WO2018026248A1 - Novel antibody against programmed cell death protein (pd-1), and use thereof - Google Patents

Abstract

The present invention relates to an antibody against human programmed cell death 1 (PD-1) or an antigen-binding fragment thereof, a nucleic acid for coding same, a vector comprising the nucleic acid, a recombinant microorganism transformed with the vector, a method for preparing the antibody or the antigen-binding fragment thereof, and a composition for cancer prevention or treatment comprising same. The novel PD-1-binding antibody or the antigen-binding fragment of thereof can bind to PD-1 and inhibit the activity thereof and is therefore useful for developing an immunotherapeutic agent for various diseases associated with PD-1.

Description

New antibodies against programmed cell death protein (PD-1) and uses thereof

The present invention provides an antibody or antigen-binding fragment thereof for human programmed cell death protein (PD-1), a nucleic acid encoding the same, a vector comprising the nucleic acid, a recombinant microorganism transformed with the vector. It relates to a method for producing the antibody or antigen-binding fragment thereof, and to a composition for preventing or treating cancer comprising the same.

In the case of the first-generation chemocancer and second-generation targeted anticancer agents, which are currently widely used, there is a problem that can be administered only to patients having high risk of side effects and drug resistance due to the toxicity of the anticancer drugs and having specific genetic mutations. Immune anticancer agents, called third-generation anticancer agents that overcome these problems, act on the signaling pathways of immune cells to activate immune cells to attack cancer cells to produce therapeutic effects. Unlike conventional anticancer drugs, the body's immune system can be used to treat various diseases including cancer, and side effects are also reported to be lower than conventional anticancer drugs.

PD-1 (also named CD279) is a 55 KD receptor protein associated with the CD28 / CTLA4 co-stimulatory / inhibitory receptor family (Blank et al., 2005 Cancer Immunol Immunother 54: 307- 314).

Cloning genes and cDNAs encoding PD-1 have been characterized in mice and humans (Ishida et al., 1992 EMBO J 11: 3887-3395; Shinohara et al., 1994 Genomics 23: 704-706) . Full length PD-1 contains 288 amino acid residues (NCBI accession number: NP_005009). The extracellular domain consists of 1-167 amino acid residues and the cytoplasmic C-terminal tail comprises 191-288 residues, which are two hypothetical immune-modulating motifs, an immunoreceptor tyrosine based inhibitory motif (ITIM; Vivier et al. , 1997 Immunol Today 18: 286-291) and immunoreceptor tyrosine switch motifs (ITSM; Chemnitz et al., 2004 J Immunol 173: 945-954).

To date, two sequence related ligands PD-L1 (B7-H1) and PD-L2 (B7-DC) specifically interact with PD-1 to induce intracellular signaling, and CD3 and CD28 mediated T- It has been shown to inhibit cell activation (Riley, 2009 Immunol Rev 229: 114-125), which eventually regulates T-cell activity, for example cell growth, IL-2 and IFN- as well as other growth factors and cytokine secretion. It is to reduce γ secretion.

Expression of PD-1 is frequently found in immune cells, such as T-cells, B-cells, monocytes, and natural killer (NK) cells, and is rarely expressed in other human tissues, such as muscle, epithelial, and neural tissues. It doesn't work. In addition, high levels of PD-1 expression are often associated with the activity of immune cells. For example, if the human T-cell line Jurkat is activated by PHA (phytohaemagglutinin) or phorbol ester (12-O-tetradecanoylphorbol-13-acetate or TPA), the expression of PD-1 is upward as shown in the Western blot. Adjusted. By stimulation of anti-CD3 antibodies, the same phenomenon was observed in stimulated mouse T- and B-lymphocytes and primary human CD4 + T cells. Increased PD-1 expression stimulates effect T cells and directs activated effect T cells in the direction of depletion and reduced immunoactivity. Thus, PD-1 mediated inhibition signals are known to play an important role in immune tolerance.

In various cancers, increased PD-1 expression of tumor-infiltrating lymphocytes (TILs) and PD-1 ligand expression of tumor cells have been reported, and other types of tissues and organs such as lung, liver, stomach, kidney, Breast, ovary, pancreas, melanocytes and esophagus. More frequently, the expression of PD-1 and PD-L1 in these cancers is associated with poor prognosis for patient survival outcomes. Transgenic mice that knocked out PD-1 genes to inhibit Xenograft cancer cell growth further explained the importance of PD-1 signaling in immune system regulation for cancer removal or tolerance.

Upregulation of PD-1 signaling leads to cancer proliferation of immune tolerance, as well as to viral infection and expansion in humans. Pandemic liver infection viruses HBV and HCV induce overexpression of PD-1 ligand in hepatocytes and activate PD-1 signaling in effect T cells, resulting in T-cell depletion and tolerance to viral infection. Likewise, HIV infection frequently evades the human immune system with similar mechanisms. Therapeutic regulation of PD-1 signaling by antagonist molecules can restore immune cells from tolerance and can be reactivated to eliminate cancer and chronic viral infections.

Drugs that target PD-1 include the monoclonal antibodies nivolumab and pembrolizumab, which are used as treatments for malignant melanoma and non-small cell lung cancer. It is reported that the largeness and the precise verification of the efficacy of treatment is required. Therefore, there is an urgent need for a new concept of PD-1 target therapy that overcomes the limitations of existing drugs.

Under this technical background, the inventors of the present application have tried to develop an anti-cancer therapeutic antibody that specifically binds to PD-1. As a result, we developed anti-PD-1 antibodies that bind to PD-1 with high affinity using phage display technology, and these anti-PD-1 antibodies prevent the formation of complexes of PD-1 / PD-L1. It was confirmed that it can significantly inhibit, the present invention was completed.

Summary of the Invention

It is an object of the present invention to provide a novel antibody or antigen binding fragment thereof against PD-1.

Another object of the present invention is to provide a nucleic acid encoding the antibody or antigen-binding fragment thereof.

It is another object of the present invention to provide a vector comprising the nucleic acid, a recombinant microorganism into which the vector is introduced, and a method of preparing the same.

Another object of the present invention to provide a composition for preventing or treating cancer comprising the antibody or antigen-binding fragment thereof.

In order to achieve the above object, the present invention is a group consisting of a heavy chain CDR1, SEQ ID NO: 31 to SEQ ID NO: 56 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: 30 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: 57-SEQ ID NO: 79 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: 198 to 222, SEQ ID NO: 223 to SEQ ID NO: 241 A light chain CDR2 comprising a sequence having 90% or more sequence homology with a sequence to be sequenced, and SEQ ID NO: 242 to SEQ ID NO: 269 It provides a sequence that is chosen as an antibody or an antigen-binding fragment that binds to 90%, PD-1 comprising a light chain variable region comprising a light chain CDR3 comprising a sequence having at least sequence homology.

The present invention also provides a nucleic acid encoding the antibody or antigen-binding fragment thereof.

The present invention also provides an expression vector comprising the nucleic acid.

The present invention also provides a cell transformed with the expression 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, containing the antibody or antigen-binding fragment thereof as an active ingredient.

1 is a schematic diagram of a PD-1 expression vector fused with a human Fc or a mouse Fc at the carboxy-terminus.

2 shows the results of PD-1 protein purification:

2A is a 10% SDS-PAGE gel of PD1-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 PD1-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 binding force of the antibody to PD-1 according to the number of panning.

Figure 4 shows the ELSIA results for measuring the binding capacity of mono phage to PD1-His.

5 shows the results of SDS-PAGE analysis for confirming the purity of the PD-1 antibody.

Figure 6 shows the results of in vitro efficacy evaluation of PD-1 antibody.

Figure 7 shows the results of the concentration-dependent in vitro efficacy evaluation of the PD-1 antibody.

Figure 8 shows the binding capacity of the antibody bound concentration-dependently to the overexpressed human PD-1 using MFI (mean fluorescence intensity).

9 shows the results of kinetic measurements between PD1-hFc, PD1-45D6, and 49A2.

10 shows the results of screening for optimized monoclones.

Figure 11 shows the results of a comparative analysis of the expression rate with the parent antibody.

Figure 12 shows the results of evaluating the in vitro efficacy of the PD1 antibody according to the present invention.

Figure 13 shows the results of evaluating the concentration-dependent in vitro efficacy of the PD1 antibody according to the present invention.

Figure 14 shows the results confirming the binding to PD-1 expressed on the cell surface of the monoclonal antibody to the selected PD1 antibody variants.

15 shows the results of binding measurements on PD-1 expressed on the cell surface of monoclonal antibodies against selected PD1 antibody variants.

Figure 16 shows the results confirming the inhibitory effect of the antibody to block the formation of PD-1 / PD-L1 or PD-1 / PD-L2 complex using enzyme immunosorbent in the selected antibody.

Figure 17 shows the kinetics measurement results between PD1-hFc protein and PD1-45D6, 49A2 and 49A2 (2B9).

18 shows a schematic of PD1 variants.

Figure 19 shows the results of confirming the binding force to the PD-1 variants through the enzyme immunosorbent of the selected phage (phage), the lower the binding force indicates a lower value.

Figure 20 shows the results confirming the binding capacity to the PD-1 variants through the enzyme immunosorbent of the selected antibody.

Figure 21 shows the results confirming the binding specificity through enzyme immunosorbent.

Figure 22 shows the result of comparing the transient expression productivity in HEK-293 cells.

Figure 23 shows the results confirmed that the increased activity in heterologous MLR (Mixed Lymphocyte Reaction) by PD1 monoclonal antibody.

Detailed description of the invention and preferred Embodiment

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 and the experimental methods described below are well known and commonly used in the art.

In one aspect, the present invention is selected from the group consisting of heavy chain CDR1, SEQ ID NO: 31 to SEQ ID NO: 56 comprising a sequence having 90% or more sequence homology with the sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 30 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: 57-SEQ ID NO: 79 A light chain CDR1 comprising a heavy chain variable region and a sequence having at least 90% sequence homology with a sequence selected from the group consisting of SEQ ID NOs: 198 to 222, and a sequence selected from the group consisting of SEQ ID NOs: 223 to SEQ ID NO: 241; 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: 242 to SEQ ID NO: 269; An antibody or antigen binding fragment thereof that binds to PD-1, comprising a light chain variable region comprising a light chain CDR3 comprising a sequence having 90% or more sequence homology.

The present inventors have tried to develop an anti-cancer therapeutic antibody that binds to PD-1, which is known to be expressed in various cancers. As a result, phage display technology was used to prepare an anti-PD-1 antibody that binds to PD-1 with high affinity, and confirmed that such anti-PD-1 antibody can inhibit the activity of PD-1.

In the present invention, "programmed cell death protein 1 (PD-1)" is a signal transduction protein, it is known to play a role in regulating the activation and function of T cells. The binding of PD-1 of T cells and PD-L1, a ligand expressed from cancer cells, in the tumor microenvironment activates the PD-1 signaling pathway, resulting in inactivation of T cells. It is found in a variety of cancers, including species, non-small cell lung cancer, and renal cell carcinoma.

As used herein, the term "antibody" refers to an anti-PD-1 antibody that specifically binds to PD-1. The scope of the present invention includes not only complete antibody forms that specifically bind PD-1, 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 (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.

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 'differs from Fab in that it has a hinge region comprising at least one cysteine residue 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.

