Anti PD-1 antibody and application of the same

Abstract

To provide a novel PD-1 protein binder that binds to programmed cell death 1 (programmed death-1; PD-1), a novel anti PD-1 antibody that binds to PD-1 and an antigen-binding fragment thereof, and a novel cross-reactive protein binder, antibody and antigen-binding fragment thereof which bind to both human PD-1 and mouse PD-1.SOLUTION: Provided are a protein binder, an antibody and an antigen-binding fragment thereof which bind to PD-1 protein, and furthermore a polynucleotide sequence which encodes the protein binder, the antibody and an antigen-binding fragment thereof, a vector and a host cell including the sequence, and a pharmaceutical composition and a kit including the protein binder, the antibody and an antigen-binding fragment thereof.SELECTED DRAWING: Figure 6

Description

[Technical field]

[0001] The present invention relates to an antibody that binds to programmed cell death-1 (PD-1) protein, an antigen-binding fragment thereof, and uses thereof. [Background technology]

[0002] The discussion in this section is intended to provide background information related to the present disclosure and is not intended to essentially constitute prior art.

[0003] Programmed cell death-1 (PD-1) (also named CD279) is a cell surface protein frequently found on immune cells such as T cells, B cells, monocytes, natural killer (NK) cells, and dendritic cells, which downregulates the immune system, modulates immune system responses, and promotes self-tolerance by suppressing T cell inflammatory activity. It may prevent autoimmune diseases but may interfere with the immune system from killing cancer cells (Syn et al., 2017 Lancet Oncol 18 (12): e731-e741).

[0004] Recently, the development of immune anticancer drugs that target the immune system and restore and promote immunity has been actively promoted. The PD-1 / PD-L1 pathway, one of the immune checkpoint proteins, has been clinically confirmed as a target for cancer immunotherapy (Patsoukis et al., 2020 Sci. Adv. 6: eabd2712). Therefore, PD-1 inhibitors have been developed to block PD-1, activate the immune system, attack tumors, and treat certain types of cancer.

[0005] Upregulation of PD-1 signaling also leads to viral infection and spread in humans. HBV and HCV, the prevalent liver-infecting viruses, induce overexpression of PD-1 ligand in hepatocytes and activate PD-1 signaling in effector T cells, causing T cell exhaustion and tolerance to viral infection (Golden-Mason et al., 2008 J Immunol 180: 3637-3641). Similarly, HIV infection frequently evades the human immune system by a similar mechanism. It has been reported that antagonistic molecules can therapeutically modulate PD-1 signaling to rescue immune cells from tolerance, reactivate them, and eliminate cancer and chronic viral infections (Okazaki et al., 2007 Int Immunol 19: 813-824).

[0006] Meanwhile, anti-PD-1 agonist antibodies are also being developed for use in treating autoimmune disorders such as rheumatoid arthritis and reducing the rejection of transplanted cells / tissues (Grebinoski and Vignali, 2020 Curr Opin Immunol 67: 1-9).

[0007] It has also been proposed that an IFN gamma-dependent systemic immune response is beneficial for the treatment of Alzheimer's disease and other central nervous system pathologies that share a neuroinflammatory component, and WO2015 / 136541 discloses the use of anti-PD-1 antibodies for the treatment of Alzheimer's disease. WO2017 / 220990 describes that blocking the PD-1 / PD-L1 inhibitory immune checkpoint pathway enhances the secretion of IFN gamma by IFN gamma-producing cells, and that the increased IFN gamma activity allows the brain's choroid plexus to selectively traffic leukocytes, infiltrate T cells and monocytes into the damaged central nervous system, and allow immune cells to home to sites of neurodegenerative pathology and neuroinflammation, making the environment less harmful, and allowing for better removal of toxic substances and rescue, regeneration, and repair of neurons.

[0008] Although the development of anti-PD-1 antibody preparations is active, there is still a pressing need to develop a variety of anti-PD-1 antibodies with more diverse indications and characteristics. In addition, to efficiently develop anti-PD-1 antibody preparations and combination drugs containing them, it is very important to have antibodies that can bind to not only human PD-1 but also mouse PD-1, and whose efficacy, pharmacokinetic properties, and toxicity can be confirmed in mice in the preclinical stage. However, since most commercialized antibodies only bind to human PD-1, it is necessary to develop new anti-PD-1 antibodies with cross-reactivity. Summary of the Invention [Problem to be solved by the invention]

[0009] One object of the present disclosure is to provide novel PD-1 protein-binding agents that bind to PD-1.

[0010] One object of the present disclosure is to provide novel anti-PD-1 antibodies and antigen-binding fragments thereof that bind to PD-1.

[0011] Another object of the present disclosure is to provide novel cross-reactive protein binding agents, antibodies, and antigen-binding fragments thereof that bind to both human PD-1 and mouse PD-1.

[0012] It is yet another object of the present disclosure to provide methods for producing novel PD-1 protein binding agents, anti-PD-1 antibodies, and antigen-binding fragments thereof.

[0013] Yet another object of the present disclosure is to provide uses of the novel PD-1 protein-binding agents, anti-PD-1 antibodies, and antigen-binding fragments thereof.

[0014] However, the problems to be solved by the present disclosure are not limited to the problems mentioned above, and other problems not mentioned will be clearly understood by a person skilled in the art from the following description. [Means for solving the problem]

[0015] In order to solve the above-mentioned problems, the inventors of the present application, after numerous experiments, have developed novel PD-1 protein binding agents, anti-PD-1 antibodies, and antigen-binding fragments thereof.

[0016] In one aspect, the present disclosure discloses anti-PD-1 antibodies and antigen-binding fragments thereof that bind to a PD-1 protein, the antibodies and antigen-binding fragments comprising a heavy chain variable region and / or a light chain variable region, the heavy chain variable region comprises a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence of SEQ ID NO:1, an HCDR2 comprising the amino acid sequence of SEQ ID NO:3, 63, 64, 65, 66 or 67, and an HCDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:5 and SEQ ID NO:68 to 82, or an HCDR variant having conservative amino acid substitutions or up to 3 amino acid mutations compared to these sequences; The light chain variable region comprises a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence of SEQ ID NO: 7, 60, 83 or 84, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 9, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 11, or an LCDR variant having conservative amino acid substitutions or not more than three amino acid mutations compared to these sequences.

[0017] In one aspect, the disclosure discloses an anti-PD-1 antibody and antigen-binding fragment thereof that binds to a PD-1 protein, the antibody comprising a heavy chain variable region and / or a light chain variable region, the heavy chain variable region comprises a sequence as set forth in SEQ ID NO: 13, 54, 56 or 58, or a variant having 1 to 10 or less amino acid mutations compared to said sequence; The light chain variable region comprises a sequence as set forth in SEQ ID NO: 15, 55, 57 or 59, or a variant having from 1 to 10 or less amino acid mutations compared to these sequences.

[0018] In one aspect, the present disclosure provides an anti-PD-1 antibody or antigen-binding fragment thereof that specifically binds to an epitope of PD-1 including P130, L128, and I126 of human PD-1 protein (SEQ ID NO:62). The anti-PD-1 antibody or antigen-binding fragment thereof may specifically bind to additional epitopes including one or more selected from the group consisting of N66, Y68, K78, A129, and A132 of SEQ ID NO:62.

[0019] In one aspect, the disclosure provides an isolated immunoglobulin heavy chain variable region polypeptide comprising a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence of SEQ ID NO:1, an HCDR2 comprising the amino acid sequence of SEQ ID NO:3, 63, 64, 65, 66 or 67, and an HCDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:5, and SEQ ID NOs:68 to 82, or an HCDR variant having conservative amino acid substitutions or not more than three amino acid mutations compared to these sequences.

[0020] In one aspect, the disclosure provides an isolated immunoglobulin light chain variable region polypeptide, wherein the light chain variable region comprises a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence of SEQ ID NO: 7, 60, 83 or 84, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 9, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 11, or an LCDR variant having conservative amino acid substitutions or not more than three amino acid mutations compared to these sequences.

[0021] In one aspect, the disclosure provides an isolated immunoglobulin heavy chain variable region polypeptide that binds to PD-1, comprising a sequence as set forth in SEQ ID NO: 13, 54, 56, or 58, or a variant having 1 to 10 or fewer amino acid mutations compared to these sequences.

[0022] In one aspect, the disclosure provides an isolated immunoglobulin light chain variable region polypeptide that binds to PD-1, comprising a sequence as set forth in SEQ ID NO: 15, 55, 57, or 59, or a variant having 1 to 10 or fewer amino acid mutations compared to these sequences.

[0023] In one aspect, the disclosure provides an isolated immunoglobulin heavy chain variable region polypeptide that binds to PD-1, having an amino acid sequence that has at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to the amino acid sequence of SEQ ID NO: 13, 54, 56 or 58.

[0024] In one aspect, the disclosure provides an isolated immunoglobulin light chain variable region polypeptide that binds to PD-1, having an amino acid sequence that has at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to the amino acid sequence of SEQ ID NO: 15, 55, 57 or 59.

[0025] In one aspect, the disclosure provides a heavy chain variable region polypeptide that binds to PD-1, having an amino acid sequence of SEQ ID NO: 13, 54, 56, or 58, including one to ten amino acid additions or deletions, conservative amino acid substitutions, or a combination thereof.

[0026] In one aspect, the disclosure provides a light chain variable region polypeptide that binds to PD-1, having an amino acid sequence of SEQ ID NO: 15, 55, 57, or 59, including one to ten amino acid additions or deletions, conservative amino acid substitutions, or a combination thereof.

[0027] In one aspect, the present disclosure provides an isolated immunoglobulin heavy chain polypeptide comprising a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence of SEQ ID NO:1, an HCDR2 comprising the amino acid sequence of SEQ ID NO:3, 63, 64, 65, 66 or 67, and an HCDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:5, and SEQ ID NOs:68 to 82, or an HCDR variant having conservative amino acid substitutions or not more than three amino acid mutations compared to these sequences, comprising a framework region and a constant region.

[0028] In one aspect, the disclosure provides an isolated immunoglobulin light chain polypeptide that binds to PD-1, wherein the light chain variable region comprises a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence of SEQ ID NO: 7, 60, 83, or 84, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 9, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 11, or an LCDR variant having conservative amino acid substitutions or no more than three amino acid mutations compared to these sequences, and the polypeptide comprises framework regions and constant regions.

[0029] In one aspect, the disclosure provides an isolated immunoglobulin heavy chain polypeptide comprising a sequence as set forth in SEQ ID NO: 13, 54, 56 or 58, or a variant having 1 to 10 or fewer amino acid mutations compared to said sequences.

[0030] In one aspect, the disclosure provides an isolated immunoglobulin light chain polypeptide comprising a sequence as set forth in SEQ ID NO: 15, 55, 57 or 59, or a variant having 1 to 10 or fewer amino acid mutations compared to said sequences.

[0031] In one aspect, the disclosure provides an isolated immunoglobulin heavy chain polypeptide having an amino acid sequence that has at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to the amino acid sequence of SEQ ID NO: 13, 54, 56 or 58.

[0032] In one aspect, the disclosure provides an isolated immunoglobulin light chain polypeptide having an amino acid sequence that has at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to the amino acid sequence of SEQ ID NO: 15, 55, 57 or 59.

[0033] In one aspect, the disclosure provides an immunoglobulin heavy chain polypeptide that binds to PD-1, having an amino acid sequence containing one to ten amino acid additions or deletions, conservative amino acid substitutions, or a combination thereof, relative to the amino acid sequence of SEQ ID NO: 13, 54, 56, or 58.

[0034] In one aspect, the disclosure provides an immunoglobulin light chain polypeptide that binds to PD-1, having an amino acid sequence containing one to ten amino acid additions or deletions, conservative amino acid substitutions, or a combination thereof, relative to the amino acid sequence of SEQ ID NO: 15, 55, 57, or 59.

[0035] In one aspect, the present disclosure provides a PD-1 protein binding agent comprising an anti-PD-1 antibody or antigen-binding fragment thereof, antibody conjugate, immunoglobulin heavy chain polypeptide and / or immunoglobulin light chain polypeptide, immunoglobulin heavy chain variable region polypeptide and / or immunoglobulin light chain variable region polypeptide according to the present disclosure.

[0036] The disclosure provides, in one aspect, a PD-1 protein-binding agent according to the disclosure that is an antibody or antigen-binding fragment thereof selected from a camelized single domain antibody, a diabody, F(ab')2, Fab', Fab, Fv, scFv, scFV dimer, BsFv, dsFv, (dsFv)2, dsFv-dsFv', Fv fragment, ds diabody, nanobody, minibody, domain antibody, bivalent domain antibody, dAb, and single-chain binding polypeptide.

