WO2018026249A1 - Antibody against programmed death-ligand 1 (pd-l1), and use thereof - Google Patents

Antibody against programmed death-ligand 1 (pd-l1), and use thereof 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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Prior art keywords
seq
light chain
variable region
heavy chain
chain variable
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PCT/KR2017/008495
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French (fr)
Korean (ko)
Inventor
박재은
최수아
이지수
이현미
이시형
백기선
김응철
박범찬
임정채
조영규
박영우
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주식회사 와이바이오로직스
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Priority to RU2019105664A priority Critical patent/RU2721582C1/en
Priority to US16/321,412 priority patent/US10919966B2/en
Priority to CA3032806A priority patent/CA3032806C/en
Priority to AU2017306507A priority patent/AU2017306507B2/en
Priority to CN201780055412.7A priority patent/CN110072889B/en
Priority to BR112019002282A priority patent/BR112019002282A2/en
Priority to JP2019528010A priority patent/JP6925421B2/en
Priority to EP17837300.7A priority patent/EP3495391A4/en
Priority claimed from KR1020170099673A external-priority patent/KR102048477B1/en
Publication of WO2018026249A1 publication Critical patent/WO2018026249A1/en

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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K39/395Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28Immunoglobulins [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

The present invention relates to an antibody against programmed death-ligand 1 (PD-L1) or an antigen-binding fragment thereof, a nucleic acid for coding same, a vector comprising the nucleic acid, a cell transformed with the vector, a method for preparing the antibody or the antigen-binding fragment thereof, and a composition, comprising same, for preventing or treating a cancer or an infectious disease.

Description

프로그램화된 세포 사멸 단백질 리간드-1 (PD-L1)에 대한 항체 및 이의 용도Antibodies to Programmed Cell Death Protein Ligand-1 (PD-L1) and Uses thereof
본 발명은 PD-L1(Programmed death-ligand 1)에 대한 항체 또는 이의 항원 결합 단편, 이를 코딩하는 핵산, 상기 핵산을 포함하는 벡터, 상기 벡터로 형질전환된 세포, 상기 항체 또는 그의 항원 결합 단편의 제조방법 및 이를 포함하는 암 또는 감염 질환의 예방 또는 치료용 조성물에 관한 것이다.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.
항원 특이 T-림프구 세포의 면역반응은 매우 복잡하고 정교하게 조절되는 과정이다. 우선 T-림프구의 활성화는 T-림프구 세포표면에 존재하는 T-림프구 항원 수용체(T-cell antigen receptor, TCR)가 항원 제시 세포(antigen presenting cell, APC)의 MHC(major histocompatibility complex, 사람의 경우 HLA (human leucocyte antigen) Class II 분자에 결합된 항원을 인지하는 것으로 시작된다. 이 때, T-림프구가 충분히 활성화하기 위해서는 항원의 인지와 동시에 자극(co-stimulatory) 신호가 필요한데 항원 제시 세포에서 발현하는 CD80, CD40 등이 T-림프구 세포 표면의 리간드에 해당하는 CD28, CD40L 등과 동시에 결합함으로써 이루어지며, 이를 통하여 사이토카인의 분비가 활성화된다. TCR-MHC/에피토프의 결합을 통한 항원의 인지가 이루어지더라도 동시 자극 신호의 신호 전달이 없을 경우 T-림프구의 활성은 이루어지지 않는다. The immune response of antigen-specific T-lymphocyte cells is a very complex and carefully regulated process. First, activation of T-lymphocytes requires that T-cell antigen receptors (TCRs) 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. 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.
그러나, 활성화된 T-림프구는 일정시간 후 비활성화가 되도록 동시에 억제(co-inhibitory) 신호 또한 활성화된다. 이를 통하여 과도한 면역자극으로 인한 조직 손상 등을 예방할 수 있다. 다양한 동시 억제 신호가 있으며 대표적으로 T-림프구의 CTLA(cytotoxic T lymphocyte antigen)-4와 PD-1(programmed death-1), 이에 상응하는 항원 제시 세포 리간드는 CD80 및 CD86과 PD-L1(programmed death-ligand 1)이 관여한다. CTLA-4의 경우 리간드인 CD80과 CD86과 결합을 통하여 naive 또는 memory T-림프구의 비활성화 기능을 주로 갖고 있으며 PD-1의 경우 PD-L1과 PD-L2를 통하여 말초조직에서 T-림프구 기능을 조절한다.However, 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. There are a variety of simultaneous suppression signals and typical cytotoxic T lymphocyte antigen (CTLA-4) and programmed death-1 (PD-1) and corresponding antigen-presenting cell ligands of T-lymphocytes are CD80 and CD86 and PD-L1 (programmed death). -ligand 1) is involved. 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.
우리 몸의 면역기능은 항원인지와 동시에 이러한 동시자극 신호 및 동시억제신호의 조절을 통하여 전체적인 T 림프구 기능을 조절한다. 이러한 조절 기전을 면역검문(immune checkpoint)라고 한다. 우리 몸의 면역기능은 종양세포에서 일어나는 돌연변이 등의 변화로 인해 발현되는 종양 특이 항원(tumor-specific neo-antigen)을 감지하고 이를 통하여 종양세포 또는 바이러스 감염원 등을 제거한다.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.
다만, 일부 종양세포는 반대로 이러한 면역공격을 회피하기 위하여 종양미세환경(tumor micro-environment)을 변화시켜 면역기능을 억제하거나 T세포 면역관용(immune tolerance) 또는 면역편집(immuno-editing) 등을 통하여 면역 회피(immune escape)를 한다. However, some tumor cells, on the contrary, in order to evade such immune attack, change the tumor micro-environment to suppress immune function, or through T-cell immune tolerance or immuno-editing. Immune escape
이러한 회피 전략의 하나로서 면역검문(immune checkpoint) 기능의 변화를 통하여 종양 특이 T 림프구 세포의 기능을 억제한다. 즉, 종양세포에서 이러한 억제 면역검문을 활성화시킴으로써 종양 특이 T-림프구 세포의 공격을 회피한다. 이와 관련하여, PD-1 또는 리간드 PD-L1에 대한 단클론항체를 이용하여 그 기능을 억제함으로써 억제된 종양 특이 T-림프구 세포 활성 및 효과를 증강시킴으로써 항종양 효과를 얻을 수 있다.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. In this regard, 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.
이러한 기술적 배경하에서, 본 출원의 발명자들은 PD-L1에 특이적으로 결합하는 항체를 개발하기 위하여 노력하였다. 그 결과, 본 발명자들은 PD-L1에 높은 친화력으로 결합하는 항-PD-L1 항체를 개발하고, 이러한 항-PD-L1 항체가 PD-1/PD-L1 복합체의 형성을 저해함으로써, 목적하는 면역항암제 또는 감염 질환 치료제의 역할을 할 수 있음을 확인하고, 본 발명을 완성하였다.Under this technical background, the inventors of the present application tried to develop an antibody that specifically binds to PD-L1. As a result, 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.
발명의 요약Summary of the Invention
본 발명의 목적은 PD-L1에 대한 신규 항체 또는 그의 항원 결합 단편을 제공하는 데 있다.It is an object of the present invention to provide a novel antibody or antigen binding fragment thereof against PD-L1.
본 발명의 다른 목적은 상기 항체 또는 그의 항원 결합 단편을 코딩하는 핵산을 제공하는 데 있다.Another object of the present invention is to provide a nucleic acid encoding the antibody or antigen-binding fragment thereof.
본 발명의 다른 목적은 상기 핵산을 포함하는 벡터, 상기 벡터로 형질전환된 세포 및 이의 제조방법을 제공하는 데 있다.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.
본 발명의 또 다른 목적은 상기 항체 또는 그의 항원 결합 단편을 포함하는 암 또는 감염 질환의 예방 또는 치료용 조성물을 제공하는 데 있다.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.
상기 목적을 달성하기 위하여, 본 발명은 서열번호 1 내지 서열번호 7로 구성된 군에서 선택되는 서열과 90% 이상의 서열 상동성을 가지는 서열을 포함하는 중쇄 CDR1, 서열번호 8 내지 서열번호 15로 구성된 군에서 선택되는 서열과 90% 이상의 서열 상동성을 가지는 서열을 포함하는 중쇄 CDR2, 및 서열번호 16 내지 서열번호 25로 구성된 군에서 선택되는 서열과 90% 이상의 서열 상동성을 가지는 서열을 포함하는 중쇄 CDR3를 포함하는 중쇄 가변영역, 및 서열번호 88 내지 서열번호 102로 구성된 군에서 선택되는 서열과 90% 이상의 서열 상동성을 가지는 서열을 포함하는 경쇄 CDR1, 서열번호 103 내지 서열번호 119로 구성된 군에서 선택되는 서열과 90% 이상의 서열 상동성을 가지는 서열을 포함하는 경쇄 CDR2, 및 서열번호 120 내지 서열번호 144로 구성된 군에서 선택되는 서열과 90% 이상의 서열 상동성을 가지는 서열을 포함하는 경쇄 CDR3을 포함하는 경쇄 가변영역을 포함하는, PD-L1에 결합하는 항체 또는 이의 항원 결합단편을 제공한다.In order to achieve the above object, 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 to 144 Provides an antibody or an antigen-binding fragment that binds to, PD-L1 comprising a light chain variable region comprising a light chain CDR3 comprising a sequence having a sequence that is at least 90% sequence homology.
본 발명은 또한, 상기 항체 또는 그의 항원 결합 단편을 코딩하는 핵산을 제공한다.The present invention also provides a nucleic acid encoding the antibody or antigen-binding fragment thereof.
본 발명은 또한, 상기 핵산을 포함하는 벡터를 제공한다.The present invention also provides a vector comprising the nucleic acid.
본 발명은 또한, 상기 벡터로 형질전환된 세포를 제공한다.The present invention also provides a cell transformed with the vector.
본 발명은 또한, 다음 단계를 포함하는 상기 항체 또는 그의 항원 결합 단편의 제조방법을 제공한다: (a) 상기 세포를 배양하는 단계; 및 (b) 상기 배양된 세포에서 항체 또는 그의 항원 결합 단편을 회수하는 단계.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.
본 발명은 또한, 상기 항체 또는 그의 항원 결합 단편을 유효성분으로 포함하는 암 또는 감염 질환의 예방 또는 치료용 조성물을 제공한다.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.
도 1은 PD-L1 발현 벡터에 대한 모식도이다. 1 is a schematic diagram of a PD-L1 expression vector.
도 2는 PD-L1 단백질 정제 결과를 나타낸 것이다:2 shows the results of PD-L1 protein purification:
도 2a는 PD-L1-hFc의 10% SDS-PAGE gel. RE (reducing)과 NR (non-reducing) 조건에서의 단백질 확인 결과를 나타낸 것이다;2A is a 10% SDS-PAGE gel of PD-L1-hFc. Results of protein identification under RE (reducing) and NR (non-reducing) conditions;
도 2b는 G-3000 SWXL SEC-HPLC 결과를 나타낸 것이다. 유속은 1 ml/min이고 전개용매는 PBS이다;Figure 2b shows the G-3000 SWXL SEC-HPLC results. Flow rate is 1 ml / min and developing solvent is PBS;
도 2c는 PD-L1-mFc의 10% SDS-PAGE gel. RE (reducing)과 NR (non-reducing) 조건에서의 단백질 확인 결과를 나타낸 것이다;2C is a 10% SDS-PAGE gel of PD-L1-mFc. Results of protein identification under RE (reducing) and NR (non-reducing) conditions;
도 2d는 G-3000 SWXL SEC-HPLC 결과를 나타낸 것이다. 유속은 1 ml/min이고 전개용매는 PBS이다.Figure 2d shows the G-3000 SWXL SEC-HPLC results. The flow rate is 1 ml / min and the developing solvent is PBS.
도 3은 패닝 횟수에 따른 PD-L1 항원에 대한 결합력 증가 결과를 나타낸 것이다. Figure 3 shows the result of increasing the binding force for PD-L1 antigen according to the number of panning.
도 4는 PD-L1-His에만 결합능이 강한 모노 파아지의 결합능 측정을 위한 ELSIA 결과를 나타낸 것이다.Figure 4 shows the ELSIA results for measuring the binding capacity of monophages strong binding capacity only PD-L1-His.
도 5는 선별된 PD-L1 항체를 SDS-PAGE를 통해 확인한 결과를 나타낸 것이다.Figure 5 shows the result of confirming the selected PD-L1 antibody through SDS-PAGE.
도 6은 PD-L1 항체의 in vitro 효능 평가 결과를 나타낸 것이다.Figure 6 shows the results of in vitro efficacy evaluation of PD-L1 antibody.
도 7은 PD-L1 항체의 농도 의존적인 in vitro 효능 평가 결과를 나타낸 것이다.Figure 7 shows the results of the concentration-dependent in vitro efficacy evaluation of PD-L1 antibody.
도 8은 PD-L1 과발현 세포에서 PD-L1 항체의 결합력 측정 결과를 나타낸 것이다.Figure 8 shows the results of measuring the binding force of the PD-L1 antibody in PD-L1 overexpressing cells.
도 9는 PD-L1-hFc와 PD-L1-16E12 간의 키네틱 (kinetics) 측정 결과를 나타낸 것이다.Figure 9 shows the results of the kinetic (kinetics) measurement between PD-L1-hFc and PD-L1-16E12.
도 10은 최적화 단일 클론을 선별한 결과를 나타낸 것이다.10 shows the results of screening for optimized monoclones.
도 11은 본 발명에 따른 PD-L1 항체의 in vitro 효능을 평가한 결과를 나타낸 것이다.Figure 11 shows the results of evaluating the in vitro efficacy of the PD-L1 antibody according to the present invention.
도 12는 본 발명에 따른 PD-L1 항체의 농도 의존적인 in vitro 효능을 평가한 결과를 나타낸 것이다.Figure 12 shows the results of evaluating the concentration-dependent in vitro efficacy of the PD-L1 antibody according to the present invention.
도 13은 PD-L1 과발현 세포에서 항체의 결합력을 측정한 결과를 나타낸 것이다.Figure 13 shows the results of measuring the binding force of the antibody in PD-L1 overexpressing cells.
도 14는 선별된 항체에서 효소면역흡착을 이용한 PD-1/PD-L1 복합체의 형성을 막는 항체의 저해 효과를 확인한 결과를 나타낸 것이다.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.
도 15는 PD-L1-hFc와 PD-L1-16E12-4F5간의 키네틱 (kinetics) 측정 결과를 나타낸 것이다.FIG. 15 shows kinetic measurements of PD-L1-hFc and PD-L1-16E12-4F5. FIG.
도 16은 PD-L1 변이체 단백질 및 단일 클론 항체의 결합 측정 결과를 나타낸 것이다.Figure 16 shows the binding measurement results of PD-L1 variant protein and monoclonal antibody.
도 17은 PD-L1 단일 클론 항체에 의해 이종 MLR (Mixed Lymphocyte Reaction)에서 활성 증가를 나타냄을 확인한 결과이다. Figure 17 shows that the PD-L1 monoclonal antibody shows an increase in activity in heterologous MLR (Mixed Lymphocyte Reaction).
도 18은 선택된 PD-L1 단일 클론 항체의 동계 (syngeneic) 암동물 모델에서의 효능 평가 결과를 나타낸 것이다.18 shows the results of efficacy evaluation of syngeneic cancer animal models of selected PD-L1 monoclonal antibodies.
도 19는 본 발명에 따른 항 PD-L1 항체의 PD-L2와 결합 여부를 확인한 결과를 나타낸 것이다. Figure 19 shows the results confirming the binding of the anti-PD-L1 antibody according to the invention with PD-L2.
발명의 상세한 설명 및 바람직한 Detailed description of the invention and preferred 구현예Embodiment
다른 식으로 정의되지 않는 한, 본 명세서에서 사용된 모든 기술적 및 과학적 용어들은 본 발명이 속하는 기술분야에서 숙련된 전문가에 의해서 통상적으로 이해되는 것과 동일한 의미를 갖는다. 일반적으로, 본 명세서에서 사용된 명명법은 본 기술분야에서 잘 알려져 있고 통상적으로 사용되는 것이다.Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.
본 발명은 일 관점에서, 서열번호 1 내지 서열번호 7로 구성된 군에서 선택되는 서열과 90% 이상의 서열 상동성을 가지는 서열을 포함하는 중쇄 CDR1, 서열번호 8 내지 서열번호 15로 구성된 군에서 선택되는 서열과 90% 이상의 서열 상동성을 가지는 서열을 포함하는 중쇄 CDR2, 및 서열번호 16 내지 서열번호 25로 구성된 군에서 선택되는 서열과 90% 이상의 서열 상동성을 가지는 서열을 포함하는 중쇄 CDR3를 포함하는 중쇄 가변영역, 및 서열번호 88 내지 서열번호 102로 구성된 군에서 선택되는 서열과 90% 이상의 서열 상동성을 가지는 서열을 포함하는 경쇄 CDR1, 서열번호 103 내지 서열번호 119로 구성된 군에서 선택되는 서열과 90% 이상의 서열 상동성을 가지는 서열을 포함하는 경쇄 CDR2, 및 서열번호 120 내지 서열번호 144로 구성된 군에서 선택되는 서열과 90% 이상의 서열 상동성을 가지는 서열을 포함하는 경쇄 CDR3을 포함하는 경쇄 가변영역을 포함하는, PD-L1에 결합하는 항체 또는 이의 항원 결합단편에 관한 것이다.In one aspect, 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, 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: 16-25 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 A light chain comprising a variable region comprising a light chain CDR3 comprising a sequence having a sequence homology, the present invention relates to an antibody or antigen-binding fragment thereof that binds to PD-L1.
본 명세서의 "PD-L1"는 활성화된 T 및 B 세포 상에서 주로 발현하는 면역억제 수용체 “프로그램된 사멸 수용체 1(PD-1)”에 대한 리간드로, PD-1과 리간드인 PD-L1 및/또는 PD-L2이 결합하는 경우 항원 수용체 시그날링을 부정적으로 조절할 수 있다. PD-1에 대한 리간드(PD-L1 및 PD-L2)는 구성적으로 발현되거나, 또는 비-조혈세포 조직 및 다양한 종양형을 포함하는 다수의 세포형으로 유도될 수 있다. PD-L1은 B 세포, T 세포, 골수세포 및 수지상 세포(DCs) 상에서 발현되지만, 또한 말초 세포, 유사 미세혈관 내피세포 및 심장, 폐 등과 같은 비-림프 기관 상에서도 발현된다. 대조적으로, PD-L2는 단지 대식세포 및 수지상 세포 상에서만 발견된다. PD-1 리간드의 발현 패턴은, 말초 내성을 유지하는데 있어 PD-1의 역할을 제시하고, 말초에서 자가-반응성 T-세포 및 B-세포 반응을 조절하는데 기여할 수 있다. 리간드 둘 다는 세포외 영역에서 IgV- 및 IgC-유사 도메인 둘 다를 포함하는 제I형 이동막 수용체이다. 리간드 둘다는 공지되지 않은 시그날링 모티프를 가진 짧은 세포질 영역을 포함한다. “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 (PD-L1 and PD-L2) 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. In contrast, 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.
다수의 연구는 PD-1과 이의 리간드의 상호작용이 시험관내 및 생체내에서 림프구 증식을 억제함을 밝혔다. PD-1/PD-L1 상호작용의 혼란은 T 세포 증식 및 사이토킨 생산을 증가시키고, 세포 주기의 진행을 차단하는 것으로 알려졌다. PD-1/PD-L1 상호작용의 차단은 증강된 종양-특이적 T-세포 면역을 유도할 수 있으므로, 면역 시스템에 의해 종양 세포를 청소하는데 도움이 될 수 있다. 또한, 만성 HIV 감염시, HIV-특이적 CD8+ T 세포는 기능적으로 손상되고, 사이토카인 및 이펙터 분자를 생산하는 능력의 감소 및 증식하는 능력의 감소를 나타내고, PD-1는 HIV 감염된 개체의 HIV 특이적 CD8+ T 세포에 고발현되는데, PD-1/PD-L1 상호작용의 차단을 통해 HIV 펩티드 자극에 반응하여 HIV 특이적 T 세포가 증식하고 사이토카인을 생산하는 능력을 향상시킴으로써, T 세포 활성 또는 항 바이러스 면역 반응을 증대시킬 수 있다.Many studies have shown that the interaction of PD-1 with its ligand inhibits lymphocyte proliferation in vitro and in vivo. Disruption of the PD-1 / PD-L1 interaction has been known to increase T cell proliferation and cytokine production and block cell cycle progression. Blocking of the PD-1 / PD-L1 interaction may induce enhanced tumor-specific T-cell immunity and may therefore help to clean up tumor cells by the immune system. In addition, upon chronic HIV infection, HIV-specific CD8 + T cells are functionally impaired, exhibiting a decrease in the ability to produce cytokine and effector molecules and a proliferation ability, while PD-1 is HIV specific in HIV infected individuals. It is highly expressed on enemy CD8 + T cells, by blocking the PD-1 / PD-L1 interaction, enhancing the ability of HIV specific T cells to proliferate and produce cytokines in response to HIV peptide stimulation, thereby enhancing T cell activity or Can increase the antiviral immune response.
본 명세서에서 사용된 용어, "항체(antibody)"는 PD-L1에 특이적으로 결합하는 항-PD-L1 항체를 의미한다. 본 발명의 범위에는 PD-L1에 특이적으로 결합하는 완전한 항체 형태 뿐 아니라, 상기 항체 분자의 항원 결합 단편도 포함된다.As used herein, the term "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.
