WO2018026249A1 - Anticorps dirigé contre le ligand 1 de mort programmée (pd-l1) et son utilisation - Google Patents

Anticorps dirigé contre le ligand 1 de mort programmée (pd-l1) et son utilisation Download PDF

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WO2018026249A1
WO2018026249A1 PCT/KR2017/008495 KR2017008495W WO2018026249A1 WO 2018026249 A1 WO2018026249 A1 WO 2018026249A1 KR 2017008495 W KR2017008495 W KR 2017008495W WO 2018026249 A1 WO2018026249 A1 WO 2018026249A1
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seq
light chain
variable region
heavy chain
chain variable
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PCT/KR2017/008495
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English (en)
Korean (ko)
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박재은
최수아
이지수
이현미
이시형
백기선
김응철
박범찬
임정채
조영규
박영우
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주식회사 와이바이오로직스
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Priority to JP2019528010A priority Critical patent/JP6925421B2/ja
Priority to AU2017306507A priority patent/AU2017306507B2/en
Priority to CA3032806A priority patent/CA3032806C/fr
Priority to BR112019002282A priority patent/BR112019002282A2/pt
Priority to CN201780055412.7A priority patent/CN110072889B/zh
Priority to EP17837300.7A priority patent/EP3495391A4/fr
Priority to RU2019105664A priority patent/RU2721582C1/ru
Priority to US16/321,412 priority patent/US10919966B2/en
Priority claimed from KR1020170099673A external-priority patent/KR102048477B1/ko
Publication of WO2018026249A1 publication Critical patent/WO2018026249A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants

Definitions

  • the present invention provides an antibody or antigen-binding fragment thereof for PD-L1 (Programmed death-ligand 1), a nucleic acid encoding the same, a vector comprising the nucleic acid, a cell transformed with the vector, the antibody or an antigen-binding fragment thereof. It relates to a manufacturing method and a composition for preventing or treating cancer or infectious disease comprising the same.
  • T-cell antigen receptors present on the surface of T-lymphocyte cells be the major histocompatibility complex (MHC) of antigen presenting cells (APCs). It begins by recognizing antigen bound to a human leucocyte antigen (HLA) Class II molecule, which requires co-stimulatory signals simultaneously with the recognition of the antigen in order for T-lymphocytes to be fully activated.
  • HLA human leucocyte antigen
  • CD80, CD40, and the like are combined with CD28, CD40L, etc., which correspond to ligands on the surface of T-lymphocytes, thereby activating the secretion of cytokines, and recognition of the antigen through the binding of TCR-MHC / epitopes. Even if there is no signal transmission of the simultaneous stimulus signal, the activity of T-lymphocytes is not achieved.
  • activated T-lymphocytes also activate a co-inhibitory signal at the same time to become inactive after a certain time. This can prevent tissue damage due to excessive immune stimulation.
  • CTLA-4 cytotoxic T lymphocyte antigen
  • PD-1 programmed death-1
  • -ligand 1 corresponding antigen-presenting cell ligands of T-lymphocytes.
  • CTLA-4 has the inactivation function of naive or memory T-lymphocytes by binding to ligands CD80 and CD86, and PD-1 regulates T-lymphocyte function in peripheral tissues through PD-L1 and PD-L2. do.
  • the body's immune function regulates T lymphocyte function through the regulation of co-stimulatory and co-inhibition signals as well as antigen recognition. This regulatory mechanism is called an immunocheckpoint.
  • the immune function of our body detects tumor-specific neo-antigens expressed by mutations such as mutations occurring in tumor cells and thereby removes tumor cells or viral infectious agents.
  • One such avoidance strategy is to inhibit the function of tumor specific T lymphocyte cells through alteration of immune checkpoint function. That is, by activating such inhibitory immunoassay in tumor cells, the attack of tumor specific T-lymphocyte cells is avoided.
  • antitumor effects can be obtained by enhancing the tumor specific T-lymphocyte cell activity and effects inhibited by inhibiting its function using monoclonal antibodies against PD-1 or ligand PD-L1.
  • the inventors of the present application tried to develop an antibody that specifically binds to PD-L1.
  • the present inventors have developed an anti-PD-L1 antibody that binds PD-L1 with high affinity, and this anti-PD-L1 antibody inhibits the formation of the PD-1 / PD-L1 complex, resulting in the desired immunity.
  • the present invention was confirmed that it can play a role of an anticancer agent or a therapeutic agent for infectious diseases.
  • Another object of the present invention is to provide a nucleic acid encoding the antibody or antigen-binding fragment thereof.
  • Another object of the present invention is to provide a vector comprising the nucleic acid, a cell transformed with the vector, and a method of manufacturing the same.
  • Still another object of the present invention is to provide a composition for preventing or treating cancer or infectious disease comprising the antibody or antigen-binding fragment thereof.
  • the present invention is a group consisting of heavy chain CDR1, SEQ ID NO: 8 to SEQ ID NO: 15 comprising a sequence having a sequence homology of 90% or more with a sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO:
  • a heavy chain CDR2 comprising a sequence having at least 90% sequence homology with a sequence selected from among and a heavy chain CDR3 comprising a sequence having at least 90% sequence homology with a sequence selected from the group consisting of SEQ ID NOs: 16 to 25
  • a heavy chain variable region comprising a light chain and a light chain CDR1 comprising a sequence having at least 90% sequence homology with a sequence selected from the group consisting of SEQ ID NOs: 88 to SEQ ID NO: 102, SEQ ID NO: 103 to SEQ ID NO: 119
  • a light chain CDR2 comprising a sequence having 90% or more sequence homology with a sequence to be sequenced, and selected from the group consisting of SEQ ID NOs: 120
  • the present invention also provides a nucleic acid encoding the antibody or antigen-binding fragment thereof.
  • the present invention also provides a vector comprising the nucleic acid.
  • the present invention also provides a cell transformed with the vector.
  • the present invention also provides a method for producing the antibody or antigen-binding fragment thereof comprising the following steps: (a) culturing the cells; And (b) recovering the antibody or antigen-binding fragment thereof from the cultured cells.
  • the present invention also provides a composition for preventing or treating cancer or infectious disease comprising the antibody or antigen-binding fragment thereof as an active ingredient.
  • FIG. 1 is a schematic diagram of a PD-L1 expression vector.
  • 2A is a 10% SDS-PAGE gel of PD-L1-hFc. Results of protein identification under RE (reducing) and NR (non-reducing) conditions;
  • Figure 2b shows the G-3000 SWXL SEC-HPLC results. Flow rate is 1 ml / min and developing solvent is PBS;
  • 2C is a 10% SDS-PAGE gel of PD-L1-mFc. Results of protein identification under RE (reducing) and NR (non-reducing) conditions;
  • Figure 2d shows the G-3000 SWXL SEC-HPLC results.
  • the flow rate is 1 ml / min and the developing solvent is PBS.
  • Figure 3 shows the result of increasing the binding force for PD-L1 antigen according to the number of panning.
  • Figure 4 shows the ELSIA results for measuring the binding capacity of monophages strong binding capacity only PD-L1-His.
  • Figure 5 shows the result of confirming the selected PD-L1 antibody through SDS-PAGE.
  • Figure 6 shows the results of in vitro efficacy evaluation of PD-L1 antibody.
  • Figure 7 shows the results of the concentration-dependent in vitro efficacy evaluation of PD-L1 antibody.
  • Figure 8 shows the results of measuring the binding force of the PD-L1 antibody in PD-L1 overexpressing cells.
  • Figure 9 shows the results of the kinetic (kinetics) measurement between PD-L1-hFc and PD-L1-16E12.
  • Figure 11 shows the results of evaluating the in vitro efficacy of the PD-L1 antibody according to the present invention.
  • Figure 12 shows the results of evaluating the concentration-dependent in vitro efficacy of the PD-L1 antibody according to the present invention.
  • Figure 13 shows the results of measuring the binding force of the antibody in PD-L1 overexpressing cells.
  • Figure 14 shows the results confirming the inhibitory effect of the antibody preventing the formation of PD-1 / PD-L1 complex using enzyme immunosorbent in the selected antibody.
  • FIG. 15 shows kinetic measurements of PD-L1-hFc and PD-L1-16E12-4F5.
  • Figure 16 shows the binding measurement results of PD-L1 variant protein and monoclonal antibody.
  • Figure 17 shows that the PD-L1 monoclonal antibody shows an increase in activity in heterologous MLR (Mixed Lymphocyte Reaction).
  • Figure 19 shows the results confirming the binding of the anti-PD-L1 antibody according to the invention with PD-L2.
  • the present invention is a heavy chain CDR1 comprising a sequence having at least 90% sequence homology with a sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 7 selected from the group consisting of SEQ ID NO: 8 to SEQ ID NO: 15
  • a heavy chain CDR2 comprising a sequence having at least 90% sequence homology with the sequence
  • a heavy chain CDR3 comprising a sequence having at least 90% sequence homology with a sequence selected from the group consisting of SEQ ID NOs: 16-25
  • a light chain CDR1 comprising a heavy chain variable region and a sequence having 90% or more sequence homology with a sequence selected from the group consisting of SEQ ID NOs: 88 to 102, and a sequence selected from the group consisting of SEQ ID NOs: 103 to 119; 90% or more of a light chain CDR2 comprising a sequence having at least 90% sequence homology, and a sequence selected from the group consisting of SEQ ID NOs: 120 to 144
  • PD-L1 as used herein is a ligand for the immunosuppressive receptor “programmed death receptor 1 (PD-1)” that is primarily expressed on activated T and B cells, and PD-1 and ligand PD-L1 and / or Or when PD-L2 binds, negatively regulates antigen receptor signaling.
  • Ligands for PD-1 may be constitutively expressed or induced into a number of cell types, including non-hematopoietic tissue and various tumor types.
  • PD-L1 is expressed on B cells, T cells, bone marrow cells and dendritic cells (DCs), but also on peripheral cells, like microvascular endothelial cells and non-lymphoid organs such as heart, lung and the like.
  • DCs dendritic cells
  • PD-L2 is found only on macrophages and dendritic cells.
  • the expression pattern of PD-1 ligand suggests the role of PD-1 in maintaining peripheral tolerance and may contribute to regulating auto-reactive T-cell and B-cell responses in the peripheral.