[0037] The present disclosure provides, in one aspect, an isolated or purified polynucleotide molecule comprising a polynucleotide sequence encoding any PD-1 protein-binding agent, anti-PD-1 antibody or antigen-binding fragment thereof, immunoglobulin heavy chain variable region polypeptide, immunoglobulin light chain variable region polypeptide, immunoglobulin heavy chain polypeptide, or immunoglobulin light chain polypeptide according to the present disclosure.

[0038] The present disclosure provides, in another aspect, an isolated or purified polynucleotide molecule comprising the polynucleotide sequence of SEQ ID NO: 14 encoding an immunoglobulin heavy chain variable region polypeptide according to the present disclosure.

[0039] The present disclosure provides, in one aspect, an isolated or purified polynucleotide molecule comprising the polynucleotide sequence of SEQ ID NO: 16, which encodes an immunoglobulin light chain variable region polypeptide according to the present disclosure.

[0040] The present disclosure provides, in one aspect, a vector comprising a polynucleotide molecule comprising a polynucleotide sequence encoding any PD-1 protein binding agent, anti-PD-1 antibody or antigen-binding fragment thereof, immunoglobulin heavy chain variable region polypeptide, immunoglobulin light chain variable region polypeptide, immunoglobulin heavy chain polypeptide, or immunoglobulin light chain polypeptide according to the present disclosure.

[0041] The present disclosure provides, in one aspect, an isolated host cell comprising a vector comprising a polynucleotide molecule comprising a polynucleotide sequence encoding any PD-1 protein binding agent, anti-PD-1 antibody or antigen-binding fragment thereof, immunoglobulin heavy chain variable region polypeptide, immunoglobulin light chain variable region polypeptide, immunoglobulin heavy chain polypeptide, or immunoglobulin light chain polypeptide according to the present disclosure.

[0042] The present disclosure provides, in one aspect, a transgenic animal engineered to express any PD-1 protein-binding agent, anti-PD-1 antibody or antigen-binding fragment thereof, immunoglobulin heavy chain variable region polypeptide, immunoglobulin light chain variable region polypeptide, immunoglobulin heavy chain polypeptide, or immunoglobulin light chain polypeptide according to the present disclosure.

[0043] In one aspect, the disclosure provides a method of expressing any PD-1 protein binding agent, anti-PD-1 antibody, or antigen-binding fragment thereof, immunoglobulin heavy chain variable region polypeptide, immunoglobulin light chain variable region polypeptide, immunoglobulin heavy chain polypeptide, or immunoglobulin light chain polypeptide according to the disclosure, comprising culturing a host cell comprising a vector comprising an isolated polynucleotide encoding said polynucleotide under conditions such that the polynucleotide is expressed.

[0044] In one aspect, the present disclosure provides a method for screening for a PD-1 mimetic substance, the method comprising the steps of reacting any PD-1 protein-binding agent, anti-PD-1 antibody or antigen-binding fragment thereof, immunoglobulin heavy chain variable region polypeptide, immunoglobulin light chain variable region polypeptide, immunoglobulin heavy chain polypeptide, or immunoglobulin light chain polypeptide according to the present disclosure with a test substance, and determining whether or not binding occurs.

[0045] In one aspect, the present disclosure provides a PD-1 mimetic screened by a method for screening PD-1 mimetics, the method comprising the steps of reacting any PD-1 protein binding agent, anti-PD-1 antibody or antigen-binding fragment thereof, immunoglobulin heavy chain variable region polypeptide, immunoglobulin light chain variable region polypeptide, immunoglobulin heavy chain polypeptide, or immunoglobulin light chain polypeptide according to the present disclosure with a test substance, and determining whether or not they bind.

[0046] In one aspect, the present disclosure provides a method for producing an anti-PD-1 antibody, comprising immunizing a PD-1 knockout mouse with a PD-1 antigen, removing the spleen, isolating B lymphocytes, and fusing the B lymphocytes with myeloma cells to obtain hybridoma cells, and selecting a hybridoma that produces an antibody that reacts with the PD-1 antigen.

[0047] In one aspect, the present disclosure provides a hybridoma producing an antibody that reacts with PD-1 antigen, the hybridoma being selected by the antibody production method according to the present disclosure.

[0048] The present disclosure provides, in one aspect, a multispecific antigen-binding molecule, immunoconjugate, chimeric antigen receptor, engineered T cell receptor, or tumor-killing virus comprising any PD-1 protein-binding agent, anti-PD-1 antibody or antigen-binding fragment thereof, immunoglobulin heavy chain variable region polypeptide, immunoglobulin light chain variable region polypeptide, immunoglobulin heavy chain polypeptide, or immunoglobulin light chain polypeptide in accordance with the present disclosure.

[0049] In one aspect, the present disclosure provides a pharmaceutical composition comprising one or more selected from the group consisting of any PD-1 protein-binding agent, anti-PD-1 antibody or antigen-binding fragment thereof, immunoglobulin heavy chain variable region polypeptide, immunoglobulin light chain variable region polypeptide, immunoglobulin heavy chain polypeptide, immunoglobulin light chain polypeptide, and multispecific antigen-binding molecules, immunoconjugates, chimeric antigen receptors, engineered T cell receptors, or tumor-killing viruses comprising same according to the present disclosure, and a pharma- ceutical acceptable excipient or carrier.

[0050] The pharmaceutical composition is also a pharmaceutical composition for preventing, ameliorating or treating a tumor, a cancer, a metastatic tumor, a metastatic cancer, an autoimmune disease, a nervous system disease, a neurodegenerative disease or an infectious disease.

[0051] In one aspect, the present disclosure provides a pharmaceutical composition further comprising a second therapeutic agent.

[0052] In one aspect, the present disclosure provides a therapeutic, diagnostic, or detection kit comprising one or more selected from the group consisting of any PD-1 protein-binding agent, anti-PD-1 antibody or antigen-binding fragment thereof, immunoglobulin heavy chain variable region polypeptide, immunoglobulin light chain variable region polypeptide, immunoglobulin heavy chain polypeptide, immunoglobulin light chain polypeptide, and multispecific antigen-binding molecules, immunoconjugates, chimeric antigen receptors, engineered T cell receptors, or tumor-killing viruses comprising same according to the present disclosure.

[0053] In one aspect, the present disclosure provides a method for preventing or treating a tumor, cancer, metastatic tumor, metastatic cancer, autoimmune disease, nervous system disease, neurodegenerative disease, or infectious disease, comprising administering to an individual one or more selected from the group consisting of any PD-1 protein-binding agent, anti-PD-1 antibody or antigen-binding fragment thereof, immunoglobulin heavy chain variable region polypeptide, immunoglobulin light chain variable region polypeptide, immunoglobulin heavy chain polypeptide, immunoglobulin light chain polypeptide, and multispecific antigen-binding molecules, immunoconjugates, chimeric antigen receptors, engineered T cell receptors, or oncolytic viruses comprising the same, according to the present disclosure.

[0054] The present disclosure also provides, in one aspect, a method of modulating an immune response in an individual comprising administering to the individual one or more selected from the group consisting of any PD-1 protein-binding agent, anti-PD-1 antibody or antigen-binding fragment thereof, immunoglobulin heavy chain variable region polypeptide, immunoglobulin light chain variable region polypeptide, immunoglobulin heavy chain polypeptide, immunoglobulin light chain polypeptide, and multispecific antigen-binding molecules, immunoconjugates, chimeric antigen receptors, engineered T cell receptors, or tumor-killing viruses according to the present disclosure.

[0055] In one aspect, the present disclosure provides the use of any PD-1 protein-binding agent, anti-PD-1 antibody or antigen-binding fragment thereof, immunoglobulin heavy chain variable region polypeptide, immunoglobulin light chain variable region polypeptide, immunoglobulin heavy chain polypeptide, immunoglobulin light chain polypeptide, or multispecific antigen-binding molecule, immunoconjugate, chimeric antigen receptor, engineered T cell receptor, or tumor-killing virus comprising same, according to the present disclosure, in the manufacture of a medicament for preventing, ameliorating, or treating a tumor, cancer, metastatic tumor, metastatic cancer, autoimmune disease, nervous system disease, neurodegenerative disease, or infectious disease.

[0056] The present disclosure also provides, in one aspect, a method of inhibiting tumor cell growth in an individual comprising administering to the individual a therapeutically effective amount of one or more selected from the group consisting of any PD-1 protein-binding agent, anti-PD-1 antibody or antigen-binding fragment thereof, immunoglobulin heavy chain variable region polypeptide, immunoglobulin light chain variable region polypeptide, immunoglobulin heavy chain polypeptide, immunoglobulin light chain polypeptide, and multispecific antigen-binding molecules, immunoconjugates, chimeric antigen receptors, engineered T cell receptors, or tumor-killing viruses according to the present disclosure to inhibit tumor cell growth.

[0057] Other aspects will be apparent from the detailed description of the present application and from the general knowledge of the art. Effect of the Invention

[0058] The PD-1 protein-binding agents, anti-PD-1 antibodies or antigen-binding fragments thereof, immunoglobulin heavy chain variable region polypeptides, immunoglobulin light chain variable region polypeptides, immunoglobulin heavy chain polypeptides, immunoglobulin light chain polypeptides, or multispecific antigen-binding molecules, immunoconjugates, chimeric antigen receptors, engineered T cell receptors, or tumor-killing viruses comprising the same according to the present disclosure bind to human PD-1 protein and can be used for immune response modulation. They are useful, for example, for targeting T cells expressing PD-1 and modulating PD-1 activity. For example, they can be used for the prevention or treatment of tumors, cancers, metastatic tumors, metastatic cancers, autoimmune diseases, nervous system diseases, neurodegenerative diseases, or infectious diseases.

[0059] Furthermore, the PD-1 protein binding agents, anti-PD-1 antibodies or antigen-binding fragments thereof, immunoglobulin heavy chain variable region polypeptides, immunoglobulin light chain variable region polypeptides, immunoglobulin heavy chain polypeptides, or immunoglobulin light chain polypeptides according to the present disclosure can bind to not only human PD-1 but also mouse PD-1, allowing the efficacy, pharmacokinetic properties, and toxicity of the antibodies to be confirmed in mice at the preclinical stage, and can play an important role in the efficient development of antibody preparations and combination preparations containing them.

[0060] The effects of the present disclosure are not limited to such literal descriptions, but include anything that a person of ordinary skill in the art can infer from the present disclosure. [Brief description of the drawings]

