WO2019137397A1 - Pd-l1抗体、其抗原结合片段及医药用途 - Google Patents

Pd-l1抗体、其抗原结合片段及医药用途 Download PDF

Info

Publication number
WO2019137397A1
WO2019137397A1 PCT/CN2019/070982 CN2019070982W WO2019137397A1 WO 2019137397 A1 WO2019137397 A1 WO 2019137397A1 CN 2019070982 W CN2019070982 W CN 2019070982W WO 2019137397 A1 WO2019137397 A1 WO 2019137397A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
cancer
variable region
chain variable
amino acid
Prior art date
Legal status (The legal status 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 status listed.)
Ceased
Application number
PCT/CN2019/070982
Other languages
English (en)
French (fr)
Inventor
顾晓玲
蒋家骅
张蕾
胡齐悦
顾津明
陶维康
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Jiangsu Hengrui Medicine Co Ltd
Shanghai Hengrui Pharmaceutical Co Ltd
Original Assignee
Jiangsu Hengrui Medicine Co Ltd
Shanghai Hengrui Pharmaceutical Co Ltd
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
Priority to AU2019206958A priority Critical patent/AU2019206958A1/en
Priority to RU2020124261A priority patent/RU2778085C2/ru
Priority to CA3087105A priority patent/CA3087105A1/en
Priority to EP19738076.9A priority patent/EP3741777A4/en
Priority to US16/960,297 priority patent/US11359021B2/en
Priority to JP2020538638A priority patent/JP2021510078A/ja
Priority to KR1020207022689A priority patent/KR20200108306A/ko
Priority to BR112020013475-9A priority patent/BR112020013475A2/pt
Application filed by Jiangsu Hengrui Medicine Co Ltd, Shanghai Hengrui Pharmaceutical Co Ltd filed Critical Jiangsu Hengrui Medicine Co Ltd
Priority to MX2020007406A priority patent/MX2020007406A/es
Priority to CN201980005432.2A priority patent/CN111278861B/zh
Publication of WO2019137397A1 publication Critical patent/WO2019137397A1/zh
Anticipated expiration legal-status Critical
Priority to ZA2020/04907A priority patent/ZA202004907B/en
Ceased legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/575—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present disclosure relates to a PD-L1 antibody and an antigen-binding fragment thereof, and further, the present disclosure also relates to a chimeric antibody, a humanized antibody comprising the CDR region of the PD-L1 antibody, and the present disclosure also relates to the PD-L1 antibody.
  • Tumor immunotherapy is a long-term hot spot in the field of cancer treatment, in which T cell tumor immunotherapy is at its core. Tumor immunotherapy is a way to fully utilize and mobilize killer T cells in tumor patients and kill tumors. It may be the most effective and safest way to treat tumors. At the same time, tumor escape is a huge obstacle to tumor immunotherapy. Tumor cells use their own inhibition of the immune system to promote rapid tumor growth.
  • tumor immunotherapy is to maximize the patient's own immune system response to the tumor. It not only activates the original immune system response in the body, but also maintains the duration of the immune system response and the intensity of the response. key.
  • T cells Activation of T cells in humans takes two signaling pathways.
  • a series of costimulatory molecules are required to provide a second signal.
  • T cells produce a normal immune response.
  • This dual-signal pathway system plays a vital role in the balance of the immune system in the body. It strictly regulates the body's immune response to both self and non-self antigens.
  • a second signal provided by a costimulatory molecule it will result in a non-responsive or sustained specific immune response of the T cell, resulting in tolerance. Therefore, the second signaling pathway plays a very important regulatory role in the whole process of the body's immune response.
  • PD-l Programmed death-l
  • CTL-4 cytotoxic T Iymphocyte antigen 4
  • PD-1 has two ligands, PD-L1 and PD-L2.
  • PD-L1 is mainly expressed on T cells, B cells, macrophages, and dendritic cells (DCs), and expression on cells after activation can be up-regulated.
  • the expression of PD-L2 is relatively limited, mainly expressed on antigen presenting cells, such as activated macrophages and dendritic cells.
  • PD-L1 inhibits the immune system by binding to PD-1 and B7-1, and many tumor cells and tumor tissue microenvironmental immune cells express PD-L1.
  • New research found high PD-L1 in human tumor tissues such as breast, lung, stomach, bowel, kidney, melanoma, non-small cell lung cancer, colon cancer, bladder cancer, ovarian cancer, pancreatic cancer, and liver cancer Protein expression, and the expression level of PD-L1 is closely related to the clinical and prognosis of patients.
  • the present disclosure provides monoclonal antibodies or antigen-binding fragments (also referred to as human PD-L1 binding molecules) that bind to the amino acid sequence or three-dimensional structure of the extracellular region of PD-L1.
  • the disclosure provides a monoclonal antibody or antigen-binding fragment thereof, which binds to human PD-L1, the monoclonal antibody or antigen-binding fragment thereof comprising:
  • a heavy chain variable region and a light chain variable region wherein the heavy chain variable region comprises an HCDR1, HCDR2 and HCDR3 region, a light chain variable region, having an amino acid sequence as set forth in SEQ ID NOs: 10, 12 and 13, respectively
  • the LCDR1, LCDR2 and LCDR3 regions having amino acid sequences as shown in SEQ ID NOS: 14, 15 and 16, respectively, wherein X 1 in the CDRs of SEQ ID NO. 12 is F or M, and X 2 is R or V, X 3 is N or H; or
  • a heavy chain variable region and a light chain variable region wherein the heavy chain variable region comprises an HCDR1, HCDR2 and HCDR3 region having an amino acid sequence as set forth in SEQ ID NOs: 11, 12 and 13, respectively, a light chain variable region
  • the LCDR1, LCDR2 and LCDR3 regions having amino acid sequences as shown in SEQ ID NOS: 14, 15 and 16, respectively, wherein X 1 in the CDRs of SEQ ID NO. 12 is F or M, and X 2 is R or V, X 3 is N or H; or
  • a heavy chain variable region and a light chain variable region wherein the heavy chain variable region comprises an HCDR1, HCDR2 and HCDR3 region, a light chain variable region having amino acid sequences as shown in SEQ ID NOs: 20, 21 and 22, respectively
  • the LCDR1, LCDR2, and LCDR3 regions having amino acid sequences as shown in SEQ ID NOS: 23, 24, and 25, respectively, wherein HCDR1, HCDR2, HCDR3, and LCDR1, LCDR2, and LCDR3 are not simultaneously SEQ ID NOs: 30, 38, 22, 23, 40 and 25,
  • X 4 is S or D
  • X 5 is Y or K
  • X 6 is H or M
  • X 7 is T
  • X 8 Is S, N or G
  • X 9 is S, L or G
  • X 10 is F, L, W or M
  • X 11 is A, P or T
  • X 12 is M, V, L or S
  • X 13 is F or Y
  • X 14 is V or A
  • X 15 is Y or N
  • X 16 is A, L or V
  • X 17 is E, F, Y or A ( These include the case where X 17 is selected from the group consisting of E, F and A and X 17 is selected from Y).
  • the monoclonal antibody or antigen-binding fragment thereof binds to human PD-L1 as described above, the monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain
  • the variable region comprises an HCDR1 represented by SEQ ID NO: 10, an HCDR2 as set forth in SEQ ID NO: 28 or 29, and an HCDR3 region as set forth in SEQ ID NO: 13, and the light chain variable region comprises an amino acid sequence
  • the LCDR1, LCDR2 and LCDR3 regions are shown as amino acid sequences of SEQ ID NOS: 14, 15 and 16, respectively.
  • the heavy chain variable region of a monoclonal antibody or antigen-binding fragment thereof as described above comprises an amino acid sequence as set forth in SEQ ID NO: 11 as shown in SEQ ID NO: 28 or HCDR2 and the HCDR3 region as set forth in SEQ ID NO: 13, the light chain variable region comprising the LCDR1, LCDR2 and LCDR3 regions having amino acid sequences as shown in SEQ ID NOs: 14, 15 and 16, respectively.
  • the heavy chain variable region of a monoclonal antibody or antigen-binding fragment thereof comprises an amino acid sequence such as HCDR1 set forth in SEQ ID NO: 30, as set forth in any one of SEQ ID NOs: 32-37 HCDR2 as shown and HCDR3 region as set forth in SEQ ID NO: 22, said light chain variable region comprising an LCDR1 of amino acid sequence SEQ ID NO: 23, such as SEQ ID NO: 39, 40, 41, 67 and 69 Either LCDR2 and LCDR3 area as SEQ ID NO: 25.
  • the heavy chain variable region of the monoclonal antibody or antigen-binding fragment thereof as described above comprises an HCDR1 having the amino acid sequence set forth in SEQ ID NO: 31, such as SEQ ID NO: 32, 33, HCDR2 as shown in any of 34, 35, 36 and 37 and HCDR3 region as set forth in SEQ ID NO: 22, said light chain variable region comprising an LCDR1 having an amino acid sequence of SEQ ID NO: 23, such as SEQ ID NO: LCDR2 of any of 39, 40, 41, 67 and 69 and LCDR3 zone of SEQ ID NO: 25.
  • the heavy chain variable region of the monoclonal antibody or antigen-binding fragment thereof comprises amino acid sequences as set forth in SEQ ID NO: 10, SEQ ID NO: 28, and SEQ ID NO: 13, respectively.
  • HCDR1, HCDR2 and HCDR3 regions, the light chain variable region comprising the LCDR1, LCDR2 and LCDR3 regions having amino acid sequences as shown in SEQ ID NOs: 14-16, respectively;
  • the heavy chain variable region of the monoclonal antibody or antigen-binding fragment thereof comprises amino acid sequences as set forth in SEQ ID NO: 11, SEQ ID NO: 28, and SEQ ID NO: 13, respectively.
  • HCDR1, HCDR2 and HCDR3 regions, the light chain variable region comprising the LCDR1, LCDR2 and LCDR3 regions having amino acid sequences as shown in SEQ ID NOs: 14-16, respectively;
  • the heavy chain variable region of the monoclonal antibody or antigen-binding fragment thereof comprises amino acid sequences as set forth in SEQ ID NO: 11, SEQ ID NO: 29, and SEQ ID NO: 13, respectively.
  • HCDR1, HCDR2 and HCDR3 regions, the light chain variable region comprising the LCDR1, LCDR2 and LCDR3 regions having amino acid sequences as shown in SEQ ID NOs: 14-16, respectively;
  • the heavy chain variable region of the monoclonal antibody or antigen-binding fragment thereof comprises amino acid sequences as set forth in SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 22, respectively.
  • HCDR1, HCDR2 and HCDR3 regions, wherein the light chain variable region comprises an LCDR1, LCDR2 and LCDR3 region having an amino acid sequence as set forth in SEQ ID NO: 23, SEQ ID NO: 39, SEQ ID NO: 25, respectively;
  • the heavy chain variable region of the monoclonal antibody or antigen-binding fragment thereof comprises amino acid sequences as set forth in SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 22, respectively.
  • HCDR1, HCDR2 and HCDR3 regions, wherein the light chain variable region comprises an LCDR1, LCDR2 and LCDR3 region having an amino acid sequence as set forth in SEQ ID NO: 23, SEQ ID NO: 40, SEQ ID NO: 25, respectively;
  • the heavy chain variable region of the monoclonal antibody or antigen-binding fragment thereof comprises amino acid sequences as set forth in SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO: 22, respectively.
  • HCDR1, HCDR2 and HCDR3 regions, wherein the light chain variable region comprises an LCDR1, LCDR2 and LCDR3 region having an amino acid sequence as set forth in SEQ ID NO: 23, SEQ ID NO: 40, SEQ ID NO: 25, respectively;
  • the heavy chain variable region of the monoclonal antibody or antigen-binding fragment thereof comprises amino acid sequences as set forth in SEQ ID NO: 30, SEQ ID NO: 34, SEQ ID NO: 22, respectively.
  • HCDR1, HCDR2 and HCDR3 regions, wherein the light chain variable region comprises an LCDR1, LCDR2 and LCDR3 region having an amino acid sequence as set forth in SEQ ID NO: 23, SEQ ID NO: 40, SEQ ID NO: 25, respectively;
  • the heavy chain variable region of the monoclonal antibody or antigen-binding fragment thereof comprises amino acid sequences as set forth in SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 22, respectively.
  • HCDR1, HCDR2 and HCDR3 regions, the light chain variable region comprising the LCDR1, LCDR2 and LCDR3 regions having the amino acid sequences set forth in SEQ ID NO: 23, SEQ ID NO: 40, SEQ ID NO: 25, respectively;
  • the heavy chain variable region of the monoclonal antibody or antigen-binding fragment thereof comprises amino acid sequences as set forth in SEQ ID NO: 31, SEQ ID NO: 32, and SEQ ID NO: 22, respectively.
  • HCDR1, HCDR2 and HCDR3 regions, the light chain variable region comprising the LCDR1, LCDR2 and LCDR3 regions of amino acid sequences as set forth in SEQ ID NO: 23, SEQ ID NO: 67 and SEQ ID NO: 25, respectively.
  • the heavy chain variable region of the monoclonal antibody or antigen-binding fragment thereof comprises amino acid sequences as set forth in SEQ ID NO: 31, SEQ ID NO: 32, and SEQ ID NO: 22, respectively.
  • HCDR1, HCDR2 and HCDR3 regions, the light chain variable region comprising the LCDR1, LCDR2 and LCDR3 regions of amino acid sequences as set forth in SEQ ID NO: 23, SEQ ID NO: 69 and SEQ ID NO: 25, respectively.
  • the heavy chain variable region of the monoclonal antibody or antigen-binding fragment thereof comprises amino acid sequences as set forth in SEQ ID NO: 31, SEQ ID NO: 32, and SEQ ID NO: 22, respectively.
  • HCDR1, HCDR2 and HCDR3 regions, the light chain variable region comprising the LCDR1, LCDR2 and LCDR3 regions having amino acid sequences as set forth in SEQ ID NO: 23, SEQ ID NO: 41 and SEQ ID NO: 25, respectively;
  • the heavy chain variable region of the monoclonal antibody or antigen thereof as described above comprises the HCDR1 having the amino acid sequence set forth in SEQ ID NO: 30, SEQ ID NO: 35, SEQ ID NO: 22, respectively.
  • a HCDR2 and an HCDR3 region wherein the light chain variable region comprises an LCDR1, LCDR2, and LCDR3 region having an amino acid sequence as set forth in SEQ ID NO: 23, SEQ ID NO: 40, and SEQ ID NO: 25, respectively;
  • the heavy chain variable region of the monoclonal antibody or antigen-binding fragment thereof comprises amino acid sequences as set forth in SEQ ID NO: 30, SEQ ID NO: 36, SEQ ID NO: 22, respectively.
  • HCDR1, HCDR2 and HCDR3 regions, wherein the light chain variable region comprises an LCDR1, LCDR2 and LCDR3 region having an amino acid sequence as set forth in SEQ ID NO: 23, SEQ ID NO: 40, SEQ ID NO: 25, respectively;
  • the heavy chain variable region of the monoclonal antibody or antigen-binding fragment thereof comprises amino acid sequences as set forth in SEQ ID NO: 30, SEQ ID NO: 37, SEQ ID NO: 22, respectively.
  • HCDR1, HCDR2 and HCDR3 regions, the light chain variable region comprising the LCDR1, LCDR2 and LCDR3 regions of amino acid sequence as set forth in SEQ ID NO: 23, SEQ ID NO: 40, SEQ ID NO: 25, respectively.
  • the affinity KD value of the monoclonal antibody or antigen-binding fragment thereof to human PD-L1 is less than 10 -9 M or 10 -10 M as previously described.
  • the monoclonal antibody or antigen-binding fragment thereof is cross-conjugated to cynomolgus monkey or rhesus monkey PD-L1 and/or mouse PD-L1 as previously described.
  • the monoclonal antibody or antigen-binding fragment comprises an amino acid sequence such as the heavy chain variable region set forth in SEQ ID NO: 17 and the light chain variable region set forth in SEQ ID NO: 19.
  • the monoclonal antibody or antigen-binding fragment comprises an amino acid sequence such as the heavy chain variable region set forth in SEQ ID NO: 18 and the light chain variable region set forth in SEQ ID NO: 19.
  • the monoclonal antibody or antigen-binding fragment comprises an amino acid sequence such as the heavy chain variable region set forth in SEQ ID NO: 26 and the light chain variable region set forth in SEQ ID NO:27.
  • the monoclonal antibody or antigen-binding fragment comprises an amino acid sequence such as the heavy chain variable region set forth in SEQ ID NO: 42 and the light chain variable region set forth in SEQ ID NO: 45.
  • the monoclonal antibody or antigen-binding fragment comprises an amino acid sequence such as the heavy chain variable region set forth in SEQ ID NO: 43 and the light chain variable region set forth in SEQ ID NO: 45.
  • the monoclonal antibody or antigen-binding fragment comprises an amino acid sequence such as the heavy chain variable region set forth in SEQ ID NO: 44 and the light chain variable region set forth in SEQ ID NO: 45.
  • the monoclonal antibody or antigen-binding fragment comprises an amino acid sequence as set forth in the heavy chain variable region set forth in SEQ ID NO: 46 and any one of SEQ ID NOs: 55, 56 and 57 Chain variable region.
  • the monoclonal antibody or antigen-binding fragment comprises a heavy chain variable region as set forth in SEQ ID NO: 47 and a light chain as set forth in any one of SEQ ID NOs: 55, 56 and 57 Variable area.
  • the monoclonal antibody or antigen-binding fragment comprises an amino acid sequence as set forth in the heavy chain variable region set forth in SEQ ID NO: 48 and any one of SEQ ID NOs: 55, 56 and 57 Chain variable region.
  • the monoclonal antibody or antigen-binding fragment comprises an amino acid sequence as set forth in the heavy chain variable region set forth in SEQ ID NO: 49 and any one of SEQ ID NOs: 55, 56 and 57 Chain variable region.
  • the monoclonal antibody or antigen-binding fragment comprises a heavy chain variable region set forth in SEQ ID NO: 50 and a light chain as set forth in any one of SEQ ID NOs: 55, 56, and 57 Variable area.
  • the monoclonal antibody or antigen-binding fragment comprises an amino acid sequence as described in SEQ ID NO: 51 and a light chain as shown in any one of SEQ ID NO: 55, 56 and 57. Chain variable region.
  • the monoclonal antibody or antigen-binding fragment comprises an amino acid sequence as set forth in the heavy chain variable region set forth in SEQ ID NO: 52 and any one of SEQ ID NOs: 55, 56 and 57 Chain variable region.
  • the monoclonal antibody or antigen-binding fragment comprises an amino acid sequence as set forth in the heavy chain variable region set forth in SEQ ID NO: 53 and any one of SEQ ID NOs: 55, 56 and 57 Chain variable region.
  • the monoclonal antibody or antigen-binding fragment comprises an amino acid sequence such as the heavy chain variable region set forth in SEQ ID NO: 54 and the light chain set forth in any one of SEQ ID NOs: 55 and 57. Variable area.
  • the monoclonal antibody or antigen-binding fragment comprises an amino acid sequence as set forth in the heavy chain variable region set forth in SEQ ID NO: 46 and any one of SEQ ID NOs: 55, 56, 57, 70 and 72. Light chain variable region as shown.
  • the monoclonal antibody or antigen-binding fragment comprises an amino acid sequence as set forth in the heavy chain variable region set forth in SEQ ID NO: 47 and any one of SEQ ID NOs: 55, 56, 57, 70 and 72. Light chain variable region as shown.
  • the monoclonal antibody or antigen-binding fragment comprises an amino acid sequence as set forth in the heavy chain variable region set forth in SEQ ID NO: 48 and any one of SEQ ID NOs: 55, 56, 57, 70 and 72. Light chain variable region as shown.
  • the monoclonal antibody or antigen-binding fragment comprises an amino acid sequence as set forth in the heavy chain variable region set forth in SEQ ID NO: 49 and any one of SEQ ID NOs: 55, 56, 57, 70 and 72. Light chain variable region as shown.
  • the monoclonal antibody or antigen-binding fragment comprises an amino acid sequence as described in SEQ ID NO: 50 and any of SEQ ID NO: 55, 56, 57, 70 and 72. Light chain variable region as shown.
  • the monoclonal antibody or antigen-binding fragment comprises an amino acid sequence as set forth in the heavy chain variable region set forth in SEQ ID NO: 51 and any one of SEQ ID NOs: 55, 56, 57, 70 and 72. Light chain variable region as shown.
  • the monoclonal antibody or antigen-binding fragment comprises an amino acid sequence as set forth in the heavy chain variable region set forth in SEQ ID NO: 52 and any one of SEQ ID NOs: 55, 56, 57, 70 and 72. Light chain variable region as shown.
  • the monoclonal antibody or antigen-binding fragment comprises an amino acid sequence as set forth in the heavy chain variable region set forth in SEQ ID NO: 53 and any one of SEQ ID NOs: 55, 56, 57, 70 and 72. Light chain variable region as shown.
  • the monoclonal antibody or antigen-binding fragment comprises an amino acid sequence as set forth in the heavy chain variable region set forth in SEQ ID NO: 66 and any one of SEQ ID NOs: 55, 56, 57, 70 and 72. Light chain variable region as shown.
  • the monoclonal antibody or antigen-binding fragment comprises an amino acid sequence as set forth in any one of the heavy chain variable regions set forth in SEQ ID NO: 54 and any one of SEQ ID NOs: 55, 57, 70 and 72.
  • Light chain variable region set forth in any one of the heavy chain variable regions set forth in SEQ ID NO: 54 and any one of SEQ ID NOs: 55, 57, 70 and 72.
  • the monoclonal antibody is a full length antibody as described above, further comprising a human antibody constant region, preferably, the heavy chain constant region of the human antibody constant region is selected from the group consisting of human IgGl, IgG2, IgG3, and IgG4 constant a region and a conventional variant thereof, wherein the light chain constant region of the human antibody constant region is selected from the group consisting of human antibody kappa and lambda chain constant regions and conventional variants thereof, preferably comprising an amino acid sequence as set forth in SEQ ID NO: 58, 60 or 65 Human antibody heavy chain constant region and human light chain constant region set forth in SEQ ID NO:59.
  • the monoclonal antibody is a full length antibody, further comprising a human antibody constant region comprising a human antibody heavy chain constant region set forth in SEQ ID NO: 58 and SEQ ID NO: 59 The human light chain constant region is shown.
  • the monoclonal antibody is a full length antibody as described above, further comprising a human antibody constant region comprising a human antibody heavy chain constant region set forth in SEQ ID NO: 60 and SEQ ID NO: 59 The human light chain constant region is shown.
  • the monoclonal antibody is a full length antibody, further comprising a human antibody constant region comprising a human antibody heavy chain constant region set forth in SEQ ID NO: 65 and SEQ ID NO: 59 The human light chain constant region is shown.
  • the antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab') 2 , single-chain antibody (scFv), dimerized V region (diabody), disulfide-stabilized An antigen-binding fragment of the V region (dsFv) and a peptide comprising a CDR.
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutically effective amount of a monoclonal antibody or antigen-binding fragment thereof according to the foregoing, and one or more pharmaceutically acceptable carriers, diluents or agents
  • the therapeutically effective amount of the monoclonal antibody or antigen-binding fragment thereof is a unit dose of 0.1 to 3000 mg/kg of the monoclonal antibody or antigen-binding fragment thereof as described above.
  • the disclosure provides a nucleic acid molecule encoding the aforementioned monoclonal antibody or antigen-binding fragment thereof.
  • the disclosure provides a recombinant vector comprising a nucleic acid molecule as described above.
  • the disclosure provides a host cell transformed with a recombinant vector according to the foregoing, the host cell being selected from the group consisting of a prokaryotic cell and a eukaryotic cell, preferably a eukaryotic cell, more preferably a mammalian cell.