In one embodiment, the antibody according to the invention is in Fv form (eg scFv), Fab or fully antibody form. 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.

As used herein, the term “heavy chain” refers to a variable region domain VH comprising an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen and a full length heavy chain comprising three constant region domains CH1, CH2 and CH3 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 VL and a constant region domain CL comprising an amino acid sequence having sufficient variable region sequence to confer specificity to the antigen. All means.

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.

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.

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.

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.

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). . VH refers to the variable domain of the heavy chain. VL refers to the variable domain of the light chain.

“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. Three CDR regions.

In the present invention, the antibody or antigen-binding fragment thereof that binds to PD-1 is heavy chain CDR1 selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 30,

A heavy chain CDR2 selected from the group consisting of SEQ ID NOs: 31 to 56, and

A heavy chain variable region comprising a heavy chain CDR3 selected from the group consisting of SEQ ID NOs: 57 to 79, and

Light chain CDR1 selected from the group consisting of SEQ ID NOs: 198 to 222,

A light chain CDR2 selected from the group consisting of SEQ ID NOs: 223 to 241, and

It may include a light chain variable region comprising a light chain CDR3 selected from the group consisting of SEQ ID NO: 242 to SEQ ID NO: 269.

Specifically, the antibody or antigen-binding fragment thereof that binds to PD-1 is a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 1, a heavy chain CDR2 of SEQ ID NO: 31, and a heavy chain CDR3 of SEQ ID NO: 57,

A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 1, a heavy chain CDR2 of SEQ ID NO: 32, and a heavy chain CDR3 of SEQ ID NO: 58,

A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 2, a heavy chain CDR2 of SEQ ID NO: 33, and a heavy chain CDR3 of SEQ ID NO: 59,

A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 2, a heavy chain CDR2 of SEQ ID NO: 33, and a heavy chain CDR3 of SEQ ID NO: 60,

A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 2, a heavy chain CDR2 of SEQ ID NO: 33, and a heavy chain CDR3 of SEQ ID NO: 61,

A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 2, a heavy chain CDR2 of SEQ ID NO: 33, and a heavy chain CDR3 of SEQ ID NO: 62,

A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 3, a heavy chain CDR2 of SEQ ID NO: 34, and a heavy chain CDR3 of SEQ ID NO: 63,

A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 4, a heavy chain CDR2 of SEQ ID NO: 35, and a heavy chain CDR3 of SEQ ID NO: 64,

A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 5, a heavy chain CDR2 of SEQ ID NO: 36, and a heavy chain CDR3 of SEQ ID NO: 65,

A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 6, a heavy chain CDR2 of SEQ ID NO: 37, and a heavy chain CDR3 of SEQ ID NO: 66,

A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 1, a heavy chain CDR2 of SEQ ID NO: 32, and a heavy chain CDR3 of SEQ ID NO: 67,

A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 7, a heavy chain CDR2 of SEQ ID NO: 38, and a heavy chain CDR3 of SEQ ID NO: 68,

A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 8, a heavy chain CDR2 of SEQ ID NO: 39, and a heavy chain CDR3 of SEQ ID NO: 69,

A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 9, a heavy chain CDR2 of SEQ ID NO: 40, and a heavy chain CDR3 of SEQ ID NO: 70,

A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 10, a heavy chain CDR2 of SEQ ID NO: 41, and a heavy chain CDR3 of SEQ ID NO: 71,

A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 11, a heavy chain CDR2 of SEQ ID NO: 42, and a heavy chain CDR3 of SEQ ID NO: 72,

A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 12, a heavy chain CDR2 of SEQ ID NO: 43, and a heavy chain CDR3 of SEQ ID NO: 73,

A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 13, a heavy chain CDR2 of SEQ ID NO: 44, and a heavy chain CDR3 of SEQ ID NO: 74,

A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 14, a heavy chain CDR2 of SEQ ID NO: 45, and a heavy chain CDR3 of SEQ ID NO: 75,

A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 15, a heavy chain CDR2 of SEQ ID NO: 46, and a heavy chain CDR3 of SEQ ID NO: 76,

A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 8, a heavy chain CDR2 of SEQ ID NO: 47, and a heavy chain CDR3 of SEQ ID NO: 77,

A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 16, a heavy chain CDR2 of SEQ ID NO: 48, and a heavy chain CDR3 of SEQ ID NO: 78,

A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 17, a heavy chain CDR2 of SEQ ID NO: 49, and a heavy chain CDR3 of SEQ ID NO: 58,

A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 18, a heavy chain CDR2 of SEQ ID NO: 50, and a heavy chain CDR3 of SEQ ID NO: 58,

A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 19, a heavy chain CDR2 of SEQ ID NO: 51, and a heavy chain CDR3 of SEQ ID NO: 58,

A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 20, a heavy chain CDR2 of SEQ ID NO: 52, and a heavy chain CDR3 of SEQ ID NO: 58,

A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 21, a heavy chain CDR2 of SEQ ID NO: 51, and a heavy chain CDR3 of SEQ ID NO: 58,

A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 22, a heavy chain CDR2 of SEQ ID NO: 53, and a heavy chain CDR3 of SEQ ID NO: 58,

A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 23, a heavy chain CDR2 of SEQ ID NO: 49, and a heavy chain CDR3 of SEQ ID NO: 58,

A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 17, a heavy chain CDR2 of SEQ ID NO: 54, and a heavy chain CDR3 of SEQ ID NO: 58,

A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 24, a heavy chain CDR2 of SEQ ID NO: 55, and a heavy chain CDR3 of SEQ ID NO: 58,

A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 21, a heavy chain CDR2 of SEQ ID NO: 51, and a heavy chain CDR3 of SEQ ID NO: 58,

A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 25, a heavy chain CDR2 of SEQ ID NO: 56, and a heavy chain CDR3 of SEQ ID NO: 57,

A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 26, a heavy chain CDR2 of SEQ ID NO: 31, and a heavy chain CDR3 of SEQ ID NO: 57,

A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 27, a heavy chain CDR2 of SEQ ID NO: 31, and a heavy chain CDR3 of SEQ ID NO: 57,

A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 28, a heavy chain CDR2 of SEQ ID NO: 31, and a heavy chain CDR3 of SEQ ID NO: 57,

A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 29, a heavy chain CDR2 of SEQ ID NO: 31, and a heavy chain CDR3 of SEQ ID NO: 57,

A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 30, a heavy chain CDR2 of SEQ ID NO: 31, and a heavy chain CDR3 of SEQ ID NO: 57,

A heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 1, the heavy chain CDR2 of SEQ ID NO: 32, and the heavy chain CDR3 of SEQ ID NO: 79, or

A heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 1, the heavy chain CDR2 of SEQ ID NO: 32, and the heavy chain CDR3 of SEQ ID NO: 57 may be included.

In addition, the antibody or antigen-binding fragment thereof that binds to PD-1 includes a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 198, a light chain CDR2 of SEQ ID NO: 223, and a light chain CDR3 of SEQ ID NO: 242,

A light chain variable region comprising a light chain CDR1 of SEQ ID NO: 199, a light chain CDR2 of SEQ ID NO: 224, and a light chain CDR3 of SEQ ID NO: 243,

A light chain variable region comprising a light chain CDR1 of SEQ ID NO: 200, a light chain CDR2 of SEQ ID NO: 225, and a light chain CDR3 of SEQ ID NO: 244,

A light chain variable region comprising a light chain CDR1 of SEQ ID NO: 201, a light chain CDR2 of SEQ ID NO: 226, and a light chain CDR3 of SEQ ID NO: 245,

A light chain variable region comprising a light chain CDR1 of SEQ ID NO: 202, a light chain CDR2 of SEQ ID NO: 227, and a light chain CDR3 of SEQ ID NO: 246,

A light chain variable region comprising a light chain CDR1 of SEQ ID NO: 203, a light chain CDR2 of SEQ ID NO: 228, and a light chain CDR3 of SEQ ID NO: 247,

A light chain variable region comprising a light chain CDR1 of SEQ ID NO: 204, a light chain CDR2 of SEQ ID NO: 229, and a light chain CDR3 of SEQ ID NO: 248,

A light chain variable region comprising a light chain CDR1 of SEQ ID NO: 205, a light chain CDR2 of SEQ ID NO: 230, and a light chain CDR3 of SEQ ID NO: 249,

A light chain variable region comprising a light chain CDR1 of SEQ ID NO: 206, a light chain CDR2 of SEQ ID NO: 231, and a light chain CDR3 of SEQ ID NO: 250,

A light chain variable region comprising a light chain CDR1 of SEQ ID NO: 207, a light chain CDR2 of SEQ ID NO: 232, and a light chain CDR3 of SEQ ID NO: 251,

A light chain variable region comprising a light chain CDR1 of SEQ ID NO: 208, a light chain CDR2 of SEQ ID NO: 230, and a light chain CDR3 of SEQ ID NO: 252,

A light chain variable region comprising a light chain CDR1 of SEQ ID NO: 209, a light chain CDR2 of SEQ ID NO: 233, and a light chain CDR3 of SEQ ID NO: 253,

A light chain variable region comprising a light chain CDR1 of SEQ ID NO: 210, a light chain CDR2 of SEQ ID NO: 234, and a light chain CDR3 of SEQ ID NO: 254,

A light chain variable region comprising a light chain CDR1 of SEQ ID NO: 211, a light chain CDR2 of SEQ ID NO: 235, and a light chain CDR3 of SEQ ID NO: 255,

A light chain variable region comprising a light chain CDR1 of SEQ ID NO: 212, a light chain CDR2 of SEQ ID NO: 236, and a light chain CDR3 of SEQ ID NO: 256,

A light chain variable region comprising a light chain CDR1 of SEQ ID NO: 213, a light chain CDR2 of SEQ ID NO: 230, and a light chain CDR3 of SEQ ID NO: 257,

A light chain variable region comprising a light chain CDR1 of SEQ ID NO: 214, a light chain CDR2 of SEQ ID NO: 237, and a light chain CDR3 of SEQ ID NO: 258,

A light chain variable region comprising a light chain CDR1 of SEQ ID NO: 215, a light chain CDR2 of SEQ ID NO: 230, and a light chain CDR3 of SEQ ID NO: 249,

A light chain variable region comprising a light chain CDR1 of SEQ ID NO: 211, a light chain CDR2 of SEQ ID NO: 238, and a light chain CDR3 of SEQ ID NO: 259,

A light chain variable region comprising a light chain CDR1 of SEQ ID NO: 216, a light chain CDR2 of SEQ ID NO: 238, and a light chain CDR3 of SEQ ID NO: 260,

A light chain variable region comprising a light chain CDR1 of SEQ ID NO: 217, a light chain CDR2 of SEQ ID NO: 239, and a light chain CDR3 of SEQ ID NO: 261,

A light chain variable region comprising a light chain CDR1 of SEQ ID NO: 218, a light chain CDR2 of SEQ ID NO: 240, and a light chain CDR3 of SEQ ID NO: 262,

A light chain variable region comprising a light chain CDR1 of SEQ ID NO: 219, a light chain CDR2 of SEQ ID NO: 241, and a light chain CDR3 of SEQ ID NO: 263,

A light chain variable region comprising a light chain CDR1 of SEQ ID NO: 220, a light chain CDR2 of SEQ ID NO: 230, and a light chain CDR3 of SEQ ID NO: 252,

