived from SEQ ID NO: 23 Human germline heavy chain IGHV1-2 sequence shown.

In other embodiments, the anti-CSF-1R antibody or antigen-binding fragment thereof as described above, wherein the humanized antibody light chain variable region sequence is as shown in SEQ ID NO: 30 or SEQ ID NO: 34 Sequence or a variant thereof; said variant preferably has an amino acid mutation of 0-10 in the variable region of the light chain, wherein the most preferred amino acid of SEQ ID NO: 30 is mutated to S31N, T37N; SEQ ID NO: 34 The most preferred amino acid mutation is F87A, F91Y or a combination thereof; and / or, the humanized antibody heavy chain variable region sequence is as SEQ ID NO: 26, SEQ ID NO: 100 or SEQ ID NO: The sequence shown in 33 or a variant thereof; said variant preferably has an amino acid mutation of 0-10 in the heavy chain variable region, wherein the preferred amino acid of SEQ ID NO: 26 is S30T, T55N, L70M or In combination, the most preferred amino acid mutation is T55N; SEQ ID NO: 33 The preferred amino acid mutation is Q1E, M34I, T55N, E89D or a combination thereof, and the most preferred amino acid mutation is M34I, T55N or a combination thereof Amino acid mutation; preferably, the sequence of the heavy chain variable region is SEQ ID NO: 31, 32, 33, or 100, and the sequence of the light chain variable region is SEQ ID NO: 34, 35 Or 3 6; Alternatively, the sequence of the heavy chain variable region is SEQ ID NO: 25, 26, 27, or 28, and the sequence of the light chain variable region is 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 constant region of a light chain and / or a heavy chain, the light chain is constant The sequence of the region is shown in SEQ ID NO: 102, and / or the sequence of the heavy chain constant region is shown in SEQ ID NO: 101.

In other embodiments, the anti-CSF-1R antibody or antigen-binding fragment thereof as described above, wherein the humanized antibody: comprises a heavy chain having the sequence shown in SEQ ID NO: 19, and the sequence is shown in SEQ ID NO: A light chain represented by 20; a heavy chain comprising a sequence shown in SEQ ID NO: 17 and a light chain comprising a sequence shown in SEQ ID NO: 18; or a heavy chain comprising a sequence shown in SEQ ID NO: 99, And the light chain shown in SEQ ID NO: 20.

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

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

In other embodiments, the murine antibody or fragment thereof as described above, the antibody heavy chain variable region further comprises the heavy chain FR region of murine IgG1, IgG2, IgG3, IgG4 or a variant thereof.

In other embodiments, a murine antibody or fragment thereof as described above, further comprising a heavy chain constant region of murine IgG1, IgG2, IgG3, IgG4, or a variant thereof.

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

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

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

In other embodiments, a CSF-1R chimeric antibody or fragment thereof as described above, further comprising a heavy chain constant region of human IgG1, IgG2, IgG3, or IgG4 or a variant thereof.

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

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

In a preferred embodiment of the present invention, a CSF-1R humanized antibody or a 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 : The sequence shown in 34, or a variant thereof.

In other embodiments, the CSF-1R humanized antibody or fragment thereof as described above, wherein the humanized antibody light chain sequence is the sequence shown in SEQ ID NO: 18 or SEQ ID NO: 20, Or a variant thereof; said humanized antibody variant preferably has an amino acid change of 0-10 in the light chain variable region; wherein the preferred amino acid of SEQ ID NO: 18 is mutated to S31N, T37N, or a combination thereof Amino acid mutation, the most preferred amino acid mutation is T37N; SEQ ID NO: 20 The preferred amino acid mutation is the amino acid mutation of F87A, F91Y, or a combination thereof.

In a preferred embodiment of the present invention, a CSF-1R humanized antibody or fragment thereof as described above, further comprising a light chain constant region of a human-derived kappa, lambda chain or a 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 the heavy chain of human IgG1, IgG2, IgG3, IgG4 or variants thereof. FR area.

In other embodiments, the CSF-1R humanized antibody or fragment thereof as described above further comprises a heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4 or a variant thereof, preferably human IgG1, IgG2 or IgG4 heavy chain constant region. More preferably, IgG1 or amino acid mutation is used to eliminate ADCC (antibody-dependent cell-mediated cytotoxicity) toxicity of IgG1.

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

In a preferred embodiment of the present invention, an antibody as described above (the antibody described here and below is the above-mentioned anti-CSF-1R antibody or the above-mentioned monoclonal antibody) or an antigen-binding fragment thereof, wherein said antigen-binding The fragments are Fab, Fv, sFv, F (ab ') 2, linear antibodies, single chain antibodies, nanobodies, domain antibodies, and multispecific antibodies.

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

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

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

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

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

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

In other embodiments, a host cell as described above is a yeast, preferably Pichia.

In other embodiments, a host cell as described above is a mammalian cell, preferably a Chinese hamster ovary (CHO) cell, a human embryonic kidney (HEK) 293 cell, or an NSO cell.

In another aspect, some embodiments of the present invention also provide an antibody-drug conjugate comprising a light chain variable region and a heavy chain variable region as described above. The antibody-drug conjugate is known in the art, and is formed by connecting antibody-linker-drug (toxin) to each other. Known linkers include cleavage linkers and split cleavage links. SPDP and the like; toxins are also known in the art, such as DM1, DM4, MMAE, MMAF and the like.

In another aspect, some embodiments of the present 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, a multispecific antibody, a single chain antibody, a nucleic acid molecule, or a pharmaceutical composition as described above is administered in cancer treatment, wherein said administration is initiated with another anti-cancer The treatment is administered before, during, substantially simultaneously, or after.

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

In other embodiments, a CSF-1R humanized antibody or a fragment thereof as described above, wherein the chemotherapeutic agent is selected from the group consisting of taxanes (palitaris, docetaxel, modified palipita (Abraxane and Opaxio)), doxorubicin, modified doxorubicin (Caelyx or Doxil), sunitinib, sorafenib and other multikinase inhibitors, oxaliplatin, cisplatin, Carboplatin, etoposide, gemcitabine, and vinblastine.

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

In another aspect, some embodiments of the invention further provide an antibody as described above or an antigen-binding fragment thereof for use in the manufacture of a medicament for the treatment of a CSF-1R or CSF-1 mediated disease or disorder; wherein The disease is preferably cancer, autoimmune disease, inflammatory disease or osteolytic bone loss; the cancer is most preferably breast cancer, endometrial cancer, squamous cell carcinoma, follicular lymphoma, renal cell carcinoma, Uveal melanoma, cervical cancer, head and neck cancer, Hodgkin's disease, stellate cell carcinoma, 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; the autoimmune disease is most preferably self Immune encephalomyelitis, systemic lupus erythematosus, multiple sclerosis, joint synovitis, psoriasis, and rheumatoid arthritis; the osteolytic bone loss is selected from osteoporosis, metastasis-induced osteolysis Mass loss and bone loss induced by rheumatoid arthritis. The present invention further provides a method for treating and preventing a CSF-1R or CSF-1 mediated disease or disorder, which method comprises administering to a patient in need thereof a therapeutically effective amount of an antibody or an antigen-binding fragment thereof as described above, or a antibody comprising the same Pharmaceutical composition; wherein the disease is preferably cancer, autoimmune disease, inflammatory disease or osteolytic bone loss; the cancer is most preferably breast cancer, endometrial cancer, squamous cell carcinoma, follicular lymphoma Tumor, renal cell carcinoma, uveal melanoma, cervical cancer, head and neck cancer, Hodgkin's disease, stellate cell carcinoma, lung adenocarcinoma, mesothelioma, chorionic carcinoma, melanoma, breast cancer, ovarian cancer, Prostate cancer, pancreatic cancer, kidney cancer, lung cancer, liver cancer, brain cancer, gastric cancer, colorectal cancer, bladder cancer, esophageal cancer, cervical cancer, multiple myeloma, leukemia, lymphoma, and glioblastoma; said self The immune disease is most preferably autoimmune encephalomyelitis, systemic lupus erythematosus, multiple sclerosis, joint synovitis, psoriasis, and rheumatoid arthritis; the osteolytic bone loss is selected from osteoporosis , Osteolytic metastasis-induced bone loss and bone loss induced by rheumatoid arthritis.

the term

To make the present invention easier to understand, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs.

The three-letter and one-letter codes for amino acids used in the present invention are described in J. Biol. Chem, 243, p3558 (1968).

The term "antibody" in the present invention refers to an immunoglobulin, which is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains connected by an interchain disulfide bond. The composition and arrangement of amino acids in the constant region of the immunoglobulin heavy chain are different, so their antigenicity is also different. According to this, immunoglobulins can be divided into five categories, or isotypes called immunoglobulins, that is, IgM, IgD, IgG, IgA, and IgE, and the corresponding heavy chains are µ, δ, γ, Alpha and epsilon chains. The same type of Ig can be divided into different subclasses according to the amino acid composition of the hinge region and the number and position of heavy chain disulfide bonds. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. The light chain is divided into a kappa chain or a lambda chain by different constant regions. Each of the five types of Ig can have a kappa chain or a lambda chain.

In the present invention, the antibody light chain variable region of the present invention may further include a light chain constant region, and the light chain constant region comprises a human or murine κ, λ chain or a variant thereof.

In the present invention, the antibody heavy chain variable region of the present invention may further include a heavy chain constant region, and the heavy chain constant region includes human or murine IgG1, 2, 3, 4 or a variant thereof.

The sequence of about 110 amino acids near the N-terminus of the heavy and light chains of the antibody varies greatly and is a variable region (V region); the remaining amino acid sequences near the C-terminus are relatively stable and are constant regions (C region). The variable region includes three hypervariable regions (HVR) and four relatively conserved backbone regions (FR). Three hypervariable regions determine the specificity of the antibody, also known as complementarity determining regions (CDRs). Each light chain variable region (VL) and heavy chain variable region (VH) consists of three CDR regions and four FR regions. The sequence from amine end to carboxyl end is: FR1, CDR1, FR2, CDR2 , FR3, CDR3, FR4. The three CDR regions of the light chain are referred to as LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain are referred to as HCDR1, HCDR2, and HCDR3. The CDR amino acid residues of the VL and VH regions of the antibody or antigen-binding fragment according to the invention conform to the known IMGT numbering rules in number and position.

The term "antigen-presenting cell" or "APC" is a cell that displays a foreign antigen complexed with MHC on its surface. T cells use the T cell receptor (TCR) to recognize this complex. Examples of APC include, but are not limited to, dendritic cells (DC), peripheral blood mononuclear cells (PBMC), monocytes, B lymphoblasts, and monocyte-derived dendritic cells (DC). 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 a MHC-I / MHC-II conjugate.