  • the disclosure provides methods for producing a monoclonal antibody or antigen-binding fragment thereof as described above, the method comprising culturing the aforementioned host cell in a culture medium to form and accumulate the aforementioned monoclonal antibody Or an antigen binding fragment thereof, and recovering the monoclonal antibody or antigen-binding fragment thereof from the culture.
  • the disclosure provides methods for immunodetection or assay of human PD-L1, the methods comprising using a monoclonal antibody or antigen-binding fragment thereof as described above.
  • the disclosure provides the use of a monoclonal antibody or antigen-binding fragment thereof as described above for the preparation of a diagnostic agent for a disease associated with human PD-L1.
  • the disclosure provides methods of treating a disease associated with human PD-L1, the method comprising administering to the subject a pharmaceutically effective amount of a monoclonal antibody or antigen-binding fragment thereof as described above, or comprising The pharmaceutical composition, or a nucleic acid molecule as described above, for treating a human PD-L1-related disease, wherein the disease is preferably a tumor or a cancer; more preferably squamous cell carcinoma, myeloma, small cell lung cancer , non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC), glioma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma (DLBCL), Follicular lymphoma, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), primary media
  • ALL
  • the disclosure provides a monoclonal antibody or antigen-binding fragment thereof as described above, or a pharmaceutical composition comprising the same, or a nucleic acid molecule as described above, in the preparation of a disease associated with human PD-L1 Use in a therapeutic agent, wherein the disease is preferably a tumor or cancer; more preferably squamous cell carcinoma, myeloma, small cell lung cancer, non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC) , glioma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, acute lymphoblastic leukemia (ALL), acute myeloid cells Leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), primary mediastinal large B-cell lymphoma, mantle cell lympho
  • ALL
  • the cancer is selected from the group consisting of squamous cell carcinoma, myeloma, small cell lung cancer, non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC), glioma, Hodgkin Lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytes Leukemia (CLL), chronic myeloid leukemia (CML), primary mediastinal large B-cell lymphoma, mantle cell lymphoma (MCL), small lymphocytic lymphoma (SLL
  • Figure 1 PD-L1 antibody promotes secretion of cellular IFNy in PBMC-T lymphocyte activation assay.
  • FIG. 2A is a comparison of IgG1 and IgG4 forms of HRP00049
  • FIG. 2B is a comparison of IgG1 and IgG4 forms of H5L11
  • FIG. 2C is a comparison of IgG1 and IgG4 forms of HRP00052
  • FIG. 2D is a comparison of IgG1 and IgG4 forms of H6L11
  • FIG. 2E is an IgG1 of H18L61.
  • Figure 2F is a comparison of the IgGl and IgG4 forms of H12L64 compared to the IgG4 format.
  • Figure 3 Effect of PD-L1 antibody on tumor volume in a mouse A375 xenograft model.
  • Figure 4 Effect of PD-L1 antibody on tumor volume in a mouse colon cancer model.
  • Figure 5 Effect of PD-L1 antibody on tumor volume in a mouse xenograft model.
  • an “antibody” as used in the present disclosure 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, and ⁇ 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 class Ig of the five classes of Ig may have a kappa chain or a lambda chain.
  • the antibody light chain of the present disclosure may further comprise a light chain constant region comprising a human or murine kappa, lambda chain or variant thereof.
  • the antibody heavy chain of the present disclosure may further comprise a heavy chain constant region comprising human or murine IgGl, IgG2, IgG3, IgG4 or variants thereof.
  • variable region The sequences of about 110 amino acids near the N-terminus of the antibody heavy and light chains vary greatly, being the variable region (Fv region); the remaining amino acid sequences near the C-terminus are relatively stable and are constant regions.
  • 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).
  • CDR complementarity determining region
  • Each of the light chain variable region (LCVR) and the heavy chain variable region (HCVR) 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 LCVR and HCVR regions of the antibodies or antigen-binding fragments of the present disclosure conform to the known Kabat numbering rules (LCDR1-3, HCDR1-3) in number and position.
  • Antibodies of the present disclosure include murine antibodies, chimeric antibodies, humanized antibodies, preferably humanized antibodies.
  • “Monoclonal antibody” refers to an antibody obtained from a population of substantially homogeneous antibodies, ie, in addition to possible variant antibodies (eg, containing naturally occurring mutations or mutations produced during the manufacture of monoclonal antibody preparations, such variants are typically In addition to being present in small amounts, the individual antibodies that make up the population are identical and/or bind to the same epitope. Unlike polyclonal antibody preparations, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation (formulation) is directed against a single determinant on the antigen.
  • monoclonal indicates the identity of an antibody as obtained from a population of substantially homogeneous antibodies and should not be construed as requiring production of the antibody by any particular method.
  • monoclonal antibodies of the present disclosure can be prepared by a variety of techniques including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods of using transgenic animals containing all or part of a human immunoglobulin locus. Such methods, as well as other exemplary methods for preparing monoclonal antibodies, are described herein.
  • murine antibody is in this disclosure a monoclonal antibody to human PD-L1 prepared according to the knowledge and skill in the art.
  • the test subject is injected with the PD-L1 antigen at the time of preparation, and then the hybridoma expressing the antibody having the desired sequence or functional property is isolated.
  • the murine PD-L1 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 variants thereof.
  • 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 first established, and then the variable region gene is cloned from the murine hybridoma cell, and the variable region gene of the human antibody is cloned as needed, and the murine variable region gene is cloned.
  • the human constant region gene is ligated into a chimeric gene, inserted into an expression vector, and finally expressed in a eukaryotic or prokaryotic system.
  • the antibody light chain of the PD-L1 chimeric antibody further comprises a light chain constant region of a human ⁇ , a ⁇ chain or a variant thereof.
  • the antibody heavy chain of the PD-L1 chimeric antibody further comprises a heavy chain constant region of human IgG1, IgG2, IgG3, IgG4 or variants thereof, preferably comprising a human IgG1, IgG2 or IgG4 heavy chain constant region, or An IgGl, IgG2 or IgG4 variant of an amino acid mutation (such as a YTE mutation or a back mutation).
  • humanized antibody also known as CDR-grafted antibody, refers to the transplantation of murine CDR sequences into human antibody variable region frameworks, ie different types of human germline antibodies An antibody produced in a framework sequence. It is possible to overcome the heterologous reaction induced by the chimeric antibody by carrying a large amount of the mouse protein component.
  • framework sequences can be obtained from public DNA databases including germline antibody gene sequences or published references.
  • the germline DNA sequences of human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database (available on the Internet at www.mrccpe.com.ac.uk/vbase), as well as in Kabat, EA, etc.
  • humanized antibodies of the present disclosure also include humanized antibodies that are further affinity matured by phage display.
  • the human antibody variable region framework is designed to be selected, wherein the heavy chain FR region sequence on the antibody heavy chain variable region is derived from the human germline heavy chain IGHV3-23*04 The combined sequence with hJH4.1, and the combined sequence of human germline light chain IGKV1-12*01 and hJK4.1.
  • the human antibody variable region can be subjected to minimal reverse mutation (reversion mutation, that is, the amino acid residue of the FR region derived from the human antibody is mutated to the original antibody. Corresponding position amino acid residues) to maintain activity.
  • minimal reverse mutation that is, the amino acid residue of the FR region derived from the human antibody is mutated to the original antibody. Corresponding position amino acid residues
  • the CDR graft can attenuate the affinity of the PD-L1 antibody or antigen-binding fragment thereof to the antigen due to the framework residues that are in contact with the antigen. Such interactions can be the result of high mutations in somatic cells. Therefore, it may still be necessary to graft such donor framework amino acids to the framework of humanized antibodies. Amino acid residues involved in antigen binding from a non-human PD-L1 antibody or antigen-binding fragment thereof can be identified by examining the murine monoclonal antibody variable region sequences and structures. Each residue in the CDR donor framework that differs from the germline can be considered to be related.
  • the sequence can be compared to a subtype consensus sequence or a consensus sequence of a murine sequence with a high percent similarity.
  • Rare framework residues are thought to be the result of high somatic mutations and thus play an important role in binding.
  • antigen-binding fragment or “functional fragment” of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind an antigen (eg, PD-L1). It has been shown that fragments of full length antibodies can be utilized for antigen binding function of antibodies.
  • binding fragment contained in the term "antigen-binding fragment" of an antibody examples include (i) a Fab fragment, a monovalent fragment consisting of VL, VH, CL and CH1 domains; (ii) a F(ab') 2 fragment, including a divalent fragment of two Fab fragments joined by a disulfide bridge on the hinge region, (iii) an Fd fragment consisting of a VH and CH1 domain; (iv) an Fv fragment consisting of a single arm VH and VL domain of the antibody (v) a single domain or dAb fragment (Ward et al, (1989) Nature 341: 544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR) or (vii) A combination of two or more separate CDRs, optionally joined by a synthetic linker.
  • CDR complementarity determining region
  • the two domains VL and VH of the Fv fragment are encoded by separate genes, they can be joined by a synthetic linker using a recombinant method such that they are capable of producing a single protein in which the VL and VH regions are paired to form a monovalent molecule.
  • Chains referred to as single-chain Fv (scFv); see, for example, Bird et al. (1988) Science 242: 423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci USA 85: 5879-5883).
  • Such single chain antibodies are also intended to be included in the term "antigen-binding fragment" of an antibody.
  • the antigen binding portion can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of the intact immunoglobulin.
  • the antibodies may be antibodies of different isotypes, for example, IgG (eg, IgGl, IgG2, IgG3 or IgG4 subtype), IgA1, IgA2, IgD, IgE or IgM antibodies.
  • Antigen-binding fragments of the present disclosure include Fab, F(ab') 2 , Fab', single-chain antibody (scFv), dimerized V region (diabody), disulfide-stabilized V region (dsFv), inclusion Peptides of CDRs, etc.
  • Fab is an antibody fragment having a molecular weight of about 50,000 and having antigen-binding activity in a fragment obtained by treating an IgG antibody molecule with a protease papain (cleaving an amino acid residue at position 224 of the H chain), wherein the N-terminal side of the H chain About half of the entire L chain is bound by a disulfide bond.
  • the Fab of the present disclosure can be produced by treating a monoclonal antibody of the present disclosure which specifically recognizes human PD-L1 and binds to the amino acid sequence of the extracellular region or a three-dimensional structure thereof with papain. Furthermore, the Fab can be produced by inserting a DNA encoding a Fab of the antibody into a prokaryotic expression vector or a eukaryotic expression vector and introducing the vector into a prokaryote or eukaryote to express a Fab.
  • F(ab') 2 is an antibody obtained by digesting the lower portion of two disulfide bonds in the IgG hinge region with an enzyme pepsin, having an molecular weight of about 100,000 and having antigen-binding activity and comprising two Fab regions linked at a hinge position. Fragment.
  • the F(ab') 2 of the present disclosure can be produced by treating a monoclonal antibody of the present disclosure which specifically recognizes human PD-L1 and binds to the amino acid sequence of the extracellular region or a three-dimensional structure thereof with pepsin. Further, the F(ab') 2 can be produced by linking the Fab' described below with a thioether bond or a disulfide bond.
  • Fab' is an antibody fragment having a molecular weight of about 50,000 and having antigen-binding activity obtained by cleaving a disulfide bond of the hinge region of the above F(ab') 2 .
  • the Fab' of the present disclosure can be produced by treating F(ab') 2 of the present disclosure which specifically recognizes PD-L1 and binds to the amino acid sequence of the extracellular region or a three-dimensional structure thereof with a reducing agent such as dithiothreitol.
  • the Fab' can be produced by inserting a DNA encoding a Fab' fragment of an antibody into a prokaryotic expression vector or a eukaryotic expression vector and introducing the vector into a prokaryote or eukaryote to express Fab'.
  • single-chain antibody single-chain Fv
  • scFv single-chain Fv
  • scFv antibody heavy chain variable domain
  • VL antibody light chain variable domain
  • scFv molecules can have the general structure: NH 2 -VL- linker -VH-COOH or NH 2 -VH- linker -VL-COOH.
  • Suitable prior art linkers consist of a repeating GGGGS amino acid sequence or variant thereof, for example using 1-4 repeat variants (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448) .
  • linkers useful in the present disclosure are by Alfthan et al. (1995), Protein Eng. 8: 725-731, Choi et al. (2001), Eur. J. Immunol. 31: 94-106, Hu et al. (1996). , Cancer Res. 56: 3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293: 41-56 and Roovers et al. (2001), Cancer Immunol.
  • the scFv of the present disclosure can be produced by obtaining the cDNA encoding the VH and VL of the monoclonal antibody of the present disclosure which specifically recognizes human PD-L1 and binds to the amino acid sequence of the extracellular region or its three-dimensional structure, and constructs the coding scFv.
  • the DNA is inserted into a prokaryotic expression vector or a eukaryotic expression vector, and the expression vector is then introduced into a prokaryote or eukaryote to express an scFv.
  • a diabody is an antibody fragment in which an scFv is dimerized, and is an antibody fragment having a bivalent antigen-binding activity.
  • the two antigens may be the same or different.
  • the diabody of the present disclosure can be produced by obtaining the coding cDNA of VH and VL of the monoclonal antibody of the present disclosure which specifically recognizes human PD-L1 and binds to the amino acid sequence of the extracellular region or its three-dimensional structure, and constructs the coding.
  • the DNA of the scFv is such that the amino acid sequence of the peptide linker is 8 residues or less in length, and the DNA is inserted into a prokaryotic expression vector or a eukaryotic expression vector, and then the expression vector is introduced into a prokaryote or true
  • the diabody is expressed in nuclear organisms.
  • dsFv is obtained by linking a polypeptide in which one of amino acid residues in each of VH and VL is substituted with a cysteine residue via a disulfide bond between cysteine residues.
  • the amino acid residue substituted with a cysteine residue can be selected based on a three-dimensional structure prediction of the antibody according to a known method (Protein Engineering, 7, 697 (1994)).
  • the dsFv of the present disclosure can be produced by obtaining the cDNA encoding the VH and VL of the monoclonal antibody of the present disclosure which specifically recognizes human PD-L1 and binds to the amino acid sequence of the extracellular region or its three-dimensional structure, and constructs the coding dsFv
  • the DNA is inserted into a prokaryotic expression vector or a eukaryotic expression vector, and the expression vector is then introduced into a prokaryote or eukaryote to express dsFv.
  • a peptide comprising a CDR is constructed by one or more regions of a CDR comprising a VH or VL. Peptides comprising a plurality of CDRs can be joined directly or via a suitable peptide linker.
  • the CDR-containing peptide of the present disclosure can be produced by constructing the encoding of the CDRs of the VH and VL of the monoclonal antibody of the present disclosure which specifically recognizes human PD-L1 and binds to the amino acid sequence of the extracellular region or its three-dimensional structure. DNA, the DNA is inserted into a prokaryotic expression vector or a eukaryotic expression vector, and the expression vector is then introduced into a prokaryote or eukaryote to express the peptide.
  • the CDR-containing peptide can also be produced by chemical synthesis methods such as the Fmoc method or the tBoc method.
  • CDR refers to one of the six hypervariable regions within the variable domain of an antibody that contribute primarily to antigen binding.
  • One of the most commonly used definitions of the six CDRs is provided by Kabat E. A. et al. (1991) Sequences of proteins of immunological interest. NIH Publication 91-3242).
  • the Kabat definition of a CDR applies to the CDR1, CDR2 and CDR3 (CDR L1, CDR L2, CDR L3 or L1, L2, L3) of the light chain variable domain, as well as the CDR1 of the heavy chain variable domain.
  • CDR2 and CDR3 CDR H1, CDR H2, CDR H3 or H2, H3.
  • antibody framework refers to a portion of the variable domain VL or VH that serves as a scaffold for the antigen binding loop (CDR) of the variable domain. Essentially, it is a variable domain that does not have a CDR.
  • Conventional variant of the human antibody heavy chain constant region and the human antibody light chain constant region refers to a heavy chain constant region or a light chain constant region derived from humans that does not alter the structure and function of the variable region of the antibody as disclosed in the prior art.
  • variants include IgGl, IgG2, IgG3 or IgG4 heavy chain constant region variants that undergo site-directed engineering and amino acid substitutions of heavy chain constant regions, specifically replacing YTE mutations known in the art, L234A and / Or L235A mutation, or S228P mutation, or mutation of the knob-into-hole structure (such that the antibody heavy chain has a combination of knob-Fc and hole-Fc), or a combination of the above mutants, these mutations have been confirmed to make the antibody new Performance, but does not alter the function of the antibody variable region.
  • epitopes refers to a site on an antigen to which an immunoglobulin or antibody specifically binds (eg, a specific site on the PD-L1 molecule).
  • Epitopes typically include at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 consecutive or non-contiguous amino acids in a unique spatial conformation. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G. E. Morris, Ed. (1996).
  • the terms “specifically binds”, “selectively binds”, “selectively binds” and “specifically binds” refers to the binding of an antibody to an epitope on a predetermined antigen. Typically, the antibody binds with an affinity (KD) of less than about 10 -8 M, such as less than about 10 - 9 M, 10 -10 M, 10 -11 M or less.
  • KD affinity
  • KD refers to the dissociation equilibrium constant for a particular antibody-antigen interaction.
  • the antibodies of the present disclosure bind PD-L1 with a dissociation equilibrium constant (KD) of less than about 10 -7 M, such as less than about 10 -8 M, 10 -9 M, or 10 -10 M or less, for example, as Determined using a surface plasmon resonance (SPR) technique in a BIACORE instrument.
  • SPR surface plasmon resonance
  • an antigen binding protein eg, an antibody or immunologically functional fragment thereof
  • a reference antigen binding protein eg, a ligand or reference antibody
  • a common antigen eg, PD-L1
  • 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 that binds to a solid surface or cell loaded with either an unlabeled antigen binding protein and a labeled reference antigen binding protein.
  • Competitive inhibition is measured by measuring the amount of label bound to a solid surface or cell in the presence of the measured antigen binding protein.
  • the antigen binding protein is tested 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. Protein, the two epitopes that interfere with each other spatially.
  • 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.
  • nucleic acid molecule refers to a DNA molecule and an RNA molecule.
  • the nucleic acid molecule may be single stranded or double stranded, but is preferably a double stranded DNA.
  • a nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence.
  • a promoter or enhancer is operably linked to the coding sequence if the promoter or enhancer affects the transcription of the coding sequence.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • the vector is a "plasmid” which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated.
  • the vector is a viral vector in which additional DNA segments can be ligated into the viral genome.
  • the vectors disclosed herein are capable of autonomous replication in a host cell into which they have been introduced (for example, a bacterial vector having an origin of replication of bacteria and an episomal mammalian vector) or can be integrated into the genome of the host cell after introduction into the host cell, thereby The host genome is replicated together (eg, a non-episomal mammalian vector).
  • a mouse can be immunized with human PD-L1 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.
  • the human FR germline sequence can be obtained from the ImMunoGeneTics (IMGT) website http://imgt.cines.fr by comparing the IMGT human antibody variable region germline gene database and MOE software, or from the Immunoglobulin Journal, 2001 ISBN 014441351. obtain.
  • IMGT ImMunoGeneTics
  • 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) and NSO cells.
  • the antibodies or antigen-binding fragments engineered in the present disclosure can be prepared and purified by conventional methods.
  • cDNA sequences encoding heavy and light chains can be cloned and recombined into GS expression vectors.