  • Both ligands are type I mobile membrane receptors that contain both IgV- and IgC-like domains in the extracellular domain. Both ligands comprise short cytoplasmic regions with unknown signaling motifs.
  • antibody refers to an anti-PD-L1 antibody that specifically binds to PD-L1.
  • the scope of the present invention includes not only complete antibody forms that specifically bind PD-L1, but also antigen binding fragments of such antibody molecules.
  • a complete antibody is a structure having two full length light chains and two full length heavy chains, each of which is linked by heavy and disulfide bonds.
  • the heavy chain constant region has gamma ( ⁇ ), mu ( ⁇ ), alpha ( ⁇ ), delta ( ⁇ ) and epsilon ( ⁇ ) types and subclasses gamma 1 ( ⁇ 1), gamma 2 ( ⁇ 2), and gamma 3 ( ⁇ 3). ), Gamma 4 ( ⁇ 4), alpha 1 ( ⁇ 1) and alpha 2 ( ⁇ 2).
  • the constant regions of the light chains have kappa ( ⁇ ) and lambda ( ⁇ ) types.
  • An antigen binding fragment or antibody fragment of an antibody means a fragment having an antigen binding function and includes Fab, F (ab '), F (ab') 2, Fv and the like.
  • Fab in the antibody fragment has a structure having a variable region of the light and heavy chains, a constant region of the light chain and the first constant region (CH1) of the heavy chain has one antigen binding site.
  • Fab ′ is a hinge region comprising one or more cysteine residues at the C-terminus of the heavy chain CH1 domain
  • F (ab ') 2 antibodies are produced by disulfide bonds of cysteine residues in the hinge region of Fab'.
  • Recombinant techniques for generating Fv fragments with minimal antibody fragments in which Fv has only heavy and light chain variable regions are described in PCT International Publication Nos. WO88 / 10649, WO88 / 106630, WO88 / 07085, WO88 / 07086 and WO88 / 09344. Is disclosed.
  • Double-chain Fv is a non-covalent bond in which a heavy chain variable region and a light chain variable region are linked, and a single chain Fv (single-chain Fv, scFv) is generally a variable region of the heavy chain and the light chain through a peptide linker. This covalent linkage or the C-terminus is directly linked to form a dimer-like structure such as a double-chain Fv.
  • Such antibody fragments can be obtained using proteolytic enzymes (e.g., restriction digestion of the entire antibody with papain yields Fab and cleavage with pepsin yields F (ab ') 2 fragments). It can also be produced by recombinant technology.
  • the antibody according to the invention is in Fv form (eg scFv) or is in the form of a complete antibody.
  • the heavy chain constant region may be selected from any one isotype of gamma ( ⁇ ), mu ( ⁇ ), alpha ( ⁇ ), delta ( ⁇ ) or epsilon ( ⁇ ).
  • the constant region is gamma 1 (IgG1), gamma 3 (IgG3) or gamma 4 (IgG4).
  • the light chain constant region may be of kappa or lambda type.
  • variable region domain V H variable region domain
  • constant region domains CH1, CH2 and CH3 constant region domains
  • amino acid sequence having sufficient variable region sequence to confer specificity to the antigen Both heavy chain and fragments thereof.
  • light chain refers to a full-length light chain and fragment thereof comprising a variable region domain V L and a constant region domain CL comprising an amino acid sequence having sufficient variable region sequence to confer specificity to the antigen. Means all.
  • Antibodies of the invention include monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, single chain Fvs (scFV), single chain antibodies, Fab fragments, F (ab ') fragments, disulfide-binding Fvs (sdFV) And anti-idiotype (anti-Id) antibodies, or epitope-binding fragments of the antibodies, and the like.
  • Said monoclonal antibody refers to the same except for possible naturally occurring mutations in which antibodies obtained from substantially homogeneous antibody populations, ie, individual antibodies in the population, may be present in trace amounts.
  • Monoclonal antibodies are highly specific and are directed against a single antigenic site. In contrast to conventional (polyclonal) antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen.
  • Epitope refers to a protein determinant to which an antibody can specifically bind.
  • Epitopes usually consist of a group of chemically active surface molecules, such as amino acids or sugar side chains, and generally have specific three dimensional structural characteristics as well as specific charge characteristics. Three-dimensional epitopes and non-stereo epitopes are distinguished in that the binding to the former is lost but not to the latter in the presence of a denatured solvent.
  • Non-human (eg murine) antibodies of the “humanized” form are chimeric antibodies that contain minimal sequences derived from non-human immunoglobulins.
  • humanized antibodies are non-human species (donor antibodies) that retain the desired specificity, affinity, and capacity for residues from the hypervariable region of the recipient, for example mice, rats, rabbits, or non-humans.
  • donor antibodies non-human species
  • Human immunoglobulins (receptor antibodies) replaced with residues from the hypervariable regions of primates.
  • human antibody refers to a molecule derived from human immunoglobulin, in which all amino acid sequences constituting the antibody including complementarity determining regions and structural regions are composed of human immunoglobulins.
  • While the heavy and / or light chain portions are the same or homologous to the corresponding sequences in an antibody derived from a particular species or belonging to a particular antibody class or subclass, the remaining chain (s) are derived from another species or another antibody class or Included are "chimeric" antibodies (immunoglobulins) that are identical or homologous to the corresponding sequences in antibodies belonging to the subclass, as well as fragments of such antibodies that exhibit the desired biological activity.
  • antibody variable domain refers to the light and heavy chain portions of an antibody molecule comprising the amino acid sequences of complementarity determining regions (CDRs; ie CDR1, CDR2, and CDR3), and framework regions (FR). .
  • CDRs complementarity determining regions
  • FR framework regions
  • V H refers to the variable domain of the heavy chain.
  • V L refers to the variable domain of the light chain.
  • CDRs Complementarity Determining Regions
  • the present invention includes a heavy chain variable region comprising a heavy chain CDR3 of SEQ ID NO: 1 and a light chain variable region comprising a light chain CDR3 of SEQ ID NO: 2.
  • the antibody or antigen-binding fragment thereof that binds to PD-L1 is a heavy chain CDR1 selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 7, heavy chain CDR2 selected from the group consisting of SEQ ID NO: 8 to SEQ ID NO: 15 And a heavy chain variable region comprising a heavy chain CDR3 selected from the group consisting of SEQ ID NOs: 16 to 25, and a light chain CDR1 selected from the group consisting of SEQ ID NOs: 88 to SEQ ID NO: 102, SEQ ID NOs: 103 to 119 Light chain CDR2 selected from the group, and light chain variable region comprising a light chain CDR3 selected from the group consisting of SEQ ID NO: 120 to SEQ ID NO: 144.
  • the antibody or antigen-binding fragment thereof that binds to PD-L1 is a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 1, a heavy chain CDR2 of SEQ ID NO: 8, and a heavy chain CDR3 of SEQ ID NO: 16,
  • a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 2, a heavy chain CDR2 of SEQ ID NO: 9, and a heavy chain CDR3 of SEQ ID NO: 17,
  • a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 2, a heavy chain CDR2 of SEQ ID NO: 9, and a heavy chain CDR3 of SEQ ID NO: 18,
  • a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 3, a heavy chain CDR2 of SEQ ID NO: 10, and a heavy chain CDR3 of SEQ ID NO: 19,
  • a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 4, a heavy chain CDR2 of SEQ ID NO: 11, and a heavy chain CDR3 of SEQ ID NO: 20,
  • a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 5, a heavy chain CDR2 of SEQ ID NO: 12, and a heavy chain CDR3 of SEQ ID NO: 21,
  • a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 6, a heavy chain CDR2 of SEQ ID NO: 13, and a heavy chain CDR3 of SEQ ID NO: 22,
  • a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 2, a heavy chain CDR2 of SEQ ID NO: 9, and a heavy chain CDR3 of SEQ ID NO: 23,
  • a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 7, a heavy chain CDR2 of SEQ ID NO: 14, and a heavy chain CDR3 of SEQ ID NO: 24,
  • a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 2, a heavy chain CDR2 of SEQ ID NO: 15, and a heavy chain CDR3 of SEQ ID NO: 25, or
  • the heavy chain variable region may include a heavy chain CDR1 of SEQ ID NO: 2, a heavy chain CDR2 of SEQ ID NO: 9, and a heavy chain CDR3 of SEQ ID NO: 17.