[0061] [Figure 1] 1 is a graph showing the results of a binding test of hybridoma 1G1 antibody according to the present disclosure to human PD-1 or mouse PD-1 on the cell surface using an enzyme-linked immunosorbent assay (ELISA). [Diagram 2]1 is a graph showing the results of a binding test of hybridoma 1G1 antibody to cell surface human PD-1 or mouse PD-1 using flow cytometry, in accordance with the present disclosure. [Diagram 3] Figure 2 shows the results of a blocking test of hybridoma 1G1 antibody of the present disclosure for human PD-L1 binding or mouse PD-L1 binding to cell surface human PD-1 or mouse PD-1, respectively, using ELISA. [Figure 4] Figure 2 shows the results of a blocking test of hybridoma 1G1 antibody of the present disclosure for human PD-L1 binding or mouse PD-L1 binding to cell surface human PD-1 or mouse PD-1, respectively, using flow cytometry. [Diagram 5] This is an SDS-PAGE result confirming purified hybridoma 1G1 antibody and Chimeric 1G1 antibody (Chimarick 1G1). [Figure 6] This is a graph showing the results of a binding test of purified hybridoma 1G1 antibody to cell surface human PD-1 or mouse PD-1 using ELISA. [Figure 7] This shows the results of ELISA testing of the selective binding of the single clone cell (hybridoma) 1G1 antibody of the present disclosure to human T cell surface immune checkpoints. [Figure 8] FIG. 1 shows the heavy chain amino acid sequences of three types of humanized 1G1 antibodies (humanized antibodies 1G1-h61, 1G1-h68, and 1G1-h70). The sequences are shown in the form of leader sequence-VH / VL (shown in bold and underlined)-hIgG4CH / hIgkappaCL. [Figure 9]FIG. 1 shows the light chain amino acid sequences of three types of humanized 1G1 antibodies (humanized antibodies 1G1-h61, 1G1-h68, and 1G1-h70). The sequences are shown in the form of leader sequence-VH / VL (shown in bold and underlined)-hIgG4CH / hIgkappaCL. [Figure 10A] 1 shows the heavy chain nucleic acid sequence of humanized antibody 1G1-h61, which is shown in the following format: leader sequence-VH / VL (indicated in bold and underlined)-hIgG4CH / hIgkappaCL-stop codon (in italics). [Figure 10B] 1 shows the heavy chain nucleic acid sequence of humanized antibody 1G1-h68, which is shown in the following format: leader sequence-VH / VL (indicated in bold and underlined)-hIgG4CH / hIgkappaCL-stop codon (in italics). [Figure 10C] 1 shows the heavy chain nucleic acid sequence of humanized antibody 1G1-h70, which is shown in the following format: leader sequence-VH / VL (indicated in bold and underlined)-hIgG4CH / hIgkappaCL-stop codon (in italics). [Figure 11] FIG. 1 shows the nucleic acid sequences of the light chains of the three humanized 1G1 antibodies (humanized antibodies 1G1-h61, 1G1-h68, and 1G1-h70), which are shown in the following format: leader sequence-VH / VL (shown in bold and underlined)-hIgG4CH / hIgkappaCL-stop codon (in italics). [Figure 12A] FIG. 1 shows the binding kinetics of humanized 1G1 antibody to human PD-1 in accordance with the present disclosure. [Figure 12B] 1 is a graph showing the affinity of humanized 1G1 antibody to human PD-1 in terms of ka value (Kon), kd value (Koff), and KD value according to the present disclosure. [Figure 13]1 is a graph showing the results of testing the selective binding (cross-reactivity) of the 1G1 antibody (chimeric 1G1 antibody, humanized 1G1 antibody) according to the present disclosure to the extracellular domain PD-1 antigen of human, mouse, rabbit, cynomolgus monkey, and rat using ELISA. [Figure 14] FIG. 1 is a diagram illustrating the schedule of animal experiments for confirming the anti-cancer effect of 1G1 antibody. [Figure 15] 1 is a graph showing the change in tumor size over time following administration of 1G1 antibody in a mouse melanoma model. [Figure 16] 1 is a graph showing the survival rate over time following administration of 1G1 antibody in a mouse melanoma model. [Figure 17] FIG. 1 shows the relative change in tumor size over time and the tumor growth inhibition rate by administration of 1G1 antibody in an MC38 colon cancer syngeneic mouse model. [Figure 18] FIG. 1 illustrates the binding regions of humanized 1G1 antibody, Keytruda, and Opdivo to human PD-1. [Figure 19A] FIG. 1 shows the binding kinetics of humanized 1G1 antibody to human PD-1 at pH 6.0 in accordance with the present disclosure. [Figure 19B] 1 is a graph showing the affinity of humanized 1G1 antibody in accordance with the present disclosure for human PD-1 at pH 6.0, expressed as ka value (Kon), kd value (Koff), and KD value. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0062] Before describing the present disclosure, it should be understood that the present disclosure is not limited to the specific methods and experimental conditions described, as such methods and conditions may vary. The terms used in this application are merely for describing specific embodiments, and are not intended to limit the scope of the claims, since the scope of the present disclosure is limited only by the appended claims.

[0063] Unless otherwise defined, 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 disclosure belongs. Any methods and materials similar or equivalent to those described herein may be used in the practice or testing of this disclosure. All references cited in this application are incorporated herein by reference in their entirety.

[0064] As used herein, the terms "programmed cell death 1," "PD-1," and "PD-1 protein" are used interchangeably and include variants, isoforms, species homologs of human PD-1, and analogs that share at least one epitope in common with PD-1. PD-1 is a T-cell co-inhibitor, also known as CD279.

[0065] The term "binding molecule" or "binding agent" in this disclosure includes antibodies, antigen-binding fragments thereof, and conjugates thereof with other molecules.

[0066] The term "antibody" in the present disclosure includes whole antibodies and any antigen-binding fragment (i.e., "antigen-binding portion") or single chains thereof. An "antibody" refers to a protein or antigen-binding portion thereof, comprising at least two heavy chains and two light chains interconnected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region and a heavy chain constant region. The heavy chain constant region is composed of three domains; CH1, CH2, and CH3. Each light chain is composed of a light chain variable region and a light chain constant region. The light chain constant region is composed of one domain; CL. The heavy chain variable region (VH) and the light chain variable region (VL) are further subdivided into regions of hypervariability called complementarity determining regions (CDRs), which are located between more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.

[0067] In the present disclosure, "antibody" refers to an immunoglobulin, or a fragment or derivative thereof, whether produced in vitro or in vivo, and includes any polypeptide that contains an antigen-binding site. The term includes, but is not limited to, polyclonal antibodies, monoclonal antibodies, monospecific antibodies, multispecific antibodies, non-specific antibodies, humanized antibodies, single-chain antibodies, chimeric antibodies, synthetic antibodies, recombinant antibodies, hybrid antibodies, mutated antibodies, and grafted antibodies. The term "antibody" also includes antibody fragments, such as Fab, Fab', F(ab')2, Fv, scFv, BsFv, dsFv, (dsFv)2, dsFv-dsFv', Fd, dAb, and other antibody fragments that retain antigen-binding function, i.e., the ability to specifically bind to PD-1. Typically, such fragments include antigen-binding fragments.

[0068] The terms "antigen-binding fragment", "antigen-binding domain" and "binding fragment" in the present disclosure refer to a portion of an antibody molecule that contains the amino acids responsible for the specific binding of the antibody to the antigen. For example, if the antigen is large, the antigen-binding fragment may only bind to a portion of the antigen. The portion of the antigen molecule responsible for the specific interaction with the antigen-binding fragment is referred to as the "epitope" or "antigenic determinant".

[0069] An antigen-binding fragment may contain an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH), but does not necessarily contain both. For example, a so-called Fd antibody fragment is composed only of the VH domain but still retains some of the antigen-binding function of an intact antibody.

[0070] The term "epitope" defines an antigenic determinant, which is specifically bound / identified by the binding fragment as defined above. The binding fragment may specifically bind / interact with a unique structural epitope or a continuous epitope for a target structure, e.g., human PD-1 and rodent PD-1. A structural or discontinuous epitope is characterized by the presence of two or more discontinuous amino acid residues that are separated in the primary sequence but are brought together on the surface of the molecule when the polypeptide folds into a native protein / antigen. The two or more discontinuous amino acid residues that contribute to an epitope are present on an isolated section of one or more polypeptide chains. The residues are brought together on the surface of the molecule when the polypeptide chain folds into a three-dimensional structure to constitute the epitope. In contrast, a continuous or linear epitope is constituted by two or more consecutive amino acid residues that are present within a single linear segment of a polypeptide chain.

[0071] The term "binds to an epitope of PD-1" refers to an antibody having specific binding to a particular epitope of PD-1, which may be defined by a linear amino acid sequence or a ternary, i.e., three-dimensional structure on a portion of the PD-1 polypeptide. The specific binding means that the affinity of the antibodies for the PD-1 portion is substantially greater than their affinity for other related polypeptides.

[0072] The term "high affinity" means that there is a measurable increase in affinity for a portion of PD-1 compared to the affinity for other related polypeptides. Desirably, the affinity is at least 1.5-fold, 2-fold, 5-fold, 10 ... 2 Double, 10 3 Double, 10 4 Double, 10 5 Double, 10 6Binding affinity can be determined by enzyme-linked immunosorbent assay (ELISA), by fluorescence activated cell sorting analysis (FACS) or by surface plasmon resonance (SPR).

[0073] The term "cross-reactivity" in the present disclosure refers to the binding of the antigen fragments described herein to the same target molecule in humans and rodents (mouse or rats). Thus, "cross-reactivity" should be understood as interspecies reactivity with the same molecule x expressed in different species, but not with other molecules other than x. For example, the cross-species specificity of a monoclonal antibody recognizing human PD-1 with rodent (mouse or rat) PD-1 can also be determined, for example, by FACS analysis.

[0074] By "individual" or "subject" in this disclosure is meant a subject in need of treatment for a disease, and more specifically means a mammal, such as a human or non-human primate, rat, mouse, dog, cat, horse, or cow.

[0075] "Treatment" in the present disclosure refers to any action in which the symptoms of a disease are ameliorated or favorably altered by administration of a pharmaceutical composition according to the present disclosure. Treatment also includes prevention. Those in need of treatment include not only those already suffering from a particular medical disorder, but also those who will eventually acquire the disorder.

[0076] By "amelioration" in this disclosure is meant any action that at least reduces a parameter associated with the condition being treated, e.g., the severity of a symptom.

[0077] In the following, the contents for carrying out the present disclosure and specific examples will be described in detail with reference to the accompanying drawings. Matters that are not different from the prior art and are not necessary for understanding the technical idea of ​​the present disclosure will be omitted from the description.

[0078] Exemplary Anti-PD-1 Antibodies and PD-1 Binding Agents In one aspect, the disclosure discloses an anti-PD-1 antibody or antigen-binding fragment thereof that binds to a PD-1 protein, the antibody or antigen-binding fragment comprising a heavy chain variable region and / or a light chain variable region, the heavy chain variable region comprises a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence of SEQ ID NO:1, an HCDR2 comprising the amino acid sequence of SEQ ID NO:3, 63, 64, 65, 66 or 67, and an HCDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:5 and SEQ ID NO:68 to 82, or an HCDR variant having conservative amino acid substitutions or up to 3 amino acid mutations compared to these sequences; The light chain variable region comprises a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence of SEQ ID NO: 7, 60, 83 or 84, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 9, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 11, or an LCDR variant having conservative amino acid substitutions or not more than three amino acid mutations compared to these sequences.

[0079] In some embodiments, the above "three or fewer amino acid mutations" refers to three, two, one or zero amino acid mutations.

[0080] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof of the present disclosure is an antibody or antigen-binding fragment thereof that has the same level of binding affinity to human PD-1 and mouse PD-1.

[0081] In some embodiments, the anti-PD-1 antibodies or antigen-binding fragments thereof disclosed herein have a KD value of 10 -7 and in some embodiments, binds to PD-1 at 10 -8 M, 10 -9 M, 10 -10 M or 10 -11 It binds to PD-1 with a KD value of less than M.

[0082] In some embodiments, the anti-PD-1 antibodies or antigen-binding fragments thereof disclosed herein have a KD value of 10 or more even in a low pH environment. -9 M or less, preferably KD value 10 -10 M or less, preferably with a KD value of 10 -11 In some embodiments, the anti-PD-1 antibodies or antigen-binding fragments thereof of the present disclosure bind to PD-1 with a binding affinity of 9x10 M or less at pH 6.0. -10 It binds to PD-1 with a KD of less than M.

[0083] In one aspect, the disclosure discloses an anti-PD-1 antibody and antigen-binding fragment thereof that binds to a PD-1 protein, the antibody comprising a heavy chain variable region and / or a light chain variable region, the heavy chain variable region comprises a sequence as set forth in SEQ ID NO: 13, 54, 56 or 58, or a variant having 1 to 10 or less amino acid mutations compared to said sequence; The light chain variable region comprises a sequence as set forth in SEQ ID NO: 15, 55, 57 or 59, or a variant having from 1 to 10 or less amino acid mutations compared to these sequences.

[0084] In one aspect, the disclosure provides an isolated immunoglobulin heavy chain variable region polypeptide comprising a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence of SEQ ID NO:1, an HCDR2 comprising the amino acid sequence of SEQ ID NO:3, 63, 64, 65, 66 or 67, and an HCDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:5, and SEQ ID NOs:68 to 82, or an HCDR variant having conservative amino acid substitutions or not more than three amino acid mutations compared to these sequences.

[0085] In one aspect, the disclosure provides an isolated immunoglobulin light chain variable region polypeptide comprising a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence of SEQ ID NO: 7, 60, 83 or 84, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 9, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 11, or an LCDR variant having conservative amino acid substitutions or not more than three amino acid mutations compared to these sequences.

[0086] In one aspect, the disclosure provides an isolated immunoglobulin heavy chain variable region polypeptide that binds to PD-1, comprising a sequence as set forth in SEQ ID NO: 13, 54, 56, or 58, or a variant having 1 to 10 or fewer amino acid mutations compared to these sequences.

[0087] In one aspect, the disclosure provides an isolated immunoglobulin light chain variable region polypeptide that binds to PD-1, comprising a sequence as set forth in SEQ ID NO: 15, 55, 57, or 59, or a variant having 1 to 10 or fewer amino acid mutations compared to these sequences.

[0088] In the present disclosure, "isolated" refers to something separated from a component of its natural environment.

[0089] In one aspect, the disclosure provides an isolated immunoglobulin heavy chain variable region polypeptide that binds to PD-1, having an amino acid sequence that has at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the amino acid sequence of SEQ ID NO: 13, 54, 56 or 58.

[0090] In one aspect, the disclosure provides an isolated immunoglobulin light chain variable region polypeptide that binds to PD-1, having an amino acid sequence that has at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the amino acid sequence of SEQ ID NO: 15, 55, 57, or 59.

