WO2019080889A1 - Anti-csf-1r antibody, antigen-binding fragment thereof and pharmaceutical use thereof - Google Patents

Anti-csf-1r antibody, antigen-binding fragment thereof and pharmaceutical use thereof

Info

Publication number
WO2019080889A1
WO2019080889A1 PCT/CN2018/111818 CN2018111818W WO2019080889A1 WO 2019080889 A1 WO2019080889 A1 WO 2019080889A1 CN 2018111818 W CN2018111818 W CN 2018111818W WO 2019080889 A1 WO2019080889 A1 WO 2019080889A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
antibody
variable region
chain variable
amino acid
Prior art date
Application number
PCT/CN2018/111818
Other languages
French (fr)
Chinese (zh)
Inventor
闫树德
黄浩
张连山
曹国庆
蒋家骅
Original Assignee
江苏恒瑞医药股份有限公司
上海恒瑞医药有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 江苏恒瑞医药股份有限公司, 上海恒瑞医药有限公司 filed Critical 江苏恒瑞医药股份有限公司
Priority to CN201880044519.6A priority Critical patent/CN110913894B/en
Publication of WO2019080889A1 publication Critical patent/WO2019080889A1/en

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates to an anti-CSF-1R antibody immunoreactive to a 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 A pharmaceutical composition comprising a human anti-CSF-1R antibody and an antigen-binding fragment thereof, and use thereof as an anticancer drug.
  • Tumor immunotherapy is a long-term hotspot in the field of cancer treatment, and T cell tumor immunotherapy is at the core. Tumor escape is a huge obstacle to tumor immunotherapy. Most tumors express antigens that can be recognized by the host immune system to varying degrees, but in many cases, due to inefficient activation of immune cells, an inadequate immune response is triggered. Cells use their own inhibition of the immune system to promote the crazy growth of the tumor. Tumor immunotherapy is to fully utilize and mobilize various immune cells in tumor patients to kill tumors through various methods such as inhibition of immune checkpoints, direct activation, and activation of tumor microenvironment.
  • Macrophages are an important cell population in the process of anti-tumor immune regulation, which can directly kill tumor cells.
  • TAM tumor-associated macrophages
  • M2 macrophages
  • TAM tumor-associated macrophages
  • Role but participate in the process of tumorigenesis, growth, invasion and metastasis.
  • TAM is derived from macrophages remaining in monocytes or tissues in the lymphatic circulation and is a major type of leukocytes infiltrating many tumor stromas. Once activated, TAM tends to be a major source of cytokines, growth factors, and proteases in the tumor microenvironment, promoting tumor growth, proliferation, angiogenesis, invasion, metastasis, and chemotherapy resistance.
  • CSF-1R colony stimulating factor-1 receptor, nicknamed FMS, C-FMS, and CD115, etc.
  • CSF-1R colony stimulating factor-1 receptor, nicknamed FMS, C-FMS, and CD115, etc.
  • CSF-1R colony stimulating factor-1 receptor, nicknamed FMS, C-FMS, and CD115, etc.
  • IL-34 interleukin-34
  • Both CSF-1 and IL-34 ligands are capable of stimulating monocyte survival, proliferation, and differentiation into macrophages.
  • CSF-1R is expressed primarily on cells of the monocyte lineage and in the female reproductive tract and placenta.
  • CSF-1 activates monocytes/M2 macrophages via CSF-1R.
  • CSF-1 levels in tumors are associated with TAM levels within the tumor, while high levels of TAM are associated with poor patient prognosis. It has been found that in human breast cancer xenografts in mice, CSF-1 promotes tumor growth and progression to metastasis.
  • 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 myeloid cells that promote differentiation to mediate disease pathology in a pathological setting.
  • various antagonists of CSF-1R receptor signaling can be used to treat various CSF-1R-mediated related diseases by blocking the activation of monocytes into M2 macrophages.
  • CSF-1R-mediated related diseases such as cancer, inflammatory conditions and autoimmune diseases.
  • W02011/123381 discloses an anti-CSF-1R antibody that internalizes CSF-1R and has ADCC activity. W02011/123381 also discloses an in vivo mouse tumor model using an anti-murine CSF-1R antibody.
  • W02011/140249 discloses an anti-CSF-1R antibody that blocks the binding of CSF-1 to CSF-1R, which is believed to be useful in the treatment of cancer.
  • W02009/112245 discloses an anti-CSF-IR IgG1 antibody that inhibits CSF-1 binding to CSF-IR, which is considered to be useful in the treatment of cancer, inflammatory bowel disease and rheumatoid arthritis.
  • W02011/131407 discloses anti-CSF-IR antibodies that inhibit CSF-1 binding to CSF-IR, which are believed to be useful in the treatment of bone loss and cancer.
  • W02011/107553 discloses anti-CSF-IR antibodies that inhibit CSF-1 binding to CSF-IR, which are believed to be useful in the treatment of bone loss and cancer.
  • W02011/070024 discloses an anti-CSF-IR antibody that binds to the human CSF-IR fragment deID4.
  • Some embodiments of the invention provide an anti-CSF-1R antibody or antigen-binding fragment thereof, comprising:
  • An antibody light chain variable region comprising at least one LCDR selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO :14, SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 96;
  • An antibody heavy chain variable region comprising at least one HCDR selected from the group consisting of 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.
  • the above anti-CSF-1R antibody or antigen-binding fragment thereof comprising an antibody light chain variable region and an antibody heavy chain variable region, wherein:
  • the antibody light chain variable region comprises 3, 2, 2 selected from the group consisting of LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 42, SEQ ID NO: 43 and SEQ ID NO: 44, respectively. Or a 0 amino acid mutated LCDR variant; said antibody heavy chain variable region comprising a base selected from the group consisting of HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 39, SEQ ID NO: 40 and SEQ ID NO: 41 HCDR variants having 3, 2, 1 or 0 amino acid mutations, respectively;
  • the antibody light chain variable region comprises 3, 2, 2 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. Or a 0 amino acid mutated LCDR variant; said antibody heavy chain variable region comprising a base selected from the group consisting of HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 47, SEQ ID NO: 48 and SEQ ID NO: HCDR variants having 3, 2, 1 or 0 amino acid mutations, respectively;
  • the antibody light chain variable region comprising 3, 2, 2 selected from the group consisting of LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 58, SEQ ID NO: 59 and SEQ ID NO: 60, respectively. Or a 0 amino acid mutated LCDR variant; said antibody heavy chain variable region selected from the group consisting of HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 55, SEQ ID NO: 56 and SEQ ID NO: 57 HCDR variants having 3, 2, 1 or 0 amino acid mutations, respectively;
  • the antibody light chain variable region comprising 3, 2, 2 selected from the group consisting of LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 66, SEQ ID NO: 67 and SEQ ID NO: 68, respectively. Or a 0 amino acid mutated LCDR variant; said antibody heavy chain variable region comprising a base selected from the group consisting of HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 63, SEQ ID NO: 64 and SEQ ID NO: 65 HCDR variants having 3, 2, 1 or 0 amino acid mutations, respectively;
  • the antibody light chain variable region comprises 3, 2, 2 selected from the group consisting of LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 74, SEQ ID NO: 75 and SEQ ID NO: 76, respectively. Or a 0 amino acid mutated LCDR variant; said antibody heavy chain variable region comprising a base selected from the group consisting of HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO:71, SEQ ID NO:72 and SEQ ID NO:73 HCDR variants having 3, 2, 1 or 0 amino acid mutations, respectively;
  • the antibody light chain variable region comprising 3, 2, 2 selected from the group consisting of LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 82, SEQ ID NO: 83 and SEQ ID NO: 84, respectively. Or a 0 amino acid mutated LCDR variant; said antibody heavy chain variable region comprising a base selected from the group consisting of HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO:79, SEQ ID NO:80 and SEQ ID NO:81 HCDR variants having 3, 2, 1 or 0 amino acid mutations, respectively;
  • the antibody light chain variable region comprising 3, 2, 2 selected from the group consisting of LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 90, SEQ ID NO: 91 and SEQ ID NO: 92, respectively. Or a 0 amino acid mutated LCDR variant; said antibody heavy chain variable region comprising a base selected from the group consisting of HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO:87, SEQ ID NO:88 and SEQ ID NO:89 HCDR variants having 3, 2, 1 or 0 amino acid mutations, respectively;
  • the antibody light chain variable region comprises 3, 2, 2 selected from the group consisting of LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively. Or a 0 amino acid mutated LCDR variant; said antibody heavy chain variable region comprising a base selected from the group consisting of HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5 HCDR variants having 3, 2, 1 or 0 amino acid mutations, respectively;
  • the antibody light chain variable region comprises 3, 2, 2 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. Or a 0 amino acid mutated LCDR variant; said antibody heavy chain variable region comprising a base selected from the group consisting of HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: HCDR variants having 3, 2, 1 or 0 amino acid mutations, respectively;
  • the antibody light chain variable region comprises 3, 2, 2 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. Or a 0 amino acid mutated LCDR variant;
  • the antibody heavy chain variable region comprises a selection of HCDR1, HCDR2 and HCDR3 selected from, for example, SEQ ID NO:98, SEQ ID NO:12 and SEQ ID NO:13, respectively HCDR variants having 3, 2, 1 or 0 amino acid mutations;
  • the antibody light chain variable region comprises 3, 2, 1 or 0 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.
  • An amino acid mutated LCDR variant; the antibody heavy chain variable region comprises 3, respectively, selected from HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO:98, SEQ ID NO:97 and SEQ ID NO:13 HCDR variant of 2, 1 or 0 amino acid mutations;
  • the antibody light chain variable region comprises 3, 2, 2 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. Or a 0 amino acid mutated LCDR variant;
  • the antibody heavy chain variable region comprises a selection of HCDR1, HCDR2 and HCDR3 selected from, for example, 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;
  • the antibody light chain variable region comprising 3, 2, 2 selected from the group consisting of LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively.
  • the antibody heavy chain variable region comprises a selection of HCDR1, HCDR2 and HCDR3 selected from, for example, SEQ ID NO:3, SEQ ID NO:95 and SEQ ID NO:5, respectively HCDR variants having 3, 2, 1 or 0 amino acid mutations;
  • the antibody light chain variable region comprises 3, 2, 2 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. Or a 0 amino acid mutated LCDR variant; the antibody heavy chain variable region comprises a selection of HCDR1, HCDR2 and HCDR3 selected from, for example, SEQ ID NO:3, SEQ ID NO:95 and SEQ ID NO:5, respectively HCDR variant having 3, 2, 1 or 0 amino acid mutations; or
  • the antibody light chain variable region comprises 3, 2, respectively, 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. 1 or 0 amino acid mutated LCDR variant; antibody heavy chain variable region comprising selected from HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 11, SEQ ID NO: 97 and SEQ ID NO: HCDR variants with 3, 2, 1 or 0 amino acid mutations, respectively.
  • an anti-CSF-1R antibody or antigen-binding fragment thereof comprising an antibody light chain variable region and an antibody heavy chain variable region, wherein:
  • the antibody light chain variable region of p comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 42, SEQ ID NO: 43 and SEQ ID NO: 44, respectively;
  • the antibody heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 39, SEQ ID NO: 40 and SEQ ID NO: 41, respectively;
  • the antibody light chain variable region comprises LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO: 50, SEQ ID NO: 51 and SEQ ID NO: 52, respectively;
  • the antibody heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 47, SEQ ID NO: 48 and SEQ ID NO: 49, respectively;
  • the antibody light chain variable region comprising LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO:58, SEQ ID NO:59 and SEQ ID NO:60, respectively; said antibody heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 55, SEQ ID NO: 56 and SEQ ID NO: 57, respectively;
  • the antibody light chain variable region of s comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 66, SEQ ID NO: 67 and SEQ ID NO: 68, respectively;
  • the antibody heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 63, SEQ ID NO: 64 and SEQ ID NO: 65, respectively;
  • the antibody light chain variable region comprises LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO: 74, SEQ ID NO: 75 and SEQ ID NO: 76, respectively;
  • the antibody heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 71, SEQ ID NO: 72 and SEQ ID NO: 73, respectively;
  • the antibody light chain variable region comprising LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO: 82, SEQ ID NO: 83 and SEQ ID NO: 84, respectively; said antibody heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 79, SEQ ID NO: 80 and SEQ ID NO: 81, respectively;
  • the antibody light chain variable region comprises LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO: 90, SEQ ID NO: 91 and SEQ ID NO: 92, respectively;
  • the antibody heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 87, SEQ ID NO: 88 and SEQ ID NO: 89, respectively;
  • the antibody light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, respectively;
  • the antibody heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, respectively;
  • the antibody light chain variable region comprises LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, respectively;
  • the antibody heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13, respectively;
  • the antibody light chain variable region comprises LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, respectively;
  • the antibody heavy chain variable region comprises: HCDR1, HCDR2 and HCDR3 set forth in SEQ ID NO: 98, SEQ ID NO: 12 and SEQ ID NO: 13;
  • the antibody light chain variable region comprises 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 comprises, for example, SEQ ID NO: 98, SEQ ID NO: 97 and SEQ ID NO: 13 HCDR1, HCDR2 and HCDR3;
  • the antibody light chain variable region of aa 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 variable region comprises: HCDR1, HCDR2 and HCDR3 set forth in SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5;
  • the antibody light chain variable region of ab comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively;
  • the antibody heavy chain variable region comprises: HCDR1, HCDR2 and HCDR3 set forth in SEQ ID NO: 3, SEQ ID NO: 95 and SEQ ID NO: 5;
  • the antibody light chain variable region of ac 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 variable region comprises: HCDR1, HCDR2 and HCDR3 set forth in SEQ ID NO: 3, SEQ ID NO: 95 and SEQ ID NO: 5;
  • the antibody light chain variable region comprises 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 comprises : HCDR1, HCDR2 and HCDR3 of SEQ ID NO: 11, SEQ ID NO: 97 and SEQ ID NO: 13.
  • 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.
  • amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 38, and the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 37;
  • amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 62
  • amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 61;
  • amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 70
  • amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 69;
  • amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 86, and the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 85;
  • the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 2 or a variant thereof, preferably having a 0-10 amino acid mutation in the light chain variable region, the most preferred amino acid mutation being N37T
  • the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 1 or a variant thereof, and the variant preferably has a 0-10 amino acid mutation in the heavy chain variable region, and the preferred amino acid mutation is N55T ;
  • the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 10
  • the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 9 or a variant thereof, preferably
  • the light chain variable region has an amino acid mutation of 0-10, and the preferred mutation is an amino acid mutation of M34I, N55T or a combination thereof.
  • 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.
  • the anti-CSF-1R antibody or antigen-binding fragment thereof as described above, wherein the light chain FR region sequence on the humanized antibody light chain variable region is derived from SEQ ID NO: Human germline light chain IGkV4-1 sequence; or human germline light chain IGkV1-13 sequence as set forth in SEQ ID NO: 24;
  • the humanized antibody heavy chain variable region further comprises a heavy chain FR region of human IgG1, IgG2, IgG3 or IgG4 or variants thereof, preferably comprising a human IgG1, IgG2 or IgG4 heavy chain FR region, More preferably comprising a human IgG1 or IgG4 heavy chain FR region; preferably, the heavy chain FR region sequence on the humanized antibody heavy chain variable region is derived from the human germline as set forth in SEQ ID NO:21.
  • the anti-CSF-1R antibody or antigen-binding fragment thereof as described above, wherein the humanized antibody light chain variable region sequence is set forth in SEQ ID NO: 30 or SEQ ID NO: 34 a sequence or variant thereof; said variant preferably having a 0-10 amino acid mutation in the light chain variable region, wherein the most preferred amino acid mutation of SEQ ID NO: 30 is S31N, T37N; SEQ ID NO: 34 is most preferred
  • the amino acid mutation is F87A, F91Y or a combination thereof;
  • the humanized antibody heavy chain variable region sequence is the sequence set forth in SEQ ID NO:26, SEQ ID NO:100 or SEQ ID NO:33 or a variant thereof;
  • the heavy chain variable region has a 0-10 amino acid mutation, 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 preferred amino acid mutation For Q1E, M34I, T55N, E89D or a combination thereof, the most preferred amino acid mutation is an amino acid mutation of M34I, T55N or a combination thereof;
  • 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;
  • 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 region sequence is set forth in SEQ ID NO: 102
  • the heavy chain constant region sequence is set forth in SEQ ID NO: 101.
  • a heavy chain comprising the sequence of SEQ ID NO: 17 and a light chain of the sequence SEQ ID NO: 18; or a heavy chain of the sequence SEQ ID NO: 99, and a sequence of SEQ ID NO: The light chain shown in 20.
  • the murine antibody or fragment thereof, as described above, wherein the antibody light chain variable region further comprises a light chain FR region of a murine kappa, lambda chain or variant thereof.
  • the murine antibody or fragment thereof as described above, further comprises a light chain constant region of a murine kappa, lambda chain or variant thereof.
  • the murine antibody or fragment thereof further comprises an antibody heavy chain variable region comprising a heavy chain FR region of murine IgGl, IgG2, IgG3, IgG4 or variants thereof.
  • the murine antibody or fragment thereof as described above, further comprises a heavy chain constant region of murine IgGl, IgG2, IgG3, IgG4 or variants thereof.
  • the anti-CSF-1R antibody or antigen-binding fragment thereof as described above, wherein the antibody is a chimeric antibody or a fragment thereof.
  • the variant has a 0-10 amino acid change in the light chain variable region, and most preferably the amino acid mutation in SEQ ID NO: 2 is N37T.
  • the CSF-1R chimeric antibody or fragment thereof as described above, wherein the chimeric antibody heavy chain variable region sequence is: SEQ ID NO: 1 or SEQ ID NO: 9, wherein SEQ The preferred amino acid mutation of ID NO: 1 is T55N; the preferred mutation of SEQ ID NO: 9 is an amino acid mutation of M34I, T55N or a combination thereof.
  • the CSF-1R chimeric antibody or fragment thereof as described above, further comprises a heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4 or variants thereof.
  • the anti-CSF-1R antibody or antigen-binding fragment thereof as described above, wherein the antibody is a humanized antibody or antigen-binding fragment thereof.
  • a CSF-1R humanized antibody or fragment thereof as described above, wherein the humanized antibody light chain variable region further comprises a light chain FR region of a human kappa, lambda chain or variant thereof.
  • a CSF-1R humanized antibody or fragment thereof wherein the humanized antibody light chain variable region sequence is SEQ ID NO: 30 or SEQ ID NO: sequence shown by 34, or a variant thereof.
  • the CSF-1R humanized antibody or fragment thereof as described above, wherein the humanized antibody light chain sequence is the sequence set forth in SEQ ID NO: 18 or SEQ ID NO: Or a variant thereof; the humanized antibody variant preferably has an amino acid change of 0-10 in the light chain variable region; wherein the preferred amino acid mutation of SEQ ID NO: 18 is an amino acid mutation of S31N, T37N or a combination thereof, most A preferred amino acid mutation is T37N; a preferred amino acid mutation of SEQ ID NO: 20 is an amino acid mutation of F87A, F91Y, or a combination thereof.
  • a CSF-1R humanized 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, preferably comprising human IgG1, IgG2 or IgG4 heavy chain constant region. More preferably, IgG1 which eliminates the toxicity of antibody-dependent cell-mediated cytotoxicity (antibody-dependent cell-mediated cytotoxicity) after IgG4 or amino acid mutation is used.
  • some embodiments of the invention also provide an isolated monoclonal antibody or antigen-binding fragment thereof that competes for binding to CSF-1R with an anti-CSF-1R antibody or antigen-binding fragment thereof as described above.
  • an antibody as described above (the antibody described herein below and the anti-CSF-1R antibody or the above monoclonal antibody) or an antigen-binding fragment thereof, wherein the antigen
  • the binding fragments are Fab, Fv, sFv, F(ab')2, linear antibodies, single chain antibodies, Nanobodies, domain antibodies and multispecific antibodies.
  • some embodiments of the invention also provide a multispecific antibody comprising a light chain variable region and/or a heavy chain variable region of an antibody or antigen binding fragment thereof as described above.
  • some embodiments of the invention further provide a single chain antibody comprising a light chain variable region and/or a heavy chain variable region of an antibody or antigen binding fragment thereof as described above.
  • 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 of the antibody or antigen-binding fragment thereof as described above Area.
  • some embodiments of the invention further provide a nucleic acid molecule encoding an antibody or antigen-binding fragment thereof as described above, a multispecific antibody as described above, or a single chain antibody as described above.
  • some embodiments of the invention also provide a DNA sequence encoding an antibody or antigen-binding fragment thereof as described above.
  • some embodiments of the invention also provide an expression vector comprising a DNA sequence as described above.
  • Also provided in some embodiments of the invention is a host cell transformed with an expression vector as described above.
  • a host cell as described above wherein the host cell is a bacterium, preferably E. coli.
  • a host cell as described above is a yeast, preferably Pichia pastoris.
  • 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.
  • CHO Chinese hamster ovary
  • HEK human embryonic kidney
  • some embodiments of the invention also provide an antibody-drug conjugate comprising a light chain variable region and a heavy chain variable region as previously described.
  • the antibody-drug conjugates are well known in the art and are formed by the interconnection of antibody-linker-drug (toxin), and known linkers include cleavage linkers, split cleavage linkers, such as linkers including, but not limited to, SMCC, SPDP Etc.; Toxins are also well known in the art, such as DM1, DM4, MMAE, MMAF, and the like.
  • some embodiments of the invention also provide a pharmaceutical composition
  • a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof as described above and a pharmaceutically acceptable buffer, excipient, diluent or carrier.
  • an antibody, or a fragment thereof, a multispecific antibody, a single chain antibody, a nucleic acid molecule, or a pharmaceutical composition, as described above is administered in the treatment of cancer, wherein the administration is at the beginning of another
  • the treatment of anticancer therapy is administered before, during, substantially simultaneously or after.
  • a CSF-1R humanized antibody or fragment thereof as described above, wherein the anti-cancer treatment is selected from the group consisting of an anti-angiogenic agent, a chemotherapeutic agent, radiation, tumor immunotherapy, or a combination thereof.
  • a CSF-1R humanized antibody or fragment thereof as described above, wherein the chemotherapeutic agent is selected from the group consisting of: docetaxel (palitax, docetaxel, modified pa Lithoace (Abraxane and Opaxio)), doxorubicin, modified doxorubicin (Caelyx or Doxil), sunitinib, sorafenib and other multi-kinase inhibitors, oxaliplatin, cis Platinum, carboplatin, etoposide, gemcitabine and vinblastine.
  • docetaxel palitax, docetaxel, modified pa Lithoace (Abraxane and Opaxio)
  • doxorubicin modified doxorubicin
  • sunitinib sunitinib
  • sorafenib sunitinib
  • oxaliplatin cis Platinum
  • carboplatin etoposide
  • gemcitabine and vinblastine.
  • a CSF-1R humanized antibody or fragment thereof as described above, wherein said tumor immunotherapy is selected from the group consisting of: a T cell engaging agent, a targeted immunosuppression, a cancer vaccine / Enhances dendritic cell function and adoptive cell transfer.
  • some embodiments of the invention further provide the use of an antibody, or antigen-binding fragment thereof, as described above, in the manufacture of a medicament for the treatment of a CSF-1R or CSF-1 mediated disease or condition;
  • 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.
  • autoimmune encephalomyelitis systemic lupus erythematosus, multiple sclerosis, joint synovitis, psoriasis, and rheumatoid arthritis
  • osteolytic bone loss is selected from the group consisting of osteoporosis, metastasis-induced dissolution Bone bone Deletions and rheumatoid arthritis-induced bone loss.
  • the invention further provides a method of treating and preventing a CSF-1R or CSF-1 mediated disease or condition, comprising administering to a patient in need thereof a therapeutically effective amount of an antibody or antigen-binding fragment thereof as described above, or comprising the same
  • 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, filtration Follicular lymphoma, renal cell carcinoma, uveal melanoma, cervical cancer, head and neck cancer, Hodgkin's disease, astrocytoma, lung adenocarcinoma, mesothelioma, choriocarcinoma, melanoma, breast cancer, ovary 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
  • Figure 1 ELISA in vitro binding assay of nine CSF-1R chimeric antibodies showed that all the antibodies except C27C had strong cross-binding with cynomolgus CSF-1R protein.
  • Figure 2 In vitro binding activity assay of humanized antibody and CHO-1R expressing CHO cells in vitro.
  • the humanized antibodies huC11 and huC19 were comparable in cell binding strength to their original chimeric antibodies C11C and C19C, respectively.
  • FIG. 3 Functional experiment of humanized antibody huC11 blocking monocyte proliferation.
  • the antibody blocks IL-34 stimulated monocyte proliferation.
  • the antibody blocks CSF-1 stimulated monocyte proliferation.
  • Figure 4 Functional experiment of humanized antibody huC19 blocking monocyte proliferation.
  • the antibody blocks IL-34 stimulated monocyte proliferation.
  • the antibody blocks CSF-1 stimulated monocyte proliferation.
  • Figure 5 In vivo pharmacodynamic experiment of humanized antibody huC19I.
  • the antibody inhibits the growth of MDA-MB-231 tumor cells.
  • antibody refers to an immunoglobulin which is a tetrapeptide chain structure in which two identical heavy chains and two identical light chains are linked by interchain disulfide bonds.
  • the immunoglobulin heavy chain constant region has different amino acid composition and arrangement order, so its antigenicity is also different. Accordingly, immunoglobulins can be classified into five classes, or isoforms of immunoglobulins, namely IgM, IgD, IgG, IgA and IgE, and the corresponding heavy chains are ⁇ chain, ⁇ chain, ⁇ chain, respectively. , ⁇ chain and ⁇ chain.
  • IgG can be classified into IgG1, IgG2, IgG3, and IgG4.
  • Light chains are classified as either a kappa chain or a lambda chain by the constant region.
  • Each of the five types of Ig may have a kappa chain or a lambda chain.
  • the antibody light chain variable region of the present invention may further comprise a light chain constant region comprising a human or murine kappa, lambda chain or a variant thereof.
  • the antibody heavy chain variable region of the present invention may further comprise a heavy chain constant region comprising human or murine IgG1, 2, 3, 4 or a variant thereof.
  • variable region The sequence of about 110 amino acids near the N-terminus of the antibody heavy and light chains varies greatly, being the variable region (V region); the remaining amino acid sequence near the C-terminus is relatively stable and is a constant region (C region).
  • the variable region includes three hypervariable regions (HVR) and four relatively conserved framework regions (FR). The three hypervariable regions determine the specificity of the antibody, also known as the complementarity determining region (CDR).
  • Each of the light chain variable region (VL) and the heavy chain variable region (VH) consists of three CDR regions and four FR regions, and the order from the amino terminus to the carboxy terminus is: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the three CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3.
  • the CDR amino acid residues of the VL and VH regions of the antibodies or antigen-binding fragments of the invention meet the known IMGT numbering rules in number and position.
  • APC antigen presenting cell
  • T cells recognize this complex using the T cell receptor (TCR).
  • APCs include, but are not limited to, dendritic cells (DC), peripheral blood mononuclear cells (PBMC), monocytes, B lymphoblasts, and monocyte-derived dendritic cells (DC).
  • DC dendritic cells
  • PBMC peripheral blood mononuclear cells
  • monocytes B lymphoblasts
  • DC monocyte-derived dendritic cells
  • 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 MHC-I/MHC-II conjugates.
  • CSF-1R refers to a colony stimulating factor-1 receptor protein, a single-pass transmembrane receptor with a C-terminal intracellular domain with an N-terminal extracellular domain and tyrosine kinase activity.
  • CSF-1R is a member of the RTK family of immunoglobulin (Ig) motifs and is characterized by a repetitive Ig domain in the extracellular portion of the receptor.
  • Ig immunoglobulin
  • Activation of CSF-1R is mediated by its ligand M-CSF or interleukin IL-34, and its signaling has important physiological roles in immune response, bone remodeling, and in the reproductive system.
  • recombinant human antibody includes human antibodies which are prepared, expressed, created or isolated by recombinant methods, and the techniques and methods involved are well known in the art, such as (1) transgenes from human immunoglobulin genes, transgenic chromosomes.
  • Such recombinant human antibodies comprise variable regions and constant regions that utilize specific human germline immunoglobulin sequences encoded by germline genes, but also include subsequent rearrangements and mutations such as occur during antibody maturation.
  • murine antibody is in the present invention a monoclonal antibody to human CSF-1R prepared according to the knowledge and skill in the art.
  • the test subject is injected with the CSF-1R antigen at the time of preparation, and then the hybridoma expressing the antibody having the desired sequence or functional properties is isolated.
  • the murine CSF-1R antibody or antigen-binding fragment thereof may further comprise a light chain constant region of a murine kappa, a lambda chain or a variant thereof, or further comprising a murine IgG1 , heavy chain constant region of IgG2, IgG3 or IgG4 or a variant thereof.
  • human antibody includes antibodies having variable and constant regions of human germline immunoglobulin sequences.
  • Human antibodies of the invention can include amino acid residues that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo).
  • human antibody does not include an antibody in which a CDR sequence derived from a germline of another mammalian species, such as a mouse, has been grafted onto a human framework sequence (ie, "humanized antibody”).
  • humanized antibody also referred to as a CDR-grafted antibody, refers to an antibody produced by grafting a CDR sequence of a mouse into a human antibody variable region framework. It is possible to overcome the strong immune response induced by chimeric antibodies by carrying a large amount of mouse protein components. To avoid a decrease in activity while reducing immunogenicity, the human antibody variable region can be subjected to minimal reverse mutation to maintain activity.
  • chimeric antibody is an antibody obtained by fusing a variable region of a murine antibody with a constant region of a human antibody, and can alleviate an immune response induced by a murine antibody.
  • a hybridoma that secretes a murine-specific monoclonal antibody is selected, and then the variable region gene is cloned from the mouse hybridoma cell, and the constant region gene of the human antibody is cloned as needed to change the mouse.
  • the region gene and the human constant region gene are ligated into a chimeric gene and inserted into a human vector, and 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 region of human IgG1, IgG2, IgG3 or IgG4 or a variant thereof, preferably comprising a human IgG2 or IgG4 heavy chain constant region, or without ADCC (antibody-dependent) after amino acid mutation Cell-mediated cytotoxicity, antibody-dependent cell-mediated cytotoxicity) toxic IgG1.
  • antigen-binding fragment refers to an antigen-binding fragment of an antibody and an antibody analog, which typically includes at least a portion of an antigen binding or variable region (eg, one or more CDRs) of a parental antibody.
  • the antibody fragment retains at least some of the binding specificity of the parent antibody. Generally, when the activity is expressed on a molar basis, the antibody fragment retains at least 10% of the parental 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 to the target.
  • 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 comprising the CH1 and CH2 domains of the antibody.
  • the two heavy chain fragments are held together by two or more disulfide bonds and by the hydrophobic action of the CH3 domain.
  • a "Fab' fragment” contains a light chain and a portion of a heavy chain comprising a VH domain and a CH1 domain and a region between the CH1 and CH2 domains, thereby being between the two heavy chains of the two Fab' fragments An interchain disulfide bond is formed to form a F(ab')2 molecule.
  • the "F(ab')2 fragment” contains two light chains and two heavy chains comprising portions of the constant region between the CH1 and CH2 domains, thereby forming an interchain disulfide bond between the two heavy chains.
  • the F(ab')2 fragment consists of two Fab' fragments held together by a disulfide bond between the two heavy chains.
  • the "Fv region” contains variable regions from both heavy and light chains, but lacks a constant region.
  • multispecific antibody is used in its broadest sense to encompass antibodies having 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; having two or more Antibodies of the VL and VH regions, each VH-VL unit binding to a different target or a different epitope of the same target; antibodies having two or more single variable regions, each single variable region Different targets or different epitopes of the same target; full-length antibodies, antibody fragments, diabodies, bispecific diabodies and triabodies, covalently or non-covalently linked Along with antibody fragments and the like.
  • single-chain antibody is a single-stranded recombinant protein joined by a heavy chain variable region (VH) and a light chain variable region (VL) of a antibody via a ligation peptide, which is the smallest with complete antigen binding sites.
  • VH heavy chain variable region
  • VL light chain variable region
  • domain antibody fragment is an immunologically functional immunoglobulin fragment containing only a heavy chain variable region or a light chain variable region chain.
  • two or more VH regions are covalently joined 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.
  • binding to CSF-1R in the present invention means that it is capable of interacting with human CSF-1R.
  • antigen binding site refers to a three-dimensional spatial site that is discrete on an antigen and is recognized by an antibody or antigen-binding fragment of the present invention.
  • amino acid mutation in a "mutant with 3, 2, 1 or 0 amino acid” means that the amino acid is mutated in the variant protein or polypeptide compared to the original protein or polypeptide, including corresponding to the original protein or polypeptide. Insertion, deletion or substitution of a number of amino acids.
  • CDR variants having a 3, 2, 1 or 0 amino acid mutation, respectively, based on "CDR1, CDR2 and CDR3 as set forth in SEQ ID NO: X, SEQ ID NO: Y and SEQ ID NO: Z"
  • a mutation to a CDR may comprise a mutation of 3, 2, 1 or 0 amino acids, and optionally the same or a different number of amino acid residues may be selected between CDR1, CDR2 and CDR3.
  • Mutation is carried out, for example, on the basis of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: X, SEQ ID NO: Y and SEQ ID NO: Z, CDR1 is subjected to 1 amino acid mutation, and CDR2 and CDR3 are subjected to 0. Amino acid mutations. When a 0 amino acid mutation is made to a certain CDR, then the CDR variant with a 0 amino acid mutation remains the CDR itself.
  • epitope refers to a site on an antigen that specifically binds to an immunoglobulin or antibody.
  • An epitope can be formed by an adjacent amino acid, or a non-adjacent amino acid juxtaposed by a tertiary folding of a protein. Epitopes formed from adjacent amino acids are typically retained after exposure to denaturing solvents, while epitopes formed by tertiary folding are typically lost after treatment with denaturing solvents. Epitopes typically include at least 3-15 amino acids in a unique spatial conformation. Methods for determining what epitope is bound by a given antibody are well known in the art, including immunoblotting and immunoprecipitation assays, and the like. Methods for determining the spatial conformation of an epitope include techniques in the art and techniques described herein, such as X-ray crystallography and two-dimensional nuclear magnetic resonance.
  • the terms “specifically bind” and “selectively bind” refer to the binding of an antibody to an epitope on a predetermined antigen.
  • the antibody has an equilibrium solution of less than about 10 -7 M or even less when measured by surface plasmon resonance (SPR) techniques in an instrument.
  • the isolating constant (K D ) binds to a predetermined antigen, and its affinity for binding to a predetermined antigen is at least twice its affinity for binding to a non-specific antigen other than the predetermined antigen or a closely related antigen (such as BSA, etc.).
  • the term “antibody recognizing an antigen” can be used interchangeably herein with the term “specifically bound antibody”.
  • the term “competition” in the context of an antigen binding protein that competes for the same epitope (eg, neutralizing an antigen binding protein or a neutralizing antibody), it means competition between antigen binding proteins, which is determined by the following assay: In the assay, the antigen binding protein (eg, an antibody or antigen-binding fragment thereof) to be detected prevents or inhibits (eg, reduces) the specific binding of a reference antigen binding protein (eg, a ligand or reference antibody) to a common antigen.
  • a reference antigen binding protein eg, a ligand or reference antibody
  • RIA solid phase direct or indirect radioimmunoassay
  • EIA solid phase direct or indirect enzyme immunoassay
  • Sandwich competition assay see, eg, Stahli et al, 1983, Methods in Enzymology 9: 242-253
  • solid phase direct biotin-avidin EIA see, eg, Kirkland et al, 1986, J. Immunol.
  • solid Direct labeling assay solid phase direct label sandwich assay (see, eg, Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press); solid phase direct labeling with I-125 label RIA (see, eg, Morel et al, 1988, Molec. Immunol. 25: 7-15); solid phase direct biotin-avidin EIA (see, eg, Cheung, et al, 1990, Virology 176: 546-552); and directly labeled RIA (Moldenhauer et al, 1990, Scand. J. Immunol. 32: 77-82).
  • the assay involves the use of a purified antigen (either on a solid surface or on the cell surface) that binds to a reference antigen binding protein with an unlabeled detection antigen binding protein and label.
  • Competitive inhibition is measured by measuring the amount of label bound to a solid surface or cell in the presence of the antigen binding protein to be tested.
  • the antigen binding protein to be tested is present in excess.
  • An antigen binding protein identified by a competitive assay comprises: an antigen binding protein that binds to the same epitope as the reference antigen binding protein; and an antigen binding that binds to a neighboring epitope that is sufficiently close to the binding epitope of the reference antigen binding protein.
  • a competing antigen binding protein when present in excess, it will inhibit (eg, reduce) at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70. -75% or 75% or more of the specific binding of the reference antigen binding protein to the common antigen. In some cases, binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97%, or 97% or more.
  • cross-reactive refers to the ability of an antibody of the invention to bind to CSF-1R from a different species.
  • an antibody of the invention that binds to human CSF-1R can also bind to another species of CSF-1R.
  • Cross-reactivity is measured by detecting specific reactivity with purified antigens in binding assays (eg, SPR and ELISA), or binding or functional interactions with cells that physiologically express CSF-1R.
  • binding assays eg, SPR and ELISA
  • Methods for determining cross-reactivity include standard binding assays as described herein, such as surface plasmon resonance (SPR) analysis, or flow cytometry.
  • SPR surface plasmon resonance
  • host cell refers to a cell into which an expression vector has been introduced.
  • Host cells can include bacterial, microbial, plant or animal cells.
  • Bacteria susceptible to transformation 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 (not limited to HEK293E cells) and NSO cells.
  • inhibiting or “blocking” are used interchangeably and encompass both partial and complete inhibition/blocking.
  • the inhibition/blocking of the ligand preferably reduces or alters the normal level or type of activity that occurs when ligand binding occurs without inhibition or blockade.
  • Inhibition and blockade are also intended to include a decrease in any measurable ligand binding affinity when contacted with an anti-CSF-1R antibody, compared to a ligand not contacted with an anti-CSF-1R antibody.
  • inhibiting growth eg, involving cells
  • inhibiting growth is intended to include any measurable reduction in cell growth.
  • inducing an immune response and “enhancing an immune response” are used interchangeably and refer to the stimulation (ie, passive or adaptive) of an immune response to a particular antigen.
  • inducing for inducing CDC or ADCC refers to stimulating a specific direct cell killing mechanism.
  • the "ADCC” described in the present invention is an antibody-dependent cell-mediated cytotoxicity, which means that a cell expressing an Fc receptor directly kills an antibody by Fc segment of the recognition antibody.
  • Target cells The ADCC effector function of the antibody can be reduced or eliminated by modification of the Fc fragment on IgG. Said modification refers to mutations in the heavy chain constant region of the antibody.
  • a mouse can be immunized with human CSF-1R or a fragment thereof, and the obtained antibody can be renatured, purified, and subjected to amino acid sequencing by a conventional method.
  • the antigen-binding fragment can also be prepared by a conventional method.
  • the antibodies or antigen-binding fragments of the invention are genetically engineered to add one or more human FR regions in a non-human CDR region. Human FR germline sequences are available on the website of ImMunoGeneTics (IMGT) http://imgt.cines.fr or from the Journal of Immunoglobulins, 2001 ISBN 014441351.
  • the engineered antibodies or antigen-binding fragments of the invention can be prepared and purified by conventional methods.
  • the cDNA sequence of the corresponding antibody can be cloned and recombined into a GS expression vector.
  • the recombinant immunoglobulin expression vector can stably transfect CHO cells.
  • mammalian expression systems result in glycosylation of antibodies, particularly at the highly conserved N-terminus of the Fc region.
  • Stable clones are obtained by expressing antibodies that specifically bind to human antigens. Positive clones were expanded in serum-free medium in a bioreactor to produce antibodies.
  • the culture medium from which the antibody is secreted can be purified and collected by a conventional technique.
  • the antibody can be concentrated by filtration in a conventional manner. Soluble mixtures and multimers can also be removed by conventional methods such as molecular sieves, ion exchange. The resulting product needs to be frozen immediately, such as -70 ° C, or lyophilized.
  • the antibody of the present invention refers to a monoclonal antibody.
  • the monoclonal antibody (mAb) of the present invention refers to an antibody obtained from a single clonal cell strain, and the cell strain is not limited to a eukaryotic, prokaryotic or phage clonal cell strain.
  • Monoclonal antibodies or antigen-binding fragments can be obtained recombinantly using, for example, hybridoma technology, recombinant techniques, phage display technology, synthetic techniques (e.g., CDR-grafting), or other prior art techniques.
  • administering when applied to an animal, human, experimental subject, cell, tissue, organ or biological fluid, refers to an exogenous drug, therapeutic agent, diagnostic agent or composition and animal, human, subject Contact of the test subject, cell, tissue, organ or biological fluid.
  • administering can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods.
  • Treatment of the cells includes contact of the reagents with the cells, and contact of the reagents with the fluid, wherein the fluids are in contact with the cells.
  • administeristering and “treating” also means treating, for example, cells in vitro and ex vivo by reagents, diagnostics, binding compositions, or by another cell.
  • Treatment when applied to a human, veterinary or research subject, refers to therapeutic treatment, prophylactic or preventive measures, research and diagnostic applications.
  • Treatment means administering to a patient a therapeutic agent for internal or external use, such as a composition comprising any of the binding compounds of the present invention, the patient having one or more symptoms of the disease, and the therapeutic agent is known to have Therapeutic effect.
  • a therapeutic agent is administered in a subject or population to be treated to effectively alleviate the symptoms of one or more diseases, whether by inducing such symptoms to degenerate or inhibiting the progression of such symptoms to any degree of clinical right measurement.
  • the amount of therapeutic agent also referred to as "therapeutically effective amount” effective to alleviate 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 a desired effect in the patient.
  • Whether the symptoms of the disease have been alleviated can be assessed by any clinical test method commonly used by a physician or other professional health care provider to assess the severity or progression of the condition.
  • Embodiments of the invention e.g., methods of treatment or preparations
  • a binding compound consisting essentially of the amino acid sequence recited may also include one or more amino acids that do not significantly affect the properties of the binding compound.
  • naturally occurring refers to the fact that the object can be found in nature.
  • a polypeptide sequence or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a natural source and that has not been intentionally modified in the laboratory by humans is naturally occurring.
  • an "effective amount” includes an amount sufficient to ameliorate or prevent a symptom or condition of a medical condition.
  • An effective amount also means an amount sufficient to allow or facilitate the diagnosis.
  • An effective amount for a particular patient or veterinary subject can vary depending on factors such as the condition to be treated, the overall health of the patient, the route and dosage of the method of administration, and the severity of the side effects.
  • An effective amount can be the maximum dose or dosing regimen that avoids significant side effects or toxic effects.
  • Exogenous refers to a substance that is produced outside of a living organism, cell, or human body depending on the background.
  • Endogenous refers to a substance produced in a cell, organism or human body depending on the background.
  • “Homology” refers to sequence similarity between two polynucleotide sequences or between two polypeptides. When positions in both comparison sequences are 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 molecule is homologous at that position .
  • the percent homology between the two sequences is a function of the number of matches or homology positions shared by the two sequences divided by the number of positions compared ⁇ 100%. For example, in the optimal alignment of sequences, if there are 6 matches or homologs in 10 positions in the two sequences, then the two sequences are 60% homologous. In general, comparisons are made when the maximum sequence of homology is obtained by aligning the two sequences.
  • “Pharmaceutical composition” means a mixture comprising one or more of the compounds described herein, or a physiologically/pharmaceutically acceptable salt or prodrug thereof, and other chemical components, as well as other components such as physiological/pharmaceutically acceptable carriers. And excipients.
  • the purpose of the pharmaceutical composition is to promote the administration of the organism, thereby facilitating the absorption of the active ingredient and thereby exerting biological activity.
  • the present invention provides anti-CSF-1R antibodies with high affinity, high selectivity, high biological activity, monoclonal antibody immunotherapy for tumor or autoimmune diseases and related applications, and other related CSF-1R positive tumors or Drugs, compositions and methods for the treatment of autoimmune diseases.
  • the human CSF-1R sequence encoding the His-Tag tag and the human CSF-1R sequence encoding the huFc tag were synthesized by Integrated DNA Technology (IDT) and cloned into the pTT5 vector (Biovector), respectively.
  • Recombinant CSF-1R protein was purified after expression in 293T cells (ATCC, CRL-3216 TM) .
  • the purified protein can be used in the experiments of the following examples.
  • the supernatant sample expressed by HEK293 cells was centrifuged at high speed to remove impurities, and the buffer was exchanged for phosphate buffer (PBS), and imidazole was added to a final concentration of 5 mM.
  • PBS phosphate buffer
  • the nickel column was equilibrated with PBS solution containing 5 mM imidazole and rinsed 2-5 column volumes. The displaced supernatant sample was applied to the column.
  • the column was washed with PBS containing 5 mM imidazole until the A280 reading dropped to baseline.
  • the column was washed with PBS + 10 mM imidazole, the non-specifically bound heteroprotein was removed, and the effluent was collected.
  • the protein of interest was eluted with PBS containing 300 mM imidazole, and the eluted peak was collected.
  • the collected eluate was further purified by ion exchange (SP column).
  • Configure solution A 0.01 M PB, pH 8.0.
  • Configure liquid B liquid A + 1 M NaCl.
  • the imidazole in PBS solution was eluted to the A solution, and the SP column was equilibrated with the A solution.
  • the concentration of the B solution was 0-100%, and 10 column volumes were eluted to collect the elution peaks.
  • the obtained protein was subjected to electrophoresis, peptide mapping, and identified by liquid chromatography-mass spectrometry (LC-MS).
  • LC-MS liquid chromatography-mass spectrometry
  • the supernatant sample expressed by HEK293 cells was centrifuged at high speed to remove impurities, and the buffer was exchanged for PBS.
  • the Protein A affinity column was equilibrated with 10 mM phosphate buffer and rinsed 2-5 column volumes. The displaced supernatant sample was applied to the column. Rinse the column with 25 column volumes of buffer until the A280 reading drops to baseline.
  • the target protein was eluted with 0.8% acetate buffer at pH 3.5, and the elution peak was collected. Immediately after the addition, the mixture was neutralized with 1 M Tris-Cl pH 8.0 buffer, and then the solution was replaced with Millipore's Amico-15 filter column. pH 6.9.
  • the obtained protein was electrophoresed, peptide map, and identified by LC-MS.
  • huCSF-1R human CSF-1R protein
  • IDT Integrated DNA Technology
  • pcDNA3.1/puro Invitrogen #V79020
  • CHO-S (ATCC) cells were cultured to 0.5 x 10 6 /ml in CD-CHO medium (Life Technologies, #10743029). 10 ⁇ g of vector encoding huCSF-1R or gene was mixed with 50 ul of LF-LTX (Life Technologies, #A12621) in 1 ml Opti-MEM medium (Life Technologies, #31985088), and incubated for 20 minutes at room temperature, then CHO cell culture solution was added.
  • Cynomolgus monkey CSF-1R-His (cynoCSF-1R-His) and mouse CSF-1R-His protein were purchased from AcroBiosciences.
  • Anti-human CSF-1R monoclonal antibodies are produced by immunizing mice.
  • the experiment used Swiss Webster white mice, female, 6 weeks old (Charles River). Feeding environment: SPF level. After the mice were purchased, the laboratory environment was kept for 1 week, 12/12 hours light/dark cycle adjustment, temperature 20-25 ° C; humidity 40-60%.
  • the immunizing antigen is an Fc-tagged human CSF-1R recombinant protein (huCSF-1R-Fc).
  • Titermax Sigma Lot Num: T2684
  • Titermax was used as an adjuvant.
  • the ratio of antigen to adjuvant (titermax) was 1:1, and the antigen was emulsified and inoculated for 0, 21, 35, 49, and 63 days.
  • IP intraperitoneal
  • IP intraperitoneal
  • ELISA enzyme linked immunosorbent assay
  • FACS fluorescence activated cell sorter
  • mice with high antibody titers in serum and titers tended to plate and spleen lymphocytes and myeloma cells Sp2/0 cells were optimized using an electrofusion procedure ( CRL-8287 (TM ) was fused to obtain hybridoma cells.
  • the culture supernatant was taken, and as described in Example 4, the hybridoma supernatant was subjected to antibody screening using CSF-1R recombinant protein.
  • the obtained positive antibody strain was further subjected to CHO-S cells stably expressing CSF-1R, and the blank CHO-S cells were compared to exclude non-specific binding antibody hybridoma strains, and screened by a flow sorting method to determine about 400 strains.
  • Example 5 using CSF-1R stable cell-based CSF-1 ligand blocking function experimental screening, about 50 hybridomas with blocker function were identified and subcloned, then collected and purified. The antibody was screened for IL-34 ligand blocking function based on CSF-1R stable cells, and 11 subclones of antibody having CSF-1 and IL-34 double blocking function were identified. Logarithmic growth phase were collected and subcloned hybridoma cells, RNA was extracted with Trizol (Invitrogen, 15596-018) and reverse transcription (PrimeScript TM Reverse Transcriptase, Takara # 2680A).
  • the cDNA obtained by reverse transcription was amplified by PCR amplification using mouse Ig-Primer Set (Novagen, TB326 Rev. B 0503), and the sequence of C11, C19, C6 of nine mouse antibodies was finally obtained after excluding the CDR region repeats. , C8, C16, C18, C23, C27 and C30.
  • the heavy and light chain variable region sequences of murine mAb C11 are as follows:
  • the heavy and light chain variable region sequences of C19 are as follows:
  • the heavy and light chain variable region sequences of murine mAb C6 are as follows:
  • the heavy and light chain variable region sequences of murine mAb C8 are as follows:
  • the heavy and light chain variable region sequences of murine mAb C16 are as follows:
  • the heavy and light chain variable region sequences of murine mAb C18 are as follows:
  • the heavy and light chain variable region sequences of murine mAb C23 are as follows:
  • the heavy and light chain variable region sequences of murine mAb C27 are as follows:
  • the heavy and light chain variable region sequences of murine mAb C30 are as follows:
  • each mouse antibody was cloned into pTT vector plasmid (Biovector) containing human IgG4 heavy chain constant region and kappa light chain constant region, respectively, and then transiently transfected into HEK293 cells to obtain anti-resistant.
  • pTT vector plasmid Biovector
  • Each chimeric antibody (C11C, C19C, C6C, C8C, C16C, C18C, C23C, C27C and C30C) of CSF-1R was purified, identified, and correlated as described in Example 2 (purification with Fc-tagged protein) Binding and functional activity detection.
  • the neutralizing avidin for binding to biotin was diluted to 1 ⁇ g/ml with PBS pH 7.4 buffer, added to a 96-well plate in a volume of 100 ⁇ l/well, and left at 4 ° C for 16 h to 20 h. After washing the plate once with PBST (pH 7.4 PBS containing 0.05% Tween-20), 120 ⁇ l/well of PBST/1% skim milk was added and incubated for 1 h at room temperature for blocking.
  • PBST pH 7.4 PBS containing 0.05% Tween-20
  • the reaction system was removed, and the plate was washed three times with PBST, and then diluted with horseradish peroxidase (HRP)-labeled anti-mouse antibody secondary antibody or anti-human antibody II with PBST/1% skim milk at 100 ⁇ l/well. Anti- (The Jackson Laboratory), incubated for 1 h at room temperature. After washing the plate 3 times with PBST, 100 ⁇ l/well of TMB was added and incubated for 5-10 min at room temperature. The reaction was stopped by the addition of 100 ⁇ l/well of 1 M H 2 SO 4 , and the absorbance was read at 450 nm, and the ELISA binding EC 50 value was calculated.
  • HRP horseradish peroxidase
  • CHO-S cells highly expressing huCSF-1R were centrifuged at 1000 rpm for 5 minutes, and the precipitate was collected and suspended in 10-15 ml of pre-cooled flow buffer, and counted.
  • the cells were collected by centrifugation at 1000 rpm for 5 minutes in a 50 ml centrifuge tube, the supernatant was discarded, and the pellet was resuspended in a pre-cooled blocking buffer at a density of 0.5-1.0 x 10 7 cells/ml. After incubating for 30 minutes at 4 ° C, it was resuspended to 100 ⁇ l per well and added to a 96-well plate.
  • a concentration gradient of anti-CSF-1R antibody (0.01 nM-670 nM) was co-incubated with CSF-1R/CHO cells, and then biotinylated CSF-1 protein or APC-tagged IL-34 protein was incubated for binding. Among them, the experiment with CSF-1 was further added to the streptavidin-PE tag. After washing with PBS, the above cells were assayed for fluorescence signal intensity corresponding to CSF-1 or IL-34 using flow cytometry.
  • Anti-CSF-1R antibody inhibits proliferation of human monocytes induced by CSF-1 or IL-34
  • PBMC Peripheral blood mononuclear cells in healthy human blood were isolated and extracted using Ficoll-Paque gradient separation kit (GE Healthcare Bio-Sciences), and purified by Percoll kit (GE Healthcare Bio-Sciences). Mononuclear cells in PBMC were extracted. The isolated monocytes were then stimulated in a blank antibody-free or concentration-concentrated anti-CSF-1R antibody using human CSF-1 or IL-34 (R&D Systems), and cultured in a carbon dioxide incubator at 37 ° C for 3 days. The ATP content in each culture sample to be tested was measured using the CellTiter-Glo kit (Promega), and the corresponding proliferation level of monocytes was finally determined based on its linear positive correlation with the number of activated monocytes.
  • the chimeric antibody blocking function data is shown in the following table.
  • Nine antibodies can effectively block the proliferation of monocytes stimulated by CSF-1 or IL-34.
  • the chimeric antibodies such as C11C, C19C, C23C and C27C have very low IC50.
  • the blocking effect is comparable or superior to the reference antibody FPA008.
  • This experiment was measured by surface plasmon resonance (SPR) method.
  • the anti-human IgG polyclonal antibody is covalently linked to a CM5 (GE) chip using a standard amino coupling method using a kit supplied by Biacore, and then the chimeric antibody or humanized antibody of the present invention to be tested is used with the antibody.
  • the antibody is captured to the stationary phase.
  • the h-CSF-1R-His protein (Example 1) having a concentration gradient of 25-800 nM diluted in the same buffer was injected at each cycle before and after the injection, and was regenerated by the regeneration reagent in the kit. Antigen-antibody binding kinetics were followed for 3 minutes and dissociation kinetics were followed for 10 minutes.
  • Humanization of murine anti-human CSF-1R monoclonal antibodies was performed as disclosed in many literatures in the art. Briefly, the human constant domain is used in place of the parental (murine antibody) constant domain, and the human antibody sequence is selected based on the homology of the murine antibody and the human antibody.
  • the present invention combines functional activity with sequence specificity and drug-forming properties. The optimal two candidate molecules C11 and C19 determined by various evaluations were humanized.
  • the heavy and light chain variable region sequences were compared with the human antibody germline database to obtain a human germline template with high homology.
  • the human germline light chain framework region is derived from a human kappa light chain gene
  • 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 is preferably a human germline heavy chain template IGHV 1-46 (SEQ ID NO: 21):
  • C11 is preferably a human germline light chain template IGkV4-1 (SEQ ID NO: 22):
  • C19 is preferably a human germline heavy chain template IGHV1-2 (SEQ ID NO: 23):
  • C19 is preferably a human germline light chain template IGkV1-13 (SEQ ID NO: 24):
  • the CDR regions of the murine antibodies C11 and C19 are grafted onto the selected corresponding humanized template, the humanized variable region is replaced, and then recombined with the IgG constant region (preferably the heavy chain is IgG4 and the light chain is ⁇ ).
  • a cDNA fragment was synthesized based on the amino acid sequences of the above humanized antibody light and heavy chains, and inserted into a pcDNA3.1 expression vector (Life Technologies Cat. No. V790-20).
  • the expression vector and the 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 placed in a CO 2 incubator for incubation 4- 5 days. After the expressed antibody was recovered by centrifugation, antibody purification was carried out by the method of Example 2 (purification with Fc-tag protein) to obtain a humanized antibody protein of the present invention.
  • the final humanized huC11 using the VH-b heavy chain and VL-b light chain
  • the huC19 antibody molecule using the VH-c heavy chain and the VL-a light chain
  • 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, and the huC19I heavy chain variable region sequence is shown in SEQ ID NO:100.
  • the respective full length sequences of huC11, huC19 and huC19I are set forth in SEQ ID NOS: 17-20 and SEQ ID NO: 99.
  • CD14 + microbeads kit The normal human peripheral blood was taken and the healthy human PBMC was isolated by density gradient centrifugation. Mononuclear cells were sorted by CD14 + microbeads kit, and CD14 + monocytes were separated according to the protocol provided by the kit, that is, 20 ul of Anti-CD14 microbeads was added per 10 7 cells, and incubated at 4 ° C for 15 minutes. Then, the cells were added to a magnetic column, and after washing three times, the cells in the magnetic column, that is, CD14 + monocytes were collected. CD14 + monocytes were cultured for 6 days in RPMI 1640 medium containing 100 ng/ml M-CSF, and induced to induce macrophag cells. The remaining cells were added to Mito-M-treated MDA-MB-231 cells, PBMC and MDA. -MB-231 was co-cultured for 6 days, and the culture solution was RPMI 1640 medium containing IL-2 and 10% FBS.
  • PBMC cells, macrophage and freshly digested MDA-MB-231 cells were mixed at a ratio of 6.25:1:50, and inoculated into each NCG mouse (Nanjing University-Nanjing Institute of Biomedical Research, adaptive feeding for 5 days) ) Under the skin.
  • NCG mouse Nemjing University-Nanjing Institute of Biomedical Research, adaptive feeding for 5 days
  • the experimental animals were kept in an independent ventilated box with constant temperature and humidity.
  • the temperature of the breeding room was 18.0-26.0 °C, the humidity was 40-70%, the ventilation was 10-20 times/hour, and the alternating time between day and night was 12h/12h.
  • the experiment was divided into human IgG1 antibody control group, reference antibody FPA8, and huC19I, and the dose was 20 mg/kg.
  • Six mice in each group were administered once every two days, intraperitoneally, and administered 10 times (see Table 1). The daily behavior of the animals was monitored daily after administration for a total of 24 days.
  • Relative tumor inhibition rate TGI (%): TGI% (1-T/C) ⁇ 100%.
  • T/C% is the relative tumor growth rate, that is, the percentage of tumor volume or tumor weight relative to the treatment group and the 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. Comparing the tumor volume and tumor weight of the treatment group with the Student's t test, there was no significant difference compared with the control group, p ⁇ 0.05 was considered to be significant difference.