완전한 항체는 2개의 전체 길이의 경쇄 및 2개의 전체 길이의 중쇄를 가지는 구조이며 각각의 경쇄는 중쇄와 다이설파이드 결합으로 연결되어 있다. 중쇄 불변영역은 감마(γ), 뮤(μ), 알파(α), 델타(δ) 및 엡실론(ε) 타입을 가지고 서브클래스로 감마1(γ1), 감마2(γ2), 감마3(γ3), 감마4(γ4), 알파1(α1) 및 알파2(α2)를 가진다. 경쇄의 불변영역은 카파(κ) 및 람다(λ) 타입을 가진다.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 (γ), mu (μ), alpha (α), delta (δ) and epsilon (ε) types and subclasses gamma 1 (γ1), gamma 2 (γ2), and gamma 3 (γ3). ), Gamma 4 (γ4), alpha 1 (α1) and alpha 2 (α2). The constant regions of the light chains have kappa (κ) and lambda (λ) types.
항체의 항원 결합 단편 또는 항체 단편이란 항원 결합 기능을 보유하고 있는 단편을 의미하며, Fab, F(ab'), F(ab')2 및 Fv 등을 포함한다. 항체 단편 중 Fab는 경쇄 및 중쇄의 가변영역과 경쇄의 불변영역 및 중쇄의 첫 번째 불변영역(CH1)을 가지는 구조로 1개의 항원 결합 부위를 가진다. Fab'는 중쇄 CH1 도메인의 C-말단에 하나 이상의 시스테인 잔기를 포함하는 힌지 영역(hingeAn 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
region)을 가진다는 점에서 Fab와 차이가 있다. F(ab')2 항체는 Fab'의 힌지 영역의 시스테인 잔기가 디설파이드 결합을 이루면서 생성된다. Fv는 중쇄 가변영역 및 경쇄 가변영역만을 가지고 있는 최소의 항체조각으로 Fv 단편을 생성하는 재조합 기술은 PCT 국제 공개특허출원 WO88/10649, WO88/106630, WO88/07085, WO88/07086 및 WO88/09344에 개시되어 있다. 이중쇄 Fv(two-chain Fv)는 비공유 결합으로 중쇄 가변영역과 경쇄 가변영역이 연결되어 있고 단쇄 Fv(single-chain Fv, scFv)는 일반적으로 펩타이드 링커를 통하여 중쇄의 가변영역과 경쇄의 가변영역이 공유결합으로 연결되거나 또는 C-말단에서 바로 연결되어 있어서 이중쇄 Fv와 같이 다이머와 같은 구조를 이룰 수 있다. 이러한 항체 단편은 단백질 가수분해 효소를 이용해서 얻을 수 있고(예를 들어, 전체 항체를 파파인으로 제한 절단하면 Fab를 얻을 수 있고 펩신으로 절단하면 F(ab')2 단편을 얻을 수 있다), 유전자 재조합 기술을 통하여 제작할 수도 있다.It differs from Fab in that it has a region. 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.
하나의 실시예에서, 본 발명에 따른 항체는 Fv 형태(예컨대, scFv)이거나, 완전한 항체 형태이다. 또한, 중쇄 불변영역은 감마(γ), 뮤(μ), 알파(α), 델타(δ) 또는 엡실론(ε) 중의 어느 한 이소타입으로부터 선택될 수 있다. 예를 들어, 불변영역은 감마1(IgG1), 감마 3(IgG3) 또는 감마 4(IgG4)이다. 경쇄 불변영역은 카파 또는 람다 형일 수 있다.In one embodiment, the antibody according to the invention is in Fv form (eg scFv) or is in the form of a complete antibody. In addition, the heavy chain constant region may be selected from any one isotype of gamma (γ), mu (μ), alpha (α), delta (δ) or epsilon (ε). For example, 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.
본 명세서에서 사용되는 용어, "중쇄"는 항원에 특이성을 부여하기 위한 충분한 가변영역 서열을 갖는 아미노산 서열을 포함하는 가변영역 도메인 VH 및 3 개의 불변영역 도메인 CH1, CH2 및 CH3을 포함하는 전체길이 중쇄 및 이의 단편을 모두 의미한다. 또한, 본 명세서에서 사용되는 용어, "경쇄"는 항원에 특이성을 부여하기 위한 충분한 가변영역 서열을 갖는 아미노산 서열을 포함하는 가변영역 도메인 VL 및 불변영역 도메인 CL을 포함하는 전체길이 경쇄 및 이의 단편을 모두 의미한다.As used herein, the term “heavy chain” means full length including variable region domain V H and three constant region domains CH1, CH2 and CH3, including an amino acid sequence having sufficient variable region sequence to confer specificity to the antigen. Both heavy chain and fragments thereof. In addition, the term "light chain" as used herein 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.
본 발명의 항체는 단일클론 항체, 다특이적 항체, 인간 항체, 인간화 항체, 키메라 항체, 단쇄 Fvs(scFV), 단쇄 항체, Fab 단편, F(ab') 단편, 다이설파이드-결합 Fvs(sdFV) 및 항-이디오타입(항-Id) 항체, 또는 상기 항체들의 에피토프-결합 단편 등을 포함하나, 이에 한정되는 것은 아니다.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.
상기 단일클론 항체는 실질적으로 동질적 항체 집단으로부터 수득한 항체, 즉 집단을 차지하고 있는 개개의 항체가 미량으로 존재할 수 있는 가능한 천연 발생적 돌연변이를 제외하고는 동일한 것을 지칭한다. 단일클론 항체는 고도로 특이적이어서, 단일 항원 부위에 대항하여 유도된다. 전형적으로 상이한 결정인자(에피토프)에 대해 지시된 상이한 항체를 포함하는 통상의 (폴리클로날) 항체 제제와는 대조적으로, 각각의 모노클로날 항체는 항원 상의 단일 결정인자에 대해 지시된다.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.
"에피토프"은 항체가 특이적으로 결합할 수 있는 단백질 결정부위 (determinant)를 의미한다. 에피토프는 통상 화학적으로 활성인 표면 분자군, 예를 들어 아미노산 또는 당 측쇄로 구성되며, 일반적으로 특정한 3차원의 구조적 특징뿐만 아니라 특정한 전하 특성을 갖는다. 입체적 에피토프 및 비입체적 에피토프는 변성 용매의 존재하에서 전자에 대한 결합은 소실되지만 후자에 대해서는 소실되지 않는다는 점에서 구별된다. "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.
상기 "인간화" 형태의 비-인간 (예: 뮤린) 항체는 비-인간 면역글로불린으로부터 유래된 최소 서열을 함유하는 키메라 항체이다. 대부분의 경우, 인간화 항체는, 수용자의 초가변 영역으로부터의 잔기를 목적하는 특이성, 친화성 및 능력을 보유하고 있는 비-인간 종 (공여자 항체), 예를 들어 마우스, 랫트, 토끼 또는 비-인간 영장류의 초가변 영역로부터의 잔기로 대체시킨 인간 면역글로불린 (수용자 항체)이다.Non-human (eg murine) antibodies of the “humanized” form are chimeric antibodies that contain minimal sequences derived from non-human immunoglobulins. In most cases, 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. Human immunoglobulins (receptor antibodies) replaced with residues from the hypervariable regions of primates.
상기 “인간 항체”는 인간 면역글로불린으로부터 유래하는 분자로서 상보성 결정영역, 구조 영역을 포함한 항체를 구성하는 모든 아미노산 서열 전체가 인간의 면역글로불린으로 구성되어 있는 것을 의미한다.The term “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.
중쇄 및/또는 경쇄 일부가 특별한 종으로부터 유래되거나 특별한 항체 부류 또는 아부류에 속하는 항체 내의 상응하는 서열과 동일하거나 이와 상동성인 반면, 나머지 쇄(들)는 또 다른 종으로부터 유래되거나 또 다른 항체 부류 또는 아부류에 속하는 항체 내의 상응하는 서열과 동일하거나 이와 상동성인 "키메라" 항체 (면역글로불린) 뿐 아니라 목적하는 생물학적 활성을 나타내는 상기 항체의 단편이 포함된다.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.
본원에 사용된 바와 같은 "항체 가변 도메인"은 상보성 결정 영역 (CDR; 즉, CDR1, CDR2, 및 CDR3), 및 골격 영역 (FR)의 아미노산 서열을 포함하는 항체 분자의 경쇄 및 중쇄 부분을 지칭한다. VH는 중쇄의 가변 도메인을 지칭한다. VL은 경쇄의 가변 도메인을 지칭한다.As used herein, “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). . V H refers to the variable domain of the heavy chain. V L refers to the variable domain of the light chain.
"상보성 결정 영역” (CDR; 즉, CDR1, CDR2, 및 CDR3)은 항원 결합을 위해 필요한 존재인, 항체 가변 도메인의 아미노산 잔기를 지칭한다. 각 가변 도메인은 전형적으로, CDR1, CDR2 및 CDR3으로서 확인된 3개의 CDR 영역을 갖는다. 본 발명은 서열번호 1의 중쇄 CDR3를 포함하는 중쇄 가변영역 및 서열번호 2의 경쇄 CDR3를 포함하는 경쇄 가변영역을 포함한다. “Complementarity Determining Regions” (CDRs; ie CDR1, CDR2, and CDR3) refer to amino acid residues of antibody variable domains that are required for antigen binding, each variable domain typically identified as CDR1, CDR2 and CDR3. 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.
본 발명에 있어, 상기 PD-L1에 결합하는 항체 또는 이의 항원 결합단편은 서열번호 1 내지 서열번호 7로 구성된 군에서 선택되는 중쇄 CDR1, 서열번호 8 내지 서열번호 15로 구성된 군에서 선택되는 중쇄 CDR2, 및 서열번호 16 내지 서열번호 25로 구성된 군에서 선택되는 중쇄 CDR3를 포함하는 중쇄 가변영역, 및 서열번호 88 내지 서열번호 102로 구성된 군에서 선택되는 경쇄 CDR1, 서열번호 103 내지 서열번호 119로 구성된 군에서 선택되는 경쇄 CDR2, 및 서열번호 120 내지 서열번호 144로 구성된 군에서 선택되는 경쇄 CDR3을 포함하는 경쇄 가변영역을 포함할 수 있다. In the present invention, 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.
구체적으로, 본 발명에 있어, 상기 PD-L1에 결합하는 항체 또는 이의 항원 결합단편은 서열번호 1의 중쇄 CDR1, 서열번호 8의 중쇄 CDR2, 및 서열번호 16의 중쇄 CDR3을 포함하는 중쇄 가변영역, Specifically, in the present invention, 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,
서열번호 2의 중쇄 CDR1, 서열번호 9의 중쇄 CDR2, 및 서열번호 17의 중쇄 CDR3을 포함하는 중쇄 가변영역,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,
서열번호 2의 중쇄 CDR1, 서열번호 9의 중쇄 CDR2, 및 서열번호 18의 중쇄 CDR3을 포함하는 중쇄 가변영역,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,
서열번호 3의 중쇄 CDR1, 서열번호 10의 중쇄 CDR2, 및 서열번호 19의 중쇄 CDR3을 포함하는 중쇄 가변영역,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,
서열번호 4의 중쇄 CDR1, 서열번호 11의 중쇄 CDR2, 및 서열번호 20의 중쇄 CDR3을 포함하는 중쇄 가변영역,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,
서열번호 5의 중쇄 CDR1, 서열번호 12의 중쇄 CDR2, 및 서열번호 21의 중쇄 CDR3을 포함하는 중쇄 가변영역,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,
서열번호 6의 중쇄 CDR1, 서열번호 13의 중쇄 CDR2, 및 서열번호 22의 중쇄 CDR3을 포함하는 중쇄 가변영역,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,
서열번호 2의 중쇄 CDR1, 서열번호 9의 중쇄 CDR2, 및 서열번호 23의 중쇄 CDR3을 포함하는 중쇄 가변영역,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,
서열번호 7의 중쇄 CDR1, 서열번호 14의 중쇄 CDR2, 및 서열번호 24의 중쇄 CDR3을 포함하는 중쇄 가변영역, 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,
서열번호 2의 중쇄 CDR1, 서열번호 15의 중쇄 CDR2, 및 서열번호 25의 중쇄 CDR3을 포함하는 중쇄 가변영역, 또는A 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
서열번호 2의 중쇄 CDR1, 서열번호 9의 중쇄 CDR2, 및 서열번호 17의 중쇄 CDR3을 포함하는 중쇄 가변영역을 포함할 수 있다. 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.
또한, 상기 PD-L1에 결합하는 항체 또는 이의 항원 결합단편은 서열번호 88의 경쇄 CDR1, 서열번호 103의 경쇄 CDR2, 및 서열번호 120의 경쇄 CDR3를 포함하는 경쇄 가변영역, In addition, 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,
서열번호 89의 경쇄 CDR1, 서열번호 104의 경쇄 CDR2, 및 서열번호 121의 경쇄 CDR3를 포함하는 경쇄 가변영역, 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,
서열번호 90의 경쇄 CDR1, 서열번호 105의 경쇄 CDR2, 및 서열번호 122의 경쇄 CDR3를 포함하는 경쇄 가변영역, 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,
서열번호 91의 경쇄 CDR1, 서열번호 106의 경쇄 CDR2, 및 서열번호 123의 경쇄 CDR3를 포함하는 경쇄 가변영역, 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,
서열번호 89의 경쇄 CDR1, 서열번호 107의 경쇄 CDR2, 및 서열번호 124의 경쇄 CDR3를 포함하는 경쇄 가변영역, 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,
서열번호 92의 경쇄 CDR1, 서열번호 108의 경쇄 CDR2, 및 서열번호 122의 경쇄 CDR3를 포함하는 경쇄 가변영역, 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,
서열번호 93의 경쇄 CDR1, 서열번호 109의 경쇄 CDR2, 및 서열번호 125의 경쇄 CDR3를 포함하는 경쇄 가변영역, 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,
서열번호 94의 경쇄 CDR1, 서열번호 110의 경쇄 CDR2, 및 서열번호 126의 경쇄 CDR3를 포함하는 경쇄 가변영역, 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,
서열번호 95의 경쇄 CDR1, 서열번호 111의 경쇄 CDR2, 및 서열번호 127의 경쇄 CDR3를 포함하는 경쇄 가변영역, 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,
서열번호 96의 경쇄 CDR1, 서열번호 112의 경쇄 CDR2, 및 서열번호 128의 경쇄 CDR3를 포함하는 경쇄 가변영역, 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,
서열번호 89의 경쇄 CDR1, 서열번호 108의 경쇄 CDR2, 및 서열번호 129의 경쇄 CDR3를 포함하는 경쇄 가변영역, 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,
서열번호 89의 경쇄 CDR1, 서열번호 105의 경쇄 CDR2, 및 서열번호 130의 경쇄 CDR3를 포함하는 경쇄 가변영역, 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,
서열번호 89의 경쇄 CDR1, 서열번호 113의 경쇄 CDR2, 및 서열번호 131의 경쇄 CDR3를 포함하는 경쇄 가변영역, 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,
서열번호 97의 경쇄 CDR1, 서열번호 104의 경쇄 CDR2, 및 서열번호 132의 경쇄 CDR3를 포함하는 경쇄 가변영역, 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,
서열번호 89의 경쇄 CDR1, 서열번호 104의 경쇄 CDR2, 및 서열번호 133의 경쇄 CDR3를 포함하는 경쇄 가변영역, 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,
서열번호 97의 경쇄 CDR1, 서열번호 114의 경쇄 CDR2, 및 서열번호 134의 경쇄 CDR3를 포함하는 경쇄 가변영역, 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,
서열번호 92의 경쇄 CDR1, 서열번호 115의 경쇄 CDR2, 및 서열번호 135의 경쇄 CDR3를 포함하는 경쇄 가변영역, 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,
서열번호 98의 경쇄 CDR1, 서열번호 104의 경쇄 CDR2, 및 서열번호 130의 경쇄 CDR3를 포함하는 경쇄 가변영역, 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,
서열번호 89의 경쇄 CDR1, 서열번호 116의 경쇄 CDR2, 및 서열번호 121의 경쇄 CDR3를 포함하는 경쇄 가변영역, 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,
서열번호 89의 경쇄 CDR1, 서열번호 108의 경쇄 CDR2, 및 서열번호 136의 경쇄 CDR3를 포함하는 경쇄 가변영역, 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,
서열번호 99의 경쇄 CDR1, 서열번호 105의 경쇄 CDR2, 및 서열번호 137의 경쇄 CDR3를 포함하는 경쇄 가변영역, 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,
서열번호 89의 경쇄 CDR1, 서열번호 117의 경쇄 CDR2, 및 서열번호 138의 경쇄 CDR3를 포함하는 경쇄 가변영역, 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,
서열번호 89의 경쇄 CDR1, 서열번호 118의 경쇄 CDR2, 및 서열번호 133의 경쇄 CDR3를 포함하는 경쇄 가변영역, 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,
서열번호 89의 경쇄 CDR1, 서열번호 119의 경쇄 CDR2, 및 서열번호 139의 경쇄 CDR3를 포함하는 경쇄 가변영역, 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,
서열번호 100의 경쇄 CDR1, 서열번호 104의 경쇄 CDR2, 및 서열번호 140의 경쇄 CDR3를 포함하는 경쇄 가변영역, 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,
서열번호 89의 경쇄 CDR1, 서열번호 108의 경쇄 CDR2, 및 서열번호 141의 경쇄 CDR3를 포함하는 경쇄 가변영역, 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,
서열번호 89의 경쇄 CDR1, 서열번호 105의 경쇄 CDR2, 및 서열번호 139의 경쇄 CDR3를 포함하는 경쇄 가변영역, 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,
서열번호 89의 경쇄 CDR1, 서열번호 104의 경쇄 CDR2, 및 서열번호 142의 경쇄 CDR3를 포함하는 경쇄 가변영역, 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,
서열번호 89의 경쇄 CDR1, 서열번호 105의 경쇄 CDR2, 및 서열번호 143의 경쇄 CDR3를 포함하는 경쇄 가변영역, 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,
서열번호 101의 경쇄 CDR1, 서열번호 104의 경쇄 CDR2, 및 서열번호 141의 경쇄 CDR3를 포함하는 경쇄 가변영역, 또는A 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
서열번호 102의 경쇄 CDR1, 서열번호 104의 경쇄 CDR2, 및 서열번호 144의 경쇄 CDR3를 포함하는 경쇄 가변영역을 포함할 수 있다.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.
본 발명에 따른 일 실시예에서, 본 발명에 따른 항체 또는 이의 항원 결합단편은 다음을 포함할 수 있다: In one embodiment according to the invention, the antibody or antigen binding fragment thereof according to the invention may comprise:
서열번호 1의 중쇄 CDR1, 서열번호 8의 중쇄 CDR2, 및 서열번호 16의 중쇄 CDR3를 포함하는 중쇄 가변영역 및 서열번호 88의 경쇄 CDR1, 서열번호 103의 경쇄 CDR2, 및 서열번호 120의 경쇄 CDR3를 포함하는 경쇄 가변영역;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;
서열번호 2의 중쇄 CDR1, 서열번호 9의 중쇄 CDR2, 및 서열번호 17의 중쇄 CDR3를 포함하는 중쇄 가변영역 및 서열번호 89의 경쇄 CDR1, 서열번호 104의 경쇄 CDR2, 및 서열번호 121의 경쇄 CDR3를 포함하는 경쇄 가변영역;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;
서열번호 2의 중쇄 CDR1, 서열번호 9의 중쇄 CDR2, 및 서열번호 18의 중쇄 CDR3를 포함하는 중쇄 가변영역 및 서열번호 90의 경쇄 CDR1, 서열번호 105의 경쇄 CDR2, 및 서열번호 122의 경쇄 CDR3를 포함하는 경쇄 가변영역;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;
서열번호 3의 중쇄 CDR1, 서열번호 10의 중쇄 CDR2, 및 서열번호 19의 중쇄 CDR3를 포함하는 중쇄 가변영역 및 서열번호 91의 경쇄 CDR1, 서열번호 106의 경쇄 CDR2, 및 서열번호 123의 경쇄 CDR3를 포함하는 경쇄 가변영역;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;
서열번호 4의 중쇄 CDR1, 서열번호 11의 중쇄 CDR2, 및 서열번호 20의 중쇄 CDR3를 포함하는 중쇄 가변영역 및 서열번호 89의 경쇄 CDR1, 서열번호 107의 경쇄 CDR2, 및 서열번호 124의 경쇄 CDR3를 포함하는 경쇄 가변영역;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;
서열번호 5의 중쇄 CDR1, 서열번호 12의 중쇄 CDR2, 및 서열번호 21의 중쇄 CDR3를 포함하는 중쇄 가변영역 및 서열번호 92의 경쇄 CDR1, 서열번호 108의 경쇄 CDR2, 및 서열번호 122의 경쇄 CDR3를 포함하는 경쇄 가변영역;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;
서열번호 6의 중쇄 CDR1, 서열번호 13의 중쇄 CDR2, 및 서열번호 22의 중쇄 CDR3를 포함하는 중쇄 가변영역 및 서열번호 93의 경쇄 CDR1, 서열번호 109의 경쇄 CDR2, 및 서열번호 125의 경쇄 CDR3를 포함하는 경쇄 가변영역;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;
서열번호 2의 중쇄 CDR1, 서열번호 9의 중쇄 CDR2, 및 서열번호 23의 중쇄 CDR3를 포함하는 중쇄 가변영역 및 서열번호 94의 경쇄 CDR1, 서열번호 110의 경쇄 CDR2, 및 서열번호 126의 경쇄 CDR3를 포함하는 경쇄 가변영역;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;
서열번호 7의 중쇄 CDR1, 서열번호 14의 중쇄 CDR2, 및 서열번호 24의 중쇄 CDR3를 포함하는 중쇄 가변영역 및 서열번호 95의 경쇄 CDR1, 서열번호 111의 경쇄 CDR2, 및 서열번호 127의 경쇄 CDR3를 포함하는 경쇄 가변영역; 또는A 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
서열번호 2의 중쇄 CDR1, 서열번호 15의 중쇄 CDR2, 및 서열번호 25의 중쇄 CDR3를 포함하는 중쇄 가변영역 및 서열번호 96의 경쇄 CDR1, 서열번호 112의 경쇄 CDR2, 및 서열번호 128의 경쇄 CDR3를 포함하는 경쇄 가변영역.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.