[0091] Sequence similarity among polypeptides is typically measured using sequence analysis software. Protein analysis software uses similarity measures assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions, to match similar sequences. For example, GCG software includes programs such as GAP and BESTFIT, which may be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides originating from different species of organisms, or between a wild-type protein and its mutein. See, for example, GCG version 6.1. Polypeptide sequences may also be compared using FASTA with default or recommended parameters, such as GCG version 6.1. FASTA (e.g., FASTA2 and FASTA3) programs provide alignment of the best overlap regions between the query and search sequences, and percent sequence identity. When comparing sequences of the present disclosure to a database containing multiple sequences originating from different organisms, another preferred algorithm is the computer program BLAST, particularly BLASTP or TBLASTN, using default parameters. See, for example, Altschul et al. (1990) J. Mol. Biol. 215: 403-410; and Altschul et al. (1997) Nucleic Acids Res. 25: 3389-3402, each of which is incorporated herein by reference.

[0092] Residue positions that are not identical also differ by, for example, conservative amino acid substitutions.

[0093] In one aspect, the disclosure provides a heavy chain variable region polypeptide that binds to PD-1, having an amino acid sequence of SEQ ID NO: 13, 54, 56, or 58, including one to ten amino acid additions or deletions, conservative amino acid substitutions, or a combination thereof.

[0094] In one aspect, the disclosure provides a light chain variable region polypeptide that binds to PD-1, having an amino acid sequence of SEQ ID NO: 15, 55, 57, or 59, including one to ten amino acid additions or deletions, conservative amino acid substitutions, or a combination thereof.

[0095] A "conservative amino acid substitution" is one in which an amino acid residue is replaced by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). Generally, such a conservative amino acid substitution does not substantially change the functional properties of a protein. When two or more amino acid sequences differ from each other by conservative substitutions, the percentage or degree of similarity may be adjusted upwards to correct for the conservative nature of the substitution. Means for such adjustments are well known to those of skill in the art. See, for example, Pearson (1994) Methods Mol. Biol. 24: 307-331, which is incorporated herein by reference. Examples of amino acid groups having side chains with similar chemical properties include: 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartic acid and glutamic acid; and 7) sulfur-containing side chains: cysteine ​​and methionine. Preferred conservative amino acid substitution groups are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Alternatively, a conservative substitution is any change that has a positive value in the PAM250 log-likelihood matrix disclosed in the incorporated herein by reference (see Gonnet et al. (1992) Science 256: 1443 45). A "moderately conservative" substitution is any change that has a nonnegative value in the PAM250 log-likelihood matrix.

[0096] In one aspect, the disclosure provides an isolated immunoglobulin heavy chain polypeptide that binds to PD-1, comprising a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence of SEQ ID NO:1, an HCDR2 comprising the amino acid sequence of SEQ ID NO:3, 63, 64, 65, 66, or 67, and an HCDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:5, and SEQ ID NOs:68-82, or an HCDR variant having conservative amino acid substitutions or not more than three amino acid mutations compared to these sequences, and comprising a framework region and a constant region.

[0097] In one aspect, the disclosure provides an isolated immunoglobulin light chain polypeptide that binds to PD-1, comprising a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence of SEQ ID NO: 7, 60, 83, or 84, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 9, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 11, or an LCDR variant having conservative amino acid substitutions or no more than three amino acid mutations compared to those sequences, and comprising framework regions and constant regions.

[0098] In one aspect, the disclosure provides an isolated immunoglobulin heavy chain polypeptide comprising a sequence as set forth in SEQ ID NO: 13, 54, 56 or 58, or a variant having 1 to 10 or fewer amino acid mutations compared to said sequences.

[0099] In one aspect, the disclosure provides an isolated immunoglobulin light chain polypeptide comprising a sequence as set forth in SEQ ID NO: 15, 55, 57 or 59, or a variant having 1 to 10 or fewer amino acid mutations compared to said sequences.

[0100] In one aspect, the disclosure provides an isolated immunoglobulin heavy chain polypeptide having an amino acid sequence that has at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the amino acid sequence of SEQ ID NO: 13, 54, 56 or 58.

[0101] In one aspect, the disclosure provides an isolated immunoglobulin light chain polypeptide having an amino acid sequence that has at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the amino acid sequence of SEQ ID NO: 15, 55, 57 or 59.

[0102] In one aspect, the disclosure provides an immunoglobulin heavy chain polypeptide that binds to PD-1, having an amino acid sequence of SEQ ID NO: 13, 54, 56, or 58, including one to ten amino acid additions or deletions, conservative amino acid substitutions, or a combination thereof.

[0103] In one aspect, the disclosure provides an immunoglobulin light chain polypeptide that binds to PD-1, having an amino acid sequence of SEQ ID NO: 15, 55, 57, or 59, including one to ten amino acid additions or deletions, conservative amino acid substitutions, or a combination thereof.

[0104] In some embodiments, the anti-PD-1 antibodies or antigen-binding fragments thereof of the present disclosure are recombinant antibodies, preferably murine antibodies, chimeric antibodies, or humanized antibodies.

[0105] In some embodiments, the heavy chain constant region of a chimeric or humanized anti-PD-1 antibody of the disclosure can be derived from human IgG1, human IgG2, human IgG3, or human IgG4, or a mutant sequence of IgG1, IgG2, IgG3, or IgG4, and the light chain constant region can be derived from a human kappa chain, human lambda chain, or a mutant sequence thereof.

[0106] In some embodiments of the anti-PD-1 antibodies or antigen-binding fragments thereof of the present disclosure, the antibody is a chimeric antibody, and the constant region of the antibody may be derived from a constant region of a human antibody or a mutant thereof.

[0107] In some embodiments of the anti-PD-1 antibodies or antigen-binding fragments thereof of the present disclosure, the antibody is a humanized antibody, and the light chain framework region (FR) and heavy chain framework region of the antibody are derived from human germline light and heavy chains, respectively, or mutant sequences thereof.

[0108] In one aspect, the present disclosure provides a PD-1 protein binding agent comprising an anti-PD-1 antibody or antigen-binding fragment thereof, antibody conjugate, immunoglobulin heavy chain polypeptide and / or immunoglobulin light chain polypeptide, immunoglobulin heavy chain variable region polypeptide and / or immunoglobulin light chain variable region polypeptide according to the present disclosure.

[0109] The PD-1 protein-binding agent can be, for example, but is not limited to, an antibody, an antibody conjugate, or an antigen-binding fragment thereof.

[0110] In some embodiments, the disclosure provides an isolated PD-1 protein-binding agent, anti-PD-1 antibody, or antigen-binding fragment thereof that competes with any of the aforementioned PD-1 protein-binding agents, anti-PD-1 antibodies, or antigen-binding fragments thereof, immunoglobulin heavy chain variable region polypeptides, immunoglobulin light chain variable region polypeptides, immunoglobulin heavy chain polypeptides, or immunoglobulin light chain polypeptides for binding to PD-1, or that binds to the same PD-1 epitope as the PD-1 protein-binding agent, anti-PD-1 antibody, or antigen-binding fragment thereof.

[0111] antigen binding fragment In one aspect, the present disclosure provides a PD-1 protein-binding agent according to the present disclosure that is an antibody or antigen-binding fragment thereof selected from, but not limited to, a camelized single domain antibody, a diabody, F(ab')2, Fab', Fab, Fv, scFv, scFV dimer, BsFv, dsFv, (dsFv)2, dsFv-dsFv', Fv fragment, ds diabody, nanobody, minibody, domain antibody, bivalent domain antibody, dAb, and single-chain binding polypeptide.

[0112] Unless otherwise specifically indicated, the term "antibody" as used herein includes not only an antibody molecule comprising two immunoglobulin heavy chains and two immunoglobulin light chains (i.e., an "intact antibody molecule"), but also an antigen-binding fragment thereof. As used herein, the terms "antigen-binding site" of an antibody, "antigen-binding fragment" of an antibody, and the like, include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen and forms a complex. As used herein, the term "antigen-binding fragment" of an antibody or "antibody fragment" refers to one or more fragments of an antibody that retain the ability to specifically bind to PD-1. The antibody fragment may also include a Fab fragment, a F(ab')2 fragment, a Fv fragment, a dAb fragment, a fragment comprising a CDR, or an isolated CDR. In certain embodiments, the term "antigen-binding fragment" refers to a polypeptide fragment of a multispecific antigen-binding molecule. Antigen-binding fragments of antibodies can be derived from intact antibody molecules using any suitable standard techniques, such as, for example, proteolytic digestion techniques or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding the antibody variable domains and (optionally) the antibody constant domains. Such DNA is known and / or may be readily available, for example, from commercial sources, DNA libraries (including, for example, phage antibody libraries), or synthesized. The DNA can be sequenced and engineered chemically or by using molecular biology techniques, for example, to place one or more variable and / or constant domains in a suitable configuration, or to incorporate codons, generate cysteine ​​residues, modify, add or delete amino acids, etc.

[0113] Non-limiting examples of antigen-binding fragments include Fab, Fab', F(ab')2, Fv, scFv, BsFv, dsFv, (dsFv)2, dsFv-dsFv', Fd, dAb, and a minimal recognition unit consisting of amino acid residues that mimic a hypervariable region of an antibody (e.g., an isolated complementarity determining region (CDR), e.g., a CDR3 peptide), or a constrained FR3-CDR3-FR4 peptide. Other engineered molecules, such as domain-specific antibodies, single domain antibodies, domain deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g. monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs) and shark variable IgNAR domains are also encompassed within the expression "antigen-binding fragment" as used herein.

[0114] Antigen-binding fragments of antibodies generally contain at least one variable domain. The variable domain can have any size or amino acid composition and generally contains at least one CDR adjacent to or in frame with one or more framework sequences. In antigen-binding fragments having a VH domain combined with a VL domain, the VH and VL domains can be positioned in any suitable arrangement relative to each other. For example, the variable region may be a dimer, including VH-VH, VH-VL or VL-VL dimers. Alternatively, antigen-binding fragments of antibodies may contain a monomeric VH domain or a monomeric VL domain.

[0115] In certain embodiments, an antigen-binding fragment of an antibody also comprises at least one variable domain covalently linked to at least one constant domain. Non-limiting, exemplary configurations of variable and constant domains that may be found within an antigen-binding fragment of an antibody of the present disclosure include (i) VH-CH1, (ii) VH-CH2, (iii) VH-CH3, (iv) VH-CH1-CH2, (v) VH-CH1-CH2-CH3, (vi) VH-CH2-CH3, (vii) VH-CL, (viii) VL-CH1, (ix) VL-CH2, (x) VL-CH3, (xi) VL-CH1-CH2, (xii) VL-CH1-CH2-CH3, (xiii) VL-CH2-CH3, and (xiv) VL-CL. In any configuration of the variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may be directly linked to each other or may be linked by a full or partial hinge or linker region. The hinge region may consist of at least two (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids that provide a flexible or semi-flexible link between adjacent variable and / or constant domains in a single polypeptide molecule. Furthermore, antigen-binding fragments of antibodies of the present disclosure may also include homodimers or heterodimers (or other multimers) of any of the configurations of the variable and constant domains listed above, non-covalently or covalently linked (e.g., by disulfide bonds) to each other and / or to one or more monomeric VH or VL domains. As with intact antibody molecules, antigen-binding fragments may be monospecific or multispecific (e.g., bispecific). A multispecific antigen-binding fragment of an antibody generally comprises at least two different variable domains, where each variable domain can specifically bind to a different epitope on a separate antigen or on the same antigen. Any multispecific antibody format, including the exemplary bispecific antibody formats disclosed herein, can be adapted for use in the context of antigen-binding fragments of antibodies of the present disclosure using routine techniques available in the art.

[0116] Nucleic Acids Encoding Exemplary Anti-PD-1 Antibodies and Binding Agents The present disclosure provides, in one aspect, an isolated or purified polynucleotide molecule comprising a polynucleotide sequence encoding any PD-1 protein-binding agent, anti-PD-1 antibody or antigen-binding fragment thereof, immunoglobulin heavy chain variable region polypeptide, immunoglobulin light chain variable region polypeptide, immunoglobulin heavy chain polypeptide, or immunoglobulin light chain polypeptide according to the present disclosure.

[0117] The present disclosure provides, in another aspect, an isolated or purified polynucleotide molecule comprising the polynucleotide sequence of SEQ ID NO: 14 encoding an immunoglobulin heavy chain variable region polypeptide according to the present disclosure.

[0118] The present disclosure provides, in one aspect, an isolated or purified polynucleotide molecule comprising the polynucleotide sequence of SEQ ID NO: 16, which encodes an immunoglobulin light chain variable region polypeptide according to the present disclosure.

[0119] Production of Exemplary Anti-PD-1 Antibodies and Binding Agents In one aspect of the present disclosure, there is provided a method for producing an antibody, comprising immunizing a PD-1 knockout mouse with a PD-1 antigen, removing the spleen, isolating B lymphocytes, and fusing the resulting B lymphocytes with myeloma cells to obtain hybridoma cells, and selecting a hybridoma that produces an antibody that reacts with the human PD-1 antigen.