  • the antibody or antigen-binding fragment thereof that binds to PD-L1 is a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 88, a light chain CDR2 of SEQ ID NO: 103, and a light chain CDR3 of SEQ ID NO: 120,
  • a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 89, a light chain CDR2 of SEQ ID NO: 104, and a light chain CDR3 of SEQ ID NO: 121,
  • a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 90, a light chain CDR2 of SEQ ID NO: 105, and a light chain CDR3 of SEQ ID NO: 122,
  • a light chain variable region comprising the light chain CDR1 of SEQ ID NO: 91, the light chain CDR2 of SEQ ID NO: 106, and the light chain CDR3 of SEQ ID NO: 123,
  • a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 89, a light chain CDR2 of SEQ ID NO: 107, and a light chain CDR3 of SEQ ID NO: 124,
  • a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 92, a light chain CDR2 of SEQ ID NO: 108, and a light chain CDR3 of SEQ ID NO: 122,
  • a light chain variable region comprising the light chain CDR1 of SEQ ID NO: 93, the light chain CDR2 of SEQ ID NO: 109, and the light chain CDR3 of SEQ ID NO: 125,
  • a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 94, a light chain CDR2 of SEQ ID NO: 110, and a light chain CDR3 of SEQ ID NO: 126,
  • a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 95, a light chain CDR2 of SEQ ID NO: 111, and a light chain CDR3 of SEQ ID NO: 127,
  • a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 96, a light chain CDR2 of SEQ ID NO: 112, and a light chain CDR3 of SEQ ID NO: 128,
  • a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 89, a light chain CDR2 of SEQ ID NO: 108, and a light chain CDR3 of SEQ ID NO: 129,
  • a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 89, a light chain CDR2 of SEQ ID NO: 105, and a light chain CDR3 of SEQ ID NO: 130,
  • a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 89, a light chain CDR2 of SEQ ID NO: 113, and a light chain CDR3 of SEQ ID NO: 131,
  • a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 97, a light chain CDR2 of SEQ ID NO: 104, and a light chain CDR3 of SEQ ID NO: 132,
  • a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 89, a light chain CDR2 of SEQ ID NO: 104, and a light chain CDR3 of SEQ ID NO: 133,
  • a light chain variable region comprising the light chain CDR1 of SEQ ID NO: 97, light chain CDR2 of SEQ ID NO: 114, and light chain CDR3 of SEQ ID NO: 134,
  • a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 92, a light chain CDR2 of SEQ ID NO: 115, and a light chain CDR3 of SEQ ID NO: 135,
  • a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 98, a light chain CDR2 of SEQ ID NO: 104, and a light chain CDR3 of SEQ ID NO: 130,
  • a light chain variable region comprising the light chain CDR1 of SEQ ID NO: 89, the light chain CDR2 of SEQ ID NO: 116, and the light chain CDR3 of SEQ ID NO: 121,
  • a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 89, a light chain CDR2 of SEQ ID NO: 108, and a light chain CDR3 of SEQ ID NO: 136,
  • a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 99, a light chain CDR2 of SEQ ID NO: 105, and a light chain CDR3 of SEQ ID NO: 137,
  • a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 89, a light chain CDR2 of SEQ ID NO: 117, and a light chain CDR3 of SEQ ID NO: 138,
  • a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 89, a light chain CDR2 of SEQ ID NO: 118, and a light chain CDR3 of SEQ ID NO: 133,
  • a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 89, a light chain CDR2 of SEQ ID NO: 119, and a light chain CDR3 of SEQ ID NO: 139,
  • a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 100, a light chain CDR2 of SEQ ID NO: 104, and a light chain CDR3 of SEQ ID NO: 140,
  • a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 89, a light chain CDR2 of SEQ ID NO: 108, and a light chain CDR3 of SEQ ID NO: 141,
  • a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 89, a light chain CDR2 of SEQ ID NO: 105, and a light chain CDR3 of SEQ ID NO: 139,
  • a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 89, a light chain CDR2 of SEQ ID NO: 104, and a light chain CDR3 of SEQ ID NO: 142,
  • a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 89, a light chain CDR2 of SEQ ID NO: 105, and a light chain CDR3 of SEQ ID NO: 143,
  • a light chain variable region comprising the light chain CDR1 of SEQ ID NO: 101, the light chain CDR2 of SEQ ID NO: 104, and the light chain CDR3 of SEQ ID NO: 141, or
  • a light chain variable region comprising the light chain CDR1 of SEQ ID NO: 102, the light chain CDR2 of SEQ ID NO: 104, and the light chain CDR3 of SEQ ID NO: 144.
  • the antibody or antigen binding fragment thereof according to the invention may comprise:
  • a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 1, the heavy chain CDR2 of SEQ ID NO: 8, and the heavy chain CDR3 of SEQ ID NO: 16, and the light chain CDR1 of SEQ ID NO: 88, the light chain CDR2 of SEQ ID NO: 103, and the light chain CDR3 of SEQ ID NO: 120;
  • a light chain variable region comprising;
  • a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 2, the heavy chain CDR2 of SEQ ID NO: 9, and the heavy chain CDR3 of SEQ ID NO: 17, and the light chain CDR1 of SEQ ID NO: 89, the light chain CDR2 of SEQ ID NO: 104, and the light chain CDR3 of SEQ ID NO: 121;
  • a light chain variable region comprising;
  • a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 2, the heavy chain CDR2 of SEQ ID NO: 9, and the heavy chain CDR3 of SEQ ID NO: 18, and the light chain CDR1 of SEQ ID NO: 90, the light chain CDR2 of SEQ ID NO: 105, and the light chain CDR3 of SEQ ID NO: 122
  • a light chain variable region comprising;
  • a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 3, the heavy chain CDR2 of SEQ ID NO: 10, and the heavy chain CDR3 of SEQ ID NO: 19, the light chain CDR1 of SEQ ID NO: 91, the light chain CDR2 of SEQ ID NO: 106, and the light chain CDR3 of SEQ ID NO: 123;
  • a light chain variable region comprising;
  • a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 4, a heavy chain CDR2 of SEQ ID NO: 11, and a heavy chain CDR3 of SEQ ID NO: 20 and a light chain CDR1 of SEQ ID NO: 89, a light chain CDR2 of SEQ ID NO: 107, and a light chain CDR3 of SEQ ID NO: 124;
  • a light chain variable region comprising;
  • a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 5, a heavy chain CDR2 of SEQ ID NO: 12, and a heavy chain CDR3 of SEQ ID NO: 21 and a light chain CDR1 of SEQ ID NO: 92, a light chain CDR2 of SEQ ID NO: 108, and a light chain CDR3 of SEQ ID NO: 122
  • a light chain variable region comprising;
  • a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 6, the heavy chain CDR2 of SEQ ID NO: 13, and the heavy chain CDR3 of SEQ ID NO: 22, the light chain CDR1 of SEQ ID NO: 93, the light chain CDR2 of SEQ ID NO: 109, and the light chain CDR3 of SEQ ID NO: 125;
  • a light chain variable region comprising;
  • a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 2, a heavy chain CDR2 of SEQ ID NO: 9, and a heavy chain CDR3 of SEQ ID NO: 23 and a light chain CDR1 of SEQ ID NO: 94, a light chain CDR2 of SEQ ID NO: 110, and a light chain CDR3 of SEQ ID NO: 126
  • a light chain variable region comprising;
  • a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 7, the heavy chain CDR2 of SEQ ID NO: 14, and the heavy chain CDR3 of SEQ ID NO: 24, and the light chain CDR1 of SEQ ID NO: 95, the light chain CDR2 of SEQ ID NO: 111, and the light chain CDR3 of SEQ ID NO: 127;
  • a light chain variable region comprising; or
  • a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 2, the heavy chain CDR2 of SEQ ID NO: 15, and the heavy chain CDR3 of SEQ ID NO: 25, the light chain CDR1 of SEQ ID NO: 96, the light chain CDR2 of SEQ ID NO: 112, and the light chain CDR3 of SEQ ID NO: 128 Light chain variable region containing.
  • an antibody was further selected through an optimization process, and the antibody or antigen-binding fragment thereof according to the present invention may include the following heavy chain variable region and light chain variable region:
  • a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 2, the heavy chain CDR2 of SEQ ID NO: 9, and the heavy chain CDR3 of SEQ ID NO: 17, the light chain CDR1 of SEQ ID NO: 89, the light chain CDR2 of SEQ ID NO: 108, and the light chain CDR3 of SEQ ID NO: 129;
  • a light chain variable region comprising;
  • a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 2, the heavy chain CDR2 of SEQ ID NO: 9, and the heavy chain CDR3 of SEQ ID NO: 17, and the light chain CDR1 of SEQ ID NO: 89, the light chain CDR2 of SEQ ID NO: 105, and the light chain CDR3 of SEQ ID NO: 130
  • a light chain variable region comprising;
  • a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 2, the heavy chain CDR2 of SEQ ID NO: 9, and the heavy chain CDR3 of SEQ ID NO: 17, the light chain CDR1 of SEQ ID NO: 89, the light chain CDR2 of SEQ ID NO: 113, and the light chain CDR3 of SEQ ID NO: 131;
  • a light chain variable region comprising;
  • a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 2, the heavy chain CDR2 of SEQ ID NO: 9, and the heavy chain CDR3 of SEQ ID NO: 17, the light chain CDR1 of SEQ ID NO: 97, the light chain CDR2 of SEQ ID NO: 104, and the light chain CDR3 of SEQ ID NO: 132;
  • a light chain variable region comprising;
  • a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 2, the heavy chain CDR2 of SEQ ID NO: 9, and the heavy chain CDR3 of SEQ ID NO: 17, and the light chain CDR1 of SEQ ID NO: 89, the light chain CDR2 of SEQ ID NO: 104, and the light chain CDR3 of SEQ ID NO: 133;
  • a light chain variable region comprising;
  • a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 2, the heavy chain CDR2 of SEQ ID NO: 9, and the heavy chain CDR3 of SEQ ID NO: 17, the light chain CDR1 of SEQ ID NO: 97, the light chain CDR2 of SEQ ID NO: 114, and the light chain CDR3 of SEQ ID NO: 134;
  • a light chain variable region comprising;
  • a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 2, the heavy chain CDR2 of SEQ ID NO: 9, and the heavy chain CDR3 of SEQ ID NO: 17, the light chain CDR1 of SEQ ID NO: 92, the light chain CDR2 of SEQ ID NO: 115, and the light chain CDR3 of SEQ ID NO: 135;
  • a light chain variable region comprising;
  • a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 2, the heavy chain CDR2 of SEQ ID NO: 9, and the heavy chain CDR3 of SEQ ID NO: 17, the light chain CDR1 of SEQ ID NO: 98, the light chain CDR2 of SEQ ID NO: 104, and the light chain CDR3 of SEQ ID NO: 130
  • a light chain variable region comprising;
  • a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 2, the heavy chain CDR2 of SEQ ID NO: 9, and the heavy chain CDR3 of SEQ ID NO: 17, the light chain CDR1 of SEQ ID NO: 89, the light chain CDR2 of SEQ ID NO: 116, and the light chain CDR3 of SEQ ID NO: 121;
  • a light chain variable region comprising;
  • a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 2, the heavy chain CDR2 of SEQ ID NO: 9, and the heavy chain CDR3 of SEQ ID NO: 17, and the light chain CDR1 of SEQ ID NO: 89, the light chain CDR2 of SEQ ID NO: 108, and the light chain CDR3 of SEQ ID NO: 136
  • a light chain variable region comprising;
  • a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 2, the heavy chain CDR2 of SEQ ID NO: 9, and the heavy chain CDR3 of SEQ ID NO: 17, the light chain CDR1 of SEQ ID NO: 99, the light chain CDR2 of SEQ ID NO: 105, and the light chain CDR3 of SEQ ID NO: 137;
  • a light chain variable region comprising;
  • a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 2, the heavy chain CDR2 of SEQ ID NO: 9, and the heavy chain CDR3 of SEQ ID NO: 17, the light chain CDR1 of SEQ ID NO: 89, the light chain CDR2 of SEQ ID NO: 117, and the light chain CDR3 of SEQ ID NO: 138;
  • a light chain variable region comprising;
  • a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 2, the heavy chain CDR2 of SEQ ID NO: 9, and the heavy chain CDR3 of SEQ ID NO: 17, the light chain CDR1 of SEQ ID NO: 89, the light chain CDR2 of SEQ ID NO: 118, and the light chain CDR3 of SEQ ID NO: 133;
  • a light chain variable region comprising;
  • a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 2, a heavy chain CDR2 of SEQ ID NO: 9, and a heavy chain CDR3 of SEQ ID NO: 17, a light chain CDR1 of SEQ ID NO: 89, a light chain CDR2 of SEQ ID NO: 119, and a light chain CDR3 of SEQ ID NO: 139;
  • a light chain variable region comprising;
  • a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 2, the heavy chain CDR2 of SEQ ID NO: 9, and the heavy chain CDR3 of SEQ ID NO: 17, the light chain CDR1 of SEQ ID NO: 100, the light chain CDR2 of SEQ ID NO: 104, and the light chain CDR3 of SEQ ID NO: 140;
  • a light chain variable region comprising;
  • a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 2, the heavy chain CDR2 of SEQ ID NO: 9, and the heavy chain CDR3 of SEQ ID NO: 17, the light chain CDR1 of SEQ ID NO: 89, the light chain CDR2 of SEQ ID NO: 108, and the light chain CDR3 of SEQ ID NO: 141;
  • a light chain variable region comprising;
  • a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 2, the heavy chain CDR2 of SEQ ID NO: 9, and the heavy chain CDR3 of SEQ ID NO: 17, and the light chain CDR1 of SEQ ID NO: 89, the light chain CDR2 of SEQ ID NO: 105, and the light chain CDR3 of SEQ ID NO: 139;
  • a light chain variable region comprising;
  • a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 2, the heavy chain CDR2 of SEQ ID NO: 9, and the heavy chain CDR3 of SEQ ID NO: 17, and the light chain CDR1 of SEQ ID NO: 89, the light chain CDR2 of SEQ ID NO: 104, and the light chain CDR3 of SEQ ID NO: 142;
  • a light chain variable region comprising;
  • a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 2, the heavy chain CDR2 of SEQ ID NO: 9, and the heavy chain CDR3 of SEQ ID NO: 17, the light chain CDR1 of SEQ ID NO: 89, the light chain CDR2 of SEQ ID NO: 105, and the light chain CDR3 of SEQ ID NO: 143;
  • a light chain variable region comprising;
  • a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 2, the heavy chain CDR2 of SEQ ID NO: 9, and the heavy chain CDR3 of SEQ ID NO: 17, the light chain CDR1 of SEQ ID NO: 101, the light chain CDR2 of SEQ ID NO: 104, and the light chain CDR3 of SEQ ID NO: 141;
  • a light chain variable region comprising; or
  • a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 2, a heavy chain CDR2 of SEQ ID NO: 9, and a heavy chain CDR3 of SEQ ID NO: 17, a light chain CDR1 of SEQ ID NO: 102, a light chain CDR2 of SEQ ID NO: 104, and a light chain CDR3 of SEQ ID NO: 144; Light chain variable region containing.