본 발명의 일 실시예에 따라, 최적화 과정을 통해 항체를 추가 선별하였으며, 본 발명에 따른 항체 또는 이의 항원 결합단편은 다음의 중쇄 가변영역 및 경쇄 가변영역을 포함할 수 있다:According to one embodiment of the present invention, 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:
서열번호 2의 중쇄 CDR1, 서열번호 9의 중쇄 CDR2, 및 서열번호 17의 중쇄 CDR3를 포함하는 중쇄 가변영역 및 서열번호 89의 경쇄 CDR1, 서열번호 108의 경쇄 CDR2, 및 서열번호 129의 경쇄 CDR3를 포함하는 경쇄 가변영역;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;
서열번호 2의 중쇄 CDR1, 서열번호 9의 중쇄 CDR2, 및 서열번호 17의 중쇄 CDR3를 포함하는 중쇄 가변영역 및 서열번호 89의 경쇄 CDR1, 서열번호 105의 경쇄 CDR2, 및 서열번호 130의 경쇄 CDR3를 포함하는 경쇄 가변영역;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;
서열번호 2의 중쇄 CDR1, 서열번호 9의 중쇄 CDR2, 및 서열번호 17의 중쇄 CDR3를 포함하는 중쇄 가변영역 및 서열번호 89의 경쇄 CDR1, 서열번호 113의 경쇄 CDR2, 및 서열번호 131의 경쇄 CDR3를 포함하는 경쇄 가변영역;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;
서열번호 2의 중쇄 CDR1, 서열번호 9의 중쇄 CDR2, 및 서열번호 17의 중쇄 CDR3를 포함하는 중쇄 가변영역 및 서열번호 97의 경쇄 CDR1, 서열번호 104의 경쇄 CDR2, 및 서열번호 132의 경쇄 CDR3를 포함하는 경쇄 가변영역;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;
서열번호 2의 중쇄 CDR1, 서열번호 9의 중쇄 CDR2, 및 서열번호 17의 중쇄 CDR3를 포함하는 중쇄 가변영역 및 서열번호 89의 경쇄 CDR1, 서열번호 104의 경쇄 CDR2, 및 서열번호 133의 경쇄 CDR3를 포함하는 경쇄 가변영역;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;
서열번호 2의 중쇄 CDR1, 서열번호 9의 중쇄 CDR2, 및 서열번호 17의 중쇄 CDR3를 포함하는 중쇄 가변영역 및 서열번호 97의 경쇄 CDR1, 서열번호 114의 경쇄 CDR2, 및 서열번호 134의 경쇄 CDR3를 포함하는 경쇄 가변영역;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;
서열번호 2의 중쇄 CDR1, 서열번호 9의 중쇄 CDR2, 및 서열번호 17의 중쇄 CDR3를 포함하는 중쇄 가변영역 및 서열번호 92의 경쇄 CDR1, 서열번호 115의 경쇄 CDR2, 및 서열번호 135의 경쇄 CDR3를 포함하는 경쇄 가변영역;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;
서열번호 2의 중쇄 CDR1, 서열번호 9의 중쇄 CDR2, 및 서열번호 17의 중쇄 CDR3를 포함하는 중쇄 가변영역 및 서열번호 98의 경쇄 CDR1, 서열번호 104의 경쇄 CDR2, 및 서열번호 130의 경쇄 CDR3를 포함하는 경쇄 가변영역;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;
서열번호 2의 중쇄 CDR1, 서열번호 9의 중쇄 CDR2, 및 서열번호 17의 중쇄 CDR3를 포함하는 중쇄 가변영역 및 서열번호 89의 경쇄 CDR1, 서열번호 116의 경쇄 CDR2, 및 서열번호 121의 경쇄 CDR3를 포함하는 경쇄 가변영역;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;
서열번호 2의 중쇄 CDR1, 서열번호 9의 중쇄 CDR2, 및 서열번호 17의 중쇄 CDR3를 포함하는 중쇄 가변영역 및 서열번호 89의 경쇄 CDR1, 서열번호 108의 경쇄 CDR2, 및 서열번호 136의 경쇄 CDR3를 포함하는 경쇄 가변영역;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;
서열번호 2의 중쇄 CDR1, 서열번호 9의 중쇄 CDR2, 및 서열번호 17의 중쇄 CDR3를 포함하는 중쇄 가변영역 및 서열번호 99의 경쇄 CDR1, 서열번호 105의 경쇄 CDR2, 및 서열번호 137의 경쇄 CDR3를 포함하는 경쇄 가변영역;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;
서열번호 2의 중쇄 CDR1, 서열번호 9의 중쇄 CDR2, 및 서열번호 17의 중쇄 CDR3를 포함하는 중쇄 가변영역 및 서열번호 89의 경쇄 CDR1, 서열번호 117의 경쇄 CDR2, 및 서열번호 138의 경쇄 CDR3를 포함하는 경쇄 가변영역;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;
서열번호 2의 중쇄 CDR1, 서열번호 9의 중쇄 CDR2, 및 서열번호 17의 중쇄 CDR3를 포함하는 중쇄 가변영역 및 서열번호 89의 경쇄 CDR1, 서열번호 118의 경쇄 CDR2, 및 서열번호 133의 경쇄 CDR3를 포함하는 경쇄 가변영역;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;
서열번호 2의 중쇄 CDR1, 서열번호 9의 중쇄 CDR2, 및 서열번호 17의 중쇄 CDR3를 포함하는 중쇄 가변영역 및 서열번호 89의 경쇄 CDR1, 서열번호 119의 경쇄 CDR2, 및 서열번호 139의 경쇄 CDR3를 포함하는 경쇄 가변영역;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;
서열번호 2의 중쇄 CDR1, 서열번호 9의 중쇄 CDR2, 및 서열번호 17의 중쇄 CDR3를 포함하는 중쇄 가변영역 및 서열번호 100의 경쇄 CDR1, 서열번호 104의 경쇄 CDR2, 및 서열번호 140의 경쇄 CDR3를 포함하는 경쇄 가변영역;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;
서열번호 2의 중쇄 CDR1, 서열번호 9의 중쇄 CDR2, 및 서열번호 17의 중쇄 CDR3를 포함하는 중쇄 가변영역 및 서열번호 89의 경쇄 CDR1, 서열번호 108의 경쇄 CDR2, 및 서열번호 141의 경쇄 CDR3를 포함하는 경쇄 가변영역;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;
서열번호 2의 중쇄 CDR1, 서열번호 9의 중쇄 CDR2, 및 서열번호 17의 중쇄 CDR3를 포함하는 중쇄 가변영역 및 서열번호 89의 경쇄 CDR1, 서열번호 105의 경쇄 CDR2, 및 서열번호 139의 경쇄 CDR3를 포함하는 경쇄 가변영역;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;
서열번호 2의 중쇄 CDR1, 서열번호 9의 중쇄 CDR2, 및 서열번호 17의 중쇄 CDR3를 포함하는 중쇄 가변영역 및 서열번호 89의 경쇄 CDR1, 서열번호 104의 경쇄 CDR2, 및 서열번호 142의 경쇄 CDR3를 포함하는 경쇄 가변영역;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;
서열번호 2의 중쇄 CDR1, 서열번호 9의 중쇄 CDR2, 및 서열번호 17의 중쇄 CDR3를 포함하는 중쇄 가변영역 및 서열번호 89의 경쇄 CDR1, 서열번호 105의 경쇄 CDR2, 및 서열번호 143의 경쇄 CDR3를 포함하는 경쇄 가변영역;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;
서열번호 2의 중쇄 CDR1, 서열번호 9의 중쇄 CDR2, 및 서열번호 17의 중쇄 CDR3를 포함하는 중쇄 가변영역 및 서열번호 101의 경쇄 CDR1, 서열번호 104의 경쇄 CDR2, 및 서열번호 141의 경쇄 CDR3를 포함하는 경쇄 가변영역; 또는 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
서열번호 2의 중쇄 CDR1, 서열번호 9의 중쇄 CDR2, 및 서열번호 17의 중쇄 CDR3를 포함하는 중쇄 가변영역 및 서열번호 102의 경쇄 CDR1, 서열번호 104의 경쇄 CDR2, 및 서열번호 144의 경쇄 CDR3를 포함하는 경쇄 가변영역.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.
구체적으로, 본 발명에 따른 항체 또는 이의 항원 결합단편은 서열번호 2의 중쇄 CDR1, 서열번호 9의 중쇄 CDR2, 및 서열번호 17의 중쇄 CDR3를 포함하는 중쇄 가변영역 및 서열번호 89의 경쇄 CDR1, 서열번호 104의 경쇄 CDR2, 및 서열번호 121의 경쇄 CDR3를 포함하는 경쇄 가변영역;Specifically, the antibody or antigen-binding fragment thereof according to the present invention 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;
서열번호 2의 중쇄 CDR1, 서열번호 9의 중쇄 CDR2, 및 서열번호 17의 중쇄 CDR3를 포함하는 중쇄 가변영역 및 서열번호 89의 경쇄 CDR1, 서열번호 105의 경쇄 CDR2, 및 서열번호 130의 경쇄 CDR3를 포함하는 경쇄 가변영역;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;
서열번호 2의 중쇄 CDR1, 서열번호 9의 중쇄 CDR2, 및 서열번호 17의 중쇄 CDR3를 포함하는 중쇄 가변영역 및 서열번호 89의 경쇄 CDR1, 서열번호 104의 경쇄 CDR2, 및 서열번호 133의 경쇄 CDR3를 포함하는 경쇄 가변영역;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;
서열번호 2의 중쇄 CDR1, 서열번호 9의 중쇄 CDR2, 및 서열번호 17의 중쇄 CDR3를 포함하는 중쇄 가변영역 및 서열번호 89의 경쇄 CDR1, 서열번호 108의 경쇄 CDR2, 및 서열번호 136의 경쇄 CDR3를 포함하는 경쇄 가변영역;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;
서열번호 2의 중쇄 CDR1, 서열번호 9의 중쇄 CDR2, 및 서열번호 17의 중쇄 CDR3를 포함하는 중쇄 가변영역 및 서열번호 89의 경쇄 CDR1, 서열번호 119의 경쇄 CDR2, 및 서열번호 139의 경쇄 CDR3를 포함하는 경쇄 가변영역;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;
서열번호 2의 중쇄 CDR1, 서열번호 9의 중쇄 CDR2, 및 서열번호 17의 중쇄 CDR3를 포함하는 중쇄 가변영역 및 서열번호 89의 경쇄 CDR1, 서열번호 105의 경쇄 CDR2, 및 서열번호 139의 경쇄 CDR3를 포함하는 경쇄 가변영역; 또는A 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
서열번호 2의 중쇄 CDR1, 서열번호 9의 중쇄 CDR2, 및 서열번호 17의 중쇄 CDR3를 포함하는 중쇄 가변영역 및 서열번호 89의 경쇄 CDR1, 서열번호 105의 경쇄 CDR2, 및 서열번호 143의 경쇄 CDR3를 포함하는 경쇄 가변영역을 포함할 수 있다. 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.
"골격 영역" (FR)은 CDR 잔기 이외의 가변 도메인 잔기이다. 각 가변 도메인은 전형적으로, FR1, FR2, FR3 및 FR4로서 확인된 4개의 FR을 가진다."Framework regions" (FRs) are variable domain residues other than CDR residues. Each variable domain typically has four FRs identified as FR1, FR2, FR3 and FR4.
본 발명의 일 실시예에 따르면, 서열번호 26 내지 서열번호 34로 구성된 군에서 선택되는 중쇄 가변영역 FR1, According to one embodiment of the present invention, the heavy chain variable region FR1 selected from the group consisting of SEQ ID NO: 26 to SEQ ID NO: 34,
서열번호 35 내지 서열번호 41로 구성된 군에서 선택되는 중쇄 가변영역 FR2, A heavy chain variable region FR2 selected from the group consisting of SEQ ID NOs: 35 to 41;
서열번호 42 내지 서열번호 49로 구성된 군에서 선택되는 중쇄 가변영역 FR3, 또는 A heavy chain variable region FR3 selected from the group consisting of SEQ ID NOs: 42 to 49, or
서열번호 50 내지 서열번호 54로 구성된 군에서 선택되는 중쇄 가변영역 FR4를 포함할 수 있다. It may comprise a heavy chain variable region FR4 selected from the group consisting of SEQ ID NO: 50 to SEQ ID NO: 54.
또한, 서열번호 145 내지 서열번호 163로 구성된 군에서 선택되는 경쇄 가변영역 FR1, In addition, the light chain variable region FR1 selected from the group consisting of SEQ ID NOs: 145 to 163,
서열번호 164 내지 서열번호 184로 구성된 군에서 선택되는 경쇄 가변영역 FR2, Light chain variable region FR2 selected from the group consisting of SEQ ID NOs: 164 to 184,
서열번호 185 내지 서열번호 210로 구성된 군에서 선택되는 경쇄 가변영역 FR3, 또는 Light chain variable region FR3 selected from the group consisting of SEQ ID NOs: 185 to 210, or
서열번호 211 내지 서열번호 216로 구성된 군에서 선택되는 경쇄 가변영역 FR4를 포함할 수 있다.The light chain variable region FR4 selected from the group consisting of SEQ ID NOs: 211 to 216 may be included.
"Fv" 단편은 완전한 항체 인식 및 결합 부위를 함유하는 항체 단편이다. 이러한 영역은 1개의 중쇄 가변 도메인과 1개의 경쇄 가변 도메인이, 예를 들어 scFv로 단단하게 사실상 공유적으로 연합된 이량체로 이루어진다."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" 단편은 경쇄의 가변 및 불변 도메인과, 중쇄의 가변 및 제1 불변 도메인 (CH1)을 함유한다. F(ab')2 항체 단편은 일반적으로 그들 사이에 힌지 시스테인에 의해 그들의 카복시 말단 근처에 공유적으로 연결되는 한 쌍의 Fab 단편을 포함한다."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.
"단일쇄 Fv" 또는 "scFv" 항체 단편은 항체의 VH 및 VL 도메인을 포함하는데, 이들 도메인은 단일 폴리펩티드 쇄 내에 존재한다. Fv 폴리펩티드는 scFv가 항원 결합을 위해 목적하는 구조를 형성할 수 있도록 하는 VH 도메인과 VL 도메인 사이에 폴리펩티드 링커를 추가로 포함할 수 있다."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.
PD-L1 항체는 1가 또는 2가이고, 단쇄 또는 이중 쇄를 포함한다. 기능적으로, PD-L1 항체의 결합 친화성은 10-5M 내지 10-12 M 범위 내에 있다. 예를 들어, PD-L1 항체의 결합 친화성은 10-6 M 내지 10-12 M, 10-7 M 내지 10-12 M, 10-8 M 내지 10-12 M, 10-9 M 내지 10-12 M, 10-5 M 내지 10-11 M, 10-6 M 내지 10-11 M, 10-7 M 내지 10-11 M, 10-8 M 내지 10-11 M, 10-9 M 내지 10-11 M, 10-10 M 내지 10-11 M, 10-5 M 내지 10-10 M, 10-6 M 내지 10-10 M, 10-7 M 내지 10-10 M, 10-8 M 내지 10-10 M, 10-9 M 내지 10-10 M, 10-5 M 내지 10-9 M, 10-6 M 내지 10-9 M, 10-7 M 내지 10-9 M, 10-8 M 내지 10-9 M, 10-5 M 내지 10-8 M, 10-6 M 내지 10-8 M, 10-7 M 내지 10-8 M, 10-5 M 내지 10-7 M, 10-6 M 내지 10-7 M 또는 10-5 M 내지 10-6 M이다.PD-L1 antibodies are monovalent or bivalent and include single or double chains. Functionally, the binding affinity of the PD-L1 antibody is in the range of 10 −5 M to 10 −12 M. For example, 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 -5 M to 10 -8 M, 10 -6 M to 10 -8 M, 10 -7 M to 10 -8 M, 10 -5 M to 10 -7 M, 10 -6 M to 10 -7 M or 10 −5 M to 10 −6 M.
상기 PD-L1에 결합하는 항체 또는 이의 항원 결합단편은 서열번호 57 내지 서열번호 87로 구성된 군에서 선택되는 서열과 90% 이상의 서열 상동성을 가지는 서열을 포함하는 중쇄 가변영역을 포함할 수 있다. 상기 PD-L1에 결합하는 항체 또는 이의 항원 결합단편은 서열번호 57 내지 서열번호 87로 구성된 군에서 선택되는 중쇄 가변영역을 포함할 수 있다. 본 발명의 일 실시예에 따르면, 서열번호 58, 68, 71, 76, 80, 83 또는 85의 중쇄 가변영역을 포함할 수 있다. 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. According to an embodiment of the present invention, the heavy chain variable region of SEQ ID NO: 58, 68, 71, 76, 80, 83 or 85 may be included.
또한, 상기 PD-L1에 결합하는 항체 또는 이의 항원 결합단편은 서열번호 217 내지 서열번호 247로 구성된 군에서 선택되는 서열과 90% 이상의 서열 상동성을 가지는 서열을 포함하는 경쇄 가변영역을 포함할 수 있다. 상기 PD-L1에 결합하는 항체 또는 이의 항원 결합단편은 서열번호 217 내지 서열번호 247로 구성된 군에서 선택되는 경쇄 가변영역을 포함할 수 있다. 본 발명의 일 실시예에 따르면, 서열번호 218, 228, 231, 236, 240, 243 또는 245의 경쇄 가변영역을 포함할 수 있다. In addition, 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. According to one embodiment of the present invention, the light chain variable region of SEQ ID NO: 218, 228, 231, 236, 240, 243 or 245 may be included.
본 발명에 따른 구체적 실시예에서, 서열번호 58의 중쇄 가변영역 및 서열번호 218의 경쇄 가변영역;In a specific embodiment according to the present invention, the heavy chain variable region of SEQ ID NO: 58 and the light chain variable region of SEQ ID NO: 218;
서열번호 68의 중쇄 가변영역 및 서열번호 228의 경쇄 가변영역;A heavy chain variable region of SEQ ID NO: 68 and a light chain variable region of SEQ ID NO: 228;
서열번호 71의 중쇄 가변영역 및 서열번호 231의 경쇄 가변영역;A heavy chain variable region of SEQ ID NO: 71 and a light chain variable region of SEQ ID NO: 231;
서열번호 76의 중쇄 가변영역 및 서열번호 236의 경쇄 가변영역;A heavy chain variable region of SEQ ID NO: 76 and a light chain variable region of SEQ ID NO: 236;
서열번호 80의 중쇄 가변영역 및 서열번호 240의 경쇄 가변영역;A heavy chain variable region of SEQ ID NO: 80 and a light chain variable region of SEQ ID NO: 240;
서열번호 83의 중쇄 가변영역 및 서열번호 243의 경쇄 가변영역; 또는A heavy chain variable region of SEQ ID NO: 83 and a light chain variable region of SEQ ID NO: 243; or
서열번호 85의 중쇄 가변영역 및 서열번호 245의 경쇄 가변영역을 포함할 수 있다. The heavy chain variable region of SEQ ID NO: 85 and the light chain variable region of SEQ ID NO: 245 may be included.
"파지 디스플레이"는 변이체 폴리펩티드를 파지, 예를 들어 섬유상 파지 입자의 표면 상에 외피 단백질의 적어도 일부와의 융합 단백질로서 디스플레이하는 기술이다. 파지 디스플레이의 유용성은 무작위화 단백질 변이체의 큰 라이브러리를 대상으로 하여, 표적 항원과 고 친화도로 결합하는 서열을 신속하고도 효율적으로 분류할 수 있다는 사실에 있다. 펩티드 및 단백질 라이브러리를 파지 상에 디스플레이하는 것은 특이적 결합 특성을 지닌 폴리펩티드를 알아보기 위해 수 백만개의 폴리펩티드를 스크리닝하는데 사용되어 왔다."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.
파지 디스플레이 기술은 특정 리간드 (예: 항원)와 결합하는 신규 단백질을 생성 및 선별하기 위한 강력한 도구를 제공하였다. 파지 디스플레이 기술을 사용하여, 단백질 변이체의 큰 라이브러리를 생성시키고, 표적 항원과 고 친화성으로 결합하는 서열을 신속하게 분류할 수 있다. 변이체 폴리펩티드를 암호화하는 핵산을 바이러스성 외피 단백질, 예를 들어 유전자 III 단백질 또는 유전자 VIII 단백질을 암호화하는 핵산 서열과 융합시킨다. 단백질 또는 폴리펩티드를 암호화하는 핵산 서열을 유전자 III 단백질의 일부를 암호화하는 핵산 서열과 융합시킨 1가 파지 디스플레이 시스템이 개발되었다. 1가 파지 디스플레이 시스템에서는, 유전자 융합물이 저 수준으로 발현되고 야생형 유전자 III 단백질이 또한 발현되어 입자 감염성이 유지된다.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.