The term "CSF-1R" refers to a colony-stimulating factor-1 receptor protein, which is a one-way 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 containing immunoglobulin (Ig) motifs and is characterized by a repeating Ig domain in the extracellular part of the receptor. The activation of CSF-1R is mediated by its ligand M-CSF or interleukin IL-34, and its signals have important physiological functions in immune response, bone reconstruction and in the reproductive system.

The term "recombinant human antibody" includes human antibodies prepared, expressed, created, or isolated by recombinant methods. The techniques and methods involved are well known in the art, such as (1) transgenes, transchromosomes from human immunoglobulin genes Antibodies isolated from animals (such as mice) or hybridomas made from them; (2) antibodies isolated from host cells transformed to express antibodies, such as transfected tumors; (3) isolated from recombinant combinatorial human antibody libraries Antibodies; and (4) antibodies prepared, expressed, created, or isolated by methods such as splicing human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies contain variable and constant regions that utilize specific human germline immunoglobulin sequences encoded by germline genes, but also include rearrangements and mutations that subsequently occur, such as during antibody maturation.

The term "murine antibody" in the present invention is a monoclonal antibody to human CSF-1R prepared according to the knowledge and skills in the art. The test subject is injected with the CSF-1R antigen during preparation, and then a hybridoma expressing an antibody having a desired sequence or functional characteristics is isolated. In a preferred embodiment of the present invention, the mouse-derived CSF-1R antibody or antigen-binding fragment thereof may further comprise a light chain constant region of a mouse-derived κ, lambda chain or a variant thereof, or further include a mouse-derived IgG1 , IgG2, IgG3, or IgG4 or a variant of the heavy chain constant region 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 that are not encoded by human germline immunoglobulin sequences (such as mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutations in vivo). However, the term "human antibody" does not include antibodies in which a CDR sequence derived from the germline of another mammalian species, such as a mouse, has been grafted onto a human backbone sequence (ie, a "humanized antibody").

The term "humanized antibody", also known as a CDR-grafted antibody, refers to an antibody produced by transplanting mouse CDR sequences into the framework of a human variable region. It can overcome the strong immune response induced by the chimeric antibody because it carries a large amount of mouse protein components. In order to avoid the decrease in activity while the decrease in immunogenicity, the human antibody variable region may be subjected to a minimum of reverse mutations to maintain the activity.

The term "chimeric antibody" is an antibody obtained by fusing the variable region of a murine antibody with the constant region of a human antibody, and can alleviate the immune response induced by the murine antibody. To establish a chimeric antibody, select a hybridoma that secretes a mouse-specific monoclonal antibody, and then copy the variable region gene from the mouse hybridoma cell. Then, copy the constant region gene of the human antibody as needed to isolate the mouse variable region. The gene is linked to the human constant region gene to form a chimeric gene and inserted into a human vector. Finally, the chimeric antibody molecule is expressed in a eukaryotic industrial system or a prokaryotic industrial system. The constant region of a human antibody may be selected from the heavy chain constant regions of human IgG1, IgG2, IgG3 or IgG4 or variants thereof, preferably including human IgG2 or IgG4 heavy chain constant regions, or without ADCC (antibody after amino acid mutation) -dependent cell-mediated cytotoxicity) Toxic IgG1.

The term "antigen-binding fragment" refers to an antigen-binding fragment and an antibody analogue of an antibody, which generally includes at least a portion of a parental antibody's antigen-binding region or variable region (eg, one or more CDRs). The antibody fragment retains at least some of the binding specificity of the parent antibody. Generally, when activity is expressed on a molar basis, antibody fragments retain at least 10% of the maternal binding activity. 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 for 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 a light chain and a heavy chain CH1 and a variable region. 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 containing the CH1 and CH2 domains of the antibody. Two heavy chain fragments are held together by two or more disulfide bonds and by the hydrophobic interaction of the CH3 domain.

A "Fab 'fragment" contains a light chain and a portion of a heavy chain containing a region between the VH and CH1 domains and between the CH1 and CH2 domains, so that it can be placed between the two heavy chains of two Fab' fragments Interchain disulfide bonds are formed to form F (ab ') 2 molecules.

The "F (ab ') 2 fragment" contains two light chains and two heavy chains containing a portion of the constant region between the CH1 and CH2 domains, thereby forming an interchain disulfide bond between the two heavy chains. Therefore, the F (ab ') 2 fragment consists of two Fab' fragments held together by a disulfide bond between the two heavy chains.

An "Fv region" contains variable regions from both heavy and light chains, but lacks a constant region.

The term "multispecific antibody" is used in its broadest sense and encompasses antibodies with multi-epitope specificity. These multispecific antibodies include, but are not limited to: antibodies comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH-VL unit has multi-epitope specificity; has two or more Antibodies for VL and VH regions, each VH-VL unit binds to a different target or different epitopes of the same target; antibodies with two or more single variable regions, each single variable region and Different targets or different epitopes of the same target bind; full-length antibodies, antibody fragments, diabodies, bispecific diabodies and triabodies, covalently or non-covalently linked together Antibody fragments and so on.

The term "single-chain antibody" is a single-chain recombinant protein composed of an antibody's heavy chain variable region (VH) and light chain variable region (VL) connected by a linker peptide. It is the smallest protein with a complete antigen-binding site. Antibody fragments.

The term "domain antibody fragment" is an immunologically functional immunoglobulin fragment containing only the heavy or light chain variable region chains. In some cases, two or more VH regions are covalently linked to a peptide linker to form a bivalent domain antibody fragment. The two VH regions of a bivalent domain antibody fragment can target the same or different antigens.

The term "binding to CSF-1R" in the present invention refers to the ability to interact with human CSF-1R. The term "antigen-binding site" in the present invention refers to a three-dimensional spatial site on the antigen that is discontinuous and recognized by the antibody or antigen-binding fragment of the present invention.

"Amino acid mutation" in the similar "having 3, 2, 1 or 0 amino acid mutations" refers to the presence of mutations in amino acids in variant proteins or polypeptides compared to the original protein or polypeptide. A corresponding number of amino acid insertions, deletions, or substitutions occur on a polypeptide basis.

CDR changes with CDR1, CDR2, and CDR3 as shown in SEQ ID NO: X, SEQ ID NO: Y, and SEQ ID NO: Z with 3, 2, 1, or 0 amino acid mutations, respectively In the description of "body", the exemplary explanation is that the mutation of the CDR may include 3, 2, 1, or 0 amino acid mutations, and the same or different numbers may be optionally selected between CDR1, CDR2, and CDR3. Mutation of the amino acid residues of CDR1, for example, based on CDR1, CDR2, and CDR3 shown in SEQ ID NO: X, SEQ ID NO: Y, and SEQ ID NO: Z. Mutations, 0 amino acid mutations to CDR2 and CDR3. When zero amino acid mutations are made to a CDR, the CDR variant with zero amino acid mutations is still the CDR itself.

The term "epitope" refers to a site on an antigen that specifically binds an immunoglobulin or antibody. Epitope can be formed by adjacent amino acids, or non-adjacent amino acids juxtaposed by the tertiary folding of a protein. An epitope formed by an adjacent amino acid is typically maintained after exposure to a denaturing solvent, while an epitope formed by tertiary folding is usually lost after treatment with a denaturing solvent. The epitope usually includes at least 3-15 amino acids in a unique steric configuration. Methods to determine what epitopes are bound by a given antibody are well known in the art and include immunoblotting and immunoprecipitation detection analysis. Methods for determining the spatial configuration of an epitope include techniques in the art and techniques described herein, such as X-ray crystal analysis and two-dimensional nuclear magnetic resonance.

The terms "specific binding" and "selective binding" used in the present invention refer to the binding of an antibody to an epitope on a predetermined antigen. Generally, when recombinant human CSF-1R is used as the analyte and the antibody is used as the ligand, as measured by surface plasmon resonance (SPR) technology in the instrument, the antibody has an equilibrium dissociation constant below about 10-7M or even less (KD) binds to a predetermined antigen, and has an affinity for binding the predetermined antigen at least twice that of a non-specific antigen (such as BSA, etc.) for binding to the predetermined antigen or a closely related antigen. The term "antibody that recognizes an antigen" is used interchangeably herein with the term "antibody that specifically binds".

When the term "competition" is used in the case of competing for antigen-binding proteins (such as neutralizing antigen-binding proteins or neutralizing antibodies) for the same epitope, it means competition between antigen-binding proteins, which is determined by the following assays: In the assay, the antigen-binding protein (eg, an antibody or antigen-binding fragment thereof) to be detected prevents or inhibits (eg, reduces) specific binding of a reference antigen-binding protein (eg, a ligand or a reference antibody) to a common antigen. Numerous types of competitive binding assays can be used to determine whether one antigen-binding protein competes with another, such as: solid-phase direct or indirect radioimmunoassay (RIA), solid-phase direct or indirect enzyme immunoassay (EIA), sandwich competition Assay (see, eg, Stahli et al., 1983, Methods in Enzymology 9: 242-253); direct biotin-avidin EIA in solid phase (see, eg, Kirkland et al., 1986, J. Immunol. 137: 3614-3619), direct labeling in the solid phase Assay, direct labelled sandwich assay with solid phase (see, for example, Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press); direct labeling of RIA with solid phase of I-125 marker (see For example, Morel et al., 1988, Molec. Immunol. 25: 7-15); solid biotin-avidin EIA (see, for example, Cheung, et al., 1990, Virology 176: 546-552); and directly labeled RIA (Moldenhauer et al.) (1990, Scand. J. Immunol. 32: 77-82). Generally the assay involves the use of a purified antigen (the antigen is on a solid surface or a cell surface) capable of binding to an unlabeled detection antigen binding protein and a labeled reference antigen binding protein. Competitive inhibition is measured by measuring the amount of label bound to a solid surface or cell in the presence of the test antigen-binding protein. Usually, the test antigen-binding protein is present in excess. Antigen-binding proteins identified by competitive assays (competing antigen-binding proteins) include: antigen-binding proteins that bind to the same epitope as the reference antigen-binding protein; and antigen-binding proteins that bind adjacent epitopes that are sufficiently close to the binding epitope of the reference antigen-binding protein Protein, the two epitopes spatially prevent each other from binding. Additional details on methods for determining competitive binding are provided in the examples of this disclosure. Usually when competing antigen-binding proteins are present in excess, they will inhibit (e.g., reduce) at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70 -75% or 75% or more specific binding of a reference antigen binding protein to a common antigen. In some cases, binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97%, or 97% or more.