[0120] In addition, one aspect of the present disclosure provides a hybridoma produced by the antibody production method.

[0121] The present disclosure provides, in one aspect, a vector comprising a polynucleotide molecule comprising a polynucleotide sequence encoding any PD-1 protein binding agent, anti-PD-1 antibody or antigen-binding fragment thereof, immunoglobulin heavy chain variable region polypeptide, immunoglobulin light chain variable region polypeptide, immunoglobulin heavy chain polypeptide, or immunoglobulin light chain polypeptide according to the present disclosure.

[0122] The present disclosure provides, in one aspect, an isolated host cell comprising a vector comprising a polynucleotide molecule comprising a polynucleotide sequence encoding any PD-1 protein binding agent, anti-PD-1 antibody or antigen-binding fragment thereof, immunoglobulin heavy chain variable region polypeptide, immunoglobulin light chain variable region polypeptide, immunoglobulin heavy chain polypeptide, or immunoglobulin light chain polypeptide according to the present disclosure.

[0123] In one embodiment, the host cell contains (e.g., is transformed with) (1) a vector comprising a polynucleotide encoding an amino acid sequence comprising an antibody VL and an amino acid sequence comprising an antibody VH according to the present disclosure, or (2) a first vector comprising a polynucleotide encoding an amino acid sequence comprising an antibody VL and a second vector comprising a polynucleotide encoding an amino acid sequence comprising an antibody VH according to the present disclosure.

[0124] In one embodiment, a method is provided for producing an anti-PD1 antibody, comprising culturing a host cell containing a polynucleotide encoding an antibody that binds PD-1, as provided supra, under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0125] Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells. For example, antibodies can be produced in bacteria, particularly when glycosylation and Fc effector functions are not required. For the expression of antibody fragments and polypeptides in bacteria, see, for example, U.S. Pat. No. 5,648,237, U.S. Pat. No. 5,789,199 and U.S. Pat. No. 5,840,523. Also, see Charlton, KA, In: Methods in Molecular Biology, Vol. 248, Lo BKC (ed.), Humana Press, Totowa, NJ (2003), pp. 245-254, which describes the expression of antibody fragments in E. coli. After expression, the antibody can be isolated from the bacterial cell paste in a soluble fraction and further purified. In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors, including fungal and yeast strains in which the glycosylation pathway has been "humanized," resulting in the production of antibodies with partially or fully human glycosylation patterns (see references: Gerngross, TU, Nat. Biotech. 22 (2004) 1409-1414; and Li, H. et al., Nat. Biotech. 24 (2006) 210-215).

[0126] Suitable host cells for the expression of glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. In particular, a number of baculovirus strains have been identified that can be used with insect cells for transfection of Spodoptera frugiperda cells. Plant cell cultures can also be used as hosts (see, for example, U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978 and 6,417,429 (PLANTIBODIES FOR PRODUCING ANTIBODIES IN TRANSGENIC PLANTS)). TM(See the following link for an explanation of the technology.)

[0127] Vertebrate cells may also be used as hosts. For example, mammalian cell lines adapted to grow in suspension are also useful. Other examples of useful mammalian host cell lines are SV40 transformed monkey kidney CV1 cell line (COS-7), human embryonic kidney cell lines (e.g., 293 cells or 293 cells as described in Graham, FL et al., J. Gen Virol. 36 (1977) 59-74); baby hamster kidney cells (BHK), mouse sertoli cells (e.g., TM4 cells as described in Mather, JP, Biol. Reprod. 23 (1980) 243-252), monkey kidney cells (CV1); African green monkey kidney cells (VERO-76), human cervical carcinoma cells (HELA), dog kidney cells (MDCK), buffalo rat hepatocytes (BRL3A), human lung cells (W138), human hepatocytes (HepG2), mouse mammary tumor cells (MMT 060562), e.g., Mather, JP et al., J. Gen Virol. 36 (1977) 59-74).

[0033] . Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells [Urlaub, G. et al., Proc. Natl. Acad. Sci. USA 77 (1980) 4216-4220], and myeloma cell lines, such as Y0, NS0 and Sp2 / 0. For a review of specific mammalian host cell lines suitable for antibody production, see, for example, [Yazaki, P. and Wu, AM, Methods in Molecular Biology, Vol. 248, Lo, BKC(ed.), Humana Press, Totowa, NJ (2004), pp. 255-268].

[0128] In one aspect, the present disclosure provides a transgenic animal engineered to express any PD-1 protein binding agent, anti-PD-1 antibody or antigen-binding fragment thereof, immunoglobulin heavy chain variable region polypeptide, immunoglobulin light chain variable region polypeptide, immunoglobulin heavy chain polypeptide, or immunoglobulin light chain polypeptide according to the present disclosure, the animal being a rodent, e.g., a mouse or a rat.

[0129] In one aspect, the disclosure provides a method of expressing any PD-1 protein binding agent, anti-PD-1 antibody, or antigen-binding fragment thereof, immunoglobulin heavy chain variable region polypeptide, immunoglobulin light chain variable region polypeptide, immunoglobulin heavy chain polypeptide, or immunoglobulin light chain polypeptide according to the disclosure, comprising culturing a host cell comprising a vector comprising an isolated polynucleotide encoding said polynucleotide under conditions such that the polynucleotide is expressed.

[0130] In one aspect, the present disclosure provides a method for screening for a PD-1 mimetic substance, the method comprising reacting any PD-1 protein-binding agent, anti-PD-1 antibody or antigen-binding fragment thereof, immunoglobulin heavy chain variable region polypeptide, immunoglobulin light chain variable region polypeptide, immunoglobulin heavy chain polypeptide, or immunoglobulin light chain polypeptide according to the present disclosure with a test substance, and measuring whether or not binding occurs.

[0131] In one aspect, the present disclosure provides a PD-1 mimetic screened by a method for screening PD-1 mimetics, the method comprising the steps of reacting a test substance with any PD-1 protein binding agent, anti-PD-1 antibody or antigen-binding fragment thereof, immunoglobulin heavy chain variable region polypeptide, immunoglobulin light chain variable region polypeptide, immunoglobulin heavy chain polypeptide, or immunoglobulin light chain polypeptide according to the present disclosure, and measuring whether or not the test substance binds.

[0132] Multispecific antigen binding molecules, immunoconjugates The present disclosure provides, in one aspect, a multispecific antigen-binding molecule comprising any PD-1 protein-binding agent, anti-PD-1 antibody or antigen-binding fragment thereof, immunoglobulin heavy chain variable region polypeptide, immunoglobulin light chain variable region polypeptide, immunoglobulin heavy chain polypeptide, or immunoglobulin light chain polypeptide, immunoconjugate, chimeric antigen receptor, engineered T cell receptor, or tumor-killing virus according to the present disclosure.

[0133] A multispecific (e.g., bispecific) antigen-binding molecule of an antibody generally comprises at least two different variable domains, where each variable domain can specifically bind to a separate antigen or to a different epitope on the same antigen. Any multispecific antibody format, including bispecific antibody formats, can be adapted for use in the context of an antigen-binding fragment of an antibody of the present disclosure using routine techniques available in the art.

[0134] In one aspect, the present disclosure includes a multispecific antigen-binding molecule or antigen-binding fragment thereof, where one specificity of the immunoglobulin is specific for the extracellular domain of PD-1 or a fragment thereof, and the other specificity of the immunoglobulin is specific for binding to a domain other than the extracellular domain of PD-1, or a second therapeutic target, or is conjugated to a therapeutic moiety. The other specificity of the immunoglobulin is also specific for a second target antigen. The second target antigen can be present on the same cell as PD-1 or on a different cell. In one embodiment, the second target cell is present on an immune cell other than a T cell, such as a B cell, an antigen-transmitting cell, a monocyte, a macrophage, or a dendritic cell. In some embodiments, the second target antigen can be present on a tumor cell, an autologous immune tissue cell, or a virus-infected cell.

[0135] In another aspect, the disclosure provides a multispecific antigen-binding molecule or antigen-binding fragment thereof that includes a first antigen-binding specificity that binds to PD-1 and a second antigen-binding specificity that binds to a T cell receptor, a B cell receptor, or an Fc receptor. In a related aspect, the disclosure provides a multispecific antigen-binding molecule or antigen-binding fragment thereof that includes a first antigen-binding specificity that binds to PD-1 and a second antigen-binding specificity that binds to a different T cell co-inhibitor, such as LAG-3, CTLA-4, BTLA, CD-28, 2B4, LY108, TIGIT, TIM3, LAIR1, ICOS, and CD160.

[0136] In yet another aspect, the disclosure provides a multispecific antigen-binding molecule or antigen-binding fragment thereof comprising a first antigen-binding specificity that binds to PD-1 and a second antigen-binding specificity that binds to an autoimmune tissue-specific antigen. In certain embodiments, the antibody is also an activating or agonist antibody.

[0137] Any of the multispecific antigen binding molecules of the present disclosure, or variants thereof, can be produced using standard molecular biology techniques (e.g., recombinant DNA and protein expression techniques) as known to those skilled in the art.

[0138] In some embodiments, PD-1 specific antibodies are generated in a bispecific format ("bispecific") in which variable regions that bind different domains of PD-1 are linked together to provide dual domain specificity within a single binding molecule. Properly designed bispecific antibodies can increase overall PD-1 inhibitory efficacy by increasing both specificity and binding avidity. Variable regions that can bind to different regions within a domain or have specificity for individual domains (e.g., segments of the N-terminal domain) are paired in a structural framework that allows each region to simultaneously bind separate epitopes or different regions within a domain.

[0139] In one example involving bispecific antibodies, a heavy chain variable region (VH) from a binder with specificity for one domain is recombined with a light chain variable region (VL) from a set of binders with specificity for a second domain to identify a non-cognate VL partner that can pair with the first VH without destroying the first specificity for the first VH. In such a manner, a single VL segment (e.g., VL1) can be combined with two different VH domains (e.g., VH1 and VH2) to generate a bispecific antibody composed of two binding "arms" (VH1-VL1 and VH2-VL1). The use of a single VL segment reduces the complexity of the system and therefore increases the efficiency of the cloning, expression and purification steps used to generate bispecific antibodies (see, e.g., US 13 / 022759 and US 2010 / 0331527).

[0140] Alternatively, one or more domains and antibodies that bind to a second target, such as, but not limited to, a second, different anti-PD-1 antibody, can be produced in a bispecific format using techniques described herein or other techniques known to those of skill in the art. Antibody variable regions that bind to different regions can be linked together, for example, with variable regions that bind to related sites on the extracellular domain of PD-1, to provide dual antigen specificity within a single binding molecule. Properly designed bispecific antibodies of such properties perform dual functions. A variable region with specificity for the extracellular domain is combined with a variable region with specificity for a domain other than the extracellular domain, paired in a structural framework that allows each variable region to bind a distinct antigen.

[0141] An exemplary bispecific antibody format that may be used in the context of the present disclosure involves the use of a first immunoglobulin (Ig) CH3 domain and a second Ig CH3 domain, where the first Ig CH3 domain and the second Ig CH3 domain differ from each other by at least one amino acid, and where the at least one amino acid difference reduces binding of the bispecific antibody to protein A compared to a bispecific antibody without the amino acid difference.

[0142] In one embodiment, the first IgCH3 domain binds protein A and the second IgCH3 domain comprises a mutation that reduces or abolishes protein A binding, such as an H95R mutation (according to IMGT exon numbering, but H435R according to EU numbering). The second CH3 may also further comprise a Y96F mutation (according to IMGT numbering, but Y436F according to EU numbering). Further mutations that may be found within the second CH3 include D16E, L18M, N44S, K52N, V57M and V82I (according to IMGT numbering, but D356E, L358M, N384S, K392N, V397M and V422I according to EU numbering) for IgG1 antibodies and N44S, K52N and V82I (according to IMGT numbering, but N422I according to EU numbering) for IgG2 antibodies. For IgG4 antibodies, the bispecific antibody formats include Q15R, N44S, K52N, V57M, R69K, E79Q and V82I (according to the IMGT numbering, but according to EU numbering, Q355R, N384S, K392N, V397M, R409K, E419Q and V422I). Variations on the foregoing bispecific antibody formats are also contemplated within the scope of the present disclosure.