Abstract

The present invention provides an anti-CSF-1R antibody, an antigen-binding fragment thereof and pharmaceutical use thereof. Further provided are a chimeric antibody and a humanized antibody comprising a CDR region of the anti-CSF-1R antibody and pharmaceutical uses thereof.

Description

抗CSF-1R抗体、其抗原结合片段及其医药用途Anti-CSF-1R antibody, antigen-binding fragment thereof and medical use thereof
本申请要求申请日为2017年10月26日的中国专利申请CN201711015488.5的优先权。本申请引用上述中国专利申请的全文。The present application claims priority from Chinese Patent Application No. CN201711015488.5, filed on Oct. 26, 2017. This application cites the entire text of the above-mentioned Chinese patent application.
技术领域Technical field
本发明涉及一种对人CSF-1R受体具有免疫反应性的抗CSF-1R抗体，以及其抗原结合片段，包含所述抗CSF-1R抗体CDR区的嵌合抗体、人源化抗体，以及包含人抗CSF-1R抗体及其抗原结合片段的药物组合物，以及其作为抗癌药物的用途。The present invention relates to an anti-CSF-1R antibody immunoreactive to a 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 A pharmaceutical composition comprising a human anti-CSF-1R antibody and an antigen-binding fragment thereof, and use thereof as an anticancer drug.
背景技术Background technique
这里的陈述仅提供与本发明有关的背景信息，而不必然地构成现有技术。The statements herein merely provide background information related to the present invention and do not necessarily constitute the prior art.
肿瘤免疫治疗是肿瘤治疗领域的长期热点，其中T细胞肿瘤免疫治疗又处于核心位置。肿瘤逃逸是肿瘤免疫治疗面临的一个巨大障碍，大部分肿瘤表达可不同程度地被宿主免疫***识别的抗原，但在许多情况下，由于免疫细胞低效活化而引发不充分的免疫反应，因此肿瘤细胞利用其自身对免疫***的抑制作用促进了肿瘤的疯狂生长。肿瘤免疫治疗即是通过免疫检查点抑制、直接激活、肿瘤微环境活化等各种方式，充分利用并调动了肿瘤患者体内的各类免疫细胞，对肿瘤进行杀伤作用。Tumor immunotherapy is a long-term hotspot in the field of cancer treatment, and T cell tumor immunotherapy is at the core. Tumor escape is a huge obstacle to tumor immunotherapy. Most tumors express antigens that can be recognized by the host immune system to varying degrees, but in many cases, due to inefficient activation of immune cells, an inadequate immune response is triggered. Cells use their own inhibition of the immune system to promote the crazy growth of the tumor. Tumor immunotherapy is to fully utilize and mobilize various immune cells in tumor patients to kill tumors through various methods such as inhibition of immune checkpoints, direct activation, and activation of tumor microenvironment.
巨噬细胞(macrophages)是抗肿瘤免疫调节过程中的一种重要细胞群，可以直接杀伤肿瘤细胞。但近年来研究证实，巨噬细胞分为M1和M2两类，肿瘤组织间质中的巨噬细胞，即肿瘤相关巨噬细胞(TAM)属于M2类，并非如同M1类巨噬细胞发挥抗肿瘤作用，而是参与肿瘤发生、生长、侵袭和转移的过程。TAM由淋巴循环中的单核细胞或组织中残留的巨噬细胞衍生而来，是浸润许多种肿瘤基质的白细胞的主要类型。一旦被激活，TAM往往成为肿瘤微环境中的细胞因子、生长因子和蛋白酶的主要来源，促进肿瘤生长、增殖、血管生成、侵袭、转移及化疗抵抗。Macrophages are an important cell population in the process of anti-tumor immune regulation, which can directly kill tumor cells. However, in recent years, studies have confirmed that macrophages are classified into M1 and M2. Macrophages in tumor stroma, tumor-associated macrophages (TAM) belong to M2 class, and do not play an anti-tumor like M1 macrophages. Role, but participate in the process of tumorigenesis, growth, invasion and metastasis. TAM is derived from macrophages remaining in monocytes or tissues in the lymphatic circulation and is a major type of leukocytes infiltrating many tumor stromas. Once activated, TAM tends to be a major source of cytokines, growth factors, and proteases in the tumor microenvironment, promoting tumor growth, proliferation, angiogenesis, invasion, metastasis, and chemotherapy resistance.
CSF-1R(集落刺激因子-1受体，别称为FMS、C-FMS和CD115等)，是一种带有N端胞外结构域(以重复Ig域为特征)和酪氨酸激酶活性的C端胞内结构域的单程跨膜受体。CSF-1或白细胞介素-34(IL-34)配体与CSF-1R的结合导致受体二聚化和酪氨酸激酶活性上调、CSF-1R酪氨酸残基磷酸化和下游信号传导等事件。CSF-1和IL-34两种配基都能够刺激单核细胞存活、增殖以及分化成为巨噬细胞。CSF-1R主要在单核细胞谱系的细胞上及在女性生殖道和胎盘中表达。CSF-1R (colony stimulating factor-1 receptor, nicknamed FMS, C-FMS, and CD115, etc.) is an N-terminal extracellular domain (characterized by a repetitive Ig domain) 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 leads to receptor dimerization and upregulation of tyrosine kinase activity, phosphorylation of CSF-1R tyrosine residues and downstream signaling And other events. Both CSF-1 and IL-34 ligands are capable of stimulating monocyte survival, proliferation, and differentiation into macrophages. CSF-1R is expressed primarily on cells of the monocyte lineage and in the female reproductive tract and placenta.
许多肿瘤细胞分泌CSF-1。CSF-1通过CSF-1R活化单核细胞/M2巨噬细胞。肿瘤内的CSF-1水平与肿瘤内的TAM水平有关，而高水平的TAM则与较差的患者预后有关。已经发现在小鼠的人乳癌异种移植物中，CSF-1促进肿瘤生长和发展成转移。另一方面，CSF-1和其受体也涉及各种炎症性和自体免疫性疾病。CSF-1R刺激促进分化的骨髓系细胞的增殖、存活、活化和成熟，且在病理学环境中刺激促进分化的骨髓系细胞介导疾病病理学的能力。因此，有关CSF-1R受体信号传导的各类拮抗剂，包括单克隆抗体等，可通过阻断单核细胞活化为M2巨噬细胞而用于治疗各种CSF-1R介导的相关性疾病，如癌症、炎症性病状和自体免疫性疾病等。Many tumor cells secrete CSF-1. CSF-1 activates monocytes/M2 macrophages via CSF-1R. CSF-1 levels in tumors are associated with TAM levels within the tumor, while high levels of TAM are associated with poor patient prognosis. It has been found that in human breast cancer xenografts in mice, CSF-1 promotes tumor growth and progression to 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 myeloid cells that promote differentiation to mediate disease pathology in a pathological setting. Therefore, various antagonists of CSF-1R receptor signaling, 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.
目前有多家跨国制药公司在研发针对CSF-1R的单克隆抗体，可通过拮抗CSF1-R抑制肿瘤微环境中的TAM达到对肿瘤细胞进行杀伤的目的，或用于相关巨噬细胞介导的自身免疫疾病等。Sheer，C.J.等人描述了抑制CSF-1活性的抗CSF-1R的抗体(Sherr，C.J.等人，1989，Blood73:1786-1793)。W02009/026303公开了结合人CSF-1R的抗CSF-1R抗体和使用抗鼠CSF-1R抗体的体内小鼠肿瘤模型。W02011/123381公开了内化CSF-1R并具有ADCC活性的抗CSF-1R抗体。W02011/123381还公开了使用抗鼠CSF-1R抗体的体内小鼠肿瘤模型。W02011/140249公开了阻断CSF-1与CSF-1R的结合的抗CSF-1R抗体，其被认为在治疗癌症中是有用的。W02009/112245公开了抑制CSF-l结合CSF-IR的抗CSF-IR IgGl抗体，其被认为在治疗癌症、炎性肠病和类风湿性关节炎中是有用的。W02011/131407公开了抑制CSF-l结合CSF-IR的抗CSF-IR抗体，其被认为在治疗骨丢失和癌症中是有用的。W02011/107553公开了抑制CSF-l结合CSF-IR的抗CSF-IR抗体，其被认为在治疗骨丢失和癌症中是有用的。W02011/070024公开了结合人CSF-IR片段deID4的抗CSF-IR抗体。虽然先前己描述了抗CSF-IR抗体在治疗某些癌症中的治疗应用，但仍然存在开发新型、更适于临床应用的抗体的需要。At present, a number of multinational pharmaceutical companies are developing monoclonal antibodies against CSF-1R, which can inhibit the killing of 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. describe antibodies against CSF-1R that inhibit CSF-1 activity (Sherr, C. J. et al., 1989, Blood 73: 1786-1793). W02009/026303 discloses an anti-CSF-1R antibody that binds to human CSF-1R and an in vivo mouse tumor model using an anti-murine CSF-1R antibody. W02011/123381 discloses an anti-CSF-1R antibody that internalizes CSF-1R and has ADCC activity. W02011/123381 also discloses an in vivo mouse tumor model using an anti-murine CSF-1R antibody. W02011/140249 discloses an anti-CSF-1R antibody that blocks the binding of CSF-1 to CSF-1R, which is believed to be useful in the treatment of cancer. W02009/112245 discloses an anti-CSF-IR IgG1 antibody that inhibits CSF-1 binding to CSF-IR, which is considered to be useful in the treatment of cancer, inflammatory bowel disease and rheumatoid arthritis. W02011/131407 discloses anti-CSF-IR antibodies that inhibit CSF-1 binding to CSF-IR, which are believed to be useful in the treatment of bone loss and cancer. W02011/107553 discloses anti-CSF-IR antibodies that inhibit CSF-1 binding to CSF-IR, which are believed to be useful in the treatment of bone loss and cancer. W02011/070024 discloses an anti-CSF-IR antibody that binds to the human CSF-IR fragment deID4. Although therapeutic applications of anti-CSF-IR antibodies in the treatment of certain cancers have previously been described, there remains a need to develop new, more clinically suitable antibodies.
发明内容Summary of the invention
本发明一些实施方案提供一种抗CSF-1R抗体或其抗原结合片段，其包含：Some embodiments of the invention provide an anti-CSF-1R antibody or antigen-binding fragment thereof, comprising:
抗体轻链可变区，所述的抗体轻链可变区包含至少1个选自如以下序列所示的LCDR：SEQ ID NO:6，SEQ ID NO:7，SEQ ID NO:8，SEQ ID NO:14，SEQ ID NO:15，SEQ ID NO:16或SEQ ID NO:96；和An antibody light chain variable region comprising at least one LCDR selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO :14, SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 96;
抗体重链可变区，所述的抗体重链可变区包含至少1个选自如以下序列所述的HCDR：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或SEQ ID NO:98。An antibody heavy chain variable region comprising at least one HCDR selected from the group consisting of 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.
在另一些实施方案中，上述抗CSF-1R抗体或其抗原结合片段，其包含抗体轻链可变区和抗体重链可变区，其中：In other embodiments, the above anti-CSF-1R antibody or antigen-binding fragment thereof, comprising an antibody light chain variable region and an antibody heavy chain variable region, wherein:
a)所述的抗体轻链可变区包含选自在如SEQ ID NO:42、SEQ ID NO:43和SEQ ID NO:44所示的LCDR1、LCDR2和LCDR3基础上分别具有3、2、1或0个氨基酸突变的LCDR变体；所述的抗体重链可变区包含选自在如SEQ ID NO:39、SEQ ID NO:40和SEQ ID NO:41所示的HCDR1、HCDR2和HCDR3基础上分别具有3、2、1或0个氨基酸突变的HCDR变体；a) The antibody light chain variable region comprises 3, 2, 2 selected from the group consisting of LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 42, SEQ ID NO: 43 and SEQ ID NO: 44, respectively. Or a 0 amino acid mutated LCDR variant; said antibody heavy chain variable region comprising a base selected from the group consisting of HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 39, SEQ ID NO: 40 and SEQ ID NO: 41 HCDR variants having 3, 2, 1 or 0 amino acid mutations, respectively;
b)所述的抗体轻链可变区包含选自在如SEQ ID NO:50、SEQ ID NO:51和SEQ ID NO:52所示的LCDR1、LCDR2和LCDR3基础上分别具有3、2、1或0个氨基酸突变的LCDR变体；所述的抗体重链可变区包含选自在如SEQ ID NO:47、SEQ ID NO:48和SEQ ID NO:49所示的HCDR1、HCDR2和HCDR3基础上分别具有3、2、1或0个氨基酸突变的HCDR变体；b) The antibody light chain variable region comprises 3, 2, 2 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. Or a 0 amino acid mutated LCDR variant; said antibody heavy chain variable region comprising a base selected from the group consisting of HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 47, SEQ ID NO: 48 and SEQ ID NO: HCDR variants having 3, 2, 1 or 0 amino acid mutations, respectively;
c)所述的抗体轻链可变区包含选自在如SEQ ID NO:58、SEQ ID NO:59和SEQ ID NO:60所示的LCDR1、LCDR2和LCDR3基础上分别具有3、2、1或0个氨基酸突变的LCDR变体；所述的抗体重链可变区选自在如SEQ ID NO:55、SEQ ID NO:56和SEQ ID NO:57所示的HCDR1、HCDR2和HCDR3基础上分别具有3、2、1或0个氨基酸突变的HCDR变体；c) the antibody light chain variable region comprising 3, 2, 2 selected from the group consisting of LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 58, SEQ ID NO: 59 and SEQ ID NO: 60, respectively. Or a 0 amino acid mutated LCDR variant; said antibody heavy chain variable region selected from the group consisting of HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 55, SEQ ID NO: 56 and SEQ ID NO: 57 HCDR variants having 3, 2, 1 or 0 amino acid mutations, respectively;
d)所述的抗体轻链可变区包含选自在如SEQ ID NO:66、SEQ ID NO:67和SEQ ID NO:68所示的LCDR1、LCDR2和LCDR3基础上分别具有3、2、1或0个氨基酸突变的LCDR变体；所述的抗体重链可变区包含选自在如SEQ ID NO:63、SEQ ID NO:64和SEQ ID NO:65所示的HCDR1、HCDR2和HCDR3基础上分别具有3、2、1或0个氨基酸突变的HCDR变体；d) the antibody light chain variable region comprising 3, 2, 2 selected from the group consisting of LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 66, SEQ ID NO: 67 and SEQ ID NO: 68, respectively. Or a 0 amino acid mutated LCDR variant; said antibody heavy chain variable region comprising a base selected from the group consisting of HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 63, SEQ ID NO: 64 and SEQ ID NO: 65 HCDR variants having 3, 2, 1 or 0 amino acid mutations, respectively;
e)所述的抗体轻链可变区包含选自在如SEQ ID NO:74、SEQ ID NO:75和SEQ ID NO:76所示的LCDR1、LCDR2和LCDR3基础上分别具有3、2、1或0个氨基酸突变的LCDR变体；所述的抗体重链可变区包含选自在如SEQ ID NO:71、SEQ ID NO:72和SEQ ID NO:73所示的HCDR1、HCDR2和HCDR3基础上分别具有3、2、1或0个氨基酸突变的HCDR变体；e) The antibody light chain variable region comprises 3, 2, 2 selected from the group consisting of LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 74, SEQ ID NO: 75 and SEQ ID NO: 76, respectively. Or a 0 amino acid mutated LCDR variant; said antibody heavy chain variable region comprising a base selected from the group consisting of HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO:71, SEQ ID NO:72 and SEQ ID NO:73 HCDR variants having 3, 2, 1 or 0 amino acid mutations, respectively;
f)所述的抗体轻链可变区包含选自在如SEQ ID NO:82、SEQ ID NO:83和SEQ ID NO:84所示的LCDR1、LCDR2和LCDR3基础上分别具有3、2、1或0个氨基酸突变的LCDR变体；所述的抗体重链可变区包含选自在如SEQ ID NO:79、SEQ ID NO:80和SEQ ID NO:81所示的HCDR1、HCDR2和HCDR3基础上分别具有3、2、1或0个氨基酸突 变的HCDR变体；f) the antibody light chain variable region comprising 3, 2, 2 selected from the group consisting of LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 82, SEQ ID NO: 83 and SEQ ID NO: 84, respectively. Or a 0 amino acid mutated LCDR variant; said antibody heavy chain variable region comprising a base selected from the group consisting of HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO:79, SEQ ID NO:80 and SEQ ID NO:81 HCDR variants having 3, 2, 1 or 0 amino acid mutations, respectively;
g)所述的抗体轻链可变区包含选自在如SEQ ID NO:90、SEQ ID NO:91和SEQ ID NO:92所示的LCDR1、LCDR2和LCDR3基础上分别具有3、2、1或0个氨基酸突变的LCDR变体；所述的抗体重链可变区包含选自在如SEQ ID NO:87、SEQ ID NO:88和SEQ ID NO:89所示的HCDR1、HCDR2和HCDR3基础上分别具有3、2、1或0个氨基酸突变的HCDR变体；g) the antibody light chain variable region comprising 3, 2, 2 selected from the group consisting of LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 90, SEQ ID NO: 91 and SEQ ID NO: 92, respectively. Or a 0 amino acid mutated LCDR variant; said antibody heavy chain variable region comprising a base selected from the group consisting of HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO:87, SEQ ID NO:88 and SEQ ID NO:89 HCDR variants having 3, 2, 1 or 0 amino acid mutations, respectively;
h)所述的抗体轻链可变区包含选自在如SEQ ID NO:6、SEQ ID NO:7和SEQ ID NO:8所示的LCDR1、LCDR2和LCDR3基础上分别具有3、2、1或0个氨基酸突变的LCDR变体；所述的抗体重链可变区包含选自在如SEQ ID NO:3、SEQ ID NO:4和SEQ ID NO:5所示的HCDR1、HCDR2和HCDR3基础上分别具有3、2、1或0个氨基酸突变的HCDR变体；h) the antibody light chain variable region comprises 3, 2, 2 selected from the group consisting of LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively. Or a 0 amino acid mutated LCDR variant; said antibody heavy chain variable region comprising a base selected from the group consisting of HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5 HCDR variants having 3, 2, 1 or 0 amino acid mutations, respectively;
i)所述的抗体轻链可变区包含选自在如SEQ ID NO:14、SEQ ID NO:15和SEQ ID NO:16所示的LCDR1、LCDR2和LCDR3基础上分别具有3、2、1或0个氨基酸突变的LCDR变体；所述的抗体重链可变区包含选自在如SEQ ID NO:11、SEQ ID NO:12和SEQ ID NO:13所示的HCDR1、HCDR2和HCDR3基础上分别具有3、2、1或0个氨基酸突变的HCDR变体；i) The antibody light chain variable region comprises 3, 2, 2 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. Or a 0 amino acid mutated LCDR variant; said antibody heavy chain variable region comprising a base selected from the group consisting of HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: HCDR variants having 3, 2, 1 or 0 amino acid mutations, respectively;
j)所述的抗体轻链可变区包含选自在如SEQ ID NO:14、SEQ ID NO:15和SEQ ID NO:16所示的LCDR1、LCDR2和LCDR3基础上分别具有3、2、1或0个氨基酸突变的LCDR变体；抗体重链可变区包含选自在如：SEQ ID NO:98、SEQ ID NO:12和SEQ ID NO:13所示的HCDR1、HCDR2和HCDR3基础上分别具有3、2、1或0个氨基酸突变的HCDR变体；j) The antibody light chain variable region comprises 3, 2, 2 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. Or a 0 amino acid mutated LCDR variant; the antibody heavy chain variable region comprises a selection of HCDR1, HCDR2 and HCDR3 selected from, for example, SEQ ID NO:98, SEQ ID NO:12 and SEQ ID NO:13, respectively HCDR variants having 3, 2, 1 or 0 amino acid mutations;
k)所述的抗体轻链可变区包含选自在如SEQ ID NO:14、SEQ ID NO15和SEQ ID NO:16所示的LCDR1、LCDR2和LCDR3基础上分别具有3、2、1或0个氨基酸突变的LCDR变体；抗体重链可变区包含选自在如：SEQ ID NO:98、SEQ ID NO:97和SEQ ID NO:13所示的HCDR1、HCDR2和HCDR3基础上分别具有3、2、1或0个氨基酸突变的HCDR变体；k) The antibody light chain variable region comprises 3, 2, 1 or 0 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. An amino acid mutated LCDR variant; the antibody heavy chain variable region comprises 3, respectively, selected from HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO:98, SEQ ID NO:97 and SEQ ID NO:13 HCDR variant of 2, 1 or 0 amino acid mutations;
l)所述的抗体轻链可变区包含选自在如SEQ ID NO:96、SEQ ID NO:7和SEQ ID NO:8所示的LCDR1、LCDR2和LCDR3基础上分别具有3、2、1或0个氨基酸突变的LCDR变体；抗体重链可变区包含选自在如：SEQ ID NO:3、SEQ ID NO:4和SEQ ID NO:5所示的HCDR1、HCDR2和HCDR3基础上分别具有3、2、1或0个氨基酸突变的HCDR变体；l) The antibody light chain variable region comprises 3, 2, 2 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. Or a 0 amino acid mutated LCDR variant; the antibody heavy chain variable region comprises a selection of HCDR1, HCDR2 and HCDR3 selected from, for example, 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;
m)所述的抗体轻链可变区包含选自在如SEQ ID NO:6、SEQ ID NO:7和SEQ ID NO:8所示的LCDR1、LCDR2和LCDR3基础上分别具有3、2、1或0个氨基酸突变的LCDR变体；抗体重链可变区包含选自在如：SEQ ID NO:3、SEQ ID NO:95和SEQ ID NO:5所示的HCDR1、HCDR2和HCDR3基础上分别具有3、2、1或0个氨基酸突变的HCDR变体；m) the antibody light chain variable region comprising 3, 2, 2 selected from the group consisting of LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively. Or a 0 amino acid mutated LCDR variant; the antibody heavy chain variable region comprises a selection of HCDR1, HCDR2 and HCDR3 selected from, for example, SEQ ID NO:3, SEQ ID NO:95 and SEQ ID NO:5, respectively HCDR variants having 3, 2, 1 or 0 amino acid mutations;
n)所述的抗体轻链可变区包含选自在如SEQ ID NO:96、SEQ ID NO:7和SEQ ID NO:8所示的LCDR1、LCDR2和LCDR3基础上分别具有3、2、1或0个氨基酸突变的LCDR变体；抗体重链可变区包含选自在如：SEQ ID NO:3、SEQ ID NO:95和SEQ ID NO:5所示的HCDR1、HCDR2和HCDR3基础上分别具有3、2、1或0个氨基酸突变的HCDR变体；或者n) The antibody light chain variable region comprises 3, 2, 2 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. Or a 0 amino acid mutated LCDR variant; the antibody heavy chain variable region comprises a selection of HCDR1, HCDR2 and HCDR3 selected from, for example, SEQ ID NO:3, SEQ ID NO:95 and SEQ ID NO:5, respectively HCDR variant having 3, 2, 1 or 0 amino acid mutations; or
o)所述的抗体轻链可变区包含选自在如：SEQ ID NO:14、SEQ ID NO:15和SEQ ID NO:16所示的LCDR1、LCDR2和LCDR3基础上分别具有3、2、1或0个氨基酸突变的LCDR变体；抗体重链可变区包含选自在如：SEQ ID NO:11、SEQ ID NO:97和SEQ ID NO:13所示的HCDR1、HCDR2和HCDR3基础上分别具有3、2、1或0个氨基酸突变的HCDR变体。o) the antibody light chain variable region comprises 3, 2, respectively, 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. 1 or 0 amino acid mutated LCDR variant; antibody heavy chain variable region comprising selected from HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 11, SEQ ID NO: 97 and SEQ ID NO: HCDR variants with 3, 2, 1 or 0 amino acid mutations, respectively.
在另一些实施方案中，如上所述的抗CSF-1R抗体或其抗原结合片段，其包含抗体轻链可变区和抗体重链可变区，其中：In other embodiments, an 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)所述的抗体轻链可变区包含分别如SEQ ID NO:42、SEQ ID NO:43和SEQ ID NO:44所示的LCDR1、LCDR2和LCDR3；所述的抗体重链可变区包含分别如SEQ ID NO:39、SEQ ID NO:40和SEQ ID NO:41所示的HCDR1、HCDR2和HCDR3；The antibody light chain variable region of p) comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 42, SEQ ID NO: 43 and SEQ ID NO: 44, respectively; the antibody heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 39, SEQ ID NO: 40 and SEQ ID NO: 41, respectively;
q)所述的抗体轻链可变区包含分别如SEQ ID NO:50、SEQ ID NO:51和SEQ ID NO:52所示的LCDR1、LCDR2和LCDR3；所述的抗体重链可变区包含分别如SEQ ID NO:47、SEQ ID NO:48和SEQ ID NO:49所示的HCDR1、HCDR2和HCDR3；q) The antibody light chain variable region comprises LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO: 50, SEQ ID NO: 51 and SEQ ID NO: 52, respectively; the antibody heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 47, SEQ ID NO: 48 and SEQ ID NO: 49, respectively;
r)所述的抗体轻链可变区包含分别如SEQ ID NO:58、SEQ ID NO:59和SEQ ID NO:60所示的LCDR1、LCDR2和LCDR3；所述的抗体重链可变区包含分别如SEQ ID NO:55、SEQ ID NO:56和SEQ ID NO:57所示的HCDR1、HCDR2和HCDR3；r) the antibody light chain variable region comprising LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO:58, SEQ ID NO:59 and SEQ ID NO:60, respectively; said antibody heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 55, SEQ ID NO: 56 and SEQ ID NO: 57, respectively;
s)所述的抗体轻链可变区包含分别如SEQ ID NO:66、SEQ ID NO:67和SEQ ID NO:68所示的LCDR1、LCDR2和LCDR3；所述的抗体重链可变区包含分别如SEQ ID NO:63、SEQ ID NO:64和SEQ ID NO:65所示的HCDR1、HCDR2和HCDR3；The antibody light chain variable region of s) comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 66, SEQ ID NO: 67 and SEQ ID NO: 68, respectively; the antibody heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 63, SEQ ID NO: 64 and SEQ ID NO: 65, respectively;
t)所述的抗体轻链可变区包含分别如SEQ ID NO:74、SEQ ID NO:75和SEQ ID NO:76所示的LCDR1、LCDR2和LCDR3；所述的抗体重链可变区包含分别如SEQ ID NO:71、 SEQ ID NO:72和SEQ ID NO:73所示的HCDR1、HCDR2和HCDR3；t) The antibody light chain variable region comprises LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO: 74, SEQ ID NO: 75 and SEQ ID NO: 76, respectively; the antibody heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 71, SEQ ID NO: 72 and SEQ ID NO: 73, respectively;
u)所述的抗体轻链可变区包含分别如SEQ ID NO:82、SEQ ID NO:83和SEQ ID NO:84所示的LCDR1、LCDR2和LCDR3；所述的抗体重链可变区包含分别如SEQ ID NO:79、SEQ ID NO:80和SEQ ID NO:81所示的HCDR1、HCDR2和HCDR3；u) the antibody light chain variable region comprising LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO: 82, SEQ ID NO: 83 and SEQ ID NO: 84, respectively; said antibody heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 79, SEQ ID NO: 80 and SEQ ID NO: 81, respectively;
v)所述的抗体轻链可变区包含分别如SEQ ID NO:90、SEQ ID NO:91和SEQ ID NO:92所示的LCDR1、LCDR2和LCDR3；所述的抗体重链可变区包含分别如SEQ ID NO:87、SEQ ID NO:88和SEQ ID NO:89所示的HCDR1、HCDR2和HCDR3；v) The antibody light chain variable region comprises LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO: 90, SEQ ID NO: 91 and SEQ ID NO: 92, respectively; the antibody heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 87, SEQ ID NO: 88 and SEQ ID NO: 89, respectively;
w)所述的抗体轻链可变区包含分别如SEQ ID NO:6、SEQ ID NO:7和SEQ ID NO:8所示的LCDR1、LCDR2和LCDR3；所述的抗体重链可变区包含分别如SEQ ID NO:3、SEQ ID NO:4和SEQ ID NO:5所示的HCDR1、HCDR2和HCDR3；w) The antibody light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, respectively; the antibody heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, respectively;
x)所述的抗体轻链可变区包含分别如SEQ ID NO:14、SEQ ID NO:15和SEQ ID NO:16所示的LCDR1、LCDR2和LCDR3；所述的抗体重链可变区包含分别如SEQ ID NO:11、SEQ ID NO:12和SEQ ID NO:13所示的HCDR1、HCDR2和HCDR3；x) The antibody light chain variable region comprises LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, respectively; the antibody heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13, respectively;
y)所述的抗体轻链可变区包含分别如SEQ ID NO:14、SEQ ID NO:15和SEQ ID NO:16所示的LCDR1、LCDR2和LCDR3；抗体重链可变区包含分别如：SEQ ID NO:98、SEQ ID NO:12和SEQ ID NO:13所示的HCDR1、HCDR2和HCDR3；y) The antibody light chain variable region comprises LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, respectively; the antibody heavy chain variable region comprises: HCDR1, HCDR2 and HCDR3 set forth in SEQ ID NO: 98, SEQ ID NO: 12 and SEQ ID NO: 13;
z)所述的抗体轻链可变区包含分别如SEQ ID NO:14、SEQ ID NO15和SEQ ID NO:16所示的LCDR1、LCDR2和LCDR3；抗体重链可变区包含分别如：SEQ ID NO:98、SEQ ID NO:97和SEQ ID NO:13所示的HCDR1、HCDR2和HCDR3；z) The antibody light chain variable region comprises 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 comprises, for example, SEQ ID NO: 98, SEQ ID NO: 97 and SEQ ID NO: 13 HCDR1, HCDR2 and HCDR3;
aa)所述的抗体轻链可变区包含分别如SEQ ID NO:96、SEQ ID NO:7和SEQ ID NO:8所示的LCDR1、LCDR2和LCDR3；抗体重链可变区包含分别如：SEQ ID NO:3、SEQ ID NO:4和SEQ ID NO:5所示的HCDR1、HCDR2和HCDR3；The antibody light chain variable region of aa) 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 variable region comprises: HCDR1, HCDR2 and HCDR3 set forth in SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5;
ab)所述的抗体轻链可变区包含分别如SEQ ID NO:6、SEQ ID NO:7和SEQ ID NO:8所示的LCDR1、LCDR2和LCDR3；抗体重链可变区包含分别如：SEQ ID NO:3、SEQ ID NO:95和SEQ ID NO:5所示的HCDR1、HCDR2和HCDR3；The antibody light chain variable region of ab) comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively; the antibody heavy chain variable region comprises: HCDR1, HCDR2 and HCDR3 set forth in SEQ ID NO: 3, SEQ ID NO: 95 and SEQ ID NO: 5;
ac)所述的抗体轻链可变区包含分别如SEQ ID NO:96、SEQ ID NO:7和SEQ ID NO:8所示的LCDR1、LCDR2和LCDR3；抗体重链可变区包含分别如：SEQ ID NO:3、SEQ ID NO:95和SEQ ID NO:5所示的HCDR1、HCDR2和HCDR3；The antibody light chain variable region of ac) 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 variable region comprises: HCDR1, HCDR2 and HCDR3 set forth in SEQ ID NO: 3, SEQ ID NO: 95 and SEQ ID NO: 5;
ad)所述的抗体轻链可变区包含分别如：SEQ ID NO:14、SEQ ID NO:15和SEQ ID NO:16所示的LCDR1、LCDR2和LCDR3；抗体重链可变区包含分别如：SEQ ID NO:11、SEQ ID NO:97和SEQ ID NO:13所示的HCDR1、HCDR2和HCDR3。Ad) The antibody light chain variable region comprises 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 comprises : HCDR1, HCDR2 and HCDR3 of SEQ ID NO: 11, SEQ ID NO: 97 and SEQ ID NO: 13.
在另一些实施方案中，如上所述的抗CSF-1R抗体或其抗原结合片段，其中所述的抗体或其抗原结合片段为鼠源抗体或者嵌合抗体。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.
在另一些实施方案中，如上所述的抗体或抗原结合片段，其中In other embodiments, the antibody or antigen-binding fragment as described above, wherein