The term "cross-reactivity" refers to the ability of the antibodies of the invention to bind to CSF-1R from different species. For example, an antibody of the invention that binds human CSF-1R can also bind CSF-1R of another species. Cross-reactivity is measured by detecting specific reactivity with purified antigens in binding assays (such as SPR and ELISA), or by binding or functional interaction with cells that physically exhibit CSF-1R. Methods to determine 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 a expression vector has been introduced. Host cells may include bacterial, microbial, plant or animal cells. Easily transformed bacteria include members of the enterobacteriaceae family, such as strains of Escherichia coli or Salmonella; Bacillaceae, such as Bacillus subtilis; Pneumococcus; Streptococcus and 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 as non-limiting HEK293E cells), and NSO cells.

The terms "inhibit" or "block" are used interchangeably and cover both partial and complete inhibition / blocking. The inhibition / blocking of the ligand preferably reduces or alters the normal degree or type of activity that occurs when ligand binding occurs without inhibition or blocking. Inhibition and blocking are also intended to include any measurable decrease in ligand binding affinity when contacted with an anti-CSF-1R antibody compared to a ligand that has not been contacted with an anti-CSF-1R antibody.

The term "inhibiting growth" (for example, involving a cell) is intended to include any measurable reduction in cell growth.

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

The "ADCC" in the present invention, that is, antibody-dependent cell-mediated cytotoxicity, refers to that the cells expressing Fc receptors are directly killed and coated with antibodies by recognizing the Fc segment of the antibody. Target cells. By modifying the Fc segment on IgG, the ADCC effector function of the antibody can be enhanced or reduced or eliminated. The modification refers to mutation in the constant region of the heavy chain 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 Cold Spring Harbor's antibody experimental technical guide, Chapters 5-8 and 15. For example, mice can be immunized with human CSF-1R or fragments thereof, and the resulting antibodies can be coated, purified, and amino acid sequenced using conventional methods. Antigen-binding fragments can also be prepared by conventional methods. The antibody or antigen-binding fragment according to the invention is genetically engineered to add one or more human FR regions to a CDR region of non-human origin. Human FR germline sequences can be obtained from the website of ImMunoGeneTics (IMGT) https://imgt.cines.fr, or from the Journal of Immunoglobulin, 2001ISBN012441351.

The engineered antibodies or antigen-binding fragments of the present invention can be prepared and purified by conventional methods. The cDNA sequence of the corresponding antibody can be copied and recombined into the GS expression vector. The recombinant immunoglobulin expression vector can stably transfect CHO cells. As a more recommended prior art, mammalian expression systems cause glycosylation of antibodies, especially the highly conserved N-terminus of the Fc region. Stable replication is obtained by antibodies that exhibit specific binding to human-derived antigens. Positive replicates were expanded in serum-free medium in the bioreactor to produce antibodies. The culture medium in which the antibody is secreted can be purified and collected by conventional techniques. The antibody can be concentrated by filtration using a conventional method. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves, ion exchange. The resulting product needs to be immediately frozen, such as -70 ° C, or lyophilized.

The antibody of the present invention refers to a monoclonal antibody. The monoclonal antibody (mAb) in the present invention refers to an antibody obtained from a single replicating cell strain, and the cell strain is not limited to a eukaryotic, prokaryotic, or phage replicating cell strain. Individual antibodies or antigen-binding fragments can be recombined using techniques such as hybridoma technology, recombinant technology, phage display technology, synthetic technology (such as CDR-grafting), or other existing technologies.

"Administration" and "treatment" when applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids refer to exogenous drugs, therapeutic agents, diagnostic agents or compositions and animals, humans, recipients Contact with a subject, cell, tissue, organ, or biological fluid. "Administration" and "treatment" may refer to, for example, treatment, pharmacokinetics, diagnosis, research, and experimental methods. Treatment of a cell includes contact of a reagent with a cell, and contact of a reagent with a fluid, wherein the fluid is in contact with the cell. "Administering" and "treating" also mean in vitro and ex vivo treatment of cells, such as cells, by an agent, diagnostic, binding composition, or by another cell. "Treatment" when applied to human, veterinary or research subjects refers to therapeutic treatment, preventive or preventative measures, research and diagnostic applications.

"Treatment" means the administration to a patient of an internal or external therapeutic agent, such as a composition comprising any of the binding compounds of the invention, said patient having one or more symptoms of a disease, and said therapeutic agent is known to have a therapeutic effect on these symptoms. Generally, a therapeutic agent is administered in a treated patient or population in an amount effective to alleviate the symptoms of one or more diseases, whether by inducing the deterioration of such symptoms or inhibiting the development of such symptoms to any clinically right degree. The amount of therapeutic agent (also known as a "therapeutically effective amount") that is effective in alleviating the symptoms of any particular disease can vary depending on a variety of factors, such as the patient's disease state, age and weight, and the ability of the drug to produce the desired effect in the patient. Evaluate whether the symptoms of the disease have been alleviated by any clinical test method that a doctor or other health care professional usually uses to assess the severity or progression of the symptoms. Exemplary embodiments of the present invention (eg, treatment methods or articles of manufacture) may not be effective in alleviating the symptoms of the target disease that each patient has, but according to any statistical test method known in the art such as Student's t-test, chi-square test, basis Mann and Whitney's U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test determined that it should reduce symptoms of the target disease in a statistically significant number of patients.

The term "consisting essentially of" or its variants used throughout the specification and claims means that it includes all the elements or groups of elements, and optionally includes other elements that are similar or different in nature to the elements, said other The element does not significantly change the basic or novel properties of a given dosing regimen, method, or composition. As a non-limiting example, a binding compound consisting essentially of the mentioned amino acid sequence may also include one or more amino acids, which does not significantly affect the properties of the binding compound.

The term “naturally occurring” applied to an object according to the present invention refers to the fact that the object can be found in nature. For example, polypeptide or polynucleotide sequences that exist in organisms (including viruses) that can be isolated from natural sources and have not been intentionally modified in the laboratory are naturally occurring.

An "effective amount" includes an amount sufficient to ameliorate or prevent the symptoms or conditions 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 can vary depending on factors such as the condition to be treated, the patient's general health, the route and dosage of administration, and the severity of the side effects. An effective amount may be the maximum dose or dosage regimen to avoid significant side effects or toxic effects.

"Exogenous" refers to substances that are produced outside the body, cell, or human depending on the context. "Endogenous" refers to a substance that is produced in a cell, organism, or human body by context.

"Homology" refers to the sequence similarity between two polynucleotide sequences or between two polypeptides. When a position in two compared sequences is occupied by the same base or amino acid monomer subunit, for example, if each position of two DNA molecules is occupied by adenine, then the molecules are identical at that position Source. 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 compared positions x 100%. For example, when the sequences are optimally aligned, if there are 6 matches or homology at 10 positions in the two sequences, then the two sequences are 60% homologous. In general, comparisons are made when the two sequences are aligned for the greatest percentage of homology.

As used herein, the expressions "cell", "cell line" and "cell culture" are used interchangeably, and all such names include their progeny. Thus, the words "transformants" and "transformed cells" include primary test cells and cultures derived from them, regardless of the number of metastases. It should also be understood that due to intentional or unintentional mutations, all offspring cannot be exactly the same in terms of DNA content. Included are mutant offspring that have the same functional or biological activity as those originally screened in the transformed cells. Where different names are meant, they are clearly visible from the context.

"Optional" or "optionally" means that the event or environment described later can, but need not, occur, and the description includes situations where the event or environment occurs or does not occur. For example, "optionally comprising 1-3 antibody heavy chain variable regions" means that an antibody heavy chain variable region of a particular sequence may, but need not, be present.

"Pharmaceutical composition" means a mixture containing one or more of the compounds described herein or a physiological / pharmaceutically acceptable salt or prodrug thereof with other chemical components, and other components such as physiological / pharmaceutically acceptable carriers And excipients. The purpose of the pharmaceutical composition is to promote the administration to the organism, which is beneficial to the absorption of the active ingredient and then exerts the biological activity.

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

detailed description

The following examples are used to further describe the present invention, but these examples do not limit the scope of the present invention. The experimental methods without specific conditions in the examples of the present invention generally follow conventional conditions, such as the manual of antibody technology experiments and molecular reproduction manuals of Cold Spring Harbor; or according to the conditions recommended by the raw material or commodity manufacturers. The reagents without specific sources are conventional reagents purchased on the market.

Example 1 Sequence and preparation of immune antigen and screening antigen

The sequence encoding the human CSF-1R with a His-Tag tag and the sequence encoding the human CSF-1R with a huFc tag were synthesized by the Integrated DNA Technology (IDT) company and copied to the pTT5 vector (Biovector). The recombinant CSF-1R protein was purified after expression in 293T cells (ATCC, CRL-3216TM). The purified protein can be used in the experiments of the following examples.

Human CSF-1R ECD-His sequence: MGSTAILGLLLAVLQGGRAIPVIEPSVPELVVKPGATVTLRCVGNGSVEWDGPPSPHWTLYSDGSSSILSTNNATFQNTGTYRCTEPGDPLGGSAAIHLYVKDPARPWNVLAQEVVVFEDQDALLPCLLTDPVLEAGVSLVRVRGRPLMRHTNYSFSPWHGFTIHRAKFIQSQDYQCSALMGGRKVMSISIRLKVQKVIPGPPALTLVPAELVRIRGEAAQIVCSASSVDVNFDVFLQHNNTKLAIPQQSDFHNNRYQKVLTLNLDQVDFQHAGNYSCVASNVQGKHSTSMFFRVVESAYLNLSSEQNLIQEVTVGEGLNLKVMVEAYPGLQGFNWTYLGPFSDHQPEPKLANATTKDTYRHTFTLSLPRLKPSEAGRYSFLARNPGGWRALTFELTLRYPPEVSVIWTFINGSGTLLCAASGYPQPNVTWLQCSGHTDRCDEAQVLQVWDDPYPEVLSQEPFHKVTVQSLLTVETLEHNQTYECRAHNSVGSGSWAFIPISAGAHTHPPDEGSHHHHHHHH SEQ ID NO: 93

Human CSF-1R-huFc sequence: MGSTAILGLLLAVLQGGRAIPVIEPSVPELVVKPGATVTLRCVGNGSVEWDGPPSPHWTLYSDGSSSILSTNNATFQNTGTYRCTEPGDPLGGSAAIHLYVKDPARPWNVLAQEVVVFEDQDALLPCLLTDPVLEAGVSLVRVRGRPLMRHTNYSFSPWHGFTIHRAKFIQSQDYQCSALMGGRKVMSISIRLKVQKVIPGPPALTLVPAELVRIRGEAAQIVCSASSVDVNFDVFLQHNNTKLAIPQQSDFHNNRYQKVLTLNLDQVDFQHAGNYSCVASNVQGKHSTSMFFRVVESAYLNLSSEQNLIQEVTVGEGLNLKVMVEAYPGLQGFNWTYLGPFSDHQPEPKLANATTKDTYRHTFTLSLPRLKPSEAGRYSFLARNPGGWRALTFELTLRYPPEVSVIWTFINGSGTLLCAASGYPQPNVTWLQCSGHTDRCDEAQVLQVWDDPYPEVLSQEPFHKVTVQSLLTVETLEHNQTYECRAHNSVGSGSWAFIPISAGAHTHPPDEGSGGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 94

Example 2 Preparation of CSF-1R recombinant protein

1. Purification steps of His-tagged CSF-1R recombinant protein: The supernatant sample of HEK293 cells was centrifuged at high speed to remove impurities, and the buffer was replaced with phosphate buffered saline (PBS), and imidazole was added to a final concentration of 5 mM. Equilibrate the nickel column with 5 mM imidazole in PBS and rinse 2-5 times the column volume. The replaced supernatant sample was applied to the column. The column was rinsed with 5 mM imidazole in PBS until the A280 reading dropped to baseline. The column was then washed with PBS + 10 mM imidazole to remove non-specifically bound heteroproteins, and the effluent was collected. The target protein was eluted with 300 mM imidazole in PBS, and the elution peak was collected.