A light chain variable region comprising a light chain CDR1 of SEQ ID NO: 215, a light chain CDR2 of SEQ ID NO: 230, and a light chain CDR3 of SEQ ID NO: 252,

A light chain variable region comprising a light chain CDR1 of SEQ ID NO: 205, a light chain CDR2 of SEQ ID NO: 230, and a light chain CDR3 of SEQ ID NO: 264,

A light chain variable region comprising a light chain CDR1 of SEQ ID NO: 205, a light chain CDR2 of SEQ ID NO: 230, and a light chain CDR3 of SEQ ID NO: 265,

A light chain variable region comprising a light chain CDR1 of SEQ ID NO: 221, a light chain CDR2 of SEQ ID NO: 230, and a light chain CDR3 of SEQ ID NO: 266,

A light chain variable region comprising a light chain CDR1 of SEQ ID NO: 205, a light chain CDR2 of SEQ ID NO: 230, and a light chain CDR3 of SEQ ID NO: 252,

A light chain variable region comprising a light chain CDR1 of SEQ ID NO: 205, a light chain CDR2 of SEQ ID NO: 230, and a light chain CDR3 of SEQ ID NO: 267,

A light chain variable region comprising a light chain CDR1 of SEQ ID NO: 205, a light chain CDR2 of SEQ ID NO: 230, and a light chain CDR3 of SEQ ID NO: 257,

A light chain variable region comprising a light chain CDR1 of SEQ ID NO: 220, a light chain CDR2 of SEQ ID NO: 230, and a light chain CDR3 of SEQ ID NO: 267,

A light chain variable region comprising a light chain CDR1 of SEQ ID NO: 222, a light chain CDR2 of SEQ ID NO: 230, and a light chain CDR3 of SEQ ID NO: 268, or

Light chain CDR1 comprising SEQ ID NO: 220, light chain CDR2 of SEQ ID NO: 230, and light chain CDR3 of SEQ ID NO: 269.

Specifically, the antibody or antigen-binding fragment thereof according to the present invention may comprise the following heavy chain variable region and light chain variable region:

A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 2, a heavy chain CDR2 of SEQ ID NO: 33, and a heavy chain CDR3 of SEQ ID NO: 62, a light chain CDR1 of SEQ ID NO: 201, a light chain CDR2 of SEQ ID NO: 226, and a light chain CDR3 of SEQ ID NO: 245; A light chain variable region comprising;

A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 3, a heavy chain CDR2 of SEQ ID NO: 34, and a heavy chain CDR3 of SEQ ID NO: 63, a light chain CDR1 of SEQ ID NO: 202, a light chain CDR2 of SEQ ID NO: 227, and a light chain CDR3 of SEQ ID NO: 246; 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: 35, and a heavy chain CDR3 of SEQ ID NO: 64, a light chain CDR1 of SEQ ID NO: 203, a light chain CDR2 of SEQ ID NO: 228, and a light chain CDR3 of SEQ ID NO: 247; 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: 36, and a heavy chain CDR3 of SEQ ID NO: 65, a light chain CDR1 of SEQ ID NO: 204, a light chain CDR2 of SEQ ID NO: 229, and a light chain CDR3 of SEQ ID NO: 248; A light chain variable region comprising;

A heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 1, the heavy chain CDR2 of SEQ ID NO: 32, and the heavy chain CDR3 of SEQ ID NO: 58, the light chain CDR1 of SEQ ID NO: 205, the light chain CDR2 of SEQ ID NO: 230, and the light chain CDR3 of SEQ ID NO: 249 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: 37, and the heavy chain CDR3 of SEQ ID NO: 66, the light chain CDR1 of SEQ ID NO: 206, the light chain CDR2 of SEQ ID NO: 231, and the light chain CDR3 of SEQ ID NO: 250; A light chain variable region comprising;

A heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 1, the heavy chain CDR2 of SEQ ID NO: 32, and the heavy chain CDR3 of SEQ ID NO: 67, the light chain CDR1 of SEQ ID NO: 207, the light chain CDR2 of SEQ ID NO: 232, and the light chain CDR3 of SEQ ID NO: 251; A light chain variable region comprising;

A heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 1, the heavy chain CDR2 of SEQ ID NO: 31, and the heavy chain CDR3 of SEQ ID NO: 57, the light chain CDR1 of SEQ ID NO: 208, the light chain CDR2 of SEQ ID NO: 230, and the light chain CDR3 of SEQ ID NO: 252; 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: 38, and the heavy chain CDR3 of SEQ ID NO: 68, the light chain CDR1 of SEQ ID NO: 209, the light chain CDR2 of SEQ ID NO: 233, and the light chain CDR3 of SEQ ID NO: 253; A light chain variable region comprising;

A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 8, a heavy chain CDR2 of SEQ ID NO: 39, and a heavy chain CDR3 of SEQ ID NO: 69, a light chain CDR1 of SEQ ID NO: 210, a light chain CDR2 of SEQ ID NO: 234, and a light chain CDR3 of SEQ ID NO: 254; A light chain variable region comprising;

A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 11, a heavy chain CDR2 of SEQ ID NO: 42, and a heavy chain CDR3 of SEQ ID NO: 72 and a light chain CDR1 of SEQ ID NO: 213, a light chain CDR2 of SEQ ID NO: 230, and a light chain CDR3 of SEQ ID NO: 257 A light chain variable region comprising;

A heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 1, the heavy chain CDR2 of SEQ ID NO: 32, and the heavy chain CDR3 of SEQ ID NO: 58, the light chain CDR1 of SEQ ID NO: 215, the light chain CDR2 of SEQ ID NO: 230, and the light chain CDR3 of SEQ ID NO: 249 A light chain variable region comprising;

A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 13, a heavy chain CDR2 of SEQ ID NO: 44, and a heavy chain CDR3 of SEQ ID NO: 74, a light chain CDR1 of SEQ ID NO: 211, a light chain CDR2 of SEQ ID NO: 238, and a light chain CDR3 of SEQ ID NO: 259; A light chain variable region comprising;

A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 15, a heavy chain CDR2 of SEQ ID NO: 46, and a heavy chain CDR3 of SEQ ID NO: 76, a light chain CDR1 of SEQ ID NO: 217, a light chain CDR2 of SEQ ID NO: 239, and a light chain CDR3 of SEQ ID NO: 336; A light chain variable region comprising; or

A heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 8, the heavy chain CDR2 of SEQ ID NO: 47, and the heavy chain CDR3 of SEQ ID NO: 77, the light chain CDR1 of SEQ ID NO: 218, the light chain CDR2 of SEQ ID NO: 240, and the light chain CDR3 of SEQ ID NO: 337 Light chain variable region containing.

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:

A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 17, a heavy chain CDR2 of SEQ ID NO: 49, and a heavy chain CDR3 of SEQ ID NO: 58, a light chain CDR1 of SEQ ID NO: 220, a light chain CDR2 of SEQ ID NO: 230, and a light chain CDR3 of SEQ ID NO: 252; A light chain variable region comprising;

A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 18, a heavy chain CDR2 of SEQ ID NO: 50, and a heavy chain CDR3 of SEQ ID NO: 58, a light chain CDR1 of SEQ ID NO: 215, a light chain CDR2 of SEQ ID NO: 230, and a light chain CDR3 of SEQ ID NO: 252; Light chain variable region containing

A heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 19, the heavy chain CDR2 of SEQ ID NO: 51, and the heavy chain CDR3 of SEQ ID NO: 58, the light chain CDR1 of SEQ ID NO: 218, the light chain CDR2 of SEQ ID NO: 240, and the light chain CDR3 of SEQ ID NO: 337 Light chain variable region containing

A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 20, a heavy chain CDR2 of SEQ ID NO: 52, and a heavy chain CDR3 of SEQ ID NO: 58, a light chain CDR1 of SEQ ID NO: 205, a light chain CDR2 of SEQ ID NO: 230, and a light chain CDR3 of SEQ ID NO: 264; Light chain variable region containing

A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 21, a heavy chain CDR2 of SEQ ID NO: 51, and a heavy chain CDR3 of SEQ ID NO: 58, a light chain CDR1 of SEQ ID NO: 205, a light chain CDR2 of SEQ ID NO: 230, and a light chain CDR3 of SEQ ID NO: 265; Light chain variable region containing

A heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 22, the heavy chain CDR2 of SEQ ID NO: 53, and the heavy chain CDR3 of SEQ ID NO: 58, the light chain CDR1 of SEQ ID NO: 221, the light chain CDR2 of SEQ ID NO: 230, and the light chain CDR3 of SEQ ID NO: 266; Light chain variable region containing,

A heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 23, the heavy chain CDR2 of SEQ ID NO: 49, and the heavy chain CDR3 of SEQ ID NO: 58, the light chain CDR1 of SEQ ID NO: 205, the light chain CDR2 of SEQ ID NO: 230, and the light chain CDR3 of SEQ ID NO: 252; Light chain variable region containing,

A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 17, a heavy chain CDR2 of SEQ ID NO: 54, and a heavy chain CDR3 of SEQ ID NO: 58, a light chain CDR1 of SEQ ID NO: 215, a light chain CDR2 of SEQ ID NO: 230, and a light chain CDR3 of SEQ ID NO: 249 Light chain variable region containing,

A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 24, a heavy chain CDR2 of SEQ ID NO: 55, and a heavy chain CDR3 of SEQ ID NO: 58, a light chain CDR1 of SEQ ID NO: 205, a light chain CDR2 of SEQ ID NO: 230, and a light chain CDR3 of SEQ ID NO: 267; Light chain variable region containing,

A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 21, a heavy chain CDR2 of SEQ ID NO: 51, and a heavy chain CDR3 of SEQ ID NO: 58, a light chain CDR1 of SEQ ID NO: 220, a light chain CDR2 of SEQ ID NO: 230, and a light chain CDR3 of SEQ ID NO: 252; Light chain variable region containing,

A heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 1, the heavy chain CDR2 of SEQ ID NO: 31, and the heavy chain CDR3 of SEQ ID NO: 57, the light chain CDR1 of SEQ ID NO: 221, the light chain CDR2 of SEQ ID NO: 230, and the light chain CDR3 of SEQ ID NO: 266 Light chain variable region containing,

A heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 1, the heavy chain CDR2 of SEQ ID NO: 31, and the heavy chain CDR3 of SEQ ID NO: 57, the light chain CDR1 of SEQ ID NO: 205, the light chain CDR2 of SEQ ID NO: 230, and the light chain CDR3 of SEQ ID NO: 252; Light chain variable region containing,

A heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 1, the heavy chain CDR2 of SEQ ID NO: 31, and the heavy chain CDR3 of SEQ ID NO: 57, the light chain CDR1 of SEQ ID NO: 205, the light chain CDR2 of SEQ ID NO: 230, and the light chain CDR3 of SEQ ID NO: 252; Light chain variable region containing,

A heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 1, the heavy chain CDR2 of SEQ ID NO: 31, and the heavy chain CDR3 of SEQ ID NO: 57, the light chain CDR1 of SEQ ID NO: 215, the light chain CDR2 of SEQ ID NO: 230, and the light chain CDR3 of SEQ ID NO: 252; Light chain variable region containing,

A heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 1, the heavy chain CDR2 of SEQ ID NO: 31, and the heavy chain CDR3 of SEQ ID NO: 57, the light chain CDR1 of SEQ ID NO: 205, the light chain CDR2 of SEQ ID NO: 230, and the light chain CDR3 of SEQ ID NO: 252; Light chain variable region containing,

A heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 1, the heavy chain CDR2 of SEQ ID NO: 31, and the heavy chain CDR3 of SEQ ID NO: 57, the light chain CDR1 of SEQ ID NO: 220, the light chain CDR2 of SEQ ID NO: 230, and the light chain CDR3 of SEQ ID NO: 252; Light chain variable region containing,

A heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 1, the heavy chain CDR2 of SEQ ID NO: 31, and the heavy chain CDR3 of SEQ ID NO: 57, the light chain CDR1 of SEQ ID NO: 205, the light chain CDR2 of SEQ ID NO: 230, and the light chain CDR3 of SEQ ID NO: 252; Light chain variable region containing,

A heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 1, the heavy chain CDR2 of SEQ ID NO: 31, and the heavy chain CDR3 of SEQ ID NO: 57, the light chain CDR1 of SEQ ID NO: 205, the light chain CDR2 of SEQ ID NO: 230, and the light chain CDR3 of SEQ ID NO: 252; Light chain variable region containing,

A heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 1, the heavy chain CDR2 of SEQ ID NO: 31, and the heavy chain CDR3 of SEQ ID NO: 57, the light chain CDR1 of SEQ ID NO: 215, the light chain CDR2 of SEQ ID NO: 230, and the light chain CDR3 of SEQ ID NO: 252; Light chain variable region containing,

A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 25, a heavy chain CDR2 of SEQ ID NO: 56, and a heavy chain CDR3 of SEQ ID NO: 57, a light chain CDR1 of SEQ ID NO: 205, a light chain CDR2 of SEQ ID NO: 230, and a light chain CDR3 of SEQ ID NO: 257 Light chain variable region containing,

A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 26, a heavy chain CDR2 of SEQ ID NO: 31, and a heavy chain CDR3 of SEQ ID NO: 57, a light chain CDR1 of SEQ ID NO: 220, a light chain CDR2 of SEQ ID NO: 230, and a light chain CDR3 of SEQ ID NO: 267; Light chain variable region containing,

A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 27, a heavy chain CDR2 of SEQ ID NO: 31, and a heavy chain CDR3 of SEQ ID NO: 57, a light chain CDR1 of SEQ ID NO: 205, a light chain CDR2 of SEQ ID NO: 230, and a light chain CDR3 of SEQ ID NO: 252; Light chain variable region containing,

A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 28, a heavy chain CDR2 of SEQ ID NO: 31, and a heavy chain CDR3 of SEQ ID NO: 57, a light chain CDR1 of SEQ ID NO: 205, a light chain CDR2 of SEQ ID NO: 230, and a light chain CDR3 of SEQ ID NO: 252; Light chain variable region containing,

A heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 29, the heavy chain CDR2 of SEQ ID NO: 31, and the heavy chain CDR3 of SEQ ID NO: 57, the light chain CDR1 of SEQ ID NO: 205, the light chain CDR2 of SEQ ID NO: 230, and the light chain CDR3 of SEQ ID NO: 252; Light chain variable region comprising, or

A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 30, a heavy chain CDR2 of SEQ ID NO: 31, and a heavy chain CDR3 of SEQ ID NO: 57, a light chain CDR1 of SEQ ID NO: 220, a light chain CDR2 of SEQ ID NO: 230, and a light chain CDR3 of SEQ ID NO: 267; Light chain variable region containing.

"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.

According to one embodiment of the present invention, the heavy chain variable region FR1 selected from the group consisting of SEQ ID NO: 80 to SEQ ID NO: 95,

A heavy chain variable region FR2 selected from the group consisting of SEQ ID NOs: 96 to 113,

A heavy chain variable region FR3 selected from the group consisting of SEQ ID NOs: 114 to 134, or

The heavy chain variable region FR4 selected from the group consisting of SEQ ID NOs: 135 to 145 may be included.

In addition, the light chain variable region FR1 selected from the group consisting of SEQ ID NO: 270 to SEQ ID NO: 294,

Light chain variable region FR2 selected from the group consisting of SEQ ID NOs: 295 to 315,

Light chain variable region FR3 selected from the group consisting of SEQ ID NOs: 316 to 355, or

Light chain variable region FR4 selected from the group consisting of SEQ ID NO: 356 to SEQ ID NO: 367.

"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 VH and VL domains of an antibody, which domains are present in a single polypeptide chain. The Fv polypeptide may further comprise a polypeptide linker between the VH domain and the VL domain that allows the scFv to form the desired structure for antigen binding.

PD-1 antibodies are monovalent or bivalent and include single or double chains. Functionally, the binding affinity of the PD-1 antibody is in the range of 10 ⁻⁵ M to 10 ⁻¹² M. For example, the binding affinity of PD-1 antibodies 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.

The antibody or antigen-binding fragment thereof that binds to PD-1 may comprise 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 NOs: 146 and 193. The antibody or antigen-binding fragment thereof that binds to PD-1 may comprise a heavy chain variable region selected from the group consisting of SEQ ID NOs: 146 to SEQ ID NO: 193. According to an embodiment of the present invention, the heavy chain variable region of SEQ ID NOs: 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 163, 165, 166, 168, 169, or 171-188 It may include.

In addition, it may include a light chain variable region comprising a sequence having 90% or more sequence homology with the sequence selected from the group consisting of SEQ ID NO: 368 to SEQ ID NO: 420. The antibody or antigen-binding fragment thereof that binds to PD-1 may include a light chain variable region selected from the group consisting of SEQ ID NOs: 368 to 420. According to an embodiment of the present invention, the light chain variable region of SEQ ID NOs: 371 to 380, 383, 385, 386, 388, 389, or 391 to 415.

In a specific embodiment according to the present invention, the heavy chain variable region of SEQ ID NO: 151 and the light chain variable region of SEQ ID NO: 371;

A heavy chain variable region of SEQ ID NO: 152 and a light chain variable region of SEQ ID NO: 372;

A heavy chain variable region of SEQ ID NO: 153 and a light chain variable region of SEQ ID NO: 373;

A heavy chain variable region of SEQ ID NO: 154 and a light chain variable region of SEQ ID NO: 374;

A heavy chain variable region of SEQ ID NO: 155 and a light chain variable region of SEQ ID NO: 375;

A heavy chain variable region of SEQ ID NO: 156 and a light chain variable region of SEQ ID NO: 376;

A heavy chain variable region of SEQ ID NO: 157 and a light chain variable region of SEQ ID NO: 377;

A heavy chain variable region of SEQ ID NO: 158 and a light chain variable region of SEQ ID NO: 378;

A heavy chain variable region of SEQ ID NO: 159 and a light chain variable region of SEQ ID NO: 379;

A heavy chain variable region of SEQ ID NO: 160 and a light chain variable region of SEQ ID NO: 380;

A heavy chain variable region of SEQ ID NO: 163 and a light chain variable region of SEQ ID NO: 383;

A heavy chain variable region of SEQ ID NO: 165 and a light chain variable region of SEQ ID NO: 385;

A heavy chain variable region of SEQ ID NO: 166 and a light chain variable region of SEQ ID NO: 386;

A heavy chain variable region of SEQ ID NO: 168 and a light chain variable region of SEQ ID NO: 388;

A heavy chain variable region of SEQ ID NO: 169 and a light chain variable region of SEQ ID NO: 389;

A heavy chain variable region of SEQ ID NO: 171 and a light chain variable region of SEQ ID NO: 391;

A heavy chain variable region of SEQ ID NO: 172 and a light chain variable region of SEQ ID NO: 392;

A heavy chain variable region of SEQ ID NO: 173 and a light chain variable region of SEQ ID NO: 393;

A heavy chain variable region of SEQ ID NO: 174 and a light chain variable region of SEQ ID NO: 394;

A heavy chain variable region of SEQ ID NO: 175 and a light chain variable region of SEQ ID NO: 395;

A heavy chain variable region of SEQ ID NO: 176 and a light chain variable region of SEQ ID NO: 396;

A heavy chain variable region of SEQ ID NO: 177 and a light chain variable region of SEQ ID NO: 397;

A heavy chain variable region of SEQ ID NO: 178 and a light chain variable region of SEQ ID NO: 398;

A heavy chain variable region of SEQ ID NO: 179 and a light chain variable region of SEQ ID NO: 399;

A heavy chain variable region of SEQ ID NO: 180 and a light chain variable region of SEQ ID NO: 400;

A heavy chain variable region of SEQ ID NO: 181 and a light chain variable region of SEQ ID NO: 401;

A heavy chain variable region of SEQ ID NO: 181 and a light chain variable region of SEQ ID NO: 402;

A heavy chain variable region of SEQ ID NO: 181 and a light chain variable region of SEQ ID NO: 403;

A heavy chain variable region of SEQ ID NO: 181 and a light chain variable region of SEQ ID NO: 404;

A heavy chain variable region of SEQ ID NO: 181 and a light chain variable region of SEQ ID NO: 405;

A heavy chain variable region of SEQ ID NO: 182 and a light chain variable region of SEQ ID NO: 406;

A heavy chain variable region of SEQ ID NO: 182 and a light chain variable region of SEQ ID NO: 407;

A heavy chain variable region of SEQ ID NO: 182 and a light chain variable region of SEQ ID NO: 408;

A heavy chain variable region of SEQ ID NO: 182 and a light chain variable region of SEQ ID NO: 409;

A heavy chain variable region of SEQ ID NO: 183 and a light chain variable region of SEQ ID NO: 410;

A heavy chain variable region of SEQ ID NO: 184 and a light chain variable region of SEQ ID NO: 411;

A heavy chain variable region of SEQ ID NO: 185 and a light chain variable region of SEQ ID NO: 412;

A heavy chain variable region of SEQ ID NO: 186 and a light chain variable region of SEQ ID NO: 413;

A heavy chain variable region of SEQ ID NO: 187 and a light chain variable region of SEQ ID NO: 414; or

A heavy chain variable region of SEQ ID NO: 188 and a light chain variable region of SEQ ID NO: 415.

In a specific embodiment, the heavy chain variable region of SEQ ID NO: 155 and the light chain variable region of SEQ ID NO: 375;

A heavy chain variable region of SEQ ID NO: 158 and a light chain variable region of SEQ ID NO: 378;

A heavy chain variable region of SEQ ID NO: 165 and a light chain variable region of SEQ ID NO: 385;

A heavy chain variable region of SEQ ID NO: 171 and a light chain variable region of SEQ ID NO: 391;

A heavy chain variable region of SEQ ID NO: 175 and a light chain variable region of SEQ ID NO: 395;

A heavy chain variable region of SEQ ID NO: 176 and a light chain variable region of SEQ ID NO: 396;

A heavy chain variable region of SEQ ID NO: 178 and a light chain variable region of SEQ ID NO: 398;

A heavy chain variable region of SEQ ID NO: 181 and a light chain variable region of SEQ ID NO: 401; or

The heavy chain variable region of SEQ ID NO: 181 and the light chain variable region of SEQ ID NO: 405 may be included.

The part indicated by X or Xaa in the sequence according to the present invention indicates other amino acids not specified, indicating that any amino acid may be included.