  • the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 2, a heavy chain CDR2 of SEQ ID NO: 9, and a heavy chain CDR3 of SEQ ID NO: 17, and a light chain CDR1 of SEQ ID NO: 89, sequence A light chain variable region comprising the light chain CDR2 of SEQ ID NO: 104 and the light chain CDR3 of SEQ ID NO: 121;
  • a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 2, the heavy chain CDR2 of SEQ ID NO: 9, and the heavy chain CDR3 of SEQ ID NO: 17, and the light chain CDR1 of SEQ ID NO: 89, the light chain CDR2 of SEQ ID NO: 105, and the light chain CDR3 of SEQ ID NO: 130
  • a light chain variable region comprising;
  • a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 2, the heavy chain CDR2 of SEQ ID NO: 9, and the heavy chain CDR3 of SEQ ID NO: 17, and the light chain CDR1 of SEQ ID NO: 89, the light chain CDR2 of SEQ ID NO: 104, and the light chain CDR3 of SEQ ID NO: 133;
  • a light chain variable region comprising;
  • a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 2, the heavy chain CDR2 of SEQ ID NO: 9, and the heavy chain CDR3 of SEQ ID NO: 17, and the light chain CDR1 of SEQ ID NO: 89, the light chain CDR2 of SEQ ID NO: 108, and the light chain CDR3 of SEQ ID NO: 136
  • a light chain variable region comprising;
  • a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 2, a heavy chain CDR2 of SEQ ID NO: 9, and a heavy chain CDR3 of SEQ ID NO: 17, a light chain CDR1 of SEQ ID NO: 89, a light chain CDR2 of SEQ ID NO: 119, and a light chain CDR3 of SEQ ID NO: 139;
  • a light chain variable region comprising;
  • a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 2, the heavy chain CDR2 of SEQ ID NO: 9, and the heavy chain CDR3 of SEQ ID NO: 17, and the light chain CDR1 of SEQ ID NO: 89, the light chain CDR2 of SEQ ID NO: 105, and the light chain CDR3 of SEQ ID NO: 139;
  • a light chain variable region comprising; or
  • a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 2, the heavy chain CDR2 of SEQ ID NO: 9, and the heavy chain CDR3 of SEQ ID NO: 17, the light chain CDR1 of SEQ ID NO: 89, the light chain CDR2 of SEQ ID NO: 105, and the light chain CDR3 of SEQ ID NO: 143; It may include a light chain variable region comprising.
  • FRs Framework regions
  • Each variable domain typically has four FRs identified as FR1, FR2, FR3 and FR4.
  • the heavy chain variable region FR1 selected from the group consisting of SEQ ID NO: 26 to SEQ ID NO: 34,
  • a heavy chain variable region FR2 selected from the group consisting of SEQ ID NOs: 35 to 41;
  • a heavy chain variable region FR3 selected from the group consisting of SEQ ID NOs: 42 to 49, or
  • It may comprise a heavy chain variable region FR4 selected from the group consisting of SEQ ID NO: 50 to SEQ ID NO: 54.
  • the light chain variable region FR1 selected from the group consisting of SEQ ID NOs: 145 to 163,
  • Light chain variable region FR2 selected from the group consisting of SEQ ID NOs: 164 to 184,
  • Light chain variable region FR3 selected from the group consisting of SEQ ID NOs: 185 to 210, or
  • the light chain variable region FR4 selected from the group consisting of SEQ ID NOs: 211 to 216 may be included.
  • Fv fragments are antibody fragments containing complete antibody recognition and binding sites. This region consists of a dimer of one heavy chain variable domain and one light chain variable domain tightly and covalently associated, for example, with scFv.
  • Fab fragments contain the variable and constant domains of the light chain and the variable and first constant domains (CH1) of the heavy chain.
  • F (ab ') 2 antibody fragments generally comprise a pair of Fab fragments covalently linked near their carboxy termini by hinge cysteines between them.
  • Single-chain Fv or “scFv” antibody fragments comprise the V H and V L domains of an antibody, which domains are present in a single polypeptide chain.
  • the Fv polypeptide may further comprise a polypeptide linker between the V H domain and the V L domain that allows the scFv to form the desired structure for antigen binding.
  • the binding affinity of the PD-L1 antibody is in the range of 10 ⁇ 5 M to 10 ⁇ 12 M.
  • the binding affinity of PD-L1 antibody is 10 -6 M to 10 -12 M, 10 -7 M to 10 -12 M, 10 -8 M to 10 -12 M, 10 -9 M to 10 -12 M, 10 -5 M to 10 -11 M, 10 -6 M to 10 -11 M, 10 -7 M to 10 -11 M, 10 -8 M to 10 -11 M, 10 -9 M to 10 -11 M, 10 -10 M to 10 -11 M, 10 -5 M to 10 -10 M, 10 -6 M to 10 -10 M, 10 -7 M to 10 -10 M, 10 -8 M to 10 -10 M, 10 -9 M to 10 -10 M, 10 -5 M to 10 -9 M, 10 -6 M to 10 -9 M, 10 -7 M to 10 -9 M, 10 -8 M to 10 -9 M, 10
  • the antibody or antigen-binding fragment thereof that binds to PD-L1 may include a heavy chain variable region comprising a sequence having 90% or more sequence homology with a sequence selected from the group consisting of SEQ ID NO: 57 to SEQ ID NO: 87.
  • the antibody or antigen binding fragment thereof that binds to PD-L1 may comprise a heavy chain variable region selected from the group consisting of SEQ ID NO: 57 to SEQ ID NO: 87.
  • the heavy chain variable region of SEQ ID NO: 58, 68, 71, 76, 80, 83 or 85 may be included.
  • the antibody or antigen-binding fragment thereof that binds to PD-L1 may comprise a light chain variable region comprising a sequence having 90% or more sequence homology with a sequence selected from the group consisting of SEQ ID NOs: 217 to 247. have.
  • the antibody or antigen-binding fragment thereof that binds to PD-L1 may comprise a light chain variable region selected from the group consisting of SEQ ID NO: 217 to SEQ ID NO: 247.
  • the light chain variable region of SEQ ID NO: 218, 228, 231, 236, 240, 243 or 245 may be included.
  • the heavy chain variable region of SEQ ID NO: 85 and the light chain variable region of SEQ ID NO: 245 may be included.
  • Phase display is a technique for displaying variant polypeptides as phage proteins, such as fusion proteins with at least a portion of the envelope protein on the surface of fibrous phage particles.
  • the utility of phage display lies in the fact that a large library of randomized protein variants can be targeted to quickly and efficiently classify sequences that bind with high affinity with a target antigen. Displaying peptide and protein libraries on phage has been used to screen millions of polypeptides to identify polypeptides with specific binding properties.
  • Phage display technology provided a powerful tool for generating and selecting new proteins that bind specific ligands (eg antigens). Phage display technology can be used to generate large libraries of protein variants and to quickly sort sequences that bind with high affinity to target antigens.
  • Nucleic acids encoding variant polypeptides are fused with nucleic acid sequences encoding viral envelope proteins, eg, gene III protein or gene VIII protein.
  • Monovalent phage display systems have been developed in which a nucleic acid sequence encoding a protein or polypeptide is fused with a nucleic acid sequence encoding a portion of a gene III protein. In monovalent phage display systems, gene fusions are expressed at low levels and wild type Gene III proteins are also expressed to maintain particle infectivity.