[0143] Other exemplary bispecific formats that may be used in the context of the present disclosure include, without limitation, for example, scFv-based or diabody bispecific formats, IgG-scFv fusions, dual variable domain (DVD)-Ig, quadroma, knobs-into-holes (KIH), common light chain (e.g., common light chain with KIH), CrossMab, CrossFab, (SEED)body, leucine zipper, duobody, IgG1 / IgG2, dual acting Fab (DAF)-IgG, and Mab2 bispecific formats (for a discussion of such formats, see, e.g., Klein et al. 2012, mAbs 4:6, 1-11, and references cited therein). Bispecific antibodies have also been generated using peptide / nucleic acid conjugates, for example, where unnatural amino acids with orthogonal chemical reactivity are used to generate site-specific antibody-oligonucleotide conjugates that subsequently self-assemble into multimeric complexes with defined composition, valency, and geometry (see, e.g., Kazane et al., J. Am. Chem. Soc. [Epub: Dec. 4, 2012]).

[0144] The present disclosure includes human anti-PD-1 monoclonal antibodies conjugated to a therapeutic moiety such as a chemotherapeutic agent or a cytotoxin to treat cancer ("immunoconjugates"). As used herein, the term "immunoconjugate" refers to an antibody chemically or biologically linked to a cytotoxin, a radioactive agent, a cytokine, an interferon, a targeting or reporter moiety, an enzyme, a toxin, a peptide or protein, or a therapeutic agent. The antibody may be linked to the cytotoxin, a radioactive agent, a cytokine, an interferon, a targeting or reporter moiety, an enzyme, a toxin, a peptide, or a therapeutic agent at any position by a molecule capable of binding to its target. Examples of such immunoconjugates include antibody-drug conjugates and antibody-toxin fusion proteins.

[0145] The types of therapeutic moieties that can be conjugated to the anti-PD-1 antibodies will take into consideration the condition being treated and the desired therapeutic effect to be achieved. Examples of materials suitable for forming immunoconjugates are known in the art, see, e.g., WO 05 / 103081.

[0146] Therapeutic Administration and Formulations In one aspect, the present disclosure provides a pharmaceutical composition comprising one or more selected from the group consisting of any PD-1 protein-binding agent, anti-PD-1 antibody or antigen-binding fragment thereof, immunoglobulin heavy chain variable region polypeptide, immunoglobulin light chain variable region polypeptide, immunoglobulin heavy chain polypeptide, immunoglobulin light chain polypeptide, and multispecific antigen-binding molecules, immunoconjugates, chimeric antigen receptors, engineered T cell receptors, or tumor-killing viruses comprising any of the above, according to the present disclosure, and a pharma- ceutical acceptable excipient or carrier.

[0147] The pharmaceutical composition is also a pharmaceutical composition for preventing, ameliorating or treating a tumor, a cancer, a metastatic tumor, a metastatic cancer, an autoimmune disease, a nervous system disease, a neurodegenerative disease or an infectious disease.

[0148] In one aspect, the present disclosure provides a pharmaceutical composition further comprising a second therapeutic agent.

[0149] In one aspect, the present disclosure provides a method for preventing, ameliorating and / or treating a tumor, cancer, metastatic tumor, metastatic cancer, autoimmune disease, nervous system disease, neurodegenerative disease or infectious disease, comprising administering to an individual one or more selected from the group consisting of any PD-1 protein-binding agent, anti-PD-1 antibody or antigen-binding fragment thereof, immunoglobulin heavy chain variable region polypeptide, immunoglobulin light chain variable region polypeptide, immunoglobulin heavy chain polypeptide, immunoglobulin light chain polypeptide, and multispecific antigen-binding molecules comprising same, immunoconjugates, chimeric antigen receptors, engineered T cell receptors, or oncolytic viruses according to the present disclosure.

[0150] Any of the PD-1 protein-binding agents, anti-PD-1 antibodies or antigen-binding fragments thereof, immunoglobulin heavy chain variable region polypeptides, immunoglobulin light chain variable region polypeptides, immunoglobulin heavy chain polypeptides, immunoglobulin light chain polypeptides, or multispecific antigen-binding molecules comprising same, immunoconjugates, chimeric antigen receptors, engineered T cell receptors, or tumor-killing viruses according to the present disclosure are particularly useful for the treatment, prevention, and / or amelioration of any disease or disorder or condition that involves or is mediated by PD-1 expression, signaling, or activity, or that can be treated by blocking the interaction of PD-1 with a PD1 ligand (e.g., PD-L1 or PD-L2), or otherwise inhibiting PD-1 activity and / or signaling.

[0151] The pharmaceutical compositions according to the present disclosure are also for preventing, treating or ameliorating a condition associated with PD-1.

[0152] The PD-1 associated condition may also be, but is not limited to, a tumor, a cancer, a metastatic tumor, a metastatic cancer, an autoimmune disease, a neurological disease, a neurodegenerative disease, or an infectious disease.

[0153] The conditions associated with PD-1 include non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, kidney cancer, liver cancer, bone cancer, skin cancer, colon cancer, rectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric carcinoma, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic carcinoma, leukemia, lymphoma, myeloma, mycosis fungoides, Merkel cell carcinoma, classical Hodgkin lymphoma (CHL), primary mediastinal large B-cell lymphoma, T-cell / histiocyte-rich B-cell lymphoma, Epstein-Barr disease, and leukemia. Other hematological cancers including Barr virus (EBV) positive and EBV negative post-transplant lymphoproliferative disease (PTLD), EBV-associated persistent large B-cell lymphoma (DLBCL), plasmablastic lymphoma, extracellular NK / T-cell lymphoma, non-pharyngeal carcinoma, human herpesvirus 8 (HHV8)-associated primary effusion lymphoma, Hodgkin's lymphoma, and neoplasms of the central nervous system including, but not limited to, primary central nervous system (CNS) lymphoma, spinal tumor, or brain stem glioma.

[0154] The pharmaceutical compositions according to the present disclosure may be used to treat early stage or late stage symptoms of cancer. In one embodiment, the antibodies or fragments thereof of the present disclosure may be used to treat metastatic cancer. The pharmaceutical compositions according to the present disclosure are useful for reducing, inhibiting or shrinking tumor growth, both solid tumors and blood cancers. In certain embodiments, treatment with the pharmaceutical compositions according to the present disclosure causes a 50% or greater reduction, a 60% or greater reduction, a 70% or greater reduction, a 80% or greater reduction, or a 90% or greater reduction in tumors in a subject. In certain embodiments, the pharmaceutical compositions may be used to prevent tumor recurrence. In certain embodiments, the pharmaceutical compositions are useful for extending overall survival in subjects with cancer. In some embodiments, the pharmaceutical compositions are useful for reducing toxicity from chemotherapy or radiation therapy while maintaining long-term survival in patients suffering from cancer.

[0155] Examples of the autoimmune disease that is a condition associated with PD-1 include lupus, systemic lupus erythematosus, Sjogren's syndrome, arthritis, rheumatoid arthritis, asthma, chronic obstructive pulmonary disease (COPD), pelvic inflammatory disease, Alzheimer's disease, inflammatory bowel disease, Crohn's disease, ulcerative colitis, Peyronie's disease, coeliac disease, gallbladder disease, pilonidal disease, peritonitis, psoriasis, psoriatic arthritis, vasculitis, surgical adhesions, stroke, type 1 diabetes, Lyme disease, meningoencephalitis, autoimmune uveitis, multiple sclerosis, Guillain-Barre syndrome, and the like. These conditions include, but are not limited to, eczema, autoimmune hepatitis, fibrosing alveolitis, Grave's disease, IgA nephropathy, idiopathic thrombocytopenic purpura, Meniere's disease, pemphigus, primary biliary cirrhosis, sarcoidosis, scleroderma, Wegener's granulomatosis, other autoimmune diseases, pancreatitis, trauma (surgery), graft-versus-host disease, transplant rejection, cardiac disease including ischemic diseases such as myocardial infarction and atherosclerosis, intravascular coagulation, bone resorption, osteoporosis, osteoarthritis, periodontitis and hypochlorhydria, infertility related to lack of fetal-maternal tolerance, leukoplakia, myasthenia gravis, or systemic sclerosis.

[0156] It has been proposed that an IFN gamma-dependent systemic immune response is beneficial for the treatment of Alzheimer's disease and other central nervous system pathologies that share a neuroinflammatory component, and WO 2015 / 136541 discloses the use of anti-PD-1 antibodies to treat Alzheimer's disease. WO 2017 / 220990 describes that blocking the PD-1 / PD-L1 inhibitory immune checkpoint pathway enhances the secretion of IFN gamma by IFN gamma-producing cells, and that the increased IFN gamma activity allows the brain's choroid plexus to selectively traffic leukocytes, infiltrate T cells and monocytes into the damaged central nervous system, and allow immune cells to home to sites of neurodegenerative pathology and neuroinflammation, making the environment less harmful and better able to remove toxic substances and rescue, regenerate, and repair nerve cells.

[0157] PD-1 is known to be associated with cognitive function, learning and memory in the central nervous system, as well as with central nervous system disorders such as brain tumors, Alzheimer's disease, stroke, spinal cord injury, multiple sclerosis, glioblastoma, melanoma, and pain (Zunli Zao et al., "Emerging role of PD-1 in the central nervous system and brain diseases", Neurosci. Bull. 2021. 04. 20, online published). It has also been reported that PD-1 is associated with retinal ganglion cells, which are known to be degenerated in neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, multiple sclerosis, and amyotrophic lateral sclerosis (ALS) (Ling Chen et al., Role of the Immune Modulator Programmed Cell Death-1 during Development and Apoptosis of Mouse Retinal Ganglion Cells, Investigative Ophthalmology & Visual Science, 2009, Vol. 50, No. 10, 4941-4948).It is known that anti-PD-1 antibodies can improve cognitive impairment and pathological features in Alzheimer's disease model mice 5×FAD and dementia model mice, and can be used for neurodegenerative diseases (Michal Schwartz et al., "Potential immunotherapy for Alzheimer disease and age-related dementia", Dialogues in clinical neuroscience, 21 (1), 21, 2019), and the anti-PD-1 antibody nivolumab has been reported to improve learning and memory (Ru-Rong Ji et al., "Anti-PD-1 treatment as a neurotherapy to enhance neuronal excitability, synaptic plasticity and memory," BioRxiv, 2019. 12. 10. Htps: / / doi.org / 10.1101.870600).

[0158] In one aspect of the present disclosure, the pharmaceutical compositions according to the present disclosure may be used to prevent, ameliorate, or treat nervous system diseases and neurodegenerative diseases that are conditions associated with PD-1, such as, but not limited to, cognitive impairment, brain tumors, Alzheimer's disease, dementia, stroke, spinal cord injury, amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, multiple sclerosis, glioblastoma, melanoma, pain, and memory decline.

[0159] In one aspect of the present disclosure, pharmaceutical compositions according to the present disclosure are useful for treating subjects suffering from chronic viral infections. In some embodiments, the present disclosure is useful for reducing viral titers and / or restoring depleted T cells in a host.

[0160] The infectious disease condition associated with PD-1 may also be a chronic viral infection including hepatitis B or C viral infection, herpes virus, Epstein-Barr virus, HIV (human immunodeficiency virus), cytomegalovirus, herpes simplex virus type I, herpes simplex virus type II, human papilloma virus, adenovirus, Kaposi-West sarcoma associated with herpes virus infection, thin ring virus (Torque teno virus), lymphocytic choriomeningitis virus (LCMV), JC virus infection, or BK virus infection.

[0161] In one embodiment, the pharmaceutical compositions of the present disclosure may be used to treat infection with Simian Immunodeficiency Virus (SIV) in a simian subject, such as a cynomolgus.

[0162] In one embodiment, the pharmaceutical compositions according to the present disclosure may be administered to alleviate, prevent, or reduce the severity of one or more symptoms or conditions of a disease or disorder. Further contemplated herein is the prophylactic use of pharmaceutical compositions according to the present disclosure in patients at risk of developing a disease or disorder, such as cancer, autoimmune diseases, and chronic viral infections.

[0163] In yet another aspect of the present disclosure, the pharmaceutical compositions according to the present disclosure may be used in adjunctive therapy with any other formulation or any other therapy known to those of skill in the art useful for the treatment of cancer, autoimmune disease, or viral infection.

[0164] Pharmaceutical compositions according to the present disclosure may be administered with suitable carriers, excipients, and other agents included in a dosage form to provide improved delivery, transmission, tolerance, and the like. Numerous suitable dosage forms may be found in formularies known to any pharmaceutical chemist (see Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA). These dosage forms include, for example, powders, pastes, ointments, jellies, waxes, oils, liquids, liquid (cationic or anionic)-containing vesicles (e.g., Lipofectin, 1997), and the like. TM ), DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, emulsions carbowax (polyethylene glycol of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax (see Powell et al. "Compendium of excipients for parenteral formulations" PDA (1998) J Pharm Sci Technol 52: 238-311).