"Phase display" is a technique for displaying variant polypeptides as fusion proteins with at least a portion of the envelope protein on the surface of a phage, eg, 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.

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.

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 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.

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.

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.

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.

Antibodies or antibody fragments of the present invention may include not only the sequences of the anti-PD-1 antibodies of the present invention described herein, but also biological equivalents thereof, as long as they specifically recognize PD-1. 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.

In another aspect, 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 not only natural nucleotides but also analogs in which sugar or base sites are modified. . 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 amino acid sequence for the antibody or antigen-binding fragment thereof of the present invention or nucleic acid encoding the same is interpreted to also include a sequence that exhibits a substantial identity with the sequence set forth 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.

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.

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.

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.

“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.

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 a 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.

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).

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.

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.

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.

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.

In another aspect, 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.

As used herein, the term “transformation” means introducing a vector containing a nucleic acid encoding a target protein into a host cell so that the protein encoded by the nucleic acid can be expressed in the host cell. May include all of them as long as they can be expressed in the host cell, whether inserted into or located outside the chromosome of the host cell, and the nucleic acid includes DNA and RNA encoding the target protein. May be introduced in any form as long as it can be introduced into and expressed in a host cell, for example, the nucleic acid may be a gene structure of an expression cassette, which is a gene construct including all elements necessary for self-expression. Can be introduced into a host cell in which the expression cassette normally operates on the nucleic acid. And a promoter, transcription termination signal, ribosomal binding site, and translation termination signal, which are possibly linked, The expression cassette may be in the form of an expression vector capable of self-replicating, and the nucleic acid may be in its own form. It may be introduced into a host cell and operably linked with a sequence required for expression in the host cell.

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.

In another aspect, the present invention relates to a composition for preventing or treating cancer, comprising the antibody or antigen-binding fragment thereof as an active ingredient.

The present invention includes, for example, (a) a pharmaceutically effective amount of an antibody against PD-1 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 comprising a pharmaceutically acceptable carrier. The present invention also relates to a method for the prevention or treatment of cancer comprising administering to a patient an effective amount necessary for an antibody against PD-1 or an antigen-binding fragment thereof according to the present invention.

In the present invention, the cancer is a group consisting of melanoma, lung cancer, liver cancer, glioblastoma, ovarian cancer, colon cancer, head and neck cancer, bladder cancer, kidney cancer, gastric cancer, breast cancer, metastatic cancer, Hodgkin's lymphoma, prostate cancer and pancreatic cancer It may be characterized in that it is selected from but is not limited thereto.

The term "treatment" in the present invention means not only inhibiting or alleviating cancer or one or more symptoms caused by administration of the composition, but also preventing the treatment of cancer or the progression of the cancer to reverse the symptoms of the disease. As used herein, the term "prevention" refers to any action that inhibits or delays the onset of cancer by administration of the composition. In the present invention, the prophylaxis or treatment of cancer is achieved by binding the antibody obtained in the present invention to PD-1, and significantly inhibits its activity by binding to PD-1 to prevent or treat cancer.

As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier or diluent that does not irritate an organism and does not inhibit the biological activity and properties of the administered compound. Acceptable pharmaceutical carriers in compositions formulated as liquid solutions are sterile and physiologically compatible, including saline, sterile water, Ringer's solution, buffered saline, albumin injectable solutions, dextrose solution, maltodextrin solution, glycerol, ethanol and One or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers and bacteriostatic agents may be added as necessary. Diluents, dispersants, surfactants, binders and lubricants may also be added in addition to formulate into injectable formulations, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like.

Cancer preventive or therapeutic composition comprising the antibody of the present invention and a pharmaceutically acceptable carrier can be applied to any formulation containing it as an active ingredient, it can be prepared in oral or parenteral formulations. Pharmaceutical formulations of the invention may be oral, rectal, nasal, topical (including the cheek and sublingual), subcutaneous, vaginal or parenteral (intramuscular, subcutaneous). And forms suitable for administration by inhalation or insufflation.

Oral dosage forms containing the composition of the present invention as an active ingredient include, for example, tablets, troches, lozenges, water-soluble or oily suspensions, preparation powders or granules, emulsions, hard or soft capsules, syrups or elixirs. can do. For formulation into tablets and capsules, lactose, saccharose, sorbitol, mannitol, starch, amylopectin, binders such as cellulose or gelatin, excipients such as dicalcium phosphate, disintegrants such as corn starch or sweet potato starch, stearic acid masne It may include a lubricating oil such as calcium, calcium stearate, sodium stearyl fumarate or polyethylene glycol wax, and in the case of a capsule, it may further contain a liquid carrier such as fatty oil in addition to the above-mentioned materials.

Formulations for parenteral administration comprising the composition of the present invention as an active ingredient, for injection, such as subcutaneous injection, intravenous injection or intramuscular injection, a suppository injection method or aerosol for spraying by inhalation through the respiratory system It can be formulated as. To formulate injectable formulations, the compositions of the present invention may be mixed in water with stabilizers or buffers to prepare solutions or suspensions, which may be formulated for unit administration of ampoules or vials. For infusion into suppositories, it may be formulated in a rectal composition such as suppositories or enemas, including conventional suppository bases such as cocoa butter or other glycerides. When formulated for spraying such as aerosols, a propellant or the like may be combined with the additives to disperse the dispersed dispersion or wet powder.

As another aspect, the present invention relates to a method for preventing or treating cancer, comprising administering a composition for preventing or treating cancer comprising the antibody.

As used herein, the term “administration” means introducing a pharmaceutical composition of the present invention to a patient in any suitable manner. It may be administered through, or specifically, can be administered in a conventional manner via oral, rectal, topical, intravenous, intraperitoneal, intramuscular, intraarterial, transdermal, nasal, inhalation, intraocular or intradermal route. .

The treatment method of the present invention includes administering a composition for preventing or treating cancer of the present invention in a pharmaceutically effective amount. It will be apparent to those skilled in the art that a suitable total daily dose may be determined by the practitioner within the correct medical judgment. The specific therapeutically effective amount for a particular patient may be based on the specific composition, including the type and severity of the reaction to be achieved, whether or not other agents are used in some cases, the age, weight, general health, sex and diet of the patient, time of administration, It is desirable to apply differently depending on the route of administration and the rate of release of the composition, the duration of treatment, and the various factors and similar factors well known in the medical arts, including drugs used with or concurrent with the specific composition. Therefore, the effective amount of the composition for preventing or treating cancer suitable for the purpose of the present invention is preferably determined in consideration of the above-mentioned matters.

In addition, the treatment method of the present invention is applicable to any animal in which diseases such as tumor development and angiogenesis may occur due to excessive activity of PD-1, and the animals are not only humans and primates, but also cattle, pigs, sheep And domestic animals such as horses, dogs and cats.

In another aspect, the present invention is a cancer diagnostic composition comprising a PD-1 antibody or an antigen-binding fragment thereof according to the present invention. The present invention also relates to a method for diagnosing cancer by treating a PD-1 antibody or antigen-binding fragment thereof according to the present invention.

Cancer can be diagnosed by measuring the level of PD-1 expression in a sample via an antibody against PD-1 according to the invention. Expression levels can be measured according to conventional immunoassay methods, radioimmunoassay using the antibody against PD-1, 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.

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.

In another aspect, the present invention relates to a cancer diagnostic kit comprising the cancer diagnostic composition. The kit according to the present invention includes an antibody against PD-1 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.

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.

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-1 expression.

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.

Example 1: PD-1 Antigen Expression and Purification

1. Construction of PD-L1 Protein Expression Vector

Cloning of PD-1 was performed using the Jurkat cell cDNA library (Stratagene, USA), using primers for PD1 containing restriction enzyme Sfi I sites at 5 'and 3' to obtain only the extracellular domain (Table 1). Amplification was carried out by polymerase chain reaction (PCR). The amplified PCR product was prepared by fusing human Fc (SEQ ID NO: 196) and mouse Fc (SEQ ID NO: 197) to the carboxy-terminus using the N293F vector (Fig. 1).

designation 5 '-> 3' sequence SEQ ID NO: PD1-F ccaggatggttcttagactcccc 194 PD1-R caccagggtttggaactggc 195

2. Expression and Purification of PD-1 Antigens

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.

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.

Purified protein was purified by SDS-PAGE gel and size exclusion chromatography (TSK-GEL G-3000 SWXL Size-exclusion chromatography (SEC) (Tosoh)).

As a result, as shown in Figures 2a to 2d, it was confirmed that the purity of the purified PD1 protein has a purity of 95% or more.

Example 2: Screening of PD-1 Human Antibodies

1. Antigen Preparation

Blocking after coating 50 μg of PD1-hFc, PD1-mFc and PD1-his (Catalog Number, 10377-H08H) protein antigen prepared in Example 1 on an immunosorbent tube Was performed.

2. Bio Panning

Human antibody library phage infected the human scFv library with a diversity of 2.7 × 10 ¹⁰ and then incubated the bacteria 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 is obtained through the panning process of infecting and eluting the eluted phages again, increasing the number of times in the PBST washing step with the amplified phages in the first round of panning. Round and three rounds of panning were performed.

As a result, as shown in Table 2, it was confirmed that the number of phage bound to the antigen in the third round panning was slightly increased compared to the input.

Panning Phage influent Phage Count 1 time 2 × 10 ¹³ 3.6 × 10 ⁷ Episode 2 1.2 × 10 ¹³ 7 × 10 ⁷ 3rd time 2 × 10 ¹³ 2 × 10 ⁹

3. Poly Phage ELISA

Polyphage enzyme linked immunoassay (ELISA) was performed to determine the specificity with the antigen for the positive poly scFv-phage antibody pool obtained through each round of panning.

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.

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.

The results are shown in FIG. According to FIG. 3, ELISA confirmed that the antigen-binding ability was enhanced in tertiary poly scFv-phage for two PD1 antigens.

4. Positive Phage Screening

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.

Each well was blocked using 4% skim milk dissolved in PBS after coating 100 ng of antigen PD1 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.

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 PD1.

5. Sequence analysis of positive phage antibody

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 15 PD1 were obtained, which are summarized in Table 3 below.

Characteristics of PD1 Monoclones Clone name VH Similarity VL Similarity group Nivolumab VH3-33 91.80% L6 98.90% Pembrolizumab VH1-2 79.60% L25 80.80% PD1-29A2 VH3-9 91.80% V2-17 86.50% One PD1-29H3 VH3-9 90.80% V1-11 88.50% 2 PD1-31F3 VH1-69 92.90% V1-13 93.90% 3 PD1-32A6 VH1-69 87.80% L5 91.60% 4 PD1-41C9 VH3-30 95.80% O1 92.10% 5 PD1-42G4 VH3-11 93.80% O2 93.80% 6 PD1-44B5 VH3-30 99.00% V1-16 85.60% 7 PD1-45D6 VH3-30 95.80% O1 93.10% 8 PD1-45F1 VH1-46 91.80% V2-13 93.80% 9 PD1-45F4 VH3-9 90.80% V2-1 77.70% 10 PD1-48A9 VH1-69 79.60% O1 96.00% 11 PD1-49B9 VH3-9 88.70% V1-2 93.90% 12 PD1-49A2 VH3-30 97.90% O1 93.10% 13 PD1-51D9 VH1-3 82.70% A3 94.00% 14 PD1-52E8 VH3-9 91.80% V2-17 90.50% 15

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.