a)所述轻链可变区的氨基酸序列如SEQ ID NO:38所示，所述重链可变区的氨基酸序列如SEQ ID NO：37所示；或者a) the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 38, and the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 37;
b)所述轻链可变区的氨基酸序列如SEQ ID NO:46所示，所述重链可变区的氨基酸序列如SEQ ID NO：45所示；或者b) the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 46, and the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 45;
c)所述轻链可变区的氨基酸序列如SEQ ID NO:54所示，所述重链可变区的氨基酸序列如SEQ ID NO：53所示；或者c) the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 54, and the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 53;
d)所述轻链可变区的氨基酸序列如SEQ ID NO:62所示，所述重链可变区的氨基酸序列如SEQ ID NO：61所示；或者d) the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 62, and the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 61;
e)所述轻链可变区的氨基酸序列如SEQ ID NO:70所示，所述重链可变区的氨基酸序列如SEQ ID NO：69所示；或者e) the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 70, and the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 69;
f)所述轻链可变区的氨基酸序列如SEQ ID NO:78所示，所述重链可变区的氨基酸序列如SEQ ID NO：77所示；或者f) the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 78, and the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 77;
g)所述轻链可变区的氨基酸序列如SEQ ID NO:86所示，所述重链可变区的氨基酸序列如SEQ ID NO：85所示；或者g) the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 86, and the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 85;
h)所述轻链可变区的氨基酸序列如SEQ ID NO:2所示或者其变体，所述变体优选在轻链可变区有0-10的氨基酸突变，最优选的氨基酸突变为N37T，所述重链可变区的氨基酸序列如SEQ ID NO：1所示或者其变体，所述变体优选在重链可变区有0-10的氨基酸突变，优选的氨基酸突变为N55T；或者h) the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 2 or a variant thereof, preferably having a 0-10 amino acid mutation in the light chain variable region, the most preferred amino acid mutation being N37T, the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 1 or a variant thereof, and the variant preferably has a 0-10 amino acid mutation in the heavy chain variable region, and the preferred amino acid mutation is N55T ;or
i)所述轻链可变区的氨基酸序列如SEQ ID NO:10所示，所述重链可变区的氨基酸序列如SEQ ID NO：9所示或者其变体，所述变体优选在轻链可变区有0-10的氨基酸突变，优选的突变为M34I、N55T或其组合的氨基酸突变。i) the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 10, and the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 9 or a variant thereof, preferably The light chain variable region has an amino acid mutation of 0-10, and the preferred mutation is an amino acid mutation of M34I, N55T or a combination thereof.
在另一些实施方案中，如上所述抗CSF-1R抗体或其抗原结合片段，其中所述的抗体或其抗原结合片段为人源化抗体或其抗原结合片段。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.
在另一些实施方案中，如上所述抗CSF-1R抗体或其抗原结合片段，其中所述人源化抗体轻链可变区上的轻链FR区序列，来源于如SEQ ID NO:22所示的人种系轻链IGkV4-1序列；或来源于如SEQ ID NO:24所示的人种系轻链IGkV1-13序列；In other embodiments, the anti-CSF-1R antibody or antigen-binding fragment thereof, as described above, wherein the light chain FR region sequence on the humanized antibody light chain variable region is derived from SEQ ID NO: Human germline light chain IGkV4-1 sequence; or human germline light chain IGkV1-13 sequence as set forth in SEQ ID NO: 24;
和/或，所述人源化抗体重链可变区进一步包含人源IgG1,IgG2,IgG3或IgG4或其变体的重链FR区，优选包含人源IgG1、IgG2或IgG4重链FR区，更优选包含人源IgG1 或IgG4重链FR区；较佳地，所述人源化抗体重链可变区上的重链FR区序列，来源于如SEQ ID NO:21所示的人种系重链IGHV1-46序列；或来源于如SEQ ID NO:23所示的人种系重链IGHV1-2序列。And/or the humanized antibody heavy chain variable region further comprises a heavy chain FR region of human IgG1, IgG2, IgG3 or IgG4 or variants thereof, preferably comprising a human IgG1, IgG2 or IgG4 heavy chain FR region, More preferably comprising a human IgG1 or IgG4 heavy chain FR region; preferably, the heavy chain FR region sequence on the humanized antibody heavy chain variable region is derived from the human germline as set forth in SEQ ID NO:21. The heavy chain IGHV1-46 sequence; or the human germline heavy chain IGHV1-2 sequence as set forth in SEQ ID NO:23.
在另一些实施方案中，如上所述抗CSF-1R抗体或其抗原结合片段，其中所述人源化抗体轻链可变区序列为如SEQ ID NO:30或SEQ ID NO:34所示的序列或其变体；所述的变体优选在轻链可变区有0-10的氨基酸突变，其中SEQ ID NO:30最优选的氨基酸突变为S31N、T37N；SEQ ID NO:34最优选的氨基酸突变为F87A、F91Y或其组合；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 set forth in SEQ ID NO: 30 or SEQ ID NO: 34 a sequence or variant thereof; said variant preferably having a 0-10 amino acid mutation in the light chain variable region, wherein the most preferred amino acid mutation of SEQ ID NO: 30 is S31N, T37N; SEQ ID NO: 34 is most preferred The amino acid mutation is F87A, F91Y or a combination thereof;
和/或，所述人源化抗体重链可变区序列为如SEQ ID NO:26、SEQ ID NO:100或SEQ ID NO:33所示的序列或其变体；所述变体优选在重链可变区有0-10的氨基酸突变，其中SEQ ID NO:26优选的氨基酸突变为S30T、T55N、L70M或其组合，最优选的氨基酸突变为T55N；SEQ ID NO:33优选的氨基酸突变为Q1E、M34I、T55N、E89D或其组合，最优选的氨基酸突变为M34I、T55N或其组合的氨基酸突变；And/or the humanized antibody heavy chain variable region sequence is the sequence set forth in SEQ ID NO:26, SEQ ID NO:100 or SEQ ID NO:33 or a variant thereof; The heavy chain variable region has a 0-10 amino acid mutation, 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 preferred amino acid mutation For Q1E, M34I, T55N, E89D or a combination thereof, the most preferred amino acid mutation is an amino acid mutation of M34I, T55N or a combination thereof;
较佳地，所述重链可变区的序列为SEQ ID NO：31、32、33或100，且所述轻链可变区的序列为SEQ ID NO：34、35或36；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;
或者，所述重链可变区的序列为SEQ ID NO：25、26、27或28，且所述轻链可变区的序列为SEQ ID NO：29或30。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.
在另一些实施方案中，如上所述抗CSF-1R抗体或其抗原结合片段，其中所述嵌合抗体或人源化抗体进一步包括轻链和/或重链的恒定区，所述轻链恒定区序列如SEQ ID NO：102所示，和/或所述重链恒定区序列如SEQ ID NO：101所示。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 being constant The region sequence is set forth in SEQ ID NO: 102, and/or the heavy chain constant region sequence is set forth in SEQ ID NO: 101.
在另一些实施方案中，如上所述抗CSF-1R抗体或其抗原结合片段，其中所述人源化抗体：In other embodiments, an anti-CSF-1R antibody or antigen-binding fragment thereof, as described above, wherein the humanized antibody:
包含序列如SEQ ID NO:19所示的重链，和序列如SEQ ID NO:20所示的轻链；a heavy chain comprising the sequence of SEQ ID NO: 19, and a light chain of the sequence SEQ ID NO: 20;
包含序列如SEQ ID NO:17所示的重链，和序列如SEQ ID NO:18所示的轻链；或者包含序列如SEQ ID NO:99所示的重链，和序列如SEQ ID NO:20所示的轻链。A heavy chain comprising the sequence of SEQ ID NO: 17 and a light chain of the sequence SEQ ID NO: 18; or a heavy chain of the sequence SEQ ID NO: 99, and a sequence of SEQ ID NO: The light chain shown in 20.
在另一些实施方案中，如上所述的鼠源抗体或其片段，其抗体轻链可变区进一步包含鼠源κ、λ链或其变体的轻链FR区。In other embodiments, the murine antibody or fragment thereof, as described above, wherein the antibody light chain variable region further comprises a light chain FR region of a murine kappa, lambda chain or variant thereof.
在另一些实施方案中，如上所述的鼠源抗体或其片段，其进一步包含鼠源κ、λ链或其变体的轻链恒定区。In other embodiments, the murine antibody or fragment thereof, as described above, further comprises a light chain constant region of a murine kappa, lambda chain or variant thereof.
在另一些实施方案中，如上所述的鼠源抗体或其片段，其抗体重链可变区进一步包含鼠源IgG1,IgG2,IgG3,IgG4或其变体的重链FR区。In other embodiments, the murine antibody or fragment thereof, as described above, further comprises an antibody heavy chain variable region comprising a heavy chain FR region of murine IgGl, IgG2, IgG3, IgG4 or variants thereof.
在另一些实施方案中，如上所述的鼠源抗体或其片段，其进一步包含鼠源IgG1,IgG2, IgG3,IgG4或其变体的重链恒定区。In other embodiments, the murine antibody or fragment thereof, as described above, further comprises a heavy chain constant region of murine IgGl, IgG2, IgG3, IgG4 or variants thereof.
在另一些实施方案中，如上所述的抗CSF-1R抗体或其抗原结合片段，其中所述的抗体为嵌合抗体或其片段。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.
在另一些实施方案中，如上所述的CSF-1R嵌合抗体或其片段，其中所述的嵌合抗体轻链可变区序列为：SEQ ID NO:2或SEQ ID NO:10。所述的变体优选在轻链可变区有0-10的氨基酸变化，最优选SEQ ID NO:2的氨基酸突变为N37T。In other embodiments, a CSF-1R chimeric antibody or fragment thereof, as described above, wherein the chimeric antibody light chain variable region sequence is: SEQ ID NO: 2 or SEQ ID NO: 10. Preferably, the variant has a 0-10 amino acid change in the light chain variable region, and most preferably the amino acid mutation in SEQ ID NO: 2 is N37T.
在另一些实施方案中，如上所述的CSF-1R嵌合抗体或其片段，其中所述的嵌合抗体重链可变区序列为：SEQ ID NO:1或SEQ ID NO:9，其中SEQ ID NO:1优选的氨基酸突变为T55N；SEQ ID NO:9优选的突变为M34I、T55N或其组合的氨基酸突变。在本发明一个优选的实施方案中，一种如上所述的CSF-1R嵌合抗体或其片段，其进一步包含人源κ、λ链或其变体的轻链恒定区。In another embodiment, the CSF-1R chimeric antibody or fragment thereof, as described above, wherein the chimeric antibody heavy chain variable region sequence is: SEQ ID NO: 1 or SEQ ID NO: 9, wherein SEQ The preferred amino acid mutation of ID NO: 1 is T55N; the 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 invention, a CSF-1R chimeric antibody or fragment thereof, as described above, further comprising a light chain constant region of a human kappa, lambda chain or variant thereof.
在另一些实施方案中，如上所述的CSF-1R嵌合抗体或其片段，其进一步包含人源IgG1,IgG2,IgG3或IgG4或其变体的重链恒定区。In other embodiments, the CSF-1R chimeric antibody or fragment thereof, as described above, further comprises a heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4 or variants thereof.
在另一些实施方案中，如上所述的抗CSF-1R抗体或其抗原结合片段，其中所述的抗体为人源化抗体或其抗原结合片段。In other embodiments, the anti-CSF-1R antibody or antigen-binding fragment thereof, as described above, wherein the antibody is a humanized antibody or antigen-binding fragment thereof.
在另一些实施方案中，如上所述的CSF-1R人源化抗体或其片段，其人源化抗体轻链可变区进一步包含人源κ、λ链或其变体的轻链FR区。In other embodiments, a CSF-1R humanized antibody or fragment thereof, as described above, wherein the humanized antibody light chain variable region further comprises a light chain FR region of a human kappa, lambda chain or variant thereof.
在本发明一个优选的实施方案中，一种如上所述的CSF-1R人源化抗体或其片段，其中所述人源化抗体轻链可变区序列为如SEQ ID NO:30或SEQ ID NO:34所示的序列，或其变体。In a preferred embodiment of the invention, a CSF-1R humanized antibody or fragment thereof, wherein the humanized antibody light chain variable region sequence is SEQ ID NO: 30 or SEQ ID NO: sequence shown by 34, or a variant thereof.
在另一些实施方案中，如上所述的CSF-1R人源化抗体或其片段，其中所述人源化抗体轻链序列为如SEQ ID NO:18或SEQ ID NO:20所示的序列，或其变体；所述人源化抗体变体优选在轻链可变区有0-10的氨基酸变化；其中SEQ ID NO:18优选的氨基酸突变为S31N、T37N或其组合的氨基酸突变，最优选的氨基酸突变为T37N；SEQ ID NO:20优选的氨基酸突变为F87A、F91Y或其组合的氨基酸突变。在本发明一个优选的实施方案中，一种如上所述的CSF-1R人源化抗体或其片段，其进一步包含人源κ、λ链或其变体的轻链恒定区。In a further embodiment, the CSF-1R humanized antibody or fragment thereof, as described above, wherein the humanized antibody light chain sequence is the sequence set forth in SEQ ID NO: 18 or SEQ ID NO: Or a variant thereof; the humanized antibody variant preferably has an amino acid change of 0-10 in the light chain variable region; wherein the preferred amino acid mutation of SEQ ID NO: 18 is an amino acid mutation of S31N, T37N or a combination thereof, most A preferred amino acid mutation is T37N; a preferred amino acid mutation of SEQ ID NO: 20 is an amino acid mutation of F87A, F91Y, or a combination thereof. In a preferred embodiment of the invention, a CSF-1R humanized antibody or fragment thereof, as described above, further comprising a light chain constant region of a human kappa, lambda chain or variant thereof.
在另一些实施方案中，如上所述的CSF-1R人源化抗体或其片段，其人源化抗体重链可变区进一步包含人源IgG1,IgG2,IgG3,IgG4或其变体的重链FR区。In other embodiments, the CSF-1R humanized antibody or fragment thereof, as described above, wherein the humanized antibody heavy chain variable region further comprises a heavy chain of human IgG1, IgG2, IgG3, IgG4 or variants thereof FR area.
在另一些实施方案中，如上所述的CSF-1R人源化抗体或其片段，其进一步包含人源IgG1,IgG2,IgG3或IgG4或其变体的重链恒定区，优选包含人源IgG1、IgG2或IgG4重 链恒定区。更优选使用IgG4或氨基酸突变后消除ADCC(antibody-dependent cell-mediated cytotoxicity，抗体依赖的细胞介导的细胞毒作用)毒性的IgG1。In other embodiments, a CSF-1R humanized 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, preferably comprising human IgG1, IgG2 or IgG4 heavy chain constant region. More preferably, IgG1 which eliminates the toxicity of antibody-dependent cell-mediated cytotoxicity (antibody-dependent cell-mediated cytotoxicity) after IgG4 or amino acid mutation is used.
另一方面，本发明的一些实施方案还提供一种分离的单克隆抗体或其抗原结合片段，其与如上所述的抗CSF-1R抗体或其抗原结合片段竞争结合CSF-1R。In another aspect, some embodiments of the invention also provide an isolated monoclonal antibody or antigen-binding fragment thereof that competes for binding to CSF-1R with an anti-CSF-1R antibody or antigen-binding fragment thereof as described above.
在本发明一个优选的实施方案中，一种如上所述的抗体(此处及以下所述的抗体为上述抗CSF-1R抗体或上述单克隆抗体)或其抗原结合片段，其中所述的抗原结合片段为Fab、Fv、sFv、F(ab’)2、线性抗体、单链抗体、纳米抗体、结构域抗体和多特异性抗体。In a preferred embodiment of the present invention, an antibody as described above (the antibody described herein below and the anti-CSF-1R antibody or the above monoclonal antibody) or an antigen-binding fragment thereof, wherein the antigen The binding fragments are Fab, Fv, sFv, F(ab')2, linear antibodies, single chain antibodies, Nanobodies, domain antibodies and multispecific antibodies.
另一方面，本发明的一些实施方案还提供一种多特异性抗体，含有如上所述的抗体或其抗原结合片段的轻链可变区和/或重链可变区。In another aspect, some embodiments of the invention also provide a multispecific antibody comprising a light chain variable region and/or a heavy chain variable region of an antibody or antigen binding fragment thereof as described above.
另一方面，本发明的一些实施方案还提供一种单链抗体，含有如上所述的抗体或其抗原结合片段的轻链可变区和/或重链可变区。In another aspect, some embodiments of the invention further provide a single chain antibody comprising a light chain variable region and/or a heavy chain variable region of an antibody or antigen binding fragment thereof as described above.
另一方面，本发明的一些实施方案还提供一种抗体-药物偶联物，其中所述的抗体含有如上所述的抗体或其抗原结合片段的轻链可变区和/或重链可变区。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 of the antibody or antigen-binding fragment thereof as described above Area.
另一方面，本发明的一些实施方案还提供一种编码如上所述的抗体或其抗原结合片段、如上所述的多特异性抗体或者如上所述的单链抗体的核酸分子。In another aspect, some embodiments of the invention further provide a nucleic acid molecule encoding an antibody or antigen-binding fragment thereof as described above, a multispecific antibody as described above, or a single chain antibody as described above.
另一方面，本发明的一些实施方案还提供一种编码如上所述的抗体或其抗原结合片段的DNA序列。In another aspect, some embodiments of the invention also provide a DNA sequence encoding an antibody or antigen-binding fragment thereof as described above.
另一方面，本发明的一些实施方案还提供一种含有如上所述的DNA序列的表达载体。In another aspect, some embodiments of the invention also provide an expression vector comprising a DNA sequence as described above.
在本发明的一些实施方案还提供一种用如上所述的表达载体转化的宿主细胞。Also provided in some embodiments of the invention is a host cell transformed with an expression vector as described above.
在一些的实施方案中，一种如上所述的宿主细胞，所述的宿主细胞为细菌，优选为大肠杆菌。In some embodiments, a host cell as described above, wherein the host cell is a bacterium, preferably E. coli.
在另一些实施方案中，一种如上所述的宿主细胞为酵母菌，优选为毕赤酵母。In other embodiments, a host cell as described above is a yeast, preferably Pichia pastoris.
在另一些实施方案中，一种如上所述的宿主细胞为哺乳动物细胞，优选为中国仓鼠卵巢(CHO)细胞、人胚肾(HEK)293细胞或NS0细胞。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.
另一方面，本发明的一些实施方案还提供一种抗体-药物偶联物，含有如前所述的轻链可变区和重链可变区。所述的抗体-药物偶联物是本领域公知的，其由抗体-接头-药物(毒素)相互连接形成，已知的接头包括裂解接头、***解接头，例如接头包括但不限于SMCC、SPDP等等；毒素也是本领域公知的，例如DM1、DM4、MMAE、MMAF等。In another aspect, some embodiments of the invention also provide an antibody-drug conjugate comprising a light chain variable region and a heavy chain variable region as previously described. The antibody-drug conjugates are well known in the art and are formed by the interconnection of antibody-linker-drug (toxin), and known linkers include cleavage linkers, split cleavage linkers, such as linkers including, but not limited to, SMCC, SPDP Etc.; Toxins are also well known in the art, such as DM1, DM4, MMAE, MMAF, and the like.
另一方面，本发明的一些实施方案还提供一种药物组合物，其含有如上所述的抗体或其抗原结合片段和可药用的缓冲剂、赋形剂、稀释剂或载体。In another aspect, some embodiments of the invention also provide a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof as described above and a pharmaceutically acceptable buffer, excipient, diluent or carrier.
在另一些实施方案中，一种如上所述的抗体或其片段、多特异性抗体、单链抗体、核酸分子或药物组合物在癌症治疗中施用，其中所述施用是在开始用另一种抗癌治疗的疗法之前、过程中、基本同时或之后施用的。In other embodiments, an antibody, or a fragment thereof, a multispecific antibody, a single chain antibody, a nucleic acid molecule, or a pharmaceutical composition, as described above, is administered in the treatment of cancer, wherein the administration is at the beginning of another The treatment of anticancer therapy is administered before, during, substantially simultaneously or after.
在另一些实施方案中，一种如上所述的CSF-1R人源化抗体或其片段，其中所述抗癌治疗选自抗血管发生剂、化疗剂、放射、肿瘤免疫疗法或其组合。In other embodiments, a CSF-1R humanized antibody or fragment thereof, as described above, wherein the anti-cancer treatment is selected from the group consisting of an anti-angiogenic agent, a chemotherapeutic agent, radiation, tumor immunotherapy, or a combination thereof.
在另一些实施方案中，一种如上所述的CSF-1R人源化抗体或其片段，其中所述化疗剂选自:紫杉炕类(帕利他赛、多因他赛、经修饰的帕利他赛(Abraxane和Opaxio))、多柔比星、经修饰的多柔比星(Caelyx或Doxil))、舒尼替尼、索拉非尼和其它多激酶抑制剂、奥沙利铂、顺铂、卡铂、依托泊苷、吉西他滨和长春碱。In other embodiments, a CSF-1R humanized antibody or fragment thereof, as described above, wherein the chemotherapeutic agent is selected from the group consisting of: docetaxel (palitax, docetaxel, modified pa Lithoace (Abraxane and Opaxio)), doxorubicin, modified doxorubicin (Caelyx or Doxil), sunitinib, sorafenib and other multi-kinase inhibitors, oxaliplatin, cis Platinum, carboplatin, etoposide, gemcitabine and vinblastine.
在另一些实施方案中，一种如上所述的CSF-1R人源化抗体或其片段，其中所述肿瘤免疫疗法选自:T细胞接合剂(engaging agent)、靶向性免疫抑制、癌症疫苗/增强树突细胞功能和过继性细胞转移。In a further embodiment, a CSF-1R humanized antibody or fragment thereof, as described above, wherein said tumor immunotherapy is selected from the group consisting of: a T cell engaging agent, a targeted immunosuppression, a cancer vaccine / Enhances dendritic cell function and adoptive cell transfer.
在另一方面，本发明的一些实施方式进一步提供一种如上所述的抗体或其抗原结合片段在制备用于治疗CSF-1R或CSF-1介导的疾病或病症的药物中的用途；其中所述的疾病优选为癌症、自身免疫疾病、炎性疾病或溶骨性骨质缺失；所述的癌症最优选为乳癌、子宫内膜癌、鳞状细胞癌、滤泡性淋巴瘤、肾细胞癌、葡萄膜黑色素瘤、子***、头颈癌、霍奇金病、星形细胞癌、肺腺癌、间皮瘤、绒毛膜癌、黑色素瘤、乳腺癌、卵巢癌、***癌、胰腺癌、肾癌、肺癌、肝癌、脑癌、胃癌、结肠直肠癌、膀胱癌、食管癌、***、多发性骨髓瘤、白血病、淋巴瘤和胶质母细胞瘤；所述的自身免疫疾病最优选为自身免疫性脑脊髓炎、***性红斑狼疮、多发性硬化、关节滑膜炎、银屑病、和类风湿关节炎；所述溶骨性骨质缺失选自骨质疏松、转移诱导的溶骨性骨质缺失和类风湿性关节炎诱导的骨质缺失。本发明进一步提供一种治疗和预防CSF-1R或CSF-1介导的疾病或病症的方法，该方法包括给予所需患者治疗有效量的如上所述的抗体或其抗原结合片段、或包含其的的药物组合物；其中所述的疾病优选为癌症、自身免疫疾病、炎性疾病或溶骨性骨质缺失；所述的癌症最优选为乳癌、子宫内膜癌、鳞状细胞癌、滤泡性淋巴瘤、肾细胞癌、葡萄膜黑色素瘤、子***、头颈癌、霍奇金病、星形细胞癌、肺腺癌、间皮瘤、绒毛膜癌、黑色素瘤、乳腺癌、卵巢癌、***癌、胰腺癌、肾癌、肺癌、肝癌、脑癌、胃癌、结肠直肠癌、膀胱癌、食管癌、***、多发性骨髓瘤、白血病、淋巴瘤和胶质母细胞瘤；所述的自身免疫疾病最优选为自身免疫性脑脊髓炎、***性红斑狼疮、多发性硬化、关节滑膜炎、银屑病、和类风湿关节炎；所述溶骨性骨质缺失选自骨质疏松、转移诱导的溶骨性骨质缺失和类风湿性关节炎诱导的骨质缺失。In another aspect, some embodiments of the invention further provide the use of an antibody, or antigen-binding fragment thereof, as described above, in the manufacture of a medicament for the treatment of a CSF-1R or CSF-1 mediated disease or condition; 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. Cancer, uveal melanoma, cervical cancer, head and neck cancer, Hodgkin's disease, astrocytoma, 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 most preferred autoimmune diseases For 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 dissolution Bone bone Deletions and rheumatoid arthritis-induced bone loss. The invention further provides a method of treating and preventing a CSF-1R or CSF-1 mediated disease or condition, comprising administering to a patient in need thereof a therapeutically effective amount of an antibody or antigen-binding fragment thereof as described above, or comprising the same 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, filtration Follicular lymphoma, renal cell carcinoma, uveal melanoma, cervical cancer, head and neck cancer, Hodgkin's disease, astrocytoma, lung adenocarcinoma, mesothelioma, choriocarcinoma, melanoma, breast cancer, ovary 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 diseases described are most preferably autoimmune encephalomyelitis, systemic lupus erythematosus, multiple sclerosis, joint synovitis, psoriasis, and rheumatoid arthritis; the osteolytic bone loss is selected from the group consisting of bone Quality , Osteolytic metastasis-induced bone loss and bone loss induced by rheumatoid arthritis.
附图说明DRAWINGS
图1：九株CSF-1R嵌合抗体的ELISA体外结合实验，显示除C27C外，其余各株抗体均与食蟹猴CSF-1R蛋白有很强的交叉结合。Figure 1: ELISA in vitro binding assay of nine CSF-1R chimeric antibodies showed that all the antibodies except C27C had strong cross-binding with cynomolgus CSF-1R protein.
图2：人源化抗体与高表达CSF-1R的CHO细胞体外结合活性实验。人源化的抗体huC11和huC19分别与其原嵌合抗体C11C及C19C的细胞结合强度相当。Figure 2: In vitro binding activity assay of humanized antibody and CHO-1R expressing CHO cells in vitro. The humanized antibodies huC11 and huC19 were comparable in cell binding strength to their original chimeric antibodies C11C and C19C, respectively.
图3：人源化抗体huC11阻断单核细胞增殖的功能实验。(A).抗体阻断IL-34刺激的单核细胞增殖。(B).抗体阻断CSF-1刺激的单核细胞增殖。Figure 3: Functional experiment of humanized antibody huC11 blocking monocyte proliferation. (A). The antibody blocks IL-34 stimulated monocyte proliferation. (B). The antibody blocks CSF-1 stimulated monocyte proliferation.
图4：人源化抗体huC19阻断单核细胞增殖的功能实验。(A).抗体阻断IL-34刺激的单核细胞增殖。(B).抗体阻断CSF-1刺激的单核细胞增殖。Figure 4: Functional experiment of humanized antibody huC19 blocking monocyte proliferation. (A). The antibody blocks IL-34 stimulated monocyte proliferation. (B). The antibody blocks CSF-1 stimulated monocyte proliferation.
图5：人源化抗体huC19I体内药效实验。抗体抑制MDA-MB-231肿瘤细胞的增长。Figure 5: In vivo pharmacodynamic experiment of humanized antibody huC19I. The antibody inhibits the growth of MDA-MB-231 tumor cells.
图6：人源化抗体huC19I体内药效实验。同对照组相比，抗CSF1R抗体对动物体重没有显著影响。Figure 6: In vivo pharmacodynamic experiment of humanized antibody huC19I. Anti-CSF1R antibodies had no significant effect on animal body weight compared to the control group.
发明详述Detailed description of the invention
一、术语First, the term
为了更容易理解本发明，以下具体定义了某些技术和科学术语。除显而易见在本文件中的它处另有明确定义，否则本文使用的所有其它技术和科学术语都具有本发明所属领域的一般技术人员通常理解的含义。In order to more easily understand the present invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined 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.
本发明所用氨基酸三字母代码和单字母代码如J.Biol.Chem,243,p3558(1968)中所述。The three-letter code and the one-letter code for amino acids used in the present invention are as described in J. Biol. Chem, 243, p3558 (1968).
本发明所述的术语“抗体”指免疫球蛋白，是由两条相同的重链和两条相同的轻链通过链间二硫键连接而成的四肽链结构。免疫球蛋白重链恒定区的氨基酸组成和排列顺序不同，故其抗原性也不同。据此，可将免疫球蛋白分为五类，或称为免疫球蛋白的同种型，即IgM，IgD，IgG，IgA和IgE，其相应的重链分别为μ链，δ链，γ链，α链和ε链。同一类Ig根据其铰链区氨基酸组成和重链二硫键的数目和位置的差别，又可分为不同的亚类，如IgG可分为IgG1，IgG2，IgG3，IgG4。轻链通过恒定区的不同分为κ链或λ链。五类Ig中第每类Ig都可以有κ链或λ链。The term "antibody" as used in the present invention refers to an immunoglobulin which is a tetrapeptide chain structure in which two identical heavy chains and two identical light chains are linked by interchain disulfide bonds. The immunoglobulin heavy chain constant region has different amino acid composition and arrangement order, so its antigenicity is also different. Accordingly, immunoglobulins can be classified into five classes, or isoforms of immunoglobulins, namely IgM, IgD, IgG, IgA and IgE, and the corresponding heavy chains are μ chain, δ chain, γ chain, respectively. , α chain and ε chain. The same type of Ig can be divided into different subclasses according to the difference in the amino acid composition of the hinge region and the number and position of heavy chain disulfide bonds. For example, IgG can be classified into IgG1, IgG2, IgG3, and IgG4. Light chains are classified as either a kappa chain or a lambda chain by the constant region. Each of the five types of Ig may have a kappa chain or a lambda chain.
在本发明中，本发明所述的抗体轻链可变区可进一步包含轻链恒定区，所述的轻链恒定区包含人源或鼠源的κ、λ链或其变体。In the present invention, the antibody light chain variable region of the present invention may further comprise a light chain constant region comprising a human or murine kappa, lambda chain or a variant thereof.
在本发明中，本发明所述的抗体重链可变区可进一步包含重链恒定区，所述的重链 恒定区包含人源或鼠源的IgG1,2,3,4或其变体。In the present invention, the antibody heavy chain variable region of the present invention may further comprise a heavy chain constant region comprising human or murine IgG1, 2, 3, 4 or a variant thereof.
抗体重链和轻链靠近N端的约110个氨基酸的序列变化很大，为可变区(V区)；靠近C端的其余氨基酸序列相对稳定，为恒定区(C区)。可变区包括3个高变区(HVR)和4个序列相对保守的骨架区(FR)。3个高变区决定抗体的特异性，又称为互补性决定区(CDR)。每条轻链可变区(VL)和重链可变区(VH)由3个CDR区4个FR区组成，从氨基端到羧基端依次排列的顺序为：FR1，CDR1，FR2，CDR2，FR3，CDR3，FR4。轻链的3个CDR区指LCDR1，LCDR2，和LCDR3；重链的3个CDR区指HCDR1，HCDR2和HCDR3。发明所述的抗体或抗原结合片段的VL区和VH区的CDR氨基酸残基在数量和位置符合已知的IMGT编号规则。The sequence of about 110 amino acids near the N-terminus of the antibody heavy and light chains varies greatly, being the variable region (V region); the remaining amino acid sequence near the C-terminus is relatively stable and is a constant region (C region). The variable region includes three hypervariable regions (HVR) and four relatively conserved framework regions (FR). The three hypervariable regions determine the specificity of the antibody, also known as the complementarity determining region (CDR). Each of the light chain variable region (VL) and the heavy chain variable region (VH) consists of three CDR regions and four FR regions, and the order from the amino terminus to the carboxy terminus is: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3. The CDR amino acid residues of the VL and VH regions of the antibodies or antigen-binding fragments of the invention meet the known IMGT numbering rules in number and position.