섬유상 파지 표면 상에서의 펩티드의 발현과 E. coli의 주변세포질에서의 기능성 항체 단편의 발현을 입증하는 것이 항체 파지 디스플레이 라이브러리를 개발하는 데에 있어 중요하다. 항체 또는 항원 결합성 폴리펩티드의 라이브러리는 수 많은 방식, 예를 들어 무작위 DNA 서열을 삽입함으로써 단일 유전자를 변경시키는 방법 또는 관련 유전자 계열을 클로닝하는 방법으로 제조하였다. 라이브러리를 대상으로 하여, 목적하는 특징을 수반한 항체 또는 항원 결합성 단백질의 발현에 관하여 스크리닝할 수 있다.Demonstrating the expression of peptides on fibrous phage surfaces and the expression of functional antibody fragments in the periplasm of E. coli is important in developing antibody phage display libraries. Libraries of antibody or antigen-binding polypeptides have been prepared in a number of ways, for example by altering a single gene by inserting random DNA sequences or by cloning related gene families. Libraries can be screened for expression of antibodies or antigen binding proteins with the desired characteristics.
파지 디스플레이 기술은 목적하는 특징을 지닌 항체를 제조하기 위한 통상적인 하이브리도마 및 재조합 방법에 비해 몇 가지 이점을 지니고 있다. 이러한 기술은 동물을 사용하지 않고서도 짧은 시간에 다양한 서열을 지닌 큰 항체 라이브러리를 생성시킬 수 있도록 한다. 하이브리도마의 제조나 인간화 항체의 제조는 수 개월의 제조기간을 필요로 할 수 있다. 또한, 면역이 전혀 요구되지 않기 때문에, 파지 항체 라이브러리는 독성이거나 항원성이 낮은 항원에 대해서도 항체를 생성시킬 수 있다. 파지 항체 라이브러리를 또한 사용하여 신규한 치료적 항체를 생성 및 확인할 수 있다.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.
파지 디스플레이 라이브러리를 사용하여 면역시킨, 비-면역시킨 인간, 생식세포계 서열, 또는 미감작 B 세포 Ig 레퍼토리 (repertory)로부터 인간 항체를 생성시키는 기술을 사용할 수 있다. 각종 림프계 조직을 사용하여, 미감작 또는 비면역 항원 결합성 라이브러리를 제조할 수 있다. Techniques for generating human antibodies from immunized, non-immunized human, germline sequences, or naïve B cell Ig repertory using immunized phage display libraries can be used. Various lymphoid tissues can be used to prepare naïve or non-immune antigen binding libraries.
파지 디스플레이 라이브러리로부터 고친화성 항체를 확인 및 분리할 수 있는 기술은 치료용 신규 항체 분리에 중요하다. 라이브러리로부터 고친화성 항체를 분리하는 것은 라이브러리의 크기, 세균성 세포 중에서의 생산 효율 및 라이브러리의 다양성에 좌우될 수 있다. 라이브러리의 크기는 항체 또는 항원 결합성 단백질의 부적절한 폴딩과 정지 코돈의 존재로 인한 비효율적 생산에 의해 감소된다. 세균성 세포에서의 발현은 항체 또는 항원 결합성 도메인이 적절하게 폴딩되지 않는 경우에는 억제될 수 있다. 발현은 가변/불변 계면의 표면이나 선별된 CDR 잔기에서의 잔기를 교대로 돌연변이시킴으로써 개선시킬 수 있다. 골격 영역의 서열은 세균성 세포에서 항체 파지 라이브러리를 생성시키는 경우에 적절한 폴딩을 제공하기 위한 하나의 요소이다.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.
고 친화성 항체 분리에서 항체 또는 항원 결합성 단백질의 다양한 라이브러리를 생성시키는 것이 중요하다. CDR3 영역은 이들이 종종 항원 결합에 참여하는 것으로 밝혀졌다. 중쇄 상의 CDR3 영역은 크기, 서열 및 구조적 입체 형태 면에서 상당히 다양하므로, 이를 이용하여 다양한 라이브러리를 제조할 수 있다.It is important to generate various libraries of antibodies or antigen binding proteins in high affinity antibody isolation. 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.
또한, 각 위치에서 20개 아미노산 모두를 사용하여 가변 중쇄 및 경쇄의 CDR 영역을 무작위화함으로써 다양성을 발생시킬 수 있다. 20개의 모든 아미노산을 사용하면 다양성이 큰 변이체 항체 서열이 생성되고 신규한 항체를 확인할 기회가 증가할 수 있다.In addition, 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.
본 발명의 항체 또는 항체 단편은 PD-L1을 특이적으로 인식할 수 있는 범위 내에서, 본 명세서에 기재된 본 발명의 항-PD-L1 항체의 서열뿐만 아니라, 이의 생물학적 균등물도 포함할 수 있다. 예를 들면, 항체의 결합 친화도 및/또는 기타 생물학적 특성을 보다 더 개선시키기 위하여 항체의 아미노산 서열에 추가적인 변화를 줄 수 있다. 이러한 변형은 예를 들어, 항체의 아미노산 서열 잔기의 결실, 삽입 및/또는 치환을 포함한다. 이러한 아미노산 변이는 아미노산 곁사슬 치환체의 상대적 유사성, 예컨대, 소수성, 친수성, 전하, 크기 등에 기초하여 이루어진다. 아미노산 곁사슬 치환체의 크기, 모양 및 종류에 대한 분석에 의하여, 아르기닌, 라이신과 히스티딘은 모두 양전하를 띤 잔기이고; 알라닌, 글라이신과 세린은 유사한 크기를 가지며; 페닐알라닌, 트립토판과 타이로신은 유사한 모양을 갖는다는 것을 알 수 있다. 따라서, 이러한 고려 사항에 기초하여, 아르기닌, 라이신과 히스티딘; 알라닌, 글라이신과 세린; 그리고 페닐알라닌, 트립토판과 타이로신은 생물학적으로 기능 균등물이라 할 수 있다.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. For example, 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. By analysis of the size, shape and type of amino acid side chain substituents, 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. Thus, based on these considerations, arginine, lysine and histidine; Alanine, glycine and serine; Phenylalanine, tryptophan and tyrosine are biologically equivalent functions.
상술한 생물학적 균등 활성을 갖는 변이를 고려한다면, 본 발명의 항체 또는 이를 코딩하는 핵산 분자는 서열번호에 기재된 서열과 실질적인 동일성(substantial identity)을 나타내는 서열도 포함하는 것으로 해석된다. 상기의 실질적인 동일성은, 상기한 본 발명의 서열과 임의의 다른 서열을 최대한 대응되도록 얼라인하고, 당업계에서 통상적으로 이용되는 알고리즘을 이용하여 얼라인된 서열을 분석한 경우에, 최소 90%의 상동성, 가장 바람직하게는 최소 95%의 상동성, 96% 이상, 97% 이상, 98% 이상, 99% 이상의 상동성을 나타내는 서열을 의미한다. 서열비교를 위한 얼라인먼트 방법은 당업계에 공지되어 있다. NCBI Basic Local Alignment Search Tool(BLAST)은 NBCI 등에서 접근 가능하며, 인터넷 상에서 blastp, blasm, blastx, tblastn 및 tblastx와 같은 서열 분석 프로그램과 연동되어 이용할 수 있다. BLSAT는 www.ncbi.nlm.nih.gov/BLAST/에서 접속 가능하다. 이 프로그램을 이용한 서열 상동성 비교 방법은 www.ncbi.nlm.nih.gov/BLAST/blast_ help.html에서 확인할 수 있다.Considering the above-described variations having biologically equivalent activity, 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. By 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. 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.
이에 기초하여, 본 발명의 항체 또는 그의 항원 결합 단편은 명세서에 기재된 명시된 서열 또는 전체와 비교하여 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 또는 그 이상의 상동성을 가질 수 있다. 이러한 상동성은 당업계에 공지된 방법에 의한 서열 비교 및/또는 정렬에 의해 결정될 수 있다. 예를 들어, 서열 비교 알고리즘(즉, BLAST 또는 BLAST 2.0), 수동 정렬, 육안 검사를 이용하여 본 발명의 핵산 또는 단백질의 퍼센트 서열 상동성을 결정할 수 있다. Based on this, 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. Such 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.
본 발명은 다른 관점에서, 상기 항체 또는 그의 항원 결합 단편을 코딩하는 핵산에 관한 것이다. In another aspect, the present invention relates to a nucleic acid encoding the antibody or antigen-binding fragment thereof.
본 발명의 항체 또는 그의 항원 결합 단편을 코딩하는 핵산을 분리하여 항체 또는 그의 항원 결합 단편을 재조합적으로 생산할 수 있다. 핵산을 분리하고, 이를 복제 가능한 벡터 내로 삽입하여 추가로 클로닝하거나 (DNA의 증폭) 또는 추가로 발현시킨다. 이를 바탕으로, 본 발명은 또 다른 관점에서 상기 핵산을 포함하는 벡터에 관한 것이다. 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.
"핵산"는 DNA(gDNA 및 cDNA) 및 RNA 분자를 포괄적으로 포함하는 의미를 가지며, 핵산에서 기본 구성단위인 뉴클레오타이드는 자연의 뉴클레오타이드 뿐만 아니라, 당 또는 염기 부위가 변형된 유사체(analogue)도 포함한다. 본 발명의 중쇄 및 경쇄 가변영역을 코딩하는 핵산의 서열은 변형될 수 있다. 상기 변형은 뉴클레오타이드의 추가, 결실, 또는 비보존적 치환 또는 보존적 치환을 포함한다."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.
상기 항체를 암호화하는 DNA는 통상적인 과정을 사용하여 (예를 들어, 항체의 중쇄와 경쇄를 암호화하는 DNA와 특이적으로 결합할 수 있는 올리고뉴클레오티드 프로브를 사용함으로써) 용이하게 분리 또는 합성한다. 많은 벡터가 입수 가능하다. 벡터 성분에는 일반적으로, 다음 중의 하나 이상이 포함되지만, 그에 제한되지 않는다: 신호 서열, 복제 기점, 하나 이상의 마커 유 전자, 증강인자 요소, 프로모터, 및 전사 종결 서열.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.
본 명세서에서 사용되는 용어, "벡터"는 숙주세포에서 목적 유전자를 발현시키기 위한 수단으로 플라스미드 벡터; 코즈미드 벡터; 박테리오파지 벡터, 아데노바이러스 벡터, 레트로바이러스 벡터 및 아데노-연관 바이러스벡터 같은 바이러스 벡터 등을 포함한다. 상기 벡터에서 항체를 코딩하는 핵산은 프로모터와 작동적으로 연결되어 있다. As used herein, 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.
“작동적으로 연결”은 핵산 발현조절서열(예: 프로모터, 시그널 서열, 또는 전사조절인자 결합 위치의 어레이)과 다른 핵산 서열사이의 기능적인 결합을 의미하며, 이에 의해 상기 조절서열은 상기 다른 핵산 서열의 전사 및/또는 해독을 조절하게 된다.“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.
원핵세포를 숙주로 하는 경우에는, 전사를 진행시킬 수 있는 강력한 프로모터(예컨대, tac 프로모터, lac 프로모터, lacUV5 프로모터, lpp 프로모터, pLλ 프로모터, pRλ프로모터, rac5 프로모터, amp 프로모터, recA 프로모터, SP6 프로모터, trp 프로모터 및 T7 프로모터 등), 해독의 개시를 위한 라이보좀 결합 자리 및 전사/해독 종결 서열을 포함하는 것이 일반적이다. 또한, 예를 들어, 진핵 세포를 숙주로 하는 경우에는, 포유동물 세포의 지놈으로부터 유래된 프로모터(예: 메탈로티오닌 프로모터, β-액틴 프로모터, 사람 헤로글로빈 프로모터 및 사람 근육 크레아틴 프로모터) 또는 포유동물 바이러스로부터 유래된 프로모터(예: 아데노바이러스 후기 프로모터, 백시니아 바이러스 7.5K 프로모터, SV40프로모터, 사이토메갈로바이러스(CMV) 프로모터, HSV의 tk 프로모터, 마우스 유방종양 바이러스(MMTV) 프로모터, HIV의 LTR 프로모터, 몰로니 바이러스의 프로모터엡스타인바 바이러스(EBV)의 프로모터 및 로우스 사코마 바이러스(RSV)의 프로모터)가 이용될 수 있으며, 전사 종결 서열로서 폴리아데닐화 서열을 일반적으로 갖는다.In the case of a prokaryotic cell as a host, powerful promoters capable of promoting transcription (e.g., tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pLλ promoter, pRλ promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter and T7 promoter, etc.), ribosome binding sites for initiation of translation, and transcription / detox termination sequences. Also, for example, when the eukaryotic cell is a host, a promoter derived from the genome of the mammalian cell (e.g., a metallothionine promoter, a β-actin promoter, a human heroglobin promoter and a human muscle creatine promoter) or 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) , Promoter of the Moroni virus Epsteinbar virus (EBV) and Loose Sacoma virus (RSV) promoter) can be used, and generally has a polyadenylation sequence as a transcription termination sequence.
경우에 따라서, 벡터는 그로부터 발현되는 항체의 정제를 용이하게 하기 위하여 다른 서열과 융합될 수도 있다. 융합되는 서열은, 예컨대 글루타티온 S-트랜스퍼라제(Pharmacia, USA), 말토스 결합 단백질(NEB, USA), FLAG(IBI, USA) 및 6x His(hexahistidine; Quiagen, USA) 등이 있다.In some cases, 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).
상기 벡터는 선택표지로서 당업계에서 통상적으로 이용되는 항생제 내성 유전자를 포함하며, 예를 들어 암피실린, 겐타마이신, 카베니실린, 클로람페니콜, 스트렙토마이신, 카나마이신, 게네티신, 네오마이신 및 테트라사이클린에 대한 내성 유전자가 있다.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.
본 발명은 또 다른 관점에서, 상기 언급된 벡터로 형질전환된 세포에 관한 것이다. 본 발명의 항체를 생성시키기 위해 사용된 세포는 원핵생물, 효모 또는 고등 진핵생물 세포일 수 있으며, 이에 제한되는 것은 아니다. In another aspect, 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.
에스케리치아 콜라이(Escherichia coli), 바실러스 서브틸리스 및 바실러스 츄린겐시스와 같은 바실러스 속 균주, 스트렙토마이세스(Streptomyces), 슈도모나스(Pseudomonas)(예를 들면, 슈도모나스 푸티다(Pseudomonas putida)), 프로테우스미라빌리스(Proteus mirabilis) 및 스타필로코쿠스(Staphylococcus)(예를 들면, 스타필로코쿠스 카르노수스(Staphylocus carnosus))와 같은 원핵 숙주세포를 이용할 수 있다.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.
다만, 동물 세포에 대한 관심이 가장 크며, 유용한 숙주 세포주의 예는 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, 또는 HT1080일 수 있으나, 이에 제한되는 것은 아니다.However, the greatest interest in animal cells, 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.
본 발명은 또 다른 관점에서, (a) 상기 세포를 배양하는 단계; 및 (b) 상기 배양된 세포에서 항체 또는 그의 항원 결합 단편을 회수하는 단계를 포함하는 상기 항체 또는 그의 항원 결합 단편의 제조방법에 관한 것이다. In another aspect, the present invention, (a) culturing the cells; And (b) recovering the antibody or antigen-binding fragment thereof from the cultured cells.
상기 세포는 각종 배지에서 배양할 수 있다. 시판용 배지 중 제한없이 배양 배지로서 사용할 수 있다. 당업자에게 공지되어 있는 기타 모든 필수 보충물이 적당한 농도로 포함될 수도 있다. 배양 조건, 예를 들어 온도, pH 등이 발현을 위해 선별된 숙주 세포와 함께 이미 사용되고 있고, 이는 당업자에게 명백할 것이다.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.
상기 항체 또는 그의 항원 결합 단편의 회수는 예를 들어 원심분리 또는 한외여과에 의해 불순물을 제거하고, 그 결과물을 예를 들어 친화 크로마토그래피 등을 이용하여 정제할 수 있다. 추가의 기타 정제 기술 예를 들어 음이온 또는 양이온 교환 크로마토그래피, 소수성 상호 작용 크로마토그래피, 히드록실아파타이트 크로마토그래피 등이 사용될 수 있다. 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.
본 발명은 또 다른 관점에서, 상기 항체를 유효성분으로 포함하는 암의 예방 또는 치료용 조성물에 관한 것이다.In still another aspect, the present invention relates to a composition for preventing or treating cancer, which comprises the antibody as an active ingredient.
본 발명은 예를 들어, (a) 본 발명에 따른 PD-L1에 대한 항체 또는 그의 항원 결합 단편의 약제학적 유효량; 및 (b) 약제학적으로 허용되는 담체를 포함하는 암 또는 감염 질환의 예방 또는 치료용 약제학적 조성물일 수 있다. 본 발명은 또한, 환자에게 필요한 유효량으로 본 발명에 따른 PD-L1에 대한 항체 또는 그의 항원 결합 단편을 투여하는 단계를 포함하는 암 또는 감염 질환의 예방 또는 치료방법에 관한 것이다.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.
상기 조성물은 상술한 본 발명의 항-PD-L1 항체 또는 그의 항원 결합 단편을 유효성분으로 이용하기 때문에, 이 둘 사이에 공통된 내용은 기재를 생략한다.Since the 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.
PD-1에 PD-L1의 결합은 자가면역 및 면역병리의 관용 및 예방에 중요한 T 세포 항원 특이적 반응을 음성적으로 조절한다. 그러나, 만성 항원 자극에 의해 유발될 수 있는 과도한 PD-L1/PD-1 상호작용은 T 세포 고갈의 특징이 되는, T 세포 항원 특이적 반응의 억제 및 T 세포의 소실을 가져올 수 있다. T 세포 고갈은 만성 감염 및 암에서 일어날 수 있는 T 세포 기능 장애의 상태이다. 이는 불량한 이펙터 기능, 억제성 수용체의 지속적 발현, 및 기능적 이펙터 또는 기억 T 세포와는 다른 전사 상태로서 정의된다. 고갈은 감염 및 종양 진행의 관리를 방해한다.The binding of PD-L1 to PD-1 negatively regulates T cell antigen specific responses important for the tolerance and prevention of autoimmunity and immunopathology. However, excessive PD-L1 / PD-1 interactions, which may be triggered by chronic antigen stimulation, can lead to inhibition of T cell antigen specific responses and loss of T cells, which are characteristic of T cell depletion. 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.
하기의 실시예에서 입증된 바와 같이, 본 발명의 항체 또는 그의 항원 결합 단편은 PD-L1에 높은 친화도로 결합하여 PD-1과 PD-L1 복합체 형성을 저해함으로써, 항-종양 T 세포 활성을 회피하는 T 세포 고갈을 유도하는 암을 치료하는 데에 유용하게 사용할 수 있다. As demonstrated in the examples below, 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.
경우에 따라서, 상기 항체 이외에 다른 항암 치료제를 병용함으로써 PD-L1을 과발현하는 종양 세포를 효과적으로 표적화하고, 항-종양 T 세포 활성을 증가시켜, 종양 세포를 표적화하는 면역 반응을 증대시킬 수 있다. 기타 항-신생물제 또는 면역원성 제제[(예를 들면, 약화된 암 세포, 종양 항원(재조합 단백질, 펩타이드 및 탄수화물 분자를 포함함), 항원 전달 세포, 예를 들면, 종양 기원된 항원 또는 핵산으로 펄스된 가지세포, 면역 자극 사이토킨(예를 들면, IL-2, IFNα2, GM-CSF), 및 면역 자극 사이토킨을 암호화하는 유전자로 형질감염된 세포(예를 들면, GM-CSF를 포함하지만 이에 제한되지 않는다)]; 표준 암 치료요법(예를 들면, 화학 치료요법, 방사선치료요법 또는 수술); 또는 기타 항체(VEGF, EGFR, Her2/neu, VEGF 수용체, 기타 성장 인자 수용체, CD20, CD40, CTLA-4, OX-40, 4-IBB, 및 ICOS를 포함하지만 이에 제한되지 않는다)와 함께 사용될 수 있다.In some cases, the use of other 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 Cell lines transfected with genes encoding immune stimulating cytokines (eg, IL-2, IFNα2, GM-CSF), and immune stimulating cytokines (eg, GM-CSF) Standard cancer therapy (eg chemotherapy, radiotherapy or surgery); or other antibodies (VEGF, EGFR, Her2 / neu, VEGF receptors, other growth factor receptors, CD20, CD40, CTLA) -4, OX-40, 4-IBB, and ICOS).
항 PD-L1 항체는 세포 사멸을 유도할 수 있다. 세포 사멸은 직접적 또는 간접적 기전에 의해 유도된다. 예를 들어, 항 PD-L1 항체에 의한 PD-L1 결합은 보체 의존성 세포독성(CDC)을 일으킬 수 있다. 경우에 따라, 항 PD-L1 항체는 PD-L1에 결합하고, PD-L1 발현 표적 세포를 사멸시킬 2차 세포 유형의 동원을 일으킨다. 항 PD-L1 항체가 2차 세포 유형의 동원에 의해 세포 사멸을 매개하는 대표적인 기전은 항체 의존성 세포독성(ADCC) 및 항체 의존성 세포 식균작용(ADCP)을 포함하나 이에 제한되지 않는다. 표적 PD-L1 발현 세포 유형은 종양 및 T 세포, 예를 들어 활성화된 T 세포를 포함한다.Anti PD-L1 antibodies can induce cell death. Cell death is induced by direct or indirect mechanisms. For example, PD-L1 binding by anti PD-L1 antibodies can result in complement dependent cytotoxicity (CDC). In some instances, 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.
또한, 본 발명의 항체 또는 항체 단편은 감염 및 감염 질환을 예방 또는 치료하는데 사용될 수 있다. In addition, the antibodies or antibody fragments of the invention can be used to prevent or treat infections and infectious diseases.