Example 3: Production of PD-1 Human Antibody

1. Conversion of scFv form to IgG form (conversion)

PCR was performed on the heavy and light chains (iCycler iQ, BIO-RAD) to convert monoclonal phage antibodies against 15 selected PD1 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.

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).

As a result, it was confirmed that the sequences of the heavy and light chains of the 15 clones for PD1 converted to total IgG match the sequences of the phage antibodies of the 15 clones of Table 3. 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. Human Antibody Production

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).

As a result, according to FIG. 5, all 15 antibodies were detected at a size of 150 kDa or more in non-reducing conditions and produced Nivolumab as a control antibody.

Example 4: Properties of PD-1 Monoclonal Antibody

1. Evaluation of the Activity of Antibodies

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). 16 PD-1 antibodies including the control antibody confirmed the activity of the antibody to recover the signal reduced by PD-1 / PD-L1 complex formation, 41C9, 45D6, 49A2 showed similar activity compared to the control antibody (Figure 6).

In order to measure the activity-dependent evaluation of the PD-1 antibodies 41C9, 45D6, 49A2, the dilution of the PD1 / PD-L1 blocking bioassay was performed in a step-diluted manner to restore the reduced signal concentration-dependently. 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. Affinity of PD1 Antibodies to Overexpressing Cells

The transgenic cell pool expressing PD-1 was transformed into HEK293E with a pcDNA3.1 plasmid containing human PD-1 (NM_005018.2), containing 150 ug / ml Zeocin (# R25001, Thermo Fisher). Screened in selective medium. Each cell pool was identified and selected by fluorescence activated cell sorting (FACS) analysis using each anti-PD-1 (# 557860, BD) and used for functional evaluation such as FACS binding assays or FACS competition assays.

Each pool of transformed cells expressing high human PD-1 was prepared with 0.5-1x10 ^ 6 cells per sample, and the antibodies were reacted with the prepared cells for 20 min at 4 ° C. by diluting the antibodies in successive dilutions. 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, all three PD-1 antibodies were specifically bound, and the binding force was determined by the equilibrium dissociation constant (Kd) through the analysis function of Graphpad Prism6.

As a result, according to FIG. 8, the binding force of the antibody bound in a concentration-dependent manner to human PD-1 overexpressed on the cell surface can be known as mean fluorescence intensity (MFI).

3. Screening PD1-45D6, 49A2 Similar Sequences in Positive Phage Antibodies

In addition to 41C9, which was evaluated for activity evaluation and binding ability in FACS, further antibody screening was performed for 45D6 and 49A2.

The same procedure as in Example 2 was further selected to have PD1-45D6, 49A2 similar sequence. Table 6 summarizes the characteristics.

Characterization of additionally screened 45D6, 49A2 like antibody clones Clone name VH Similarity VL Similarity 2 group PD1-72D10 VH3-30 96.90% 01 94.10% One PD1-74A11 VH3-30 97.96% 01 95.05% 2 PD1-75C10 VH3-30 96.94% 01 95.05% 3 PD1-74A01 VH3-30 95.92% 01 92.08% 4 PD1-74H12 VH3-30 94.90% 01 92.08% 5

4. Affinity of PD1 Antibody Using ProteOn XPR36

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 PD1-hFc dissolved at 5 ug / ml in buffer were injected and immobilized on a GLC sensor chip.

1 M ethanolamine was flowed to inactivate activated carboxyl groups that remained unreacted with PD1 protein, and 10 mM glycine, pH 2.0, was injected to wash 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).

The equilibrium dissociation constant (KD) was calculated by plotting and fitting the sensogram data at equilibrium according to the concentration, and showed a high affinity for PD1 antigen with 45D6 of 0.001 nM and 49A2 of 0.019 nM ( 9).

Example 5: Antibody Optimization for PD1 Antibody 45D6, 49A2

1. Library construction for optimization of PD1-45D6 and 49A2 antibodies

Antibody optimization involves the addition of 10 ⁵ -10 ⁶ light chain (LC) pools retained by WBIOLOGICS to construct a new LC shuffling library, and LC shuffling, hydrophobicity of heavy chains. Mutations to conserved residues were compared with residues of structurally important sites such as hydrophobic cores, exposed residues, charge clusters, salt bridges, etc. Core packing + LC shuffling followed by LC shuffling, mutations in antibody variable regions can be frequently mutated during in vivo affinity maturation Mutational hot spots were randomly mutated, followed by three methods: CDR hotspot + LC shuffling.

To prepare LC shuffling libraries, LC genes of 45D6 and 49A2 antibodies were digested with BstX I and used as vectors, and library pools retained by WBIOLOGICS were digested with BstX I and used as inserts. 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 1.5 × 10 7 were obtained. As a result of sequencing, all of the HC sequences were identical and the LC sequences were different.

To construct the core packing + LC shuffling library, the frame work (FR) portion of the 45D6, 49A2 antibody was replaced with a conserved amanoic acid sequence, followed by cutting the LC gene with BstX I and using it as a vector. The library pool retained by Biologics was cut with BstX I and used as an insert. 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 8.4 × 10 6 were obtained. The sequencing analysis showed that the FR region of HC was replaced with the amanoic acid sequence that was conserved. This different thing was confirmed.

In order to construct a CDR hotspot + LC shuffling library, the frame work (FR) portion of the 45D6 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 electroporation transformation taxa. 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.

Example 6: Screening of PD1 Human Antibodies

1. Antigen Preparation

Blocking was performed after coating PD1-hFc, PD1-mFc produced by YBIOLOGICS and 50 ug of PD1-his (Catalog Number, 10377-H08H) protein antigen purchased from Sino Biological Inc. in an immunosorb tube. .

2. Bio-panning

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. As a result, it was confirmed that the number of phages bound to the antigen in the first round panning was slightly increased compared to the input as shown in Table 7.

Titer Comparison of Antibodies by Optimization Panning Sample Phage influent Phage Count 45D6 (LS) 1.3 × 10 ¹³ 2.8 × 10 ⁷ 45D6 (Core packing + LS) 1.1 × 10 ¹³ 4.8 × 10 ⁷ 45D6 (CDR hotspot + LS) 1.1 × 10 ¹³ 3.6 × 10 ⁶ 49A2 (LS) 1.3 × 10 ¹³ 1.8 × 10 ⁷ 49A2 (CDR hotspot + LS) 1.1 × 10 ¹³ 1.6 × 10 ⁶

3. Positive Phage Screening

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.

Each well was blocked using 4% skim milk dissolved in PBS after coating 100 ng of antigen PD1 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.

As a result, dozens of single phage clones having stronger binding capacity to each antigen than the parent antibody (49A2 or 45D6) were obtained, as shown in FIG.

4. Sequencing of Positive Phage Antibodies

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 was carried out using the Ig BLAST program of / to obtain 25 specific phage antibodies with higher binding capacity than the parent antibody (49A2: 10, 45D6: 15), which are summarized in Table 8 below.

The antibodies comprising the heavy and light chain CDRs, FR sequences, and heavy and variable chain and light chain variable regions of the selected antibodies are shown in Tables 9 and 10 below.

Example 7: Production of PD1 Human Antibody

1. Conversion of scFv form to IgG form (conversion)

PCR was performed on the heavy and light chains (iCycler iQ, BIO-RAD) to convert monoclonal phage antibodies against 25 selected PD1 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.

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).

As a result, it was confirmed that the sequences of the heavy and light chains of 25 clones for PD1 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. Human Antibody Production

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 25 antibodies were loaded by 5 ug each in 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 antibody expression rate was increased than the parent antibody, the results can be seen in FIG.

Example 8: Properties of PD1 Monoclonal Antibody

1. Evaluation of the Activity of Antibodies

The activity evaluation experiments of the selected antibodies were conducted using the PD1 / 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-four PD-1 antibodies confirmed the activity of the antibody to restore the reduced signal due to PD-1 / PD-L1 complex formation, and 45D6-3D2, 45D6-3H7, 45D6-5B2, 45D6 in the 45D6 antibody. 49A2-1B2, 49A2-1H8, 49A2-2A6, 49A2-2B9 increased the activity against the parent antibody and showed similar activity to the control antibody in the 49A2 antibody (FIG. 12).

Activity-determining the Activity Assessment Again for 8 PD-1 Antibodies (45D6-3D2, 45D6-3H7, 45D6-5B2, 45D6-5B5, 49A2-1B2, 49A2-1H8, 49A2-2A6, 49A2-2B9) As a result of the dilution, the PD1 / PD-L1 blocking bioassay was performed again to recover the reduced signal 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. 49A2-1B2 showed the highest recovery ability of in vitro efficacy inhibition of EC50 (Fig. 13, Table). 11).

Activity of Monoclonals of Selected PD1 Antibody Variants Antibodies EC50 [nM] PD-1-41C9 3.91 PD-1-45D6 2 PD-1-45D6-A-3D2 2.16 PD-1-45D6-A-3G1 0.98 PD-1-45D6-A-3H4 1.67 PD-1-45D6-A-3H6 1.49 PD-1-45D6-A-3H7 1.98 PD-1-45D6-A-4C1 1.76 PD-1-45D6-A-4C9 2.72 PD-1-45D6-A-4D4 1.74 PD-1-45D6-A-4H6 1.19 PD-1-45D6-A-5A6 0.92 PD-1-45D6-A-5B12 0.8 PD-1-45D6-A-5B2 1.48 PD-1-45D6-A-5B5 1.68 PD-1-45D6-A-5C8 1.44 PD-1-45D6-A-5H9 2 PD-1-48A9 8.64 PD-1-49A2 3.78 PD-1-49A2-A-1B2 1.3 PD-1-49A2-A-1D11 2.89 PD-1-49A2-A-1F12 1.03 PD-1-49A2-A-1H4 0.84 PD-1-49A2-A-1H8 1.21 PD-1-49A2-A-2A11 0.79 PD-1-49A2-A-2A6 1.51 PD-1-49A2-A-2B10 0.65 PD-1-49A2-A-2B9 1.23 PD-1-51D9 ~ 4.695e + 010 PD-1-52E8 8.43 PD-1-62E1 2.73 PD1-72D10 1.21 PD1-74A11 1.31 PD1-75C10 0.81 PD1-74A01 1.01 PD1-74H12 0.98

2. Affinity of PD1 Antibodies to Overexpressing Cells

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 pool of transformed cells expressing human PD1 was prepared with 0.5-1 × 10 6 cells per sample, and the antibodies were reacted with the prepared cells for 20 min at 4 ° C. by diluting the antibodies in successive dilutions. 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).

Each pool of transformed cells expressing human PD-1 was prepared with 0.5x10 ^ 6 cells per sample, and the antibodies were continuously diluted with a constant dilution factor, 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). The binding force was obtained from the equilibrium dissociation constant (Kd) through the analysis function of Graphpad Prism6. According to FIG. 15, the binding force of the antibody bound in a concentration-dependent manner to human PD-1 overexpressed on the cell surface can be known as mean fluorescence intensity (MFI). According to FIG. 14 and FIG. 15, the antibodies except for 49A2 (Parent 49A2) showed similar binding strength.