术语“抗原呈递细胞”或“APC”是在其表面上展示与MHC复合的外来抗原的细胞。T细胞利用T细胞受体(TCR)识别这种复合物。APC的实例包括但不限于树突细胞(DC)、外用血单个核细胞(PBMC)、单核细胞、B淋巴母细胞和单核细胞衍生的树突细胞(DC)。术语“抗原呈递”是指APC捕获抗原和使它们能够被T细胞识别的过程，例如作为MHC-I/MHC-II偶联物的组分。The term "antigen presenting cell" or "APC" is a cell which displays a foreign antigen complexed with MHC on its surface. T cells recognize this complex using the T cell receptor (TCR). Examples of APCs include, but are not limited to, dendritic cells (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 MHC-I/MHC-II conjugates.
术语“CSF-1R”指集落刺激因子-1受体蛋白，是一种带有N端胞外结构域和酪氨酸激酶活性的C端胞内结构域的单程跨膜受体。CSF-1R为含有免疫球蛋白(Ig)基序的RTK家族的一名成员，以受体的胞外部分中的重复Ig域为特征。CSF-1R的激活由其配体M-CSF或白介素IL-34介导，其信号在免疫应答、骨重建及在生殖***中都具有重要的生理学作用。The term "CSF-1R" refers to a colony stimulating factor-1 receptor protein, a single-pass transmembrane receptor with a C-terminal intracellular domain with an N-terminal extracellular domain and tyrosine kinase activity. CSF-1R is a member of the RTK family of immunoglobulin (Ig) motifs and is characterized by a repetitive Ig domain in the extracellular portion of the receptor. Activation of CSF-1R is mediated by its ligand M-CSF or interleukin IL-34, and its signaling has important physiological roles in immune response, bone remodeling, and in the reproductive system.
术语“重组人抗体”包括通过重组方法制备、表达、创建或分离的人抗体，所涉及的技术和方法在本领域中是熟知的，诸如(1)从人免疫球蛋白基因的转基因、转染色体动物(例如小鼠)或由其制备的杂交瘤中分离的抗体；(2)从经转化以表达抗体的宿主细胞如转染瘤中分离的抗体；(3)从重组组合人抗体文库中分离的抗体；以及(4)通过将人免疫球蛋白基因序列剪接到其他DNA序列等方法制备、表达、创建或分离的抗体。此类重组人抗体包含可变区和恒定区，这些区域利用特定的由种系基因编码的人种系免疫球蛋白序列，但也包括随后诸如在抗体成熟过程中发生的重排和突变。The term "recombinant human antibody" includes human antibodies which are prepared, expressed, created or isolated by recombinant methods, and the techniques and methods involved are well known in the art, such as (1) transgenes from human immunoglobulin genes, transgenic chromosomes. An antibody isolated from an animal (eg, a mouse) or a hybridoma prepared therefrom; (2) an antibody isolated from a host cell transformed with an antibody, such as a transfectoma; (3) isolated from a recombinant combinatorial human antibody library And (4) an antibody prepared, expressed, created or isolated by splicing a human immunoglobulin gene sequence to other DNA sequences. Such recombinant human antibodies comprise variable regions and constant regions that utilize specific human germline immunoglobulin sequences encoded by germline genes, but also include subsequent rearrangements and mutations such as occur during antibody maturation.
术语“鼠源抗体”在本发明中为根据本领域知识和技能制备的对人CSF-1R的单克隆抗体。制备时用CSF-1R抗原注射试验对象，然后分离表达具有所需序列或功能特性的抗体的杂交瘤。在本发明一个优选的实施方案中，所述的鼠源CSF-1R抗体或其抗原结合片段，可进一步包含鼠源κ、λ链或其变体的轻链恒定区，或进一步包含鼠源IgG1,IgG2,IgG3或IgG4或其变体的重链恒定区。The term "murine antibody" is in the present invention a monoclonal antibody to human CSF-1R prepared according to the knowledge and skill in the art. The test subject is injected with the CSF-1R antigen at the time of preparation, and then the hybridoma expressing the antibody having the desired sequence or functional properties is isolated. In a preferred embodiment of the present invention, the murine CSF-1R antibody or antigen-binding fragment thereof may further comprise a light chain constant region of a murine kappa, a lambda chain or a variant thereof, or further comprising a murine IgG1 , heavy chain constant region of IgG2, IgG3 or IgG4 or a variant thereof.
术语“人抗体”包括具有人种系免疫球蛋白序列的可变和恒定区的抗体。本发明的人抗体可包括不由人种系免疫球蛋白序列编码的氨基酸残基(如通过体外随机或位点特异性诱变或通过体内体细胞突变所引入的突变)。然而，术语“人抗体”不包括这样的抗体，即其中已将衍生自另一种哺乳动物物种(诸如小鼠)种系的CDR序列移植到人骨架序列上(即“人源化抗体”)。The term "human antibody" includes antibodies having variable and constant regions of human germline immunoglobulin sequences. Human antibodies of the invention can include amino acid residues that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody" does not include an antibody in which a CDR sequence derived from a germline of another mammalian species, such as a mouse, has been grafted onto a human framework sequence (ie, "humanized antibody"). .
术语“人源化抗体(humanized antibody)”，也称为CDR移植抗体(CDR-grafted antibody)，是指将小鼠的CDR序列移植到人的抗体可变区框架中产生的抗体。可以克服嵌合抗体由于携带大量小鼠蛋白成分，从而诱导的强烈的免疫应答反应。为避免在免疫原性下降的同时引起活性的下降，可对所述的人抗体可变区可进行最少反向突变，以保持活性。The term "humanized antibody", also referred to as a CDR-grafted antibody, refers to an antibody produced by grafting a CDR sequence of a mouse into a human antibody variable region framework. It is possible to overcome the strong immune response induced by chimeric antibodies by carrying a large amount of mouse protein components. To avoid a decrease in activity while reducing immunogenicity, the human antibody variable region can be subjected to minimal reverse mutation to maintain activity.
术语“嵌合抗体(chimeric antibody)”，是将鼠源性抗体的可变区与人抗体的恒定区融合而成的抗体，可以减轻鼠源性抗体诱发的免疫应答反应。建立嵌合抗体，要选建立分泌鼠源性特异性单抗的杂交瘤，然后从小鼠杂交瘤细胞中克隆可变区基因，再要据需要克隆人抗体的恒定区基因，将小鼠可变区基因与人恒定区基因连接成嵌合基因后***人载体中，最后在真核工业***或原核工业***中表达嵌合抗体分子。人抗体的恒定区可选自人源IgG1,IgG2,IgG3或IgG4或其变体的重链恒定区，优选包含人源IgG2或IgG4重链恒定区，或者使用氨基酸突变后无ADCC(antibody-dependent cell-mediated cytotoxicity，抗体依赖的细胞介导的细胞毒作用)毒性的IgG1。The term "chimeric antibody" is an antibody obtained by fusing a variable region of a murine antibody with a constant region of a human antibody, and can alleviate an immune response induced by a murine antibody. To establish a chimeric antibody, a hybridoma that secretes a murine-specific monoclonal antibody is selected, and then the variable region gene is cloned from the mouse hybridoma cell, and the constant region gene of the human antibody is cloned as needed to change the mouse. The region gene and the human constant region gene are ligated into a chimeric gene and inserted into a human vector, and 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 region of human IgG1, IgG2, IgG3 or IgG4 or a variant thereof, preferably comprising a human IgG2 or IgG4 heavy chain constant region, or without ADCC (antibody-dependent) after amino acid mutation Cell-mediated cytotoxicity, antibody-dependent cell-mediated cytotoxicity) toxic IgG1.
术语“抗原结合片段”是指抗体的抗原结合片段及抗体类似物，其通常包括至少部分母体抗体(parental antibody)的抗原结合区或可变区(例如一个或多个CDR)。抗体片段保留母体抗体的至少某些结合特异性。通常，当基于摩尔来表示活性时，抗体片段保留至少10％的母体结合活性。优选地，抗体片段保留至少20％、50％、70％、80％、90％、95％或100％或更多的母体抗体对靶标的结合亲和力。抗原结合片段实例包括但不限于：Fab、Fab′、F(ab′)2、Fv片段、线性抗体(linear antibody)、单链抗体、纳米抗体、结构域抗体和多特异性抗体。工程改造的抗体变体综述于Holliger和Hudson(2005)Nat.Biotechnol.23：1126-1136中。The term "antigen-binding fragment" refers to an antigen-binding fragment of an antibody and an antibody analog, which typically includes at least a portion of an antigen binding or variable region (eg, one or more CDRs) of a parental antibody. The antibody fragment retains at least some of the binding specificity of the parent antibody. Generally, when the activity is expressed on a molar basis, the antibody fragment retains at least 10% of the parental 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 to the target. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv fragments, linear antibodies, single chain antibodies, Nanobodies, domain antibodies, and multispecific antibodies. Engineered antibody variants are reviewed in Holliger and Hudson (2005) Nat. Biotechnol. 23: 1126-1136.
“Fab片段”由一条轻链和一条重链的CH1及可变区组成。Fab分子的重链不能与另一个重链分子形成二硫键。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.
“Fc”区含有包含抗体的CH1和CH2结构域的两个重链片段。两个重链片段由两个或多个二硫键并通过CH3结构域的疏水作用保持在一起。The "Fc" region contains two heavy chain fragments comprising the CH1 and CH2 domains of the antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by the hydrophobic action of the CH3 domain.
“Fab′片段”含有一条轻链和包含VH结构域和CH1结构域以及CH1和CH2结构域 之间区域的一条重链的部分，由此可在两个Fab′片段的两条重链之间形成链间二硫键以形成F(ab′)2分子。A "Fab' fragment" contains a light chain and a portion of a heavy chain comprising a VH domain and a CH1 domain and a region between the CH1 and CH2 domains, thereby being between the two heavy chains of the two Fab' fragments An interchain disulfide bond is formed to form a F(ab')2 molecule.
“F(ab′)2片段”含有两条轻链和两条包含CH1和CH2结构域之间的恒定区的部分的重链，由此在两条重链间形成链间二硫键。因此，F(ab′)2片段由通过两条重链间的二硫键保持在一起的两个Fab′片段组成。The "F(ab')2 fragment" contains two light chains and two heavy chains comprising portions of the constant region between the CH1 and CH2 domains, thereby forming an interchain disulfide bond between the two heavy chains. Thus, the F(ab')2 fragment consists of two Fab' fragments held together by a disulfide bond between the two heavy chains.
“Fv区”包含来自重链和轻链二者的可变区，但缺少恒定区。The "Fv region" contains variable regions from both heavy and light chains, but lacks a constant region.
术语“多特异性抗体”按其最广义使用，涵盖具有多表位特异性的抗体。这些多特异性抗体包括但不限于：包含重链可变区(VH)和轻链可变区(VL)的抗体，其中该VH-VL单元具有多表位特异性；具有两个或多个VL和VH区的抗体，每个VH-VL单元与不同的靶点或同一个靶点的不同表位结合；具有两个或更多个单可变区的抗体，每个单可变区与不同的靶点或同一个靶点的不同的表位结合；全长抗体、抗体片段、双抗体(diabodies)、双特异性双抗体和三抗体(triabodies)、己共价或非共价连接在一起的抗体片段等。The term "multispecific antibody" is used in its broadest sense to encompass antibodies having 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; having two or more Antibodies of the VL and VH regions, each VH-VL unit binding to a different target or a different epitope of the same target; antibodies having two or more single variable regions, each single variable region Different targets or different epitopes of the same target; full-length antibodies, antibody fragments, diabodies, bispecific diabodies and triabodies, covalently or non-covalently linked Along with antibody fragments and the like.
术语“单链抗体”是由抗体的重链可变区(VH)和轻链可变区(VL)通过一段连接肽连接而成的单链重组蛋白，它是具有完全抗原结合位点的最小抗体片段。The term "single-chain antibody" is a single-stranded recombinant protein joined by a heavy chain variable region (VH) and a light chain variable region (VL) of a antibody via a ligation peptide, which is the smallest with complete antigen binding sites. Antibody fragment.
术语“结构域抗体片段”是仅含有重链可变区或轻链可变区链的具有免疫学功能的免疫球蛋白片段。在某些情况下，两个或多个VH区与肽接头共价连接以形成二价结构域抗体片段。二价结构域抗体片段的两个VH区可靶向相同或不同抗原。The term "domain antibody fragment" is an immunologically functional immunoglobulin fragment containing only a heavy chain variable region or a light chain variable region chain. In some cases, two or more VH regions are covalently joined 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.
本发明的术语“与CSF-1R结合”，指能与人CSF-1R相互作用。本发明的术语“抗原结合位点”指抗原上不连续的，由本发明抗体或抗原结合片段识别的三维空间位点。The term "binding to CSF-1R" in the present invention means that it is capable of interacting with human CSF-1R. The term "antigen binding site" as used in the present invention refers to a three-dimensional spatial site that is discrete on an antigen and is recognized by an antibody or antigen-binding fragment of the present invention.
在类似“具有3、2、1或0个氨基酸突变”中“氨基酸突变”是指相较于原蛋白质或多肽，变体蛋白质或多肽存在氨基酸的突变，包括在原蛋白质或多肽的基础上发生相应数目氨基酸的***、缺失或替换。"Amino acid mutation" in a "mutant with 3, 2, 1 or 0 amino acid" means that the amino acid is mutated in the variant protein or polypeptide compared to the original protein or polypeptide, including corresponding to the original protein or polypeptide. Insertion, deletion or substitution of a number of amino acids.
在类似“在如SEQ ID NO:X、SEQ ID NO:Y和SEQ ID NO:Z所示的CDR1、CDR2和CDR3的基础上分别具有3、2、1或0个氨基酸突变的CDR变体”的描述中，示例性的解释是对CDR的突变可以包含3个、2个、1个或0个氨基酸的突变，CDR1、CDR2和CDR3之间可以任选地选择相同或不同数目的氨基酸残基进行突变，例如在如SEQ ID NO:X、SEQ ID NO:Y和SEQ ID NO:Z所示的CDR1、CDR2和CDR3的基础上，对CDR1进行1个氨基酸的突变，对CDR2和CDR3进行0个氨基酸突变。当对某个CDR进行0个氨基酸突变时，则该具有0个氨基酸突变的CDR变体仍是该CDR本身。CDR variants having a 3, 2, 1 or 0 amino acid mutation, respectively, based on "CDR1, CDR2 and CDR3 as set forth in SEQ ID NO: X, SEQ ID NO: Y and SEQ ID NO: Z" In the description, an exemplary explanation is that a mutation to a CDR may comprise a mutation of 3, 2, 1 or 0 amino acids, and optionally the same or a different number of amino acid residues may be selected between CDR1, CDR2 and CDR3. Mutation is carried out, for example, on the basis of CDR1, CDR2 and CDR3 as shown in SEQ ID NO: X, SEQ ID NO: Y and SEQ ID NO: Z, CDR1 is subjected to 1 amino acid mutation, and CDR2 and CDR3 are subjected to 0. Amino acid mutations. When a 0 amino acid mutation is made to a certain CDR, then the CDR variant with a 0 amino acid mutation remains the CDR itself.
术语“表位”是指抗原上与免疫球蛋白或抗体特异性结合的位点。表位可以由相邻的氨基酸、或通过蛋白质的三级折叠而并列的不相邻的氨基酸形成。由相邻的氨基酸形成 的表位通常在暴露于变性溶剂后保持，而通过三级折叠形成的表位通常在变性溶剂处理后丧失。表位通常以独特的空间构象包括至少3-15个氨基酸。确定什么表位由给定的抗体结合的方法在本领域中是熟知的，包括免疫印迹和免疫沉淀检测分析等。确定表位的空间构象的方法包括本领域中的技术和本文所述的技术，例如X射线晶体分析法和二维核磁共振等。The term "epitope" refers to a site on an antigen that specifically binds to an immunoglobulin or antibody. An epitope can be formed by an adjacent amino acid, or a non-adjacent amino acid juxtaposed by a tertiary folding of a protein. Epitopes formed from adjacent amino acids are typically retained after exposure to denaturing solvents, while epitopes formed by tertiary folding are typically lost after treatment with denaturing solvents. Epitopes typically include at least 3-15 amino acids in a unique spatial conformation. Methods for determining what epitope is bound by a given antibody are well known in the art, including immunoblotting and immunoprecipitation assays, and the like. Methods for determining the spatial conformation of an epitope include techniques in the art and techniques described herein, such as X-ray crystallography and two-dimensional nuclear magnetic resonance.
本发明所用的术语“特异性结合”、“选择性结合”是指抗体与预定的抗原上的表位结合。通常，当使用重组人CSF-1R作为分析物并使用抗体作为配体，在仪器中通过表面等离子体共振(SPR)技术测定时，抗体以大约低于10 -7M或甚至更小的平衡解离常数(K D)与预定的抗原结合，并且其与预定抗原结合的亲和力是其与预定抗原或紧密相关的抗原之外的非特异性抗原(如BSA等)结合的亲和力的至少两倍。术语“识别抗原的抗体”在本文中可以与术语“特异性结合的抗体”互换使用。 As used herein, the terms "specifically bind" and "selectively bind" refer to the binding of an antibody to an epitope on a predetermined antigen. Typically, when recombinant human CSF-1R is used as an analyte and an antibody is used as a ligand, the antibody has an equilibrium solution of less than about 10 -7 M or even less when measured by surface plasmon resonance (SPR) techniques in an instrument. The isolating constant (K D ) binds to a predetermined antigen, and its affinity for binding to a predetermined antigen is at least twice its affinity for binding to a non-specific antigen other than the predetermined antigen or a closely related antigen (such as BSA, etc.). The term "antibody recognizing an antigen" can be used interchangeably herein with the term "specifically bound antibody".
当术语“竞争”用于竞争相同表位的抗原结合蛋白(例如中和抗原结合蛋白或中和抗体)的情况中时，意指在抗原结合蛋白之间竞争，其通过以下测定法来测定：在所述测定法中，待检测的抗原结合蛋白(例如抗体或其抗原结合片段)防止或抑制(例如降低)参考抗原结合蛋白(例如配体或参考抗体)与共同抗原的特异性结合。众多类型的竞争性结合测定可用于确定一种抗原结合蛋白是否与另一种竞争，这些测定例如：固相直接或间接放射免疫测定(RIA)、固相直接或间接酶免疫测定(EIA)、夹心竞争测定(参见例如Stahli等，1983，Methodsin Enzymology 9：242-253)；固相直接生物素-亲和素EIA(参见例如Kirkland等，1986，J.Immunol.137：3614-3619)、固相直接标记测定、固相直接标记夹心测定(参见例如Harlow和Lane，1988，Antibodies，A Laboratory Manual(抗体，实验室手册)，Cold Spring Harbor Press)；用I-125标记物的固相直接标记RIA(参见例如Morel等，1988，Molec.Immunol.25：7-15)；固相直接生物素-亲和素EIA(参见例如Cheung，等，1990，Virology176：546-552)；和直接标记的RIA(Moldenhauer等，1990，Scand.J.Immunol.32：77-82)。通常所述测定法涉及使用能与带有未标记的检测抗原结合蛋白及标记的参考抗原结合蛋白结合的纯化抗原(所述抗原在固态表面或细胞表面)。在待测抗原结合蛋白存在下，测量结合于固态表面或细胞的标记的量，来测量竞争性抑制。通常，待测抗原结合蛋白是过量存在的。由竞争性测定(竞争抗原结合蛋白)鉴定的抗原结合蛋白包括：结合与参考抗原结合蛋白同一表位的抗原结合蛋白；和结合充分接近参考抗原结合蛋白的结合表位的邻近表位的抗原结合蛋白，所述两个表位在空间上互相妨碍发生结合。在本公开实施例中提供关于用于测定竞争性结合的方法的其它详细资料。通常当竞争的抗原结合蛋白过量存在时，其将抑制(例如降低)至少40-45％、45-50％、50-55％、55-60％、60-65％、 65-70％、70-75％或75％或更多参考抗原结合蛋白与共同抗原的特异性结合。在某些情况下，结合被抑制至少80-85％、85-90％、90-95％、95-97％或97％或更多。When the term "competition" is used in the context of an antigen binding protein that competes for the same epitope (eg, neutralizing an antigen binding protein or a neutralizing antibody), it means competition between antigen binding proteins, which is determined by the following assay: In the assay, the antigen binding protein (eg, an antibody or antigen-binding fragment thereof) to be detected prevents or inhibits (eg, reduces) the specific binding of a reference antigen binding protein (eg, a ligand or reference antibody) to a common antigen. Numerous types of competitive binding assays can be used to determine whether an antigen binding protein competes with another assay 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); solid phase direct biotin-avidin EIA (see, eg, Kirkland et al, 1986, J. Immunol. 137: 3614-3619), solid Direct labeling assay, solid phase direct label sandwich assay (see, eg, Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press); solid phase direct labeling with I-125 label RIA (see, eg, Morel et al, 1988, Molec. Immunol. 25: 7-15); solid phase direct biotin-avidin EIA (see, eg, Cheung, et al, 1990, Virology 176: 546-552); and directly labeled RIA (Moldenhauer et al, 1990, Scand. J. Immunol. 32: 77-82). Typically, the assay involves the use of a purified antigen (either on a solid surface or on the cell surface) that binds to a reference antigen binding protein with an unlabeled detection antigen binding protein and label. Competitive inhibition is measured by measuring the amount of label bound to a solid surface or cell in the presence of the antigen binding protein to be tested. Usually, the antigen binding protein to be tested is present in excess. An antigen binding protein identified by a competitive assay (competing antigen binding protein) comprises: an antigen binding protein that binds to the same epitope as the reference antigen binding protein; and an antigen binding that binds to a neighboring epitope that is sufficiently close to the binding epitope of the reference antigen binding protein. Protein, the two epitopes that interfere with each other spatially. Additional details regarding methods for determining competitive binding are provided in embodiments of the present disclosure. Typically, when a competing antigen binding protein is present in excess, it will inhibit (eg, reduce) at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70. -75% or 75% or more of the specific binding of the reference antigen binding protein to the common antigen. In some cases, binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97%, or 97% or more.
术语“交叉反应”是指本发明的抗体与来自不同物种的CSF-1R结合的能力。例如，结合人CSF-1R的本发明的抗体也可以结合另一物种的CSF-1R。交叉反应性是通过在结合测定(例如SPR和ELISA)中检测与纯化抗原的特异性反应性，或与生理表达CSF-1R的细胞的结合或功能性相互作用来测量。确定交叉反应性的方法包括如本文所述的标准结合测定，例如表面等离子体共振(SPR)分析，或流式细胞术。The term "cross-reactive" refers to the ability of an antibody of the invention to bind to CSF-1R from a different species. For example, an antibody of the invention that binds to human CSF-1R can also bind to another species of CSF-1R. Cross-reactivity is measured by detecting specific reactivity with purified antigens in binding assays (eg, SPR and ELISA), or binding or functional interactions with cells that physiologically express CSF-1R. Methods for determining cross-reactivity include standard binding assays as described herein, such as surface plasmon resonance (SPR) analysis, or flow cytometry.
术语“宿主细胞”是指已向其中引入了表达载体的细胞。宿主细胞可包括细菌、微生物、植物或动物细胞。易于转化的细菌包括肠杆菌科(enterobacteriaceae)的成员，例如大肠杆菌(Escherichia coli)或沙门氏菌(Salmonella)的菌株；芽孢杆菌科(Bacillaceae)例如枯草芽孢杆菌(Bacillus subtilis)；肺炎球菌(Pneumococcus)；链球菌(Streptococcus)和流感嗜血菌(Haemophilus influenzae)。适当的微生物包括酿酒酵母(Saccharomyces cerevisiae)和毕赤酵母(Pichia pastoris)。适当的动物宿主细胞系包括CHO(中国仓鼠卵巢细胞系)、HEK293细胞(非限制性示为HEK293E细胞)和NS0细胞。The term "host cell" refers to a cell into which an expression vector has been introduced. Host cells can include bacterial, microbial, plant or animal cells. Bacteria susceptible to transformation 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 (not limited to HEK293E cells) and NSO cells.
术语“抑制”或“阻断”可互换使用，并涵盖部分和完全抑制/阻断这两者。配体的抑制/阻断优选地降低或改变无抑制或阻断的情况下发生配体结合时出现活性的正常水平或类型。抑制和阻断也旨在包括与抗CSF-1R抗体接触时，与未与抗CSF-1R抗体接触的配体相比，任何可测量的配体结合亲和力降低。The terms "inhibiting" or "blocking" are used interchangeably and encompass both partial and complete inhibition/blocking. The inhibition/blocking of the ligand preferably reduces or alters the normal level or type of activity that occurs when ligand binding occurs without inhibition or blockade. Inhibition and blockade are also intended to include a decrease in any measurable ligand binding affinity when contacted with an anti-CSF-1R antibody, compared to a ligand not contacted with an anti-CSF-1R antibody.
术语“抑制生长”(例如涉及细胞)旨在包括细胞生长任何可测量的降低。The term "inhibiting growth" (eg, involving cells) is intended to include any measurable reduction in cell growth.
术语“诱导免疫应答”和“增强免疫应答”可互换使用，并指免疫应答对特定抗原的剌激(即，被动或适应性的)。针对诱导CDC或ADCC的术语“诱导”是指剌激特定的直接细胞杀伤机制。The terms "inducing an immune response" and "enhancing an immune response" are used interchangeably and refer to the stimulation (ie, passive or adaptive) of an immune response to a particular antigen. The term "inducing" for inducing CDC or ADCC refers to stimulating a specific direct cell killing mechanism.
本发明中所述的“ADCC”，即antibody-dependent cell-mediated cytotoxicity，抗体依赖的细胞介导的细胞毒作用，是指表达Fc受体的细胞通过识别抗体的Fc段直接杀伤被抗体包被的靶细胞。可通过对IgG上Fc段的修饰，增强或降低降低或消除抗体的ADCC效应功能。所述的修饰指在抗体的重链恒定区进行突变。The "ADCC" described in the present invention, that is, antibody-dependent cell-mediated cytotoxicity, is an antibody-dependent cell-mediated cytotoxicity, which means that a cell expressing an Fc receptor directly kills an antibody by Fc segment of the recognition antibody. Target cells. The ADCC effector function of the antibody can be reduced or eliminated by modification of the Fc fragment on IgG. Said modification refers to mutations in the heavy chain constant region of the antibody.
生产和纯化抗体和抗原结合片段的方法在现有技术中熟知和能找到，如冷泉港的抗体实验技术指南，5-8章和15章。如，老鼠可以用人CSF-1R或其片段免疫，所得到的抗体能被复性，纯化，并且可以用常规的方法进行氨基酸测序。抗原结合片段同样可以用常规方法制备。发明所述的抗体或抗原结合片段用基因工程方法在非人源的CDR区加上一个或多个人FR区。人FR种系序列可以从ImMunoGeneTics(IMGT)的网站 http://imgt.cines.fr得到，或者从免疫球蛋白杂志，2001ISBN012441351上获得。Methods for producing and purifying antibodies and antigen-binding fragments are well known and can be found in the prior art, such as the Cold Spring Harbor Antibody Technical Guide, Chapters 5-8 and 15. For example, a mouse can be immunized with human CSF-1R or a fragment thereof, and the obtained antibody can be renatured, purified, and subjected to amino acid sequencing by a conventional method. The antigen-binding fragment can also be prepared by a conventional method. The antibodies or antigen-binding fragments of the invention are genetically engineered to add one or more human FR regions in a non-human CDR region. Human FR germline sequences are available on the website of ImMunoGeneTics (IMGT) http://imgt.cines.fr or from the Journal of Immunoglobulins, 2001 ISBN 014441351.
本发明工程化的抗体或抗原结合片段可用常规方法制备和纯化。相应抗体的cDNA序列可以克隆并重组至GS表达载体。重组的免疫球蛋白表达载体可以稳定地转染CHO细胞。作为一种更推荐的现有技术，哺乳动物类表达***会导致抗体的糖基化，特别是在Fc区的高度保守N端。通过表达与人源抗原特异性结合的抗体得到稳定的克隆。阳性的克隆在生物反应器的无血清培养基中扩大培养以生产抗体。分泌了抗体的培养液可以用常规技术纯化、收集。抗体可用常规方法进行过滤浓缩。可溶的混合物和多聚体，也可以用常规方法去除，比如分子筛，离子交换。得到的产物需立即冷冻，如-70℃，或者冻干。The engineered antibodies or antigen-binding fragments of the invention can be prepared and purified by conventional methods. The cDNA sequence of the corresponding antibody can be cloned and recombined into a GS expression vector. The recombinant immunoglobulin expression vector can stably transfect CHO cells. As a more preferred prior art, mammalian expression systems result in glycosylation of antibodies, particularly at the highly conserved N-terminus of the Fc region. Stable clones are obtained by expressing antibodies that specifically bind to human antigens. Positive clones were expanded in serum-free medium in a bioreactor to produce antibodies. The culture medium from which the antibody is secreted can be purified and collected by a conventional technique. The antibody can be concentrated by filtration in a conventional manner. Soluble mixtures and multimers can also be removed by conventional methods such as molecular sieves, ion exchange. The resulting product needs to be frozen immediately, such as -70 ° C, or lyophilized.
本发明的抗体指单克隆抗体。本发明所述的单克隆抗体(mAb)，指由单一的克隆细胞株得到的抗体，所述的细胞株不限于真核的，原核的或噬菌体的克隆细胞株。单克隆抗体或抗原结合片段可以用如杂交瘤技术、重组技术、噬菌体展示技术，合成技术(如CDR-grafting)，或其它现有技术进行重组得到。The antibody of the present invention refers to a monoclonal antibody. The monoclonal antibody (mAb) of the present invention refers to an antibody obtained from a single clonal cell strain, and the cell strain is not limited to a eukaryotic, prokaryotic or phage clonal cell strain. Monoclonal antibodies or antigen-binding fragments can be obtained recombinantly using, for example, hybridoma technology, recombinant techniques, phage display technology, synthetic techniques (e.g., CDR-grafting), or other prior art techniques.