  • the recombinant immunoglobulin expression vector can stably transfect CHO cells.
  • mammalian expression systems result in glycosylation of antibodies, particularly at the highly conserved N-terminal site of the Fc region.
  • Stable clones were obtained by expressing antibodies that specifically bind to human PD-L1. 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 by a conventional technique.
  • purification is carried out using an A or G Sepharose FF column containing an adjusted buffer.
  • the non-specifically bound components are washed away.
  • the bound antibody was eluted by a pH gradient method, and the antibody fragment was detected by SDS-PAGE and collected.
  • 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.
  • 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, for example a composition comprising any of the binding compounds of the present disclosure, 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 to induce such symptoms to degenerate or to inhibit 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 present disclosure may be ineffective in ameliorating the symptoms of each target disease
  • any statistical test methods known in the art such as Student's t test, chi-square test, according to Mann and Whitney 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.
  • Constantly modified refers to amino acids in other amino acid substitution proteins having similar characteristics (eg, charge, side chain size, hydrophobicity/hydrophilicity, backbone conformation and rigidity, etc.), such that Changes are made without altering the biological activity of the protein. It will be appreciated by those skilled in the art that, in general, a single amino acid substitution in a non-essential region of a polypeptide does not substantially alter biological activity (see, for example, Watson et al. (1987) Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co., Page 224, (4th edition)). In addition, substitution of structurally or functionally similar amino acids is unlikely to disrupt biological activity.
  • 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 methodological route and dosage of the 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 being, cell or human, depending on the situation.
  • Endogenous refers to a substance produced in a cell, organism or human body, depending on the circumstances.
  • “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 x 100.
  • the expression "cell”, “cell line” and “cell culture” are used interchangeably and all such names include progeny.
  • 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.
  • PCR polymerase chain reaction
  • oligonucleotide primers can be designed; these primers are identical or similar in sequence to the corresponding strand of the template to be amplified.
  • the 5' terminal nucleotides of the two primers may coincide with the ends of the material to be amplified.
  • PCR can be used to amplify specific RNA sequences, specific DNA sequences from total genomic DNA, and cDNA, phage or plasmid sequences transcribed from total cellular RNA, and the like. See generally, Mullis et al. (1987) Cold Spring Harbor Symp. Ouant. Biol. 51:263; Erlich ed., (1989) PCR TECHNOLOGY (Stockton Press, N.Y.).
  • PCR used herein is considered as an example, but not the only example, of a nucleic acid polymerase reaction method for amplifying a nucleic acid test sample, which comprises using a known nucleic acid and a nucleic acid polymerase as a primer to amplify or Produce a specific portion of the nucleic acid.
  • “Pharmaceutical composition” means a mixture comprising one or more compounds described herein, or a physiologically/pharmaceutically acceptable salt or prodrug thereof, with other chemical components, such as physiological/pharmaceutically acceptable Carrier and excipients.
  • the purpose of the pharmaceutical composition is to promote the administration of the organism, which facilitates the absorption of the active ingredient and thereby exerts biological activity.
  • the present disclosure includes an agent for treating a disease associated with PD-L1 positive cells, the agent comprising the monoclonal antibody of the present disclosure or an antibody fragment thereof as an active ingredient.
  • the disease associated with PD-L1 there is no limitation on the disease associated with PD-L1 as long as it is a disease associated with PD-L1, for example, by using the molecularly induced therapeutic response of the present disclosure by binding to human PD-L1, and then repressing PD-L1 and its ligand Binding of PD-1, B7-1, or killing of tumor cells overexpressing PD-L1.
  • the molecules of the present disclosure are very useful for people who have a tumor or cancer, preferably melanoma, colon cancer, breast cancer, lung cancer, stomach cancer, when in preparations and formulations suitable for therapeutic applications. Intestinal cancer, kidney cancer, non-small cell lung cancer, bladder cancer, etc.
  • the present disclosure relates to methods for immunodetection or assay of PD-L1, reagents for immunodetection or assay of PD-L1, methods for immunodetection or assay of cells expressing PD-L1, and for diagnosis and PD-
  • a diagnostic agent for a disease associated with an L1-positive cell comprising a monoclonal antibody or antibody fragment of the present disclosure which specifically recognizes human PD-L1 and binds to an amino acid sequence of an extracellular region or a three-dimensional structure thereof as an active ingredient.
  • the method for detecting or measuring the amount of PD-L1 may be any known method.
  • it includes immunodetection or assay methods.
  • the immunodetection or assay method is a method of detecting or measuring the amount of an antibody or the amount of an antigen using a labeled antigen or antibody.
  • immunoassay or assay methods include radioactive substance labeling immunological antibody method (RIA), enzyme immunoassay (EIA or ELISA), fluorescent immunoassay (FIA), luminescent immunoassay, protein immunoblotting, physicochemical methods Wait.
  • the above-mentioned diseases associated with PD-L1 positive cells can be diagnosed by detecting or measuring cells expressing PD-L1 using the monoclonal antibodies or antibody fragments of the present disclosure.
  • a known immunodetection method can be used, and immunoprecipitation, fluorescent cell staining, immunohistochemical staining, or the like is preferably used. Further, a fluorescent antibody staining method or the like using the FMAT8100HTS system (Applied Biosystem) can be used.
  • a living sample for detecting or measuring PD-L1 is not particularly limited as long as it has a possibility of including cells expressing PD-L1, such as tissue cells, blood, plasma, serum, pancreatic juice, urine. Liquid, feces, tissue fluid or culture fluid.
  • the diagnostic agent containing the monoclonal antibody of the present disclosure or an antibody fragment thereof may further contain an agent for performing an antigen-antibody reaction or an agent for detecting a reaction, depending on a desired diagnostic method.
  • Agents for performing antigen-antibody reactions include buffers, salts, and the like.
  • the reagents for detection include reagents commonly used in immunoassays or assay methods, such as labeled secondary antibodies that recognize the monoclonal antibodies, antibody fragments or conjugates thereof, substrates corresponding to the labels, and the like.
  • the present disclosure obtains a PD-L1 antibody having higher affinity, stronger tumor killing activity, lower immunogenicity and the like.
  • an affinity mature yeast library of scFv antibody was designed and prepared based on two antibodies of HRP00052 and HRP00049, and a new human PD-L1 antibody was selected therefrom.
  • the CDR and light heavy chain variable region sequences of HRP00052 and HRP00049 are both from WO2017084495A1. The specific sequence is as follows:
  • HRP00049 antibody heavy chain sequence (SEQ ID NO: 1)
  • HRP00049 antibody light chain sequence (SEQ ID NO: 3)
  • HRP00052 antibody heavy chain sequence (SEQ ID NO: 5)
  • HRP00052 antibody heavy chain sequence encoding gene sequence (SEQ ID NO: 6)
  • HRP00052 antibody light chain sequence (SEQ ID NO: 7)
  • HRP00052 antibody light chain sequence encoding gene sequence (SEQ ID NO: 8)
  • the underlined portion of the above antibody sequence is the variable region portion of the antibody, and the other normal portion is the antibody constant region portion.
  • Yeast Library Construction of Yeast Library: Degenerate primers were designed and the designed mutant amino acids were introduced into the library of HRP00049 and HRP00052 antibodies by PCR. The QC of the library was verified by the second-generation sequencing method, and seven antibody yeast libraries with a library capacity of 10 9 were constructed based on HRP00049 and HRP00052 sequences, respectively.
  • the human PD-L1-IgG1Fc fusion protein was designed and synthesized, and purified by affinity column of Protein A to obtain high-purity recombinant PD-L1-Fc protein for detecting binding of anti-PD-L1 antibody to antigen.
  • Human PD-L1-IgG1Fc (SEQ ID NO: 9)
  • HRP00052 library using biotinylated human PD-L1-hIgG1Fc antigen after two rounds of MACS screening (streptomycin beads, Invitrogen), and two screening FACS (BD FACSAria TM FUSION).
  • MACS screening streptomycin beads, Invitrogen
  • FACS BD FACSAria TM FUSION
  • a total of about 400 yeast monoclonal cultures and induced expression were selected, and FACS (BD FACSCanto II) was used to detect the binding of yeast monoclonal to human PD-L1-hIgG1 Fc antigen, and yeast monoclonal sequencing with higher affinity than wild-type HRP00052 antibody was selected.
  • the redundant sequences are removed, and the non-redundant sequences are converted into full-length IgG ( ⁇ 1, ⁇ ) for mammalian cell expression.
  • Full-length antibody after affinity purification using BIAcore TM X-100 (GE Life Sciences) for affinity assays.
  • the HRP00049 library utilizes biotinylated human PD-L1-hIgG1 Fc antigen and biotinylated mouse PD-L1-hIgG1Fc, after three rounds of MACS screening and three rounds of FACS screening. A total of about 400 yeast monoclonal cultures and induced expression were selected. FACS was used to detect the binding of yeast monoclonal to human PD-L1-hIgG1Fc antigen and mouse PD-L1-hIgG1Fc antigen, and the simultaneous binding of human PD-L1-hIgG1Fc antigen was selected. Yeast monoclonal sequencing validation of mouse PD-L1-hIgG1 Fc antigen.
  • the clones selected based on the HRP00049 sequence-based mutant library differed from HRP00049 in HCDR1 and HCDR2.
  • Related CDR sequences or formulas and their corresponding heavy chain variable regions are described below.
  • HCDR1 is DGSAYWS (SEQ ID NO: 10) or NDYWT (SEQ ID NO: 11)
  • the relevant light chain variable region sequence was obtained as follows: (SEQ ID NO: 19)
  • the clones selected based on the HRP00052 sequence-based mutant library differed from HRP00052 on HCDR1 and HCDR2 and LCDR2.
  • the related HCDR1 and HCDR2 and LCDR2 formulas and their corresponding heavy light chain variable regions are described below.
  • X 4 is selected from the group consisting of S and D
  • X 5 is selected from Y and K
  • X 6 is selected from H and M
  • X 7 is selected from the group consisting of T, S, H and G
  • X 8 is selected from S.
  • N and G X 9 is selected from S, L and G
  • X 10 is selected from F, L, W and M
  • X 11 is selected from A, P and T
  • X 12 is selected from M, V, L and S, X 13 Selected from F and Y.
  • X 14 is selected from V and A
  • X 15 is selected from Y and N
  • X 16 is selected from A, L and V
  • X 17 is selected from the group consisting of E, F, Y and A (including the case where X 17 is selected from the group consisting of E, F and A and X 17 is selected from Y).
  • the specific correlation sequences obtained include, but are not limited to, those described in Tables 1 and 2:
  • the light and heavy chain variable region of the above antibody is selected from the constant region of the human heavy chain IgG1/light chain kappa, and the heavy chain variable region and the light chain variable region are respectively combined to form a complete antibody heavy chain and light chain, and the specific heavy chain
  • the sequences of the constant region and the light chain constant region are as follows:
  • IgG1 heavy chain constant region (SEQ ID NO: 58)
  • Kappa light chain constant region (SEQ ID NO: 59)
  • the heavy light chain variable region derived from the HRP00049 and HRP00052 mutant antibody libraries as described in Table 5-1 and Table 5-2 when joined to the heavy light chain constant region to form an intact antibody, it is indicated that the human IgG1 heavy chain constant region (SEQ ID NO: 58) and the kappa light chain constant region (SEQ ID NO: 59) are ligated to form an intact antibody, for example, H15L61 means that H15 and IgG1 heavy chain constant region are joined to form a heavy body.
  • the chain and L61 are linked to the light chain constant region of kappa to form a light chain, and the light and heavy chains are combined to form an intact antibody, and other naming analogies.
  • HRP00052-IgG1 refers to an intact antibody formed by replacing the heavy chain constant region of HRP00052 (IgG4 subclass) with the above IgG1 heavy chain constant region (SEQ ID NO: 58).
  • the full length antibodies of Tables 6-1 and 6-2 are screened from the above-described HRP00049 and HRP00052 mutant antibody libraries.
  • the heavy light chain variable region is ligated to the human IgG4 heavy chain constant region (shown as SEQ ID NO: 60, containing the S228P and 234A235A mutations) and the kappa light chain constant region (same as SEQ ID NO: 59), respectively, to form a complete
  • An antibody such as H15L61-IgG4, refers to a heavy chain formed by H15 binding to the heavy chain constant region of IgG4 and a light chain linked to the kappa light chain constant region by L61. The light and heavy chains combine to form an intact antibody, and other naming analogs.
  • Human IgG4 heavy chain constant region (SEQ ID NO: 60)
  • the full length antibodies of Tables 7-1 and 7-2 are the heavy and light chain variable regions selected from the above-described HRP00049 and HRP00052 mutant antibody libraries, respectively, and are heavy with the following human IgG4 S228P
  • the chain constant region shown as SEQ ID NO: 65, containing the S228P mutation
  • the kappa light chain constant region as SEQ ID NO: 59
  • H15L61-IgG4 refers to the heavy chain constant region from H15 and IgG4.
  • the ligation forms a heavy chain and L61 is linked to the kappa light chain constant region to form a light chain, and the light and heavy chains combine to form an intact antibody, and other naming analogies.
  • the heavy chain constant region sequence of IgG4 S228P is as follows: (SEQ ID NO: 65)
  • Table 7-2 Full-length antibody names following heavy chain variable region joining human IgG4 S228P heavy chain constant region (SEQ ID NO: 65) and light chain variable region linked to kappa light chain constant region (SEQ ID NO: 59)
  • the present disclosure uses Merck's PD-L1 antibody Avelumab (A09) and/or Genetech's 3280A as positive controls, wherein the light heavy chain amino acid sequence of A09 (derived from US20140341917) is as follows:
  • the light and heavy chain amino acid sequences of 3280A are as follows:
  • the PD-L1 antibody was obtained by a conventional method.
  • the antibodies in the following examples are full length antibodies.
  • *10 ⁇ g/ml, 5 ⁇ g/ml and 1 ⁇ g/ml are the concentrations of PD-L1 in different blocking assays, respectively.
  • the PD-L1 antibody of the present disclosure can block the binding of PD-L1 to PD1, and can also block the binding of PD-L1 to B7-1, and the H5L11, H6L11 antibody with HRP00049 mutation has Mouse PD-L1 cross-binding activity blocks the ability of mouse PD-L1 to bind to PD-1.
  • Human anti-capture antibody was covalently coupled to biosensor chip CM5 (Cat.#BR-1000-12, GE) according to the method described in the Human Anti-Capture Kit (Cat. #BR-1008-39, GE). On the chip, and thus affinity capture a certain amount of human PD-L1 (Cat. #10084-H08H, Sino Biological), monkey PD-L1 (Cat. #90251-C08H, Sino Biological), mouse PD-L1 (Cat. #50010-M08H, Sino Biological). The reaction affinities of the PD-L1 antibody and PD-L1 were measured using a Biacore X100, GE instrument.
  • test used HBS-EP+10X buffer solution (Cat.#BR-1006-69, GE) diluted to 1 ⁇ (pH7.4) with DIWater, using BIAevaluation version 4.1, GE software (1:1) Langmuir The model fits the data and the affinity values are obtained. The results are shown in Table 9.
  • PBMC peripheral blood mononuclear cells
  • Fresh blood PBMC were obtained by Ficoll-Hypaque density gradient centrifugation (Stem Cell Technologies), and cultured in RPMI 1640 medium supplemented with 10% (v/v) FBS, and cultured at 37 ° C under 5% CO 2 .
  • the freshly purified PBMC was adjusted to a density of 2 ⁇ 10 6 /ml in RPMI 1640 medium, and 25 ⁇ l of tuberculin was added to 20 mL of the cell suspension, and cultured in a 37 ° C, 5% CO 2 incubator for 5 days. On the sixth day, the cultured cells were collected and centrifuged, washed once with PBS, resuspended in fresh RPMI 1640 medium, adjusted to a density of 1 ⁇ 10 6 /ml, and seeded into a 96-well cell culture plate at 90 ⁇ l per well. A gradient diluted antibody sample (diluted with PBS) or an equivalent amount of isotype IgG was added as a blank control, 10 ⁇ l per well.
  • the cell culture plates were incubated at 37 ° C for 3 days in a 5% CO 2 incubator.
  • the cell culture plate was taken out, and the cell culture supernatant was collected by centrifugation (4000 rpm, 10 min), and the level of IFN- ⁇ was measured by an ELISA method (human IFN- ⁇ detection kit, Xinbosheng, EHC 102g.96).
  • ELISA method human IFN- ⁇ detection kit, Xinbosheng, EHC 102g.96.
  • Example 7 Exemplary antibody-mediated ADCC effect of PD-L1+ cells
  • PBMCs were resuspended in RPMI-1640 medium containing 5% Low IgG FBS (BIOS UN, BS-0007-500) for cell counting.
  • CHO-S/PD-L1 cells were resuspended in RPMI1640 medium containing 5% Low IgG FBS and counted, seeded in 96-well plates at a density of 10,000 cells per well, and then incubated with gradient-diluted PD-L1 antibody. After 15 minutes, 300,000 human PBMC cells were added to each well for 4 hours. The medium control was set, the CHO-S/PD-L1 cells spontaneously released the control, the PBMC cells spontaneously released the control and the CHO-S/PD-L1 cells were maximally lysed. Control.
  • the 96-well plate was centrifuged, and 50 ⁇ L of the supernatant was transferred to another 96-well plate, and 50 ⁇ L of CytoTox96 Non-Radioactive Cytotoxicity Assay (Promega, G1780) was added to each well, and the mixture was incubated at room temperature for 30 minutes in the dark, and a stop solution was added. The absorbance was read at a wavelength of 490 nm. The data were analyzed according to the following formula, and the cleavage rate was calculated. The EC 50 value of the ADCC effect of the antibody was calculated by Graphpad Prism software according to the respective concentrations of the antibody and the corresponding cleavage rate.
  • % lysis rate (sample hole 490nm - PBMC cells spontaneously released 490nm - CHO-S/PD-L1 cells spontaneously released 490nm ) / (CHO-S/PD-L1 cells maximally lysed 490nm - CHO-S/PD-L1 cells spontaneously Release 490nm ) ⁇ 100
  • Example 8 Role of exemplary antibodies in human melanoma A375 xenograft models
  • the cultured A375 cells were mixed with PBMC and inoculated subcutaneously in NOG mice.
  • the experiment was divided into PBMC+PBS group, PBMC+H12L64 20 mg/kg group, PBMC+H12L64-IgG4-20 mg/kg group and PBMC+HRP0005220 mg/kg group.
  • Six rats in each group were administered once every two days, intraperitoneally, and administered 11 times in succession. After the end of the administration experiment, the tumor growth inhibition rate TGI (%) and the relative tumor growth rate T/C (%) were used for evaluation of the therapeutic effect.
  • the results are shown in Figure 3 and Table 10.
  • the tumor suppressive rate of H12L64-IgG4 20mg/kg group was 82.15%.
  • the results showed that H12L64 20 mg/kg group and H12L64-IgG4 20 mg/kg and HRP00052 20 mg/kg were effective in inhibiting the growth of A375 (p ⁇ 0.05).
  • H12L64, H12L64-IgG4 and HRP00052 tumor-bearing mice showed no weight loss or animal death, indicating that the mice were well tolerated by H12L64, H12L64-IgG4 and HRP00052 at this dose.
  • TGI% tumor inhibition rate
  • T/C% tumor growth rate
  • CR tumor regression.
  • mice 100 ⁇ l of MC38-hPD-L1 cells (hPD-L1, 4 ⁇ 10 5 cells expressed on the surface of cells after transforming hPD-L1 into mouse colon cancer cells MC38) were inoculated into the right flank of 50 C57 mice, 7 days later.
  • the mice were oversized and the tumor volume was too large.
  • the mice were randomly divided into three groups: PD-L1 antibody single-use group and negative control group, with a total tumor volume of about 52 mm 3 , a total of 4 groups of 10 rats each.
  • each drug was administered to the abdominal cavity (the control group was injected with an equal volume of PBS) three times a week for 12 times, the drug administration cycle was 28 days, and the monitoring of the tumor-bearing mice was completed two days after the drug withdrawal.
  • the tumor volume was measured twice a week, the body weight was weighed, and data was recorded.
  • the grouping and administration are shown in the table below. Body weight, tumor volume, and tumor weight of each group were expressed as mean ⁇ standard deviation (Mean ⁇ SEM) and plotted using Graphpad Prism 5 and Excel software, and statistical analysis was performed using student t test.
  • Tumor volume (TV) 1/2 ⁇ L length ⁇ L short 2
  • Tumor proliferation rate T/C% (T-T0)/(C-C0) ⁇ 100%
  • the tumor-bearing mice were euthanized and weighed and weighed.
  • the tumor weight was basically consistent with the tumor volume: in the three drug groups, the average tumor weight was the smallest in the H6L11 group, followed by the A09 group, and the average tumor weight of HRP00052-IgG1 was the largest. There was a significant difference between the two groups of H6L11 and A09 and the control group (p ⁇ 0.001). Tumor-bearing mice are well tolerated by various drugs, and no symptoms such as weight loss are caused by drugs.
  • the tumor volume was measured twice a week, the body weight was weighed, and data was recorded. The grouping and administration are shown in the table below. Body weight, tumor volume and tumor weight of each group were expressed as mean ⁇ standard deviation (Mean ⁇ SEM) and plotted with Graphpad Prism 6 and Excel software, using statistical analysis of student t test.
  • Tumor volume (TV) 1/2 ⁇ L length ⁇ L short 2
  • Tumor proliferation rate T/C% (T-T0)/(C-C0) ⁇ 100%
  • the tumor volume of the PD-L1 monoclonal antibody (H5L11-IgG4 S228P group) which was cross-reactive with mouse PD-L1 was significantly smaller than that of the control group, and it started at about one week after administration and the control group. There was a statistical difference between them.
  • the tumor-bearing mice were euthanized and weighed and weighed. There was a certain similarity between the tumor weight and the tumor volume. During the experiment, there was no significant difference in body weight between the drug-administered group and the control group, and the mice were well tolerated for each administered antibody.