The collected eluate was further purified by ion exchange (SP column). Solution A: 0.01M PB, pH 8.0. Configure liquid B: liquid A + 1M NaCl. Firstly, the target protein eluting with the PBS solution of imidazole was replaced with liquid A, and the SP column was equilibrated with liquid A, and the concentration of liquid B was gradientd from 0 to 100%, and the column volume was eluted at 10 times. The obtained proteins were identified by electrophoresis, peptide mapping, and liquid chromatography-mass spectrometry (LC-MS).

2. Purification steps of Fc-tagged CSF-1R recombinant protein (h-CSF-1R-Fc): The supernatant sample expressed by HEK293 cells was centrifuged at high speed to remove impurities, and the buffer was replaced with PBS. The Protein A affinity column was equilibrated with 10 mM phosphate buffer, and the column volume was washed 2-5 times. The replaced supernatant sample was applied to the column. Flush the column with 25 volumes of column buffer until the A280 reading drops to baseline. The target protein was eluted with 0.8% acetic acid buffer at pH 3.5, and the eluted peaks were collected. Immediately after aliquoting, 1M Tris-Cl pH8.0 buffer was added to neutralize, and then the solution was replaced with Millipore's Amico-15 filter column to PBS. pH6.9. The obtained protein was used for electrophoresis, peptide mapping, and LC-MS identification.

3. Preparation of CHO stable 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 was copied to the pcDNA3.1 vector and pcDNA3, respectively. .1 / puro (Invitrogen # V79020). CHO-S (ATCC) cells were cultured in CD-CHO medium (Life Technologies, # 10743029) to 0.5 Í ^{10 6} / ml. 10 μg of the vector encoding huCSF-1R or gene was mixed with 50 ul of LF-LTX (Life Technologies, # A12621) in 1 ml of Opti-MEM medium (Life Technologies, # 31985088). After incubating at room temperature for 20 minutes, it was added to the CHO cell culture solution. And put in a carbon dioxide incubator. After 24 hours, the medium was replaced with new medium and 10 μg / ml puromycin was added. After that, a new culture medium was replaced every 2-3 days, and a stable CHO-S cell pool was obtained after screening for 10-12 days.

4. Cynomolgus monkey CSF-1R-His (cynoCSF-1R-His) and mouse CSF-1R-His protein were purchased from AcroBiosciences.

Example 3 Preparation of Antibodies

Anti-human CSF-1R monoclonal antibodies were produced by immunizing mice. Swiss Webster white mice, female, 6 weeks of age (Charles River) were used in the experiments. Rearing environment: SPF level. After the mice were purchased, they were kept in a laboratory environment for 1 week, with a 12/12 hour light / dark cycle adjustment, a temperature of 20-25 ° C, and a humidity of 40-60%. The immune antigen was a human CSF-1R recombinant protein (huCSF-1R-Fc) with an Fc tag. Titermax (sigma Lot Num: T2684) was used as an adjuvant. The ratio of antigen to adjuvant (titermax) is 1: 1. The antigen is emulsified and inoculated for days 0, 21, 35, 49, and 63. On day 0, 15 μg + 25 / foot of post-emulsified antigen were injected intraperitoneally (IP). 21, 35, 49, 63 days Intraperitoneal (IP) injection of 15 μg + footpad 15 / post-emulsified antigen, to strengthen immunity 3 days before splenocyte fusion, intraperitoneal (IP) injection of 15 μg + footpad (footpad) ) 15 / antigen solution in normal saline. Blood tests were performed on days 42, 56, and 70. Enzyme linked immunosorbent assay (ELISA) and fluorescence activated cell sorter (FACS) were used to detect mouse serum. Antibody titer in mouse serum. After the 5th immunization, mice with high antibody titers and plateaus in the serum were selected for splenocyte fusion, and spleen lymphocytes were splenocytes and myeloma cells Sp2 / 0 cells (ATCC® CRL) using an optimized electrofusion procedure. -8287 ™) for fusion to obtain hybridoma cells.

After fusion, the hybridoma cells are 30,000-50,000 cells / well. After 7-14 days of culture, the culture supernatant is taken. As described in Example 4, using the CSF-1R recombinant protein and ELISA experiments for antibody screening of the hybridoma supernatant, The obtained positive antibody strains further used CHO-S cells stably expressing CSF-1R, compared with blank CHO-S cells to exclude non-specifically bound antibody hybridoma strains, and were screened by flow sorting to determine about 400 strains. Hybridomas that bind to recombinant proteins and also to cells that express antigens. Subsequently, as described in Example 5, using CSF-1R-based CSF-1 ligand blocking function experimental screening based on CSF-1R stably transformed cells, about 50 hybridomas with blocking agent function were identified and sub-replicated, and then collected and purified. Antibodies were screened based on the IL-34 ligand blocking function of CSF-1R-stably transfected cells, and 11 strains of antibodies with double blocking function of CSF-1 and IL-34 were identified. The logarithmic growth phase hybridoma subreplicating cells were collected, and RNA was extracted using Trizol (Invitrogen, 15596-018) and reverse transcribed (PrimeScript ™ Reverse Transcriptase, Takara # 2680A). The reverse transcripted cDNA was amplified by PCR using mouse Ig-Primer Set (Novagen, TB326 Rev. B 0503) and sequenced. After CDR region repeats were excluded, the sequences of nine mouse antibodies C11, C19, C6, and C8, C16, C18, C2, C27, and C30.

The sequence of the variable region of the heavy and light chains of the mouse monoclonal antibody C11 is as follows:

C11 HCVR EVQLQQSGPELVKPGASVKISCKASGYTFSNYYMNWVNQRHGERLEWIGEMNPNNGDSSYNQKFKGKATLTVDKSSSTAYMRSRSSESEAVYYCARRSPWWFFDVWGPGTTVIVSS SEQ ID NO: 1

C11 LCVR DIVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNSLTWYQQKPGQPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQTEDLAVYYCQNDYTYPFTFGTGTKLEIK SEQ ID NO: 2

It contains the following CDR sequences:

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

C19 HCVR EVQLQQSGPELVKPGASVKISCTASAITFTDYYMNWVKQSPGKGLEWIGDIYPNNGGTTYNQKFKGKATLTVDKSSTTAYMELRSLTSEDSAVYYCAREKITMEYYYAMDYWGQGTSVTVSS SEQ ID NO: 9

C19 LCVR DIVLTQSPASLAVSLGQRATISCRASESVSSHDIHLIHWYQQKPGQSPKLLIYAASSLESGVPARFSGSGSATDFTLNIHPVEEEDAATYFCQQSIEDPPTFGGGTKLEIK SEQ ID NO: 10

It contains the following CDR sequences:

The sequence of the variable region of the heavy and light chains of murine monoclonal antibody C6 is as follows:

C6HCVR EVRLQQSGSGPELVKPGASVKISCKASGYTFTGYYMHWVKQSPEKSLEWIGEINPNTGSCTYNQKFTGKATLTVDKSSSTAYMQLTSLTSEDSAVYYCARLNYYWYFDVWGAGTTVTVSS SEQID NO: 37

C6LCVR DIVMTQSPSSLAMSVGQKVTMSCKSSQTLLNSNDQKNYLAWYQQKPGQSPKLLVYFASTRDSGVPDRFTGSGSGTDFTLTITSVQTEDLAVYYCQQDYSTPFTFGSGTKLEIQ SEQID NO: 38

It contains the following CDR sequences:

The sequence of the variable region of the heavy and light chains of murine monoclonal antibody C8 is as follows:

C8HCVR QVQLQQSGTELVQPGASVKMTCKTSGYTFTNYWIAWVKQRPGQGLEWIGEMYPGGGGDNHHEKFKNKASLTVDMSSSTAYMQLSRLTFEDSAVYYCARRDYGNPCFDYWGQGTSLTVSS SEQID NO: 45

C8LCVR DIVLTQSPASLIVSLGQRATISCRASQGVTTSSHSYMHWYQQKLGQSPKLLIKYASNLESGVPARFSGSGSGTDFTLNIHPVEEEDTATYYCQHSWEIPYTFGGGTKLEIK SEQID NO: 46

It contains the following CDR sequences:

The sequence of the variable region of the heavy and light chains of murine monoclonal antibody C16 is as follows:

C16HCVR QVQLQQSGPELVKPGASVKLSCKASGYTFTTYDINWVKQRPGQGLEWIGWVYPRDGSTKYNEKFKGKATLTVDTSSRTVYMEFHSLTSEDSAVYFCARSGLTGSPFAYWGQGTLVTVSA SEQID NO: 53

C16LCVR KIVLTQSPASLAVSLRQRATISCRASESFDSYGNTFMHWFQQKSGQPPKLLIYRASNLESGVPARFSGSRTRTTLTIDPVEADDAATYYCQQNNEYPLTFGSGTKLELK SEQID NO: 54

It contains the following CDR sequences:

The sequence of the variable region of the heavy and light chains of murine monoclonal antibody C18 is as follows:

C18HCVR EVQLQQSGPELVKPGASVKISCKASAITFTDYYMNWVKQSPGKGLEWIGDINPNNGGTTYNQKFKGKATLTVDKSSTTAYMELRSLTSEDSAVYYCAREKISMEYYYAMDYWGQGTSVTVSS SEQID NO: 61

C18LCVR DIVLTQSPASLAVSLGQRATISCRASESVSSHDIHLMHWYQQKPGQSPKLLIYAASNLESGVPARFSGSGSATDFTLNIHPVEEEDAATYFCQQSIEDPPTFGGGTKLEIK SEQID NO: 62

It contains the following CDR sequences:

The sequence of the variable region of the heavy and light chains of mouse monoclonal antibody C23 is as follows:

C23HCVR EVQLQQSGPELVKPGTSVKISCQASGYTFTVYYMNWVKQSHGKSLEWIGDIDPNTGDSTYNQKFRGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARYDGYIDYWGQGTTLTVSS SEQID NO: 69