“给予”和“处理”当应用于动物、人、实验受试者、细胞、组织、器官或生物流体时，是指外源性药物、治疗剂、诊断剂或组合物与动物、人、受试者、细胞、组织、器官或生物流体的接触。“给予”和“处理”可以指例如治疗、药物代谢动力学、诊断、研究和实验方法。细胞的处理包括试剂与细胞的接触，以及试剂与流体的接触，其中所述流体与细胞接触。“给予”和“处理”还意指通过试剂、诊断、结合组合物或通过另一种细胞体外和离体处理例如细胞。“处理”当应用于人、兽医学或研究受试者时，是指治疗处理、预防或预防性措施，研究和诊断应用。"Administration" and "treatment" when applied to an animal, human, experimental subject, cell, tissue, organ or biological fluid, refers to an exogenous drug, therapeutic agent, diagnostic agent or composition and animal, human, subject Contact of the test subject, cell, tissue, organ or biological fluid. "Administration" and "treatment" can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Treatment of the cells includes contact of the reagents with the cells, and contact of the reagents with the fluid, wherein the fluids are in contact with the cells. "Administering" and "treating" also means treating, for example, cells in vitro and ex vivo by reagents, diagnostics, binding compositions, or by another cell. "Treatment", when applied to a human, veterinary or research subject, refers to therapeutic treatment, prophylactic or preventive measures, research and diagnostic applications.
“治疗”意指给予患者内用或外用治疗剂，诸如包含本发明的任一种结合化合物的组合物，所述患者具有一种或多种疾病症状，而已知所述治疗剂对这些症状具有治疗作用。通常，在受治疗患者或群体中以有效缓解一种或多种疾病症状的量给予治疗剂，无论是通过诱导这类症状退化还是抑制这类症状发展到任何临床右测量的程度。有效缓解任何具体疾病症状的治疗剂的量(也称作“治疗有效量”)可根据多种因素变化，例如患者的疾病状态、年龄和体重，以及药物在患者产生需要疗效的能力。通过医生或其它专业卫生保健人士通常用于评价该症状的严重性或进展状况的任何临床检测方法，可评价疾病症状是否已被减轻。尽本发明的实施方案(例如治疗方法或制品)在缓解每个患者都有的目标疾病症状方面可能无效，但是根据本领域已知的任何统计学检验方法如Student t检验、卡方检验、依据Mann和Whitney的U检验、Kruskal-Wallis检验(H检验)、Jonckheere-Terpstra检验和Wilcoxon检验确定，其在统计学显著数目的患者中应当 减轻目标疾病症状。"Treatment" means administering to a patient a therapeutic agent for internal or external use, such as a composition comprising any of the binding compounds of the present invention, the patient having one or more symptoms of the disease, and the therapeutic agent is known to have Therapeutic effect. Generally, a therapeutic agent is administered in a subject or population to be treated to effectively alleviate the symptoms of one or more diseases, whether by inducing such symptoms to degenerate or inhibiting the progression of such symptoms to any degree of clinical right measurement. The amount of therapeutic agent (also referred to as "therapeutically effective amount") effective to alleviate 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 a desired effect in the patient. Whether the symptoms of the disease have been alleviated can be assessed by any clinical test method commonly used by a physician or other professional health care provider to assess the severity or progression of the condition. Embodiments of the invention (e.g., methods of treatment or preparations) may be ineffective in alleviating the symptoms of the target disease in each patient, but according to any statistical test methods 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 the target disease symptoms should be alleviated in a statistically significant number of patients.
整个说明书和权利要求书中使用的术语“基本上由……组成”或其变形表示包括所有所述元件或元件组，并且任选包括与所述元件类似或不同性质的其它元件，所述其它元件非显著改变指定给药方案、方法或组合物的基本性质或新性质。作为非限制性例子，基本上由所提及的氨基酸序列组成的结合化合物还可以包括一种或多种氨基酸，其不显著影响结合化合物的性质。The term "consisting essentially of" or variations thereof, as used throughout the specification and claims, includes all such elements or groups of elements, and optionally includes other elements that are similar or different in nature to the elements, the other The element does not significantly alter the essential or novel properties of a given dosage regimen, method or composition. As a non-limiting example, a binding compound consisting essentially of the amino acid sequence recited may also include one or more amino acids that do not significantly affect the properties of the binding compound.
本发明所述的应用于某个对象的术语“天然存在的”是指这样的事实，即该对象可在自然界中发现。例如存在于可从自然界来源分离得到的生物体(包括病毒)、且未经人工在实验室中有意修饰的多肽序列或多核苷酸序列即是天然存在的。The term "naturally occurring" as applied to an object according to the invention refers to the fact that the object can be found in nature. For example, a polypeptide sequence or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a natural source and that has not been intentionally modified in the laboratory by humans is naturally occurring.
“有效量”包含足以改善或预防医字病症的症状或病症的量。有效量还意指足以允许或促进诊断的量。用于特定患者或兽医学受试者的有效量可依据以下因素而变化：如待治疗的病症、患者的总体健康情况、给药的方法途径和剂量以及副作用严重性。有效量可以是避免显著副作用或毒性作用的最大剂量或给药方案。An "effective amount" includes an amount sufficient to ameliorate or prevent a symptom or condition of a medical condition. An effective amount also means an amount sufficient to allow or facilitate the diagnosis. An effective amount for a particular patient or veterinary subject can vary depending on factors such as the condition to be treated, the overall health of the patient, the route and dosage of the method of administration, and the severity of the side effects. An effective amount can be the maximum dose or dosing regimen that avoids significant side effects or toxic effects.
“外源性”指要据背景在生物、细胞或人体外产生的物质。“内源性”指根据背景在细胞、生物或人体内产生的物质。"Exogenous" refers to a substance that is produced outside of a living organism, cell, or human body depending on the background. "Endogenous" refers to a substance produced in a cell, organism or human body depending on the background.
“同源性”是指两个多核苷酸序列之间或两个多肽之间的序列相似性。当两个比较序列中的位置均被相同碱基或氨基酸单体亚基占据时，例如如果两个DNA分子的每一个位置都被腺嘌呤占据时，那么所述分子在该位置是同源的。两个序列之间的同源性百分率是两个序列共有的匹配或同源位置数除以比较的位置数×100％的函数。例如，在序列最佳比对时，如果两个序列中的10个位置有6个匹配或同源，那么两个序列60％同源。一般而言，当比对两个序列而得到最大的同源性百分率时进行比较。"Homology" refers to sequence similarity between two polynucleotide sequences or between two polypeptides. When positions in both comparison sequences are 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 molecule is homologous at that position . The percent homology between the two sequences is a function of the number of matches or homology positions shared by the two sequences divided by the number of positions compared × 100%. For example, in the optimal alignment of sequences, if there are 6 matches or homologs in 10 positions in the two sequences, then the two sequences are 60% homologous. In general, comparisons are made when the maximum sequence of homology is obtained by aligning the two sequences.
本文使用的表述“细胞”、“细胞系”和“细胞培养物”可互换使用，并且所有这类名称都包括其后代。因此，单词“转化体”和“转化细胞”包括原代受试细胞和由其衍生的培养物，而不考虑转移数目。还应当理解的是，由于故意或非有意的突变，所有后代在DNA含量方面不可能精确相同。包括具有与最初转化细胞中筛选的相同的功能或生物学活性的突变后代。在意指不同名称的情况下，其由上下文清楚可见。The expression "cell", "cell line" and "cell culture" as used herein are used interchangeably and all such names include their progeny. Thus, the words "transformants" and "transformed cells" include primary test cells and cultures derived therefrom, regardless of the number of transfers. It should also be understood that all offspring may not be exactly identical in terms of DNA content due to intentional or unintentional mutations. Mutant progeny having the same function or biological activity as screened for in the originally transformed cell are included. In the case of a different name, it is clearly visible from the context.
“任选”或“任选地”意味着随后所描述地事件或环境可以但不必发生，该说明包括该事件或环境发生或不发生地场合。例如，“任选包含1-3个抗体重链可变区”意味着特定序列的抗体重链可变区可以但不必须存在。"Optional" or "optionally" means that the subsequently described event or environment may, but need not, occur, including where the event or environment occurs or does not occur. For example, "optionally comprising 1-3 antibody heavy chain variable regions" means that the antibody heavy chain variable region of a particular sequence may, but need not, be present.
“药物组合物”表示含有一种或多种本文所述化合物或其生理学上/可药用的盐或前体药物与其他化学组分的混合物，以及其他组分例如生理学/可药用的载体和赋形剂。药 物组合物的目的是促进对生物体的给药，利于活性成分的吸收进而发挥生物活性。"Pharmaceutical composition" means a mixture comprising one or more of the compounds described herein, or a physiologically/pharmaceutically acceptable salt or prodrug thereof, and other chemical components, as well as other components such as physiological/pharmaceutically acceptable carriers. And excipients. The purpose of the pharmaceutical composition is to promote the administration of the organism, thereby facilitating the absorption of the active ingredient and thereby exerting biological activity.
本发明提供有着高亲和力，高选择性，高生物活性的抗CSF-1R抗体，用于肿瘤或自身免疫疾病的单克隆抗体免疫疗法及其相关应用，以及其它相关用于CSF-1R阳性肿瘤或自身免疫疾病治疗的药物、组合物以及方法。The present invention provides anti-CSF-1R antibodies with high affinity, high selectivity, high biological activity, monoclonal antibody immunotherapy for tumor or autoimmune diseases and related applications, and other related CSF-1R positive tumors or Drugs, compositions and methods for the treatment of autoimmune diseases.
具体实施方式Detailed ways
以下结合实施例用于进一步描述本发明，但这些实施例并非限制着本发明的范围。本发明实施例中未注明具体条件的实验方法，通常按照常规条件，如冷泉港的抗体技术实验手册，分子克隆手册；或按照原料或商品制造厂商所建议的条件。未注明具体来源的试剂，为市场购买的常规试剂。The invention is further described in the following examples, but these examples are not intended to limit the scope of the invention. The experimental methods in the examples of the present invention which do not specify the specific conditions are usually in accordance with conventional conditions, such as the cold spring harbor antibody technology experiment manual, molecular cloning manual; or according to the conditions recommended by the raw material or commodity manufacturer. Reagents without specific source are routine reagents purchased from the market.
实施例1免疫抗原、筛选抗原的序列及制备Example 1 Sequence of immunizing antigen, screening antigen and preparation thereof
编码带His-Tag标签的人CSF-1R序列、编码带huFc标签的人CSF-1R序列由Integrated DNA Technology(IDT)公司合成，分别克隆到pTT5载体上(Biovector)。重组的CSF-1R蛋白在293T细胞(ATCC，CRL-3216 TM)表达后进行纯化。纯化的蛋白可用于下述各实施例实验中。 The human CSF-1R sequence encoding the His-Tag tag and the human CSF-1R sequence encoding the huFc tag were synthesized by Integrated DNA Technology (IDT) and cloned into the pTT5 vector (Biovector), respectively. Recombinant CSF-1R protein was purified after expression in 293T cells (ATCC, CRL-3216 TM) . The purified protein can be used in the experiments of the following examples.
Human CSF-1R ECD-His的序列:Human CSF-1R ECD-His sequence:
Figure PCTCN2018111818-appb-000001
Figure PCTCN2018111818-appb-000001
Human CSF-1R-huFc的序列：Human CSF-1R-huFc sequence:
Figure PCTCN2018111818-appb-000002
Figure PCTCN2018111818-appb-000002
Figure PCTCN2018111818-appb-000003
Figure PCTCN2018111818-appb-000003
实施例2 CSF-1R重组蛋白制备Example 2 Preparation of CSF-1R recombinant protein
1、带His标签的CSF-1R重组蛋白的纯化步骤：1. Purification steps of His-tagged CSF-1R recombinant protein:
将HEK293细胞表达的上清样品高速离心去除杂质，并将缓冲液换置换为磷酸缓冲液(PBS)，加入咪唑至终浓度为5mM。用含有5mM咪唑的PBS溶液平衡镍柱，冲洗2-5倍柱体积。将置换后的上清样品上柱。用含有5mM咪唑的PBS溶液冲洗柱子，至A280读数降至基线。后用PBS+10mM咪唑冲洗层析柱，除去非特异结合的杂蛋白，并收集流出液。再用含有300mM咪唑的PBS溶液洗脱目的蛋白，并收集洗脱峰。The supernatant sample expressed by HEK293 cells was centrifuged at high speed to remove impurities, and the buffer was exchanged for phosphate buffer (PBS), and imidazole was added to a final concentration of 5 mM. The nickel column was equilibrated with PBS solution containing 5 mM imidazole and rinsed 2-5 column volumes. The displaced supernatant sample was applied to the column. The column was washed with PBS containing 5 mM imidazole until the A280 reading dropped to baseline. The column was washed with PBS + 10 mM imidazole, the non-specifically bound heteroprotein was removed, and the effluent was collected. The protein of interest was eluted with PBS containing 300 mM imidazole, and the eluted peak was collected.
收集的洗脱液用离子交换(SP柱)进一步纯化。配置A液：0.01M PB，pH8.0。配置B液：A液+1M NaCl。先将咪唑的PBS溶液洗脱目的蛋白置换到A液，并使用A液平衡SP柱，上样，B液浓度梯度0-100％，10倍柱体积洗脱，收集各洗脱峰。所得到的蛋白经电泳，肽图，经液相色谱-质谱联用(LC-MS)鉴定后分装备用。The collected eluate was further purified by ion exchange (SP column). Configure solution A: 0.01 M PB, pH 8.0. Configure liquid B: liquid A + 1 M NaCl. First, the imidazole in PBS solution was eluted to the A solution, and the SP column was equilibrated with the A solution. The concentration of the B solution was 0-100%, and 10 column volumes were eluted to collect the elution peaks. The obtained protein was subjected to electrophoresis, peptide mapping, and identified by liquid chromatography-mass spectrometry (LC-MS).
2、带Fc标签的CSF-1R重组蛋白(h-CSF-1R-Fc)的纯化步骤：2. Purification step of Fc-tagged CSF-1R recombinant protein (h-CSF-1R-Fc):
将HEK293细胞表达的上清样品高速离心去除杂质，并将缓冲液换置换为PBS。用含有10mM磷酸缓冲液平衡Protein A亲和力柱，冲洗2-5倍柱体积。将置换后的上清样品上柱。用含有25倍柱体积缓冲液冲洗柱子，至A280读数降至基线。再用pH 3.5的0.8％醋酸缓冲液洗脱目的蛋白，并收集洗脱峰，分装后立刻加入1M Tris-Cl pH8.0缓冲液中和，然后使用Millipore’s Amico-15滤柱置换溶液为PBS pH6.9。所得到的蛋白经电泳， 肽图，LC-MS鉴定后分装备用。The supernatant sample expressed by HEK293 cells was centrifuged at high speed to remove impurities, and the buffer was exchanged for PBS. The Protein A affinity column was equilibrated with 10 mM phosphate buffer and rinsed 2-5 column volumes. The displaced supernatant sample was applied to the column. Rinse the column with 25 column volumes of buffer until the A280 reading drops to baseline. The target protein was eluted with 0.8% acetate buffer at pH 3.5, and the elution peak was collected. Immediately after the addition, the mixture was neutralized with 1 M Tris-Cl pH 8.0 buffer, and then the solution was replaced with Millipore's Amico-15 filter column. pH 6.9. The obtained protein was electrophoresed, peptide map, and identified by LC-MS.
3、表达人CSF-1R抗原的CHO稳转细胞株制备：3. Preparation of CHO stable transfected cell lines expressing human CSF-1R antigen:
编码人CSF-1R蛋白(huCSF-1R)的全长序列由Integrated DNA Technology(IDT)公司合成，分别克隆到pcDNA3.1载体上，pcDNA3.1/puro(Invitrogen#V79020)。CHO-S(ATCC)细胞于CD-CHO培养基(Life Technologies,#10743029)内培养至0.5×10 6/ml。将10μg编码huCSF-1R或基因的载体与50ul LF-LTX(Life Technologies,#A12621)在1ml Opti-MEM培养基(Life Technologies,#31985088)中混合，室温孵育20分钟后，加入CHO细胞培养液中并放入二氧化碳培养箱培养。24小时后更换新培养基并加入10μg/ml嘌呤霉素。之后每2-3天更换一次新培养液，经过10-12天筛选后得到稳定CHO-S细胞池。 The full-length sequence encoding the human CSF-1R protein (huCSF-1R) was synthesized by Integrated DNA Technology (IDT) and cloned into the pcDNA3.1 vector, pcDNA3.1/puro (Invitrogen #V79020). CHO-S (ATCC) cells were cultured to 0.5 x 10 6 /ml in CD-CHO medium (Life Technologies, #10743029). 10 μg of vector encoding huCSF-1R or gene was mixed with 50 ul of LF-LTX (Life Technologies, #A12621) in 1 ml Opti-MEM medium (Life Technologies, #31985088), and incubated for 20 minutes at room temperature, then CHO cell culture solution was added. It is placed in a carbon dioxide incubator for cultivation. After 24 hours, the new medium was replaced and 10 μg/ml of puromycin was added. After that, the new culture solution was changed every 2-3 days, and after 10-12 days of screening, a stable CHO-S cell pool was obtained.
4、食蟹猕猴CSF-1R-His(cynoCSF-1R-His)和小鼠CSF-1R-His蛋白均购自AcroBiosciences公司。4. Cynomolgus monkey CSF-1R-His (cynoCSF-1R-His) and mouse CSF-1R-His protein were purchased from AcroBiosciences.
实施例3抗体的制备Example 3 Preparation of Antibodies
抗人CSF-1R单克隆抗体通过免疫小鼠产生。实验用Swiss Webster白小鼠，雌性，6周龄(Charles River公司)。饲养环境：SPF级。小鼠购进后，实验室环境饲养1周，12/12小时光/暗周期调节，温度20-25℃；湿度40-60％。免疫抗原为带Fc标签的人CSF-1R重组蛋白(huCSF-1R-Fc)。用Titermax(sigma Lot Num：T2684)为佐剂。抗原与佐剂(titermax)比例为1:1，抗原乳化后进行接种，时间为第0、21、35、49、63天。第0天腹膜内(IP)注射15μg+爪垫(footpad)25/只的乳化后抗原。21,35,49,63天腹膜内(IP)注射15μg+爪垫(footpad)15/只的乳化后抗原,在进行脾细胞融合前3天加强免疫，腹膜内(IP)注射15μg+爪垫(footpad)15/只的生理盐水配制的抗原溶液。于第42,56,70天进行血检，用酶联免疫吸附剂测定(enzyme linked immunosorbent assay，ELISA)及荧光激活细胞分离仪(fluorescence activated cell sorter，FACS)方法检测小鼠血清，确定小鼠血清中的抗体滴度。在第5次免疫以后，选择血清中抗体滴度高并且滴度趋于平台的小鼠进行脾细胞融合，采用优化的电融合步骤将脾淋巴细胞与骨髓瘤细胞Sp2/0细胞(
Figure PCTCN2018111818-appb-000004
CRL-8287 TM)进行融合得到杂交瘤细胞。 Anti-human CSF-1R monoclonal antibodies are produced by immunizing mice. The experiment used Swiss Webster white mice, female, 6 weeks old (Charles River). Feeding environment: SPF level. After the mice were purchased, the laboratory environment was kept for 1 week, 12/12 hours light/dark cycle adjustment, temperature 20-25 ° C; humidity 40-60%. The immunizing antigen is an Fc-tagged human CSF-1R recombinant protein (huCSF-1R-Fc). Titermax (sigma Lot Num: T2684) was used as an adjuvant. The ratio of antigen to adjuvant (titermax) was 1:1, and the antigen was emulsified and inoculated for 0, 21, 35, 49, and 63 days. Day 0 intraperitoneal (IP) injection of 15 μg + footpad 25 / emulsified antigen. 21, 35, 49, 63 days of intraperitoneal (IP) injection of 15 μg + footpad 15 / emulsified antigen, boosted 3 days before spleen cell fusion, intraperitoneal (IP) injection 15μg + claw pad (footpad 15% of the antigen solution prepared in physiological saline. On the 42nd, 56th, and 70th day, blood tests were performed, and the mouse serum was determined by enzyme linked immunosorbent assay (ELISA) and fluorescence activated cell sorter (FACS) method to determine the mice. Antibody titer in serum. After the fifth immunization, spleen cell fusion was performed in mice with high antibody titers in serum and titers tended to plate, and spleen lymphocytes and myeloma cells Sp2/0 cells were optimized using an electrofusion procedure (
Figure PCTCN2018111818-appb-000004
CRL-8287 (TM ) was fused to obtain hybridoma cells.
融合后的杂交瘤细胞30,000-50,000细胞/孔，培养7-14天后，取培养基上清，如实施例4所描述，使用CSF-1R重组蛋白，ELISA实验对杂交瘤上清进行抗体筛选，得到的阳性抗体株进一步使用稳转表达CSF-1R的CHO-S细胞，对比空白CHO-S细胞以排除非特异性结合抗体杂交瘤株，用流式分选方法进行筛选，从而确定约400株结合重组蛋白且也结合细胞表达抗原的杂交瘤。随后如实施例5所描述，使用基于CSF-1R稳转细胞的CSF-1配基阻 断功能实验筛选，鉴定出约50株具备阻断剂功能的杂交瘤并将其亚克隆，然后收集纯化抗体进行基于CSF-1R稳转细胞的IL-34配基阻断功能实验筛选，鉴定到11株具备CSF-1和IL-34双阻断功能的抗体亚克隆。收集对数生长期杂交瘤亚克隆细胞，用Trizol(Invitrogen,15596-018)提取RNA并反转录(PrimeScript TMReverse Transcriptase，Takara#2680A)。将反转录得到的cDNA采用mouse Ig-Primer Set(Novagen,TB326 Rev.B 0503)进行PCR扩增后测序，排除CDR区域重复序列后，最终得到九株鼠源抗体的序列C11，C19，C6，C8，C16，C18，C23，C27和C30。 After the fused hybridoma cells were 30,000-50,000 cells/well, after 7-14 days of culture, the culture supernatant was taken, and as described in Example 4, the hybridoma supernatant was subjected to antibody screening using CSF-1R recombinant protein. The obtained positive antibody strain was further subjected to CHO-S cells stably expressing CSF-1R, and the blank CHO-S cells were compared to exclude non-specific binding antibody hybridoma strains, and screened by a flow sorting method to determine about 400 strains. A recombinant protein that also binds to a cell expressing an antigen. Subsequently, as described in Example 5, using CSF-1R stable cell-based CSF-1 ligand blocking function experimental screening, about 50 hybridomas with blocker function were identified and subcloned, then collected and purified. The antibody was screened for IL-34 ligand blocking function based on CSF-1R stable cells, and 11 subclones of antibody having CSF-1 and IL-34 double blocking function were identified. Logarithmic growth phase were collected and subcloned hybridoma cells, RNA was extracted with Trizol (Invitrogen, 15596-018) and reverse transcription (PrimeScript TM Reverse Transcriptase, Takara # 2680A). The cDNA obtained by reverse transcription was amplified by PCR amplification using mouse Ig-Primer Set (Novagen, TB326 Rev. B 0503), and the sequence of C11, C19, C6 of nine mouse antibodies was finally obtained after excluding the CDR region repeats. , C8, C16, C18, C23, C27 and C30.
鼠单抗C11的重链和轻链可变区序列如下：The heavy and light chain variable region sequences of murine mAb C11 are as follows:
C11 HCVRC11 HCVR
Figure PCTCN2018111818-appb-000005
Figure PCTCN2018111818-appb-000005
C11 LCVRC11 LCVR
Figure PCTCN2018111818-appb-000006
Figure PCTCN2018111818-appb-000006
其含有下列CDR序列：It contains the following CDR sequences:
名称name 序列sequence 编号Numbering
HCDR1HCDR1 GYTFSNYYMNGYTFSNYYMN SEQ ID NO：3SEQ ID NO: 3
HCDR2HCDR2 EMNPNNGDSSYNQKFKGEMNPNNGDSSYNQKFKG SEQ ID NO：4SEQ ID NO: 4
HCDR3HCDR3 RSPWWFFDVRSPWWFFDV SEQ ID NO：5SEQ ID NO: 5
LCDR1LCDR1 KSSQSLLNSGNQKNSLTKSSQSLLNSGNQKNSLT SEQ ID NO：6SEQ ID NO: 6
LCDR2LCDR2 WASTRESWASTRES SEQ ID NO：7SEQ ID NO:7
LCDR3LCDR3 QNDYTYPFTQNDYTYPFT SEQ ID NO：8SEQ ID NO:8
C19的重链和轻链可变区序列如下：The heavy and light chain variable region sequences of C19 are as follows:
C19 HCVRC19 HCVR
Figure PCTCN2018111818-appb-000007
Figure PCTCN2018111818-appb-000007
C19 LCVRC19 LCVR
Figure PCTCN2018111818-appb-000008
Figure PCTCN2018111818-appb-000008
其含有下列CDR序列：It contains the following CDR sequences:
名称name 序列sequence 编号Numbering
HCDR1HCDR1 AITFTDYYMNAITFTDYYMN SEQ ID NO：11SEQ ID NO: 11
HCDR2HCDR2 DIYPNNGGTTYNQKFKGDIYPNNGGTTYNQKFKG SEQ ID NO：12SEQ ID NO: 12
HCDR3HCDR3 EKITMEYYYAMDYEKITMEYYYAMDY SEQ ID NO：13SEQ ID NO: 13
LCDR1LCDR1 RASESVSSHDIHLIHRASESVSSHDIHLIH SEQ ID NO：14SEQ ID NO: 14
LCDR2LCDR2 AASSLESAASSLES SEQ ID NO：15SEQ ID NO: 15
LCDR3LCDR3 QQSIEDPPTQQSIEDPPT SEQ ID NO：16SEQ ID NO: 16
鼠单抗C6的重链和轻链可变区序列如下：The heavy and light chain variable region sequences of murine mAb C6 are as follows:
C6 HCVRC6 HCVR
Figure PCTCN2018111818-appb-000009
Figure PCTCN2018111818-appb-000009
C6 LCVRC6 LCVR
Figure PCTCN2018111818-appb-000010
Figure PCTCN2018111818-appb-000010
其含有下列CDR序列：It contains the following CDR sequences:
名称name 序列sequence 编号Numbering
HCDR1HCDR1 GYTFTGYYMHGYTFTGYYMH SEQID NO：39SEQID NO: 39
HCDR2HCDR2 EINPNTGSCTYNQKFTGEINPNTGSCTYNQKFTG SEQID NO：40SEQID NO: 40
HCDR3HCDR3 LNYYWYFDVLNYYWYFDV SEQID NO：41SEQID NO: 41
LCDR1LCDR1 KSSQTLLNSNDQKNYLAKSSQTLLNSNDQKNYLA SEQID NO：42SEQID NO: 42
LCDR2LCDR2 FASTRDSFASTRDS SEQID NO：43SEQID NO: 43
LCDR3LCDR3 QQDYSTPFTQQDYSTPFT SEQID NO：44SEQID NO: 44
鼠单抗C8的重链和轻链可变区序列如下：The heavy and light chain variable region sequences of murine mAb C8 are as follows:
C8 HCVRC8 HCVR
Figure PCTCN2018111818-appb-000011
Figure PCTCN2018111818-appb-000011
C8 LCVRC8 LCVR
Figure PCTCN2018111818-appb-000012
Figure PCTCN2018111818-appb-000012
其含有下列CDR序列：It contains the following CDR sequences:
名称name 序列sequence 编号Numbering
HCDR1HCDR1 GYTFTNYWIAGYTFTNYWIA SEQID NO：47SEQID NO: 47
HCDR2HCDR2 EMYPGGGGDNHHEKFKNEMYPGGGGDNHHEKFKN SEQID NO：48SEQID NO: 48
HCDR3HCDR3 RDYGNPCFDYRDYGNPCFDY SEQID NO：49SEQ ID NO: 49
LCDR1LCDR1 RASQGVTTSSHSYMHRASQGVTTSSHSYMH SEQID NO：50SEQID NO: 50
LCDR2LCDR2 YASNLESYASNLES SEQID NO：51SEQID NO: 51
LCDR3LCDR3 QHSWEIPYTQHSWEIPYT SEQID NO：52SEQID NO: 52
鼠单抗C16的重链和轻链可变区序列如下：The heavy and light chain variable region sequences of murine mAb C16 are as follows:
C16 HCVRC16 HCVR
Figure PCTCN2018111818-appb-000013
Figure PCTCN2018111818-appb-000013
C16 LCVRC16 LCVR
Figure PCTCN2018111818-appb-000014
Figure PCTCN2018111818-appb-000014
其含有下列CDR序列：It contains the following CDR sequences:
名称name 序列sequence 编号Numbering
HCDR1HCDR1 GYTFTTYDINGYTFTTYDIN SEQID NO：55SEQID NO: 55
HCDR2HCDR2 WVYPRDGSTKYNEKFKGWVYPRDGSTKYNEKFKG SEQID NO：56SEQID NO: 56
HCDR3HCDR3 SGLTGSPFAYSGLTGSPFAY SEQID NO：57SEQ ID NO: 57
LCDR1LCDR1 RASESFDSYGNTFMHRASESFDSYGNTFMH SEQID NO：58SEQID NO: 58
LCDR2LCDR2 RASNLESRASNLES SEQID NO：59SEQID NO: 59
LCDR3LCDR3 QQNNEYPLTQQNNEYPLT SEQID NO：60SEQID NO: 60
鼠单抗C18的重链和轻链可变区序列如下：The heavy and light chain variable region sequences of murine mAb C18 are as follows:
C18 HCVRC18 HCVR
Figure PCTCN2018111818-appb-000015
Figure PCTCN2018111818-appb-000015
C18 LCVRC18 LCVR
Figure PCTCN2018111818-appb-000016
Figure PCTCN2018111818-appb-000016
其含有下列CDR序列：It contains the following CDR sequences:
名称name 序列sequence 编号Numbering
HCDR1HCDR1 AITFTDYYMNAITFTDYYMN SEQID NO：63SEQ ID NO: 63
HCDR2HCDR2 DINPNNGGTTYNQKFKGDINPNNGGTTYNQKFKG SEQID NO：64SEQID NO: 64
HCDR3HCDR3 EKISMEYYYAMDYEKISMEYYYAMDY SEQID NO：65SEQ ID NO: 65
LCDR1LCDR1 RASESVSSHDIHLMHRASESVSSHDIHLMH SEQID NO：66SEQ ID NO: 66
LCDR2LCDR2 AASNLESAASNLES SEQID NO：67SEQ ID NO: 67
LCDR3LCDR3 QQSIEDPPTQQSIEDPPT SEQID NO：68SEQID NO: 68
鼠单抗C23的重链和轻链可变区序列如下：The heavy and light chain variable region sequences of murine mAb C23 are as follows:
C23 HCVRC23 HCVR
Figure PCTCN2018111818-appb-000017
Figure PCTCN2018111818-appb-000017
C23 LCVRC23 LCVR
Figure PCTCN2018111818-appb-000018
Figure PCTCN2018111818-appb-000018
其含有下列CDR序列：It contains the following CDR sequences:
名称name 序列sequence 编号Numbering
HCDR1HCDR1 GYTFTVYYMNGYTFTVYYMN SEQID NO：71SEQID NO: 71
HCDR2HCDR2 DIDPNTGDSTYNQKFRGDIDPNTGDSTYNQKFRG SEQID NO：72SEQID NO: 72
HCDR3HCDR3 YDGYIDYYDGYIDY SEQID NO：73SEQ ID NO:73
LCDR1LCDR1 RSSQSIVHSNRHTYLERSSQSIVHSNRHTYLE SEQID NO：74SEQ ID NO: 74
LCDR2LCDR2 GVSNRFSGVSNRFS SEQID NO：75SEQID NO: 75
LCDR3LCDR3 FQGTHVPLTFQGTHVPLT SEQID NO：76SEQID NO: 76
鼠单抗C27的重链和轻链可变区序列如下：The heavy and light chain variable region sequences of murine mAb C27 are as follows:
C27 HCVRC27 HCVR
Figure PCTCN2018111818-appb-000019