Landscapes

  • Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Hematology (AREA)
  • Biomedical Technology (AREA)
  • Urology & Nephrology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Physics & Mathematics (AREA)
  • Cell Biology (AREA)
  • General Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Food Science & Technology (AREA)
  • Analytical Chemistry (AREA)
  • Pathology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Hospice & Palliative Care (AREA)
  • Oncology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

一种新的PD-L1抗体、其抗原结合片段及医药用途。包含所述PD-L1抗体CDR区的人源化抗体，以及包含PD-L1抗体及其抗原结合片段的药物组合物，以及其作为药物的用途。一种人源化的PD-L1抗体在制备用于治疗PD-L1相关的疾病或病症的药物中的用途。

Description

PD-L1抗体、其抗原结合片段及医药用途
本申请要求申请日为2018年01月10日的中国专利申请CN201810023267.0的优先权。本申请引用上述中国专利申请的全文。
技术领域
本公开涉及PD-L1抗体以及其抗原结合片段，进一步地，本公开还涉及包含所述PD-L1抗体CDR区的嵌合抗体、人源化抗体，本公开还涉及包含所述PD-L1抗体及其抗原结合片段的药物组合物，以及其作为PD-L1相关疾病诊断剂和治疗药物的用途。
背景技术
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。
肿瘤免疫治疗是肿瘤治疗领域一个长时期的热点，其中T细胞的肿瘤免疫治疗又处于其核心位置。肿瘤免疫治疗是充分利用、调动肿瘤患者体内的杀伤性T细胞，对肿瘤进行杀伤作用，它可能是最有效的也是最安全的治疗肿瘤的途径。与此同时，肿瘤逃逸是肿瘤免疫治疗面临的一个巨大障碍，肿瘤细胞利用其自身对免疫系统的抑制作用促进了肿瘤的快速生长。
肿瘤的免疫逃逸机制与机体对肿瘤的免疫应答之间存在着极为复杂的关系。肿瘤免疫治疗早期肿瘤特异性的杀伤性T细胞是有其生物活性的，但随着肿瘤生长后期失去了杀伤的功能。所以肿瘤免疫治疗是为了最大限度的提高患者自身对肿瘤的免疫系统反应，它不但要在体内激活原有的免疫系统反应，更要维持免疫系统反应的持续时间和反应强度，才是免疫治疗肿瘤的关键。
人体内T细胞的活化采取了两条信号通路系统，除了需要通过抗原呈递细胞递呈MHC-抗原肽给T细胞提供第一信号外，还需要一系列协同刺激分子提供第二信号，进而才能使T细胞产生正常的免疫应答。这个双信号通路系统对体内免疫系统的平衡起着至关重要的作用，它严格调控机体对自身和非自身抗原产生不同的免疫应答。如果缺少协同刺激分子提供的第二信号，将会导致T细胞的无应答或持续特异性免疫应答，从而产生耐受。因此，第二信号通路在机体免疫应答的整个过程中起着非常关键的调节作用。
程序性死亡分子1(programmed death-l,PD-l)是1992年发现的表达在T细胞表面的一个蛋白受体，参与到细胞的凋亡过程之中。PD-l属于CD28家族，与细胞毒性T淋巴细胞抗原4(cytotoxic T Iymphocyte antigen 4,CTLA-4)具有23％的氨基酸同源性，但其表 达却与CTLA不同，主要表达在活化的T细胞、B细胞和髓系细胞上。PD-1有两个配体，分别为PD-L1和PD-L2。PD-L1主要表达于T细胞、B细胞、巨噬细胞和树突状细胞(dendritic cell,DC)上，在活化后细胞上的表达能够进行上调。而PD-L2的表达相对较局限，主要表达在抗原呈递细胞上，如活化的巨噬细胞和树突状细胞。
PD-L1通过和PD-1及B7-1的结合抑制免疫系统，很多肿瘤细胞及肿瘤组织微环境的免疫细胞表达PD-L1。新的研究发现乳腺癌、肺癌、胃癌、肠癌、肾癌、黑素瘤、非小细胞肺癌、结肠癌、膀胱癌、卵巢癌、胰腺癌及肝癌等人类肿瘤组织中检测到高PD-L1蛋白的表达，且PD-L1的表达水平和患者的临床及预后紧密相关。
由于PD-L1起到第二信号通路抑制T细胞增殖的作用，所以阻断PD-L1/PD-1之间结合成为了肿瘤免疫治疗领域一个非常有潜力的新兴靶点。
目前有多家跨国制药公司在研发针对PD-L1的单克隆抗体，它通过阻断PD-L1/PD-1之间结合，最大限度的提高患者自身对肿瘤的免疫系统反应，从而达到对肿瘤细胞进行杀伤的目的。相关的专利如：WO0139722、WO2013173223、WO2014195852、WO2013181634、WO2015048520、WO2015036511、US2014335093、WO2014100079、WO2014055897、US6803192B1、WO2014022758、US8617546B2和WO2010089411A2。
发明内容
本公开提供与PD-L1的胞外区的氨基酸序列或三维结构结合的单克隆抗体或抗原结合片段(也可称人PD-L1结合分子)。
在一些可选的实施方案中，本公开提供一种单克隆抗体或其抗原结合片段，所述单克隆抗体或抗原结合片段结合人PD-L1，所述单克隆抗体或其抗原结合片段包含：
(i)重链可变区和轻链可变区，其中重链可变区包含氨基酸序列分别如SEQ ID NO：10、12和13所示的HCDR1、HCDR2和HCDR3区，轻链可变区包含氨基酸序列分别如SEQ ID NO：14、15和16所示的LCDR1、LCDR2和LCDR3区，所述SEQ ID NO.12所示的HCDR2中X 1为F或M，X 2为R或V，X 3为N或H；或
(ii)重链可变区和轻链可变区，其中重链可变区包含氨基酸序列分别如SEQ ID NO：11、12和13所示的HCDR1、HCDR2和HCDR3区，轻链可变区包含氨基酸序列分别如SEQ ID NO：14、15和16所示的LCDR1、LCDR2和LCDR3区，所述SEQ ID NO.12所示的HCDR2中X 1为F或M，X 2为R或V，X 3为N或H；或
(iii)重链可变区和轻链可变区，其中重链可变区包含氨基酸序列分别如SEQ ID NO：20、21和22所示的HCDR1、HCDR2和HCDR3区，轻链可变区包含氨基酸序列分别如 SEQ ID NO：23、24和25所示的LCDR1、LCDR2和LCDR3区，其中HCDR1、HCDR2、HCDR3和LCDR1、LCDR2、LCDR3依次不同时为SEQ ID NO：30、38、22、23、40和25，
所述SEQ ID NO.20和21所示的HCDR1和HCDR2中X 4为S或D，X 5为Y或K，X 6为H或M，X 7为T、S、H或G，X 8为S、N或G，X 9为S、L或G，X 10为F、L、W或M，X 11为A、P或T，X 12为M、V、L或S，X 13为F或Y，所述SEQ ID NO：24所示的LCDR2中X 14为V或A，X 15为Y或N，X 16为A、L或V，X 17为E、F、Y或A(其中包括X 17选自E、F和A与X 17选自Y的情况)。
在一些实施方案中，如前所述单克隆抗体或其抗原结合片段结合人PD-L1，所述单克隆抗体或其抗原结合片段包含重链可变区和轻链可变区，其中重链可变区包含氨基酸序列如SEQ ID NO：10所示的HCDR1、如SEQ ID NO：28或29所示的HCDR2和如SEQ ID NO：13所示的HCDR3区，轻链可变区包含氨基酸序列分别如SEQ ID NO：14、15和16氨基酸序列所示的LCDR1、LCDR2和LCDR3区。
在一些实施方案中，如前所述单克隆抗体或其抗原结合片段的所述重链可变区包含氨基酸序列如SEQ ID NO：11所示的HCDR1、如SEQ ID NO：28或29所示的HCDR2和如SEQ ID NO：13所示的HCDR3区，所述轻链可变区包含氨基酸序列分别如SEQ ID NO：14、15和16所示的LCDR1、LCDR2和LCDR3区。
在一些实施方案中，如前所述单克隆抗体或其抗原结合片段的所述重链可变区包含氨基酸序列如SEQ ID NO：30所示的HCDR1、如SEQ ID NO：32至37任一所示的HCDR2和如SEQ ID NO：22所示的HCDR3区，所述轻链可变区包含氨基酸序列如SEQ ID NO：23的LCDR1、如SEQ ID NO：39、40、41、67和69任一的LCDR2和如SEQ ID NO：25的LCDR3区。
在一些实施方案中，如前的所述单克隆抗体或其抗原结合片段的所述重链可变区包含氨基酸序列如SEQ ID NO：31所示的HCDR1、如SEQ ID NO：32、33、34、35、36和37任一所示的HCDR2和如SEQ ID NO：22所示的HCDR3区，所述轻链可变区包含氨基酸序列如SEQ ID NO：23的LCDR1、如SEQ ID NO：39、40、41、67和69任一的LCDR2和如SEQ ID NO：25的LCDR3区。
在一些实施方案中，如前所述单克隆抗体或其抗原结合片段所述重链可变区包含氨基酸序列分别如SEQ ID NO：10、SEQ ID NO：28、SEQ ID NO：13所示的HCDR1、HCDR2和HCDR3区，所述轻链可变区包含氨基酸序列分别如SEQ ID NO：14-16所示的LCDR1、LCDR2和LCDR3区；或
在一些实施方案中，如前所述单克隆抗体或其抗原结合片段所述重链可变区包含氨基酸序列分别如SEQ ID NO：11、SEQ ID NO：28、SEQ ID NO：13所示的HCDR1、HCDR2和HCDR3区，所述轻链可变区包含氨基酸序列分别如SEQ ID NO：14-16所示的LCDR1、LCDR2和LCDR3区；或
在一些实施方案中，如前所述单克隆抗体或其抗原结合片段所述重链可变区包含氨基酸序列分别如SEQ ID NO：11、SEQ ID NO：29、SEQ ID NO：13所示的HCDR1、HCDR2和HCDR3区，所述轻链可变区包含氨基酸序列分别如SEQ ID NO：14-16所示的LCDR1、LCDR2和LCDR3区；或
在一些实施方案中，如前所述单克隆抗体或其抗原结合片段所述重链可变区包含氨基酸序列分别如SEQ ID NO：30、SEQ ID NO：32、SEQ ID NO：22所示的HCDR1、HCDR2和HCDR3区，所述轻链可变区包含氨基酸序列分别如SEQ ID NO：23、SEQ ID NO：39、SEQ ID NO：25所示的LCDR1、LCDR2和LCDR3区；或
在一些实施方案中，如前所述单克隆抗体或其抗原结合片段所述重链可变区包含氨基酸序列分别如SEQ ID NO：30、SEQ ID NO：32、SEQ ID NO：22所示的HCDR1、HCDR2和HCDR3区，所述轻链可变区包含氨基酸序列分别如SEQ ID NO：23、SEQ ID NO：40、SEQ ID NO：25所示的LCDR1、LCDR2和LCDR3区；或
在一些实施方案中，如前所述单克隆抗体或其抗原结合片段所述重链可变区包含氨基酸序列分别如SEQ ID NO：30、SEQ ID NO：33、SEQ ID NO：22所示的HCDR1、HCDR2和HCDR3区，所述轻链可变区包含氨基酸序列分别如SEQ ID NO：23、SEQ ID NO：40、SEQ ID NO：25所示的LCDR1、LCDR2和LCDR3区；或
在一些实施方案中，如前所述单克隆抗体或其抗原结合片段所述重链可变区包含氨基酸序列分别如SEQ ID NO：30、SEQ ID NO：34、SEQ ID NO：22所示的HCDR1、HCDR2和HCDR3区，所述轻链可变区包含氨基酸序列分别如SEQ ID NO：23、SEQ ID NO：40、SEQ ID NO：25所示的LCDR1、LCDR2和LCDR3区；或
在一些实施方案中，如前所述单克隆抗体或其抗原结合片段所述重链可变区包含氨基酸序列分别如SEQ ID NO：31、SEQ ID NO：32、SEQ ID NO：22所示的HCDR1、HCDR2和HCDR3区，轻链可变区包含氨基酸序列分别如SEQ ID NO：23、SEQ ID NO：40、SEQ ID NO：25所示的LCDR1、LCDR2和LCDR3区；或
在一些实施方案中，如前所述单克隆抗体或其抗原结合片段所述重链可变区包含氨基酸序列分别如SEQ ID NO：31、SEQ ID NO：32和SEQ ID NO：22所示的HCDR1、HCDR2和HCDR3区，所述轻链可变区包含氨基酸序列分别如SEQ ID NO：23、SEQ ID  NO：67和SEQ ID NO：25所示的LCDR1、LCDR2和LCDR3区。
在一些实施方案中，如前所述单克隆抗体或其抗原结合片段所述重链可变区包含氨基酸序列分别如SEQ ID NO：31、SEQ ID NO：32和SEQ ID NO：22所示的HCDR1、HCDR2和HCDR3区，所述轻链可变区包含氨基酸序列分别如SEQ ID NO：23、SEQ ID NO：69和SEQ ID NO：25所示的LCDR1、LCDR2和LCDR3区。
在一些实施方案中，如前所述单克隆抗体或其抗原结合片段所述重链可变区包含氨基酸序列分别如SEQ ID NO：31、SEQ ID NO：32和SEQ ID NO：22所示的HCDR1、HCDR2和HCDR3区，所述轻链可变区包含氨基酸序列分别如SEQ ID NO：23、SEQ ID NO：41和SEQ ID NO：25所示的LCDR1、LCDR2和LCDR3区；
或在一些实施方案中，如前所述单克隆抗体或其抗原所述重链可变区包含氨基酸序列分别如SEQ ID NO：30、SEQ ID NO：35、SEQ ID NO：22所示的HCDR1、HCDR2和HCDR3区，所述轻链可变区包含氨基酸序列分别如SEQ ID NO：23、SEQ ID NO：40、SEQ ID NO：25所示的LCDR1、LCDR2和LCDR3区；或
在一些实施方案中，如前所述单克隆抗体或其抗原结合片段所述重链可变区包含氨基酸序列分别如SEQ ID NO：30、SEQ ID NO：36、SEQ ID NO：22所示的HCDR1、HCDR2和HCDR3区，所述轻链可变区包含氨基酸序列分别如SEQ ID NO：23、SEQ ID NO：40、SEQ ID NO：25所示的LCDR1、LCDR2和LCDR3区；或
在一些实施方案中，如前所述单克隆抗体或其抗原结合片段所述重链可变区包含氨基酸序列分别如SEQ ID NO：30、SEQ ID NO：37、SEQ ID NO：22所示的HCDR1、HCDR2和HCDR3区，所述轻链可变区包含氨基酸序列分别如SEQ ID NO：23、SEQ ID NO：40、SEQ ID NO：25所示的LCDR1、LCDR2和LCDR3区。
在一些实施方案中，如前所述单克隆抗体或其抗原结合片段与人PD-L1的亲和力KD值小于10 -9M或10 -10M。
在一些实施方案中，如前所述单克隆抗体或其抗原结合片段交叉结合食蟹猴或恒河猴PD-L1和/或小鼠PD-L1。
在一些实施方案中，如前所述单克隆抗体或抗原结合片段包含氨基酸序列如SEQ ID NO：17所示的重链可变区和SEQ ID NO：19所示的轻链可变区。
在一些实施方案中，如前所述单克隆抗体或抗原结合片段包含氨基酸序列如SEQ ID NO：18所示的重链可变区和SEQ ID NO：19所示的轻链可变区。
在一些实施方案中，如前所述单克隆抗体或抗原结合片段包含氨基酸序列如SEQ ID NO：26所示的重链可变区和SEQ ID NO：27所示的轻链可变区。
在一些实施方案中，如前所述单克隆抗体或抗原结合片段包含氨基酸序列如SEQ ID NO：42所示的重链可变区和SEQ ID NO：45所示的轻链可变区。
在一些实施方案中，如前所述单克隆抗体或抗原结合片段包含氨基酸序列如SEQ ID NO：43所示的重链可变区和SEQ ID NO：45所示的轻链可变区。
在一些实施方案中，如前所述单克隆抗体或抗原结合片段包含氨基酸序列如SEQ ID NO：44所示的重链可变区和SEQ ID NO：45所示的轻链可变区。
在一些实施方案中，如前所述单克隆抗体或抗原结合片段包含氨基酸序列如SEQ ID NO：46所示的重链可变区和SEQ ID NO：55、56和57任一所示的轻链可变区。
在一些实施方案中，所述单克隆抗体或抗原结合片段包含氨基酸序列如SEQ ID NO：47所示的重链可变区和SEQ ID NO：55、56和57任一所示的轻链可变区。
在一些实施方案中，如前所述单克隆抗体或抗原结合片段包含氨基酸序列如SEQ ID NO：48所示的重链可变区和SEQ ID NO：55、56和57任一所示的轻链可变区。
在一些实施方案中，如前所述单克隆抗体或抗原结合片段包含氨基酸序列如SEQ ID NO：49所示的重链可变区和SEQ ID NO：55、56和57任一所示的轻链可变区。
在一些实施方案中，所述单克隆抗体或抗原结合片段包含氨基酸序列如SEQ ID NO：50所示的重链可变区和SEQ ID NO：55、56和57任一所示的轻链可变区。
在一些实施方案中，如前所述单克隆抗体或抗原结合片段包含氨基酸序列如SEQ ID NO：51所示的重链可变区和SEQ ID NO：55、56和57任一所示的轻链可变区。
在一些实施方案中，如前所述单克隆抗体或抗原结合片段包含氨基酸序列如SEQ ID NO：52所示的重链可变区和SEQ ID NO：55、56和57任一所示的轻链可变区。
在一些实施方案中，如前所述单克隆抗体或抗原结合片段包含氨基酸序列如SEQ ID NO：53所示的重链可变区和SEQ ID NO：55、56和57任一所示的轻链可变区。
在一些实施方案中，如前所述单克隆抗体或抗原结合片段包含氨基酸序列如SEQ ID NO：54所示的重链可变区和SEQ ID NO：55和57任一所示的轻链可变区。
在一些实施方案中，如前所述单克隆抗体或抗原结合片段包含氨基酸序列如SEQ ID NO：46所示的重链可变区和SEQ ID NO：55、56、57、70和72任一所示的轻链可变区。
在一些实施方案中，如前所述单克隆抗体或抗原结合片段包含氨基酸序列如SEQ ID NO：47所示的重链可变区和SEQ ID NO：55、56、57、70和72任一所示的轻链可变区。
在一些实施方案中，如前所述单克隆抗体或抗原结合片段包含氨基酸序列如SEQ ID NO：48所示的重链可变区和SEQ ID NO：55、56、57、70和72任一所示的轻链可变区。
在一些实施方案中，如前所述单克隆抗体或抗原结合片段包含氨基酸序列如SEQ ID  NO：49所示的重链可变区和SEQ ID NO：55、56、57、70和72任一所示的轻链可变区。
在一些实施方案中，如前所述单克隆抗体或抗原结合片段包含氨基酸序列如SEQ ID NO：50所示的重链可变区和SEQ ID NO：55、56、57、70和72任一所示的轻链可变区。
在一些实施方案中，如前所述单克隆抗体或抗原结合片段包含氨基酸序列如SEQ ID NO：51所示的重链可变区和SEQ ID NO：55、56、57、70和72任一所示的轻链可变区。
在一些实施方案中，如前所述单克隆抗体或抗原结合片段包含氨基酸序列如SEQ ID NO：52所示的重链可变区和SEQ ID NO：55、56、57、70和72任一所示的轻链可变区。
在一些实施方案中，如前所述单克隆抗体或抗原结合片段包含氨基酸序列如SEQ ID NO：53所示的重链可变区和SEQ ID NO：55、56、57、70和72任一所示的轻链可变区。
在一些实施方案中，如前所述单克隆抗体或抗原结合片段包含氨基酸序列如SEQ ID NO：66所示的重链可变区和SEQ ID NO：55、56、57、70和72任一所示的轻链可变区。
在一些实施方案中，如前所述单克隆抗体或抗原结合片段包含氨基酸序列如SEQ ID NO：54所示的重链可变区和SEQ ID NO：55、57、70和72任一所示的轻链可变区。
在一些实施方案中，如前所述单克隆抗体为全长抗体，进一步包括人抗体恒定区，优选地，所述人抗体恒定区的重链恒定区选自人IgG1、IgG2、IgG3和IgG4恒定区及其常规变体，所述人抗体恒定区的轻链恒定区选自人抗体κ和λ链恒定区及其常规变体，优选包含氨基酸序列如SEQ ID NO:58、60或65所示的人抗体重链恒定区和SEQ ID NO:59所示的人轻链恒定区。
在一些实施方案中，如前所述单克隆抗体为全长抗体，进一步包括人抗体恒定区，包含氨基酸序列如SEQ ID NO:58所示的人抗体重链恒定区和SEQ ID NO:59所示的人轻链恒定区。
在一些实施方案中，如前所述单克隆抗体为全长抗体，进一步包括人抗体恒定区，包含氨基酸序列如SEQ ID NO:60所示的人抗体重链恒定区和SEQ ID NO:59所示的人轻链恒定区。
在一些实施方案中，如前所述单克隆抗体为全长抗体，进一步包括人抗体恒定区，包含氨基酸序列如SEQ ID NO:65所示的人抗体重链恒定区和SEQ ID NO:59所示的人轻链恒定区。
在一些实施方案中，所述抗原结合片段是选自Fab、Fab'、F(ab') 2、单链抗体(scFv)、二聚化的V区(双抗体)、二硫键稳定化的V区(dsFv)和包含CDR的肽的抗原结合片段。
另一方面，本公开提供一种药物组合物，其含有治疗有效量的根据前述所述的单克 隆抗体或其抗原结合片段，以及一种或多种药学上可接受的载体、稀释剂或赋形剂，优选地，所述治疗有效量的单克隆抗体或其抗原结合片段为单位剂量0.1-3000mg/kg的如前所述单克隆抗体或其抗原结合片段。
在一些方面，本公开提供一种核酸分子，其编码前述的单克隆抗体或其抗原结合片段。
在一些方面，本公开提供一种重组载体，其包含如前所述的核酸分子。
在一些方面，本公开提供一种用根据如前所述的重组载体转化的宿主细胞，所述宿主细胞选自原核细胞和真核细胞，优选为真核细胞，更优选哺乳动物细胞。
在一些方面，本公开提供用于生产如前所述的单克隆抗体或其抗原结合片段的方法，所述方法包括将前述的宿主细胞在培养基中进行培养以形成并积累前述的单克隆抗体或其抗原结合片段，以及从培养物回收所述单克隆抗体或其抗原结合片段。
在一些方面，本公开提供用于免疫检测或测定人PD-L1的方法，所述方法包括使用如前所述的单克隆抗体或其抗原结合片段。
在一些方面，本公开提供如前所述的单克隆抗体或其抗原结合片段在制备与人PD-L1相关的疾病的诊断剂中的应用。
在一些方面，本公开提供与人PD-L1相关的疾病的治疗方法，所述方法包括向受试者施用药物有效量的如前所述的单克隆抗体或其抗原结合片段，或包含如前所述的药物组合物，或如前所述的核酸分子，以治疗人PD-L1相关的疾病，其中所述疾病优选为肿瘤或癌症；更优选为鳞状细胞癌、骨髓瘤、小细胞肺癌、非小细胞肺癌(NSCLC)、头和颈鳞状细胞癌(HNSCC)、神经胶质瘤、何杰金淋巴瘤、非何杰金淋巴瘤、弥漫性大B-细胞淋巴瘤(DLBCL)、滤泡性淋巴瘤、急性成淋巴细胞性白血病(ALL)、急性髓细胞样白血病(AML)、慢性淋巴细胞性白血病(CLL)、慢性髓细胞样白血病(CML)、原发性纵隔大B-细胞淋巴瘤、套细胞淋巴瘤(MCL)、小淋巴细胞性淋巴瘤(SLL)、富含T-细胞/组织细胞的大B-细胞淋巴瘤、多发性骨髓瘤、髓样细胞白血病-1蛋白(Mcl-1)、骨髓异常增生综合征(MDS)、胃肠(道)癌、肾癌、卵巢癌、肝癌、成淋巴细胞性白血病、淋巴细胞白血病、结肠直肠癌、子宫内膜癌、肾癌、前列腺癌、甲状腺癌、黑素瘤、软骨肉瘤、神经母细胞瘤、胰腺癌、多形性成胶质细胞瘤、胃癌、骨癌、尤因氏肉瘤、子宫颈癌、脑癌、胃癌、膀胱癌、肝细胞瘤、乳腺癌、结肠癌、肝细胞癌(HCC)、透明细胞肾细胞癌(RCC)、头和颈癌、咽喉癌、肝胆癌(hepatobiliary cancer)、中枢神经系统癌、食管癌、恶性胸膜间皮瘤、全身性轻链淀粉样变性、淋巴浆细胞性淋巴瘤(lymphoplasmacytic lymphoma)、骨髓异常增生综合征、骨髓增生性肿瘤、神经内分泌 肿瘤、梅克尔细胞癌、睾丸癌和皮肤癌，最优选为PD-L1阳性的鳞状细胞癌、骨髓瘤、小细胞肺癌、非小细胞肺癌(NSCLC)、头和颈鳞状细胞癌(HNSCC)、神经胶质瘤、何杰金淋巴瘤、非何杰金淋巴瘤、弥漫性大B-细胞淋巴瘤(DLBCL)、滤泡性淋巴瘤、急性成淋巴细胞性白血病(ALL)、急性髓细胞样白血病(AML)、慢性淋巴细胞性白血病(CLL)、慢性髓细胞样白血病(CML)、原发性纵隔大B-细胞淋巴瘤、套细胞淋巴瘤(MCL)、小淋巴细胞性淋巴瘤(SLL)、富含T-细胞/组织细胞的大B-细胞淋巴瘤、多发性骨髓瘤、髓样细胞白血病-1蛋白(Mcl-1)、骨髓异常增生综合征(MDS)、胃肠(道)癌、肾癌、卵巢癌、肝癌、成淋巴细胞性白血病、淋巴细胞白血病、结肠直肠癌、子宫内膜癌、肾癌、前列腺癌、甲状腺癌、黑素瘤、软骨肉瘤、神经母细胞瘤、胰腺癌、多形性成胶质细胞瘤、胃癌、骨癌、尤因氏肉瘤、子宫颈癌、脑癌、胃癌、膀胱癌、肝细胞瘤、乳腺癌、结肠癌、肝细胞癌(HCC)、透明细胞肾细胞癌(RCC)、头和颈癌、咽喉癌、肝胆癌(hepatobiliary cancer)、中枢神经系统癌、食管癌、恶性胸膜间皮瘤、全身性轻链淀粉样变性、淋巴浆细胞性淋巴瘤(lymphoplasmacytic lymphoma)、骨髓异常增生综合征、骨髓增生性肿瘤、神经内分泌肿瘤、梅克尔细胞癌、睾丸癌和皮肤癌。
在一些方面，本公开提供如前所述的单克隆抗体或其抗原结合片段，或包含如前所述的药物组合物，或如前所述的核酸分子在制备与人PD-L1相关的疾病的治疗剂中的应用，其中所述疾病优选为肿瘤或癌症；更优选为鳞状细胞癌、骨髓瘤、小细胞肺癌、非小细胞肺癌(NSCLC)、头和颈鳞状细胞癌(HNSCC)、神经胶质瘤、何杰金淋巴瘤、非何杰金淋巴瘤、弥漫性大B-细胞淋巴瘤(DLBCL)、滤泡性淋巴瘤、急性成淋巴细胞性白血病(ALL)、急性髓细胞样白血病(AML)、慢性淋巴细胞性白血病(CLL)、慢性髓细胞样白血病(CML)、原发性纵隔大B-细胞淋巴瘤、套细胞淋巴瘤(MCL)、小淋巴细胞性淋巴瘤(SLL)、富含T-细胞/组织细胞的大B-细胞淋巴瘤、多发性骨髓瘤、髓样细胞白血病-1蛋白(Mcl-1)、骨髓异常增生综合征(MDS)、胃肠(道)癌、肾癌、卵巢癌、肝癌、成淋巴细胞性白血病、淋巴细胞白血病、结肠直肠癌、子宫内膜癌、肾癌、前列腺癌、甲状腺癌、黑素瘤、软骨肉瘤、神经母细胞瘤、胰腺癌、多形性成胶质细胞瘤、胃癌、骨癌、尤因氏肉瘤、子宫颈癌、脑癌、胃癌、膀胱癌、肝细胞瘤、乳腺癌、结肠癌、肝细胞癌(HCC)、透明细胞肾细胞癌(RCC)、头和颈癌、咽喉癌、肝胆癌(hepatobiliary cancer)、中枢神经系统癌、食管癌、恶性胸膜间皮瘤、全身性轻链淀粉样变性、淋巴浆细胞性淋巴瘤(lymphoplasmacytic lymphoma)、骨髓异常增生综合征、骨髓增生性肿瘤、神经内分泌肿瘤、梅克尔细胞癌、睾丸癌和皮肤癌，最优选为PD-L1阳性的鳞状细胞癌、骨髓瘤、小细胞肺癌、非小细胞肺癌(NSCLC)、头和颈鳞状细胞癌(HNSCC)、神经 胶质瘤、何杰金淋巴瘤、非何杰金淋巴瘤、弥漫性大B-细胞淋巴瘤(DLBCL)、滤泡性淋巴瘤、急性成淋巴细胞性白血病(ALL)、急性髓细胞样白血病(AML)、慢性淋巴细胞性白血病(CLL)、慢性髓细胞样白血病(CML)、原发性纵隔大B-细胞淋巴瘤、套细胞淋巴瘤(MCL)、小淋巴细胞性淋巴瘤(SLL)、富含T-细胞/组织细胞的大B-细胞淋巴瘤、多发性骨髓瘤、髓样细胞白血病-1蛋白(Mcl-1)、骨髓异常增生综合征(MDS)、胃肠(道)癌、肾癌、卵巢癌、肝癌、成淋巴细胞性白血病、淋巴细胞白血病、结肠直肠癌、子宫内膜癌、肾癌、前列腺癌、甲状腺癌、黑素瘤、软骨肉瘤、神经母细胞瘤、胰腺癌、多形性成胶质细胞瘤、胃癌、骨癌、尤因氏肉瘤、子宫颈癌、脑癌、胃癌、膀胱癌、肝细胞瘤、乳腺癌、结肠癌、肝细胞癌(HCC)、透明细胞肾细胞癌(RCC)、头和颈癌、咽喉癌、肝胆癌(hepatobiliary cancer)、中枢神经系统癌、食管癌、恶性胸膜间皮瘤、全身性轻链淀粉样变性、淋巴浆细胞性淋巴瘤(lymphoplasmacytic lymphoma)、骨髓异常增生综合征、骨髓增生性肿瘤、神经内分泌肿瘤、梅克尔细胞癌、睾丸癌和皮肤癌。
作为药物的如前所述单克隆抗体或其抗原结合片段，或包含如前所述的药物组合物，或如前所述的核酸分子。