C23LCVR DVVMTQTPLSLPVSLGDQASISCRSSQSIVHSNRHTYLEWYLQKPGQSLKLLIYGVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDMGIYYCFQGTHVPLTFGAGTKLELK SEQID NO: 70

It contains the following CDR sequences:

The sequence of the variable region of the heavy and light chains of mouse monoclonal antibody C27 is as follows:

C27HCVR EVQLQQSGAELVRPGALVKLSCKASGFNIKDYYMHWVKQRPEQGLEWIGRFDPENGDTIYDWKFQDKAIITSDTSSNTAYLHLSSLTSEDTAVYYCARSGDYMFDYWGQGTLVTVSA SEQID NO: 77

C27LCVR ETTVTQSPASLSMAIGEKVTIRCITSTGVDDDFNWYQQRPGEPPKLLISEGNTLRPGVPSRFSSSGYGTDFVFTIENMLSEDVADYFCLQSDHLPFTFGSGTKLEIK SEQID NO: 78

It contains the following CDR sequences:

The sequence of the variable region of the heavy and light chains of mouse monoclonal antibody C30 is as follows:

C30HCVR QVQLQQSGAELMKPGASVKISCKATGYTFSSYWIEWVKQRPGHGLEWIGEILPGSGSTNYNEKFKGKATFTADTSSNTAYMQLNSLTSEDSAVYYCARNYDGSLYPMDYWGQGTSVTVSS SEQID NO: 85

C30LCVR DIVLTQSPASLAVSLGQRATISCRTSESVSIHGTHLMHWYQQKPGQPPKLLIYAASNLESGVPARFSGSGSETDFTLNIHPVEEEDAATYFCQQSIEDPPTFGGGTKLEIK SEQID NO: 86

It contains the following CDR sequences:

The heavy and light chain variable regions of each mouse antibody were copied into pTT vector plasmids (Biovector) containing human IgG4 heavy chain constant region and kappa light chain constant region, respectively, and then transiently transfected into HEK293 cells to obtain antibody resistance. Each chimeric antibody of CSF-1R (C11C, C19C, C6C, C8C, C16C, C18C, C23C, C27C, and C30C) was purified, identified, and related according to the method described in Example 2 (Fc tag protein purification) Binding and functional activity testing.

Example 4 Determination of in vitro binding activity of antibodies

The neutral avidin used for binding with biotin was diluted to 1 µg / ml with PBS pH 7.4 buffer solution, added to a 96-well plate at a volume of 100 µl / well, and left at 4 ° C for 16h-20h. After washing the plate once with PBST (pH 7.4 PBS containing 0.05% Tween-20) buffer, add 120 μl / well PBST / 1% skim milk and incubate at room temperature for 1 h to block. After washing the plate with PBST buffer once, add 1µg / ml biotin-labeled h-CSF-1R-his (or monkey and mouse CSF-1R protein to be tested) diluted with PBST / 1% skimmed milk, and set the temperature Incubate for 1 h. After washing the plate 3 times with PBST buffer, add the CSF-1R antibody (mouse antibody / chimeric antibody / humanized antibody) to be tested diluted with PBST / 1% milk to an appropriate concentration, and incubate at room temperature for 1.5h. Remove the reaction system and wash the plate 3 times with PBST. Then add 100 µl / well of diluted anti-mouse antibody or anti-human antibody labeled with Horseradish Peroxidase (HRP) labeled with PBST / 1% skim milk. Antibody (The Jackson Laboratory), incubate at room temperature for 1h. After washing the plate 3 times with PBST, add 100 µl / well of TMB and incubate at room temperature for 5-10 min. The reaction was stopped by adding 100 µl / well of 1M H ₂ SO ₄ , the absorption value was read at 450 nm, and the ELISA binding EC ₅₀ value was calculated.

CHO-S cells with high performance huCSF-1R were centrifuged at 1000 rpm for 5 minutes, the pellet was collected and suspended in 10-15 ml of pre-chilled flow buffer, and the cells were counted. The cells were collected by centrifugation in a 50 ml centrifuge tube at 1000 rpm for 5 minutes, the supernatant was discarded, and the pellet was resuspended in a pre-cooled blocking buffer with a density of 0.5-1.0 × 10 ⁷ cells / ml. After incubating at 4 ° C for 30 minutes, resuspend in 100 μl per well and add to a 96-well plate. The 96-well plate was centrifuged at 1500 rpm for 5 minutes, and the supernatant was discarded. Add 100 μl of the test antibody to each well with a concentration gradient of 0.085nM to 670nM. Resuspend the cells and incubate at 4 ° C in the dark for 60 minutes. The supernatant was discarded by centrifugation, and 100 µl of 1: 400 diluted FITC-labeled secondary antibody (BD Biosciences) was added. The cells were resuspended and incubated at 4 ° C in the dark for 60 minutes. Cells were washed twice with flow buffer and resuspended in 1% paraformaldehyde to fix the cells for flow cytometry.

The same ELISA method uses CSF-1R-His protein to detect the cross-binding of each antibody and cynomolgus monkey CSF-1R. The result data is shown in Figure 1. Except that C27C does not bind to the monkey antigen protein, the remaining antibodies and FPA008 reference are available. Strong cross-binding activity with monkey CSF-1R.

Example 5: Test of in vitro blocking function of antibodies

1.Anti-CSF-1R antibodies blocking CSF-1 or IL-34 binding on CSF-1R / CHO cells

The concentration gradient anti-CSF-1R antibody (0.01nM-670nM) was incubated with CSF-1R / CHO cells, and then biotinylated CSF-1 protein or APC-labeled IL-34 protein was added for incubation and binding. The experiment with CSF-1 was further added by streptavidin-PE tag binding. After washing with PBS, the above cells were tested for the fluorescence signal intensity of CSF-1 or IL-34 using flow cytometry.

2.Anti-CSF-1R antibody blocking human monocyte proliferation induced by CSF-1 or IL-34

Peripheral blood mononuclear cells (PBMC) in healthy human blood were first extracted and separated using Ficoll-Paque gradient separation kit (GE Healthcare Bio-Sciences), and then purified using Percoll kit (GE Healthcare Bio-Sciences) Mononuclear cells were extracted from PBMC. The isolated monocytes were then stimulated with human CSF-1 or IL-34 (R & D Systems) in a blank antibody-free or concentration-gradient anti-CSF-1R antibody, and cultured in a carbon dioxide incubator at 37 ° C for 3 days. The CellTiter-Glo kit (Promega) was used to measure the ATP content in each culture sample to be tested, and the corresponding proliferation degree of monocytes was finally measured according to its linear positive correlation with the number of activated monocytes.

The results of the binding and preliminary functional screening experiments of 30 strains of sub-replicating mouse anti-FivePrime reference antibody FPA008 are summarized in the table below. After sequencing, the nine selected murine antibodies are shown in bold.

The chimeric antibody blocking function data is shown in the table below. All 9 antibodies can effectively block the proliferation of monocytes stimulated by CSF-1 or IL-34. Among them, chimeric antibodies such as C11C, C19C, C23C and C27C have very low IC50. The blocking effect is equivalent or better than the reference antibody FPA008.

Example 6 In vitro binding affinity and kinetic experiments

This experiment uses surface plasmon resonance (SPR) method. Using a kit provided by Biacore, a standard amine coupling method was used to covalently link an anti-human IgG multi-replication antibody to a CM5 (GE) chip, and then this antibody was used to chimeric or humanize the test antibody of the present invention. Antibodies are captured to the stationary phase. H-CSF-1R-His protein (Example 1) diluted in the same buffer solution with a concentration gradient of 25-800 nM (Example 1) was injected before and after each cycle. After the injection, the reagent was regenerated with a regeneration reagent in the kit. The antigen-antibody binding kinetics were followed for 3 minutes and the dissociation kinetics were followed for 10 minutes. The data obtained using GE ’s BIAevaluation software was analyzed with a 1: 1 (Langmuir) binding model. The k _a (k _on ), kd (k _off ), and K _D values of each chimeric antibody determined by this method are shown in the table below.

Example 7 Humanization Experiment of Mouse Antibodies

Humanization of the murine anti-human CSF-1R monoclonal antibody was performed as described in many literatures in the art. In short, the human constant domain is used instead of the parent (murine antibody) constant domain, and the human antibody sequence is selected based on the homology of the mouse antibody and the human antibody. The present invention integrates functional activity with sequence specificity and drug The optimal candidate strains C11 and C19 identified in various aspects such as sexuality were humanized.

Based on the obtained typical structure of the mouse-derived antibody VH / VL CDR, the heavy and light chain variable region sequences were compared with human-derived antibody germline databases to obtain human germline templates with high homology. The human germline light chain framework region is derived from a human kappa light chain gene, and the human germline heavy chain framework region is derived from a human heavy chain. The antibody of the present invention is preferably a human germline antibody template shown below.

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

C11 preferred human germline light chain template IGkV4-1 (SEQ ID NO: 22): DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVP.DRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYYYP

C19 preferred human germline heavy chain template IGHV1-2 (SEQ ID NO: 23): QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCAR

C19 preferred human germline light chain template IGkV1-13 (SEQ ID NO: 24): AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPGKAPKLLIYDASSLES GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYP

The CDR regions of the murine antibodies C11 and C19 were transplanted onto the corresponding selected humanized template, the humanized variable region was replaced, and then recombined with the IgG constant region (preferably the heavy chain was IgG4 and the light chain was κ). Then, based on the three-dimensional structure of the mouse-derived antibody, back-mutate the embedded residues, the residues that directly interact with the CDR regions, and the residues that have an important influence on the conformation of VL and VH, and CDR regions Optimization of chemically unstable asparagine residue mutations, in which HCDR2 of mouse antibody C11: EMNPNNGDSSYNQKFKG (SEQID NO: 4) is optimized to: EMNPTNGDSSYNQKFKG (SEQID NO: 95), LCDR1: KSSQSLLNSGNQKNSLT (SEQID NO: 6) is optimized to: KSSQSLLNSGNQKTS (SEQID NO: 96); HCDR2 of mouse antibody C19: DIYPNNGGTTYNQKFKG (SEQID NO: 12) is optimized as: DIYPNTGGTTYNQKFKG (SEQID NO: 97). Thus, an antibody composed of the following humanized light and heavy chain variable region sequences was designed and detected.