"예방"은 본 발명에 따른 조성물의 투여로 암 또는 감염 질환을 억제시키거나 진행을 지연시키는 모든 행위를 의미하며, "치료"는 암의 발전의 억제, 암의 경감 또는 암의 제거, 또는 감염질환의 억제, 감염질환의 경감 또는 감염질환의 제거를 의미한다."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, and "treatment" means inhibiting cancer development, reducing or eliminating cancer, or infection Inhibition of disease, reduction of infectious disease or removal of infectious disease.
상기 조성물에 적용되는 질환인 암은 전형적으로 면역치료요법에 반응하는 암, 및 지금까지 면역요법에 관련되지 않은 암을 포함한다. 치료용으로 바람직한 암의 비-제한적인 예는 흑색종(예를 들면, 전이성 악성 흑색종), 신장암(예를 들면, 투명세포암종), 전립선암(예를 들면, 호르몬 불응 전립선샘암종), 췌장샘암종, 유방암, 결장암, 폐암(예를 들면, 비-소세포 폐암), 식도암, 두경부편평세포암종, 간암, 난소암, 자궁경부암, 갑상샘암, 아교모세포종, 신경아교종, 백혈병, 림프종, 및 기타 신생물암종을 포함한다. 추가로, 본 발명은 본 발명의 항체를 사용하여 성장을 억제할 수 있는 불응 또는 재발 암을 포함한다. 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. In addition, the present invention includes refractory or recurrent cancer that can inhibit growth using the antibodies of the present invention.
항체 또는 항체 단편은 단독, 또는 백신과 함께 사용되어 병원체, 독소, 및 자가-항원에 대한 면역 반응을 자극시킬 수 있다. 항체 또는 이의 항원-결합 단편은 예를 들어, 사람 면역결핍 바이러스, 간염 바이러스 부류 A, B 및 C, 엡스테인 바르 바이러스(Eppstein Barr virus), 사람 사이토메갈로바이러스(cytomegalovirus), 사람 파필로마(papilloma) 바이러스, 헤르페스 바이러스를 포함하지만, 이에 제한되지 않는 사람을 전염시키는 바이러스에 대한 면역 반응을 자극시키는데 사용될 수 있다. 항체 또는 이의 항원-결합 단편은 세균 또는 진균 기생체, 및 기타 병원체의 감염에 대한 면역 반응을 자극시키는데 사용될 수 있다.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.
본 발명의 조성물에 포함되는 약제학적으로 허용되는 담체는 제제시에 통상적으로 이용되는 것으로서, 락토오스, 덱스트로오스, 수크로오스, 솔비톨, 만니톨, 전분, 아카시아 고무, 인산 칼슘, 알기네이트, 젤라틴, 규산칼슘, 미세결정성 셀룰로오스, 폴리비닐피롤리돈, 물, 시럽, 메틸 셀룰로스, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 활석, 스테아르산 마그네슘 및 미네랄 오일 등을 포함하나, 이에 한정되는 것은 아니다. 본 발명의 조성물은 상기 성분들 이외에 윤활제, 습윤제, 감미제, 향미제, 유화제, 현탁제, 보존제 등을 추가로 포함할 수 있다. Pharmaceutically acceptable carriers included in the 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.
본 발명의 약제학적 조성물은 경구 또는 비경구로 투여할 수 있고, 비경구 투여인 경우에는 정맥내 주입, 피하 주입, 근육 주입, 복강 주입, 내피 투여, 국소 투여, 비내 투여, 폐내 투여 및 직장 내 투여 등으로 투여할 수 있다.The pharmaceutical 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.
경구 투여시, 단백질 또는 펩타이드는 소화가 되기 때문에 경구용 조성물은 활성 약제를 코팅하거나 위에서의 분해로부터 보호되도록 제형화 되어야 한다. 또한, 약제학적 조성물은 활성 물질이 표적 세포로 이동할 수 있는 임의의 장치에 의해 투여될 수 있다.In oral administration, because proteins or peptides are digested, oral compositions should be formulated to coat the active agent or to protect it from degradation in the stomach. In addition, the pharmaceutical composition may be administered by any device in which the active agent may migrate to the target cell.
본 발명에 따른 조성물의 적합한 투여량은 제제화 방법, 투여 방식, 환자의 연령, 체중, 성, 병적 상태, 음식, 투여 시간, 투여 경로, 배설 속도 및 반응 감응성과 같은 요인들에 의해 다양하며, 보통으로 숙련된 의사는 소망하는 치료 또는 예방에 효과적인 투여량을 용이하게 결정 및 처방할 수 있다. 예를 들어, 본 발명의 약제학적 조성물의 1일 투여량은 0.0001-100 ㎎/㎏이다. 본 명세서에서 용어 "약제학적 유효량"은 암을 예방 또는 치료하는 데 충분한 양을 의미한다.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. For example, the daily dose of the pharmaceutical composition of the present invention is 0.0001-100 mg / kg. The term "pharmaceutically effective amount" as used herein means an amount sufficient to prevent or treat cancer.
본 발명의 약제학적 조성물은 당해 발명이 속하는 기술분야에서 통상의 지식을 가진 자가 용이하게 실시할 수 있는 방법에 따라, 약제학적으로 허용되는 담체 및/또는 부형제를 이용하여 제제화 함으로써 단위 용량 형태로 제조되거나 또는 다용량 용기 내에 내입시켜 제조될 수 있다. 이 때 제형은 오일 또는 수성 매질중의 용액, 현탁액 또는 유화액 형태이거나 엑스제, 산제, 좌제, 분말제, 과립제, 정제 또는 캅셀제 형태일 수도 있으며, 분산제 또는 안정화제를 추가적으로 포함할 수 있다.The pharmaceutical 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. In this case, 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.
본 발명의 조성물은 개별 치료제로 투여하거나 다른 치료제와 병용하여 투여될 수 있고 종래의 치료제와는 순차적 또는 동시에 투여될 수 있다.The 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.
또 다른 관점에서, 본 발명은 본 발명에 따른 PD-L1에 대한 항체를 포함하는 암 진단용 조성물이다. 본 발명은 또한, 본 발명에 따른 PD-L1에 대한 항체 또는 그의 항원 결합 단편을 처리하여 암을 진단하는 방법에 관한 것이다. In another aspect, 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.
본 발명에 따른 PD-L1에 대한 항체를 통해 샘플에서의 PD-L1 발현 수준을 측정함으로써 암을 진단할 수 있다. 발현 수준은 통상적인 면역분석 방법에 따라 측정할 수 있으며, 상기 PD-L1에 대한 항체를 이용한 방사능면역분석, 방사능면역침전, 면역침전, 면역조직화학염색, ELISA (enzyme-linked immunosorbant assay), 캡처-ELISA, 억제 또는 경재 분석, 샌드위치 분석, 유세포 분석(flow cytometry), 면역형광염색 및 면역친화성 정제를 통해 측정할 수 있으나, 이에 한정되는 것은 아니다.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.
상기 면역분석 과정에 의한 최종적인 시그널의 세기를 분석함으로써, 암을 진단할 수 있다. 즉, 생물학적 시료에서 본 발명의 마커의 단백질이 고발현 되어 시그널이 정상 생물학적 시료(예컨대, 정상 위조직, 혈액, 혈장 또는 혈청) 보다 강하게 나오는 경우에는 암으로 진단된다.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.
또 다른 관점에서, 본 발명은 상기 암 진단용 조성물을 포함하는 암 진단용 키트에 관한 것이다. 본 발명에 따른 키트는 본 발명에 따른 PD-L1에 대한 항체를 포함하고, 시료와 항체가 반응함으로써 나타내는 시그널을 분석하여, 암을 진단할 수 있다. 이 때, 상기 시그널은 항체에 결합된 효소 예를 들어 알칼린 포스파타아제, β-갈락토시다아제, 호스 래디쉬 퍼옥시다아제, 루시퍼라아제 또는 사이토크롬 P450을 포함할 수 있으나 이에 제한되는 것은 아니며, 이 때 효소에 대한 기질은 효소로서 알칼린 포스파타아제가 이용되는 경우에는, 기질로서 브로모클로로인돌일 포스페이트 (BCIP), 니트로 블루 테트라졸리움 (NBT), 나프톨-AS-B1-포스페이트 (naphthol-AS-B1-phosphate) 및 ECF (enhanced chemifluorescence)와 같은 발색반응 기질이 이용되고, 호스 래디쉬 퍼옥시다아제가 이용되는 경우에는 클로로나프톨, 아미노에틸카바졸, 디아미노벤지딘, D-루시페린, 루시게닌 (비스-N-메틸아크리디늄 니트레이트), 레소루핀 벤질 에테르, 루미놀, 암플렉스 레드 시약(10-아세틸-3,7-디하이드록시페녹사진), HYR (p-phenylenediamine-HCl and pyrocatechol), TMB (tetramethylbenzidine), ABTS (2,2'-Azine-di[3-ethylbenzthiazoline sulfonate]), o-페닐렌디아민 (OPD) 및 나프톨/파이로닌, 글루코스 옥시다아제와 t-NBT (nitroblue tetrazolium) 및 m-PMS (phenzaine methosulfate)과 같은 기질이 이용될 수 있으나, 이에 제한되는 것은 아니다. In another aspect, 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. In this case, the signal may include, but is not limited to, an enzyme bound to the antibody, for example, alkaline phosphatase, β-galactosidase, horse radish peroxidase, luciferase or cytochrome P450. In this case, when the alkaline phosphatase is used as the enzyme, the substrate for the enzyme is bromochloroindolyl phosphate (BCIP), nitro blue tetrazolium (NBT), naphthol-AS-B1-phosphate (naphthol) as the substrate. 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) And m-PMS (phenzaine methosulfate) may be used, but is not limited thereto.
또한, 본 발명에 따른 키트는 검출 가능한 시그널을 발생시키는 레이블을 포함할 수 있으며, 상기 레이블은 화학물질 (예컨대, 바이오틴), 효소 (알칼린 포스파타아제, β-갈락토시다아제, 호스 래디쉬 퍼옥시다아제 및 사이토크롬 P450), 방사능물질(예컨대, C14, I125, P32 및 S35), 형광물질 (예컨대, 플루오레신), 발광물질, 화학발광물질 (chemiluminescent) 및 FRET (fluorescence resonance energy transfer)을 포함할 수 있으나, 이에 제한되는 것은 아니다.In addition, the 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, β-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.
암 진단을 위해 사용되는 효소의 활성 측정 또는 시그널의 측정은 당업계에 공지된 다양한 방법에 따라 실시될 수 있다. 이를 통해 PD-L1 발현을 정성적 또는 정량적으로 분석할 수 있다. 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.
실시예Example
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 예시하기 위한 것으로서, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지는 않는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.
실시예 1. PD-L1 항원 발현 및 정제Example 1. PD-L1 Antigen Expression and Purification
1. PD-L1 단백질 발현 벡터 제작1. Construction of PD-L1 Protein Expression Vector
PD-L1의 클로닝은 Jurkat cells cDNA 라이브러리 (Stratagene, 미국)를 가지고 세포외 도메인만을 얻기 위해 5'과 3'에 제한효소 SfiⅠ 사이트가 포함된 PD-L1에 대한 프라이머 (표 1)를 이용하여 중합효소연쇄반응 (polymerase chain reaction, PCR)에 의해 증폭시켰고, 증폭된 PCR 산물은 N293F 벡터를 이용하여 카복시-말단에 인간 Fc (서열번호 248), 마우스 Fc (서열번호 249)를 융합시켜 제작되었다 (도 1).Cloning of PD-L1 using a Jurkat cells cDNA library (Stratagene, USA) using primers for PD-L1 with restriction enzyme Sfi I sites at 5 'and 3' to obtain only the extracellular domain (Table 1) Amplified by polymerase chain reaction (PCR), the amplified PCR product was prepared by fusing human Fc (SEQ ID NO: 248) and mouse Fc (SEQ ID NO: 249) at the carboxy-terminus using N293F vector. (FIG. 1).
Figure PCTKR2017008495-appb-T000001
Figure PCTKR2017008495-appb-T000001
2. PD-L1 항원의 발현 및 정제2. Expression and Purification of PD-L1 Antigen
항원을 동물세포에서 발현시키기 위해서 HEK-293F 세포에 플라스미드 DNA를 형질감염 (transfection)시켰다. 형질감염을 수행하기 위한 폴리플렉스 (polyplex) 반응액은 프리스타일 293 발현 배지 3 ml에 플라스미드 DNA를 25 μg을 넣어 섞어준 후, 추가로 2 mg/ml PEI (Polyethylenimine) (polyplUSA-형질감염, USA) 50 μl을 넣어 다시 한 번 섞어 주었다. 폴리플렉스 반응액은 15분 동안 상온에서 반응시킨 후, 1×106 cells/ml로 배양된 40 ml의 배양액에 넣어 120 rpm으로 37℃, 8% CO2에서 24시간 배양하였다. 형질감염 24시간 후에 보강제 (supplement)인 Soytone (BD, USA)을 최종 농도가 10 g/L가 되도록 첨가한다. 7일 동안 HEK-293F를 이용한 임시발현시스템을 이용하여 항체를 생산하였다. 배양액에서 항원을 얻기 위해 친화도 크로마토그래피 (affinity chromatography)를 수행하였다. 7일째 되는 날 회수한 배양액에서 세포와 세포 잔여물 (debris)을 제거하기 위해 5000 rpm에서 10분간 원심분리하여 상층액을 얻었다. 상층액을 DPBS로 세척한 재조합 단백질 A 아가로스 레진과 4℃에서 16시간 동안 반응시켰다. 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 μg of plasmid DNA in 3 ml of Freestyle 293 expression medium and further adding 2 mg / ml PEI (Polyethylenimine) (polyplUSA-transfection, USA). ) 50 μl 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. After 24 hours of transfection, a supplement Soytone (BD, USA) is added to a final concentration of 10 g / L. Antibodies were produced using a transient expression system using HEK-293F for 7 days. Affinity chromatography was performed to obtain the antigen in the culture. The supernatant was obtained by centrifugation at 5000 rpm for 10 minutes to remove cells and cell debris from the culture solution recovered on the 7th day. The supernatant was reacted with recombinant Protein A agarose resin washed with DPBS for 16 hours at 4 ° C.
재조합 단백질 A 아가로스 레진을 사용한 경우, 0.1M 글리신 (Glycine)으로 단백질을 용출시켰고 1M Tris-HCl 500 μl로 중화시켜 1차 정제하였고, 1차 정제된 단백질은 Superdex 200 (1.5cm*100cm) 겔 여과 크로마토그래피를 이용하여 2차 정제를 하였다. When using recombinant Protein A agarose resin, the protein was eluted with 0.1M glycine and neutralized with 500 μl 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.
정제된 단백질은 SDS-PAGE gel과 크기 배제 크로마토그래피 (TSK-GEL G-3000 SWXL Size-exclusion chromatography (SEC) (Tosoh)) 를 이용하여 순도를 확인하였다. Purified protein was purified by SDS-PAGE gel and size exclusion chromatography (TSK-GEL G-3000 SWXL Size-exclusion chromatography (SEC) (Tosoh)).
그 결과, 도 2a 내지 도 2d에 나타난 바와 같이, 정제된 PD-L1 단백질의 순도는 95% 이상의 순도를 가지는 것을 확인할 수 있었다. As a result, as shown in Figures 2a to 2d, it was confirmed that the purity of the purified PD-L1 protein has a purity of 95% or more.
실시예 2. PD-L1 인간항체의 선별Example 2 Screening of PD-L1 Human Antibodies
1. 항원 준비1. Antigen Preparation
실시예 1에서 제작한 PD-L1-hFc, PD-L1-mFc와 Sino Biological Inc.에서 구입한 PD-L1-his (Catalog Number, 10084-H08H) 단백질 항원 50ug을 면역 흡착(immunosorb) 튜브에 코팅한 후 블로킹 (blocking)을 수행하였다.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.
2. 바이오패닝2. Bio Panning
인간항체 라이브러리 파아지는 2.7×1010의 다양성을 가진 인간 scFv 라이브러리를 박테리아에 감염시킨 후 박테리아를 30℃에서 16 시간 배양하였다. 배양 후 원심 분리하여 상층액을 PEG로 농축한 다음, 이를 PBS 완충용액에 녹여 인간항체 라이브러리를 준비하였다. 면역튜브에 라이브러리 파아지를 넣은 후, 실온에서 2시간 반응한 다음, 1XPBS/T와 1XPBS로 워싱 후 항원에 특이적으로 결합한 scFv-파아지들만 용출하였다. 용출된 파아지를 다시 대장균에 감염시켜 증폭시키는 패닝과정을 통해 양성 파아지의 pool을 얻고, 첫 번째 라운드의 패닝에서 증폭된 파아지를 가지고 PBST 워싱 단계에서 횟수만 늘리고, 나머지는 동일한 방법으로 2라운드와 3라운드 패닝을 수행하였다. 그 결과, 표 2와 같이 3라운드 패닝에서 항원에 결합한 파아지 수가 인풋 대비 아웃풋이 다소 증가였음을 확인하였다.Human Antibody Library 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.In the first round of panning, 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.
Figure PCTKR2017008495-appb-T000002
Figure PCTKR2017008495-appb-T000002
3. 폴리 파아지 ELISA3. Poly Phage ELISA
각 1차부터 3차까지 패닝하여 얼려두었던 세포 스톡(stock)을 5 ml의 2xYTCM, 2% 글루코오스, 5 mM MgCl2 배지에 OD600에서 0.1 되게 넣어준 다음, 37℃에서 2~3시간(OD600=0.5~0.7) 배양 후 M1 헬퍼 파지를 감염하여 2xYTCMK, 5 mM MgCl2 1mM IPTG 배지에 30℃에서 16시간 배양하였다. 배양한 세포를 원심분리 (4500 rpm, 15 min, 4℃) 후 상층액을 새 튜브로 옮겼다. (1차~3차 패닝 poly scFv-phage) 96 웰 면역-플레이트 (NUNC 439454)에 두 종류의 항원 각각을 웰 당 100 ng씩 4℃에서 16시간 정도 코팅버퍼 (coating buffer)로 코팅한 후 PBS에 녹인 4% 스킴 밀크 (skim milk)를 사용하여 각 웰을 블로킹 (blocking)하였다. The cell stocks, which were panned from each first to third, were frozen in 5 ml of 2xYTCM, 2% glucose, and 5 mM MgCl 2 medium at OD 600 of 0.1, followed by 2-3 hours at 37 ° C. 600 = 0.5 ~ 0.7) and then cultured with M1 helper phage was incubated for 16 hours at 30 ℃ in 2xYTCMK, 5 mM MgCl 2 1mM IPTG medium. The cultured cells were centrifuged (4500 rpm, 15 min, 4 ° C.) and the supernatant was transferred to a new tube. (1st ~ 3rd panning poly scFv-phage) PBS was coated with 96 ng immuno-plate (NUNC 439454), each of two antigens, at 100 ° C per well for 16 hours at 4 ° C. Each well was blocked using 4% skim milk dissolved in.
각 웰 마다 PBS/T 0.2 ㎖ 사용하여 씻어준 다음 1차~3차 패닝 폴리 scFv-파지를 각 웰에 100 ㎕씩 넣고 상온에서 2시간 동안 반응시켰다. 다시 각 웰마다 PBS/T 0.2 ㎖을 사용하여 4번 씻어준 후 이차 항체인 항-M13-HRP (Amersham 27-9421-01)를 1:2000으로 희석하여 실온에서 1시간 동안 반응하였다. PBS/T로 씻어준 후에 OPD tablet (sigma. 8787-TAB)을 PC 버퍼를 만들어 100 ul/well씩 넣어 10분 동안 발색 시킨 다음 흡광도 490 nm에서 분광광도계 (spectrophotometer : MolecularDevice)로 측정하였다. Each well was washed with 0.2 ml of PBS / T, and then 100 μl 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.
그 결과를 도 3에 나타내었다. 도 3에 따르면, 항원에 대한 결합능이 두 가지 PD-L1 항원에 대해 3차 poly scFv-phage에서 증강(enrichment)됨을 ELISA로 확인하였다. The results are shown in FIG. According to FIG. 3, it was confirmed by ELISA that the binding ability to the antigen is enhanced in tertiary poly scFv-phage for two PD-L1 antigens.
4. 양성 파아지 선별4. Positive Phage Screening
결합능이 큰 다클론 파아지 항체군 (3차 패닝)에서 얻은 콜로니를 2xYTCM, 2% 글루코스, 5 mM MgCl2 배지에 1 ml 96-깊은 웰 플레이트 (바이오니아 90030)에 37℃에서 16시간 배양하였다. 이렇게 키운 세포에서 OD600에서 값이 0.1이 되도록 100~200 ul를 취해 1 ml의 2xYTCM, 2% 글루코스, 5 mM MgCl2 배지에 넣은 다음, 96-깊은 웰 플레이트에 37℃에서 OD600에서 그 값이 0.5~0.7 되도록 2~3 시간 배양 하였다. M1 헬퍼 파지를 MOI값이 1:20 되도록 감염하여 2xYTCMK, 5 mM MgCl2 1 mM IPTG 배지에 30℃에서 16시간 배양 하였다. Colonies from high-binding polyclonal phage antibody groups (tertiary panning) 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.
96웰 면역 플레이트에 항원 PD-L1을 웰당 100 ng씩을 4℃에서 16시간 코팅한 후 PBS에 녹인 4% 스킴 밀크를 사용하여 각 웰을 블로킹하였다. 각 웰마다 0.2 ㎖ PBS/T 사용하여 씻어준 16시간 동안 배양한 단일클론 scFv-파지 (각각 100 scFv-phage)를 각 웰에 100 ㎕씩 넣고 상온에서 2시간 동안 반응시켰다. 다시 각 웰마다 0.2 ㎖ PBS/T을 사용하여 4번 씻어준 후 2차 항체인 항-M13-HRP를 1/2000로 희석하여 실온에서 1시간 동안 반응하였다. 0.2 ㎖ PBS/T로 씻어준 후에 발색하여 흡광도 490 nm에서 측정하였다. 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 μl 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.