作为药物的根据如前所述的单克隆抗体或其抗原结合片段，或包含如前所述的药物组合物，或如前所述的核酸分子，所述药物用于治疗PD-L1阳性的肿瘤或癌症，优选地，所述癌症选自鳞状细胞癌、骨髓瘤、小细胞肺癌、非小细胞肺癌(NSCLC)、头和颈鳞状细胞癌(HNSCC)、神经胶质瘤、何杰金淋巴瘤、非何杰金淋巴瘤、弥漫性大B-细胞淋巴瘤(DLBCL)、滤泡性淋巴瘤、急性成淋巴细胞性白血病(ALL)、急性髓细胞样白血病(AML)、慢性淋巴细胞性白血病(CLL)、慢性髓细胞样白血病(CML)、原发性纵隔大B-细胞淋巴瘤、套细胞淋巴瘤(MCL)、小淋巴细胞性淋巴瘤(SLL)、富含T-细胞/组织细胞的大B-细胞淋巴瘤、多发性骨髓瘤、髓样细胞白血病-1蛋白(Mcl-1)、骨髓异常增生综合征(MDS)、胃肠(道)癌、肾癌、卵巢癌、肝癌、成淋巴细胞性白血病、淋巴细胞白血病、结肠直肠癌、子宫内膜癌、肾癌、前列腺癌、甲状腺癌、黑素瘤、软骨肉瘤、神经母细胞瘤、胰腺癌、多形性成胶质细胞瘤、胃癌、骨癌、尤因氏肉瘤、子宫颈癌、脑癌、胃癌、膀胱癌、肝细胞瘤、乳腺癌、结肠癌、肝细胞癌(HCC)、透明细胞肾细胞癌(RCC)、头和颈癌、咽喉癌、肝胆癌(hepatobiliary cancer)、中枢神经系统癌、食管癌、恶性胸膜间皮瘤、全身性轻链淀粉样变性、淋巴浆细胞性淋巴瘤(lymphoplasmacytic lymphoma)、骨髓异常增生综合征、骨髓增生性肿瘤、神经内分泌肿瘤、梅克尔细胞癌、睾丸癌和皮肤癌。
附图说明
图1：PD-L1抗体在PBMC-T淋巴细胞激活实验中对细胞IFNγ的分泌促进作用。
图2：不同PD-L1抗体的IgG1形式与IgG4形式的ADCC效应比较。图2A是HRP00049的IgG1和IgG4形式比较，图2B是H5L11的IgG1和IgG4形式比较，图2C是HRP00052的IgG1和IgG4形式比较，图2D是H6L11的IgG1和IgG4形式比较，图2E是H18L61的IgG1和IgG4形式比较，图2F是H12L64的IgG1和IgG4形式比较。
图3：PD-L1抗体对小鼠A375异种移植模型肿瘤体积的影响。
图4：PD-L1抗体对小鼠结肠癌模型肿瘤体积的影响。
图5：PD-L1抗体对小鼠移植瘤模型肿瘤体积的影响。
具体实施方案
一.术语
为了更容易理解本公开，以下具体定义了某些技术和科学术语。除非在本文中另有明确定义，本文使用的所有其它技术和科学术语都具有本公开所属领域的一般技术人员通常理解的含义。
本公开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。
本公开所述的“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中每类Ig都可以有κ链或λ链。
在本公开中，本公开所述的抗体轻链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。
在本公开中，本公开所述的抗体重链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、IgG2、IgG3、IgG4或其变体。
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(Fv区)；靠近C端的其余氨基酸序列相对稳定，为恒定区。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(LCVR)和重链可变区(HCVR)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。本公开所述的抗体或抗原结合片段的LCVR区和HCVR区的CDR氨基酸残基在数量和位置符合已知的Kabat编号规则(LCDR1-3，HCDR1-3)。
本公开的抗体包括鼠源抗体、嵌合抗体、人源化抗体，优选人源化抗体。
“单克隆抗体”是指从基本上均质抗体的群体获得的抗体，即除可能的变体抗体(例如含有天然存在的突变或在制造单克隆抗体制剂的期间产生的突变，这些变体通常以少量存在)之外，构成所述群体的个别抗体相同和/或结合相同表位。与通常包含针对不同决定簇(表位)的不同抗体的多克隆抗体制备物不同，单克隆抗体制备物(制剂)的每个单克隆抗体是针对抗原上的单一决定簇的。因此，修饰语“单克隆”指示如从基本上均质抗体群体获得的抗体的特性，且不应解释为需要通过任何特定方法来制造抗体。例如，本公开的单克隆抗体可通过各种技术制备，所述技术包括但不限于杂交瘤方法、重组DNA方法、噬菌体展示方法以及利用含有全部或部分人免疫球蛋白基因座的转基因动物的方法，此类方法以及用于制备单克隆抗体的其他示例性方法在本文中进行描述。
术语“鼠源抗体”在本公开中为根据本领域知识和技能制备的对人PD-L1的单克隆抗体。制备时用PD-L1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。在本公开一个优选的实施方案中，所述的鼠源PD-L1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或其变体的重链恒定区。
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，要先建立分泌鼠源性特异性单抗的杂交瘤，然后从鼠杂交瘤细胞中克隆可变区基因，再根据需要克隆人抗体的恒定区基因，将鼠可变区基因与人恒定区基因连接成嵌合基因后插入表达载体中，最后在真核系统或原核系统中表达嵌合抗体分子。在本公开一个优选的实施方案中，所述的PD-L1嵌合抗体的抗体轻链进一步包含人源κ、λ链或其变体的轻链恒定区。所述的PD-L1嵌合抗体的抗体重链进一步包含人源IgG1、IgG2、IgG3、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，或者使用氨基酸突变(如YTE突变或回复突变)的IgG1、IgG2或IgG4变体。
术语“人源化抗体(humanized antibody)”，也称为CDR移植抗体(CDR-grafted antibody)，是指将鼠的CDR序列移植到人的抗体可变区框架，即不同类型的人种系抗体框架序列中产生的抗体。可以克服嵌合抗体由于携带大量鼠蛋白成分，从而诱导的异 源性反应。此类构架序列可以从包括种系抗体基因序列的公共DNA数据库或公开的参考文献获得。如人重链和轻链可变区基因的种系DNA序列可以在“VBase”人种系序列数据库(在因特网www.mrccpe.com.ac.uk/vbase可获得)，以及在Kabat，E.A.等人，1991Sequences of Proteins of Immunological Interest，第5版中找到。为避免免疫原性下降的同时，引起的活性下降，可对所述的人抗体可变区框架序列进行最少反向突变或回复突变，以保持活性。本公开的人源化抗体也包括进一步由噬菌体展示对CDR进行亲和力成熟后的人源化抗体。在本公开一个优选的实施方案中，人的抗体可变区框架经过设计选择，其中所述抗体重链可变区上的重链FR区序列，来源于人种系重链IGHV3-23*04和hJH4.1的组合序列，和人种系轻链IGKV1-12*01和hJK4.1的组合序列。为避免免疫原性下降的同时，引起的活性下降，可对所述的人抗体可变区可进行最少反向突变(回复突变，即将人抗体来源的FR区氨基酸残基突变成原始来源抗体对应位置的氨基酸残基)，以保持活性。
CDR的移植可由于与抗原接触的构架残基而导致产生的PD-L1抗体或其抗原结合片段对抗原的亲和力减弱。此类相互作用可以是体细胞高度突变的结果。因此，可能仍然需要将此类供体构架氨基酸移植至人源化抗体的构架。来自非人PD-L1抗体或其抗原结合片段的参与抗原结合的氨基酸残基可通过检查鼠单克隆抗体可变区序列和结构来鉴定。CDR供体构架中与种系不同的的各残基可被认为是相关的。如果不能确定最接近的种系，那么可将序列与亚型共有序列或具有高相似性百分数的鼠序列的共有序列相比较。稀有构架残基被认为可能是体细胞高度突变的结果，从而在结合中起着重要作用。
术语抗体的“抗原结合片段”或“功能片段”是指抗体的保持特异性结合抗原(例如，PD-L1)的能力的一个或多个片段。已显示可利用全长抗体的片段来进行抗体的抗原结合功能。术语抗体的“抗原结合片段”中包含的结合片段的实例包括(i)Fab片段，由VL、VH、CL和CH1结构域组成的单价片段；(ii)F(ab') 2片段，包含通过铰链区上的二硫桥连接的两个Fab片段的二价片段，(iii)由VH和CH1结构域组成的Fd片段；(iv)由抗体的单臂的VH和VL结构域组成的Fv片段；(v)单结构域或dAb片段(Ward等人，(1989)Nature341：544-546)，其由VH结构域组成；和(vi)分离的互补决定区(CDR)或(vii)可任选地通过合成的接头连接的两个或更多个分离的CDR的组合。此外，虽然Fv片段的两个结构域VL和VH由分开的基因编码，但可使用重组方法，通过合成的接头连接它们，从而使得其能够产生为其中VL和VH区配对形成单价分子的单个蛋白质链(称为单链Fv(scFv)；参见，例如，Bird等人(1988)Science242:423-426；和Huston等人(1988)Proc.Natl.Acad.Sci USA85:5879-5883)。此类单链抗体也意欲包括在术语抗体的 “抗原结合片段”中。使用本领域技术人员已知的常规技术获得此类抗体片段，并且以与对于完整抗体的方式相同的方式就功用性筛选片段。可通过重组DNA技术或通过酶促或化学断裂完整免疫球蛋白来产生抗原结合部分。抗体可以是不同同种型的抗体，例如，IgG(例如，IgG1，IgG2，IgG3或IgG4亚型)，IgA1，IgA2，IgD，IgE或IgM抗体。
本公开的抗原结合片段包括Fab、F(ab') 2、Fab'、单链抗体(scFv)、二聚化的V区(双抗体)、二硫键稳定化的V区(dsFv)、包含CDR的肽等。
Fab是通过用蛋白酶木瓜蛋白酶(切割H链的224位的氨基酸残基)处理IgG抗体分子所获得的片段中的具有约50,000的分子量并具有抗原结合活性的抗体片段，其中H链N端侧的约一半和整个L链通过二硫键结合在一起。
本公开的Fab可以通过用木瓜蛋白酶处理本公开的特异性识别人PD-L1并与胞外区的氨基酸序列或其三维结构结合的单克隆抗体来生产。此外，可以通过将编码所述抗体的Fab的DNA插入到原核生物表达载体或真核生物表达载体中并将载体导入到原核生物或真核生物中以表达Fab来生产所述Fab。
F(ab') 2是通过用酶胃蛋白酶消化IgG铰链区中两个二硫键的下方部分而获得的分子量为约100,000并具有抗原结合活性并包含在铰链位置相连的两个Fab区的抗体片段。
本公开的F(ab') 2可以通过用胃蛋白酶处理本公开的特异性识别人PD-L1并与胞外区的氨基酸序列或其三维结构结合的单克隆抗体来生产。此外，可以通过用硫醚键或二硫键连接下面描述的Fab'来生产所述F(ab') 2。
Fab'是通过切割上述F(ab') 2的铰链区的二硫键而获得的分子量为约50,000并具有抗原结合活性的抗体片段。本公开的Fab'可以通过用还原剂例如二硫苏糖醇处理本公开的特异性识别PD-L1并与胞外区的氨基酸序列或其三维结构结合的F(ab') 2来生产。
此外，可以通过将编码抗体的Fab'片段的DNA插入到原核生物表达载体或真核生物表达载体中并将载体导入到原核生物或真核生物中以表达Fab'来生产所述Fab'。
术语“单链抗体”、“单链Fv”或“scFv”意指包含通过接头连接的抗体重链可变结构域(或区域；VH)和抗体轻链可变结构域(或区域；VL)的分子。此类scFv分子可具有一般结构：NH 2-VL-接头-VH-COOH或NH 2-VH-接头-VL-COOH。合适的现有技术接头由重复的GGGGS氨基酸序列或其变体组成，例如使用1-4个重复的变体(Holliger等人(1993)，Proc.Natl.Acad.Sci.USA90:6444-6448)。可用于本公开的其他接头由Alfthan等人(1995)，Protein Eng.8:725-731，Choi等人(2001)，Eur.J.Immuno l.31:94-106，Hu等人(1996)，Cancer Res.56:3055-3061，Kipriyanov等人(1999)，J.Mol.Biol.293:41-56和 Roovers等人(2001)，Cancer Immunol.描述。
本公开的scFv可以通过以下步骤来生产：获得本公开的特异性识别人PD-L1并与胞外区的氨基酸序列或其三维结构结合的单克隆抗体的VH和VL的编码cDNA，构建编码scFv的DNA，将所述DNA插入到原核生物表达载体或真核生物表达载体中，然后将所述表达载体导入到原核生物或真核生物中以表达scFv。
双抗体是其中scFv被二聚体化的抗体片段，是具有二价抗原结合活性的抗体片段。在二价抗原结合活性中，两个抗原可以是相同或不同的。
本公开的双抗体可以通过以下步骤来生产：获得本公开的特异性识别人PD-L1并与胞外区的氨基酸序列或其三维结构结合的单克隆抗体的VH和VL的编码cDNA，构建编码scFv的DNA以使肽接头的氨基酸序列长度为8个残基或更少，将所述DNA插入到原核生物表达载体或真核生物表达载体中，然后将所述表达载体导入到原核生物或真核生物中以表达双抗体。
dsFv是通过将其中每个VH和VL中的一个氨基酸残基被半胱氨酸残基取代的多肽经由半胱氨酸残基之间的二硫键相连而获得的。可以按照已知方法(Protein Engineering,7,697(1994))基于抗体的三维结构预测来选择被半胱氨酸残基取代的氨基酸残基。
本公开的dsFv可以通过以下步骤来生产：获得本公开的特异性识别人PD-L1并与胞外区的氨基酸序列或其三维结构结合的单克隆抗体的VH和VL的编码cDNA，构建编码dsFv的DNA，将所述DNA插入到原核生物表达载体或真核生物表达载体中，然后将所述表达载体导入到原核生物或真核生物中以表达dsFv。
包含CDR的肽是通过包含VH或VL的CDR中的一个或多个区域而构成的。包含多个CDR的肽可以被直接相连或经由适合的肽接头相连。
本公开的包含CDR的肽可以通过以下步骤来生产：构建本公开的特异性识别人PD-L1并与胞外区的氨基酸序列或其三维结构结合的单克隆抗体的VH和VL的CDR的编码DNA，将所述DNA插入到原核生物表达载体或真核生物表达载体中，然后将所述表达载体导入到原核生物或真核生物中以表达所述肽。也可以通过化学合成方法例如Fmoc方法或tBoc方法来生产所述包含CDR的肽。
术语“CDR”是指抗体的可变结构域内主要促成抗原结合的6个高变区之一。所述6个CDR的最常用的定义之一由Kabat E.A.等人，(1991)Sequences of proteins of immunological interest.NIH Publication91-3242)提供。如本文中使用的，CDR的Kabat定义应用于轻链可变结构域的CDR1、CDR2和CDR3(CDR L1、CDR L2、CDR L3或L1、 L2、L3)，以及重链可变结构域的CDR1、CDR2和CDR3(CDR H1、CDR H2、CDR H3或H2、H3)。
本文中使用的术语“抗体框架”，是指可变结构域VL或VH的一部分，其用作该可变结构域的抗原结合环(CDR)的支架。从本质上讲，其是不具有CDR的可变结构域。
人抗体重链恒定区和人抗体轻链恒定区的“常规变体”是指现有技术已公开的来源于人的不改变抗体可变区结构和功能的重链恒定区或轻链恒定区的变体，示例性变体包括对重链恒定区进行定点改造和氨基酸替换的IgG1、IgG2、IgG3或IgG4重链恒定区变体，具体替换如现有技术已知的YTE突变，L234A和/或L235A突变，或S228P突变，或获得knob-into-hole结构的突变(使得抗体重链具有knob-Fc和hole-Fc组合)，或上述突变体的组合，这些突变已被证实使得抗体具有新的性能，但不改变抗体可变区的功能。
术语“表位”或“抗原决定簇”是指抗原上免疫球蛋白或抗体特异性结合的部位(例如，PD-L1分子上的特定部位)。表位通常以独特的空间构象包括至少3，4，5，6，7，8，9，10，11，12，13，14或15个连续或非连续的氨基酸。参见，例如，Epitope Mapping Protocols in Methods in Molecular B iology，第66卷，G.E.Morris，Ed.(1996)。
术语“特异性结合”、“选择性结合”、“选择性地结合”和“特异性地结合”是指抗体对预先确定的抗原上的表位的结合。通常，抗体以大约小于10 -8M，例如大约小于10 - 9M、10 -10M、10 -11M或更小的亲和力(KD)结合。
术语“KD”或“Kd”是指特定抗体-抗原相互作用的解离平衡常数。通常，本公开的抗体以小于大约10 -7M，例如小于大约10 -8M、10 -9M或10 -10M或更小的解离平衡常数(KD)结合PD-L1，例如，如使用表面等离子体共振(SPR)技术在BIACORE仪中测定的。
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(例如抗体或其免疫学功能片段)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(例如PD-L1抗原或其片段)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结合荷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任一种的固态表面或细胞的纯化的抗原。通过测量在所测抗原结合蛋白存在下结合固态表面或细胞的标记的量来测量竞争性抑制。通常所测抗原结合蛋白过量存在。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。在本文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。
本文中使用的术语“核酸分子”是指DNA分子和RNA分子。核酸分子可以是单链或双链的，但优选是双链DNA。当将核酸与另一个核酸序列置于功能关系中时，核酸是“有效连接的”。例如，如果启动子或增强子影响编码序列的转录，那么启动子或增强子有效地连接至所述编码序列。
术语“载体”是指能够运输已与其连接的另一个核酸的核酸分子。在一个实施方案中，载体是“质粒”，其是指可将另外的DNA区段连接至其中的环状双链DNA环。在另一个实施方案中，载体是病毒载体，其中可将另外的DNA区段连接至病毒基因组中。本文中公开的载体能够在已引入它们的宿主细胞中自主复制(例如，具有细菌的复制起点的细菌载体和附加型哺乳动物载体)或可在引入宿主细胞后整合入宿主细胞的基因组，从而随宿主基因组一起复制(例如，非附加型哺乳动物载体)。
现有技术中熟知生产和纯化抗体和抗原结合片段的方法，如冷泉港的抗体实验技术指南，5-8章和15章。例如，鼠可以用人PD-L1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源FR区。人FR种系序列可以通过比对IMGT人类抗体可变区种系基因数据库和MOE软件，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。
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)和NS0细胞。
本公开工程化的抗体或抗原结合片段可用常规方法制备和纯化。比如，编码重链和轻链的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，哺乳动物类表达系统会导致抗体的糖基化，特别是在Fc区的高度保守N端位点。通过表达与人PD-L1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。比如，用含调整过的缓冲液的A或G Sepharose FF柱进行纯化。洗去非特异性结合的组分。再用pH梯度法洗脱结合的抗体，用SDS-PAGE检测抗体片段，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。
“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。
“治疗”意指给予患者内用或外用治疗剂，例如包含本公开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，以诱导这类症状退化或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。尽管本公开的实施方案(例如治疗方法或制品)在缓解每个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。
“保守修饰”或“保守置换或取代”是指具有类似特征(例如电荷、侧链大小、疏水性/亲水性、主链构象和刚性等)的其它氨基酸置换蛋白中的氨基酸，使得可频繁进行改变而不改变蛋白的生物学活性。本领域技术人员知晓，一般而言，多肽的非必需区域中的单个氨基酸置换基本上不改变生物学活性(参见例如Watson等(1987)Molecular Biology of the Gene,The Benjamin/Cummings Pub.Co.,第224页，(第4版))。另外，结构或功能类似的氨基酸的置换不大可能破环生物学活性。
“有效量”包含足以改善或预防医学疾病的症状或病症的量。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：例如，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。
“外源性”指根据情况在生物、细胞或人体外产生的物质。“内源性”指根据情况在细胞、生物或人体内产生的物质。
“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，那么两个序列为60％同源；如果两个序列中的100个位置有95个匹配或同源，那么两个序列为95％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。
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。
本文使用的“聚合酶链式反应”或“PCR”是指其中微量的特定部分的核酸、RNA和/或DNA如在例如美国专利号4,683,195中所述扩增的程序或技术。一般来说，需要获得来自目标区域末端或之外的序列信息，使得可以设计寡核苷酸引物；这些引物在序列方面与待扩增模板的对应链相同或相似。2个引物的5’末端核苷酸可以与待扩增材料的末端一致。PCR可用于扩增特定的RNA序列、来自总基因组DNA的特定DNA序列和由总细胞RNA转录的cDNA、噬菌体或质粒序列等。一般参见Mullis等(1987)Cold Spring  Harbor Symp.Ouant.Biol.51:263；Erlich编辑，(1989)PCR TECHNOLOGY(Stockton Press,N.Y.)。本文使用的PCR被视为用于扩增核酸测试样品的核酸聚合酶反应法的一个实例，但不是唯一的实例，所述方法包括使用作为引物的已知核酸和核酸聚合酶，以扩增或产生核酸的特定部分。
“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的场合。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。
“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，所述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。
此外，本公开包括用于治疗与PD-L1阳性细胞相关的疾病的药剂，所述药剂包含本公开的单克隆抗体或其抗体片段作为活性成分。
对与PD-L1相关的疾病没有限制，只要它是与PD-L1相关的疾病即可，例如利用本公开的分子诱导的治疗反应可通过结合人类PD-L1，然后阻遏PD-L1与其配体PD-1、B7-1的结合，或杀伤过表达PD-L1的肿瘤细胞。因此，当处于适于治疗应用的制备物和制剂中时，本公开的分子对这样一些人是非常有用的，他们患有肿瘤或癌症，优选黑色素瘤、结肠癌、乳腺癌、肺癌、胃癌、肠癌、肾癌、非小细胞肺癌、膀胱癌等。
此外，本公开涉及用于免疫检测或测定PD-L1的方法、用于免疫检测或测定PD-L1的试剂、用于免疫检测或测定表达PD-L1的细胞的方法和用于诊断与PD-L1阳性细胞相关的疾病的诊断剂，其包含本公开的特异性识别人PD-L1并与胞外区的氨基酸序列或其三维结构结合的单克隆抗体或抗体片段作为活性成分。
在本公开中，用于检测或测定PD-L1的量的方法可以是任何已知方法。例如，它包括免疫检测或测定方法。
免疫检测或测定方法是使用标记的抗原或抗体检测或测定抗体量或抗原量的方法。免疫检测或测定方法的实例包括放射性物质标记的免疫抗体方法(RIA)、酶免疫测定法(EIA或ELISA)、荧光免疫测定法(FIA)、发光免疫测定法、蛋白质免疫印迹法、物理化学方法等。
上述与PD-L1阳性细胞相关的疾病可以通过用本公开的单克隆抗体或抗体片段检测或测定表达PD-L1的细胞来诊断。
为了检测表达多肽的细胞，可以使用已知的免疫检测方法，并优选使用免疫沉淀法、荧光细胞染色法、免疫组织染色法等。此外，可以使用利用FMAT8100HTS系统(Applied  Biosystem)的荧光抗体染色法等。
在本公开中，对用于检测或测定PD-L1的活体样品没有特别限制，只要它具有包含表达PD-L1的细胞的可能性即可，例如组织细胞、血液、血浆、血清、胰液、尿液、粪便、组织液或培养液。
根据所需的诊断方法，含有本公开的单克隆抗体或其抗体片段的诊断剂还可以含有用于执行抗原-抗体反应的试剂或用于检测反应的试剂。用于执行抗原-抗体反应的试剂包括缓冲剂、盐等。用于检测的试剂包括通常用于免疫检测或测定方法的试剂，例如识别所述单克隆抗体、其抗体片段或其结合物的标记的第二抗体和与所述标记对应的底物等。
通过改造，本公开获得了亲和力更高、肿瘤杀伤活性更强、免疫原性更低等效果的PD-L1抗体。
二.实施例与测试例
以下结合实施例进一步描述本公开，但这些实施例并非限制着本公开的范围。本公开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。
实施例1.PD-L1抗体的亲和力成熟酵母文库的构建以及文库的验证
为获得药效更佳的抗人PD-L1抗体，在HRP00052和HRP00049两个抗体的基础上设计并制备scFv抗体的亲和力成熟酵母文库，并从中筛选新的人PD-L1抗体。HRP00052和HRP00049的CDR和轻重链可变区序列均来自WO2017084495A1。具体序列如下：
HRP00049：9-2(H2/L10)IgG4(AA)(S228P)
重链：HRP00049抗体重链序列：(SEQ ID NO：1)
Figure PCTCN2019070982-appb-000001
HRP00049抗体重链序列编码基因序列：(SEQ ID NO：2)
Figure PCTCN2019070982-appb-000002
Figure PCTCN2019070982-appb-000003
轻链：HRP00049抗体轻链序列：(SEQ ID NO：3)
Figure PCTCN2019070982-appb-000004
HRP00049抗体轻链序列编码基因序列：(SEQ ID NO：4)
Figure PCTCN2019070982-appb-000005
Figure PCTCN2019070982-appb-000006
HRP00052：24D5(GF)IgG4(AA)(S228P)
重链：HRP00052抗体重链序列：(SEQ ID NO：5)
Figure PCTCN2019070982-appb-000007
HRP00052抗体重链序列编码基因序列：(SEQ ID NO：6)
Figure PCTCN2019070982-appb-000008
Figure PCTCN2019070982-appb-000009
轻链：HRP00052抗体轻链序列：(SEQ ID NO：7)
Figure PCTCN2019070982-appb-000010
HRP00052抗体轻链序列编码基因序列：(SEQ ID NO：8)
Figure PCTCN2019070982-appb-000011
Figure PCTCN2019070982-appb-000012
上述抗体序列中下划线部分为抗体的可变区部分，其他正体部分为抗体恒定区部分。
酵母文库的构建:设计简并引物，通过PCR的方法将设计的突变氨基酸引入到HRP00049和HRP00052抗体的文库中。再通过二代测序的方法对文库的QC进行验证，其中基于HRP00049和HRP00052序列分别各构建了7个库容为10 9级别的抗体酵母文库。
实施例2.抗原的制备
设计并合成人PD-L1-IgG1Fc融合蛋白，经Protein A的亲和柱纯化，可获得高纯度的重组PD-L1-Fc蛋白，用于检测抗PD-L1抗体与抗原的结合。
人PD-L1-IgG1Fc：(SEQ ID NO：9)
Figure PCTCN2019070982-appb-000013
注释： 信号肽+胞外区+hIgG1Fc。
实施例3.抗体的筛选