  • Phage display technology has several advantages over conventional hybridoma and recombinant methods for preparing antibodies with the desired characteristics. This technique allows the production of large antibody libraries with various sequences in a short time without the use of animals. The preparation of hybridomas or the production of humanized antibodies may require months of preparation. In addition, since no immunity is required at all, phage antibody libraries can produce antibodies against antigens that are toxic or low antigenic. Phage antibody libraries can also be used to generate and identify novel therapeutic antibodies.
  • Techniques for generating human antibodies from immunized, non-immunized human, germline sequences, or na ⁇ ve B cell Ig repertory using immunized phage display libraries can be used.
  • Various lymphoid tissues can be used to prepare na ⁇ ve or non-immune antigen binding libraries.
  • the ability to identify and isolate high affinity antibodies from phage display libraries is important for the isolation of novel therapeutic antibodies. Separation of high affinity antibodies from the library may depend on the size of the library, the efficiency of production in bacterial cells, and the diversity of the library.
  • the size of the library is reduced by inefficient folding of the antibody or antigen binding protein and inefficient production due to the presence of stop codons. Expression in bacterial cells can be inhibited if the antibody or antigen binding domain is not properly folded. Expression can be improved by alternating mutations at the surface of the variable / constant interface or at selected CDR residues.
  • the sequence of the backbone region is one element to provide proper folding when generating antibody phage libraries in bacterial cells.
  • CDR3 regions have been found to often participate in antigen binding.
  • the CDR3 regions on the heavy chains vary considerably in size, sequence, and structural conformation, and thus can be used to prepare a variety of libraries.
  • diversity can be generated by randomizing the CDR regions of the variable heavy and light chains using all 20 amino acids at each position.
  • the use of all twenty amino acids can result in highly variable variant antibody sequences and increase the chance of identifying new antibodies.
  • the antibody or antibody fragment of the present invention may include not only the sequences of the anti-PD-L1 antibodies of the present invention, but also biological equivalents thereof, as long as they specifically recognize PD-L1.
  • further changes can be made to the amino acid sequence of the antibody to further improve the binding affinity and / or other biological properties of the antibody.
  • Such modifications include, for example, deletions, insertions and / or substitutions of amino acid sequence residues of the antibody.
  • Such amino acid variations are made based on the relative similarity of amino acid side chain substituents such as hydrophobicity, hydrophilicity, charge, size, and the like.
  • arginine, lysine and histidine are all positively charged residues; Alanine, glycine and serine have similar sizes; It can be seen that phenylalanine, tryptophan and tyrosine have a similar shape.
  • arginine, lysine and histidine; Alanine, glycine and serine; Phenylalanine, tryptophan and tyrosine are biologically equivalent functions.
  • the antibody or nucleic acid molecule encoding the same of the present invention is interpreted to include a sequence that exhibits substantial identity with the sequence described in SEQ ID NO.
  • the above substantial identity is at least 90% when the sequences of the present invention are aligned as closely as possible with any other sequences, and the aligned sequences are analyzed using algorithms commonly used in the art.
  • a homology most preferably at least 95% homology, at least 96%, at least 97%, at least 98%, at least 99% homology. Alignment methods for sequence comparison are known in the art.
  • BLAST The NCBI Basic Local Alignment Search Tool (BLAST) is accessible from NBCI and the like and can be used in conjunction with sequence analysis programs such as blastp, blasm, blastx, tblastn and tblastx on the Internet.
  • BLSAT is accessible at www.ncbi.nlm.nih.gov/BLAST/. Sequence homology comparisons using this program can be found at www.ncbi.nlm.nih.gov/BLAST/blast_help.html.
  • the antibody or antigen-binding fragment thereof of the present invention is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% compared to the specified sequence or all described in the specification. , 99%, or more homology.
  • homology can be determined by sequence comparison and / or alignment by methods known in the art. For example, sequence comparison algorithms (ie, BLAST or BLAST 2.0), manual alignment, visual inspection can be used to determine the percent sequence homology of nucleic acids or proteins of the invention.
  • the present invention relates to a nucleic acid encoding the antibody or antigen-binding fragment thereof.
  • the nucleic acid encoding the antibody or antigen-binding fragment thereof of the present invention can be isolated to recombinantly produce the antibody or antigen-binding fragment thereof.
  • the nucleic acid is isolated and inserted into a replicable vector for further cloning (amplification of DNA) or for further expression. Based on this, the present invention relates to a vector comprising the nucleic acid in another aspect.
  • Nucleic acid is meant to encompass DNA (gDNA and cDNA) and RNA molecules inclusively, and the nucleotides that are the basic building blocks of nucleic acids include natural nucleotides as well as analogs with modified sugar or base sites. .
  • the sequences of nucleic acids encoding heavy and light chain variable regions of the invention can be modified. Such modifications include addition, deletion, or non-conservative or conservative substitutions of nucleotides.
  • the DNA encoding the antibody is readily isolated or synthesized using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to the DNA encoding the heavy and light chains of the antibody).
  • Many vectors are available.
  • Vector components generally include, but are not limited to, one or more of the following: signal sequence, origin of replication, one or more marker genes, enhancer elements, promoters, and transcription termination sequences.
  • the term "vector” refers to a plasmid vector as a means for expressing a gene of interest in a host cell; Cosmid vector; Viral vectors such as bacteriophage vectors, adenovirus vectors, retrovirus vectors, and adeno-associated virus vectors, and the like.
  • the nucleic acid encoding the antibody in the vector is operably linked with a promoter.
  • “Operatively linked” means a functional binding between a nucleic acid expression control sequence (eg, an array of promoters, signal sequences, or transcriptional regulator binding sites) and another nucleic acid sequence, whereby the regulatory sequence is the other nucleic acid. To control transcription and / or translation of the sequence.
  • a nucleic acid expression control sequence eg, an array of promoters, signal sequences, or transcriptional regulator binding sites
  • promoters capable of promoting transcription e.g., tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pL ⁇ promoter, pR ⁇ promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter and T7 promoter, etc.
  • ribosome binding sites for initiation of translation e.g., amp promoter, recA promoter, SP6 promoter, trp promoter and T7 promoter, etc.
  • a promoter derived from the genome of the mammalian cell e.g., a metallothionine promoter, a ⁇ -actin promoter, a human heroglobin promoter and a human muscle creatine promoter
  • a mammal Promoters derived from animal viruses e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus (CMV) promoter, tk promoter of HSV, mouse breast tumor virus (MMTV) promoter, LTR promoter of HIV
  • a promoter derived from the genome of the mammalian cell e.g., a metallothionine promoter, a ⁇ -actin promoter, a human heroglobin promoter and a human muscle creatine promoter
  • a mammal Promoters derived from animal viruses e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter
  • the vector may be fused with other sequences to facilitate purification of the antibody expressed therefrom.
  • Sequences to be fused include, for example, glutathione S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA), FLAG (IBI, USA) and 6x His (hexahistidine; Quiagen, USA).
  • Such vectors include antibiotic resistance genes commonly used in the art as selectable markers and include, for example, ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin and tetracycline. There is a resistance gene.
  • the present invention relates to a cell transformed with the above-mentioned vector.
  • the cells used to produce the antibodies of the invention can be prokaryote, yeast or higher eukaryote cells, but are not limited thereto.
  • Bacillus strains such as Escherichia coli, Bacillus subtilis and Bacillus thuringiensis, Streptomyces, Pseudomonas (e.g. Pseudomonas putida), Proteus Prokaryotic host cells such as Proteus mirabilis and Staphylococcus (eg, Staphylocus carnosus) can be used.
  • Pseudomonas e.g. Pseudomonas putida
  • Proteus Prokaryotic host cells such as Proteus mirabilis and Staphylococcus (eg, Staphylocus carnosus) can be used.
  • examples of useful host cell lines are COS-7, BHK, CHO, CHOK1, DXB-11, DG-44, CHO / -DHFR, CV1, COS-7, HEK293, BHK, TM4, VERO, HELA, MDCK, BRL 3A, W138, Hep G2, SK-Hep, MMT, TRI, MRC 5, FS4, 3T3, RIN, A549, PC12, K562, PER.C6, SP2 / 0, NS-0 , U20S, or HT1080, but is not limited thereto.
  • the present invention (a) culturing the cells; And (b) recovering the antibody or antigen-binding fragment thereof from the cultured cells.
  • the cells can be cultured in various media. It can be used as a culture medium without limitation among commercial media. All other necessary supplements known to those skilled in the art may be included at appropriate concentrations. Culture conditions, such as temperature, pH, and the like, are already in use with host cells selected for expression, which will be apparent to those skilled in the art.
  • the recovery of the antibody or antigen-binding fragment thereof can be removed by, for example, centrifugation or ultrafiltration, and the resultant can be purified using, for example, affinity chromatography or the like. Further other purification techniques such as anion or cation exchange chromatography, hydrophobic interaction chromatography, hydroxylapatite chromatography and the like can be used.
  • the present invention relates to a composition for preventing or treating cancer, which comprises the antibody as an active ingredient.
  • the present invention includes, for example, (a) a pharmaceutically effective amount of an antibody against PD-L1 or an antigen binding fragment thereof according to the present invention; And (b) it may be a pharmaceutical composition for the prevention or treatment of cancer or infectious diseases comprising a pharmaceutically acceptable carrier.
  • the present invention also relates to a method for the prophylaxis or treatment of cancer or infectious disease comprising administering to a patient an effective amount necessary for an antibody against PD-L1 or an antigen-binding fragment thereof.
  • composition uses the above-described anti-PD-L1 antibody or antigen-binding fragment thereof as an active ingredient, the description in common between the two is omitted.
  • T cell depletion is a condition of T cell dysfunction that can occur in chronic infections and cancer. It is defined as poor effector function, sustained expression of inhibitory receptors, and transcriptional states that are different from functional effector or memory T cells. Depletion interferes with the management of infection and tumor progression.
  • the antibodies or antigen-binding fragments thereof of the present invention bind to PD-L1 with high affinity and inhibit the formation of PD-1 and PD-L1 complexes, thereby avoiding anti-tumor T cell activity. It can be usefully used to treat cancer that induces T cell depletion.
  • anticancer therapeutic agents in addition to the above antibodies can effectively target tumor cells overexpressing PD-L1, increase anti-tumor T cell activity, and enhance the immune response targeting tumor cells.