그 결과, 도 4에 나타난 바와 같이, 각 항원에 대한 결합능이 강한 단일 파아지 클론들은 PD-L1에 대해 총 수 십개의 단일 파아지 클론을 얻었다.As a result, as shown in Figure 4, single phage clones with strong binding ability to each antigen obtained a total of several dozen single phage clones for PD-L1.
5. 양성 파아지 항체의 염기서열 분석5. Sequence analysis of positive phage antibody
상기 선별된 단일 클론에 대해 DNA 정제 키트 (Qiagen, 독일)를 이용하여 DNA-prep을 수행하여 DNA를 얻어 서열 분석을 의뢰하였다 (솔젠트). 서열 분석 결과를 보고, 선별된 항체의 VH와 VL의 CDR 부위를 확인하였고 이들 항체와 점라인 (germ line) 항체군의 유사성을 NCBI의 웹페이지 http://www.ncbi.nlm.nih.gov/igblast/의 Ig BLAST 프로그램을 이용하여 조사하였다. 그 결과 10종의 PD-L1에 특이적인 파아지 항체를 얻었고 이는 하기 표 3에 정리하여 제시하였다. 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.
선별된 항체의 중쇄 및 경쇄 CDR, FR서열 및 이를 포함하는 중쇄 가변영역 및 경쇄 가변영역을 포함하는 항체는 다음 표 4 및 표 5와 같다. 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.
Figure PCTKR2017008495-appb-T000004
Figure PCTKR2017008495-appb-T000004
Figure PCTKR2017008495-appb-I000001
Figure PCTKR2017008495-appb-I000001
Figure PCTKR2017008495-appb-T000005
Figure PCTKR2017008495-appb-T000005
실시예 3: PD-L1 인간항체의 생산Example 3: Production of PD-L1 Human Antibody
1. scFv 형태를 IgG 형태로 전환 (conversion)1. Conversion of scFv form to IgG form (conversion)
선별된 1110종의 PD-L1에 대한 단일 클론 파아지 항체들을 파아지에서 IgG 전체 벡터 (whole vector)로 전환하기 위해 중쇄와 경쇄에 대해 PCR (iCycler iQ, BIO-RAD)을 수행 하였다. 그 결과, 중쇄와 경쇄를 얻었고, 제한 효소로 벡터와 각 클론의 중쇄, 경쇄를 절단하였다. 벡터와 중쇄 각각은 DNA-gel extraction kit (Qiagen)으로 DNA를 용출하였다. 라이게이션 (Ligation)은 vector 1 ul (10 ng) 중쇄 (100~200 ng) 15 ul, 10x Buffer 2 ul, ligase (1 U/ul) 1 ul, 증류수를 혼합하여 실온에서 1~2시간 방치 후, 형질전환 세포 (competent cell) (XL1-blue)에 넣어 얼음에 5분간 놓고, 42℃에서 90초간 열 충격 (heat shock)을 주었다. 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. As a result, 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 ℃ for 90 seconds.
열 충격 후 배지 1 ml을 넣은 뒤 1시간 동안 37℃에서 키운 후, LB Amp 플레이트에 스프레딩 (spreading)하여 37℃에 16시간 동안 배양하였다. 이렇게 얻은 콜로니를 취해 LB Amp 배지 5 ml을 접종하여 37℃에 16시간 동안 배양 후, DNA-prep kit (Nuclogen) 이용하여 DNA-prep을 수행하였다. 얻은 DNA는 서열 분석을 의뢰하였다 (솔젠트). After heat shock, 1 ml of medium was added, and then grown at 37 ° C. for 1 hour, and then spread on LB Amp plates and incubated at 37 ° C. for 16 hours. The colonies thus obtained were inoculated with 5 ml of LB Amp medium and incubated at 37 ° C. for 16 hours, followed by DNA-prep using a DNA-prep kit (Nuclogen). The obtained DNA was requested for sequencing (solgent).
그 결과, 전체 IgG로 전환한 PD-L1에 대한 11개의 클론의 중쇄와 경쇄의 서열이 파아지 항체의 서열과 일치됨을 확인하였다. HEK 293F 세포에 형질감염 하기 위해, 전체 IgG로 전환한 각 클론의 중쇄와 경쇄는 LB Amp 100 ml 배지에 키워 midi-prep kit (QIAgen)을 이용하여 DNA를 얻었다.As a result, it was confirmed that the sequences of the heavy and light chains of 11 clones for PD-L1 converted to total IgG match the sequences of phage antibodies. In order to transfect HEK 293F cells, the heavy and light chains of each clone converted to total IgG were grown in LB Amp 100 ml medium to obtain DNA using midi-prep kit (QIAgen).
2. 인간항체 생산2. Human Antibody Production
클로닝된 pNATVH와 pNATVL 벡터는 6:4의 비율로 HEK293F 세포에 동시 형질감염(co-형질감염)하여 7일차 상층액을 수거하여 원심분리와 0.22 ㎛ Top-필터를 통해 세포와 부유물질을 제거한 후, 상층액을 모아 단백질 A 친화도 크로마토그래피를 수행하여 IgG 항체를 정제하였다. 정제 후 글리신 버퍼 (glycine buffer)를 통해 항체를 분리하고, 최종 재서스펜션 버퍼 (resuspension buffer)는 PBS가 되도록 버퍼를 교환하였다. 정제된 항체를 BCA 및 나노 드랍 (nano drop)을 통해 정량하였고, 15종의 항체를 환원, 비환원 조건에서 각 5 ug씩 로딩하여 SDS-PAGE 분석하여 정제 단백질의 순도 및 이동도 (mobility) 상태를 확인하였다 (도 5). 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 μm 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).
그 결과, 도 5에 따르면, 10종 항체 모두 비환원 조건에서는 150 kDa이상의 크기에서 검출되었다.As a result, according to Figure 5, all 10 antibodies were detected at a size of 150 kDa or more in non-reducing conditions.
실시예 4: PD-L1 단일 클론 항체의 특성Example 4: Properties of PD-L1 Monoclonal Antibody
1. 항체의 활성 평가1. Evaluation of the Activity of Antibodies
선별된 항체의 활성 평가 실험은 PD1/PD-L1 차단 바이오어세이 키트 (promega, J1250)을 사용하여 진행하였다. PD-L1이 고발현되어 있는 CHO 세포주를 96-웰 플레이트에 도말하고 16시간 이상 배양 후, 일정 농도로 연속 희석된 각 항체들을 처리하고, 인간 PD-1이 고발현되는 Jurkat 세포주를 6시간 동안 함께 배양하였다. 항체의 저해 회복 정도는 루시퍼라제 (luciferase)가 기질을 분해하여 나오는 발광세기로 알 수 있고 분광광도계 (SpectraMax M5 spectraphotometer, Molecular Devices, 미국)로 측정하였다. 10종의 PD-L1 항체는 PD-1/PD-L1 복합체 형성으로 감소되어 있던 시그널을 회복시키는 값으로 항체의 활성을 확인하였고, 16E12가 대조 항체 대비 비슷한 활성도를 보였다 (도 6).Activity evaluation experiments of selected antibodies were conducted using the 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).
PD-L1 항체 16E12의 활성 평가를 농도 의존적으로 측정하기 위해 단계 희석을 하여 PD-1/PD-L1 차단 바이오어세이를 다시 진행한 결과 감소되어 있던 시그널을 농도 구배 의존적으로 회복시켰다. 그 회복 정도는 EC50 (effective concentration of mAb at 50% level of Recovery signal)으로 나타낼 수 있고 Graphpad Prism6를 이용하여 분석하였으며 EC50의 in vitro efficacy 저해회복능력은 도 7과 같다.In order to determine the activity-dependent evaluation of the activity of PD-L1 antibody 16E12, 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.
2. 과발현 세포에 대한 PD-L1 항체의 친화도 2. Affinity of PD-L1 Antibody to Overexpressing Cells
PD-L1을 고발현하는 형질전환 세포 풀은 인간 PD-L1을 포함하고 있는 pcDNA3.1 플라스미드를 HEK293E에 형질전환 시키고, 150 ug/ml Zeocin (#R25001, Thermo Fisher)이 들어있는 선택적 배지에서 선별하였다. 각 세포 풀은 각 anti-PD-L1을 이용한 FACS (fluorescence activated cell sorting) 분석을 통해 확인되고 선택되었고, FACS 결합 어세이나 FACS 경쟁 어세이와 같은 기능 평가법에 사용되었다.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.
인간 PD-L1을 고발현하는 형질전환 세포 풀 각각을 시료당 0.5-1x10^6의 세포를 준비하고, 항체들을 각각 일정한 희석 배수로 연속적으로 희석하여 준비된 세포와 4℃에서 20분간 반응시켰다. 그 후, 세포는 2% 소태아혈청 (fetal bovine serum)이 포함된 PBS (#LB001-02, welgene)로 3차례 수세하고, FITC (fluorescein isothiocyanate) 형광물질이 결합된 항-인간 IgG 항체 (#FI-3000, Vectorlabs)를 사용하여 4℃에서 20분간 반응 후, 동일한 수세과정을 거치고 이후 0.5 ml의 2% FBS(#26140-079, Thermo fisher)가 든 PBS로 현탁 시킨 후, 유세포 분석기인 FACSCanto II flow cytometer (BD Biosciences, 미국)을 사용하여 분석하였다. 그 결과로 PD-L1 항체 16E12가 특이적으로 결합하였고, 그 결합력은 평형해리상수(equilibrium dissociation constant, Kd)를 Graphpad Prism6의 분석함수를 통하여 구하였다. 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. for 20 minutes, followed by the same washing process, and then suspended in PBS containing 0.5 ml of 2% FBS (# 26140-079, Thermo fisher), followed by flow cytometry FACSCanto. The analysis was performed using a II flow cytometer (BD Biosciences, USA). As a result, PD-L1 antibody 16E12 was specifically bound, and the binding force was calculated by the equilibrium dissociation constant (Kd) through the analysis function of Graphpad Prism6.
그 결과, 도 8에 따르면, 세포표면에 과발현된 인간 PD-L1에 대해 농도 의존적으로 결합된 항체의 결합력을 MFI(mean fluorescence intensity)로 알 수 있다.As a result, according to FIG. 8, 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).
3. ProteOn XPR36을 이용한 PD-L1 항체의 친화도3. Affinity of PD-L1 Antibody Using ProteOn XPR36
ProteOn XPR36 (BioRad) 기기를 통해 수행하였다. GLC 센서칩 (BioRad)을 기기에 장착하고 PBST 완충용액으로 세척을 한 후, EDC/sulfo-NHS 혼합액으로 카르복시메틸 덱스트란 표면을 활성화시켰다. 10 mM 소듐 아세테이트 (sodium acetate), pH 5.0, 완충액에 5 ug/ml 농도로 녹인 PD-L1-hFc을 주입시켜 GLC 센서칩에 고정화시켰다.It was performed on a ProteOn XPR36 (BioRad) instrument. The GLC sensor chip (BioRad) was mounted on the instrument and washed with PBST buffer, and then the surface of carboxymethyl dextran was activated with EDC / sulfo-NHS mixture. 10 mM sodium acetate, pH 5.0, and PD-L1-hFc dissolved at 5 ug / ml in buffer were injected and immobilized on a GLC sensor chip.
PD-L1 단백질과 반응하지 않고 남아있는 활성화된 카르복시 그룹을 비활성화시키기 위해 1 M 에탄올아민을 흘려주었고, 센서칩에 결합되지 않은 단백질을 세척하기 위해 10 mM glycine, pH2.0을 주입하였다. 이후 PBST 완충액을 이용하여 항체를 농도별 (30 nM ~ 0.123 nM)로 30 ul/min 유속으로 10분 흘려주면서 시간에 따른 결합과 해리과정 중의 센소그램 (sensogram) 데이터를 수집하였다.1 M ethanolamine was flowed to inactivate activated carboxyl groups remaining unreacted with PD-L1 protein, and 10 mM glycine, pH2.0, was injected to wash proteins not bound to the sensor chip. Then, 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).
평형상태에서의 센소그램 데이터를 농도에 따라 플로팅 (plotting) 및 피팅 (fitting)하여 평형 해리상수 (KD) 계산한 결과 0.045 nM로 PD-L1 항원에 대해 높은 친화도를 보였다 (도 9).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).
실시예 5: PD-L1항체 16E12에 대한 항체 최적화Example 5: Antibody Optimization for PD-L1 Antibody 16E12
1. PD-L1-16E12 항체의 최적화를 위한 라이브러리 제작1. Preparation of Library for Optimization of PD-L1-16E12 Antibody
항체 최적화는 중쇄는 고정하고 와이바이오로직스에서 보유하고 있는 105-106 경쇄 (LC) 풀 (pool)을 넣어 새로운 LC 셔플링 라이브러리 (LC shuffling library)를 제작하고, LC 셔플링, 중쇄의 소수성 코어 (hydrophobic core), 노출 잔기 (exposed residue), 전하 클러스터 (charge cluster), 염 브릿지 (salt bridge)등과 같이 구조적으로 중요한 부위의 잔기들과 비교분석을 하여 보존된 잔기 (conserved residue)로 변이시킨 뒤 LC 셔플링을 진행하는 코어 패킹 (core packing) + LC 셔플링, 항체 가변 영역 (antibody variable region)의 DNA는 in vivo 친화도 성숙 (affinity maturation) 과정에서 빈번하게 변이 (mutation) 될 수 있는 변이 핫스팟 (mutational hot spot)을 랜덤하게 변이시킨 뒤 LC 셔플링을 진행하는 CDR 핫스팟 + LC 셔플링등 3가지 방법으로 진행을 하였다. 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.
LC 셔플링 라이브러리를 제작하기 위해 16E12 항체의 LC 유전자를 BstX I으로 절단한 다음 벡터로 사용하고, 와이바이오로직스에서 보유하고 있는 라이브러리 풀을 BstX I으로 절단하여 인서트로 사용하였다. 리가제로 라이게이션 후, 전기천공 형질전환용 세표를 이용하여 형질 전환을 수행하였다. 사각 접시 (square plate)에 형질 전환된 세포를 모아 항체 라이브러리를 제조한 결과 약 1.5×107 의 다양한 라이브러리를 얻었고 염기 서열 분석 결과 HC의 서열은 모두 같으며 LC의 서열이 서로 다른 것을 확인하였다.To prepare an LC shuffling library, 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.
Core 패킹 + LC 셔플링 라이브러리를 제작하기 위해 16E12 항체의 frame work (FR) 부분을 보존 (conserved)된 아마노산 서열로 치환한 뒤 LC 유전자를 BstX I으로 절단 한 다음 벡터로 사용하고, 와이바이오로직스에서 보유하고 있는 라이브러리 풀을 BstX I으로 절단하여 인서트로 사용하였다. 리가제로 라이게인션 후, 전기천공 형질전환용 세표를 이용하여 형질전환을 수행하였다. 사각 접시 (square plate)에 형질전환된 세포를 모아 항체 라이브러리를 제조한 결과 약 8.4×106 의 다양한 라이브러리를 얻었고 염기 서열 분석 결과 HC의 FR 부위가 보존 (conserved)된 아마노산 서열로 치환되었고 LC의 서열이 서로 다른 것을 확인하였다.To construct the Core packing + LC shuffling library, 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. 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 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 핫스팟 + LC 셔플링 라이브러리를 제작하기 위해 16E12 항체의 frame work (FR) 부분을 보존 (conserved)된 아마노산 서열로 치환한 뒤 CDR1의 핫스팟 라이브러리를 Sfi I으로 절단 한 다음 인서트로 사용하고, 와이바이오로직스에서 보유하고 있는 library pool을 Sfi I으로 절단하여 벡터로 사용하였다. 리가제로 라이게인션 후, 전기천공 형질전환용 세포를 이용하여 형질 전환을 수행 하였다. 사각 접시 (square plate)에 형질 전환 된 세포를 모아 항체 라이브러리를 제조한 결과 약 5.6x106 의 다양한 라이브러리를 얻었고 염기 서열 분석 결과 HC의 FR 부위가 보존 (conserved)된 아마노산 서열로 치환되었고 CDR1의 핫스팟 서열의 아미노산이 랜덤하게 변이되었고 LC의 서열이 서로 다른 것을 확인하였다.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.
실시예 6: PD-L1 인간 항체의 선별Example 6: Screening of PD-L1 Human Antibodies
1. 항원 준비1. Antigen Preparation
와이바이오로직스에서 생산한 PD-L1-hFc, PD-L1-mFc와 Sino Biological Inc.에서 구입한 PD-L1-his (Catalog Number, 10377-H08H) 단백질 항원 50 ug을 immunosorb tube에서 코팅한 후 블로킹 (blocking)을 수행하였다. 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.
2. 바이오-패닝2. Bio-panning
인간항체 라이브러리 파아지는 2.7×1010의 다양성을 가진 인간 scFv 라이브러리를 박테리아에 감염시킨 후 박테리아를 30℃에 16 시간 배양하였다. 배양 후 원심 분리하여 상층액을 PEG로 농축한 다음, 이를 PBS 완충용액에 녹여 인간항체 라이브러리를 준비하였다. 면역튜브에 라이브러리 파아지를 넣은 후, 실온에서 2시간 반응한 다음, 1XPBS/T와 1XPBS로 워싱 후 항원에 특이적으로 결합한 scFv-파아지들만 용출하였다. 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.
용출된 파아지를 다시 대장균에 감염시켜 증폭시키는 패닝과정을 통해 양성 파아지의 풀 (pool)을 얻었고, 항체 최적화를 위한 패닝은 첫 번째 라운드만 진행하였다. 그 결과, 표 6과 같이 1라운드 패닝에서 항원에 결합한 파아지 수가 유입 (input) 대비 결합 (output)이 다소 증가였음을 확인 하였다. 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. As a result, as shown in Table 6, the number of phages bound to the antigen in the first round panning was slightly increased compared to the input.
Figure PCTKR2017008495-appb-T000006
Figure PCTKR2017008495-appb-T000006
3. 양성 파아지 선별3. Positive Phage Screening
패닝에서 얻은 콜로니를 2xYTCM, 2% 글루코스, 5 mM MgCl2 배지에 1 ml 96-깊은 웰 플레이트 (96-deep well plate: 바이오니아 90030)에 37℃에서 16시간 배양 하였다. 이렇게 키운 세포에서 OD600에서 값이 0.1이 되도록 100~200 ul를 취해 1 ml의 2xYTCM, 2% glucose, 5 mM MgCl2 배지에 넣은 다음, 96-깊은 웰 플레이트에 37℃에서 OD600에서 그 값이 0.5~0.7 되도록 2~3 시간 배양하였다. M1 헬퍼 파지를 MOI값이 1:20 되도록 감염하여 2xYTCMK, 5 mM MgCl2 1 mM IPTG 배지에 30℃에서 16시간 배양하였다. 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.
96웰 면역 플레이트에 항원 PD-L1을 well당 100 ng씩을 4 ℃에서 16시간 코팅한 후 PBS에 녹인 4% 스킴 밀크를 사용하여 각 웰을 블로킹하였다. 각 웰마다 0.2 ㎖ PBS/T 사용하여 씻어준 16시간 동안 배양한 단일클론 scFv-파지 (각각 100 scFv-phage)를 각 well에 1 ㎕씩 넣고 상온에서 2시간 동안 반응시켰다. 다시 각 웰마다 0.2 ㎖ PBS/T을 사용하여 4번 씻어준 후 2차 항체인 anti-M13-HRP를 1/2000로 희석하여 실온에서 1시간 동안 반응하였다. 0.2 ㎖ PBS/T로 씻어준 후에 발색하여 흡광도 490 nm에서 측정하였다. 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. Monoclonal scFv-phage (100 scFv-phage), which was incubated for 16 hours washed with 0.2 ml PBS / T in each well, 1 μl of each well was added to each well and reacted 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.
그 결과, 모항체 (16E12, 빨간색표시-6D) 각 항원에 대한 결합능이 강한 수 십개의 단일 파아지 클론을 얻었고, 도 10과 같다. As a result, dozens of single phage clones with strong binding ability to each antigen of the parent antibody (16E12, red-6D) were obtained, as shown in FIG. 10.
4. 양성 파아지 항체의 염기서열 분석4. Sequencing of Positive Phage Antibodies
선별된 단일 클론에 대해 DNA 정제 키트 (Qiagen, 독일)를 이용하여 DNA-prep을 수행하여 DNA를 얻어 서열 분석을 의뢰하였다 (솔젠트). 서열 분석 결과를 보고, 선별된 항체의 VH와 VL 의 CDR region을 확인하였고 이들 항체와 germ line 항체군의 유사성을 NCBI 의 웹페이지 http://www.ncbi.nlm.nih.gov/igblast/ 의 Ig BLAST 프로그램을 이용하여 조사하여 그 결과 21종의 모항체보다 결합력이 높은 특이적인 파아지 항체를 얻었고 이는 하기 표 7에서 정리하여 제시하였다. 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.
Figure PCTKR2017008495-appb-T000007
Figure PCTKR2017008495-appb-T000007
선별된 항체의 중쇄 및 경쇄 CDR, FR서열 및 이를 포함하는 중쇄 가변영역 및 경쇄 가변영역을 포함하는 항체는 다음 표 8 및 표 9와 같다. 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.