Figure PCTCN2018111818-appb-000019
C27 LCVRC27 LCVR
Figure PCTCN2018111818-appb-000020
Figure PCTCN2018111818-appb-000020
其含有下列CDR序列：It contains the following CDR sequences:
名称name 序列sequence 编号Numbering
HCDR1HCDR1 GFNIKDYYMHGFNIKDYYMH SEQID NO：79SEQID NO: 79
HCDR2HCDR2 RFDPENGDTIYDWKFQDRFDPENGDTIYDWKFQD SEQID NO：80SEQID NO: 80
HCDR3HCDR3 SGDYMFDYSGDYMFDY SEQID NO：81SEQID NO: 81
LCDR1LCDR1 ITSTGVDDDFNITSTGVDDDFN SEQID NO：82SEQ ID NO: 82
LCDR2LCDR2 EGNTLRPEGNTLRP SEQID NO：83SEQID NO: 83
LCDR3LCDR3 LQSDHLPFTLQSDHLPFT SEQID NO：84SEQ ID NO: 84
鼠单抗C30的重链和轻链可变区序列如下：The heavy and light chain variable region sequences of murine mAb C30 are as follows:
C30 HCVRC30 HCVR
Figure PCTCN2018111818-appb-000021
Figure PCTCN2018111818-appb-000021
C30 LCVRC30 LCVR
Figure PCTCN2018111818-appb-000022
Figure PCTCN2018111818-appb-000022
其含有下列CDR序列：It contains the following CDR sequences:
名称name 序列sequence 编号Numbering
HCDR1HCDR1 GYTFSSYWIEGYTFSSYWIE SEQID NO：87SEQ ID NO: 87
HCDR2HCDR2 EILPGSGSTNYNEKFKGEILPGSGSTNYNEKFKG SEQID NO：88SEQ ID NO: 88
HCDR3HCDR3 NYDGSLYPMDYNYDGSLYPMDY SEQID NO：89SEQID NO: 89
LCDR1LCDR1 RTSESVSIHGTHLMHRTSESVSIHGTHLMH SEQID NO：90SEQID NO: 90
LCDR2LCDR2 AASNLESAASNLES SEQID NO：91SEQID NO: 91
LCDR3LCDR3 QQSIEDPPTQQSIEDPPT SEQID NO：92SEQID NO: 92
将每株鼠抗的重链和轻链可变区分别克隆进入含人IgG4重链恒定区和κ轻链恒定区的pTT载体质粒(Biovector)，然后瞬转转染入HEK293细胞，得到了抗CSF-1R的各个嵌合抗体(C11C，C19C，C6C，C8C，C16C，C18C，C23C，C27C和C30C)，按实施例2(带Fc标签蛋白纯化)所描述的方法纯化、鉴定，并进行相关结合与功能活性检测。The heavy and light chain variable regions of each mouse antibody were cloned into pTT vector plasmid (Biovector) containing human IgG4 heavy chain constant region and kappa light chain constant region, respectively, and then transiently transfected into HEK293 cells to obtain anti-resistant. Each chimeric antibody (C11C, C19C, C6C, C8C, C16C, C18C, C23C, C27C and C30C) of CSF-1R was purified, identified, and correlated as described in Example 2 (purification with Fc-tagged protein) Binding and functional activity detection.
实施例4抗体的体外结合活性测定In vitro binding activity assay of the antibody of Example 4
用PBS pH7.4缓冲液将用于和生物素结合的中和亲和素稀释至1μg/ml，以100μl/孔的体积加于96孔板中，于4℃放置16h-20h。用PBST(PH7.4PBS含0.05％Tween-20)缓冲液洗板1次后，加入120μl/孔PBST/1％脱脂奶，室温孵育1h进行封闭。PBST缓冲液洗板1次后，加入用PBST/1％脱脂奶稀释的1μg/ml的生物素标记h-CSF-1R-his(或猴、鼠CSF-1R蛋白等待测样)，置室温孵育1h。PBST缓冲液洗板3次后，加入用PBST/1％milk稀释至合适浓度的待测CSF-1R抗体(鼠抗体/嵌合抗体/人源化抗体)，置室温孵育1.5h。移去反应体系，用PBST洗板3次后，以100μl/孔加入用PBST/1％脱脂奶稀释辣根过氧化物酶(Horseradish Peroxidase，HRP)标记的抗鼠抗体二抗或抗人抗体二抗(The Jackson  Laboratory)，室温孵育1h。PBST洗板3次后，加入100μl/孔TMB，于室温孵育5-10min。加入100μl/孔1M H 2SO 4终止反应，在450nm处读取吸收值，计算ELISA结合EC 50值。 The neutralizing avidin for binding to biotin was diluted to 1 μg/ml with PBS pH 7.4 buffer, added to a 96-well plate in a volume of 100 μl/well, and left at 4 ° C for 16 h to 20 h. After washing the plate once with PBST (pH 7.4 PBS containing 0.05% Tween-20), 120 μl/well of PBST/1% skim milk was added and incubated for 1 h at room temperature for blocking. After washing the plate once with PBST buffer, add 1 μg/ml biotin-labeled h-CSF-1R-his diluted with PBST/1% skim milk (or monkey, mouse CSF-1R protein waiting for the sample), incubate at room temperature. 1h. After washing the plate three times with PBST buffer, the CSF-1R antibody (murine antibody/chimeric antibody/humanized antibody) to be diluted with PBST/1% milk was added to an appropriate concentration, and incubated at room temperature for 1.5 h. The reaction system was removed, and the plate was washed three times with PBST, and then diluted with horseradish peroxidase (HRP)-labeled anti-mouse antibody secondary antibody or anti-human antibody II with PBST/1% skim milk at 100 μl/well. Anti- (The Jackson Laboratory), incubated for 1 h at room temperature. After washing the plate 3 times with PBST, 100 μl/well of TMB was added and incubated for 5-10 min at room temperature. The reaction was stopped by the addition of 100 μl/well of 1 M H 2 SO 4 , and the absorbance was read at 450 nm, and the ELISA binding EC 50 value was calculated.
将高表达huCSF-1R的CHO-S细胞以1000rpm的转速离心5分钟，收集沉淀并用10-15ml的预冷的流式缓冲液悬浮，细胞计数。用50ml的离心管中以1000rpm的转速离心5分钟收集细胞，丢掉上清，沉淀用预冷封闭缓冲液重悬，密度为0.5-1.0×10 7细胞/毫升。4℃孵育30分钟后，重悬以每孔100μl加入到96孔板。96孔板在1500rpm的转速下离心5分钟后，弃上清。向每个孔加入100μl待测抗体，浓度梯度为0.085nM至670nM，将细胞重悬，4℃避光孵育60分钟。离心弃上清，加入100μl的l:400稀释的FITC标记二抗(BD Biosciences)。将细胞重悬，4℃避光孵育60分钟。用流式缓冲液洗两次细胞，并用1％的多聚甲醛重悬细胞进行固定，进行流式检测。 CHO-S cells highly expressing huCSF-1R were centrifuged at 1000 rpm for 5 minutes, and the precipitate was collected and suspended in 10-15 ml of pre-cooled flow buffer, and counted. The cells were collected by centrifugation at 1000 rpm for 5 minutes in a 50 ml centrifuge tube, the supernatant was discarded, and the pellet was resuspended in a pre-cooled blocking buffer at a density of 0.5-1.0 x 10 7 cells/ml. After incubating for 30 minutes at 4 ° C, it was resuspended to 100 μl per well and added to a 96-well plate. After centrifugation of the 96-well plate at 1500 rpm for 5 minutes, the supernatant was discarded. 100 μl of the antibody to be tested was added to each well at a concentration gradient of 0.085 nM to 670 nM, the cells were resuspended, and incubated at 4 ° C for 60 minutes in the dark. The supernatant was discarded by centrifugation, and 100 μl of a 1:400 diluted FITC-labeled secondary antibody (BD Biosciences) was added. The cells were resuspended and incubated for 60 minutes at 4 ° C in the dark. The cells were washed twice with flow buffer and resuspended in 1% paraformaldehyde for flow cytometry.
同样的ELISA方法，使用CSF-1R-His蛋白检测各抗体与食蟹猴CSF-1R的交叉结合，结果数据如图1所示，除C27C与猴抗原蛋白无结合，其余抗体及FPA008参照都有与猴CSF-1R很强的交叉结合活性。The same ELISA method, using CSF-1R-His protein to detect the cross-binding of each antibody to cynomolgus CSF-1R, the results of the data shown in Figure 1, except C27C and monkey antigen protein no binding, the rest of the antibody and FPA008 reference Strong cross-binding activity with monkey CSF-1R.
实施例5抗体的体外阻断功能测定实验In vitro blocking function assay of the antibody of Example 5
1、抗CSF-1R抗体在CSF-1R/CHO细胞上对CSF-1或IL-34结合的阻断实验1. Blocking of CSF-1 or IL-34 binding by anti-CSF-1R antibody on CSF-1R/CHO cells
将浓度梯度的抗CSF-1R抗体(0.01nM-670nM)与CSF-1R/CHO细胞共孵育，再加入生物素化的CSF-1蛋白或APC标签的IL-34蛋白孵育结合。其中与CSF-1的实验再进一步加入链霉亲和素-PE标签结合。PBS洗后，将以上细胞使用流式细胞检测方法检测CSF-1或IL-34对应的荧光信号强度。A concentration gradient of anti-CSF-1R antibody (0.01 nM-670 nM) was co-incubated with CSF-1R/CHO cells, and then biotinylated CSF-1 protein or APC-tagged IL-34 protein was incubated for binding. Among them, the experiment with CSF-1 was further added to the streptavidin-PE tag. After washing with PBS, the above cells were assayed for fluorescence signal intensity corresponding to CSF-1 or IL-34 using flow cytometry.
2、抗CSF-1R抗体对CSF-1或IL-34诱导的人源单核细胞增殖阻断实验2. Anti-CSF-1R antibody inhibits proliferation of human monocytes induced by CSF-1 or IL-34
首先使用Ficoll-Paque梯度分离试剂盒(GE Healthcare Bio-Sciences)分离提取健康人血中的外周血单个核细胞(Peripheral blood mononuclear cell，PBMC)，再用Percoll试剂盒(GE Healthcare Bio-Sciences)纯化提取PBMC中的单核细胞。然后将分离得到的单核细胞在空白无抗体或浓度梯度的抗CSF-1R抗体中使用人CSF-1或IL-34(R&D Systems)刺激，37℃下于二氧化碳培养箱中培养3天。使用CellTiter-Glo试剂盒(Promega)测量各个待测培养样品中的ATP含量，并依据其与活化单核细胞数量的线性正相关来最终测算出单核细胞的对应增殖水平。Peripheral blood mononuclear cells (PBMC) in healthy human blood were isolated and extracted using Ficoll-Paque gradient separation kit (GE Healthcare Bio-Sciences), and purified by Percoll kit (GE Healthcare Bio-Sciences). Mononuclear cells in PBMC were extracted. The isolated monocytes were then stimulated in a blank antibody-free or concentration-concentrated anti-CSF-1R antibody using human CSF-1 or IL-34 (R&D Systems), and cultured in a carbon dioxide incubator at 37 ° C for 3 days. The ATP content in each culture sample to be tested was measured using the CellTiter-Glo kit (Promega), and the corresponding proliferation level of monocytes was finally determined based on its linear positive correlation with the number of activated monocytes.
30株亚克隆鼠抗及FivePrime公司的参照抗体FPA008的结合及初步功能筛选实验结果汇总见下表。测序后选定的9株鼠源抗体以粗体显示。The results of the binding and preliminary functional screening experiments of 30 subcloning mouse anti-Freprim reference antibody FPA008 are summarized in the table below. The 9 selected murine antibodies selected after sequencing were shown in bold.
Figure PCTCN2018111818-appb-000023
Figure PCTCN2018111818-appb-000023
Figure PCTCN2018111818-appb-000024
Figure PCTCN2018111818-appb-000024
嵌合抗体阻断功能数据如下表所示，9株抗体均能有效阻断CSF-1或IL-34刺激单核细胞增殖，其中C11C、C19C、C23C和C27C等嵌合抗体具有很低的IC50，阻断效果相当或优于参照抗体FPA008。The chimeric antibody blocking function data is shown in the following table. Nine antibodies can effectively block the proliferation of monocytes stimulated by CSF-1 or IL-34. The chimeric antibodies such as C11C, C19C, C23C and C27C have very low IC50. The blocking effect is comparable or superior to the reference antibody FPA008.
Figure PCTCN2018111818-appb-000025
Figure PCTCN2018111818-appb-000025
实施例6体外结合亲和力和动力学实验Example 6 In vitro binding affinity and kinetics experiments
本实验采用表面等离子共振(SPR)方法测定。利用由Biacore提供的试剂盒，采用标准氨基偶联法将抗人IgG多克隆抗体共价连接至CM5(GE)芯片上，然后用此抗体将本发明待测的嵌合抗体或人源化抗体抗体捕捉至固定相。将稀释于同样缓冲液中的25-800nM浓度梯度的h-CSF-1R-His蛋白(实施例1)于前后各个循环进样，进样后均以试剂盒内配再生试剂再生。追踪抗原-抗体结合动力学3分钟并追踪解离动力学10分钟。使用GE的BIAevaluation软件以1:1(Langmuir)结合模型分析所得数据，以此法测定各个嵌合抗体的k a(k on)、kd(k off)和K D值显示如下表。 This experiment was measured by surface plasmon resonance (SPR) method. The anti-human IgG polyclonal antibody is covalently linked to a CM5 (GE) chip using a standard amino coupling method using a kit supplied by Biacore, and then the chimeric antibody or humanized antibody of the present invention to be tested is used with the antibody. The antibody is captured to the stationary phase. The h-CSF-1R-His protein (Example 1) having a concentration gradient of 25-800 nM diluted in the same buffer was injected at each cycle before and after the injection, and was regenerated by the regeneration reagent in the kit. Antigen-antibody binding kinetics were followed for 3 minutes and dissociation kinetics were followed for 10 minutes. The data obtained by analyzing the 1:1 (Langmuir) binding model using GE's BIAevaluation software, the k a (k on ), kd (k off ) and K D values of each chimeric antibody were determined by the following table.
Figure PCTCN2018111818-appb-000026
Figure PCTCN2018111818-appb-000026
Figure PCTCN2018111818-appb-000027
Figure PCTCN2018111818-appb-000027
实施例7小鼠抗体人源化实验Example 7 Humanization Experiment of Mouse Antibody
鼠源抗人CSF-1R单克隆抗体人源化如本领域许多文献公示的方法进行。简言之，使用人恒定结构域替代亲本(鼠源抗体)恒定结构域，根据鼠源抗体和人抗体的同源性选择人种抗体序列，本发明将综合功能活性与序列特异性、成药性等各方面评价确定的最优两株候选分子C11和C19进行人源化。Humanization of murine anti-human CSF-1R monoclonal antibodies was performed as disclosed in many literatures in the art. Briefly, the human constant domain is used in place of the parental (murine antibody) constant domain, and the human antibody sequence is selected based on the homology of the murine antibody and the human antibody. The present invention combines functional activity with sequence specificity and drug-forming properties. The optimal two candidate molecules C11 and C19 determined by various evaluations were humanized.
在所获得的鼠源抗体VH/VL CDR典型结构的基础上，将重、轻链可变区序列与人源抗体种系数据库比较，获得同源性高的人种系模板。其中人类种系轻链框架区来自人κ轻链基因，人类种系重链框架区来自人重链，本发明抗体优选以下所示的人种系抗体模版。Based on the typical structure of the obtained murine antibody VH/VL CDR, the heavy and light chain variable region sequences were compared with the human antibody germline database to obtain a human germline template with high homology. Wherein 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, and the antibody of the present invention is preferably a human germline antibody template shown below.
C11优选人种系重链模版IGHV1-46(SEQ ID NO:21):C11 is preferably a human germline heavy chain template IGHV 1-46 (SEQ ID NO: 21):
Figure PCTCN2018111818-appb-000028
Figure PCTCN2018111818-appb-000028
C11优选人种系轻链模板IGkV4-1(SEQ ID NO:22)：C11 is preferably a human germline light chain template IGkV4-1 (SEQ ID NO: 22):
Figure PCTCN2018111818-appb-000029
Figure PCTCN2018111818-appb-000029
C19优选人种系重链模版IGHV1-2(SEQ ID NO:23):C19 is preferably a human germline heavy chain template IGHV1-2 (SEQ ID NO: 23):
Figure PCTCN2018111818-appb-000030
Figure PCTCN2018111818-appb-000030
C19优选人种系轻链模板IGkV1-13(SEQ ID NO:24)：C19 is preferably a human germline light chain template IGkV1-13 (SEQ ID NO: 24):
Figure PCTCN2018111818-appb-000031
Figure PCTCN2018111818-appb-000031
将鼠源抗体C11和C19的CDR区移植到选择好的相应人源化模板上，替换人源化可变区，再与IgG恒定区(优选重链为IgG4，轻链为κ)重组。然后，以鼠源抗体的三维结构为基础，对包埋残基、与CDR区有直接相互作用的残基，以及对VL和VH的构象有重要影响的残基进行回复突变，并对CDR区化学不稳定天冬酰胺残基突变优化，其中鼠抗体C11的HCDR2：EMNPNNGDSSYNQKFKG(SEQ ID NO：4)优化为：EMNPNTGDSSYNQKFKG(SEQ ID NO：95)，LCDR1：KSSQSLLNSGNQKNSLT(SEQ  ID NO：6)优化为：KSSQSLLNSGNQKTSLT(SEQ ID NO：96)；鼠抗体C19的HCDR2：DIYPNNGGTTYNQKFKG(SEQ ID NO：12)优化为：DIYPNTGGTTYNQKFKG(SEQ ID NO：97)。从而设计并检测了由如下人源化轻重链可变区序列组合而成的抗体。The CDR regions of the murine antibodies C11 and C19 are grafted onto the selected corresponding humanized template, the humanized variable region is replaced, and then recombined with the IgG constant region (preferably the heavy chain is IgG4 and the light chain is κ). Then, based on the three-dimensional structure of the murine antibody, the residues which have an important interaction with the CDRs and the residues of the CDRs, and the residues which have an important influence on the conformation of VL and VH are subjected to back mutation and the CDR regions are Mutation optimization of chemically unstable asparagine residues, wherein HCDR2:EMNPNNGDSSYNQKFKG (SEQ ID NO:4) of murine antibody C11 was optimized to be: EMNPNTGDSSYNQKFKG (SEQ ID NO: 95), and LCDR1: KSSQSLLNSGNQKNSLT (SEQ ID NO: 6) was optimized to : KSSQSLLNSGNQKTSLT (SEQ ID NO: 96); HCDR2: DIYPNNGGTTYNQKFKG (SEQ ID NO: 12) of murine antibody C19 was optimized to: DIYPNTGGTTYNQKFKG (SEQ ID NO: 97). Thus, an antibody composed of the following humanized light heavy chain variable region sequences was designed and tested.
huC11-VH-a(SEQ ID NO:25)：huC11-VH-a (SEQ ID NO: 25):
Figure PCTCN2018111818-appb-000032
Figure PCTCN2018111818-appb-000032
huC11-VH-b(SEQ ID NO:26)：huC11-VH-b (SEQ ID NO: 26):
Figure PCTCN2018111818-appb-000033
Figure PCTCN2018111818-appb-000033
huC11-VH-c(SEQ ID NO:27)：huC11-VH-c (SEQ ID NO: 27):
Figure PCTCN2018111818-appb-000034
Figure PCTCN2018111818-appb-000034
huC11-VH-d(SEQ ID NO:28)：huC11-VH-d (SEQ ID NO: 28):
Figure PCTCN2018111818-appb-000035
Figure PCTCN2018111818-appb-000035
huC11-VL-a(SEQ ID NO:29)：huC11-VL-a (SEQ ID NO: 29):
Figure PCTCN2018111818-appb-000036
Figure PCTCN2018111818-appb-000036
huC11-VL-b(SEQ ID NO:30)：huC11-VL-b (SEQ ID NO: 30):
Figure PCTCN2018111818-appb-000037
Figure PCTCN2018111818-appb-000037
huC19-VH-a(SEQ ID NO:31)：huC19-VH-a (SEQ ID NO: 31):
Figure PCTCN2018111818-appb-000038
Figure PCTCN2018111818-appb-000038
huC19-VH-b(SEQ ID NO:32)：huC19-VH-b (SEQ ID NO: 32):
Figure PCTCN2018111818-appb-000039
Figure PCTCN2018111818-appb-000039
Figure PCTCN2018111818-appb-000040
Figure PCTCN2018111818-appb-000040
huC19-VH-c(SEQ ID NO:33)：huC19-VH-c (SEQ ID NO: 33):
Figure PCTCN2018111818-appb-000041
Figure PCTCN2018111818-appb-000041
huC19-VL-a(SEQ ID NO:34)：huC19-VL-a (SEQ ID NO: 34):
Figure PCTCN2018111818-appb-000042
Figure PCTCN2018111818-appb-000042
huC19-VL-b(SEQ ID NO:35)：huC19-VL-b (SEQ ID NO: 35):
Figure PCTCN2018111818-appb-000043
Figure PCTCN2018111818-appb-000043
huC19-VL-c(SEQ ID NO:36)：huC19-VL-c (SEQ ID NO: 36):
Figure PCTCN2018111818-appb-000044
Figure PCTCN2018111818-appb-000044
根据以上各人源化抗体轻链和重链的氨基酸序列合成cDNA片段，***到pcDNA3.1表达载体(Life Technologies Cat.No.V790-20)中。将表达载体和转染试剂PEI(Polysciences,Inc.Cat.No.23966)以1:2的比例转染HEK293细胞(Life Technologies Cat.No.11625019)，并置于CO 2孵育箱中孵育4-5天。表达的抗体通过离心回收后，按实施例2方法(带Fc标签蛋白纯化)进行抗体纯化，得到本发明的人源化抗体蛋白。 A cDNA fragment was synthesized based on the amino acid sequences of the above humanized antibody light and heavy chains, and inserted into a pcDNA3.1 expression vector (Life Technologies Cat. No. V790-20). The expression vector and the 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 placed in a CO 2 incubator for incubation 4- 5 days. After the expressed antibody was recovered by centrifugation, antibody purification was carried out by the method of Example 2 (purification with Fc-tag protein) to obtain a humanized antibody protein of the present invention.
经表达测试和回复突变数量对比，选择出最终的人源化huC11(使用VH-b重链和VL-b轻链)和huC19抗体分子(使用VH-c重链和VL-a轻链)。The final humanized huC11 (using the VH-b heavy chain and VL-b light chain) and the huC19 antibody molecule (using the VH-c heavy chain and the VL-a light chain) were selected for expression test and back mutation number comparison.
进一步地，对于huC19人源化抗体，将其HCDR1：AITFTDYYMN(SEQ ID NO:11)突变为：AITFTDYYIN(SEQ ID NO:98)从而去除了化学不稳定的甲硫氨酸残基位点，最终得到人源化抗体huC19I，huC19I重链可变区序列如SEQ ID NO:100所示。huC11、huC19和huC19I的各自全长序列如SEQ ID NO:17-20和SEQ ID NO:99所示。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, and the huC19I heavy chain variable region sequence is shown in SEQ ID NO:100. The respective full length sequences of huC11, huC19 and huC19I are set forth in SEQ ID NOS: 17-20 and SEQ ID NO: 99.
huC19-VH-dhuC19-VH-d
Figure PCTCN2018111818-appb-000045
Figure PCTCN2018111818-appb-000045
huC11 HChuC11 HC
Figure PCTCN2018111818-appb-000046
Figure PCTCN2018111818-appb-000046
huC11 LChuC11 LC
Figure PCTCN2018111818-appb-000047
Figure PCTCN2018111818-appb-000047
huC19 HChuC19 HC
Figure PCTCN2018111818-appb-000048
Figure PCTCN2018111818-appb-000048
huC19 LC/huC19I LChuC19 LC/huC19I LC
Figure PCTCN2018111818-appb-000049
Figure PCTCN2018111818-appb-000049
Figure PCTCN2018111818-appb-000050
Figure PCTCN2018111818-appb-000050
huC19I HChuC19I HC
Figure PCTCN2018111818-appb-000051
Figure PCTCN2018111818-appb-000051
其中，人源化抗体的重链恒定区序列(SEQ ID NO：101)Wherein the heavy chain constant region sequence of the humanized antibody (SEQ ID NO: 101)
Figure PCTCN2018111818-appb-000052
Figure PCTCN2018111818-appb-000052
人源化抗体的轻链恒定区序列(SEQ ID NO：102)Light chain constant region sequence of a humanized antibody (SEQ ID NO: 102)
Figure PCTCN2018111818-appb-000053
Figure PCTCN2018111818-appb-000053
实施例8人源化抗体活性测定Example 8 Humanized Antibody Activity Assay
对huC11和huC19抗体在体外进行了以下实验测定：The following experimental assays were performed on huC11 and huC19 antibodies in vitro:
1、细胞结合实验(方法步骤同实施例4)，结果如图2所示，人源化后的抗体huC11和huC19与高表达CSF-1R的CHO细胞均有阳性结合，且结合能力与对应的嵌合抗体C11C和C19C相当，而C19的嵌合抗体及各人源化抗体版本均强于C11对应版本。1. Cell binding assay (method procedure is the same as in Example 4). The results are shown in Figure 2. The humanized antibodies huC11 and huC19 positively bind to CHO cells with high expression of CSF-1R, and the binding ability and corresponding The chimeric antibodies C11C and C19C were comparable, while the chimeric antibodies of C19 and the humanized antibody versions were stronger than the C11 counterpart.
2、亲和力动力学实验(方法步骤同实施例6)，结果如下表所示，最终优选的人源化抗体huC11和huC19对人CSF-1R抗原蛋白均保有很强的亲和力，其中huC19的亲和力比参照抗体FPA008更强。2. Affinity kinetics experiment (method procedure is 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 human CSF-1R antigen protein, and the affinity of huC19 is higher. The reference antibody FPA008 is stronger.
Figure PCTCN2018111818-appb-000054
Figure PCTCN2018111818-appb-000054
3.体外阻断功能活性实验(方法步骤同实施例6)，结果如图3、图4和下表所示，人源化后的抗体huC11、huC19及其突变体huC19I在对CSF-1和IL-34诱导的单核细胞增殖功能上都显示出很强的阻断功能，且其中huC19的阻断功能活性比FPA008参照抗体相当或略优，而其突变体huC19I稍有下降至与FPA008相近。3. In vitro blocking functional activity assay (method steps are the same as in Example 6). The results are shown in Figure 3, Figure 4 and the table below. The humanized antibody huC11, huC19 and its mutant huC19I are in CSF-1 and IL-34-induced monocyte proliferation showed strong blocking function, and the blocking function of huC19 was comparable or slightly better than that of FPA008 reference antibody, while its mutant huC19I decreased slightly to similar to FPA008. .
huC11的体外阻断功能活性实验In vitro blocking function activity assay of huC11
Figure PCTCN2018111818-appb-000055
Figure PCTCN2018111818-appb-000055
huC19及其突变体huC19I的体外阻断功能活性实验In vitro blocking function activity assay of huC19 and its mutant huC19I
Figure PCTCN2018111818-appb-000056
Figure PCTCN2018111818-appb-000056
实施例9抗CSF1R抗体在人源化肿瘤模型中的药效Example 9 Efficacy of anti-CSF1R antibody in a humanized tumor model
取正常人外周血，用密度梯度离心法分离健康人PBMC。用CD14 +microbeads试剂盒分选单核细胞，按照试剂盒所提供的protocol进行CD14 +单核细胞的分离，即每10 7个细胞加入20ul Anti-CD14 microbeads，4℃孵育15分钟。然后，将细胞加入磁柱中，洗三次后，收集磁柱中的细胞，即CD14 +单核细胞。CD14 +单核细胞中加入含100ng/ml M-CSF的RPMI 1640培养液培养6天，进行macrophag细胞的诱导培养，剩余的细胞加入经Mitomycin C处理的MDA-MB-231细胞中，PBMC与MDA-MB-231共培养6天，培养液为含IL-2和10％FBS的RPMI 1640培养液。 The normal human peripheral blood was taken and the healthy human PBMC was isolated by density gradient centrifugation. Mononuclear cells were sorted by CD14 + microbeads kit, and CD14 + monocytes were separated according to the protocol provided by the kit, that is, 20 ul of Anti-CD14 microbeads was added per 10 7 cells, and incubated at 4 ° C for 15 minutes. Then, the cells were added to a magnetic column, and after washing three times, the cells in the magnetic column, that is, CD14 + monocytes were collected. CD14 + monocytes were cultured for 6 days in RPMI 1640 medium containing 100 ng/ml M-CSF, and induced to induce macrophag cells. The remaining cells were added to Mito-M-treated MDA-MB-231 cells, PBMC and MDA. -MB-231 was co-cultured for 6 days, and the culture solution was RPMI 1640 medium containing IL-2 and 10% FBS.
6天后收集macrophage细胞，取1×10 5个细胞，CD68，CD115，CD163，CD206染色，FACS分析是否成功诱导macrophage。将PBMC细胞，macrophage和新鲜消化下来的MDA-MB-231细胞(ATCC)按6.25:1:50比例混合，接种于每只NCG小鼠(南京大学-南京生物医药研究院，适应性饲养5天)皮下。实验动物均饲养于恒温恒湿的独立通风盒内，饲养室温度18.0-26.0℃，湿度40-70％，10-20次/小时换气，昼夜明暗交替时间12h/12h。 After 6 days, macrophage cells were collected, 1×10 5 cells, CD68, CD115, CD163, CD206 staining, and FACS analysis successfully induced macrophage. PBMC cells, macrophage 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 Biomedical Research, adaptive feeding for 5 days) ) Under the skin. The experimental animals were kept in an independent ventilated box with constant temperature and humidity. The temperature of the breeding room was 18.0-26.0 °C, the humidity was 40-70%, the ventilation was 10-20 times/hour, and the alternating time between day and night was 12h/12h.
实验分为人IgG1抗体对照组，参照抗体FPA8，和huC19I，给药剂量均为20mg/kg。每组6只小鼠，每两天一次，腹腔注射给药，给药10次(见表1)。给药后每天监测动物日常行为表现，共进行24天。整个实验过程中，用游标卡尺每周测量2次肿瘤长径和宽径，肿瘤体积(mm 3)＝0.5×(肿瘤长径×肿瘤短径 2)计算。相对肿瘤抑制率TGI(％)：TGI％＝(1-T/C)×100％。T/C％为相对肿瘤增值率，即在某一时间点，治疗组和对照组相对肿瘤体积或瘤重的百分比值。T和C分别为治疗组和IgG1对照组在某一特定时间点的肿瘤体积(TV)或瘤重(TW)。所有数据均采用Mean±SEM表示，用student’s t test比较治疗组肿瘤体积和瘤重与对照组相比有无显著性差异，p<0.05为具有显著性差异。 The experiment was divided into human IgG1 antibody control group, reference antibody FPA8, and huC19I, and the dose was 20 mg/kg. Six mice in each group were administered once every two days, intraperitoneally, and administered 10 times (see Table 1). The daily behavior of the animals was monitored daily after administration for a total of 24 days. Throughout the experiment, the long diameter and the broad diameter of the tumor were measured twice a week using a vernier caliper, and the tumor volume (mm 3 ) = 0.5 × (long diameter of the tumor × short diameter of the tumor 2 ) was calculated. Relative tumor inhibition rate TGI (%): TGI% = (1-T/C) × 100%. T/C% is the relative tumor growth rate, that is, the percentage of tumor volume or tumor weight relative to the treatment group and the 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. Comparing the tumor volume and tumor weight of the treatment group with the Student's t test, there was no significant difference compared with the control group, p<0.05 was considered to be significant difference.
如图5，图6所示，在Macrophage-PBMC-MDA-MB-231肿瘤模型中，CSF1R抗体huC19I表现出抗肿瘤作用，各组终末平均肿瘤体积分别为：294.32mm 3、230.46mm 3、131.19mm 3、；FPA8组和huC19I组抑瘤率(TGI)分别为22.62％和55.95％。各给药组均没有出现明显的NCG小鼠体重下降情况，表明NCG小鼠对该剂量下的CSF1R抗体耐受性良好。 As shown in FIG. 5, FIG. 6, in Macrophage-PBMC-MDA-MB- 231 tumor model, CSFIR huC19I antibodies exhibit anti-tumor effect, the mean tumor volume of each group were terminally: 294.32mm 3, 230.46mm 3, The tumor inhibition rate (TGI) of the 131.19 mm 3 , FPA8 and huC19I groups was 22.62% and 55.95%, respectively. There was no significant decrease in body weight of NCG mice in each of the drug-administered groups, indicating that NCG mice were well tolerated with CSF1R antibodies at this dose.
实验分组及给药方案见下表：The experimental grouping and dosing schedule are shown in the following table:
Figure PCTCN2018111818-appb-000057
Figure PCTCN2018111818-appb-000057