huC11-VH-a (SEQ ID NO: 25): EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYMNWVRQAPGQRLEWMGEMNPNTGDSSYNQKFKGKATLTVDKSTSTAYMELSSLRSEDTAVYYCARRSPWWFFDVWGQGTMVTVSS

huC11-VH-b (SEQ ID NO: 26): EVQLVQSGAEVKKPGASVKVSCKASGYTFSNYYMNWVRQAPGQGLEWMGEMNPNTGDSSYNQKFKGRVTLTVDKSTSTAYMELSSLRSEDTAVYYCARRSPWWFFDVWGQGTMVTVSS

huC11-VH-c (SEQ ID NO: 27): EVQLVQSGAEVKKPGASVKVSCKASGYTFSNYYMNWVRQAPGQGLEWMGEMNPNTGDSSYNQKFKGRVTMTVDKSTSTAYMELSSLRSEDTAVYYCARRSPWWFFDV WGQGTMVTVSS

huC11-VH-d (SEQ ID NO: 28): EVQLVQSGAEVKKPGASVKVSCKASGYTFSNYYMNWVRQAPGQGLEWMGEMNPNTGDSSYNQKFKGRVTMTRDKSTSTVYMELSSLRSEDTAVYYCARRSPWWFFDVWGQGTMVTVSS

huC11-VL-a (SEQ ID NO: 29): DIVMTQSPDSLAVSLGERATINCKSSQSLLNSGNQKTSLTWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYTYPFTFGQGTKLEIK

huC11-VL-b (SEQ ID NO: 30): DIVMTQSPDSLAVSLGERATINCKSSQSLLSSGNQKTSLTWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYTYPFTFGQGTKLEIK

huC19-VH-a (SEQ ID NO: 31): EVQLVQSGAEVKKPGASVKVSCKASAITFTDYYMNWVKQAPGQGLEWIGDIYPNTGGTTYNQKFKGRVTLTVDTSISTAYMELSRLRSDDTAVYYCAREKITMEYYYAMDYWGQGTLVTVSS

huC19-VH-b (SEQ ID NO: 32): EVQLVQSGPEVKKPGASVKVSCKASAITFTDYYMNWVRQAPGQGLEWMGDIYPNTGGTTYNQKFKGRATLTVDTSISTAYMELSRLRSDDTAVYYCAREKITMEYYYAMDYWGQGTLVTVSS

huC19-VH-c (SEQ ID NO: 33): QVQLVQSGAEVKKPGASVKVSCKASAITFTDYYMNWVKQAPGQGLEWIGDIYPNTGGTTYNQKFKGRVTLTVDTSISTAYMELSRLRSEDTAVYYCAREKITMEYYYAMDYWGQGTLVTVSS

huC19-VL-a (SEQ ID NO: 34): DIQLTQSPSSLSASVGDRVTITCRASESVSSHDIHLIHWYQQKPGKAPKLLIYAASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQSIEDPPTFGGGTKVEIK

huC19-VL-b (SEQ ID NO: 35): DIQLTQSPSSLSASVGDRVTITCRASESVSSHDIHLIHWYQQKPGKAPKLLIYAASSLESGVPSRFSGSGSGTDFTLTISSLQPEDAATYYCQQSIEDPPTFGGGTKVEIK

huC19-VL-c (SEQ ID NO: 36): DIQLTQSPSSLSASVGDRVTISCRASESVSSHDIHLIHWYQQKPGKSPKLLIYAASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQSIEDPPTFGGGTKVEIK

A cDNA fragment was synthesized based on the amino acid sequences of the light and heavy chains of each humanized antibody, and inserted into a pcDNA3.1 expression vector (Life Technologies Cat. No. V790-20). The expression vector and transfection reagent PEI (Polysciences, Inc. Cat. No. 23966) were transfected into HEK293 cells (Life Technologies Cat. No. 11625019) at a ratio of 1: 2, and incubated in a CO ₂ incubator for 4-5 day. After the expressed antibody is recovered by centrifugation, the antibody is purified according to the method of Example 2 (purification of Fc-tagged protein) to obtain the humanized antibody protein of the present invention.

After performance testing and comparison of the number of back mutations, 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.

Further, for the huC19 humanized antibody, its HCDR1: AITFTDYYMN (SEQ ID NO: 11) was mutated to: AITFTDYYIN (SEQ ID NO: 98) to remove the chemically unstable methionine residue site, and finally The humanized antibody huC19I was obtained. The sequence of the huC19I heavy chain variable region is shown in SEQ ID NO: 100. The respective full-length sequences of huC11, huC19 and huC19I are shown in SEQ ID NOs: 17-20 and SEQ ID NO: 99.

huC19-VH-d QVQLVQSGAEVKKPGASVKVSCKASAITFTDYYINWVKQAPGQGLEWIGDIYPNTGGTTYNQKFKGRVTLTVDTSISTAYMELSRLRSEDTAVYYCAREKITMEYYYAMDYWGQGTLVTVSS SEQ ID NO: 100

huC11 HC EVQLVQSGAEVKKPGASVKVSCKASGYTFSNYYMNWVRQAPGQGLEWMGEMNPNTGDSSYNQKFKGRVTLTVDKSTSTAYMELSSLRSEDTAVYYCARRSPWWFFDVWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 17

huC11 LC DIVMTQSPDSLAVSLGERATINCKSSQSLLSSGNQKTSLTWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYTYPFTFGQGTKLEIKRTVAAPSVFIFPVQQQQQQQQQQQQQQQQQQQQQQQQQQQQQK

huC19 HC QVQLVQSGAEVKKPGASVKVSCKASAITFTDYYMNWVKQAPGQGLEWIGDIYPNTGGTTYNQKFKGRVTLTVDTSISTAYMELSRLRSEDTAVYYCAREKITMEYYYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 19

huC19 LC / huC19I LC DIQLTQSPSSLSASVGDRVTITCRASESVSSHDIHLIHWYQQKPGKAPKLLIYAASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQSIEDPPTFGGGTKVEIKRTVAAPSVSSQTLQQTLQQTLQVQQQQQQQQQQQQQQQQQQQ

huC19I HC QVQLVQSGAEVKKPGASVKVSCKASAITFTDYYINWVKQAPGQGLEWIGDIYPNTGGTTYNQKFKGRVTLTVDTSISTAYMELSRLRSEDTAVYYCAREKITMEYYYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 99

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

Humanized antibody light chain constant region sequence (SEQ ID NO: 102) RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRNRC

Example 8 Humanized antibody activity determination

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

1. Cell binding experiment (method steps are the same as in Example 4). The results are shown in Figure 2. The humanized antibodies huC11 and huC19 have positive binding to CHO cells with high expression of CSF-1R. The chimeric antibodies C11C and C19C are equivalent, and the chimeric antibody and each humanized antibody version of C19 are stronger than the corresponding version of C11.

2. Affinity kinetics experiment (method steps are the same as in Example 6). The results are shown in the following table. The final preferred humanized antibodies huC11 and huC19 have strong affinity for the human CSF-1R antigen protein, among which huC19 has an affinity ratio The reference antibody FPA008 is stronger.

3. In vitro blocking functional activity experiment (method steps are the same as in Example 6). The results are shown in Figure 3, Figure 4 and the table below. The humanized antibodies huC11, huC19 and their mutants huC19I were tested against IL-34-induced monocyte proliferation showed a strong blocking function, and the blocking function activity of huC19 was comparable or slightly better than the FPA008 reference antibody, while its mutant huC19I slightly decreased to similar to FPA008. . HuC11 in vitro blocking functional activity test In vitro blocking functional activity of huC19 and its mutant huC19I

Example 9 Pharmacological Effect of Anti-CSF1R Antibody in Humanized Tumor Model

Peripheral blood from normal subjects was collected and PBMCs from healthy subjects were isolated by density gradient centrifugation. Using CD14 ⁺ microbeads kit sub menu nucleated cells, CD14 ⁺ monocytes isolated cells according to the protocol provided in the kit, i.e., per 10 ⁷ cells were added 20ul Anti-CD14 microbeads, incubated for 15 minutes 4 ℃. Then, the cells were added to the magnetic column, and after washing three times, the cells in the magnetic column, that is, CD14 ⁺ monocytes were collected. CD14 ⁺ monocytes were added with RPMI 1640 medium containing 100ng / ml M-CSF for 6 days, and macrophages were induced. The remaining cells were added to MDA-MB-231 cells treated with Mitomycin C. PBMC and MDA-MB-231 was co-cultured for 6 days. The culture medium was RPMI 1640 medium containing IL-2 and 10% FBS.

After 6 days, macrophages were collected, 1 × 10 ⁵ cells were collected, and CD68, CD115, CD163, and CD206 stained. FACS analysis was performed to determine whether macrophages were successfully induced. PBMC cells, macrophages and freshly digested MDA-MB-231 cells (ATCC) were mixed at a ratio of 6.25: 1:50 and inoculated into each NCG mouse (Nanjing University-Nanjing Institute of Biomedicine, adaptive breeding) 5 days) subcutaneously. The experimental animals were kept in an independent ventilation box with constant temperature and humidity. The temperature of the breeding room was 18.0-26.0 ℃, the humidity was 40-70%, the ventilation was 10-20 times / hour, and the day and night light and dark alternating time was 12h / 12h.

The experiment was divided into a human IgG1 antibody control group, reference antibodies FPA8, and huC19I, both at a dose of 20 mg / kg. Six mice in each group were administered by intraperitoneal injection once every two days, 10 times (see Table 1). The daily behavior of the animals was monitored daily after the administration for 24 days. During the entire experiment, the long and wide diameters of the tumors were measured twice a week using a vernier caliper, and the tumor volume (mm ³ ) = 0.5 × (tumor long diameter x tumor short diameter ² ) was calculated. Relative tumor suppression rate TGI (%): TGI% = (1-T / C) × 100%. T / C% is the relative tumor increment rate, that is, the percentage of tumor volume or tumor weight in the treatment group and control group at a certain time point. T and C are the tumor volume (TV) or tumor weight (TW) of the treatment group and the IgG1 control group at a specific time point, respectively. All data were expressed by Mean ± SEM. Student's t test was used to compare the tumor volume and tumor weight of the treatment group with the control group. There was a significant difference compared with the control group. P <0.05 was a significant difference.

As shown in Figures 5 and 6, in the Macrophage-PBMC-MDA-MB-231 tumor model, the CSF1R antibody huC19I showed antitumor effects. The terminal average tumor volumes of each group were: 294.32 mm ³ , 230.46 mm ³ , 131.19 The tumor inhibition rates (TGI) of mm ³ , FPA8 group and huC19I group were 22.62% and 55.95%, respectively. There was no significant weight loss of NCG mice in each administration group, indicating that NCG mice were well tolerated to the CSF1R antibody at this dose.