[0165] The dose of the antibody is also variable depending on the age and size of the subject to be administered, the target disease, the condition, the route of administration, etc. When the antibody of the present disclosure is used to treat a disease or disorder or to prevent such a disease in an adult patient, it is generally advantageous to administer the antibody of the present disclosure at a single dose of about 0.1 to about 60 mg per kg of body weight, more preferably about 5 to about 60 mg, about 10 to about 50 mg, or about 20 to about 50 mg per kg of body weight. The frequency and duration of treatment can be adjusted depending on the severity of the condition. In certain embodiments, the antibody or antigen-binding fragment of the present disclosure can be administered at an initial dose of at least about 0.1 mg to about 800 mg, about 1 to about 500 mg, about 5 to about 300 mg, or about 10 to about 200 mg, or about 10 to about 100 mg, or about 10 to about 50 mg. In certain embodiments, the initial dose also leads to the administration of a second dose or multiple subsequent doses of the antibody or antigen-binding fragment thereof that are approximately the same as or less than the initial dose, where said subsequent doses are separated by at least 1 to 3 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 12 weeks, or at least 14 weeks.

[0166] The pharmaceutical compositions of the present disclosure may be administered through a variety of delivery systems, such as liposomal encapsulation, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, receptor-mediated endocytosis (see, e.g., Wu et al. (1987) J. Biol. Chem. 262: 4429-4432). Methods of introduction include, but are not limited to, intradermal, transdermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compositions may also be administered by any common route, such as by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and may be administered together with other biologically active agents. The administration may be systemic or local.

[0167] The pharmaceutical compositions of the present disclosure can also be delivered to vesicles, particularly liposomes (see, for example, Langer (1990) Science 249: 1527-1533). The use of nanoparticles delivering the antibodies of the present disclosure can also be considered. Antibody-conjugated nanoparticles are used for both therapeutic and diagnostic applications. Antibody-conjugated nanoparticles, their preparation methods, and their applications are described in detail in Arruebo, M., et al. 2009, "Antibody-conjugated nanoparticles for biomedical applications" in J. Nanomat. Volume 2009, Article ID 439389, 24 pages, doi: 10.1155 / 2009 / 439389, which is incorporated herein by reference. Nanoparticles can be developed and conjugated to antibodies contained in the pharmaceutical composition to target tumor cells, autoimmune tissue cells, or virus-infected cells. Nanoparticles for drug delivery are also described, for example, in US Pat. No. 8,257,740 or US Pat. No. 8,246,995, each of which is incorporated herein in its entirety.

[0168] In certain circumstances, the pharmaceutical compositions of the present disclosure may be delivered in a controlled release system. In one embodiment, a pump may be used. In another embodiment, a polymeric material may be used. In yet another embodiment, the controlled release system may be placed in close proximity to the target of the composition, requiring only a portion of the systemic dose. The injectable formulations also include dosage forms for intravenous, subcutaneous, intradermal, intracranial, intraperitoneal and intramuscular injections, drip infusions, and the like. These injectable formulations may be prepared by methods formally known in the art. The injectable formulations may be prepared, for example, by dissolving, suspending or emulsifying the antibody or its salt in a sterile aqueous medium or an oily medium commonly used for injections. As the aqueous medium for injection, for example, physiological saline, isotonic solution containing glucose, and other auxiliary agents can be used together with a suitable solubilizing agent, for example, alcohol (e.g., ethanol), polyhydric alcohol (e.g., propylene glycol, polyethylene glycol), nonionic surfactant (e.g., polysorbate 80, hydrogenated castor oil HCO-50 (polyoxyethylene (50 mol) additive) and the like. As the oily medium, for example, sesame oil, soybean oil and the like can be used together with a solubilizing agent, for example, benzyl benzoate, benzyl alcohol and the like. The injection solution prepared thereby is preferably filled into a suitable ampoule.

[0169] The pharmaceutical compositions of the present disclosure may be delivered subcutaneously or intravenously with a standard needle and syringe. For subcutaneous delivery, a pen delivery device may also be conveniently used to deliver the pharmaceutical compositions of the present disclosure. Such pen delivery devices may be reusable or single use. Reusable pen delivery devices generally utilize a replaceable cartridge containing the pharmaceutical composition. Once all of the pharmaceutical composition in the cartridge has been administered and the cartridge is empty, the empty cartridge may be conveniently discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen delivery device may then be reused. In single use pen delivery devices, there is no replaceable cartridge. Instead, single use pen delivery devices are pre-filled with the pharmaceutical composition contained in a reservoir within the device. Once the reservoir is depleted of pharmaceutical composition, the entire device is discarded. A number of reusable pen and autoinjector delivery devices are used for subcutaneous delivery of the pharmaceutical compositions of the present disclosure. For example, the AUTOPEN TM , Owen Mumford, Inc., Woodstock, UK), DISETRONIC TM ) Pen (Disetronic Medical Systems, Burghdorf, Switzerland), HUMALOG MIX 75 / 25 TM Pen, HUMALOG TM ) Pen, HUMALIN 70 / 30 TM Eli Lilly and Co., Indianapolis, IN, NOVOPEN TM ) I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR TM )(Novo Nordisk, Copenhagen, Denmark), BD TM Becton Dickinson, Franklin Lakes, NJ, OPTIPEN TM), OPTIPEN PRO TM ), OPTIPEN STARLET TM ) and OPTICLICL TM ) (Sanofi-Aventis, Frankfurt, Germany). Single-use pen delivery devices that may be used for subcutaneous delivery of the pharmaceutical compositions of the present disclosure include, but are not necessarily limited to, SOLOSTAR TM ) Pen (Sanofi-Aventis), FlexPen (FLEXPEN TM )(Novo Nordisk), KWIKPEN TM )(Eli Lilly), SURECLICK TM ) autoinjector (Amgen, Thousand Oaks, CA), PENLET TM ) (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, LP) and HUMIRA TM ) pens (Abbott Labs, Abbott Park, IL), but are not necessarily limited to these.

[0170] Conveniently, the aforementioned pharmaceutical compositions for oral or parenteral use are prepared in a unit dose dosage form suitable for fitting a dose of the active ingredient. Such unit dose dosage forms include, for example, tablets, pills, capsules, injection solutions (ampoules), suppositories, etc. The amount of antibody contained is generally about 5 to about 500 mg per unit dose, particularly for injection dosage forms, and desirably about 5 to about 100 mg of antibody is contained, and about 10 to about 250 mg for other dosage forms.

[0171] The present disclosure also provides, in one aspect, a method of modulating an immune response in an individual comprising administering to the individual one or more selected from the group consisting of any PD-1 protein-binding agent, anti-PD-1 antibody or antigen-binding fragment thereof, immunoglobulin heavy chain variable region polypeptide, immunoglobulin light chain variable region polypeptide, immunoglobulin heavy chain polypeptide, immunoglobulin light chain polypeptide, and multispecific antigen-binding molecules comprising same, immunoconjugates, chimeric antigen receptors, engineered T cell receptors, or tumor-killing viruses according to the present disclosure.

[0172] In one aspect, the present disclosure provides the use of any PD-1 protein-binding agent, anti-PD-1 antibody or antigen-binding fragment thereof, immunoglobulin heavy chain variable region polypeptide, immunoglobulin light chain variable region polypeptide, immunoglobulin heavy chain polypeptide, immunoglobulin light chain polypeptide, or multispecific antigen-binding molecule comprising same, immunoconjugate, chimeric antigen receptor, engineered T cell receptor or tumor-killing virus according to the present disclosure in the manufacture of an medicament for the prevention, amelioration or treatment of a tumor, cancer, metastatic tumor, metastatic cancer, autoimmune disease, nervous system disease, neurodegenerative disease or infectious disease.

[0173] The present disclosure also provides, in one aspect, a method of inhibiting tumor cell growth in an individual comprising administering to the individual a therapeutically effective amount of one or more selected from the group consisting of any PD-1 protein-binding agent, anti-PD-1 antibody or antigen-binding fragment thereof, immunoglobulin heavy chain variable region polypeptide, immunoglobulin light chain variable region polypeptide, immunoglobulin heavy chain polypeptide, immunoglobulin light chain polypeptide, and multispecific antigen-binding molecules comprising same, immunoconjugates, chimeric antigen receptors, engineered T cell receptors, or tumor-killing viruses according to the present disclosure to inhibit tumor cell growth.

[0174] Coadministration In one aspect of the present disclosure, a pharmaceutical composition according to the present disclosure is provided which further comprises a second therapeutic agent.

[0175] In various embodiments, the second therapeutic agent includes an antibody against PD-L1, a second antibody against PD-1 (e.g., nivolumab), a LAG-3 inhibitor, a CTLA-4 inhibitor (e.g., iprimumab), a TIM3 inhibitor, a BTLA inhibitor, a TIGIT inhibitor, a CD47 inhibitor, an antagonist of another T cell co-inhibitor or ligand (e.g., an antibody against CD-28, 2B4, LY108, LAIR1, ICOS, CD160, or VISTA), an indoleamine-2,3-deoxygenase (IDO) inhibitor, a vascular endothelial growth factor (VEGF) antagonist [e.g., "VEGF-Trap," see, e.g., U.S. Pat. No. 7,087,411, or other VEGF-inhibitory fusion proteins, anti-VEGF antibodies or antigen-binding fragments thereof (e.g., bevacizumab or ranibizumab), or small molecule kinase inhibitors of VEGF receptors (e.g., sunitinib, sorafenib, or pazopanib)], Ang2 inhibitors (e.g., nesvacumab), transforming growth factor beta (TGFβ) inhibitors, epidermal growth factor receptor (EGFR) inhibitors (e.g., erlotinib, cetuximab), agents against costimulatory receptors (e.g., agents against glucocorticoid-induced TNFR-associated protein), antibodies against tumor-specific antigens (e.g., CA9, CA125, melanoma-associated antigen 3 (MAGE3), carcinoembryonic antigen (CEA), vimentin, tumor-M2-PK, prostate-specific antigen (PSA), mucin-1, MART-1, and CA19-9), vaccines (e.g., BCG (Bacillus Calmette-Guerin, cancer vaccines), adjuvants to enhance antigen delivery (e.g., granulocyte-macrophage colony-stimulating factor), bispecific antibodies (e.g., CD3xCD20 bispecific antibody, PSMAxCD3 bispecific antibody), cytotoxins, chemotherapeutic agents (e.g., dacarbazine, temozolomide, cyclophosphamide, docetaxel, doxorubicin, daunorubicin, cisplatin, carboplatin, gemcitabine, methotrexate, mitoxantrone, oxaliplatin, paclitaxel, and vincristine), cyclophosphamide, radiation therapy, IL-6R inhibitors (e.g., sarilumab), IL-4R inhibitors (e.g., dupilumab), IL-10 inhibitors, cytokines (e.g., IL-2, IL-7, IL-21, and IL-15), antibody-drug conjugates (ADCs) (e.g., anti-CD19-DM4 ADC and anti-DS6-DM4 ADC). ADCs), anti-inflammatory drugs (e.g., corticosteroids and nonsteroidal anti-inflammatory drugs), dietary supplements (e.g., antioxidants, or any palliative care for cancer treatment).

[0176] In certain embodiments, the second therapeutic agent is also a cancer vaccine, including dendritic cell vaccines, oncolytic viruses, tumor cell vaccines, etc. to increase anti-tumor responses. Examples of cancer vaccines include MAGE3 vaccines for melanoma and bladder cancer, MUC1 vaccines for breast cancer, EGFRv3 (e.g., rindopepimut) for brain cancers (including glioblastoma multiforme) or ALVAC-CEA (for CEA+ cancers).

[0177] Methods and kits for diagnosis and detection In one aspect, the present disclosure provides a kit for the treatment, diagnosis, or detection of a disease comprising one or more selected from the group consisting of any PD-1 protein-binding agent, anti-PD-1 antibody or antigen-binding fragment thereof, immunoglobulin heavy chain variable region polypeptide, immunoglobulin light chain variable region polypeptide, immunoglobulin heavy chain polypeptide, immunoglobulin light chain polypeptide, and multispecific antigen-binding molecules, immunoconjugates, chimeric antigen receptors, engineered T cell receptors, or tumor-killing viruses comprising same according to the present disclosure.

[0178] The anti-PD-1 antibodies of the present disclosure can be used to detect and / or measure PD-1 from a sample, e.g., for diagnostic purposes. Some embodiments contemplate the use of one or more antibodies of the present disclosure in assays to detect a disease or disorder, such as cancer, an autoimmune disease, or a chronic viral infection. An exemplary diagnostic assay for PD-1 includes, for example, contacting a sample obtained from a patient with an anti-PD-1 antibody of the present disclosure, where the anti-PD-1 antibody is labeled with a detectable label or reporter molecule or is used as a capture ligand to selectively isolate PD-1 from the patient sample.