  • Other anti-neoplastic or immunogenic agents eg, weakened cancer cells, tumor antigens (including recombinant proteins, peptides and carbohydrate molecules), antigen-transmitting cells, eg, tumor-derived antigens or nucleic acids
  • VEGF VEGF, EGFR, Her2 / neu, VEGF receptors,
  • Anti PD-L1 antibodies can induce cell death.
  • Cell death is induced by direct or indirect mechanisms.
  • PD-L1 binding by anti PD-L1 antibodies can result in complement dependent cytotoxicity (CDC).
  • CDC complement dependent cytotoxicity
  • the anti-PD-L1 antibody binds to PD-L1 and results in the recruitment of secondary cell types that will kill PD-L1 expressing target cells.
  • Representative mechanisms by which anti-PD-L1 antibodies mediate cell death by recruitment of secondary cell types include, but are not limited to, antibody dependent cytotoxicity (ADCC) and antibody dependent cell phagocytosis (ADCP).
  • Target PD-L1 expressing cell types include tumors and T cells, for example activated T cells.
  • antibodies or antibody fragments of the invention can be used to prevent or treat infections and infectious diseases.
  • Prevention means any action that inhibits or delays the progression of cancer or infectious disease by administration of a composition according to the present invention
  • treatment means inhibiting cancer development, reducing or eliminating cancer, or infection Inhibition of disease, reduction of infectious disease or removal of infectious disease.
  • Cancers which are diseases to which the composition is applied include cancers that typically respond to immunotherapy, and cancers that are not related to immunotherapy so far.
  • Non-limiting examples of preferred cancers for treatment include melanoma (eg metastatic malignant melanoma), kidney cancer (eg clear cell carcinoma), prostate cancer (eg hormonal refractory prostate carcinoma), Pancreatic adenocarcinoma, breast cancer, colon cancer, lung cancer (eg, non-small cell lung cancer), esophageal cancer, head and neck squamous cell carcinoma, liver cancer, ovarian cancer, cervical cancer, thyroid cancer, glioblastoma, glioma, leukemia, lymphoma, and other kidneys Includes biological carcinoma.
  • the present invention includes refractory or recurrent cancer that can inhibit growth using the antibodies of the present invention.
  • the antibody or antibody fragment can be used alone or in combination with a vaccine to stimulate an immune response to pathogens, toxins, and self-antigens.
  • Antibodies or antigen-binding fragments thereof include, for example, human immunodeficiency virus, hepatitis virus classes A, B and C, Eppstein Barr virus, human cytomegalovirus, human papilloma Virus, Herpes Virus, can be used to stimulate an immune response against a virus that infects a human, including but not limited to.
  • Antibodies or antigen-binding fragments thereof can be used to stimulate an immune response to infection of bacterial or fungal parasites, and other pathogens.
  • compositions of the present invention are those commonly used in the preparation, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate , Microcrystalline cellulose, polyvinylpyrrolidone, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like.
  • the composition of the present invention may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives and the like in addition to the above components.
  • composition of the present invention may be administered orally or parenterally, and in the case of parenteral administration, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, endothelial administration, topical administration, intranasal administration, pulmonary administration and rectal administration Or the like.
  • oral compositions should be formulated to coat the active agent or to protect it from degradation in the stomach.
  • the pharmaceutical composition may be administered by any device in which the active agent may migrate to the target cell.
  • Suitable dosages of the compositions according to the invention vary depending on factors such as the method of formulation, mode of administration, age, weight, sex, morbidity, condition of the patient, food, time of administration, route of administration, rate of excretion and reaction sensitivity, and usually The skilled practitioner can readily determine and prescribe a dosage effective for the desired treatment or prophylaxis.
  • the daily dose of the pharmaceutical composition of the present invention is 0.0001-100 mg / kg.
  • pharmaceutically effective amount as used herein means an amount sufficient to prevent or treat cancer.
  • compositions of the present invention may be prepared in unit dosage form by formulating with a pharmaceutically acceptable carrier and / or excipient according to methods which can be easily carried out by those skilled in the art. Or may be prepared by incorporation into a multi-dose container.
  • the formulation may be in the form of a solution, suspension, or emulsion in an oil or aqueous medium, or may be in the form of extracts, powders, suppositories, powders, granules, tablets, or capsules, and may further include a dispersant or stabilizer.
  • compositions of the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents and may be administered sequentially or simultaneously with conventional therapeutic agents.
  • the present invention is a cancer diagnostic composition comprising an antibody against PD-L1 according to the present invention.
  • the present invention also relates to a method for diagnosing cancer by treating an antibody against PD-L1 or an antigen-binding fragment thereof according to the present invention.
  • Cancer can be diagnosed by measuring PD-L1 expression levels in a sample via an antibody against PD-L1 according to the invention.
  • Expression levels can be measured according to conventional immunoassay methods, radioimmunoassay using the antibody against PD-L1, radioimmunoprecipitation, immunoprecipitation, immunohistochemical staining, enzyme-linked immunosorbant assay (ELISA), capture -ELISA, inhibition or hardwood analysis, sandwich analysis, flow cytometry, immunofluorescence staining and immunoaffinity purification can be measured, but is not limited thereto.
  • cancer By analyzing the final signal intensity by the immunoassay process, cancer can be diagnosed. That is, if the protein of the marker of the present invention is expressed high in a biological sample and the signal is stronger than that of the normal biological sample (eg, normal gastric tissue, blood, plasma or serum), the cancer is diagnosed.
  • the normal biological sample eg, normal gastric tissue, blood, plasma or serum
  • the present invention relates to a cancer diagnostic kit comprising the cancer diagnostic composition.
  • the kit according to the present invention comprises an antibody against PD-L1 according to the present invention, and can diagnose cancer by analyzing a signal indicated by the reaction between the sample and the antibody.
  • the signal may include, but is not limited to, an enzyme bound to the antibody, for example, alkaline phosphatase, ⁇ -galactosidase, horse radish peroxidase, luciferase or cytochrome P450.
  • the substrate for the enzyme is bromochloroindolyl phosphate (BCIP), nitro blue tetrazolium (NBT), naphthol-AS-B1-phosphate (naphthol) as the substrate.
  • BCIP bromochloroindolyl phosphate
  • NBT nitro blue tetrazolium
  • naphthol-AS-B1-phosphate naphthol
  • Chloronaphthol aminoethylcarbazole, diaminobenzidine, D-luciferin, lucigenin when color development reaction substrates such as -AS-B1-phosphate) and enhanced chemifluorescence (ECF) are used, and horse radish peroxidase is used (Bis-N-methylacridinium nitrate), resorphin benzyl ether, luminol, amplex red reagent (10-acetyl-3,7-dihydroxyphenoxazine), HYR (p-phenylenediamine-HCl and pyr ocatechol), TMB (tetramethylbenzidine), ABTS (2,2'-Azine-di [3-ethylbenzthiazoline sulfonate]), o-phenylenediamine (OPD) and naphthol / pyronine, glucose oxidase and t-NBT (nitroblue tetrazolium)
  • kits according to the invention may comprise a label which generates a detectable signal, said label comprising a chemical (eg biotin), an enzyme (alkaline phosphatase, ⁇ -galactosidase, horse radish). Peroxidase and cytochrome P450), radioactive materials (eg C14, I125, P32 and S35), fluorescent materials (eg fluorescein), luminescent materials, chemiluminescent and fluorescence resonance energy transfer (FRET) It may include, but is not limited thereto.
  • a chemical eg biotin
  • an enzyme alkaline phosphatase, ⁇ -galactosidase, horse radish
  • Peroxidase and cytochrome P450 cytochrome P450
  • radioactive materials eg C14, I125, P32 and S35
  • fluorescent materials eg fluorescein
  • luminescent materials eg fluorescein
  • FRET fluorescence resonance energy transfer
  • Measurement of the activity or signal of an enzyme used for cancer diagnosis can be carried out according to various methods known in the art. This allows for either qualitative or quantitative analysis of PD-L1 expression.
  • Plasmid DNA was transfected into HEK-293F cells to express the antigen in animal cells.
  • the polyplex reaction solution for transfection was performed by mixing 25 ⁇ g of plasmid DNA in 3 ml of Freestyle 293 expression medium and further adding 2 mg / ml PEI (Polyethylenimine) (polyplUSA-transfection, USA). ) 50 ⁇ l and mix again.
  • the polyplex reaction solution was reacted at room temperature for 15 minutes, and then placed in a 40 ml culture medium incubated at 1 ⁇ 10 6 cells / ml and incubated at 120 rpm at 37 ° C. and 8% CO 2 for 24 hours.
  • the protein When using recombinant Protein A agarose resin, the protein was eluted with 0.1M glycine and neutralized with 500 ⁇ l of 1M Tris-HCl, and the first purified protein was a Superdex 200 (1.5 cm * 100 cm) gel. Secondary purification was performed using filtration chromatography.
  • Purified protein was purified by SDS-PAGE gel and size exclusion chromatography (TSK-GEL G-3000 SWXL Size-exclusion chromatography (SEC) (Tosoh)).
  • Example 1 50 ug of PD-L1-hFc, PD-L1-mFc and PD-L1-his (Catalog Number, 10084-H08H) protein antigen prepared in Example 1 were coated on an immunosorbent tube After blocking was performed.
  • the phage were infected with bacteria with a human scFv library with a variety of 2.7 ⁇ 10 10 , and the bacteria were incubated at 30 ° C. for 16 hours. After incubation, the supernatant was concentrated by PEG, and then dissolved in PBS buffer to prepare a human antibody library. After the library phage was put into the immunotube, the reaction was carried out at room temperature for 2 hours, and then washed with 1XPBS / T and 1XPBS to elute only scFv-phage specifically bound to the antigen.
  • the pool of positive phages is obtained through the panning process of infecting and eluting the eluted phages again.
  • the amplified phages are increased and only the number of times in the PBST washing step is used. Round panning was performed. As a result, it was confirmed that the output of the phage bound to the antigen was slightly increased compared to the input in the third round panning as shown in Table 2.