HRP00052文库利用生物素化的人PD-L1-hIgG1Fc抗原，经过两轮MACS筛选(链霉素磁珠，Invitrogen)和两轮FACS筛选(BD FACSAria TM FUSION)。再挑选400个左右的酵母单克隆培养和诱导表达，FACS(BD FACSCanto II)检测酵母单克隆对人PD-L1-hIgG1Fc抗原的结合，挑选出比野生型HRP00052抗体亲和力高的酵母单克隆测序验证，经过对测序克隆进行比对分析，去除冗余序列之后，将非冗余序列转换成全长IgG(γ1,κ)进行哺乳动物细胞表达。亲和纯化之后的全长抗体采用BIAcore TM X-100(GE Life Sciences)进行亲合力测定。
HRP00049文库利用生物素化的人PD-L1-hIgG1Fc抗原和生物素化的小鼠PD-L1-hIgG1Fc，经过三轮MACS筛选和三轮FACS筛选。再挑选400个左右的酵母单克隆培养 和诱导表达，FACS检测酵母单克隆对人PD-L1-hIgG1Fc抗原和小鼠PD-L1-hIgG1Fc抗原的结合，挑选出同时结合人PD-L1-hIgG1Fc抗原和小鼠PD-L1-hIgG1Fc抗原的酵母单克隆测序验证。去除冗余序列之后，将非冗余序列转换成全长IgG(γ1,κ)进行哺乳动物细胞表达。亲和纯化之后的全长抗体采用BIAcore TM X-100(GE Life Sciences)进行亲合力测定。
经筛选确认选择抗体CDR区序列。
来自HRP00049突变文库的抗体
基于HRP00049序列的突变文库所挑选的克隆与HRP00049在HCDR1和HCDR2上有差异。相关的CDR序列或通式及其相应重链可变区如下所描述。
HCDR1 为DGSAYWS(SEQ ID NO：10)或NDYWT(SEQ ID NO：11)
HCDR2 X 1ISX 2AGSTYX 3TPSLKG              SEQ ID NO：12
HCDR3 SGGWLAPFDY                      SEQ ID NO：13
LCDR1 KSSQSLFYHSNQKHSLA               SEQ ID NO：14
LCDR2 GASTRES                         SEQ ID NO：15
LCDR3 QQYYGYPYT                       SEQ ID NO：16
进而获得相关的重链可变区序列通式如下：
Figure PCTCN2019070982-appb-000014
或
QVQLQESGPGLVKPSQTLSLTCTVSGGSIS NDYWTWIRQHPGKGLEYIG X 1ISX 2AG STYX 3TPSLKG RVTISRDTSKNQFSLKLSSVTAADTAVYYCAR SGGWLAPFDYWGRGTLVTVSS(SEQ ID NO：18)。上述SEQ ID NO.12所示的HCDR2和SEQ ID NO.17或18所示的重链可变区中X 1选自F或M，X 2选自R或V，X 3选自N或H。
获得相关的轻链可变区序列如下：(SEQ ID NO：19)
Figure PCTCN2019070982-appb-000015
来自HRP00052突变文库的抗体
基于HRP00052序列的突变文库所挑选的克隆与HRP00052在HCDR1和HCDR2及LCDR2上有差异。相关的HCDR1和HCDR2及LCDR2通式及其相应重轻链可变区如下所描述。
HCDR1 X 4X 5WMX 6                         SEQ ID NO：20
HCDR2 RIX 7PX 8X 9GX 10X 11X 12YNEKX 13KN       SEQ ID NO：21
HCDR3 GGSSYDYFDY                      SEQ ID NO：22
LCDR1 RASESVSIHGTHLMH                 SEQ ID NO：23
LCDR2 X 14ASX 15X 16X 17S                   SEQ ID NO：24
LCDR3 QQSFEDPLT                       SEQ ID NO：25
进而获得相关的重链可变区序列通式如下：(SEQ ID NO：26)
QVQLVQSGAEVKKPGASVKVSCKASGYTFT X 4X 5WMX 6 WVRQAPGQGLEWMG R IX 7PX 8X 9GX 10X 11X 12YNEKX 13KN RVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR GGSS YDYFDYWGQGTTVTVSS，上述SEQ ID NO.20和21所示的HCDR1和HCDR2及SEQ ID NO.26所示的重链可变区中X 4选自S和D，X 5选自Y和K，X 6选自H和M，X 7选自T，S，H和G，X 8选自S，N和G，X 9选自S，L和G，X 10选自F，L，W和M，X 11选自A，P和T，X 12选自M，V，L和S，X 13选自F和Y。
进而获得相关的轻链可变区序列通式如下：(SEQ ID NO：27)
Figure PCTCN2019070982-appb-000016
上述SEQ ID NO：24所示的LCDR2及SEQ ID NO.27所示的轻链可变区中X 14选自V和A，X 15选自Y和N，X 16选自A，L和V，X 17选自E，F，Y和A(其中包括X 17选自E，F和A与X 17选自Y的情况)。获得的具体相关序列包括但不限于表1和表2所述：
表1.亲和力筛选所确定的重链可变区序列
Figure PCTCN2019070982-appb-000017
Figure PCTCN2019070982-appb-000018
表2.亲和力筛选所确定的轻链可变区序列
Figure PCTCN2019070982-appb-000019
Figure PCTCN2019070982-appb-000020
衍生自HRP00049抗体突变文库的抗体轻重链可变区具体序列：
9-2 H5重链可变区(SEQ ID NO：42)
Figure PCTCN2019070982-appb-000021
9-2 H6重链可变区(SEQ ID NO：43)
Figure PCTCN2019070982-appb-000022
9-2 H7重链可变区(SEQ ID NO：44)
Figure PCTCN2019070982-appb-000023
9-2 L11轻链可变区(SEQ ID NO：45)
Figure PCTCN2019070982-appb-000024
衍生自HRP00052抗体突变文库的抗体轻重链可变区具体序列：
24D5 H12重链可变区(SEQ ID NO：46)
Figure PCTCN2019070982-appb-000025
24D5 H13重链可变区(SEQ ID NO：47)
Figure PCTCN2019070982-appb-000026
24D5 H14重链可变区(SEQ ID NO：48)
Figure PCTCN2019070982-appb-000027
24D5 H15重链可变区(SEQ ID NO：49)
Figure PCTCN2019070982-appb-000028
24D5 H16重链可变区(SEQ ID NO：50)
Figure PCTCN2019070982-appb-000029
24D5 H17重链可变区(SEQ ID NO：51)
Figure PCTCN2019070982-appb-000030
24D5 H18重链可变区(SEQ ID NO：52)
Figure PCTCN2019070982-appb-000031
24D5 H19重链可变区(SEQ ID NO：53)
Figure PCTCN2019070982-appb-000032
24D5 H20重链可变区(SEQ ID NO：54)
Figure PCTCN2019070982-appb-000033
24D5 H21重链可变区(SEQ ID NO：66)
Figure PCTCN2019070982-appb-000034
24D5 L64轻链可变区(SEQ ID NO：55)
Figure PCTCN2019070982-appb-000035
24D5 L61轻链可变区(SEQ ID NO：56)
Figure PCTCN2019070982-appb-000036
24D5 L66轻链可变区(SEQ ID NO：57)
Figure PCTCN2019070982-appb-000037
24D5 L67轻链可变区(SEQ ID NO：70)
Figure PCTCN2019070982-appb-000038
24D5 L68轻链可变区(SEQ ID NO：71)
Figure PCTCN2019070982-appb-000039
24D5 L69轻链可变区(SEQ ID NO：72)
Figure PCTCN2019070982-appb-000040
上述抗体的轻重链可变区，选用人重链IgG1/轻链kappa的恒定区分别与各重链可变区和轻链可变区组合，形成完整的抗体重链和轻链，具体重链恒定区和轻链恒定区序列如下：
IgG1重链恒定区(SEQ ID NO：58)
Figure PCTCN2019070982-appb-000041
kappa轻链恒定区(SEQ ID NO：59)
Figure PCTCN2019070982-appb-000042
表3 由HRP00049突变衍生的完整抗体的轻重链可变区组合
抗体名称 重轻链可变区组合
H5L11 9-2 H5和9-2 L11
H6L11 9-2 H6和9-2 L11
H7L11 9-2 H7和9-2 L11
表4-1 由HRP00052突变衍生的完整抗体的轻重链可变区组合
Figure PCTCN2019070982-appb-000043
表4-2 由HRP00052突变衍生的完整抗体的轻重链可变区组合
可变区名称 24D5 L61 24D5 L64 24D5 L66 24D5 L67 24D5 L68 24D5 L69
24D5 H12 H12L61 H12L64 H12L66 H12L67 H12L68 H12L69
24D5 H13 H13L61 H13L64 H13L66 H13L67 H13L68 H13L69
24D5 H14 H14L61 H14L64 H14L66 H14L67 H14L68 H14L69
24D5 H15 H15L61 H15L64 H15L66 H15L67 H15L68 H15L69
24D5 H16 H16L61 H16L64 H16L66 H16L67 H16L68 H16L69
24D5 H17 H17L61 H17L64 H17L66 H17L67 H17L68 H17L69
24D5 H18 H18L61 H18L64 H18L66 H18L67 H18L68 H18L69
24D5 H19 H19L61 H19L64 H19L66 H19L67 H19L68 H19L69
24D5 H20 H20L61 H20L64 H20L66 H20L67 H20L68 H20L69
24D5 H21 H21L61 H21L64 H21L66 H21L67 H21L68 H21L69
在本公开具体实施方案中，如表5-1和表5-2所述衍生自HRP00049和HRP00052突变抗体文库中的重轻链可变区，在与重轻链恒定区连接形成完整抗体时，均表示与上述人IgG1重链恒定区(SEQ ID NO：58)和kappa轻链恒定区(SEQ ID NO：59)连接形成完整抗体，如H15L61即指由H15与IgG1重链恒定区连接形成重链和L61与kappa轻链恒定区连接形成轻链，轻重链组合形成完整抗体，其他命名类推。
HRP00052-IgG1则指将HRP00052的重链恒定区(IgG4亚类)替换为上述IgG1重链恒定区(SEQ ID NO：58)后形成的完整抗体。
表5-1 重链可变区连接IgG1重链恒定区(SEQ ID NO：58)和轻链可变区连接kappa轻链恒定区(SEQ ID NO：59)后的全长抗体名称
可变区名称 24D5 L61 24D5 L64 24D5 L66 24D5 L67 24D5 L68 24D5 L69
24D5 H12 H12L61 H12L64 H12L66 H12L67 H12L68 H12L69
24D5 H13 H13L61 H13L64 H13L66 H13L67 H13L68 H13L69
24D5 H14 H14L61 H14L64 H14L66 H14L67 H14L68 H14L69
24D5 H15 H15L61 H15L64 H15L66 H15L67 H15L68 H15L69
24D5 H16 H16L61 H16L64 H16L66 H16L67 H16L68 H16L69
24D5 H17 H17L61 H17L64 H17L66 H17L67 H17L68 H17L69
24D5 H18 H18L61 H18L64 H18L66 H18L67 H18L68 H18L69
24D5 H19 H19L61 H19L64 H19L66 H19L67 H19L68 H19L69
24D5 H20 H20L61 H20L64 H20L66 H20L67 H20L68 H20L69
24D5 H21 H21L61 H21L64 H21L66 H21L67 H21L68 H21L69
表5-2 重链可变区连接IgG1重链恒定区(SEQ ID NO：58)和轻链可变区连接kappa轻链恒定区(SEQ ID NO：59)后的全长抗体名称
可变区名称 9-2 H5 9-2 H6 9-2 H7
9-2 L11 H5L11 H6L11 H7L11
为了与连接人IgG1重链恒定区的完整抗体进行对比，某些具体实施方案中，表6-1和表6-2中的全长抗体，是上述衍生自HRP00049和HRP00052突变抗体文库中筛选获得的重轻链可变区分别与下述人IgG4重链恒定区(如SEQ ID NO：60所示，含S228P和234A235A突变)和kappa轻链恒定区(同SEQ ID NO：59)连接形成完整抗体，如H15L61-IgG4即指由H15与IgG4重链恒定区连接形成重链和L61与kappa轻链恒定区连接形成轻链，轻重链组合形成完整抗体，其他命名类推。
人IgG4重链恒定区：(SEQ ID NO：60)
Figure PCTCN2019070982-appb-000044
表6-1 重链可变区连接人IgG4重链恒定区(SEQ ID NO：60)和轻链可变区连接kappa轻链恒定区(SEQ ID NO：59)后的全长抗体名称
Figure PCTCN2019070982-appb-000045
Figure PCTCN2019070982-appb-000046
表6-2 重链可变区连接人IgG4重链恒定区(SEQ ID NO：60)和轻链可变区连接kappa轻链恒定区(SEQ ID NO：59)后的全长抗体名称
可变区名称 9-2 H5 9-2 H6 9-2 H7
9-2 L11 H5L11-IgG4 H6L11-IgG4 H7L11-IgG4
某些具体实施方案中，表7-1和表7-2中的全长抗体，是上述衍生自HRP00049和HRP00052突变抗体文库中筛选获得的重轻链可变区分别与下述人IgG4 S228P重链恒定区(如SEQ ID NO：65所示，含S228P突变)和kappa轻链恒定区(同SEQ ID NO：59)连接形成完整抗体，如H15L61-IgG4即指由H15与IgG4重链恒定区连接形成重链和L61与kappa轻链恒定区连接形成轻链，轻重链组合形成完整抗体，其他命名类推。IgG4 S228P的重链恒定区序列如下：(SEQ ID NO：65)
Figure PCTCN2019070982-appb-000047
表7-1 重链可变区连接人IgG4 S228P重链恒定区(SEQ ID NO：65)和轻链可变区连接kappa轻链恒定区(SEQ ID NO：59)后的全长抗体名称
Figure PCTCN2019070982-appb-000048
Figure PCTCN2019070982-appb-000049
表7-2 重链可变区连接人IgG4 S228P重链恒定区(SEQ ID NO：65)和轻链可变区连接kappa轻链恒定区(SEQ ID NO：59)后的全长抗体名称
可变区名称 9-2 H5 9-2 H6 9-2 H7
9-2 L11 H5L11-IgG4 S228P H6L11-IgG4 S228P H7L11-IgG4 S228P
具体轻重链恒定区并非作为本公开抗体恒定区的限制，也可选用本领域其它已知的轻重链恒定区及其突变体来增加其性能。
本公开以Merck的PD-L1抗体Avelumab(A09)和/或Genetech的3280A为阳性对照，其中A09的轻重链氨基酸序列(来源自US20140341917)如下：
>A09重链：(SEQ ID NO：61)
Figure PCTCN2019070982-appb-000050
>A09轻链：(SEQ ID NO：62)
Figure PCTCN2019070982-appb-000051
3280A(Genetech，Atezolizumab，WHO Drug Information,Vol.28,No.4,2014，P488)的轻重链氨基酸序列如下：
>3280A重链:(SEQ ID NO：63)
Figure PCTCN2019070982-appb-000052
>3280A轻链:(SEQ ID NO：64)
Figure PCTCN2019070982-appb-000053
PD-L1抗体经常规方法纯化获得。
以下实施例中抗体均为全长抗体
实施例4.抗体和配体阻断试验
深入研究了本品对PD-L1与PD1结合的阻断作用，同时与在临床试验中的同类产品进行了比较。方法参照WO2017084495A1测试例2和3，在小鼠PD-L1/PD-1结合阻断试验中使用人PD-L1抗体阻断小鼠PD-L1/PD-1的结合。数据详见下表8。
表8 PD-L1抗体阻断PD-L1与其配体的结合测试
Figure PCTCN2019070982-appb-000054
Figure PCTCN2019070982-appb-000055
*10μg/ml，5μg/ml和1μg/ml分别是不同阻断试验中PD-L1的浓度。
配体阻断实验证明，本公开PD-L1抗体能够阻断PD-L1与PD1的结合，也能够阻断PD-L1与B7-1的结合，并且由HRP00049突变而来的H5L11，H6L11抗体具有小鼠PD-L1交叉结合活性，可阻断小鼠PD-L1与PD-1结合的能力。
实施例5.实例性抗体的BIAcore抗体亲和力实验
按照人抗捕获试剂盒(Cat.#BR-1008-39，GE)说明书中的方法将人抗捕获抗体共价偶联于生物传感芯片CM5(Cat.#BR-1000-12，GE)生物芯片上，从而亲和捕获一定量的人PD-L1(Cat.#10084-H08H，Sino Biological),猴PD-L1(Cat.#90251-C08H，Sino Biological),小鼠PD-L1(Cat.#50010-M08H，Sino Biological)。用Biacore X100，GE仪器测定PD-L1抗体和PD-L1的反应亲和力。试验使用的是HBS-EP+10X缓冲溶液(Cat.#BR-1006-69，GE)用D.I.Water稀释至1×(pH7.4)，用BIAevaluation version 4.1，GE软件以(1:1)Langmuir模型拟合数据，得出亲和力数值，结果见表9。
表9-1 HRP00049和HRP00052及其突变抗体与不同物种PD-L1结合的Biacore亲和力(同批次试验)
Figure PCTCN2019070982-appb-000056
Figure PCTCN2019070982-appb-000057
表9-2 HRP00052及其突变抗体与不同物种PD-L1结合的Biacore亲和力(同批次试验)
Figure PCTCN2019070982-appb-000058
结果显示，筛选自HRP00052突变抗体文库的抗体(除H21L68外)与人PD-L1的亲和力均较HRP00052有所提高，且均高于阳性对照抗体。而筛选自HRP00049突变抗体文库的抗体H5L11，H6L11，H7L11则产生较强的鼠PD-L1交叉亲和活性。
实施例6.PD-L1抗体在PBMC-T淋巴细胞激活实验中对细胞IFNγ的分泌作用
为了研究PD-L1抗体对人原代T淋巴细胞功能的影响，收集和纯化人外周血单核细胞(PBMC)，采用结核菌素(TB)体外刺激5天后，检测细胞因子IFNγ分泌水平。实验过程简单描述如下：
新鲜血液利用Ficoll-Hypaque密度梯度离心(Stem Cell Technologies)得到PBMC，于RPMI 1640培养基中培养，该培养基中添加10％(v/v)FBS，37℃、5％CO 2条件下培养。
新鲜分离纯化的PBMC以RPMI 1640培养基调整密度为2×10 6/ml，20mL细胞悬液中加入25μl结核菌素，37℃、5％CO 2培养箱培养5天。第6天，收集上述培养的细胞离心，用PBS洗一次，重悬至新鲜的RPMI 1640培养基中，调整密度为1×10 6/ml，接种至96孔细胞培养板，每孔90μl。同时加入梯度稀释的抗体样品(用PBS稀释)或等量的同型IgG作为空白对照,每孔10μl。细胞培养板置于37℃，5％CO 2培养箱孵育3 天。取出细胞培养板，离心(4000rpm，10min)收集细胞培养上清，采用ELISA的方法(人IFN-γ检测试剂盒，欣博盛，EHC102g.96)检测IFN-γ的水平。具体操作参考试剂说明书。
结果(见图1)显示，H18L61，H12L64，H5L11，H6L11均能较强地刺激细胞IFNγ的分泌，其中H12L64，H5L11，H6L11在促进细胞IFNγ分泌时，呈现出剂量依赖关系，上述四种抗体在某些剂量或所有剂量上优于HRP00052-IgG1的作用。
实施例7.实例性抗体介导的PD-L1+细胞的ADCC效应
将人全血用等体积的PBS缓冲液进行稀释后，加入已有适量Lymphoprep(STEMCELL Technologies Inc.,07851)的SepMate(STEMCELL Technologies Inc.,15460)管底部，室温下以1200g离心10分钟。将管中上层液体倒入新的50ml离心管中，用PBS缓冲液洗涤细胞，并以300g离心8分钟，得到PBMC。用含有5％Low IgG FBS(BIOSUN，BS-0007-500)的RPMI-1640培养液重悬PBMC，进行细胞计数。
将CHO-S/PD-L1细胞用含有5％Low IgG FBS的RPMI1640培养基重悬并计数，以每孔10,000个细胞的密度接种在96孔板中，然后加入梯度稀释的PD-L1抗体孵育15分钟，再在每孔中加入300,000个人PBMC细胞作用4小时，设置培养基对照，CHO-S/PD-L1细胞自发释放对照，PBMC细胞自发释放对照和CHO-S/PD-L1细胞最大裂解对照。4小时后，将96孔板离心，每孔取50μL上清转移至另一96孔板，每孔加入50μL CytoTox96Non-Radioactive Cytotoxicity Assay(Promega，G1780)，避光室温孵育30分钟，加入终止液，在490nm波长处读取吸光值。按照下列公式分析数据，计算出裂解率，用Graphpad Prism软件根据抗体各浓度与相应的裂解率计算抗体ADCC效应的EC 50值。
裂解率％＝(样品孔 490nm-PBMC细胞自发释放 490nm-CHO-S/PD-L1细胞自发释放 490nm)/(CHO-S/PD-L1细胞最大裂解 490nm-CHO-S/PD-L1细胞自发释放 490nm)×100
结果显示(图2A至图2F所示)，不同PD-L1抗体HRP00049-IgG1，H5L11，HRP00052-IgG1，H6L11，H18L61，H12L64的IgG1形式都有较强的ADCC效应,并且这些分子的ADCC效应均明显优于各PD-L1抗体的IgG4形式。
实施例8.实例性抗体在人的黑色素瘤A375异种移植模型中的作用
将培养的A375细胞与PBMC混合接种于NOG小鼠皮下。实验分为PBMC+PBS组、PBMC+H12L64 20mg/kg组、PBMC+H12L64-IgG4-20mg/kg组和PBMC+HRP0005220mg/kg组。每组6只，每两天一次，腹腔注射给药，连续给药11次。给药实验结束后，使用肿瘤生长抑制率TGI(％)和相对肿瘤增值率T/C(％)进行疗效评价。
结果见图3和表10，H12L64 20mg/kg组TGI为98.15％，与PBMC+PBS组相比有 显著性差异(p＝0.0018)；H12L64-IgG4 20mg/kg组肿瘤抑制率TGI为82.15％，与PBMC+PBS组相比有显著性差异(p＝0.0064)；HRP00052 20mg/kg组肿瘤抑制率TGI为68.84％，与PBMC+PBS组相比有显著性差异(p＝0.0291)。该结果表明，H12L64 20mg/kg组和H12L64-IgG4 20mg/kg及HRP00052 20mg/kg能有效抑制A375的生长(p<0.05)。在整个实验过程中，H12L64，H12L64-IgG4和HRP00052荷瘤小鼠均没有出现体重下降或动物死亡情况，表明小鼠对该剂量下的H12L64，H12L64-IgG4和HRP00052耐受性良好。
表10 PD-L1抗体在人源化A375皮下移植瘤模型的疗效
Figure PCTCN2019070982-appb-000059
TGI％：抑瘤率；T/C％:肿瘤增值率；CR：肿瘤消退。
实施例9.PD-L1抗体在小鼠结肠癌模型MC38-hPD-L1中的作用
将MC38-hPD-L1细胞(向小鼠结肠癌细胞MC38转化hPD-L1后在细胞表面可表达hPD-L1，4×10 5个)100μl接种至50只C57小鼠右肋部皮下，7天后去除肿瘤体积过大过小动物，按平均肿瘤体积约52mm 3将小鼠随机分为：3个PD-L1抗体单用组和阴性对照组，共4组，每组10只。分组后腹腔给与各药物(对照组注射等量体积的PBS)，每周天三次，共12次，给药周期28天，停药后两天结束对荷瘤小鼠的监测。每周测2次瘤体积，称体重，记录数据。分组及给药情况见下表。各组动物体重、肿瘤体积以及瘤重均用平均值±标准差(Mean±SEM)表示，并用Graphpad Prism 5和Excel软件作图，使用student t test统计分析。
肿瘤体积(TV)＝1/2×L 长×L 短 2
肿瘤增殖率T/C％＝(T-T0)/(C-C0)×100％
抑瘤率％TGI＝1-T/C％
G 组别 动物只数 给药剂量(mg/kg) 给药途径
Group1 对照组(PBS) 10 - ip
Group2 HRP00052-IgG1-10mpk 10 10 ip
Group3 H6L11-10mpk 10 10 ip
Group4 A09-10mpk 10 10 ip
实验结果(见图4)显示，H6L11，A09都能显著抑制MC38-hPD-L1小鼠皮下移植瘤的生长。与阴性对照组相比，两组瘤体积从给药后3天都出现统计学差异(p＜0.05)，实验结束时(给药后27d)，两组抑瘤率分别为106.74％、103.47％，与阴性对照组有极显著差异(p＜0.001)，约80％-90％的肿瘤能完全消退，H6L11比A09抑瘤效果更好，但二者没有统计学差异(见图4)。HRP00052-IgG1在实验结束时，抑瘤率为29.10％。
实验结束后将荷瘤鼠安乐死并剥瘤称重，瘤重结果与瘤体积大小基本相符：三个药物组中，H6L11组平均瘤重最小，A09组次之，HRP00052-IgG1的平均瘤重最大；H6L11，A09两组与对照组之间都存在显著差异(p＜0.001)。荷瘤小鼠对各药物均能很好耐受，无药物致体重减轻等症状发生。
实施例10.PD-L1抗体在小鼠结肠癌模型MC38-hPD-L1中的作用
将MC38-hPD-L1细胞以5.8×10 5个/100μl/只接种到C57/BL-6小鼠皮下，荷瘤模型确立后，测量肿瘤体积，去除体重、肿瘤过大和过小的动物，按照肿瘤体积大小将荷瘤小鼠随机分为2组(n＝7)：IgG-PBS(C25-IgG4)对照组、H5L11-IgG4 S228P实验组，以分组给药日期设定为D0。分组后腹腔给与各药物，每周三次，共10次，给药周期18天，停药后两天结束对荷瘤小鼠的监测。每周测2次瘤体积，称体重，记录数据。分组及给药情况见下表。各组动物体重、肿瘤体积以及瘤重均用平均值±标准差(Mean±SEM)表示，并用Graphpad Prism 6和Excel软件作图，使用student t test统计分析。
肿瘤体积(TV)＝1/2×L 长×L 短 2
肿瘤增殖率T/C％＝(T-T0)/(C-C0)×100％
抑瘤率％TGI＝1-T/C％
如图5结果显示，与小鼠PD-L1存在交叉反应的PD-L1单抗(H5L11-IgG4 S228P组)实验组肿瘤体积显著小于对照组，并且在给药后一周左右开始即与对照组之间出现统计学差异。
实验结束后对荷瘤小鼠进行安乐死并剥瘤称重，瘤重结果与瘤体积大小存在一定相似性。实验过程中，给药组与对照组动物体重未出现明显差异，小鼠对各给药抗体耐受良好。
虽然为了清楚的理解，已经借助于附图和实例详细描述了上述发明，但是描述和实例不应当解释为限制本公开的范围。本文中引用的所有专利和科学文献的公开内容通过引用完整地清楚结合。