Figure PCTKR2017008495-appb-T000008
Figure PCTKR2017008495-appb-T000008
Figure PCTKR2017008495-appb-I000002
Figure PCTKR2017008495-appb-I000002
Figure PCTKR2017008495-appb-T000009
Figure PCTKR2017008495-appb-T000009
Figure PCTKR2017008495-appb-I000003
Figure PCTKR2017008495-appb-I000003
실시예 7: PD-L1 인간항체의 생산Example 7: Production of PD-L1 Human Antibody
1. scFv 형태를 IgG 형태로 전환 (conversion)1. Conversion of scFv form to IgG form (conversion)
선별된 21종의 PD-L1에 대한 단일 클론 파아지 항체들을 파아지에서 IgG whole vector로 전환하기 위해 중쇄와 경쇄에 대해 PCR (iCycler iQ, BIO-RAD)을 수행하였다. 그 결과, 중쇄와 경쇄를 얻었고, 제한 효소로 벡터와 각 클론들의 중쇄, 경쇄를 절단하였다. 벡터와 중쇄 각각은 DNA-겔 추출 키트 (DNA-gel extraction kit : Qiagen)로 DNA 용출하였다. 라이게이션 (ligation)은 벡터 1 ul (10 ng) 중쇄 (100~200 ng) 15 ul, 10x 버퍼 2 ul, 리가아제 (1 U/ul) 1 ul, 증류수를 혼합하여 실온에서 1~2시간 방치 후, 형질전환 세포 (competent cell : XL1-blue)에 넣어 얼음에 5분간 놓고, 42℃에서 90초간 열 충격 (heat shock)을 주었다. PCR (iCycler iQ, BIO-RAD) was performed on the heavy and light chains to convert monoclonal phage antibodies against 21 selected PD-L1 phages into IgG whole vectors. As a result, 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 ℃ for 90 seconds.
열 충격 후 배지 1 ml을 넣은 뒤 1시간 동안 37℃에서 키운 후, LB Amp 플레이트에 스프레딩하여 37℃ 에 16시간 동안 배양하였다. 이렇게 얻은 콜로니를 취해 LB Amp 배지 5 ml을 접종하여 37℃에 16시간 동안 배양 후, DNA-prep kit (Nuclogen) 이용하여 DNA-prep을 수행하였다. 얻은 DNA는 서열 분석을 의뢰하였다 (솔젠트). After heat shock, 1 ml of medium was added, and then grown at 37 ° C. for 1 hour, and then spread on an LB Amp plate and incubated at 37 ° C. for 16 hours. The colonies thus obtained were inoculated with 5 ml of LB Amp medium and incubated at 37 ° C. for 16 hours, followed by DNA-prep using a DNA-prep kit (Nuclogen). The obtained DNA was requested for sequencing (solgent).
그 결과, 전체 IgG로 전환한 PD-L1에 대한 21개의 클론의 중쇄와 경쇄의 서열이 파아지 항체의 서열과 일치됨을 확인하였다. HEK 293F 세포에 형질감염 하기 위해, 전체 IgG로 전환한 각 클론의 중쇄와 경쇄는 LB Amp 100 ml 배지에 키워 midi-prep kit (QIAgen)을 이용하여 DNA를 얻었다. As a result, it was confirmed that the sequences of the heavy and light chains of 21 clones for PD-L1 converted to total IgG match the sequences of phage antibodies. In order to transfect HEK 293F cells, the heavy and light chains of each clone converted to total IgG were grown in LB Amp 100 ml medium to obtain DNA using midi-prep kit (QIAgen).
2. 인간항체 생산2. Human Antibody Production
클로닝된 pNATVH와 pNATVL 벡터는 6:4의 비율로 HEK293F 세포에 동시 형질감염(co-형질감염)하여 7일차 상층액을 수거하여 원심분리와 0.22 ㎛ Top-필터를 통해 세포와 부유물질을 제거한 후, 상층액을 모아 단백질 A 친화도 크로마토그래피를 수행하여 IgG 항체를 정제하였다. 정제 후 글리신 버퍼 (glycine buffer)를 통해 항체를 분리하고, 최종 재서스펜션 버퍼 (resuspension buffer)는 PBS가 되도록 버퍼를 교환하였다. 정제된 항체를 BCA 및 나노 드랍 (nano drop)을 통해 정량하였고, 21종의 항체를 환원, 비-환원 조건에서 각 5 ug씩 로딩하여 SDS-PAGE 분석하여 정제 단백질의 순도 및 이동도 (mobility) 상태를 확인하였다. 또한 상층액의 일부는 모항체와의 발현율을 비교하기 위해 SDS-PAGE에 로딩하였고 대부분 항체가 모항체보다 발현율이 증가하였다.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 μm 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.
실시예 8: PD-L1 단일 클론 항체의 특성Example 8: Properties of PD-L1 Monoclonal Antibody
1. 항체의 활성 평가1. Evaluation of the Activity of Antibodies
선별된 항체의 활성 평가 실험은 PD-1/PD-L1 차단 바이오어세이 키트 (blockade bioassay kit :promega, J1250)을 사용하여 진행하였다. PD-L1이 고발현되어 있는 CHO 세포주를 96-웰 플레이트에 도말하고 16시간 이상 배양 후, 일정 농도로 연속 희석된 각 항체들을 처리하고, 인간 PD-1이 고발현되는 Jurkat 세포주를 6시간 동안 함께 배양하였다. 항체의 저해 회복 정도는 루시퍼라제가 기질을 분해하여 나오는 발광세기로 알 수 있고 분광광도계 (SpectraMax M5 spectraphotometer, Molecular Devices, 미국)로 측정하였다. 21종의 PD-L1 항체는 PD-1/PD-L1 복합체 형성으로 감소되어 있던 시그널을 회복시키는 값으로 항체의 활성을 확인하였고, 4A7, 4A11, 4C9, 4F5, 4H5, 4H8이 모항체 대비 활성이 증가하였고 대조 항체 대비 비슷한 활성도를 보였다 (도 11 및 표 10).Activity evaluation experiments of the selected antibodies were conducted using a 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). Twenty-one 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).
Figure PCTKR2017008495-appb-T000010
Figure PCTKR2017008495-appb-T000010
PD-L1 항체 6종(4A7, 4A11, 4C9, 4F5, 4H5, 4H8)에 대해 다시 활성 평가를 농도 의존적으로 측정하기 위해 단계 희석을 하여 PD-1/PD-L1 차단 바이오어세이를 다시 진행한 결과 감소되어 있던 시그널을 농도 구배 의존적으로 회복시켰다. 그 회복 정도는 EC50 (effective concentration of mAb at 50% level of Recovery signal)으로 나타낼 수 있고 Graphpad Prism6를 이용하여 분석하였으며 EC50의 In vitro efficacy 저해 회복 능력은 4F5가 가장 높게 나왔다 (도 12). Six PD-L1 antibodies (4A7, 4A11, 4C9, 4F5, 4H5, 4H8) were subjected to step dilution to concentration-determining activity evaluation again and re-run PD-1 / PD-L1 blocking bioassay. As a result, the reduced signal was recovered in a concentration gradient dependent manner. The degree of recovery can be expressed by the effective concentration of mAb at 50% level of recovery signal (EC50), and analyzed using Graphpad Prism6, and 4F5 showed the highest recovery ability of in vitro efficacy inhibition of EC50 (FIG. 12).
2. 과발현 세포에 대한 PD-L1 항체의 친화도 2. Affinity of PD-L1 Antibody to Overexpressing Cells
인간 PD-1을 고발현하는 형질전환 세포 풀은 인간 PD-1(NM_005018.2) 혹은 인간 PD-L1(NM_014143.2)을 포함하고 있는 pcDNA3.1 플라스미드를 HEK293E에 형질전환시키고, 400 ug/ml Zeocin (#R25001, Thermo Fisher)이 들어있는 선택적 배지에서 선별하였다. 각 세포 풀은 각각 anti-PD-1 (#557860, BD)을 이용한 FACs(fluorescence activated cell sorting) 분석을 통해 확인되고 선택되었고, FACs 결합 어세이나 FACs 경쟁 어세이 (Competition assay)와 같은 기능 평가법에 사용되었다. 인간 PD-L1을 고발현하는 형질전환 세포 풀 각각을 시료당 0.5-1x10^6의 세포를 준비하고, 항체들을 각각 일정한 희석 배수로 연속적으로 희석하여 준비된 세포와 4℃에서 20분간 반응시켰다. 그 후, 세포는 2% 소태아혈청 (fetal bovine serum)이 포함된 PBS (#LB001-02, welgene)로 3차례 수세하고, FITC (fluorescein isothiocyanate) 형광물질이 결합된 항-인간 IgG 항체 (#FI-3000, Vectorlabs)를 사용하여 4℃에서 20분간 반응 후, 동일한 수세과정을 거치고 이후 0.5 ml의 2% FBS(#26140-079, Thermo fisher)가 든 PBS로 현탁 시킨 후, 유세포 분석기인 FACSCanto II flow cytometer (BD Biosciences, 미국)을 사용하여 분석하였다 (표 11). 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. 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. for 20 minutes, followed by the same washing process, and then suspended in PBS containing 0.5 ml of 2% FBS (# 26140-079, Thermo fisher), followed by flow cytometry FACSCanto. Analysis was performed using a II flow cytometer (BD Biosciences, USA) (Table 11).
Figure PCTKR2017008495-appb-T000011
Figure PCTKR2017008495-appb-T000011
인간의 PD-L1을 고발현하는 형질전환 세포 풀 각각을 시료당 0.5x10^6의 세포를 준비하고, 항체들을 각각 일정한 희석배수로 연속적으로 희석하여 준비된 세포와 4℃에서 20분간 반응시켰다. 그 후, 세포는 2% 소태아혈청 (fetal bovine serum)이 포함된 PBS (#LB001-02, welgene)로 3차례 수세하고, FITC (fluorescein isothiocyanate) 형광물질이 결합된 항-인간 IgG 항체 (#FI-3000, Vectorlabs)를 사용하여 4℃에서 20분간 반응 후, 동일한 수세과정을 거치고 이후 0.5 ml의 2% FBS(#26140-079, Thermo fisher)가 든 PBS로 현탁시킨 후, 유세포 분석기인 FACsCanto II flow cytometer(BD Biosciences, 미국)을 사용하여 분석하였다 (도 13).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. for 20 minutes, followed by the same washing process, and then suspended in PBS containing 0.5 ml of 2% FBS (# 26140-079, Thermo fisher), followed by flow cytometry FACsCanto. Analysis was performed using a II flow cytometer (BD Biosciences, USA) (FIG. 13).
3. 효소면역흡착을 이용한 PD-1/PD-L1 복합체의 형성을 막는 항체의 저해능력3. Inhibitory ability of antibodies to prevent the formation of PD-1 / PD-L1 complex using enzyme immunosorbent
인간 PD-1-Fc(S1420, Y-Biologics)를 96-웰 면역 마이크로플레이트 (96-well immuno microplate: #439454, Thermo)의 웰에 4℃에서 16시간 동안 고정시키고, 0.05% tween-20(#P9416, Sigma-Aldrich)이 들어있는 PBS로 3번 세척 후, 4% 스킴 밀크(#232120, Becton, Dickinson and Company)가 포함된 세척액으로 상온에서 1시간동안 방치함으로써 비특이적 결합을 차단하였다. 그 사이 일정 희석배수로 연속 희석된 각 항체와 인간 PD-L1-His (S1479, Y-Biologics)를 상온에서 1시간 동안 반응시킨 후, 준비된 마이크로플레이트에 넣어 상온에서 1시간 동안 방치시킨다. 동일한 세척방법을 적용한 이후, anti-Biotin-His 항체 (#MA1-21315-BTIN, Thermo)를 1:2000으로 희석하여 마이크로플레이트의 웰에 넣고 상온에서 1시간 반응시킨 후, 동일한 방법으로 세척한 뒤 Streptavidin poly-HRP 항체(#21140, Pierce)를 1: 5000으로 희석하여 마이크로플레이트의 웰에 넣고 상온에서 1시간 반응시킨 후, 동일한 방법으로 세척하였다. 100 ul TMB 기질용액 (#T0440, Sigma-Aldrich)을 넣고 빛을 차단한 뒤 상온에서 3분간 방치 후 50 ul 2.5M 황산(#S1478, Samchun)을 넣어 반응을 중단시키고 분광광도계(#GM3000, Glomax® Discover System Promega)를 이용하여 450nm에서 흡광도를 측정하였다. 그 결과를 도 14에 나타내었다.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). Meanwhile, each antibody serially diluted with a constant dilution factor and human PD-L1-His (S1479, Y-Biologics) are reacted at room temperature for 1 hour, and then placed in a prepared microplate for 1 hour at room temperature. After applying the same washing method, 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 results are shown in FIG.
4. ProteOn XPR36을 이용한 PD-L1 항체의 친화도 4. Affinity of PD-L1 Antibody Using ProteOn XPR36
ProteOn XPR36 (BioRad) 기기를 통해 수행하였다. GLC 센서칩 (BioRad)을 기기에 장착하고 PBST 완충용액으로 세척을 한 후, EDC/sulfo-NHS 혼합액으로 카르복시메틸 덱스트란 표면을 활성화 시켰다. 10 mM 소듐 아세테이트 (sodium acetate), pH 5.0, 완충액에 5 ug/ml 농도로 녹인 PD-L1-hFc을 주입시켜 GLC 센서칩에 고정화시켰다.It was performed on a ProteOn XPR36 (BioRad) instrument. The GLC sensor chip (BioRad) was mounted on the device, washed with PBST buffer solution, and activated with carboxymethyl dextran surface with EDC / sulfo-NHS mixture. 10 mM sodium acetate, pH 5.0, and PD-L1-hFc dissolved at 5 ug / ml in buffer were injected and immobilized on a GLC sensor chip.
PD-L1 단백질과 반응하지 않고 남아있는 활성화된 카르복시 그룹을 비활성화시키기 위해 1 M 에탄올아민을 흘려주었고, 센서칩에 결합되지 않은 단백질을 세척하기 위해 10 mM 글리신 (glycine), pH2.0을 주입하였다. 이후 PBST 완충액을 이용하여 항체를 농도별 (30 nM ~ 0.123 nM)로 30 ul/min 유속으로 10분 흘려주면서 시간에 따른 결합과 해리과정 중의 센소그램 (sensogram) 데이터를 수집하였다. 1 M ethanolamine was flowed to inactivate activated carboxyl groups that remained unreacted with PD-L1 protein, and 10 mM glycine, pH 2.0 was injected to wash off proteins that were not bound to the sensor chip. . Then, 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).
평형상태에서의 센소그램 데이터를 농도에 따라 플로팅 (plotting) 및 피팅 (fitting)을 하여 평형 해리상수 (KD)계산한 결과 16E12(4F5)은 0.001 nM로 PD-L1 항원에 대해 높은 친화도를 보였다 (도 15).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).
PDL1-16E12, LS, 4F5의 사람, 원숭이, 생쥐 PD-L1 단백질의 결합능 비교 결과는 표 12에 기재한 바와 같다.PDL1-16E12, LS, 4F5 human, monkey, mouse PD-L1 protein binding results are shown in Table 12.
Figure PCTKR2017008495-appb-T000012
Figure PCTKR2017008495-appb-T000012
실시예 9: PD-L1 단일 클론 항체의 에피토프 (epitope) 결정Example 9: Epitope Determination of PD-L1 Monoclonal Antibody
96-웰 면역-플레이트에 항원 PD-L1 wild type (WT) 또는 여러 변이체 (mutants)를 웰 당 100 ng씩 4 ℃에서 16시간 코딩한 후 PBS에 녹인 4% 스킴 밀크를 사용하여 각 웰을 차단 (blocking)하였다. 각 웰마다 0.2 ㎖ PBS/T 사용하여 씻어준 뒤 16시간 동안 배양한 단일클론 scFv-phage (each 100 scFv-phage)를 각 well에 100 ㎕씩 넣고 상온에서 2시간 동안 반응시켰다. 다시 각 웰마다 0.2 ㎖ PBS/T을 사용하여 4번 씻어준 후 2차 항체 (second antibody)인 anti-Fab를 1/2000로 희석하여 실온에서 1시간 동안 반응하였다. 0.2 ㎖ PBS/T로 씻어준 후에 발색하여 흡광도 490 nm에서 측정하였다. Block each well with 4% skim milk dissolved in PBS after encoding antigen PD-L1 wild type (WT) or multiple variants at 100 ° C per well for 16 hours at 4 ° C in a 96-well immuno-plate. (blocking). After washing with 0.2 ml PBS / T for each well, monoclonal scFv-phage (each 100 scFv-phage), which was incubated for 16 hours, was added to 100 μl of each well and reacted at room temperature for 2 hours. Each well was washed four times with 0.2 ml PBS / T, and then the anti-Fab, a secondary antibody, 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.
그 결과, 대조 항체와 PD-L1 변이체들에 대해 다른 결합양상을 보임으로 다른 에피토프를 가진다는 것을 확인할 수 있었다 (도 16).As a result, it was confirmed that different epitopes were shown by showing different binding patterns for the control antibody and PD-L1 variants (FIG. 16).
실시예 10: PD-L1 단일 클론 항체의 이종 MLR (allogenic MLR) 반응에서의 활성 증가Example 10 Increasing Activity in an Allogenic MLR Reaction of PD-L1 Monoclonal Antibody
서로 다른 인간으로부터 분리된 단핵구 유래 수지상 세포 (monocyte derived dendritic cell)에 T 세포를 1: 10의 비율로 섞은 후에 5일 배양 후 배양액의 인터페론 감마의 양을 재어 보면 16E12의 모항체를 넣어준 것에서 농도에 의존적으로 인터페론 감마의 양이 증가함을 확인하였다 (도 17).T cells were mixed at a ratio of 1: 10 to monocyte-derived dendritic cells isolated from different humans, and after 5 days of cultivation, the amount of interferon gamma in the culture was measured. It was confirmed that the amount of interferon gamma increases depending on (Fig. 17).
실시예 11: PD-L1 단일 클론 항체의 동계 (syngeneic) 암동물 모델에서의 효능 평가Example 11 Evaluation of Efficacy of PD-L1 Monoclonal Antibodies in Syngeneic Cancer Animal Models
16E12-2B9 PD-L1 단일 클론 항체의 생체내 효능을 확인하기 위하여 BAlb/C 마우스에 옆구리 피하에 대장암 세포인 CT-26 세포를 8×106개 투여하고 종양의 크기가 좁쌀만할 때부터 3주간 주 2회씩 5mg/kg의 용량으로 투여하면서 종양의 성장을 관찰하였을 경우, PD-L1 단일 클론 항체 투여 군에서 현격한 종양 크기의 증가 감소가 관찰되었다(도 18). To confirm the in vivo efficacy of 16E12-2B9 PD-L1 monoclonal antibody, BAlb / C mice were administered 8 × 10 6 CT-26 cells, which are colon cancer cells under the flank subcutaneously, and the tumor size was narrow. When tumor growth was observed with administration of 5 mg / kg twice a week for 3 weeks, a marked decrease in tumor size was observed in the PD-L1 monoclonal antibody administration group (FIG. 18).
실시예 11: PD-L1 단일 클론 항체의 열 안정성 테스트 Example 11: Thermal Stability Test of PD-L1 Monoclonal Antibody
항체 단백질을 DPBS 에 희석하여 3uM, 45 ul를 만들고, 200x sypro orange dye(#S6650, Thermo) 5 ul와 섞어서 qPCR Tube(#B77009, B57651, bioplastics)에 50 ul씩 분주한다. Biorad CFX96 real time PCR 기기를 사용하여 qPCR을 실시하였다. qPCR 조건은 다음과 같이 25도에서 30초 반응 후, 99도까지 1도씩 증가시키되 각 온도에 1분간 반응시키고 마지막 25도 10초 반응을 시켜 마무리 하였다. 항체 구조가 풀리는 속도 상수로는 Tm (Melting temperature, 용융 온도)을 사용하였다. 그 결과는 아래 표(표13)와 같다.Dilute the antibody protein in DPBS to make 3 uM, 45 ul, mix with 5 ul of 200x sypro orange dye (# S6650, Thermo) and dispense 50 ul into qPCR Tube (# B77009, B57651, bioplastics). QPCR was performed using a Biorad CFX96 real time PCR instrument. The qPCR condition was 30 seconds after the reaction at 25 degrees, and then increased by 1 degree to 99 degrees, followed by reaction for 1 minute at each temperature. Tm (Melting temperature, melting temperature) was used as the rate constant of the antibody structure is released. The results are shown in the table below (Table 13).