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

no

Figure 1: ELISA in vitro binding experiments of nine CSF-1R chimeric antibodies, showing that all antibodies except C27C have strong cross-binding with cynomolgus CSF-1R protein. Figure 2: In vitro binding activity experiments of humanized antibodies and CHO cells with high expression of CSF-1R. The humanized antibodies huC11 and huC19 have comparable cell-binding strength to their original chimeric antibodies C11C and C19C, respectively. Figure 3: Functional experiment of humanized antibody huC11 blocking monocyte proliferation. (A). Antibodies block IL-34-stimulated monocyte proliferation. (B). Antibodies block CSF-1 stimulated monocyte proliferation. Figure 4: Functional experiment of humanized antibody huC19 blocking monocyte proliferation. (A). Antibodies block IL-34-stimulated monocyte proliferation. (B). Antibodies block CSF-1 stimulated monocyte proliferation. Figure 5: In vivo pharmacodynamic experiment of humanized antibody huC19I. The antibody inhibited the growth of MDA-MB-231 tumor cells. Figure 6: In vivo pharmacodynamic experiment of humanized antibody huC19I. Compared with the control group, the anti-CSF1R antibody had no significant effect on animal body weight.

Claims (24)

An anti-CSF-1R antibody or an antigen-binding fragment thereof comprising an antibody light chain variable region and an antibody heavy chain variable region, wherein: a) the antibody light chain variable region comprises a member selected from the group consisting of SEQ ID NO: 42 LCDR variants having 3, 2, 1, or 0 amino acid mutations on the basis of LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 43, and SEQ ID NO: 44, respectively; the antibody heavy chain variable region Comprising an HCDR variant selected from the group consisting of HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 39, SEQ ID NO: 40 and SEQ ID NO: 41 with 3, 2, 1 or 0 amino acid mutations, respectively; b) the variable region of the antibody light chain comprises a member selected from the group consisting of LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 50, SEQ ID NO: 51, and SEQ ID NO: 52, respectively; 0 amino acid-mutated LCDR variants; the antibody heavy chain variable region comprises a base selected from the group consisting of HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 47, SEQ ID NO: 48, and SEQ ID NO: 49 HCDR variants having 3, 2, 1 or 0 amino acid mutations on the above; c) the variable region of the light chain of the antibody comprises a member selected from the group consisting of SEQ ID NO: 58, SEQ ID NO: 59 and SEQ ID NO : LCDR1 shown at 60 LCDR2 and LCDR3 have LCDR variants having 3, 2, 1 or 0 amino acid mutations respectively; the antibody heavy chain variable region is selected from the group consisting of SEQ ID NO: 55, SEQ ID NO: 56 and SEQ ID HCDR1, HCDR2 and HCDR3 shown in NO: 57 are HCDR variants having 3, 2, 1 or 0 amino acid mutations, respectively; d) the variable region of the light chain of the antibody comprises : 66. LCDR variants having 3, 2, 1, or 0 amino acid mutations on the basis of LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 67 and SEQ ID NO: 68, respectively; the heavy chain of the antibody may be The variable region comprises a HCDR variant selected from HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 63, SEQ ID NO: 64, and SEQ ID NO: 65, respectively, having 3, 2, 1, or 0 amino acid mutations, respectively. E) the variable region of the light chain of the antibody comprises a member selected from the group consisting of LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 74, SEQ ID NO: 75, and SEQ ID NO: 76 having 3, 2, respectively 1 or 0 amino acid-mutated LCDR variants; the antibody heavy chain variable region comprises a member selected from the group consisting of HCDR1, HCDR2, and SEQ ID NO: 71, SEQ ID NO: 72, and SEQ ID NO: 73 HCDR3 has 3, 2, 1 respectively Or 0 amino acid mutant HCDR variants; f) the variable region of the antibody light chain comprises LCDR1, LCDR2 selected from the group consisting of SEQ ID NO: 82, SEQ ID NO: 83, and SEQ ID NO: 84 LCDR variants having 3, 2, 1 or 0 amino acid mutations on the basis of LCDR3 and LCDR3 respectively; the variable region of the heavy chain of the antibody comprises a group selected from SEQ ID NO: 79, SEQ ID NO: 80 and SEQ ID HCDR1, HCDR2, and HCDR3 shown in NO: 81 are HCDR variants having 3, 2, 1, or 0 amino acid mutations, respectively; g) the variable region of the light chain of the antibody comprises : 90. LCDR variants having 3, 2, 1 or 0 amino acid mutations on the basis of LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 91 and SEQ ID NO: 92, respectively; the heavy chain of the antibody may be The variable region comprises a HCDR variant selected from the group consisting of HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 87, SEQ ID NO: 88, and SEQ ID NO: 89 with 3, 2, 1, or 0 amino acid mutations, respectively. H) the variable region of the antibody light chain comprises a member selected from the group consisting of LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8 having 3, 2, respectively LCDR variants with 1 or 0 amino acid mutations The variable region of the antibody heavy chain comprises 3, 2, 1 or 0 selected from HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, respectively. Amino acid mutated HCDR variants; i) the antibody light chain variable region comprises a base selected from the group consisting of LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16 LCDR variants with 3, 2, 1 or 0 amino acid mutations respectively; the variable region of the antibody heavy chain comprises a selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13 HCDR1, HCDR2, and HCDR3 are shown as HCDR variants having 3, 2, 1, or 0 amino acid mutations, respectively; j) the variable region of the antibody light chain comprises a region selected from SEQ ID NO: 14, LCDR variants having 3, 2, 1 or 0 amino acid mutations on the basis of LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 15 and SEQ ID NO: 16 respectively; the antibody heavy chain variable region comprises : HCDR variants having 3, 2, 1, or 0 amino acid mutations on the basis of HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 98, SEQ ID NO: 12, and SEQ ID NO: 13, respectively; k) Antibody light chain variable region LCDR variants with 3, 2, 1 or 0 amino acid mutations on the basis of LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, respectively; antibody weight The chain variable region comprises 3, 2, 1 or 0 amino acid mutations selected from HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 98, SEQ ID NO: 97, and SEQ ID NO: 13, respectively. HCDR variants; l) the antibody light chain variable region comprises a member selected from the group consisting of LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 96, SEQ ID NO: 7, and SEQ ID NO: 8, respectively; , 2, 1 or 0 amino acid-mutated LCDR variants; the antibody heavy chain variable region comprises HCDR1, HCDR2 selected from SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5 And HCDR3 based on HCDR variants having 3, 2, 1 or 0 amino acid mutations, respectively; m) the variable region of the antibody light chain comprises a group selected from SEQ ID NO: 6, SEQ ID NO: 7 and LCDR variants having 3, 2, 1 or 0 amino acid mutations on the basis of LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 8 respectively; the variable region of the antibody heavy chain comprises a sequence selected from, for example, SEQ ID NO: 3 HCD as shown in SEQ ID NO: 95 and SEQ ID NO: 5 HCDR variants having 3, 2, 1 or 0 amino acid mutations on the basis of R1, HCDR2 and HCDR3, respectively; n) the variable region of the antibody light chain comprises a group selected from SEQ ID NO: 96, SEQ ID NO : LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 8 with 3, 2, 1, or 0 amino acid mutations, respectively; the variable region of the antibody heavy chain comprises a sequence selected from: SEQ ID NO: 3, HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 95 and SEQ ID NO: 5 with 3, 2, 1, or 0 amino acid mutations, respectively; or o) The variable region of the antibody light chain comprises 3, 2, 1 or 0 amine groups 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. Acid-mutated LCDR variant; the antibody heavy chain variable region comprises a member selected from HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 11, SEQ ID NO: 97, and SEQ ID NO: 13 having 3, 2 respectively , 1 or 0 amino acid mutant HCDR variants. An anti-CSF-1R antibody or an 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 SEQ ID NO: 42, The LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 43 and SEQ ID NO: 44; the antibody heavy chain variable region comprises as shown in SEQ ID NO: 39, SEQ ID NO: 40, and SEQ ID NO: 41, respectively HCDR1, HCDR2 and HCDR3 shown in the figure; q) the variable region of the antibody light chain comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 50, SEQ ID NO: 51 and SEQ ID NO: 52, respectively; said The antibody heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in 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 shown in SEQ ID NO: 58, SEQ ID NO: 59, and SEQ ID NO: 60; the variable region of the antibody heavy chain comprises SEQ ID NO: 55, SEQ ID NO: 56, and HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 57; s) The variable region of the antibody light chain comprises LCDR1, shown in SEQ ID NO: 66, SEQ ID NO: 67, and SEQ ID NO: 68, respectively LCDR2 and LCDR3; described The antibody heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 63, SEQ ID NO: 64 and SEQ ID NO: 65, respectively; t) the antibody light chain variable region comprises IDR: 74, SEQ ID NO: 75, and SEQ ID NO: 76; LCDR1, LCDR2, and LCDR3; the variable region of the antibody heavy chain comprises SEQ ID NO: 71, SEQ ID NO: 72, and SEQ, respectively. HCDR1, HCDR2, and HCDR3 shown in ID NO: 73; u) the variable region of the light chain of the antibody comprises LCDR1, LCDR2 shown in SEQ ID NO: 82, SEQ ID NO: 83, and SEQ ID NO: 84, respectively And LCDR3; the variable region of the antibody heavy chain comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 79, SEQ ID NO: 80, and SEQ ID NO: 81, respectively; v) the light chain of the antibody may be The variable region includes LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 90, SEQ ID NO: 91, and SEQ ID NO: 92, respectively; the variable region of the antibody heavy chain includes as shown in SEQ ID NO: 87, SEQ, HCDR1, HCDR2, and HCDR3 shown in ID NO: 88 and SEQ ID NO: 89; w) the variable region of the light chain of the antibody comprises SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8 respectively LCDR1, LCDR2 and LCDR3 shown; said reactance The variable region of the weight chain comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5 respectively; x) the variable region of the antibody light chain comprises as shown in SEQ ID NO: 14, LCD ID1, LCDR2 and LCDR3 shown in SEQ ID NO: 15 and SEQ ID NO: 16; the variable region of the antibody heavy chain comprises SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID, respectively HCDR1, HCDR2 and HCDR3 shown in NO: 13; y) the variable region of the light chain of the antibody comprises LCDR1, LCDR2 and LCDR1 shown in SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16 respectively; LCDR3; the antibody heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 98, SEQ ID NO: 12 and SEQ ID NO: 13, respectively; z) 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 respectively; the antibody heavy chain variable region contains SEQ ID NO: 98, SEQ ID NO: 97, and HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO: 13; aa) the antibody light chain variable region comprises LCDR1, shown in SEQ ID NO: 96, SEQ ID NO: 7 and SEQ ID NO: 8 respectively; LCDR2 and LCDR3; antibody heavy chain variable region contains For example: HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5; ab) the variable region of the light chain of the antibody comprises as shown in SEQ ID NO: 6, SEQ LCDR1, LCDR2, and LCDR3 shown in ID NO: 7 and SEQ ID NO: 8; the variable region of the antibody heavy chain includes HCDR1 shown in SEQ ID NO: 3, SEQ ID NO: 95, and SEQ ID NO: 