Claims (24)

  1. 一种抗CSF-1R抗体或其抗原结合片段，其包含抗体轻链可变区和抗体重链可变区，其中：An anti-CSF-1R antibody or antigen-binding fragment thereof comprising an antibody light chain variable region and an antibody heavy chain variable region, wherein:
    a)所述的抗体轻链可变区包含选自在如SEQ ID NO:42、SEQ ID NO:43和SEQ ID NO:44所示的LCDR1、LCDR2和LCDR3基础上分别具有3、2、1或0个氨基酸突变的LCDR变体；所述的抗体重链可变区包含选自在如SEQ ID NO:39、SEQ ID NO:40和SEQ ID NO:41所示的HCDR1、HCDR2和HCDR3基础上分别具有3、2、1或0个氨基酸突变的HCDR变体；a) The antibody light chain variable region comprises 3, 2, 2 selected from the group consisting of LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 42, SEQ ID NO: 43 and SEQ ID NO: 44, respectively. Or a 0 amino acid mutated LCDR variant; said antibody heavy chain variable region comprising a base selected from the group consisting of HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 39, SEQ ID NO: 40 and SEQ ID NO: 41 HCDR variants having 3, 2, 1 or 0 amino acid mutations, respectively;
    b)所述的抗体轻链可变区包含选自在如SEQ ID NO:50、SEQ ID NO:51和SEQ ID NO:52所示的LCDR1、LCDR2和LCDR3基础上分别具有3、2、1或0个氨基酸突变的LCDR变体；所述的抗体重链可变区包含选自在如SEQ ID NO:47、SEQ ID NO:48和SEQ ID NO:49所示的HCDR1、HCDR2和HCDR3基础上分别具有3、2、1或0个氨基酸突变的HCDR变体；b) The antibody light chain variable region comprises 3, 2, 2 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. Or a 0 amino acid mutated LCDR variant; said antibody heavy chain variable region comprising a base selected from the group consisting of HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 47, SEQ ID NO: 48 and SEQ ID NO: HCDR variants having 3, 2, 1 or 0 amino acid mutations, respectively;
    c)所述的抗体轻链可变区包含选自在如SEQ ID NO:58、SEQ ID NO:59和SEQ ID NO:60所示的LCDR1、LCDR2和LCDR3基础上分别具有3、2、1或0个氨基酸突变的LCDR变体；所述的抗体重链可变区选自在如SEQ ID NO:55、SEQ ID NO:56和SEQ ID NO:57所示的HCDR1、HCDR2和HCDR3基础上分别具有3、2、1或0个氨基酸突变的HCDR变体；c) the antibody light chain variable region comprising 3, 2, 2 selected from the group consisting of LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 58, SEQ ID NO: 59 and SEQ ID NO: 60, respectively. Or a 0 amino acid mutated LCDR variant; said antibody heavy chain variable region selected from the group consisting of HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 55, SEQ ID NO: 56 and SEQ ID NO: 57 HCDR variants having 3, 2, 1 or 0 amino acid mutations, respectively;
    d)所述的抗体轻链可变区包含选自在如SEQ ID NO:66、SEQ ID NO:67和SEQ ID NO:68所示的LCDR1、LCDR2和LCDR3基础上分别具有3、2、1或0个氨基酸突变的LCDR变体；所述的抗体重链可变区包含选自在如SEQ ID NO:63、SEQ ID NO:64和SEQ ID NO:65所示的HCDR1、HCDR2和HCDR3基础上分别具有3、2、1或0个氨基酸突变的HCDR变体；d) the antibody light chain variable region comprising 3, 2, 2 selected from the group consisting of LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 66, SEQ ID NO: 67 and SEQ ID NO: 68, respectively. Or a 0 amino acid mutated LCDR variant; said antibody heavy chain variable region comprising a base selected from the group consisting of HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 63, SEQ ID NO: 64 and SEQ ID NO: 65 HCDR variants having 3, 2, 1 or 0 amino acid mutations, respectively;
    e)所述的抗体轻链可变区包含选自在如SEQ ID NO:74、SEQ ID NO:75和SEQ ID NO:76所示的LCDR1、LCDR2和LCDR3基础上分别具有3、2、1或0个氨基酸突变的LCDR变体；所述的抗体重链可变区包含选自在如SEQ ID NO:71、SEQ ID NO:72和SEQ ID NO:73所示的HCDR1、HCDR2和HCDR3基础上分别具有3、2、1或0个氨基酸突变的HCDR变体；e) The antibody light chain variable region comprises 3, 2, 2 selected from the group consisting of LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 74, SEQ ID NO: 75 and SEQ ID NO: 76, respectively. Or a 0 amino acid mutated LCDR variant; said antibody heavy chain variable region comprising a base selected from the group consisting of HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO:71, SEQ ID NO:72 and SEQ ID NO:73 HCDR variants having 3, 2, 1 or 0 amino acid mutations, respectively;
    f)所述的抗体轻链可变区包含选自在如SEQ ID NO:82、SEQ ID NO:83和SEQ ID NO:84所示的LCDR1、LCDR2和LCDR3基础上分别具有3、2、1或0个氨基酸突变的 LCDR变体；所述的抗体重链可变区包含选自在如SEQ ID NO:79、SEQ ID NO:80和SEQ ID NO:81所示的HCDR1、HCDR2和HCDR3基础上分别具有3、2、1或0个氨基酸突变的HCDR变体；f) the antibody light chain variable region comprising 3, 2, 2 selected from the group consisting of LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 82, SEQ ID NO: 83 and SEQ ID NO: 84, respectively. Or a 0 amino acid mutated LCDR variant; said antibody heavy chain variable region comprising a base selected from the group consisting of HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO:79, SEQ ID NO:80 and SEQ ID NO:81 HCDR variants having 3, 2, 1 or 0 amino acid mutations, respectively;
    g)所述的抗体轻链可变区包含选自在如SEQ ID NO:90、SEQ ID NO:91和SEQ ID NO:92所示的LCDR1、LCDR2和LCDR3基础上分别具有3、2、1或0个氨基酸突变的LCDR变体；所述的抗体重链可变区包含选自在如SEQ ID NO:87、SEQ ID NO:88和SEQ ID NO:89所示的HCDR1、HCDR2和HCDR3基础上分别具有3、2、1或0个氨基酸突变的HCDR变体；g) the antibody light chain variable region comprising 3, 2, 2 selected from the group consisting of LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 90, SEQ ID NO: 91 and SEQ ID NO: 92, respectively. Or a 0 amino acid mutated LCDR variant; said antibody heavy chain variable region comprising a base selected from the group consisting of HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO:87, SEQ ID NO:88 and SEQ ID NO:89 HCDR variants having 3, 2, 1 or 0 amino acid mutations, respectively;
    h)所述的抗体轻链可变区包含选自在如SEQ ID NO:6、SEQ ID NO:7和SEQ ID NO:8所示的LCDR1、LCDR2和LCDR3基础上分别具有3、2、1或0个氨基酸突变的LCDR变体；所述的抗体重链可变区包含选自在如SEQ ID NO:3、SEQ ID NO:4和SEQ ID NO:5所示的HCDR1、HCDR2和HCDR3基础上分别具有3、2、1或0个氨基酸突变的HCDR变体；h) the antibody light chain variable region comprises 3, 2, 2 selected from the group consisting of LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively. Or a 0 amino acid mutated LCDR variant; said antibody heavy chain variable region comprising a base selected from the group consisting of HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5 HCDR variants having 3, 2, 1 or 0 amino acid mutations, respectively;
    i)所述的抗体轻链可变区包含选自在如SEQ ID NO:14、SEQ ID NO:15和SEQ ID NO:16所示的LCDR1、LCDR2和LCDR3基础上分别具有3、2、1或0个氨基酸突变的LCDR变体；所述的抗体重链可变区包含选自在如SEQ ID NO:11、SEQ ID NO:12和SEQ ID NO:13所示的HCDR1、HCDR2和HCDR3基础上分别具有3、2、1或0个氨基酸突变的HCDR变体；i) The antibody light chain variable region comprises 3, 2, 2 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. Or a 0 amino acid mutated LCDR variant; said antibody heavy chain variable region comprising a base selected from the group consisting of HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: HCDR variants having 3, 2, 1 or 0 amino acid mutations, respectively;
    j)所述的抗体轻链可变区包含选自在如SEQ ID NO:14、SEQ ID NO:15和SEQ ID NO:16所示的LCDR1、LCDR2和LCDR3基础上分别具有3、2、1或0个氨基酸突变的LCDR变体；抗体重链可变区包含选自在如：SEQ ID NO:98、SEQ ID NO:12和SEQ ID NO:13所示的HCDR1、HCDR2和HCDR3基础上分别具有3、2、1或0个氨基酸突变的HCDR变体；j) The antibody light chain variable region comprises 3, 2, 2 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. Or a 0 amino acid mutated LCDR variant; the antibody heavy chain variable region comprises a selection of HCDR1, HCDR2 and HCDR3 selected from, for example, SEQ ID NO:98, SEQ ID NO:12 and SEQ ID NO:13, respectively HCDR variants having 3, 2, 1 or 0 amino acid mutations;
    k)所述的抗体轻链可变区包含选自在如SEQ ID NO:14、SEQ ID NO15和SEQ ID NO:16所示的LCDR1、LCDR2和LCDR3基础上分别具有3、2、1或0个氨基酸突变的LCDR变体；抗体重链可变区包含选自在如：SEQ ID NO:98、SEQ ID NO:97和SEQ ID NO:13所示的HCDR1、HCDR2和HCDR3基础上分别具有3、2、1或0个氨基酸突变的HCDR变体；k) The antibody light chain variable region comprises 3, 2, 1 or 0 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. An amino acid mutated LCDR variant; the antibody heavy chain variable region comprises 3, respectively, selected from HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO:98, SEQ ID NO:97 and SEQ ID NO:13 HCDR variant of 2, 1 or 0 amino acid mutations;
    l)所述的抗体轻链可变区包含选自在如SEQ ID NO:96、SEQ ID NO:7和SEQ ID NO:8所示的LCDR1、LCDR2和LCDR3基础上分别具有3、2、1或0个氨基酸突变的LCDR变体；抗体重链可变区包含选自在如：SEQ ID NO:3、SEQ ID NO:4和SEQ ID NO:5所示 的HCDR1、HCDR2和HCDR3基础上分别具有3、2、1或0个氨基酸突变的HCDR变体；l) The antibody light chain variable region comprises 3, 2, 2 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. Or a 0 amino acid mutated LCDR variant; the antibody heavy chain variable region comprises a selection of HCDR1, HCDR2 and HCDR3 selected from, for example, 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;
    m)所述的抗体轻链可变区包含选自在如SEQ ID NO:6、SEQ ID NO:7和SEQ ID NO:8所示的LCDR1、LCDR2和LCDR3基础上分别具有3、2、1或0个氨基酸突变的LCDR变体；抗体重链可变区包含选自在如：SEQ ID NO:3、SEQ ID NO:95和SEQ ID NO:5所示的HCDR1、HCDR2和HCDR3基础上分别具有3、2、1或0个氨基酸突变的HCDR变体；m) the antibody light chain variable region comprising 3, 2, 2 selected from the group consisting of LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively. Or a 0 amino acid mutated LCDR variant; the antibody heavy chain variable region comprises a selection of HCDR1, HCDR2 and HCDR3 selected from, for example, SEQ ID NO:3, SEQ ID NO:95 and SEQ ID NO:5, respectively HCDR variants having 3, 2, 1 or 0 amino acid mutations;
    n)所述的抗体轻链可变区包含选自在如SEQ ID NO:96、SEQ ID NO:7和SEQ ID NO:8所示的LCDR1、LCDR2和LCDR3基础上分别具有3、2、1或0个氨基酸突变的LCDR变体；抗体重链可变区包含选自在如：SEQ ID NO:3、SEQ ID NO:95和SEQ ID NO:5所示的HCDR1、HCDR2和HCDR3基础上分别具有3、2、1或0个氨基酸突变的HCDR变体；或者n) The antibody light chain variable region comprises 3, 2, 2 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. Or a 0 amino acid mutated LCDR variant; the antibody heavy chain variable region comprises a selection of HCDR1, HCDR2 and HCDR3 selected from, for example, SEQ ID NO:3, SEQ ID NO:95 and SEQ ID NO:5, respectively HCDR variant having 3, 2, 1 or 0 amino acid mutations; or
    o)所述的抗体轻链可变区包含选自在如：SEQ ID NO:14、SEQ ID NO:15和SEQ ID NO:16所示的LCDR1、LCDR2和LCDR3基础上分别具有3、2、1或0个氨基酸突变的LCDR变体；抗体重链可变区包含选自在如：SEQ ID NO:11、SEQ ID NO:97和SEQ ID NO:13所示的HCDR1、HCDR2和HCDR3基础上分别具有3、2、1或0个氨基酸突变的HCDR变体。o) the antibody light chain variable region comprises 3, 2, respectively, 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. 1 or 0 amino acid mutated LCDR variant; antibody heavy chain variable region comprising selected from HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 11, SEQ ID NO: 97 and SEQ ID NO: HCDR variants with 3, 2, 1 or 0 amino acid mutations, respectively.
  2. 一种抗CSF-1R抗体或其抗原结合片段，其包含抗体轻链可变区和抗体重链可变区，其中：An anti-CSF-1R antibody or antigen-binding fragment thereof comprising an antibody light chain variable region and an antibody heavy chain variable region, wherein:
    p)所述的抗体轻链可变区包含分别如SEQ ID NO:42、SEQ ID NO:43和SEQ ID NO:44所示的LCDR1、LCDR2和LCDR3；所述的抗体重链可变区包含分别如SEQ ID NO:39、SEQ ID NO:40和SEQ ID NO:41所示的HCDR1、HCDR2和HCDR3；The antibody light chain variable region of p) comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 42, SEQ ID NO: 43 and SEQ ID NO: 44, respectively; the antibody heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 39, SEQ ID NO: 40 and SEQ ID NO: 41, respectively;
    q)所述的抗体轻链可变区包含分别如SEQ ID NO:50、SEQ ID NO:51和SEQ ID NO:52所示的LCDR1、LCDR2和LCDR3；所述的抗体重链可变区包含分别如SEQ ID NO:47、SEQ ID NO:48和SEQ ID NO:49所示的HCDR1、HCDR2和HCDR3；q) The antibody light chain variable region comprises LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO: 50, SEQ ID NO: 51 and SEQ ID NO: 52, respectively; the antibody heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 47, SEQ ID NO: 48 and SEQ ID NO: 49, respectively;
    r)所述的抗体轻链可变区包含分别如SEQ ID NO:58、SEQ ID NO:59和SEQ ID NO:60所示的LCDR1、LCDR2和LCDR3；所述的抗体重链可变区包含分别如SEQ ID NO:55、SEQ ID NO:56和SEQ ID NO:57所示的HCDR1、HCDR2和HCDR3；r) the antibody light chain variable region comprising LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO:58, SEQ ID NO:59 and SEQ ID NO:60, respectively; said antibody heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 55, SEQ ID NO: 56 and SEQ ID NO: 57, respectively;
    s)所述的抗体轻链可变区包含分别如SEQ ID NO:66、SEQ ID NO:67和SEQ ID NO:68所示的LCDR1、LCDR2和LCDR3；所述的抗体重链可变区包含分别如SEQ ID NO:63、SEQ ID NO:64和SEQ ID NO:65所示的HCDR1、HCDR2和HCDR3；The antibody light chain variable region of s) comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 66, SEQ ID NO: 67 and SEQ ID NO: 68, respectively; the antibody heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 63, SEQ ID NO: 64 and SEQ ID NO: 65, respectively;
    t)所述的抗体轻链可变区包含分别如SEQ ID NO:74、SEQ ID NO:75和SEQ ID NO:76所示的LCDR1、LCDR2和LCDR3；所述的抗体重链可变区包含分别如SEQ ID NO:71、SEQ ID NO:72和SEQ ID NO:73所示的HCDR1、HCDR2和HCDR3；t) The antibody light chain variable region comprises LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO: 74, SEQ ID NO: 75 and SEQ ID NO: 76, respectively; the antibody heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 71, SEQ ID NO: 72 and SEQ ID NO: 73, respectively;
    u)所述的抗体轻链可变区包含分别如SEQ ID NO:82、SEQ ID NO:83和SEQ ID NO:84所示的LCDR1、LCDR2和LCDR3；所述的抗体重链可变区包含分别如SEQ ID NO:79、SEQ ID NO:80和SEQ ID NO:81所示的HCDR1、HCDR2和HCDR3；u) the antibody light chain variable region comprising LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO: 82, SEQ ID NO: 83 and SEQ ID NO: 84, respectively; said antibody heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 79, SEQ ID NO: 80 and SEQ ID NO: 81, respectively;
    v)所述的抗体轻链可变区包含分别如SEQ ID NO:90、SEQ ID NO:91和SEQ ID NO:92所示的LCDR1、LCDR2和LCDR3；所述的抗体重链可变区包含分别如SEQ ID NO:87、SEQ ID NO:88和SEQ ID NO:89所示的HCDR1、HCDR2和HCDR3；v) The antibody light chain variable region comprises LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO: 90, SEQ ID NO: 91 and SEQ ID NO: 92, respectively; the antibody heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 87, SEQ ID NO: 88 and SEQ ID NO: 89, respectively;
    w)所述的抗体轻链可变区包含分别如SEQ ID NO:6、SEQ ID NO:7和SEQ ID NO:8所示的LCDR1、LCDR2和LCDR3；所述的抗体重链可变区包含分别如SEQ ID NO:3、SEQ ID NO:4和SEQ ID NO:5所示的HCDR1、HCDR2和HCDR3；w) The antibody light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, respectively; the antibody heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, respectively;
    x)所述的抗体轻链可变区包含分别如SEQ ID NO:14、SEQ ID NO:15和SEQ ID NO:16所示的LCDR1、LCDR2和LCDR3；所述的抗体重链可变区包含分别如SEQ ID NO:11、SEQ ID NO:12和SEQ ID NO:13所示的HCDR1、HCDR2和HCDR3；x) The antibody light chain variable region comprises LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, respectively; the antibody heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13, respectively;
    y)所述的抗体轻链可变区包含分别如SEQ ID NO:14、SEQ ID NO:15和SEQ ID NO:16所示的LCDR1、LCDR2和LCDR3；抗体重链可变区包含分别如：SEQ ID NO:98、SEQ ID NO:12和SEQ ID NO:13所示的HCDR1、HCDR2和HCDR3；y) The antibody light chain variable region comprises LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, respectively; the antibody heavy chain variable region comprises: HCDR1, HCDR2 and HCDR3 set forth in SEQ ID NO: 98, SEQ ID NO: 12 and SEQ ID NO: 13;
    z)所述的抗体轻链可变区包含分别如SEQ ID NO:14、SEQ ID NO15和SEQ ID NO:16所示的LCDR1、LCDR2和LCDR3；抗体重链可变区包含分别如：SEQ ID NO:98、SEQ ID NO:97和SEQ ID NO:13所示的HCDR1、HCDR2和HCDR3；z) The antibody light chain variable region comprises 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 comprises, for example, SEQ ID NO: 98, SEQ ID NO: 97 and SEQ ID NO: 13 HCDR1, HCDR2 and HCDR3;
    aa)所述的抗体轻链可变区包含分别如SEQ ID NO:96、SEQ ID NO:7和SEQ ID NO:8所示的LCDR1、LCDR2和LCDR3；抗体重链可变区包含分别如：SEQ ID NO:3、SEQ ID NO:4和SEQ ID NO:5所示的HCDR1、HCDR2和HCDR3；The antibody light chain variable region of aa) 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 variable region comprises: HCDR1, HCDR2 and HCDR3 set forth in SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5;
    ab)所述的抗体轻链可变区包含分别如SEQ ID NO:6、SEQ ID NO:7和SEQ ID NO:8所示的LCDR1、LCDR2和LCDR3；抗体重链可变区包含分别如：SEQ ID NO:3、SEQ ID NO:95和SEQ ID NO:5所示的HCDR1、HCDR2和HCDR3；The antibody light chain variable region of ab) comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively; the antibody heavy chain variable region comprises: HCDR1, HCDR2 and HCDR3 set forth in SEQ ID NO: 3, SEQ ID NO: 95 and SEQ ID NO: 5;
    ac)所述的抗体轻链可变区包含分别如SEQ ID NO:96、SEQ ID NO:7和SEQ ID NO:8所示的LCDR1、LCDR2和LCDR3；抗体重链可变区包含分别如：SEQ ID NO:3、SEQ ID NO:95和SEQ ID NO:5所示的HCDR1、HCDR2和HCDR3；或者The antibody light chain variable region of ac) 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 variable region comprises: HCDR1, HCDR2 and HCDR3 of SEQ ID NO: 3, SEQ ID NO: 95 and SEQ ID NO: 5;
    ad)所述的抗体轻链可变区包含分别如：SEQ ID NO:14、SEQ ID NO:15和SEQ ID  NO:16所示的LCDR1、LCDR2和LCDR3；抗体重链可变区包含分别如：SEQ ID NO:11、SEQ ID NO:97和SEQ ID NO:13所示的HCDR1、HCDR2和HCDR3。Ad) The antibody light chain variable region comprises 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 comprises : HCDR1, HCDR2 and HCDR3 of SEQ ID NO: 11, SEQ ID NO: 97 and SEQ ID NO: 13.
  3. 如权利要求1或2所述的抗CSF-1R抗体或其抗原结合片段，其中所述的抗体或其抗原结合片段为鼠源抗体或者嵌合抗体。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.
  4. 如权利要求3所述的抗体或抗原结合片段，其中The antibody or antigen-binding fragment of claim 3, wherein
    a)所述轻链可变区的氨基酸序列如SEQ ID NO:38所示，所述重链可变区的氨基酸序列如SEQ ID NO：37所示；或者a) the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 38, and the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 37;
    b)所述轻链可变区的氨基酸序列如SEQ ID NO:46所示，所述重链可变区的氨基酸序列如SEQ ID NO：45所示；或者b) the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 46, and the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 45;
    c)所述轻链可变区的氨基酸序列如SEQ ID NO:54所示，所述重链可变区的氨基酸序列如SEQ ID NO：53所示；或者c) the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 54, and the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 53;
    d)所述轻链可变区的氨基酸序列如SEQ ID NO:62所示，所述重链可变区的氨基酸序列如SEQ ID NO：61所示；或者d) the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 62, and the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 61;
    e)所述轻链可变区的氨基酸序列如SEQ ID NO:70所示，所述重链可变区的氨基酸序列如SEQ ID NO：69所示；或者e) the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 70, and the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 69;
    f)所述轻链可变区的氨基酸序列如SEQ ID NO:78所示，所述重链可变区的氨基酸序列如SEQ ID NO：77所示；或者f) the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 78, and the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 77;
    g)所述轻链可变区的氨基酸序列如SEQ ID NO:86所示，所述重链可变区的氨基酸序列如SEQ ID NO：85所示；或者g) the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 86, and the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 85;
    h)所述轻链可变区的氨基酸序列如SEQ ID NO:2所示或者其变体，所述变体优选在轻链可变区有0-10的氨基酸突变，最优选的氨基酸突变为N37T，所述重链可变区的氨基酸序列如SEQ ID NO：1所示或者其变体，所述变体优选在重链可变区有0-10的氨基酸突变，优选的氨基酸突变为N55T；或者h) the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 2 or a variant thereof, preferably having a 0-10 amino acid mutation in the light chain variable region, the most preferred amino acid mutation being N37T, the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 1 or a variant thereof, and the variant preferably has a 0-10 amino acid mutation in the heavy chain variable region, and the preferred amino acid mutation is N55T ;or
    i)所述轻链可变区的氨基酸序列如SEQ ID NO:10所示，所述重链可变区的氨基酸序列如SEQ ID NO：9所示或者其变体，所述变体优选在轻链可变区有0-10的氨基酸突变，优选的突变为M34I、N55T或其组合的氨基酸突变。i) the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 10, and the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 9 or a variant thereof, preferably The light chain variable region has an amino acid mutation of 0-10, and the preferred mutation is an amino acid mutation of M34I, N55T or a combination thereof.
  5. 如权利要求1或2所述的抗CSF-1R抗体或其抗原结合片段，其中所述的抗体或其抗原结合片段为人源化抗体或其抗原结合片段。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.
  6. 如权利要求5所述的抗CSF-1R抗体或其抗原结合片段，其中所述人源化抗体轻链可变区上的轻链FR区序列，来源于如SEQ ID NO:22所示的人种系轻链IGkV4-1序列；或来源于如SEQ ID NO:24所示的人种系轻链IGkV1-13序列；The anti-CSF-1R antibody or antigen-binding fragment thereof according to claim 5, wherein the light chain FR region sequence on the variable region of the humanized antibody light chain is derived from the human as set forth in SEQ ID NO: a germline light chain IGkV4-1 sequence; or a human germline light chain IGkV1-13 sequence as set forth in SEQ ID NO:24;
    和/或，所述人源化抗体重链可变区进一步包含人源IgG1,IgG2,IgG3或IgG4或其变体的重链FR区，优选包含人源IgG1、IgG2或IgG4重链FR区，更优选包含人源IgG1或IgG4重链FR区；较佳地，所述人源化抗体重链可变区上的重链FR区序列，来源于如SEQ ID NO:21所示的人种系重链IGHV1-46序列；或来源于如SEQ ID NO:23所示的人种系重链IGHV1-2序列。And/or the humanized antibody heavy chain variable region further comprises a heavy chain FR region of human IgG1, IgG2, IgG3 or IgG4 or variants thereof, preferably comprising a human IgG1, IgG2 or IgG4 heavy chain FR region, More preferably comprising a human IgGl or IgG4 heavy chain FR region; preferably, the heavy chain FR region sequence on the humanized antibody heavy chain variable region is derived from the human germline as set forth in SEQ ID NO:21. The heavy chain IGHV1-46 sequence; or the human germline heavy chain IGHV1-2 sequence as set forth in SEQ ID NO:23.
  7. 如权利要求6所述的抗CSF-1R抗体或其抗原结合片段，其中所述人源化抗体轻链可变区序列为如SEQ ID NO:30或SEQ ID NO:34所示的序列或其变体；所述的变体优选在轻链可变区有0-10的氨基酸突变，其中SEQ ID NO:30最优选的氨基酸突变为S31N、T37N；SEQ ID NO:34最优选的氨基酸突变为F87A、F91Y或其组合；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 set forth in SEQ ID NO: 30 or SEQ ID NO: 34 or Variant; the variant preferably has a 0-10 amino acid mutation in the light chain variable region, wherein the most preferred amino acid mutation of SEQ ID NO: 30 is S31N, T37N; the most preferred amino acid mutation of SEQ ID NO: 34 is F87A, F91Y or a combination thereof;
    和/或，所述人源化抗体重链可变区序列为如SEQ ID NO:26、SEQ ID NO:100或SEQ ID NO:33所示的序列或其变体；所述变体优选在重链可变区有0-10的氨基酸突变，其中SEQ ID NO:26优选的氨基酸突变为S30T、T55N、L70M或其组合，最优选的氨基酸突变为T55N；SEQ ID NO:33优选的氨基酸突变为Q1E、M34I、T55N、E89D或其组合，最优选的氨基酸突变为M34I、T55N或其组合的氨基酸突变；And/or the humanized antibody heavy chain variable region sequence is the sequence set forth in SEQ ID NO:26, SEQ ID NO:100 or SEQ ID NO:33 or a variant thereof; The heavy chain variable region has a 0-10 amino acid mutation, 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 preferred amino acid mutation For Q1E, M34I, T55N, E89D or a combination thereof, the most preferred amino acid mutation is an amino acid mutation of M34I, T55N or a combination thereof;
    较佳地，所述重链可变区的序列为SEQ ID NO：31、32、33或100，且所述轻链可变区的序列为SEQ ID NO：34、35或36；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;
    或者，所述重链可变区的序列为SEQ ID NO：25、26、27或28，且所述轻链可变区的序列为SEQ ID NO：29或30。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.
  8. 如权利要求3～7任一项所述的抗CSF-1R抗体或其抗原结合片段，其中所述嵌合抗体或人源化抗体进一步包括轻链和/或重链的恒定区，所述轻链恒定区序列如SEQ ID NO：102所示，和/或所述重链恒定区序列如SEQ ID NO：101所示。The anti-CSF-1R antibody or antigen-binding fragment thereof according to any one of claims 3 to 7, wherein the chimeric antibody or humanized antibody further comprises a constant region of a light chain and/or a heavy chain, the light The chain constant region sequence is set forth in SEQ ID NO: 102, and/or the heavy chain constant region sequence is set forth in SEQ ID NO: 101.
  9. 如权利要求8所述的抗CSF-1R抗体或其抗原结合片段，其中所述人源化抗体：The anti-CSF-1R antibody or antigen-binding fragment thereof according to claim 8, wherein the humanized antibody:
    包含序列如SEQ ID NO:19所示的重链，和序列如SEQ ID NO:20所示的轻链；a heavy chain comprising the sequence of SEQ ID NO: 19, and a light chain of the sequence SEQ ID NO: 20;
    包含序列如SEQ ID NO:17所示的重链，和序列如SEQ ID NO:18所示的轻链；或者包含序列如SEQ ID NO:99所示的重链，和序列如SEQ ID NO:20所示的轻链。A heavy chain comprising the sequence of SEQ ID NO: 17 and a light chain of the sequence SEQ ID NO: 18; or a heavy chain of the sequence SEQ ID NO: 99, and a sequence of SEQ ID NO: The light chain shown in 20.
  10. 一种分离的单克隆抗体或其抗原结合片段，其与权利要求1～9任一项所述的抗CSF-1R抗体或其抗原结合片段竞争结合CSF-1R。An isolated monoclonal antibody or antigen-binding fragment thereof which competes with CSF-1R for binding to an anti-CSF-1R antibody or antigen-binding fragment thereof according to any one of claims 1-9.
  11. 一种多特异性抗体，含有如权利要求1～10任一项所述的体或其抗原结合片段的轻链可变区和/或重链可变区。A multispecific antibody comprising a light chain variable region and/or a heavy chain variable region of the human or antigen-binding fragment thereof according to any one of claims 1 to 10.
  12. 一种单链抗体，含有如权利要求1～10任一项所述的抗体或其抗原结合片段的轻链可变区和/或重链可变区。A single-chain antibody comprising a light chain variable region and/or a heavy chain variable region of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10.
  13. 一种抗体-药物偶联物，其中所述的抗体含有如权利要求1～10任一项所述的抗体或其抗原结合片段的轻链可变区和/或重链可变区。An antibody-drug conjugate, wherein the antibody comprises a light chain variable region and/or a heavy chain variable region of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10.
  14. 一种编码如权利要求1～10任一项所述的抗体或其抗原结合片段、如权利要求11所述的多特异性抗体或者如权利要求12所述的单链抗体的核酸分子。A nucleic acid molecule encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, the multispecific antibody of claim 11, or the single-chain antibody of claim 12.
  15. 一种含有如权利要求14所述的核酸的表达载体。An expression vector comprising the nucleic acid of claim 14.
  16. 一种用如权利要求15所述的表达载体转化的宿主细胞。A host cell transformed with the expression vector of claim 15.
  17. 如权利要求16所述的宿主细胞，其特征在于，所述的宿主细胞为细菌，优选大肠杆菌；The host cell according to claim 16, wherein said host cell is a bacterium, preferably Escherichia coli;
    或者，所述的宿主细胞为酵母菌，优选毕赤酵母；Alternatively, the host cell is a yeast, preferably Pichia pastoris;
    或者，所述的宿主细胞为动物细胞，优选中国仓鼠卵巢(CHO)细胞、人胚肾(HEK)293细胞或NS0细胞。Alternatively, the host cell is an animal cell, preferably a Chinese hamster ovary (CHO) cell, a human embryonic kidney (HEK) 293 cell or an NSO cell.
  18. 一种用于制备权利要求1～10任一项所述的抗体或其抗原结合片段、如权利要求11所述的多特异性抗体或者如权利要求12所述的单链抗体的方法，包括在权利要求16或17所述的宿主细胞中表达所述抗体或其抗原结合片段、所述多特异性抗体或者所述单链抗体，并自该宿主细胞中分离所述抗体或其抗原结合片段、所述多特异性抗体或者所述单链抗体。A method for the preparation of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, the multispecific antibody of claim 11, or the single-chain antibody of claim 12, comprising Or expressing the antibody or antigen-binding fragment thereof, the multispecific antibody or the single-chain antibody in the host cell according to claim 16 or 17, and isolating the antibody or antigen-binding fragment thereof from the host cell, The multispecific antibody or the single chain antibody.
  19. 一种药物组合物，其含有如权利要求1～10任一项所述的抗体或其抗原结合片段、如权利要求11所述的多特异性抗体或者如权利要求12所述的单链抗体以及可药用的缓冲剂、赋形剂、稀释剂或载体。A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, the multispecific antibody according to claim 11 or the single-chain antibody according to claim 12; A pharmaceutically acceptable buffer, excipient, diluent or carrier.
  20. 如权利要求1～10任一项所述的抗体或其抗原结合片段、如权利要求11所述的多特异性抗体、如权利要求12所述的单链抗体、如权利要求13所述的抗体-药物偶联物、如权利要求14所述的核酸分子或者如权利要求19所述的药物组合物在抗癌治疗中的应用，所述的抗体或其抗原结合片段、所述的多特异性抗体、所述的单链抗体、所述的抗体-药物偶联物、所述的核酸分子或者所述的药物组合物是在开始用另一种抗癌治疗的疗法之前、过程中、基本同时或之后施用的。The antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, the multispecific antibody according to claim 11, the single-chain antibody according to claim 12, the antibody according to claim 13, or the antibody according to claim Use of a drug conjugate, a nucleic acid molecule according to claim 14 or a pharmaceutical composition according to claim 19 in anticancer therapy, said antibody or antigen-binding fragment thereof, said multispecific The antibody, the single chain antibody, the antibody-drug conjugate, the nucleic acid molecule or the pharmaceutical composition is before, during, and substantially simultaneously with the initiation of treatment with another anticancer treatment Or applied later.
  21. 如权利要求20所述的应用，其中所述另一种抗癌治疗的疗法选自抗血管发生剂、化疗剂、放射、肿瘤免疫疗法或其组合。The use according to claim 20, wherein said another anti-cancer treatment therapy is selected from the group consisting of an anti-angiogenic agent, a chemotherapeutic agent, radiation, tumor immunotherapy or a combination thereof.
  22. 如权利要求21所述的应用，其中所述化疗剂选自:紫杉烷类(紫杉醇注射液、多西他赛、紫杉醇白蛋白等)、多柔比星、经修饰的多柔比星(Caelyx或Doxil))、舒尼替尼、索拉非尼和其它多激酶抑制剂、奥沙利铂、顺铂、卡铂、依托泊苷、吉西他滨和长春碱中的一种或多种；和/或，The use according to claim 21, wherein said chemotherapeutic agent is selected from the group consisting of: taxanes (paclitaxel injection, docetaxel, paclitaxel albumin, etc.), doxorubicin, modified doxorubicin ( Caelyx or Doxil)), one or more of sunitinib, sorafenib and other multi-kinase inhibitors, oxaliplatin, cisplatin, carboplatin, etoposide, gemcitabine and vinblastine; /or,
    所述肿瘤免疫疗法选自:T细胞接合剂(engaging agent)、靶向性免疫抑制、癌症疫苗/增强树突细胞功能和过继性细胞转移。The tumor immunotherapy is selected from the group consisting of: a T cell engaging agent, targeted immunosuppression, a cancer vaccine/enhanced dendritic cell function, and adoptive cell transfer.
  23. 如权利要求1～10任一项所述的抗体或其抗原结合片段、如权利要求11所述的多特异性抗体、如权利要求12所述的单链抗体、如权利要求13所述的抗体-药物偶联物、如权利要求14所述的核酸分子或者如权利要求19所述的药物组合物在制备用于治疗CSF-1R或CSF-1介导的疾病或病症的药物中的用途；其中所述的疾病优选为癌症、自身免疫疾病、炎性疾病或溶骨性骨质缺失；所述的癌症最优选为乳癌、子宫内膜癌、鳞状细胞癌、滤泡性淋巴瘤、肾细胞癌、葡萄膜黑色素瘤、子***、头颈癌、霍奇金病、星形细胞癌、肺腺癌、间皮瘤、绒毛膜癌、黑色素瘤、乳腺癌、卵巢癌、***癌、胰腺癌、肾癌、肺癌、肝癌、脑癌、胃癌、结肠直肠癌、膀胱癌、食管癌、***、多发性骨髓瘤、白血病、淋巴瘤和胶质母细胞瘤；所述的自身免疫疾病最优选为自身免疫性脑脊髓炎、***性红斑狼疮、多发性硬化、关节滑膜炎、银屑病、和类风湿关节炎；所述溶骨性骨质缺失选自骨质疏松、转移诱导的溶骨性骨质缺失和类风湿性关节炎诱导的骨质缺失。The antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, the multispecific antibody according to claim 11, the single-chain antibody according to claim 12, the antibody according to claim 13, or the antibody according to claim Use of a drug conjugate, a nucleic acid molecule according to claim 14 or a pharmaceutical composition according to claim 19 for the manufacture of a medicament for the treatment of a CSF-1R or CSF-1 mediated disease or condition; The disease described therein 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, kidney Cell carcinoma, uveal melanoma, cervical cancer, head and neck cancer, Hodgkin's disease, astrocytoma, lung adenocarcinoma, mesothelioma, choriocarcinoma, melanoma, breast cancer, ovarian cancer, prostate cancer, pancreas 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; Autoimmune cerebrospinal Myelitis, systemic lupus erythematosus, multiple sclerosis, joint synovitis, psoriasis, and rheumatoid arthritis; said osteolytic bone loss is selected from osteoporosis, metastasis-induced osteolytic bone loss And bone loss induced by rheumatoid arthritis.
  24. 一种防治CSF-1R或CSF-1介导的疾病或病症的方法，该方法包括给予所需患者治疗有效量的如权利要求1～10任一项所述的抗体或其抗原结合片段、如权利要求11所述的多特异性抗体、如权利要求12所述的单链抗体、如权利要求13所述的抗体-药物偶联物、如权利要求14所述的核酸分子或者如权利要求19所述的药物组合物，其中所述的疾病优选为癌症、自身免疫疾病、炎性疾病或溶骨性骨质缺失；所述的癌症最优选为乳癌、子宫内膜癌、鳞状细胞癌、滤泡性淋巴瘤、肾细胞癌、葡萄膜黑色素瘤、子***、头颈癌、霍奇金病、星形细胞癌、肺腺癌、间皮瘤、绒毛膜癌、黑色素瘤、乳腺癌、卵巢癌、***癌、胰腺癌、肾癌、肺癌、肝癌、脑癌、胃癌、结肠直肠癌、膀胱癌、食管癌、***、多发性骨髓瘤、白血病、淋巴瘤和胶质母细胞瘤；所述的自身免疫疾病最优选为自身免疫性脑脊髓炎、***性红斑狼疮、多发性硬化、关节滑膜炎、银屑病、和类风湿关节炎；所述溶骨性骨质缺失选自骨质疏松、转移诱导的溶骨性骨质缺失和类风湿性关节炎诱导的骨质缺失。A method of preventing a CSF-1R or CSF-1 mediated disease or condition, comprising administering a therapeutically effective amount of the antibody or antigen-binding fragment thereof of any one of claims 1 to 10, such as The multispecific antibody of claim 11, the single chain antibody of claim 12, the antibody-drug conjugate of claim 13, the nucleic acid molecule of claim 14, or the claim 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, Follicular lymphoma, renal cell carcinoma, uveal melanoma, cervical cancer, head and neck cancer, Hodgkin's disease, astrocytoma, 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; Autoimmune disease Most preferred are 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 and metastasis induction. Osteolytic bone loss and osteoid loss induced by rheumatoid arthritis.
PCT/CN2018/111818 2017-10-26 2018-10-25 Anti-csf-1r antibody, antigen-binding fragment thereof and pharmaceutical use thereof WO2019080889A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201880044519.6A CN110913894B (en) 2017-10-26 2018-10-25 anti-CSF-1R antibodies, antigen binding fragments thereof and medical uses thereof