Figure PCTKR2017008495-appb-T000013
Figure PCTKR2017008495-appb-T000013
실시예 13: PD-L2와의 결합력 여부Example 13: Adhesion with PD-L2
항 PD-L1 항체의 PD-L2와 결합 여부를 확인하기 위해 인간 PD-L2-Fc(#10292-H02H, Sino)를 96-웰 면역 마이크로플레이트 (96-well immuno microplate: #439454, Thermo)의 웰에 4℃에서 16시간 동안 고정시키고, 0.05% tween-20(#P9416, Sigma-Aldrich)이 들어있는 PBS로 3번 세척 후, 4% 스킴 밀크(#232120, Becton, Dickinson and Company)가 포함된 세척액으로 상온에서 1시간동안 방치함으로써 비특이적 결합을 차단하였다. 그 사이 일정 희석 배수로 연속 희석된 각 항체 혹은 양성 대조로 사용된 인간 PD-1-His (S1352, Y-Biologics)를 상온에서 1시간 동안 반응시킨 후, 준비된 마이크로플레이트에 넣어 상온에서 1시간 동안 방치시킨다. 동일한 세척 방법을 적용한 이후, anti-Biotin-His 항체 (#MA1-21315-BTIN, Thermo)를 1:2000으로 희석하여 마이크로플레이트의 웰에 넣고 상온에서 1시간 반응시킨 후, 동일한 방법으로 세척한 뒤 Streptavidin poly-HRP 항체(#21140, Pierce)를 1: 5000으로 희석하여 마이크로플레이트의 웰에 넣고 상온에서 1시간 반응시킨 후, 동일한 방법으로 세척하였다. 100 ul TMB 기질용액 (#T0440, Sigma-Aldrich)을 넣고 빛을 차단한 뒤 상온에서 3분간 방치 후 50 ul 2.5M 황산(#S1478, Samchun)을 넣어 반응을 중단시키고 분광광도계(#GM3000, Glomax® Discover System Promega)를 이용하여 450nm에서 흡광도를 측정하였다. 그 결과를 도 19 에 나타내었다Human PD-L2-Fc (# 10292-H02H, Sino) was subjected to 96-well immuno microplate (# 439454, Thermo) to confirm anti-PD-L1 antibody binding to PD-L2. The wells were fixed at 4 ° C. for 16 hours, washed three times with PBS containing 0.05% tween-20 (# P9416, Sigma-Aldrich) followed by 4% Scheme Milk (# 232120, Becton, Dickinson and Company). The non-specific binding was blocked by leaving the washed solution at room temperature for 1 hour. In the meantime, 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. After applying the same washing method, 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.
본 발명에 따른 PD-L1에 결합하는 항체 또는 이의 항원 결합단편은 PD-L1에 높은 친화력으로 결합하면서도, PD-1/PD-L1 복합체의 형성을 저해함으로써, PD-1/PD-L1 매개 T 세포 활성을 회피하는 T 세포 고갈을 억제할 수 있다. 이를 통해, 본 발명에 따른 PD-L1에 결합하는 항체 또는 이의 항원 결합단편은 목적하는 암 또는 감염 질환의 예방 또는 치료에 유용하게 사용될 수 있다. 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. Through this, 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.
이상으로 본 발명의 내용의 특정한 부분을 상세히 기술하였는바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적 기술은 단지 바람직한 실시양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서, 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다고 할 것이다.As described above in detail a specific part of the content of the present invention, for those skilled in the art, such a specific description is only a preferred embodiment, which is not limited by the scope of the present invention Will be obvious. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.
전자파일 첨부하였음.Electronic file attached.

Claims (12)

  1. 서열번호 1 내지 서열번호 7로 구성된 군에서 선택되는 서열과 90% 이상의 서열 상동성을 가지는 서열을 포함하는 중쇄 CDR1, A heavy chain CDR1 comprising a sequence having 90% or more sequence homology with a sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 7,
    서열번호 8 내지 서열번호 15로 구성된 군에서 선택되는 서열과 90% 이상의 서열 상동성을 가지는 서열을 포함하는 중쇄 CDR2, 및 A heavy chain CDR2 comprising a sequence having at least 90% sequence homology with a sequence selected from the group consisting of SEQ ID NOs: 8 to 15, and
    서열번호 16 내지 서열번호 25로 구성된 군에서 선택되는 서열과 90% 이상의 서열 상동성을 가지는 서열을 포함하는 중쇄 CDR3를 포함하는 중쇄 가변영역, 및 A heavy chain variable region comprising 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, and
    서열번호 88 내지 서열번호 102로 구성된 군에서 선택되는 서열과 90% 이상의 서열 상동성을 가지는 서열을 포함하는 경쇄 CDR1, 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 102,
    서열번호 103 내지 서열번호 119로 구성된 군에서 선택되는 서열과 90% 이상의 서열 상동성을 가지는 서열을 포함하는 경쇄 CDR2, 및A light chain CDR2 comprising a sequence having 90% or more sequence homology with a sequence selected from the group consisting of SEQ ID NOs: 103 through 119, and
    서열번호 120 내지 서열번호 144로 구성된 군에서 선택되는 서열과 90% 이상의 서열 상동성을 가지는 서열을 포함하는 경쇄 CDR3을 포함하는 경쇄 가변영역을 포함하는, PD-L1에 결합하는 항체 또는 이의 항원 결합단편.An antibody binding to PD-L1 or an antigen binding thereof, comprising a light chain variable region comprising a light chain CDR3 comprising a sequence having at least 90% sequence homology with a sequence selected from the group consisting of SEQ ID NOs: 120 to 144 snippet.
  2. 제1항에 있어서, The method of claim 1,
    서열번호 1의 중쇄 CDR1, 서열번호 8의 중쇄 CDR2, 및 서열번호 16의 중쇄 CDR3을 포함하는 중쇄 가변영역, 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,
    서열번호 2의 중쇄 CDR1, 서열번호 9의 중쇄 CDR2, 및 서열번호 17의 중쇄 CDR3을 포함하는 중쇄 가변영역,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,
    서열번호 2의 중쇄 CDR1, 서열번호 9의 중쇄 CDR2, 및 서열번호 18의 중쇄 CDR3을 포함하는 중쇄 가변영역,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,
    서열번호 3의 중쇄 CDR1, 서열번호 10의 중쇄 CDR2, 및 서열번호 19의 중쇄 CDR3을 포함하는 중쇄 가변영역,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,
    서열번호 4의 중쇄 CDR1, 서열번호 11의 중쇄 CDR2, 및 서열번호 20의 중쇄 CDR3을 포함하는 중쇄 가변영역,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,
    서열번호 5의 중쇄 CDR1, 서열번호 12의 중쇄 CDR2, 및 서열번호 21의 중쇄 CDR3을 포함하는 중쇄 가변영역,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,
    서열번호 6의 중쇄 CDR1, 서열번호 13의 중쇄 CDR2, 및 서열번호 22의 중쇄 CDR3을 포함하는 중쇄 가변영역,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,
    서열번호 2의 중쇄 CDR1, 서열번호 9의 중쇄 CDR2, 및 서열번호 23의 중쇄 CDR3을 포함하는 중쇄 가변영역,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,
    서열번호 7의 중쇄 CDR1, 서열번호 14의 중쇄 CDR2, 및 서열번호 24의 중쇄 CDR3을 포함하는 중쇄 가변영역, 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,
    서열번호 2의 중쇄 CDR1, 서열번호 15의 중쇄 CDR2, 및 서열번호 25의 중쇄 CDR3을 포함하는 중쇄 가변영역, 또는A 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
    서열번호 2의 중쇄 CDR1, 서열번호 9의 중쇄 CDR2, 및 서열번호 17의 중쇄 CDR3을 포함하는 중쇄 가변영역을 포함하는 항체 또는 이의 항원 결합단편.An antibody or antigen-binding fragment thereof 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.
  3. 제1항에 있어서, The method of claim 1,
    서열번호 88의 경쇄 CDR1, 서열번호 103의 경쇄 CDR2, 및 서열번호 120의 경쇄 CDR3를 포함하는 경쇄 가변영역, 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,
    서열번호 89의 경쇄 CDR1, 서열번호 104의 경쇄 CDR2, 및 서열번호 121의 경쇄 CDR3를 포함하는 경쇄 가변영역, 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,
    서열번호 90의 경쇄 CDR1, 서열번호 105의 경쇄 CDR2, 및 서열번호 122의 경쇄 CDR3를 포함하는 경쇄 가변영역, 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,
    서열번호 91의 경쇄 CDR1, 서열번호 106의 경쇄 CDR2, 및 서열번호 123의 경쇄 CDR3를 포함하는 경쇄 가변영역, 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,
    서열번호 89의 경쇄 CDR1, 서열번호 107의 경쇄 CDR2, 및 서열번호 124의 경쇄 CDR3를 포함하는 경쇄 가변영역, 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,
    서열번호 92의 경쇄 CDR1, 서열번호 108의 경쇄 CDR2, 및 서열번호 122의 경쇄 CDR3를 포함하는 경쇄 가변영역, 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,
    서열번호 93의 경쇄 CDR1, 서열번호 109의 경쇄 CDR2, 및 서열번호 125의 경쇄 CDR3를 포함하는 경쇄 가변영역, 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,
    서열번호 94의 경쇄 CDR1, 서열번호 110의 경쇄 CDR2, 및 서열번호 126의 경쇄 CDR3를 포함하는 경쇄 가변영역, 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,
    서열번호 95의 경쇄 CDR1, 서열번호 111의 경쇄 CDR2, 및 서열번호 127의 경쇄 CDR3를 포함하는 경쇄 가변영역, 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,
    서열번호 96의 경쇄 CDR1, 서열번호 112의 경쇄 CDR2, 및 서열번호 128의 경쇄 CDR3를 포함하는 경쇄 가변영역, 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,
    서열번호 89의 경쇄 CDR1, 서열번호 108의 경쇄 CDR2, 및 서열번호 129의 경쇄 CDR3를 포함하는 경쇄 가변영역, 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,
    서열번호 89의 경쇄 CDR1, 서열번호 105의 경쇄 CDR2, 및 서열번호 130의 경쇄 CDR3를 포함하는 경쇄 가변영역, 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,
    서열번호 89의 경쇄 CDR1, 서열번호 113의 경쇄 CDR2, 및 서열번호 131의 경쇄 CDR3를 포함하는 경쇄 가변영역, 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,
    서열번호 97의 경쇄 CDR1, 서열번호 104의 경쇄 CDR2, 및 서열번호 132의 경쇄 CDR3를 포함하는 경쇄 가변영역, 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,
    서열번호 89의 경쇄 CDR1, 서열번호 104의 경쇄 CDR2, 및 서열번호 133의 경쇄 CDR3를 포함하는 경쇄 가변영역, 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,
    서열번호 97의 경쇄 CDR1, 서열번호 114의 경쇄 CDR2, 및 서열번호 134의 경쇄 CDR3를 포함하는 경쇄 가변영역, 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,
    서열번호 92의 경쇄 CDR1, 서열번호 115의 경쇄 CDR2, 및 서열번호 135의 경쇄 CDR3를 포함하는 경쇄 가변영역, 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,
    서열번호 98의 경쇄 CDR1, 서열번호 104의 경쇄 CDR2, 및 서열번호 130의 경쇄 CDR3를 포함하는 경쇄 가변영역, 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,
    서열번호 89의 경쇄 CDR1, 서열번호 116의 경쇄 CDR2, 및 서열번호 121의 경쇄 CDR3를 포함하는 경쇄 가변영역, 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,
    서열번호 89의 경쇄 CDR1, 서열번호 108의 경쇄 CDR2, 및 서열번호 136의 경쇄 CDR3를 포함하는 경쇄 가변영역, 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,
    서열번호 99의 경쇄 CDR1, 서열번호 105의 경쇄 CDR2, 및 서열번호 137의 경쇄 CDR3를 포함하는 경쇄 가변영역, 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,
    서열번호 89의 경쇄 CDR1, 서열번호 117의 경쇄 CDR2, 및 서열번호 138의 경쇄 CDR3를 포함하는 경쇄 가변영역, 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,
    서열번호 89의 경쇄 CDR1, 서열번호 118의 경쇄 CDR2, 및 서열번호 133의 경쇄 CDR3를 포함하는 경쇄 가변영역, 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,
    서열번호 89의 경쇄 CDR1, 서열번호 119의 경쇄 CDR2, 및 서열번호 139의 경쇄 CDR3를 포함하는 경쇄 가변영역, 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,
    서열번호 100의 경쇄 CDR1, 서열번호 104의 경쇄 CDR2, 및 서열번호 140의 경쇄 CDR3를 포함하는 경쇄 가변영역, 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,
    서열번호 89의 경쇄 CDR1, 서열번호 108의 경쇄 CDR2, 및 서열번호 141의 경쇄 CDR3를 포함하는 경쇄 가변영역, 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,
    서열번호 89의 경쇄 CDR1, 서열번호 105의 경쇄 CDR2, 및 서열번호 139의 경쇄 CDR3를 포함하는 경쇄 가변영역, 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,
    서열번호 89의 경쇄 CDR1, 서열번호 104의 경쇄 CDR2, 및 서열번호 142의 경쇄 CDR3를 포함하는 경쇄 가변영역, 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,
    서열번호 89의 경쇄 CDR1, 서열번호 105의 경쇄 CDR2, 및 서열번호 143의 경쇄 CDR3를 포함하는 경쇄 가변영역, 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,
    서열번호 101의 경쇄 CDR1, 서열번호 104의 경쇄 CDR2, 및 서열번호 141의 경쇄 CDR3를 포함하는 경쇄 가변영역, 또는A 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
    서열번호 102의 경쇄 CDR1, 서열번호 104의 경쇄 CDR2, 및 서열번호 144의 경쇄 CDR3를 포함하는 경쇄 가변영역을 포함하는 항체 또는 이의 항원 결합단편.An antibody or antigen-binding fragment thereof comprising a light chain variable region comprising 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.
  4. 제1항에 있어서, 서열번호 26 내지 서열번호 34로 구성된 군에서 선택되는 중쇄 가변영역 FR1, The method according to claim 1, wherein heavy chain variable region FR1 selected from the group consisting of SEQ ID NO: 26 to SEQ ID NO: 34,
    서열번호 35 내지 서열번호 41로 구성된 군에서 선택되는 중쇄 가변영역 FR2, A heavy chain variable region FR2 selected from the group consisting of SEQ ID NOs: 35 to 41;
    서열번호 42 내지 서열번호 49로 구성된 군에서 선택되는 중쇄 가변영역 FR3, 또는 A heavy chain variable region FR3 selected from the group consisting of SEQ ID NOs: 42 to 49, or
    서열번호 50 내지 서열번호 54로 구성된 군에서 선택되는 중쇄 가변영역 FR4를 포함하는 항체 또는 이의 항원 결합단편.An antibody or antigen-binding fragment thereof comprising a heavy chain variable region FR4 selected from the group consisting of SEQ ID NO: 50 to SEQ ID NO: 54.
  5. 제1항에 있어서, 서열번호 145 내지 서열번호 163로 구성된 군에서 선택되는 경쇄 가변영역 FR1, According to claim 1, Light chain variable region FR1 selected from the group consisting of SEQ ID NO: 145 to SEQ ID NO: 163,
    서열번호 164 내지 서열번호 184로 구성된 군에서 선택되는 경쇄 가변영역 FR2, Light chain variable region FR2 selected from the group consisting of SEQ ID NOs: 164 to 184,
    서열번호 185 내지 서열번호 210로 구성된 군에서 선택되는 경쇄 가변영역 FR3, 또는 Light chain variable region FR3 selected from the group consisting of SEQ ID NOs: 185 to 210, or
    서열번호 211 내지 서열번호 216로 구성된 군에서 선택되는 경쇄 가변영역 FR4를 포함하는 항체 또는 이의 항원 결합단편.An antibody or antigen-binding fragment thereof comprising a light chain variable region FR4 selected from the group consisting of SEQ ID NOs: 211 to 216.
  6. 제1항에 있어서, 서열번호 57 내지 서열번호 87로 구성된 군에서 선택되는 서열과 90% 이상의 서열 상동성을 가지는 서열을 포함하는 중쇄 가변영역을 포함하는 항체 또는 이의 항원 결합단편.The antibody or antigen-binding fragment thereof according to claim 1, comprising a heavy chain variable region comprising a sequence having at least 90% sequence homology with a sequence selected from the group consisting of SEQ ID NO: 57 to SEQ ID NO: 87.
  7. 제1항에 있어서, 서열번호 217 내지 서열번호 247로 구성된 군에서 선택되는 서열과 90% 이상의 서열 상동성을 가지는 서열을 포함하는 경쇄 가변영역을 포함하는 항체 또는 이의 항원 결합단편.The antibody or antigen-binding fragment thereof according to claim 1, comprising a light chain variable region comprising a sequence having at least 90% sequence homology with a sequence selected from the group consisting of SEQ ID NOs: 217 to 247.
  8. 제 1 항 내지 제 7 항 중 어느 한 항의 항체 또는 이의 항원 결합단편을 코딩하는 핵산.A nucleic acid encoding the antibody of claim 1 or an antigen binding fragment thereof.
  9. 제8항의 핵산을 포함하는 발현벡터.An expression vector comprising the nucleic acid of claim 8.
  10. 제9항의 발현 벡터로 형질전환된 세포.A cell transformed with the expression vector of claim 9.
  11. 다음 단계를 포함하는 PD-L1에 결합하는 항체 또는 그의 항원 결합 단편의 제조방법:Method for producing an antibody or antigen-binding fragment thereof that binds to PD-L1, comprising the following steps:
    (a) 제10항의 세포를 배양하는 단계; 및(a) culturing the cells of claim 10; And
    (b) 상기 배양된 세포에서 항체 또는 그의 항원 결합 단편을 회수하는 단계.(b) recovering the antibody or antigen-binding fragment thereof from the cultured cells.
  12. 제 1 항 내지 제 7 항 중 어느 한 항의 항체 또는 그의 항원 결합 단편을 유효성분으로 포함하는 암 또는 감염 질환의 예방 또는 치료용 조성물.A composition for preventing or treating cancer or infectious disease comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 7 as an active ingredient.
PCT/KR2017/008495 2016-08-05 2017-08-07 Antibody against programmed death-ligand 1 (pd-l1), and use thereof WO2018026249A1 (en)

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RU2019105664A RU2721582C1 (en) 2016-08-05 2017-08-07 Antibodies against ligand-1 programmed death (pd-l1) and application thereof
US16/321,412 US10919966B2 (en) 2016-08-05 2017-08-07 Antibody to programmed death-ligand 1 (PD-L1) and use thereof
CA3032806A CA3032806C (en) 2016-08-05 2017-08-07 Antibody to programmed death-ligand 1 (pd-l1) and use thereof
AU2017306507A AU2017306507B2 (en) 2016-08-05 2017-08-07 Antibody to programmed death-ligand 1 (PD-L1) and use thereof
CN201780055412.7A CN110072889B (en) 2016-08-05 2017-08-07 Antibodies against programmed cell death ligand 1 (PD-L1) and uses thereof
BR112019002282A BR112019002282A2 (en) 2016-08-05 2017-08-07 pd-11 binding antibody or antibody antigen-binding fragment, method for producing the same and composition for the prevention or treatment of cancer or infectious diseases
JP2019528010A JP6925421B2 (en) 2016-08-05 2017-08-07 Antibodies to programmed cell death protein ligand-1 (PD-L1) and their uses
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2021517904A (en) * 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 Cell Engagement Binding Molecules
WO2023187460A1 (en) * 2022-04-01 2023-10-05 Mabtree Biologics Ag Human antibody or antigen binding fragment thereof specific against pd-l1 to enhance t-cell function

Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1988001649A1 (en) 1986-09-02 1988-03-10 Genex Corporation Single polypeptide chain binding molecules
WO1988006630A1 (en) 1987-03-02 1988-09-07 Genex Corporation Method for the preparation of binding molecules
WO1988007085A1 (en) 1987-03-20 1988-09-22 Creative Biomolecules, Inc. Process for the purification of recombinant polypeptides
WO1988007086A1 (en) 1987-03-20 1988-09-22 Creative Biomolecules, Inc. Leader sequences for the production of recombinant proteins
WO1988009344A1 (en) 1987-05-21 1988-12-01 Creative Biomolecules, Inc. Targeted multifunctional proteins
US20090055944A1 (en) * 2005-07-01 2009-02-26 Medarex, Inc. Human monoclonal antibodies to be programmed death ligand 1 (pd-l1)
WO2010077634A1 (en) * 2008-12-09 2010-07-08 Genentech, Inc. Anti-pd-l1 antibodies and their use to enhance t-cell function
WO2015061668A1 (en) * 2013-10-25 2015-04-30 Dana-Farber Cancer Institute, Inc. Anti-pd-l1 monoclonal antibodies and fragments thereof
WO2016061142A1 (en) * 2014-10-14 2016-04-21 Novartis Ag Antibody molecules to pd-l1 and uses thereof

Patent Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1988001649A1 (en) 1986-09-02 1988-03-10 Genex Corporation Single polypeptide chain binding molecules
WO1988006630A1 (en) 1987-03-02 1988-09-07 Genex Corporation Method for the preparation of binding molecules
WO1988007085A1 (en) 1987-03-20 1988-09-22 Creative Biomolecules, Inc. Process for the purification of recombinant polypeptides
WO1988007086A1 (en) 1987-03-20 1988-09-22 Creative Biomolecules, Inc. Leader sequences for the production of recombinant proteins
WO1988009344A1 (en) 1987-05-21 1988-12-01 Creative Biomolecules, Inc. Targeted multifunctional proteins
US20090055944A1 (en) * 2005-07-01 2009-02-26 Medarex, Inc. Human monoclonal antibodies to be programmed death ligand 1 (pd-l1)
US20110209230A1 (en) * 2005-07-01 2011-08-25 Korman Alan J Human Monoclonal Antibodies To Programmed Death Ligand 1 (PD-L1)
WO2010077634A1 (en) * 2008-12-09 2010-07-08 Genentech, Inc. Anti-pd-l1 antibodies and their use to enhance t-cell function
WO2015061668A1 (en) * 2013-10-25 2015-04-30 Dana-Farber Cancer Institute, Inc. Anti-pd-l1 monoclonal antibodies and fragments thereof
WO2016061142A1 (en) * 2014-10-14 2016-04-21 Novartis Ag Antibody molecules to pd-l1 and uses thereof

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2021517904A (en) * 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 Cell Engagement Binding Molecules
JP7076571B2 (en) 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 Cell engagement binding molecule
WO2023187460A1 (en) * 2022-04-01 2023-10-05 Mabtree Biologics Ag Human antibody or antigen binding fragment thereof specific against pd-l1 to enhance t-cell function

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