  • each well was washed with 0.2 ml of PBS / T, and then 100 ⁇ l of the first to third panning poly scFv-phage was added to each well and allowed to react at room temperature for 2 hours. Again, each well was washed four times with 0.2 ml of PBS / T, and then the secondary antibody anti-M13-HRP (Amersham 27-9421-01) was diluted 1: 2000 and reacted at room temperature for 1 hour. After washing with PBS / T, OPD tablet (sigma. 8787-TAB) was made into PC buffer, 100 ul / well each color developed for 10 minutes, and absorbance was measured with a spectrophotometer (Molecular Device) at 490 nm.
  • OPD tablet sigma. 8787-TAB
  • Colonies from high-binding polyclonal phage antibody groups were incubated for 16 hours at 37 ° C. in 1 ml 96-deep well plates (Bionia 90030) in 2 ⁇ YTCM, 2% glucose, 5 mM MgCl 2 medium. Take 100-200 ul of cells so that the value is 0.1 at OD 600 in these grown cells and place in 1 ml of 2xYTCM, 2% glucose, 5 mM MgCl 2 medium, and then measure the value at OD 600 at 37 ° C in a 96-deep well plate. Incubate for 2-3 hours so that it is 0.5-0.7. M1 helper phages were infected with a MOI of 1:20 and incubated for 16 hours at 30 ° C. in 2 ⁇ YTCMK, 5 mM MgCl 2 1 mM IPTG medium.
  • Each well was blocked using 4% skim milk dissolved in PBS after coating 100 ng of antigen PD-L1 per well for 16 hours at 4 ° C. in a 96 well immune plate.
  • 100 ⁇ l of monoclonal scFv-phage (100 scFv-phage) incubated for 16 hours washed with 0.2 ml PBS / T in each well was added to each well and allowed to react at room temperature for 2 hours.
  • Each well was washed four times with 0.2 ml PBS / T, and then the second antibody, anti-M13-HRP, was diluted to 1/2000 and reacted at room temperature for 1 hour. After washing with 0.2 ml PBS / T, color development was measured at 490 nm.
  • the selected clones were subjected to DNA-prep using a DNA purification kit (Qiagen, Germany) to obtain DNA and to request sequencing (solgent). Based on the results of the sequencing analysis, the CDR sites of V H and V L of the selected antibodies were identified, and the similarity between these antibodies and the germ line antibody group was determined by NCBI's web page http: //www.ncbi.nlm.nih.
  • the Ig BLAST program at .gov / igblast / was used to investigate. As a result, phage antibodies specific for 10 PD-L1 were obtained, which are summarized in Table 3 below.
  • the antibodies comprising the heavy and light chain CDRs, FR sequences, and the heavy chain variable region and the light chain variable region comprising the selected antibodies are shown in Tables 4 and 5.
  • PCR (iCycler iQ, BIO-RAD) was performed on the heavy and light chains to convert monoclonal phage antibodies against selected 1110 PD-L1 phages into IgG whole vectors.
  • heavy and light chains were obtained, and the heavy and light chains of the vector and each clone were cleaved with restriction enzymes.
  • the vector and heavy chains were each eluted with a DNA-gel extraction kit (Qiagen).
  • Ligation is a mixture of vector 1 ul (10 ng) heavy chain (100-200 ng) 15 ul, 10x Buffer 2 ul, ligase (1 U / ul) 1 ul, distilled water and left at room temperature for 1-2 hours. , Transformed into cells (competent cells) (XL1-blue) and placed on ice for 5 minutes, gave a heat shock (heat shock) at 42 °C for 90 seconds.
  • the cloned pNATVH and pNATVL vectors were cotransfected (co-transfected) into HEK293F cells at a ratio of 6: 4, and the supernatant was collected on day 7 to remove cells and suspended solids through centrifugation and a 0.22 ⁇ m top-filter. The supernatants were collected and protein A affinity chromatography was performed to purify IgG antibodies. After purification, the antibody was isolated through glycine buffer, and the buffer was exchanged so that the final resuspension buffer became PBS. Purified antibodies were quantified by BCA and nano drop, and 15 antibodies were loaded at 5 ug each under reduced and non-reduced conditions, and subjected to SDS-PAGE analysis for purity and mobility of purified proteins. It was confirmed (Fig. 5).
  • PD1 / PD-L1 blocking bioassay kit Promega, J1250.
  • CHO cell line with high expression of PD-L1 was plated in a 96-well plate, cultured for 16 hours or more, treated with each antibody serially diluted to a constant concentration, and Jurkat cell line with high expression of human PD-1 for 6 hours. Incubated together.
  • the degree of recovery of inhibition of the antibody was determined by the intensity of luminescence produced by luciferase decomposing the substrate and measured by a spectrophotometer (SpectraMax M5 spectraphotometer, Molecular Devices, USA).
  • Ten PD-L1 antibodies confirmed the activity of the antibody to restore the signal reduced by PD-1 / PD-L1 complex formation, 16E12 showed similar activity compared to the control antibody (Fig. 6).
  • the dilution of the PD-1 / PD-L1 blocking bioassay was carried out to recover the reduced signal in a concentration gradient dependent manner.
  • the degree of recovery can be represented as EC50 (effective concentration of mAb at 50% level of Recovery signal) and analyzed using Graphpad Prism6 of EC50 in vitro efficacy inhibit recovery capability is shown in FIG.
  • Transgenic cell pools expressing PD-L1 were transformed to HEK293E with a pcDNA3.1 plasmid containing human PD-L1 and screened in selective medium containing 150 ug / ml Zeocin (# R25001, Thermo Fisher). It was. Each cell pool was identified and selected by fluorescence activated cell sorting (FACS) analysis using each anti-PD-L1, and used for functional evaluation methods such as FACS binding assays and FACS competition assays.
  • FACS fluorescence activated cell sorting
  • Each transgenic cell pool expressing human PD-L1 was prepared with 0.5-1x10 ⁇ 6 cells per sample, and the antibodies were continuously diluted in constant dilution multiples and reacted with the prepared cells at 4 ° C. for 20 minutes. The cells were then washed three times with PBS (# LB001-02, welgene) containing 2% fetal bovine serum, and the anti-human IgG antibody (#ITC) bound to the fluorescein isothiocyanate (FITC) phosphor FI-3000, Vectorlabs) and reacted at 4 ° C.
  • PBS # LB001-02, welgene
  • ITC anti-human IgG antibody
  • FITC fluorescein isothiocyanate
  • the binding force of the antibody bound in a concentration-dependent manner to human PD-L1 overexpressed on the cell surface can be known as mean fluorescence intensity (MFI).
  • PBST buffer was used to collect the sensogram data during the binding and dissociation process over time while flowing the antibody for 10 minutes at a flow rate of 30 ul / min at different concentrations (30 nM to 0.123 nM).
  • the sensogram data at the equilibrium were plotted and plotted according to the concentration to calculate the equilibrium dissociation constant (K D ), showing a high affinity for the PD-L1 antigen at 0.045 nM (FIG. 9).
  • Antibody optimization was performed by constructing a new LC shuffling library by fixing the heavy chain and adding the 105-106 light chain (LC) pool possessed by YBIOLOGICS, and the LC shuffling, hydrophobic core of the heavy chain (LCs were converted to conserved residues by comparison with residues of structurally important sites such as hydrophobic cores, exposed residues, charge clusters and salt bridges.
  • Core packing + LC shuffling undergoing shuffling, DNA of antibody variable region is in vivo There are three ways to proceed: affinity maturation, CDR hotspot + LC shuffling, which randomly mutates mutational hot spots that can be frequently muted. It was.
  • the LC gene of the 16E12 antibody was cleaved with BstX I and used as a vector, and the library pool retained by WBIOLOGICS was cut with BstX I and used as an insert. After ligation with ligase, transformation was performed using electroporation transformation taxa.
  • the antibody libraries were prepared by collecting the transformed cells in a square plate, and various libraries of about 1.5 ⁇ 10 7 were obtained. As a result of sequencing analysis, the sequences of HC were all identical and the sequences of LCs were different.
  • the frame work (FR) portion of the 16E12 antibody was replaced with a conserved amanoic acid sequence, followed by cleavage of the LC gene with BstX I and use as a vector.
  • the library pool retained at was cut with BstX I and used as an insert.
  • transformation was performed using electroporation transformation taxa.
  • the antibody libraries were prepared by collecting the transformed cells in a square plate, and various libraries of about 8.4 ⁇ 10 6 were obtained.
  • the sequencing analysis showed that the FR sites of HC were substituted with conserved amanoic acid sequences. It was confirmed that the sequence of is different.
  • CDR hotspot + LC shuffling library To construct a CDR hotspot + LC shuffling library, the frame work (FR) portion of the 16E12 antibody was replaced with a conserved amanoic acid sequence, the CDR1 hotspot library was cleaved with Sfi I and used as an insert, and then The library pool possessed by Biologics was cut into Sfi I and used as a vector. After ligation with ligase, transformation was performed using the cells for electroporation transformation. The antibody libraries were prepared by collecting the transformed cells in a square plate, and various libraries of about 5.6x106 were obtained, and sequencing showed that the FR sites of HC were substituted with conserved amanoic acid sequences and hotspots of CDR1. It was confirmed that the amino acids of the sequence was randomly changed and the sequence of the LC is different.
  • Blocking after coating 50 ug of PD-L1-hFc, PD-L1-mFc and PD-L1-his (Catalog Number, 10377-H08H) protein antigen produced by YBIOLOGICS in immunosorb tube blocking was performed.
  • Human Antibody Library Phage was infected with bacteria with a human scFv library of 2.7 ⁇ 10 10 diversity and then incubated at 30 ° C. for 16 hours. After incubation, the supernatant was concentrated by PEG, and then dissolved in PBS buffer to prepare a human antibody library. After the library phage was put into the immunotube, the reaction was carried out at room temperature for 2 hours, and then washed with 1XPBS / T and 1XPBS to elute only scFv-phage specifically bound to the antigen.
  • a pool of positive phages was obtained through a panning process in which the eluted phages were again infected with E. coli and amplified, and panning for antibody optimization was performed only the first round.
  • the number of phages bound to the antigen in the first round panning was slightly increased compared to the input.