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201711015488.5 2017-10-26
CN201711015488 2017-10-26

Publications (1)

Publication Number Publication Date
WO2019080889A1 true WO2019080889A1 (en) 2019-05-02

Family

ID=66247753

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2018/111818 WO2019080889A1 (en) 2017-10-26 2018-10-25 Anti-csf-1r antibody, antigen-binding fragment thereof and pharmaceutical use thereof

Country Status (3)

Country Link
CN (1) CN110913894B (en)
TW (1) TWI714895B (en)
WO (1) WO2019080889A1 (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023168432A3 (en) * 2022-03-04 2024-03-14 Coagulant Therapeutics Corporation Human antibodies against activated protein c and uses thereof

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111690063A (en) * 2020-05-25 2020-09-22 北京大学 anti-GM-CSF nano antibody and preparation method and application thereof
CN114426580B (en) * 2022-04-07 2022-06-24 北京科诺信诚科技有限公司 anti-CSF-1R antibodies, products, methods and uses thereof
CN116199772B (en) * 2022-11-16 2023-10-17 怡道生物科技(苏州)有限公司 HPV31 type capsid protein L1 monoclonal antibody, preparation method and application

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104159921A (en) * 2011-12-15 2014-11-19 霍夫曼-拉罗奇有限公司 Antibodies against human csf-1r and uses thereof
CN104271158A (en) * 2012-03-08 2015-01-07 霍夫曼-拉罗奇有限公司 Combination therapy of antibodies against human CSF-1R and uses thereof
CN107011438A (en) * 2010-05-04 2017-08-04 戊瑞治疗有限公司 With reference to CSF1R antibody

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102020639A (en) * 2009-09-14 2011-04-20 上海恒瑞医药有限公司 6-amido quinazoline or 3-cyano quinoline derivative, preparation method thereof and application of derivative to medicament

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107011438A (en) * 2010-05-04 2017-08-04 戊瑞治疗有限公司 With reference to CSF1R antibody
CN104159921A (en) * 2011-12-15 2014-11-19 霍夫曼-拉罗奇有限公司 Antibodies against human csf-1r and uses thereof
CN104271158A (en) * 2012-03-08 2015-01-07 霍夫曼-拉罗奇有限公司 Combination therapy of antibodies against human CSF-1R and uses thereof

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023168432A3 (en) * 2022-03-04 2024-03-14 Coagulant Therapeutics Corporation Human antibodies against activated protein c and uses thereof

Also Published As

Publication number Publication date
CN110913894A (en) 2020-03-24
TW201917136A (en) 2019-05-01
CN110913894B (en) 2023-03-10
TWI714895B (en) 2021-01-01

Similar Documents

Publication Publication Date Title
CN109843927B (en) anti-B7-H3 antibodies, antigen binding fragments thereof, and medical uses thereof
US11472882B2 (en) Anti-B7-H4 antibody, antigen-binding fragment thereof and pharmaceutical use thereof
JP2022502051A (en) SIRPα binding protein and its usage
US11525005B2 (en) Anti-CD40 antibody, antigen binding fragment thereof and medical use thereof
TWI714895B (en) Anti-csf-1r antibody, antigen-binding fragment thereof and pharmaceutical use thereof
WO2019141268A1 (en) Anti-4-1bb antibody, antigen-binding fragment thereof and medical use thereof
CN112243443B (en) anti-TROP-2 antibodies, antigen-binding fragments thereof, and medical uses thereof
WO2019184935A1 (en) Anti-cd27 antibody, antigen-binding fragment thereof and medical use thereof
TWI685504B (en) Anti-gitr antibody, antigen-binding fragments and pharmaceutical use thereof
CN115298216A (en) Antibody or antigen binding fragment thereof, preparation method and medical application thereof
WO2024088386A1 (en) Antibody, antigen-binding fragment thereof, and pharmaceutical use thereof
WO2022121941A1 (en) Anti-human msln antibody and application thereof
WO2022078424A1 (en) Anti-trop-2 antibody, antigen-binding fragment thereof or mutant thereof, and medical use thereof

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 18870191

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 18870191

Country of ref document: EP

Kind code of ref document: A1