3. Inhibition of Antibody to Form PD-1 / PD-L1 or PD-1 / PD-L2 Complex by Enzyme Immunosorbent

Human PD-1-Fc (S1420, Y-Biologics) or PD-L2-Fc (# 10292-H02H, Sino) was added to the wells of a 96-well immunomicroplate (# 439454, Thermo). Fixed for 16 hours at ℃, washed three times with PBS containing 0.05% tween-20 (# P9416, Sigma-Aldrich), followed by a wash solution containing 4% skim milk (# 232120, Becton, Dickinson and Company) Nonspecific binding was blocked by standing at room temperature for 1 hour. In the meantime, each antibody serially diluted with a constant dilution factor and human PD-L1-His (S1479, Y-Biologics) or PD-1-His (S1352, Y-Biologics) were reacted at room temperature for 1 hour, and then prepared microplates. And leave 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, stop the reaction by adding 50 ul 2.5M sulfuric acid (# S1478, Samchun) and stop the reaction with spectrophotometer (# GM3000, Glomax ® Discover System Promega) was used to measure absorbance at 450 nm. The results are shown in FIG.

4. Affinity of PD1 Antibody Using ProteOn XPR36

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 PD1-hFc dissolved at 5 ug / ml in buffer were injected and immobilized on a GLC sensor chip.

1 M ethanolamine was flowed to deactivate the activated carboxyl groups that remained unreacted with the PD1 protein, and 10 mM glycine, pH 2.0 was injected to wash the protein that was 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).

The equilibrium dissociation constant (KD) was calculated by plotting and plotting the sensogram data at equilibrium according to the concentration, and 49A2 (2B9) showed 0.001 nM with high affinity for the PD1 antigen (Fig. 17).

The affinity for the human PD-1 protein of other antibodies, the affinity for the PD-1 protein of monkeys, and the affinity for the PD-1 protein of mice are shown in Tables 12-14.

Binding kinetics between human PD1 protein and selected antibodies Ab KD (M) Ka (1 / Ms) Kd (1 / s) PD1 49A2 2.371 × 10 ^-10 3.953 × 10 ⁵ 9.372 × 10 ^-5 PD1 49A2 1H8 1.245 × 10 ^-11 2.201 × 10 ⁵ 2.741 × 10 ^-6 PD1 49A2 1B2 1.659 × 10 ^-10 3.853 × 10 ⁵ 6.390 × 10 ^-5 PD1 49A2 2A6 6.907 × 10 ^-11 3.101 × 10 ⁵ 2.142 × 10 ^-5 PD1 49A2 2B9 1.0 × 10 ^-12 3.774 × 10 ⁵ 1.0 × 10 ^-7 PD1 45D6 1.0 × 10 ^-12 2.701 × 10 ⁵ 1.07 × 10 ^-7 PD1 45D6 3D2 1.0 × 10 ^-12 1.81 × 10 ⁵ 1.0 × 10 ^-7 PD1 45D6 3H7 2.53 × 10 ^-11 3.076 × 10 ⁵ 7.781 × 10 ^-6 PD1 45D6 5B2 5.818 × 10 ^-11 3.315 × 10 ⁵ 1.928 × 10 ^-5 PD1 45D6 5B5 4.773 × 10 ^-11 2.96 × 10 ⁵ 1.413 × 10 ^-5

Binding kinetics between monkey's PD1 protein and selected antibodies Ab KD (M) Ka (1 / Ms) Kd (1 / s) PD-1-49A2 1.0 × 10 ^-12 1.683 × 10 ⁵ 1.0 × 10 ^-7 PD-1-49A2-2B9 1.0 × 10 ^-12 1.853 × 10 ⁵ 1.0 × 10 ^-7 PD-1-45D6 1.0 × 10 ^-12 1.535 × 10 ⁵ 1.0 × 10 ^-7 PD-1-45D6-5B2 1.0 × 10 ^-12 2.078 × 10 ⁵ 1.0 × 10 ^-7

Binding kinetics measurement between PD1 protein and selected antibodies in mice Ab KD (M) Ka (1 / Ms) Kd (1 / s) PD-1-49A2-2B9 4.231 × 10 ^-9 1.478 × 10 ⁵ 6.252 × 10 ^-4 PD-1-45D6 2.391 × 10 ^-9 5.544 × 10 ⁴ 1.326 × 10 ^-4 PD-1-45D6-5B2 4.590 × 10 ^-9 1.879 × 10 ⁵ 8.626 × 10 ^-4

Example 9: Epitope Determination of PD1 Monoclonal Antibodies

Monoclonal scFv-phage binding to the PD1 antigen was incubated for 16 hours at 37 ℃ in 2xYTCM, 2% glucose (glucose), 5 mM MgCl ₂ medium. Cells thus grown were placed in 2xYTCM, 2% glucose, 5 mM MgCl ₂ medium to a value of 0.1 at OD ₆₀₀ , and then incubated for 2 to 3 hours at 37 ° C. to a value of 0.5 to 0.7 at OD 6 ₀₀ . 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 digested using 4% skim milk dissolved in PBS after encoding antigen PD1 wild type (WT) or various variants (mutants: FIG. 18) at 4 ° C. for 16 hours in a 96-well immune-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. Again, each well was washed four times with 0.2 ml PBS / T, and then a 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.

As a result, different binding patterns were shown for the selected monoclonal scFv-phage, control scFv-phage, and PD-1 variants, indicating that they had different epitopes (FIG. 19).

Each well using 4% skim milk dissolved in PBS after coating the antigen PD1 wild type (WT) or several variants (FIG. 18) at 96 ° C. for 16 hours at 4 ° C. in a 96-well immune-plate. Was blocked. Each well was washed with 0.2 ml PBS / T, and 100 μl (1ug / ml) of the monoclonal antibody incubated for 16 hours was added to each well for 2 hours at room temperature. Each well was washed four times with 0.2 ml PBS / T, and then the secondary antibody anti-Fab 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.

As a result, different binding patterns were shown for the control antibody and the PD-1 variants, so that they had different epitopes (FIG. 20).

Example 10 Specific Binding of PD1 Parent Antibody

Over 90 unspecific antigens purified for binding to the PD1 parent antibody and the control antibody (Nivolumab) in addition to the PD1 antigen (Table 15) were placed in a 96 well immune-plate at 100 ng per well at 4 ° C. Each well was blocked using 4% skim milk dissolved in PBS after 16 hours of coating. After washing with 0.2 ml PBS / T in each well, 100 ng of the parent antibody and the control antibody were 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 secondary antibody anti-kappa-HRP was diluted to 1/1000 and reacted at room temperature for 1 hour. After washing with 0.2 ml PBS / T, color development was measured at 490 nm.

As a result, it was confirmed that both the control antibody and the parent antibody specifically bind only to the PD1 antigen without binding to the unspecific antigen (FIG. 21).

Example 11: Comparison of Productivity of Temporary Expression Systems of PD1 Monoclonal Antibodies

It can be seen that the optimized antibody has a relatively uniform and high yield due to its excellent physical properties during production and purification through a temporary expression system. It can be seen that some antibodies have higher production than previously commercialized antibodies (FIG. 22).

Example 12 Increasing Activity of a PD1 Monoclonal Antibody in an Allogenic MLR Response

After mixing T cells at a ratio of 1: 10 to monocyte-derived dendritic cells isolated from different humans, and measuring the amount of interferon gamma in the culture medium after 5 days, 45D6 and 49A2 parent antibodies were added. It was confirmed that the amount of interferon gamma increases depending on the concentration (Fig. 23).

Example 13: Thermal Stability Testing of PD1 Monoclonal Antibodies

Dilute 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. qPCR conditions were 30 seconds after the reaction at 25 ℃, increased by 1 degree to 99 ℃ by 1 minute at each temperature and finished by the last 25 ℃ 10 seconds reaction. Tm (Melting temperature, melting temperature) was used as the rate constant of the antibody structure is released. The results are shown in Table 16 below.

Sample Tm Pembrolizumab 63 Opdivo 64 45D6 63 49A2 63 49A2-2B9 64

The novel antibodies or antigen-binding fragments thereof that bind to PD-1 of the present invention are useful in the development of immunotherapeutic agents for various diseases associated with PD-1 because they can bind to PD-1 and inhibit its activity.

The specific parts of the present invention have been described in detail above, and it is apparent to those skilled in the art that such specific descriptions are merely preferred embodiments, and thus the scope of the present invention is not limited thereto. something to do. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Electronic file attached.

Claims (13)