Claims (18)

  1. 一种单克隆抗体或其抗原结合片段，所述单克隆抗体或抗原结合片段结合人PD-L1，所述单克隆抗体或其抗原结合片段包含重链可变区和轻链可变区，其中：
    (i)所述重链可变区包含氨基酸序列分别如SEQ ID NO：10、12和13所示的HCDR1、HCDR2和HCDR3区，且所述轻链可变区包含氨基酸序列分别如SEQ ID NO：14、15和16所示的LCDR1、LCDR2和LCDR3区，所述SEQ ID NO.12所示的HCDR2中X 1为F或M，X 2为R或V，X 3为N或H；或
    (ii)所述重链可变区包含氨基酸序列分别如SEQ ID NO：11、12和13所示的HCDR1、HCDR2和HCDR3区，且所述轻链可变区包含氨基酸序列分别如SEQ ID NO：14、15和16所示的LCDR1、LCDR2和LCDR3区，所述SEQ ID NO.12所示的HCDR2中X 1为F或M，X 2为R或V，X 3为N或H；或
    (iii)所述重链可变区包含氨基酸序列分别如SEQ ID NO：20、21和22所示的HCDR1、HCDR2和HCDR3区，且所述轻链可变区包含氨基酸序列分别如SEQ ID NO：23、24和25所示的LCDR1、LCDR2和LCDR3区，其中HCDR1、HCDR2、HCDR3和LCDR1、LCDR2、LCDR3依次不同时为SEQ ID NO：30、38、22、23、40和25，
    所述SEQ ID NO.20和21中的X 4为S或D，X 5为Y或K，X 6为H或M，X 7为T、S、H或G，X 8为S、N或G，X 9为S、L或G，X 10为F、L、W或M，X 11为A、P或T，X 12为M、V、L或S，X 13为F或Y，所述SEQ ID NO：24所示的LCDR2中X 14为V或A，X 15为Y或N，X 16为A、L或V，X 17为E、F、Y或A。
  2. 一种单克隆抗体或其抗原结合片段，所述单克隆抗体或其抗原结合片段结合人PD-L1，所述单克隆抗体或其抗原结合片段包含重链可变区和轻链可变区，其中：
    所述重链可变区包含氨基酸序列如SEQ ID NO：10所示的HCDR1、如SEQ ID NO：28或29所示的HCDR2和如SEQ ID NO：13所示的HCDR3区，所述轻链可变区包含氨基酸序列分别如SEQ ID NO：14、15和16所示的LCDR1、LCDR2和LCDR3区；或
    所述重链可变区包含氨基酸序列如SEQ ID NO：11所示的HCDR1、如SEQ ID NO：28或29所示的HCDR2和如SEQ ID NO：13所示的HCDR3区，所述轻链可变区包含氨基酸序列分别如SEQ ID NO：14、15和16所示的LCDR1、LCDR2和LCDR3区；或
    所述重链可变区包含氨基酸序列如SEQ ID NO：30所示的HCDR1、如SEQ ID NO：32至37任一所示的HCDR2和如SEQ ID NO：22所示的HCDR3区，所述轻链可变区包含氨基酸序列如SEQ ID NO：23的LCDR1、如SEQ ID NO：39、40、41、67和69任一 的LCDR2和如SEQ ID NO：25的LCDR3区；或
    所述重链可变区包含氨基酸序列如SEQ ID NO：31所示的HCDR1、如SEQ ID NO：32至37任一所示的HCDR2和如SEQ ID NO：22所示的HCDR3区，所述轻链可变区包含氨基酸序列如SEQ ID NO：23的LCDR1、如SEQ ID NO：39、40、41、67和69任一的LCDR2和如SEQ ID NO：25的LCDR3区。
  3. 根据权利要求2所述的单克隆抗体或其抗原结合片段，其中：
    所述重链可变区包含氨基酸序列分别如SEQ ID NO：10、SEQ ID NO：28和SEQ ID NO：13所示的HCDR1、HCDR2和HCDR3区，所述轻链可变区包含氨基酸序列分别如SEQ ID NO：14、15和16所示的LCDR1、LCDR2和LCDR3区；
    所述重链可变区包含氨基酸序列分别如SEQ ID NO：11、SEQ ID NO：28和SEQ ID NO：13所示的HCDR1、HCDR2和HCDR3区，所述轻链可变区包含氨基酸序列分别如SEQ ID NO：14、15和16所示的LCDR1、LCDR2和LCDR3区；
    所述重链可变区包含氨基酸序列分别如SEQ ID NO：11、SEQ ID NO：29和SEQ ID NO：13所示的HCDR1、HCDR2和HCDR3区，所述轻链可变区包含氨基酸序列分别如SEQ ID NO：14、15和16所示的LCDR1、LCDR2和LCDR3区；
    所述重链可变区包含氨基酸序列分别如SEQ ID NO：30、SEQ ID NO：32和SEQ ID NO：22所示的HCDR1、HCDR2和HCDR3区，所述轻链可变区包含氨基酸序列分别如SEQ ID NO：23、SEQ ID NO：39和SEQ ID NO：25所示的LCDR1、LCDR2和LCDR3区；
    所述重链可变区包含氨基酸序列分别如SEQ ID NO：30、SEQ ID NO：32和SEQ ID NO：22所示的HCDR1、HCDR2和HCDR3区，所述轻链可变区包含氨基酸序列分别如SEQ ID NO：23、SEQ ID NO：40和SEQ ID NO：25所示的LCDR1、LCDR2和LCDR3区；
    所述重链可变区包含氨基酸序列分别如SEQ ID NO：30、SEQ ID NO：33和SEQ ID NO：22所示的HCDR1、HCDR2和HCDR3区，所述轻链可变区包含氨基酸序列分别如SEQ ID NO：23、SEQ ID NO：40和SEQ ID NO：25所示的LCDR1、LCDR2和LCDR3区；
    所述重链可变区包含氨基酸序列分别如SEQ ID NO：30、SEQ ID NO：34和SEQ ID NO：22所示的HCDR1、HCDR2和HCDR3区，所述轻链可变区包含氨基酸序列分别如SEQ ID NO：23、SEQ ID NO：40和SEQ ID NO：25所示的LCDR1、LCDR2和LCDR3区；
    所述重链可变区包含氨基酸序列分别如SEQ ID NO：31、SEQ ID NO：32和SEQ ID NO：22所示的HCDR1、HCDR2和HCDR3区，所述轻链可变区包含氨基酸序列分别如SEQ ID NO：23、SEQ ID NO：40和SEQ ID NO：25所示的LCDR1、LCDR2和LCDR3区；
    所述重链可变区包含氨基酸序列分别如SEQ ID NO：31、SEQ ID NO：32和SEQ ID NO：22所示的HCDR1、HCDR2和HCDR3区，所述轻链可变区包含氨基酸序列分别如SEQ ID NO：23、SEQ ID NO：67和SEQ ID NO：25所示的LCDR1、LCDR2和LCDR3区；
    所述重链可变区包含氨基酸序列分别如SEQ ID NO：31、SEQ ID NO：32和SEQ ID NO：22所示的HCDR1、HCDR2和HCDR3区，所述轻链可变区包含氨基酸序列分别如SEQ ID NO：23、SEQ ID NO：69和SEQ ID NO：25所示的LCDR1、LCDR2和LCDR3区；
    所述重链可变区包含氨基酸序列分别如SEQ ID NO：31、SEQ ID NO：32和SEQ ID NO：22所示的HCDR1、HCDR2和HCDR3区，所述轻链可变区包含氨基酸序列分别如SEQ ID NO：23、SEQ ID NO：41和SEQ ID NO：25所示的LCDR1、LCDR2和LCDR3区；
    所述重链可变区包含氨基酸序列分别如SEQ ID NO：30、SEQ ID NO：35和SEQ ID NO：22所示的HCDR1、HCDR2和HCDR3区，所述轻链可变区包含氨基酸序列分别如SEQ ID NO：23、SEQ ID NO：40和SEQ ID NO：25所示的LCDR1、LCDR2和LCDR3区；
    所述重链可变区包含氨基酸序列分别如SEQ ID NO：30、SEQ ID NO：36和SEQ ID NO：22所示的HCDR1、HCDR2和HCDR3区，所述轻链可变区包含氨基酸序列分别如SEQ ID NO：23、SEQ ID NO：40和SEQ ID NO：25所示的LCDR1、LCDR2和LCDR3区；或
    所述重链可变区包含氨基酸序列分别如SEQ ID NO：30、SEQ ID NO：37和SEQ ID NO：22所示的HCDR1、HCDR2和HCDR3区，所述轻链可变区包含氨基酸序列分别如SEQ ID NO：23、SEQ ID NO：40和SEQ ID NO：25所示的LCDR1、LCDR2和LCDR3区。
  4. 如权利要求1或2所述的单克隆抗体或其抗原结合片段，其中所述单克隆抗体或抗原结合片段包含：
    氨基酸序列如SEQ ID NO：17所示的重链可变区和SEQ ID NO：19所示的轻链可 变区；
    氨基酸序列如SEQ ID NO：18所示的重链可变区和SEQ ID NO：19所示的轻链可变区；或
    氨基酸序列如SEQ ID NO：26所示的重链可变区和SEQ ID NO：27所示的轻链可变区。
  5. 如权利要求4所述的单克隆抗体或其抗原结合片段，其中所述单克隆抗体或抗原结合片段包含：
    氨基酸序列如SEQ ID NO：42所示的重链可变区和SEQ ID NO：45所示的轻链可变区；
    氨基酸序列如SEQ ID NO：43所示的重链可变区和SEQ ID NO：45所示的轻链可变区；
    氨基酸序列如SEQ ID NO：44所示的重链可变区和SEQ ID NO：45所示的轻链可变区；
    氨基酸序列如SEQ ID NO：46所示的重链可变区和SEQ ID NO：55、56、57、70和72任一所示的轻链可变区；
    氨基酸序列如SEQ ID NO：47所示的重链可变区和SEQ ID NO：55、56、57、70和72任一所示的轻链可变区；
    氨基酸序列如SEQ ID NO：48所示的重链可变区和SEQ ID NO：55、56、57、70和72任一所示的轻链可变区；
    氨基酸序列如SEQ ID NO：49所示的重链可变区和SEQ ID NO：55、56、57、70和72任一所示的轻链可变区；
    氨基酸序列如SEQ ID NO：50所示的重链可变区和SEQ ID NO：55、56、57、70和72任一所示的轻链可变区；
    氨基酸序列如SEQ ID NO：51所示的重链可变区和SEQ ID NO：55、56、57、70和72任一所示的轻链可变区；
    氨基酸序列如SEQ ID NO：52所示的重链可变区和SEQ ID NO：55、56、57、70和72任一所示的轻链可变区；
    氨基酸序列如SEQ ID NO：53所示的重链可变区和SEQ ID NO：55、56、57、70和72任一所示的轻链可变区；或
    氨基酸序列如SEQ ID NO：66所示的重链可变区和SEQ ID NO：55、56、57、70和72任一所示的轻链可变区。
  6. 如权利要求1至5任一项所述的单克隆抗体或其抗原结合片段，其中所述抗体为全长抗体，进一步包括人抗体恒定区，优选地，所述人抗体恒定区的重链恒定区选自人IgG1、IgG2、IgG3和IgG4恒定区及其常规变体，所述人抗体恒定区的轻链恒定区选自人抗体κ和λ链恒定区及其常规变体，更优选所述全长抗体包含氨基酸序列如SEQ ID NO:58、60或65所示的人抗体重链恒定区和SEQ ID NO:59所示的人轻链恒定区。
  7. 如权利要求1至5任一项所述的单克隆抗体或其抗原结合片段，其中所述抗原结合片段是选自Fab、Fab'、F(ab') 2、单链抗体(scFv)、二聚化的V区(双抗体)、二硫键稳定化的V区(dsFv)和包含CDR的肽的抗原结合片段。
  8. 一种药物组合物，其含有治疗有效量的根据权利要求1至7任一项所述的单克隆抗体或其抗原结合片段，以及一种或多种药学上可接受的载体、稀释剂或赋形剂。
  9. 一种核酸分子，其编码权利要求1至7任一项所述的单克隆抗体或其抗原结合片段。
  10. 一种重组载体，其包含权利要求9所述的核酸分子。
  11. 一种用如权利要求10所述的重组载体转化的宿主细胞，所述宿主细胞选自原核细胞和真核细胞，优选真核细胞，更优选哺乳动物细胞。
  12. 用于生产如权利要求1至7任一项所述的单克隆抗体或其抗原结合片段的方法，所述方法包括将如权利要求11所述的宿主细胞在培养基中进行培养以形成并积累权利要求1至7任一项所述的单克隆抗体或其抗原结合片段，以及从培养物回收所述单克隆抗体或其抗原结合片段。
  13. 用于免疫检测或测定人PD-L1的方法，所述方法包括使用如权利要求1至7任一项所述的单克隆抗体或其抗原结合片段。
  14. 如权利要求1至7任一项所述的单克隆抗体或其抗原结合片段在制备与人PD-L1相关的疾病的诊断剂中的应用。
  15. 与人PD-L1相关的疾病的治疗方法，所述方法包括向受试者施用药物有效量的如权利要求1至7任一项所述的单克隆抗体或其抗原结合片段，或包含权利要求8所述的药物组合物，或权利要求9所述的核酸分子，以治疗人PD-L1相关的疾病，其中所述疾病优选肿瘤或癌症；更优选PD-L1阳性的鳞状细胞癌、骨髓瘤、小细胞肺癌、非小细胞肺癌(NSCLC)、头和颈鳞状细胞癌(HNSCC)、神经胶质瘤、何杰金淋巴瘤、非何杰金淋巴瘤、弥漫性大B-细胞淋巴瘤(DLBCL)、滤泡性淋巴瘤、急性成淋巴细胞性白血病(ALL)、急性髓细胞样白血病(AML)、慢性淋巴细胞性白血病(CLL)、慢性髓细胞样白血病(CML)、原发性纵隔大B-细胞淋巴瘤、套细胞淋巴瘤(MCL)、小淋巴 细胞性淋巴瘤(SLL)、富含T-细胞/组织细胞的大B-细胞淋巴瘤、多发性骨髓瘤、髓样细胞白血病-1蛋白(Mcl-1)、骨髓异常增生综合征(MDS)、胃肠(道)癌、肾癌、卵巢癌、肝癌、成淋巴细胞性白血病、淋巴细胞白血病、结肠直肠癌、子宫内膜癌、肾癌、前列腺癌、甲状腺癌、黑素瘤、软骨肉瘤、神经母细胞瘤、胰腺癌、多形性成胶质细胞瘤、胃癌、骨癌、尤因氏肉瘤、子宫颈癌、脑癌、胃癌、膀胱癌、肝细胞瘤、乳腺癌、结肠癌、肝细胞癌(HCC)、透明细胞肾细胞癌(RCC)、头和颈癌、咽喉癌、肝胆癌(hepatobiliary cancer)、中枢神经系统癌、食管癌、恶性胸膜间皮瘤、全身性轻链淀粉样变性、淋巴浆细胞性淋巴瘤(lymphoplasmacytic lymphoma)、骨髓异常增生综合征、骨髓增生性肿瘤、神经内分泌肿瘤、梅克尔细胞癌、睾丸癌和皮肤癌。
  16. 如权利要求1至7任一项所示的单克隆抗体或其抗原结合片段，或包含权利要求8所述的药物组合物，或权利要求9所述的核酸分子在制备与人PD-L1相关的疾病的治疗剂中的应用，其中所述疾病优选肿瘤或癌症；更优选PD-L1阳性的鳞状细胞癌、骨髓瘤、小细胞肺癌、非小细胞肺癌(NSCLC)、头和颈鳞状细胞癌(HNSCC)、神经胶质瘤、何杰金淋巴瘤、非何杰金淋巴瘤、弥漫性大B-细胞淋巴瘤(DLBCL)、滤泡性淋巴瘤、急性成淋巴细胞性白血病(ALL)、急性髓细胞样白血病(AML)、慢性淋巴细胞性白血病(CLL)、慢性髓细胞样白血病(CML)、原发性纵隔大B-细胞淋巴瘤、套细胞淋巴瘤(MCL)、小淋巴细胞性淋巴瘤(SLL)、富含T-细胞/组织细胞的大B-细胞淋巴瘤、多发性骨髓瘤、髓样细胞白血病-1蛋白(Mcl-1)、骨髓异常增生综合征(MDS)、胃肠(道)癌、肾癌、卵巢癌、肝癌、成淋巴细胞性白血病、淋巴细胞白血病、结肠直肠癌、子宫内膜癌、肾癌、前列腺癌、甲状腺癌、黑素瘤、软骨肉瘤、神经母细胞瘤、胰腺癌、多形性成胶质细胞瘤、胃癌、骨癌、尤因氏肉瘤、子宫颈癌、脑癌、胃癌、膀胱癌、肝细胞瘤、乳腺癌、结肠癌、肝细胞癌(HCC)、透明细胞肾细胞癌(RCC)、头和颈癌、咽喉癌、肝胆癌(hepatobiliary cancer)、中枢神经系统癌、食管癌、恶性胸膜间皮瘤、全身性轻链淀粉样变性、淋巴浆细胞性淋巴瘤(lymphoplasmacytic lymphoma)、骨髓异常增生综合征、骨髓增生性肿瘤、神经内分泌肿瘤、梅克尔细胞癌、睾丸癌和皮肤癌。
  17. 作为药物的如权利要求1至7任一项所述的单克隆抗体或其抗原结合片段，或包含权利要求8所述的药物组合物，或权利要求9所述的核酸分子。
  18. 作为药物的如权利要求1至7任一项所述的单克隆抗体或其抗原结合片段，或包含权利要求8所述的药物组合物，或权利要求9所述的核酸分子，所述药物用于治疗PD-L1阳性的肿瘤或癌症，优选地，所述癌症选自鳞状细胞癌、骨髓瘤、小细胞肺癌、非小细胞肺癌(NSCLC)、头和颈鳞状细胞癌(HNSCC)、神经胶质瘤、何杰金淋巴瘤、 非何杰金淋巴瘤、弥漫性大B-细胞淋巴瘤(DLBCL)、滤泡性淋巴瘤、急性成淋巴细胞性白血病(ALL)、急性髓细胞样白血病(AML)、慢性淋巴细胞性白血病(CLL)、慢性髓细胞样白血病(CML)、原发性纵隔大B-细胞淋巴瘤、套细胞淋巴瘤(MCL)、小淋巴细胞性淋巴瘤(SLL)、富含T-细胞/组织细胞的大B-细胞淋巴瘤、多发性骨髓瘤、髓样细胞白血病-1蛋白(Mcl-1)、骨髓异常增生综合征(MDS)、胃肠(道)癌、肾癌、卵巢癌、肝癌、成淋巴细胞性白血病、淋巴细胞白血病、结肠直肠癌、子宫内膜癌、肾癌、前列腺癌、甲状腺癌、黑素瘤、软骨肉瘤、神经母细胞瘤、胰腺癌、多形性成胶质细胞瘤、胃癌、骨癌、尤因氏肉瘤、子宫颈癌、脑癌、胃癌、膀胱癌、肝细胞瘤、乳腺癌、结肠癌、肝细胞癌(HCC)、透明细胞肾细胞癌(RCC)、头和颈癌、咽喉癌、肝胆癌(hepatobiliary cancer)、中枢神经系统癌、食管癌、恶性胸膜间皮瘤、全身性轻链淀粉样变性、淋巴浆细胞性淋巴瘤(lymphoplasmacytic lymphoma)、骨髓异常增生综合征、骨髓增生性肿瘤、神经内分泌肿瘤、梅克尔细胞癌、睾丸癌和皮肤癌。
PCT/CN2019/070982 2018-01-10 2019-01-09 Pd-l1抗体、其抗原结合片段及医药用途 Ceased WO2019137397A1 (zh)