  • Colonies from panning were incubated for 16 hours at 37 ° C. in 1 ml 96-deep well plate (Biononia 90030) in 2 ⁇ YTCM, 2% glucose, 5 mM MgCl 2 medium. Take 100-200 ul of cells so that the value is 0.1 at OD 600 in these grown cells and place in 1 ml of 2xYTCM, 2% glucose, 5 mM MgCl 2 medium, and then measure the value at OD 600 at 37 ° C in a 96-deep well plate. It was incubated for 2-3 hours so that it is 0.5-0.7. M1 helper phages were infected with a MOI of 1:20 and incubated for 16 hours at 30 ° C. in 2 ⁇ YTCMK, 5 mM MgCl 2 1 mM IPTG medium.
  • the selected monoclones were subjected to DNA-prep using a DNA purification kit (Qiagen, Germany) to obtain DNA and to request sequencing (solgent). Based on the results of the sequencing analysis, the CDR regions of V H and V L of the selected antibodies were identified, and the similarity between these antibodies and germ line antibody group was determined by NCBI's web page http://www.ncbi.nlm.nih.gov/igblast Investigation using the Ig BLAST program of / resulted in a specific phage antibody having a higher binding capacity than the 21 parent antibodies, which are summarized in Table 7 below.
  • the antibodies comprising the heavy and light chain CDRs, FR sequences, and heavy chain variable regions and light chain variable regions comprising the selected antibodies are shown in Tables 8 and 9 below.
  • PCR iCycler iQ, BIO-RAD
  • iCycler iQ BIO-RAD
  • PCR was performed on the heavy and light chains to convert monoclonal phage antibodies against 21 selected PD-L1 phages into IgG whole vectors.
  • heavy and light chains were obtained, and the restriction enzymes cut the heavy and light chains of the vector and the individual clones.
  • Each vector and heavy chain was eluted with a DNA-gel extraction kit (Qiagen).
  • Ligation is a vector of 1 ul (10 ng) heavy chain (100-200 ng) 15 ul, 10x buffer 2 ul, ligase (1 U / ul) 1 ul, distilled water and mixed for 1 to 2 hours at room temperature Then, put into transformed cells (competent cells: XL1-blue) and placed on ice for 5 minutes, gave a heat shock (heat shock) at 42 °C for 90 seconds.
  • the cloned pNATVH and pNATVL vectors were cotransfected (co-transfected) into HEK293F cells at a ratio of 6: 4, and the supernatant was collected on day 7 to remove cells and suspended solids through centrifugation and a 0.22 ⁇ m top-filter.
  • the supernatants were collected and protein A affinity chromatography was performed to purify IgG antibodies. After purification, the antibody was isolated through glycine buffer, and the buffer was exchanged so that the final resuspension buffer became PBS.
  • Purified antibodies were quantified by BCA and nano drop, and 21 antibodies were loaded by 5 ug each for reducing and non-reducing conditions, followed by SDS-PAGE analysis for purity and mobility of purified proteins. The state was confirmed. In addition, a portion of the supernatant was loaded on SDS-PAGE to compare the expression rate with the parent antibody, and most of the antibodies showed higher expression rate than the parent antibody.
  • PD-1 / PD-L1 block bioassay kit Promega, J1250.
  • CHO cell line with high expression of PD-L1 was plated in a 96-well plate, cultured for 16 hours or more, treated with each antibody serially diluted to a constant concentration, and Jurkat cell line with high expression of human PD-1 for 6 hours. Incubated together.
  • the extent of recovery of inhibition of the antibody was determined by the luminescence intensity of luciferase by breaking down the substrate and measured by spectrophotometer (SpectraMax M5 spectraphotometer, Molecular Devices, USA).
  • PD-L1 antibodies confirmed the activity of the antibody to restore the signal reduced by PD-1 / PD-L1 complex formation, 4A7, 4A11, 4C9, 4F5, 4H5, 4H8 compared to the parent antibody This increased and showed similar activity compared to the control antibody (FIG. 11 and Table 10).
  • the transgenic cell pool expressing human PD-1 was transformed into HEK293E with a pcDNA3.1 plasmid containing human PD-1 (NM_005018.2) or human PD-L1 (NM_014143.2), and 400 ug / Selection was made in selective medium containing ml Zeocin (# R25001, Thermo Fisher). Each cell pool was identified and selected by fluorescence activated cell sorting (FACs) analysis using anti-PD-1 (# 557860, BD), respectively, and used for functional evaluation methods such as FACs binding assays or FACs competition assays. Was used.
  • FACs fluorescence activated cell sorting
  • Each transgenic cell pool expressing human PD-L1 was prepared with 0.5-1x10 ⁇ 6 cells per sample, and the antibodies were continuously diluted in constant dilution multiples and reacted with the prepared cells at 4 ° C. for 20 minutes. The cells were then washed three times with PBS (# LB001-02, welgene) containing 2% fetal bovine serum, and the anti-human IgG antibody (#ITC) bound to the fluorescein isothiocyanate (FITC) phosphor FI-3000, Vectorlabs) and reacted at 4 ° C.
  • PBS # LB001-02, welgene
  • ITC anti-human IgG antibody
  • FITC fluorescein isothiocyanate
  • Each transgenic cell pool expressing human PD-L1 was prepared with 0.5x10 ⁇ 6 cells per sample, and the antibodies were reacted with the prepared cells for 20 min at 4 ° C. by diluting the antibodies serially with a constant dilution factor. The cells were then washed three times with PBS (# LB001-02, welgene) containing 2% fetal bovine serum, and the anti-human IgG antibody (#ITC) bound to the fluorescein isothiocyanate (FITC) phosphor FI-3000, Vectorlabs) and reacted at 4 ° C.
  • PBS # LB001-02, welgene
  • ITC anti-human IgG antibody
  • FITC fluorescein isothiocyanate
  • Human PD-1-Fc (S1420, Y-Biologics) was fixed in a well of a 96-well immuno microplate (96-well immunoplate, # 439454, Thermo) at 4 ° C. for 16 hours, and 0.05% tween-20 ( After washing three times with PBS containing # P9416, Sigma-Aldrich), non-specific binding was blocked by standing at room temperature for 1 hour with a wash solution containing 4% skim milk (# 232120, Becton, Dickinson and Company).
  • the equilibrium dissociation constant (KD) was calculated by plotting and plotting the sensogram data at equilibrium according to the concentration, and 16E12 (4F5) showed 0.001 nM with high affinity for the PD-L1 antigen. ( Figure 15).
  • each antibody serially diluted in a constant dilution multiple or human PD-1-His (S1352, Y-Biologics) used as a positive control was reacted at room temperature for 1 hour, and then placed in a prepared microplate for 1 hour at room temperature. Let's do it.
  • the anti-Biotin-His antibody (# MA1-21315-BTIN, Thermo) was diluted 1: 2000 and put in the well of the microplate and reacted at room temperature for 1 hour, and then washed in the same manner
  • Streptavidin poly-HRP antibody (# 21140, Pierce) was diluted 1: 5000, put into a well of a microplate, and reacted at room temperature for 1 hour, and washed in the same manner.
  • Add 100 ul TMB substrate solution (# T0440, Sigma-Aldrich), block the light, leave at room temperature for 3 minutes, and stop by adding 50 ul 2.5M sulfuric acid (# S1478, Samchun) to stop the reaction.
  • ® Discover System Promega was used to measure absorbance at 450 nm. The result is shown in FIG.
  • the antibody or antigen-binding fragment thereof that binds to PD-L1 according to the present invention binds to PD-L1 with high affinity, while inhibiting the formation of PD-1 / PD-L1 complex, thereby preventing PD-1 / PD-L1 mediated T. T cell depletion that avoids cellular activity can be suppressed.
  • the antibody or antigen-binding fragment thereof that binds to PD-L1 according to the present invention can be usefully used for the prevention or treatment of the desired cancer or infectious disease.

Abstract

La présente invention concerne un anticorps dirigé contre le ligand 1 de mort programmée (PD-L1) ou un fragment de liaison à l'antigène de celui-ci, un acide nucléique pour le codage de celui-ci, un vecteur comprenant l'acide nucléique, une cellule transformée avec le vecteur, un procédé de préparation de l'anticorps ou du fragment de liaison à l'antigène de celui-ci, et une composition le comprenant, pour la prévention ou le traitement d'un cancer ou d'une maladie infectieuse.
PCT/KR2017/008495 2016-08-05 2017-08-07 Anticorps dirigé contre le ligand 1 de mort programmée (pd-l1) et son utilisation WO2018026249A1 (fr)

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JP2019528010A JP6925421B2 (ja) 2016-08-05 2017-08-07 プログラム化された細胞死蛋白質リガンド−１（ｐｄ−ｌ１）に対する抗体及びその用途
AU2017306507A AU2017306507B2 (en) 2016-08-05 2017-08-07 Antibody to programmed death-ligand 1 (PD-L1) and use thereof
CA3032806A CA3032806C (fr) 2016-08-05 2017-08-07 Anticorps dirige contre le ligand 1 de mort programmee (pd-l1) et son utilisation
BR112019002282A BR112019002282A2 (pt) 2016-08-05 2017-08-07 anticorpo que se liga a pd-l1 ou um fragmento de ligação ao antígeno do anticorpo, método para produzir o mesmo e composição para prevenção ou tratamento de câncer ou doenças infecciosas
CN201780055412.7A CN110072889B (zh) 2016-08-05 2017-08-07 抗程序性细胞死亡配体1(pd-l1)的抗体及其用途
EP17837300.7A EP3495391A4 (fr) 2016-08-05 2017-08-07 Anticorps dirigé contre le ligand 1 de mort programmée (pd-l1) et son utilisation
RU2019105664A RU2721582C1 (ru) 2016-08-05 2017-08-07 Антитела против лиганда-1 запрограммированной смерти (PD-L1) и их применение
US16/321,412 US10919966B2 (en) 2016-08-05 2017-08-07 Antibody to programmed death-ligand 1 (PD-L1) and use thereof

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JP2021517904A (ja) * 2018-04-10 2021-07-29 ワイ−バイオロジクス・インコーポレイテッド 細胞エンゲージ結合分子
JP7076571B2 (ja) 2018-04-10 2022-05-27 ワイ－バイオロジクス・インコーポレイテッド 細胞エンゲージ結合分子
WO2023187460A1 (fr) * 2022-04-01 2023-10-05 Mabtree Biologics Ag Anticorps humain ou fragment de liaison à l'antigène de celui-ci spécifique contre pd-l1 pour améliorer la fonction des lymphocytes t

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