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: 30, A heavy chain CDR2 comprising a sequence having 90% or more sequence homology with a sequence selected from the group consisting of SEQ ID NOs: 31 to 56, and 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: 57-79, 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: 198 to 222, A light chain CDR2 comprising a sequence having at least 90% sequence homology with a sequence selected from the group consisting of SEQ ID NOs: 223 to 241, and An antibody binding to PD-1 or 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: 242 to 269 snippet. The method of claim 1, A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 1, a heavy chain CDR2 of SEQ ID NO: 31, and a heavy chain CDR3 of SEQ ID NO: 57, A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 1, a heavy chain CDR2 of SEQ ID NO: 32, and a heavy chain CDR3 of SEQ ID NO: 58, A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 2, a heavy chain CDR2 of SEQ ID NO: 33, and a heavy chain CDR3 of SEQ ID NO: 59, A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 2, a heavy chain CDR2 of SEQ ID NO: 33, and a heavy chain CDR3 of SEQ ID NO: 60, A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 2, a heavy chain CDR2 of SEQ ID NO: 33, and a heavy chain CDR3 of SEQ ID NO: 61, A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 2, a heavy chain CDR2 of SEQ ID NO: 33, and a heavy chain CDR3 of SEQ ID NO: 62, A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 3, a heavy chain CDR2 of SEQ ID NO: 34, and a heavy chain CDR3 of SEQ ID NO: 63, A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 4, a heavy chain CDR2 of SEQ ID NO: 35, and a heavy chain CDR3 of SEQ ID NO: 64, A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 5, a heavy chain CDR2 of SEQ ID NO: 36, and a heavy chain CDR3 of SEQ ID NO: 65, A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 6, a heavy chain CDR2 of SEQ ID NO: 37, and a heavy chain CDR3 of SEQ ID NO: 66, A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 1, a heavy chain CDR2 of SEQ ID NO: 32, and a heavy chain CDR3 of SEQ ID NO: 67, A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 7, a heavy chain CDR2 of SEQ ID NO: 38, and a heavy chain CDR3 of SEQ ID NO: 68, A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 8, a heavy chain CDR2 of SEQ ID NO: 39, and a heavy chain CDR3 of SEQ ID NO: 69, A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 9, a heavy chain CDR2 of SEQ ID NO: 40, and a heavy chain CDR3 of SEQ ID NO: 70, A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 10, a heavy chain CDR2 of SEQ ID NO: 41, and a heavy chain CDR3 of SEQ ID NO: 71, A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 11, a heavy chain CDR2 of SEQ ID NO: 42, and a heavy chain CDR3 of SEQ ID NO: 72, A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 12, a heavy chain CDR2 of SEQ ID NO: 43, and a heavy chain CDR3 of SEQ ID NO: 73, A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 13, a heavy chain CDR2 of SEQ ID NO: 44, and a heavy chain CDR3 of SEQ ID NO: 74, A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 14, a heavy chain CDR2 of SEQ ID NO: 45, and a heavy chain CDR3 of SEQ ID NO: 75, A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 15, a heavy chain CDR2 of SEQ ID NO: 46, and a heavy chain CDR3 of SEQ ID NO: 76, A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 8, a heavy chain CDR2 of SEQ ID NO: 47, and a heavy chain CDR3 of SEQ ID NO: 77, A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 16, a heavy chain CDR2 of SEQ ID NO: 48, and a heavy chain CDR3 of SEQ ID NO: 78, A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 17, a heavy chain CDR2 of SEQ ID NO: 49, and a heavy chain CDR3 of SEQ ID NO: 58, A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 18, a heavy chain CDR2 of SEQ ID NO: 50, and a heavy chain CDR3 of SEQ ID NO: 58, A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 19, a heavy chain CDR2 of SEQ ID NO: 51, and a heavy chain CDR3 of SEQ ID NO: 58, A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 20, a heavy chain CDR2 of SEQ ID NO: 52, and a heavy chain CDR3 of SEQ ID NO: 58, A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 21, a heavy chain CDR2 of SEQ ID NO: 51, and a heavy chain CDR3 of SEQ ID NO: 58, A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 22, a heavy chain CDR2 of SEQ ID NO: 53, and a heavy chain CDR3 of SEQ ID NO: 58, A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 23, a heavy chain CDR2 of SEQ ID NO: 49, and a heavy chain CDR3 of SEQ ID NO: 58, A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 17, a heavy chain CDR2 of SEQ ID NO: 54, and a heavy chain CDR3 of SEQ ID NO: 58, A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 24, a heavy chain CDR2 of SEQ ID NO: 55, and a heavy chain CDR3 of SEQ ID NO: 58, A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 21, a heavy chain CDR2 of SEQ ID NO: 51, and a heavy chain CDR3 of SEQ ID NO: 58, A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 25, a heavy chain CDR2 of SEQ ID NO: 56, and a heavy chain CDR3 of SEQ ID NO: 57, A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 26, a heavy chain CDR2 of SEQ ID NO: 31, and a heavy chain CDR3 of SEQ ID NO: 57, A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 27, a heavy chain CDR2 of SEQ ID NO: 31, and a heavy chain CDR3 of SEQ ID NO: 57, A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 28, a heavy chain CDR2 of SEQ ID NO: 31, and a heavy chain CDR3 of SEQ ID NO: 57, A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 29, a heavy chain CDR2 of SEQ ID NO: 31, and a heavy chain CDR3 of SEQ ID NO: 57, A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 30, a heavy chain CDR2 of SEQ ID NO: 31, and a heavy chain CDR3 of SEQ ID NO: 57, A heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 1, the heavy chain CDR2 of SEQ ID NO: 32, and the heavy chain CDR3 of SEQ ID NO: 79, or An antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 1, a heavy chain CDR2 of SEQ ID NO: 32, and a heavy chain CDR3 of SEQ ID NO: 57. The method of claim 1, A light chain variable region comprising a light chain CDR1 of SEQ ID NO: 198, a light chain CDR2 of SEQ ID NO: 223, and a light chain CDR3 of SEQ ID NO: 242, A light chain variable region comprising a light chain CDR1 of SEQ ID NO: 199, a light chain CDR2 of SEQ ID NO: 224, and a light chain CDR3 of SEQ ID NO: 243, A light chain variable region comprising a light chain CDR1 of SEQ ID NO: 200, a light chain CDR2 of SEQ ID NO: 225, and a light chain CDR3 of SEQ ID NO: 244, A light chain variable region comprising a light chain CDR1 of SEQ ID NO: 201, a light chain CDR2 of SEQ ID NO: 226, and a light chain CDR3 of SEQ ID NO: 245, A light chain variable region comprising a light chain CDR1 of SEQ ID NO: 202, a light chain CDR2 of SEQ ID NO: 227, and a light chain CDR3 of SEQ ID NO: 246, A light chain variable region comprising a light chain CDR1 of SEQ ID NO: 203, a light chain CDR2 of SEQ ID NO: 228, and a light chain CDR3 of SEQ ID NO: 247, A light chain variable region comprising a light chain CDR1 of SEQ ID NO: 204, a light chain CDR2 of SEQ ID NO: 229, and a light chain CDR3 of SEQ ID NO: 248, A light chain variable region comprising a light chain CDR1 of SEQ ID NO: 205, a light chain CDR2 of SEQ ID NO: 230, and a light chain CDR3 of SEQ ID NO: 249, A light chain variable region comprising a light chain CDR1 of SEQ ID NO: 206, a light chain CDR2 of SEQ ID NO: 231, and a light chain CDR3 of SEQ ID NO: 250, A light chain variable region comprising a light chain CDR1 of SEQ ID NO: 207, a light chain CDR2 of SEQ ID NO: 232, and a light chain CDR3 of SEQ ID NO: 251, A light chain variable region comprising a light chain CDR1 of SEQ ID NO: 208, a light chain CDR2 of SEQ ID NO: 230, and a light chain CDR3 of SEQ ID NO: 252, A light chain variable region comprising a light chain CDR1 of SEQ ID NO: 209, a light chain CDR2 of SEQ ID NO: 233, and a light chain CDR3 of SEQ ID NO: 253, A light chain variable region comprising a light chain CDR1 of SEQ ID NO: 210, a light chain CDR2 of SEQ ID NO: 234, and a light chain CDR3 of SEQ ID NO: 254, A light chain variable region comprising a light chain CDR1 of SEQ ID NO: 211, a light chain CDR2 of SEQ ID NO: 235, and a light chain CDR3 of SEQ ID NO: 255, A light chain variable region comprising a light chain CDR1 of SEQ ID NO: 212, a light chain CDR2 of SEQ ID NO: 236, and a light chain CDR3 of SEQ ID NO: 256, A light chain variable region comprising a light chain CDR1 of SEQ ID NO: 213, a light chain CDR2 of SEQ ID NO: 230, and a light chain CDR3 of SEQ ID NO: 257, A light chain variable region comprising a light chain CDR1 of SEQ ID NO: 214, a light chain CDR2 of SEQ ID NO: 237, and a light chain CDR3 of SEQ ID NO: 258, A light chain variable region comprising a light chain CDR1 of SEQ ID NO: 215, a light chain CDR2 of SEQ ID NO: 230, and a light chain CDR3 of SEQ ID NO: 249, A light chain variable region comprising a light chain CDR1 of SEQ ID NO: 211, a light chain CDR2 of SEQ ID NO: 238, and a light chain CDR3 of SEQ ID NO: 259, A light chain variable region comprising a light chain CDR1 of SEQ ID NO: 216, a light chain CDR2 of SEQ ID NO: 238, and a light chain CDR3 of SEQ ID NO: 260, A light chain variable region comprising a light chain CDR1 of SEQ ID NO: 217, a light chain CDR2 of SEQ ID NO: 239, and a light chain CDR3 of SEQ ID NO: 261, A light chain variable region comprising a light chain CDR1 of SEQ ID NO: 218, a light chain CDR2 of SEQ ID NO: 240, and a light chain CDR3 of SEQ ID NO: 262, A light chain variable region comprising a light chain CDR1 of SEQ ID NO: 219, a light chain CDR2 of SEQ ID NO: 241, and a light chain CDR3 of SEQ ID NO: 263, A light chain variable region comprising a light chain CDR1 of SEQ ID NO: 220, a light chain CDR2 of SEQ ID NO: 230, and a light chain CDR3 of SEQ ID NO: 252, A light chain variable region comprising a light chain CDR1 of SEQ ID NO: 215, a light chain CDR2 of SEQ ID NO: 230, and a light chain CDR3 of SEQ ID NO: 252, A light chain variable region comprising a light chain CDR1 of SEQ ID NO: 205, a light chain CDR2 of SEQ ID NO: 230, and a light chain CDR3 of SEQ ID NO: 264, A light chain variable region comprising a light chain CDR1 of SEQ ID NO: 205, a light chain CDR2 of SEQ ID NO: 230, and a light chain CDR3 of SEQ ID NO: 265, A light chain variable region comprising a light chain CDR1 of SEQ ID NO: 221, a light chain CDR2 of SEQ ID NO: 230, and a light chain CDR3 of SEQ ID NO: 266, A light chain variable region comprising a light chain CDR1 of SEQ ID NO: 205, a light chain CDR2 of SEQ ID NO: 230, and a light chain CDR3 of SEQ ID NO: 252, A light chain variable region comprising a light chain CDR1 of SEQ ID NO: 205, a light chain CDR2 of SEQ ID NO: 230, and a light chain CDR3 of SEQ ID NO: 267, A light chain variable region comprising a light chain CDR1 of SEQ ID NO: 205, a light chain CDR2 of SEQ ID NO: 230, and a light chain CDR3 of SEQ ID NO: 257, A light chain variable region comprising a light chain CDR1 of SEQ ID NO: 220, a light chain CDR2 of SEQ ID NO: 230, and a light chain CDR3 of SEQ ID NO: 267, A light chain variable region comprising a light chain CDR1 of SEQ ID NO: 222, a light chain CDR2 of SEQ ID NO: 230, and a light chain CDR3 of SEQ ID NO: 268, or An antibody or antigen-binding fragment thereof comprising a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 220, a light chain CDR2 of SEQ ID NO: 230, and a light chain CDR3 of SEQ ID NO: 269. The method according to claim 1, wherein heavy chain variable region FR1 selected from the group consisting of SEQ ID NO: 80 to SEQ ID NO: 95, A heavy chain variable region FR2 selected from the group consisting of SEQ ID NOs: 96 to 113, A heavy chain variable region FR3 selected from the group consisting of SEQ ID NOs: 114 to 134, or An antibody or antigen-binding fragment thereof comprising a heavy chain variable region FR4 selected from the group consisting of SEQ ID NOs: 135 to 145. According to claim 1, Light chain variable region FR1 selected from the group consisting of SEQ ID NO: 270 to SEQ ID NO: 294, Light chain variable region FR2 selected from the group consisting of SEQ ID NOs: 295 to 315, Light chain variable region FR3 selected from the group consisting of SEQ ID NOs: 316 to 355, or An antibody or antigen-binding fragment thereof comprising a light chain variable region FR4 selected from the group consisting of SEQ ID NOs: 356 to 367. 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 NOs: 146 and 193. The antibody or antigen-binding fragment thereof according to claim 1, comprising 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: 368 to 420. A nucleic acid encoding the antibody of claim 1 or an antigen binding fragment thereof. An expression vector comprising the nucleic acid of claim 8. A cell transformed with the expression vector of claim 9. A method for producing an antibody or antigen-binding fragment thereof that binds to PD-1, comprising the following steps: (a) culturing the cells of claim 10; And (b) recovering the antibody or antigen-binding fragment thereof from the cultured cells. A composition for preventing or treating cancer, comprising the antibody of claim 1 or an antigen-binding fragment thereof as an active ingredient. The method of claim 12, wherein the cancer is selected from the group consisting of melanoma, lung cancer, liver cancer, glioblastoma, ovarian cancer, colon cancer, head and neck cancer, bladder cancer, kidney cell cancer, gastric cancer, breast cancer, metastatic cancer, prostate cancer and pancreatic cancer. Cancer preventive or therapeutic composition, characterized in that.

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