5, respectively , HCDR2 and HCDR3; ac) The variable region of the antibody light chain comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 96, SEQ ID NO: 7 and SEQ ID NO: 8, respectively; the antibody heavy chain is variable The region comprises HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 3, SEQ ID NO: 95, and SEQ ID NO: 5, respectively; or the antibody light chain variable region described in ad) includes, respectively, SEQ ID NO: : 14, LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 15 and SEQ ID NO: 16; the variable region of the antibody heavy chain includes, respectively, SEQ ID NO: 11, SEQ ID NO: 97, and SEQ ID NO: 13 HCDR1, HCDR2 and HCDR3 shown. The anti-CSF-1R antibody or antigen-binding fragment thereof according to 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 according to claim 3, wherein a) the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 38, and the amino acid sequence of the heavy chain variable region is SEQ ID NO: 37; or b) the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 46, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 45 Or c) the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 54, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 53; or d ) The amino acid sequence of the light chain variable region is shown in SEQ ID NO: 62, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 61; or e) the light chain The amino acid sequence of the variable region is shown in SEQ ID NO: 70, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 69; or f) the amine of the light chain variable region The amino acid sequence is shown in SEQ ID NO: 78, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 77; or g) the amino acid sequence of the light chain variable region is shown in SEQ. ID NO: 86, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 85; or h) the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 2 or a variant thereof, and the variant preferably has 0-10 in the light chain variable region The most preferred amino acid mutation is N37T. The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 1 or a variant thereof, and the variant is preferably in the heavy chain. The variable region has an amino acid mutation of 0-10, and the preferred amino acid mutation is N55T; or i) the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 10, and the heavy chain may be The amino acid sequence of the variable region is shown in SEQ ID NO: 9 or a variant thereof. The variant preferably has a 0-10 amino acid mutation in the light chain variable region, and the preferred mutation is M34I, N55T or Combination of amino acid mutations. The anti-CSF-1R antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the antibody or antigen-binding fragment thereof is a humanized antibody or an antigen-binding fragment thereof. The anti-CSF-1R antibody or antigen-binding fragment thereof according to claim 5, wherein the light chain FR region sequence on the light chain variable region of the humanized antibody is derived from a human as shown in SEQ ID NO: 22 Germline light chain IGkV4-1 sequence; or derived from the human germline light chain IGkV1-13 sequence as shown in SEQ ID NO: 24; and / or, the humanized antibody heavy chain variable region further comprises human origin The heavy chain FR region of IgG1, IgG2, IgG3 or IgG4 or a variant thereof preferably comprises a human IgG1, IgG2 or IgG4 heavy chain FR region, and more preferably a human IgG1 or IgG4 heavy chain FR region; preferably, said The heavy chain FR region sequence on the humanized antibody heavy chain variable region is derived from the human germline heavy chain IGHV1-46 sequence as shown in SEQ ID NO: 21; or is derived as shown in SEQ ID NO: 23 Human germline heavy chain IGHV1-2 sequence. The anti-CSF-1R antibody or antigen-binding fragment thereof according to claim 6, wherein the humanized antibody light chain variable region sequence is the sequence shown in SEQ ID NO: 30 or SEQ ID NO: 34 or Variants; said variants preferably have amino acid mutations of 0-10 in the variable region of the light chain, of which the most preferred amino acid mutations of SEQ ID NO: 30 are S31N, T37N; the most preferred is SEQ ID NO: 34 Amino acid mutation of F87A, F91Y or a combination thereof; and / or, the sequence of the humanized antibody heavy chain variable region is as shown in SEQ ID NO: 26, SEQ ID NO: 100 or SEQ ID NO: 33 Or a variant thereof; said variant preferably has an amino acid mutation of 0-10 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, The most preferred amino acid mutation is T55N; SEQ ID NO: 33 The preferred amino acid mutation is Q1E, M34I, T55N, E89D or a combination thereof, and the most preferred amino acid mutation is an amino group of M34I, T55N or a combination thereof Acid mutation; preferably, the sequence of the heavy chain variable region is SEQ ID NO: 31, 32, 33, or 100, and the sequence of the light chain variable region is SEQ ID NO: 34, 35, or 36; Or, said The sequence of the heavy chain variable region is SEQ ID NO: 25, 26, 27, or 28, and the sequence of the light chain variable region is SEQ ID NO: 29 or 30. The anti-CSF-1R antibody or antigen-binding fragment thereof according to any one of claims 3-7, wherein the chimeric antibody or humanized antibody further includes a constant region of a light chain and / or a heavy chain, the The light chain constant region sequence is shown in SEQ ID NO: 102, and / or the heavy chain constant region sequence is shown in SEQ ID NO: 101. The anti-CSF-1R antibody or antigen-binding fragment thereof according to claim 8, wherein the humanized antibody: comprises a heavy chain having the sequence shown in SEQ ID NO: 19, and the sequence is shown in SEQ ID NO: 20 A light chain comprising a sequence shown in SEQ ID NO: 17 and a light chain comprising a sequence shown in SEQ ID NO: 18; or a heavy chain comprising a sequence shown in SEQ ID NO: 99 and a sequence such as The light chain shown in SEQ ID NO: 20. An isolated monoclonal antibody or an antigen-binding fragment thereof, which competes with the anti-CSF-1R antibody or the antigen-binding fragment thereof according to any one of claims 1-9 to bind CSF-1R. A multispecific antibody comprising the light chain variable region and / or the heavy chain variable region of the antibody or antigen-binding fragment thereof according to any one of claims 1-10. A single-chain antibody comprising a light chain variable region and / or a heavy chain variable region of the antibody or the antigen-binding fragment thereof according to any one of claims 1-10. An antibody-drug conjugate, wherein the antibody contains the light chain variable region and / or the heavy chain variable region of the antibody or antigen-binding fragment thereof according to any one of claims 1-10. A nucleic acid molecule encoding the antibody or the antigen-binding fragment thereof according to any one of claims 1-10, the multispecific antibody according to claim 11, or the single-chain antibody according to claim 12. A expression vector comprising the nucleic acid according to claim 14. A host cell transformed with the expression vector of claim 15. The host cell according to claim 16, wherein the host cell is a bacterium, preferably Escherichia coli; or the host cell is a yeast, preferably Pichia pastoris; or the host cell is an animal cell, Chinese hamster ovary cells, human embryonic kidney 293 cells or NSO cells are preferred. A method for preparing the antibody or the antigen-binding fragment thereof according to any one of claims 1 to 10, the multispecific antibody according to claim 11, or the single-chain antibody according to claim 12, comprising: The host cell according to claim 16 or 17, expressing the antibody or antigen-binding fragment thereof, the multispecific antibody or the single chain antibody, and isolating the antibody or antigen-binding fragment thereof from the host cell, The multispecific antibody or the single chain antibody. A pharmaceutical composition comprising the antibody or the antigen-binding fragment thereof according to any one of claims 1-10, the multispecific antibody according to claim 11, or the single-chain antibody according to claim 12, and A pharmaceutically acceptable buffer, excipient, diluent or carrier. 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, or a claim 13 Use of an antibody-drug conjugate, a nucleic acid molecule according to claim 14 or a pharmaceutical composition according to claim 19 in anticancer treatment, wherein said antibody or antigen-binding fragment thereof, said The multispecific antibody, the single-chain antibody, the antibody-drug conjugate, the nucleic acid molecule, or the pharmaceutical composition is before, during, starting treatment with another anti-cancer therapy, Applied substantially simultaneously or afterwards. Use according to claim 20, wherein said another anti-cancer therapy is selected from the group consisting of anti-angiogenic agents, chemotherapeutic agents, radiation, tumor immunotherapy or a combination thereof. The use according to claim 21, wherein the chemotherapeutic agent is selected from the group consisting of taxanes (paclitaxel injection, docetaxel, paclitaxel albumin, etc.), doxorubicin, modified doxorubicin ( Caelyx or Doxil)), sunitinib, sorafenib, and other multikinase inhibitors, oxaliplatin, cisplatin, carboplatin, etoposide, gemcitabine, and vinblastine; and / or The tumor immunotherapy is selected from the group consisting of: T-cell binders, targeted immunosuppression, cancer vaccine / enhancing dendritic cell function, and adoptive cell metastasis. 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, or a claim 13 An antibody-drug conjugate, a nucleic acid molecule according to claim 14, or a pharmaceutical composition according to claim 19 in the manufacture of a medicament for the treatment of a CSF-1R or CSF-1 mediated disease or disorder Use; wherein the disease is preferably cancer, autoimmune disease, inflammatory disease or osteolytic bone loss; the cancer is most preferably breast cancer, endometrial cancer, squamous cell carcinoma, follicular lymphoma, Renal cell carcinoma, uveal melanoma, cervical cancer, head and neck cancer, Hodgkin's disease, stellate cell carcinoma, lung adenocarcinoma, mesothelioma, choriocarcinoma, melanoma, breast cancer, ovarian cancer, prostate cancer , Pancreatic cancer, kidney cancer, lung cancer, liver cancer, brain cancer, gastric cancer, colorectal cancer, bladder cancer, esophageal cancer, cervical cancer, multiple myeloma, leukemia, lymphoma and glioblastoma; said autoimmune disease Most preferred is autoimmune encephalomyelitis Systemic lupus erythematosus, multiple sclerosis, joint synovitis, psoriasis, and rheumatoid arthritis; the osteolytic bone loss is selected from the group consisting of osteoporosis, metastasis-induced osteolytic bone loss, and rheumatoid arthritis Bone loss. A method for preventing or treating a CSF-1R or CSF-1 mediated disease or condition, the method comprising administering to a patient in need thereof a therapeutically effective amount of the antibody or antigen-binding fragment thereof according to any one of claims 1-10, such as 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 request 19 The pharmaceutical composition, wherein the disease is preferably cancer, autoimmune disease, inflammatory disease or osteolytic bone loss; the cancer is most preferably breast cancer, endometrial cancer, squamous cell carcinoma, filter Bubble lymphoma, renal cell carcinoma, uveal melanoma, cervical cancer, head and neck cancer, Hodgkin's disease, stellate cell carcinoma, lung adenocarcinoma, mesothelioma, chorionic carcinoma, melanoma, breast cancer, Ovarian cancer, prostate cancer, pancreatic cancer, kidney cancer, lung cancer, liver cancer, brain cancer, gastric cancer, colorectal cancer, bladder cancer, esophageal cancer, cervical cancer, multiple myeloma, leukemia, lymphoma and glioblastoma; The autoimmune disease described above is most preferably Immune encephalomyelitis, systemic lupus erythematosus, multiple sclerosis, joint synovitis, psoriasis, and rheumatoid arthritis; the osteolytic bone loss is selected from osteoporosis, metastasis-induced osteolytic bone loss, and Rheumatoid arthritis-induced bone loss.