Priority Applications (11)

Application Number Priority Date Filing Date Title
KR1020207022689A KR20200108306A (ko) 2018-01-10 2019-01-09 Pd-l1 항체 또는 이의 항원-결합 단편, 및 이의 약학적 용도
CA3087105A CA3087105A1 (en) 2018-01-10 2019-01-09 Pd-l1 antibody, antigen-binding fragment thereof, and pharmaceutical use thereof
EP19738076.9A EP3741777A4 (en) 2018-01-10 2019-01-09 PD-L1 ANTIBODIES, ANTIGIBODY FRAGMENT THEREOF, AND PHARMACEUTICAL USE THEREOF
US16/960,297 US11359021B2 (en) 2018-01-10 2019-01-09 PD-L1 antibody, antigen-binding fragment thereof, and pharmaceutical use thereof
JP2020538638A JP2021510078A (ja) 2018-01-10 2019-01-09 Ｐｄ−ｌ１抗体、その抗原結合フラグメント、及びその製薬学的使用
AU2019206958A AU2019206958A1 (en) 2018-01-10 2019-01-09 PD-L1 antibody, antigen-binding fragment thereof, and pharmaceutical use thereof
CN201980005432.2A CN111278861B (zh) 2018-01-10 2019-01-09 Pd-l1抗体、其抗原结合片段及医药用途
BR112020013475-9A BR112020013475A2 (pt) 2018-01-10 2019-01-09 Anticorpo de pd-l1, fragmento de ligação ao antígeno do mesmo, e uso farmacêutico do mesmo
MX2020007406A MX2020007406A (es) 2018-01-10 2019-01-09 Anticuerpo pd-l1, fragmento de union al antigeno del mismo y uso farmaceutico del mismo.
RU2020124261A RU2778085C2 (ru) 2018-01-10 2019-01-09 Антитело к pd-l1, его антигенсвязывающий фрагмент и их фармацевтическое применение
ZA2020/04907A ZA202004907B (en) 2018-01-10 2020-08-07 Pd-l1 antibody, antigen-binding fragment thereof, and pharmaceutical use thereof

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201810023267.0 2018-01-10
CN201810023267 2018-01-10

Publications (1)

Publication Number Publication Date
WO2019137397A1 true WO2019137397A1 (zh) 2019-07-18

Family

ID=67219311

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2019/070982 Ceased WO2019137397A1 (zh) 2018-01-10 2019-01-09 Pd-l1抗体、其抗原结合片段及医药用途

Country Status (12)

Country Link
US (1) US11359021B2 (zh)
EP (1) EP3741777A4 (zh)
JP (1) JP2021510078A (zh)
KR (1) KR20200108306A (zh)
CN (1) CN111278861B (zh)
AU (1) AU2019206958A1 (zh)
BR (1) BR112020013475A2 (zh)
CA (1) CA3087105A1 (zh)
MX (1) MX2020007406A (zh)
TW (1) TWI717678B (zh)
WO (1) WO2019137397A1 (zh)
ZA (1) ZA202004907B (zh)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022121846A1 (zh) * 2020-12-08 2022-06-16 博际生物医药科技(杭州)有限公司 Pd-l1抗体及其应用
CN115181180A (zh) * 2021-03-03 2022-10-14 山东先声生物制药有限公司 抗人程序性死亡配体-1（pd-l1）的抗体及其应用
WO2023001025A1 (zh) * 2021-07-23 2023-01-26 南京吉盛澳玛生物医药有限公司 Pd-l1抗体及其用途
CN115925937A (zh) * 2022-08-05 2023-04-07 赛灵药业科技集团股份有限公司北京分公司 一种抗体或其片段及其制药用途
WO2025201522A1 (zh) * 2024-03-29 2025-10-02 江苏恒瑞医药股份有限公司 抗Nectin-4抗体药物偶联物治疗肿瘤
WO2025252203A1 (zh) * 2024-06-06 2025-12-11 江苏恒瑞医药股份有限公司 抗Nectin-4抗体药物偶联物治疗肿瘤的用途

Families Citing this family (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA3004804A1 (en) * 2015-11-17 2017-05-26 Jiangsu Hengrui Medicine Co., Ltd. Pd-l1 antibody, antigen-binding fragment thereof and medical application thereof
CN110760517B (zh) * 2019-10-09 2022-04-29 天津大学 拮抗pd-1骆驼抗体类似物ap基因及蛋白和应用
TWI789679B (zh) * 2021-01-12 2023-01-11 大陸商江蘇先聲藥業有限公司 抗人程序性死亡配體-1（pd-l1）的抗體及其用途
WO2023046113A1 (zh) * 2021-09-24 2023-03-30 广东菲鹏制药股份有限公司 一种抗人pd-l1人源化抗体或其抗原结合片段及其应用
CN115785269B (zh) * 2022-11-01 2023-09-22 四川大学 抗pd-l1的抗体及其应用
CN120081942A (zh) * 2024-04-16 2025-06-03 赛业（苏州）生物科技有限公司 一种pd-l1抗体及其制备方法和应用
CN119954956B (zh) * 2025-02-05 2025-08-22 可蓝赛生物医药(上海)有限公司 一种nk细胞的制备方法及其在治疗癌症中的应用

Citations (21)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4683195A (en) 1986-01-30 1987-07-28 Cetus Corporation Process for amplifying, detecting, and/or-cloning nucleic acid sequences
WO2001039722A2 (en) 1999-11-30 2001-06-07 Mayo Foundation For Medical Education And Research B7-h1, a novel immunoregulatory molecule
US6803192B1 (en) 1999-11-30 2004-10-12 Mayo Foundation For Medical Education And Research B7-H1, a novel immunoregulatory molecule
CN101104640A (zh) * 2006-07-10 2008-01-16 苏州大学 抗人pd-l1单克隆抗体制备及应用
WO2010089411A2 (en) 2009-02-09 2010-08-12 Universite De La Mediterranee Pd-1 antibodies and pd-l1 antibodies and uses thereof
WO2013173223A1 (en) 2012-05-15 2013-11-21 Bristol-Myers Squibb Company Cancer immunotherapy by disrupting pd-1/pd-l1 signaling
WO2013181634A2 (en) 2012-05-31 2013-12-05 Sorrento Therapeutics Inc. Antigen binding proteins that bind pd-l1
US8617546B2 (en) 2008-10-02 2013-12-31 Seoul National University Industry Foundation Anticancer agent comprising anti-PD-1 antibody or anti-PD-L1 antibody
WO2014022758A1 (en) 2012-08-03 2014-02-06 Dana-Farber Cancer Institute, Inc. Single agent anti-pd-l1 and pd-l2 dual binding antibodies and methods of use
WO2014055897A2 (en) 2012-10-04 2014-04-10 Dana-Farber Cancer Institute, Inc. Human monoclonal anti-pd-l1 antibodies and methods of use
WO2014100079A1 (en) 2012-12-21 2014-06-26 Merck Sharp & Dohme Corp. Antibodies that bind to human programmed death ligand 1 (pd-l1)
WO2014151006A2 (en) * 2013-03-15 2014-09-25 Genentech, Inc. Biomarkers and methods of treating pd-1 and pd-l1 related conditions
US20140341917A1 (en) 2011-11-28 2014-11-20 Merck Patent Gmbh Anti-pd-l1 antibodies and uses thereof
WO2014195852A1 (en) 2013-06-03 2014-12-11 Glaxosmithkline Intellectual Property (No.2) Limited Combinations of an anti-pd-l1 antibody and a mek inhibitor and/or a braf inhibitor
WO2015036511A1 (en) 2013-09-12 2015-03-19 F. Hoffmann-La Roche Ag Combination therapy of antibodies against human csf-1r and antibodies against human pd-l1
WO2015048520A1 (en) 2013-09-27 2015-04-02 Genentech, Inc. Anti-pdl1 antibody formulations
CN105777906A (zh) * 2014-12-19 2016-07-20 苏州丁孚靶点生物技术有限公司 抗pd-l1全人抗体及其应用
CN106243225A (zh) * 2015-06-11 2016-12-21 智翔（上海）医药科技有限公司 新型抗-pd-l1抗体
CN106699891A (zh) * 2017-01-25 2017-05-24 北京天广实生物技术股份有限公司 一种抗pd‑l1 抗体、其药物组合物及其用途
WO2017084495A1 (zh) 2015-11-17 2017-05-26 江苏恒瑞医药股份有限公司 Pd-l1抗体、其抗原结合片段及其医药用途
WO2017196867A1 (en) * 2016-05-09 2017-11-16 Igm Biosciences, Inc. Anti-pd-l1 antibodies

Family Cites Families (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2612241C (en) * 2005-07-01 2018-11-06 Medarex, Inc. Human monoclonal antibodies to programmed death ligand 1 (pd-l1)
PL2342226T3 (pl) * 2008-09-26 2017-01-31 Dana-Farber Cancer Institute, Inc. Ludzkie przeciwciała anty-PD-1, PD-L1 i PD-L2 oraz ich zastosowania
EP4209510B1 (en) * 2008-12-09 2024-01-31 F. Hoffmann-La Roche AG Anti-pd-l1 antibodies and their use to enhance t-cell function
NO2504364T3 (zh) * 2009-11-24 2018-01-06
JP6909153B2 (ja) * 2014-08-05 2021-07-28 アポロミクス インコーポレイテッド 抗ｐｄ−ｌ１抗体
CN108112254B (zh) 2015-03-13 2022-01-28 西托姆克斯治疗公司 抗-pdl1抗体、可活化的抗-pdl1抗体、及其使用方法
CN107216389B (zh) * 2016-03-18 2022-03-29 和迈生物科技有限公司 抗pd-l1纳米抗体及其编码序列和用途
CN107365383B (zh) * 2017-07-12 2019-04-30 苏州大学附属第一医院 一种人源化抗人pd-1单克隆抗体及其制备方法、用途与药物
WO2019129136A1 (zh) 2017-12-27 2019-07-04 信达生物制药(苏州)有限公司 抗pd-l1抗体及其用途

Patent Citations (23)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4683195B1 (zh) 1986-01-30 1990-11-27 Cetus Corp
US4683195A (en) 1986-01-30 1987-07-28 Cetus Corporation Process for amplifying, detecting, and/or-cloning nucleic acid sequences
WO2001039722A2 (en) 1999-11-30 2001-06-07 Mayo Foundation For Medical Education And Research B7-h1, a novel immunoregulatory molecule
US6803192B1 (en) 1999-11-30 2004-10-12 Mayo Foundation For Medical Education And Research B7-H1, a novel immunoregulatory molecule
CN101104640A (zh) * 2006-07-10 2008-01-16 苏州大学 抗人pd-l1单克隆抗体制备及应用
US8617546B2 (en) 2008-10-02 2013-12-31 Seoul National University Industry Foundation Anticancer agent comprising anti-PD-1 antibody or anti-PD-L1 antibody
US20140335093A1 (en) 2009-02-09 2014-11-13 Universite De La Mediterranee PD-1 Antibodies and PD-L1 Antibodies and Uses Thereof
WO2010089411A2 (en) 2009-02-09 2010-08-12 Universite De La Mediterranee Pd-1 antibodies and pd-l1 antibodies and uses thereof
US20140341917A1 (en) 2011-11-28 2014-11-20 Merck Patent Gmbh Anti-pd-l1 antibodies and uses thereof
WO2013173223A1 (en) 2012-05-15 2013-11-21 Bristol-Myers Squibb Company Cancer immunotherapy by disrupting pd-1/pd-l1 signaling
WO2013181634A2 (en) 2012-05-31 2013-12-05 Sorrento Therapeutics Inc. Antigen binding proteins that bind pd-l1
WO2014022758A1 (en) 2012-08-03 2014-02-06 Dana-Farber Cancer Institute, Inc. Single agent anti-pd-l1 and pd-l2 dual binding antibodies and methods of use
WO2014055897A2 (en) 2012-10-04 2014-04-10 Dana-Farber Cancer Institute, Inc. Human monoclonal anti-pd-l1 antibodies and methods of use
WO2014100079A1 (en) 2012-12-21 2014-06-26 Merck Sharp & Dohme Corp. Antibodies that bind to human programmed death ligand 1 (pd-l1)
WO2014151006A2 (en) * 2013-03-15 2014-09-25 Genentech, Inc. Biomarkers and methods of treating pd-1 and pd-l1 related conditions
WO2014195852A1 (en) 2013-06-03 2014-12-11 Glaxosmithkline Intellectual Property (No.2) Limited Combinations of an anti-pd-l1 antibody and a mek inhibitor and/or a braf inhibitor
WO2015036511A1 (en) 2013-09-12 2015-03-19 F. Hoffmann-La Roche Ag Combination therapy of antibodies against human csf-1r and antibodies against human pd-l1
WO2015048520A1 (en) 2013-09-27 2015-04-02 Genentech, Inc. Anti-pdl1 antibody formulations
CN105777906A (zh) * 2014-12-19 2016-07-20 苏州丁孚靶点生物技术有限公司 抗pd-l1全人抗体及其应用
CN106243225A (zh) * 2015-06-11 2016-12-21 智翔（上海）医药科技有限公司 新型抗-pd-l1抗体
WO2017084495A1 (zh) 2015-11-17 2017-05-26 江苏恒瑞医药股份有限公司 Pd-l1抗体、其抗原结合片段及其医药用途
WO2017196867A1 (en) * 2016-05-09 2017-11-16 Igm Biosciences, Inc. Anti-pd-l1 antibodies
CN106699891A (zh) * 2017-01-25 2017-05-24 北京天广实生物技术股份有限公司 一种抗pd‑l1 抗体、其药物组合物及其用途

Non-Patent Citations (25)

* Cited by examiner, † Cited by third party
Title
"Epitope Mapping Protocols in Methods in Molecular Biology", vol. 66, 1996
"PCR TECHNOLOGY", 1989, STOCKTON PRESS
"WHO Drug Information", vol. 28, 2014, GENETECH, article "Atezolizumab", pages: 488
ALFTHAN ET AL., PROTEIN ENG., vol. 8, 1995, pages 725 - 731
BIRD ET AL., SCIENCE, vol. 242, 1988, pages 423 - 426
CHEUNG ET AL., VIROLOGY, vol. l76, 1990, pages 546 - 552
CHOI ET AL., EUR.J.IMMUNO, vol. 1.31, 2001, pages 94 - 106
HAMANISHI, J.: "Safety and Antitumor Activity of Anti - PD-1 Antibody, Nivolumab, in Patients With Platinum-Resistant Ovarian Cancer", JOURNAL OF CLINICAL ONCOLOGY, vol. 33, no. 34, 1 December 2015 (2015-12-01), pages 4015 - 4022, XP055625077 *
HOLLIGER ET AL., PROC. NATL. ACAD. SCI. USA, vol. 90, 1993, pages 6444 - 6448
HU ET AL., CANCER RES., vol. 56, 1996, pages 3055 - 3061
HUSTON ET AL., PROC. NATL. ACAD. SCI USA, vol. 85, 1988, pages 5879 - 5883
J. BIOL. CHEM, vol. 243, 1968, pages 3558
JOURNAL OF IMMUNOGLOBULINS, 2001, ISBN: 012441351
KABAT E.A. ET AL.: "Sequences of proteins of immunological interest", 1991, NIH
KIPRIYANOV ET AL., J. MOL. BIOL., vol. 293, 1999, pages 41 - 56
KIRKLAND ET AL., J. IMMUNOL., vol. 137, 1986, pages 3614 - 3619
MOLDENHAUER ET AL., SCAND.J.IMMUNOL., vol. 32, 1990, pages 77 - 82
MOREL ET AL., MOLEC. IMMUNOL., vol. 25, 1988, pages 7 - 15
MULLIS ET AL., COLD SPRING HARBOR SYMP. OUANT. BIOL., vol. 51, 1987, pages 263
PROTEIN ENGINEERING, vol. 7, 1994, pages 697
ROOVERS ET AL., CANCER IMMUNOL., 2001
See also references of EP3741777A4
STAHLI ET AL., METHODSIN ENZYMOLOGY, vol. 9, 1983, pages 242 - 253
WARD ET AL., NATURE, vol. 341, 1989, pages 544 - 546
ZHAN, M.M.: "From monoclonal antibodies to small molecules: the develo- pment of inhibitors targeting the PD-1/PD-L1 pathway", DRUG DISCOVERY TODAY, vol. 21, no. 6, 16 April 2016 (2016-04-16), pages 1027 - 1036, XP029565582 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022121846A1 (zh) * 2020-12-08 2022-06-16 博际生物医药科技(杭州)有限公司 Pd-l1抗体及其应用
CN115181180A (zh) * 2021-03-03 2022-10-14 山东先声生物制药有限公司 抗人程序性死亡配体-1（pd-l1）的抗体及其应用
WO2023001025A1 (zh) * 2021-07-23 2023-01-26 南京吉盛澳玛生物医药有限公司 Pd-l1抗体及其用途
CN115925937A (zh) * 2022-08-05 2023-04-07 赛灵药业科技集团股份有限公司北京分公司 一种抗体或其片段及其制药用途
WO2025201522A1 (zh) * 2024-03-29 2025-10-02 江苏恒瑞医药股份有限公司 抗Nectin-4抗体药物偶联物治疗肿瘤
WO2025252203A1 (zh) * 2024-06-06 2025-12-11 江苏恒瑞医药股份有限公司 抗Nectin-4抗体药物偶联物治疗肿瘤的用途

Also Published As

Publication number Publication date
RU2020124261A (ru) 2022-01-25
CN111278861A (zh) 2020-06-12
EP3741777A1 (en) 2020-11-25
TW201930360A (zh) 2019-08-01
CN111278861B (zh) 2022-05-27
MX2020007406A (es) 2020-09-14
US11359021B2 (en) 2022-06-14
JP2021510078A (ja) 2021-04-15
US20200339692A1 (en) 2020-10-29
KR20200108306A (ko) 2020-09-17
CA3087105A1 (en) 2019-07-18
AU2019206958A1 (en) 2020-07-16
TWI717678B (zh) 2021-02-01
ZA202004907B (en) 2022-10-26
BR112020013475A2 (pt) 2020-12-08
EP3741777A4 (en) 2021-10-13
RU2020124261A3 (zh) 2022-01-25

Similar Documents

Publication Publication Date Title
US11365255B2 (en) PD-1 antibody, antigen-binding fragment thereof, and medical application thereof
TWI717678B (zh) Pd-l1抗體、其抗原結合片段及醫藥用途
JP7317272B2 (ja) Ｔｉｇｉｔ抗体、その抗原結合断片及びその医療用途 本願は、２０１９年９月２９日に出願された出願番号ｃｎ２０１７１０９０８５６５．３に基づいたものであり、その優先権を主張する。その開示は、その全体が参照により本明細書に組み込まれる。
TWI758558B (zh) Cd96抗體、其抗原結合片段及醫藥用途
TWI843799B (zh) 抗pd-1抗體、其抗原結合片段及醫藥用途
TWI699376B (zh) 抗cd40抗體、其抗原結合片段及其醫藥用途
WO2017084495A1 (zh) Pd-l1抗体、其抗原结合片段及其医药用途
CN110790839B (zh) 抗pd-1抗体、其抗原结合片段及医药用途
JP2020508658A (ja) Ｔｉｍ−３抗体、その抗原結合断片、及びそれらの医学的使用
JP2022502417A (ja) 抗ｏｘ４０抗体、その抗原結合フラグメント、および医薬用途
WO2021098822A1 (zh) 一种双特异性抗体
WO2019076277A1 (zh) 抗pd-1抗体和抗lag-3抗体联合在制备治疗肿瘤的药物中的用途
WO2021209066A1 (zh) 特异性抗原结合分子，其制备方法及医药用途
RU2807484C2 (ru) Антитело к pd-1, его антигенсвязывающий фрагмент и его фармацевтическое применение
HK40051455A (zh) 抗pd-1抗体、其抗原结合片段及医药用途
HK40032196B (zh) Pd-l1抗体、其抗原结合片段及医药用途
HK40032196A (zh) Pd-l1抗体、其抗原结合片段及医药用途

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: 19738076

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 3087105

Country of ref document: CA

ENP Entry into the national phase

Ref document number: 2020538638

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2019206958

Country of ref document: AU

Date of ref document: 20190109

Kind code of ref document: A

ENP Entry into the national phase

Ref document number: 20207022689

Country of ref document: KR

Kind code of ref document: A

ENP Entry into the national phase

Ref document number: 2019738076

Country of ref document: EP

Effective date: 20200810

REG Reference to national code

Ref country code: BR

Ref legal event code: B01A

Ref document number: 112020013475

Country of ref document: BR

ENP Entry into the national phase

Ref document number: 112020013475

Country of ref document: BR

Kind code of ref document: A2

Effective date: 20200630