[0179] Alternatively, unlabeled anti-PD-1 antibodies may be used for diagnostic purposes in conjunction with a secondary antibody that is itself detectably labeled. The detectable label or reporter molecule may be a radioisotope, e.g., 3H, 14C, 32P, 35S, or 125I; a fluorescent or chemiluminescent moiety, such as fluorescein isothiocyanate or rhodamine; or an enzyme, such as alkaline phosphatase, β-galactosidase, horseradish peroxidase, or luciferase.

[0180] Specific exemplary assays that can be used to detect or measure PD-1 in a sample include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence-activated cell sorting (FACS).

[0181] Samples that may be used in PD-1 diagnostic assays according to the present disclosure include any tissue or fluid sample that may be obtained from a patient that contains a detectable amount of PD-1 protein or fragments thereof under normal or pathological conditions. Generally, PD-1 levels are measured in a particular sample obtained from a healthy patient (e.g., a patient not suffering from cancer or an autoimmune disease) to initially establish a baseline or standard level of PD-1. Such baseline levels of PD-1 can then be compared to levels of PD-1 measured from samples obtained from individuals suspected of having a cancer-related condition or symptoms associated with such a condition.

[0182] The polypeptides, PD-1 binding agents, antibodies, etc. according to the present disclosure specific for PD-1 may contain no additional labels or moieties, or may contain N-terminal or C-terminal labels or moieties. In one embodiment, the label or moiety is biotin. In binding assays, the location of the label (if present) can determine the orientation of the peptide relative to the surface to which it is bound. For example, if a surface is coated with avidin, a peptide containing an N-terminal biotin will be oriented such that the C-terminal portion of the peptide is located away from the surface.

[0183] One aspect of the present disclosure relates to the use of the disclosed antibodies as markers for predicting the prognosis of cancer or an autoimmune disorder in a patient. Polypeptides, PD-1-binding agents, antibodies, etc., according to the present disclosure can be used in diagnostic assays to assess cancer prognosis and predict survival in a patient.

[0184] The following provides a more detailed explanation of antibody production methods according to specific embodiments of the invention, but these are presented as examples of the invention and do not limit the scope of the invention. It will be obvious to those skilled in the art that various modifications of the embodiments are possible within the scope of the invention.

[0185] Working Example Example 1. Preparation of immunological center and establishment of cell lines Protein antigens The extracellular domain (ECD) of human PD-1 was synthesized by polymerase chain reaction (PCR) from pCMV3-C-FLAG vector (Sino) containing cDNA and inserted into pEM.CMV-SF-IRES-EGFP vector. The constructed vector was transfected into CHOS cell line, and cells expressing human PD-1 protein extracellular domain were selected by flow cytometry based on EGFP expression. Human PD-1 protein was purified and quantified in the cell culture medium using FLAG tag affinity chromatography method.

[0186] cell line antigen The human PD-1 cDNA-containing pCMV3-C-FLAG vector (Sino) was transfected into CT26 cell line derived from colon cancer in BALB / c mice. Human PD-1 expressing cells were selected by flow cytometry using APC-cy7 anti-human PD-1 antibody. Single clones were obtained by limiting dilution in 96-well plates.

[0187] Acquisition of control arm (Keytruda, Opdivo) The control antibody, Keytruda (human IgG4 (S228P) isotype, cat. No. hpd1pe-mab14), was purchased from InvivoGen or clinical products from MSD. Opdivo was purchased from BMS clinical products.

[0188] Establishment of cell lines CHOS cell lines were transfected with pCMV3-C-FLAG vector (Sino) containing human PD-1 or mouse PD-1 cDNA. Cells expressing human PD-1 or mouse PD-1 were selected by flow cytometry using APC-cy7 anti-human PD-1 antibody or APC-cy7 anti-mouse PD-1 antibody. Single clones were obtained by limiting dilution in 96-well plates.

[0189] Example 2. Generation of PD-1 knockout mice PD-1 knockout mice were generated from mice (C57BL / 6N) using the CRISPR-CAS system. Guide RNA specific to the mouse PD-1 gene sequence was generated, and after confirming whether it could degrade mouse PD-1 DNA in vitro, the guide RNA and Cas9 protein were microinjected into zygotes. Surviving zygotes injected with the guide RNA were selected and transplanted into surrogate oviducts. After transplantation, the tails of mice born about two weeks after transplantation were cut, genomic DNA was extracted, and the deletion of the mouse PD-1 gene was confirmed by PCR.

[0190] Example 3. Generation of antibody hybridomas Immunization of mice Eight-week-old female PD-1 knockout mice were immunized with human PD-1 antigens to induce antibody production, using purified human PD-1 protein antigens and CT26-derived human PD-1 cell line antigens.

[0191] For protein antigen immunization, 50 μg of antigen protein per mouse was mixed with 50 μg of TiterMax Gold adjuvant and injected subcutaneously (sc) into the left and right dorsal sites of the mice. For cell line antigen immunization, the cell line antigen was irradiated with X-rays one day before immunization to suppress cell growth, and 1x10 6Cells were administered intraperitoneally (ip). Immunizations were performed at 3-week intervals, and 10 days after immunization, blood was collected from each mouse by submandibular bleeding. Antibody titers produced using serum isolated from the blood were measured by ELISA.

[0192] Cell fusion and generation of single clone cells (hybridomas) Spleens were removed from mice immunized with human PD-1, and B lymphocytes were isolated and fused with cultured myeloma cells (sp2 / 0). The fused cells were cultured in a medium containing hypoxanthine, aminopterin, and thymidine (HAT medium), and hybridomas in which only myeloma and B lymphocytes were fused were selectively selected and cultured.

[0193] Among the obtained hybridoma cells, hybridomas producing antibodies that react with the human PD-1 antigen were identified by performing protein-based ELISA analysis. The hybridomas reacting with the human PD-1 antigen were repeatedly cloned using the limiting dilution method to produce a single clone cell (hybridoma) (1G1) that produces antibodies that react with the human PD-1 antigen.

[0194] Example 4. Binding test of hybridoma 1G1 to PD-1 To confirm the binding between the single clone cell (hybridoma) (1G1) prepared in Example 3 and the constitutive PD-1 protein expressed on the cell surface, cell-based ELISA and flow cytometry were performed. Keytruda (Invivogen) was used as the anti-hPD-1 antibody control.

[0195] Briefly, 10,000 CHO-S cells expressing human PD-1 or mouse PD-1 were coated on collagen-coated 96-well plates (Thermo Fisher) overnight at 37°C. The coated cells were fixed with 8% paraformaldehyde for 15 min at room temperature. After blocking and washing, the Keytruda culture supernatant or hybridoma culture supernatant was added to the coated plates and incubated at room temperature for 2 h. After washing, the secondary antibody was added and incubated overnight at 4°C. The secondary antibody used was mouse anti-human IgG Fc HRP (GenScript) in the Keytruda-added wells and goat anti-mouse IgG Fc HRP (Thermo Fisher) in the hybridoma supernatant-added wells. After washing, TMB substrate (Abcam) was added and the color reaction was stopped with STOP solution (Abcam). The absorbance at 450 / 650 nm was confirmed using a microplate reader (Thermo Fisher).

[0196] Briefly, for flow cytometry, CHO-S cells expressing human PD-1 or mouse PD-1 were cultured at 1 × 10 6 Cells were loaded at a density of 1000 cells / well into a 96-well V-bottom plate (Corning), and Keytruda culture supernatant or hybridoma culture supernatant was added and incubated at 4°C for 1 hour. After washing with 1x PBS / 2% BSA, secondary antibodies were added and incubated with the cells for 1 hour at 4°C. The secondary antibodies used were PE anti-human IgGFc (Biolegend) for Keytruda-added wells and AF647 goat anti-mouse IgG (H+L) (Thermo Fisher) for hybridoma culture supernatant-added wells. The cells were then washed, resuspended in 1x PBS / 2% BSA, and analyzed using a flow cytometer (BD) and FlowJo software.

[0197] Figure 1 is a graph showing the results of a binding test to human PD-1 or mouse PD-1 on the cell surface of the hybridoma antibody 1G1 produced in Example 3 using ELISA. Figure 2 is a graph showing the results of a binding test to human PD-1 or mouse PD-1 on the cell surface of the hybridoma antibody produced in Example 3 using flow cytometry. The 1G1 antibody bound to human PD-1 and mouse PD-1 with high affinity.

[0198] In addition, KEYTRUDA, which was used as a control, bound only to cell surface human PD-1, while the 1G1 hybridoma antibody produced in Example 3 bound to both cell surface human PD-1 and mouse PD-1, demonstrating cross-reactivity.

[0199] Example 5. Ligand blocking test of hybridoma 1G1 antibody To verify whether the single clone cell (hybridoma) 1G1 prepared in Example 3 blocks the binding of PD-L1 to cell surface PD-1, cell-based ELISA and flow cytometry were performed. Keytruda (Invivogen) was used as the anti-hPD-1 antibody control.

[0200] Briefly, for the ELISA analysis, 10,000 CHO-S cells expressing human PD-1 or mouse PD-1 were coated on collagen-coated 96-well plates overnight at 37℃. The coated cells were fixed with 8% paraformaldehyde for 15 minutes at room temperature. After blocking and washing, human PD-L1 llama Fc protein was added to the human PD-1 CHO-S cell-coated wells, and mouse PD-L1 human Fc protein was added to the mouse PD-1 CHO-S cell-coated wells and incubated for 20 minutes at room temperature. Keytruda culture supernatant or hybridoma culture supernatant was added and incubated for 1 hour and 20 minutes at room temperature.

[0201] After washing, secondary antibodies were added and incubated overnight at 4°C. Mouse anti-llama IgG2 / IgG3 HRP (Antibody onlines) was used for the wells containing human PD-L1 llama Fc protein, and mouse anti-human IgG Fc HRP (Genscript) was used for the wells containing mouse PD-L1 human Fc protein. After washing, TMB substrate (Abcam) was added and the color reaction was stopped with STOP solution (Abcam). The absorbance at 450 / 650 nm was confirmed using a microplate reader (Thermo Fisher).

[0202] Briefly, for flow cytometry, CHO-S cells expressing human PD-1 or mouse PD-1 were cultured at 1x10 6 Cells were loaded at a density of 100 cells / well into a 96-well V-bottom plate (Corning), and a mixture of PD-L1 protein and Keytruda culture supernatant or hybridoma culture supernatant was added and incubated at 4℃ for 1 hour. Human PD-1 CHO-S cell loading wells were added with human PD-L1 llama Fc protein and antibody mixture, and mouse PD-1 CHO-S cell loading wells were added with mouse PD-L1 human Fc protein and antibody mixture. After washing with 1× PBS / 2% BSA, secondary antibody was added and incubated at 4℃ for 1 hour.

[0203] The secondary antibodies used were FITC goat anti-llama IgG (Abcam) for wells containing human PD-L1 llama Fc protein and antibody mixture, and PE anti-human IgGFc (Biolegend) for wells containing mouse PD-L1 human Fc protein and antibody mixture. Cells were washed, resuspended in 1× PBS / 2% BSA, and analyzed using a flow cytometer (BD) and FlowJo software.

[0204] Figure 3 shows the graphical results of a blocking test of the single clone cell (hybridoma) 1G1 antibody produced in Example 3 for human PD-L1 binding or mouse PD-L1 binding to cell surface human PD-1 or mouse PD-1, respectively, using ELISA.

[0205] FIG. 4 shows the results of a blocking test of the single clone cell (hybridoma) 1G1 antibody prepared in Example 3 for human PD-L1 binding or mouse PD-L1 binding to cell surface human PD-1 or mouse PD-1, respectively, using flow cytometry.

[0206] The control drug, Keytruda, only blocked the binding between cell surface human PD-1 and PD-L1, while the 1G1 hybridoma antibody blocked the binding of both human PD-1 / PD-L1 and mouse PD-1 / PD-L1.

[0207] Example 6. Binding test of purified hybridoma 1G1 antibody to PD-1 (ELISA) To produce antibodies in the hybridomas, the hybridomas were cultured in DMEM (Dulbecco Modified Eagle Medium) (HyClone) (Cytiva) medium containing 3% low-IgG FBS (Gibco) for one week, centrifuged, and then filtered using a 0.22 μm filter (Millipore) to collect the cell culture medium.

[0208] Antibody proteins were isolated from hybridoma cell culture medium using affinity chromatography. The hybridoma cell culture medium was loaded onto a chromatography column (Bio Rad) containing Protein G beads (Cytiva), and eluted with IgG Elution Buffer (Thermo Scientific). To minimize damage to the protein caused by the low pH of the IgG Elution Buffer (pH 2.5-3.0), the antibodies were eluted into a tube containing 1M Tris-HCL solution (pH 8.0), and the pH of the neutralized protein was measured using pH indicator strips.

[0209] The purified antibodies were concentrated using an Amicon 100K centrifugal filter (Merck), and the proteins were confirmed and quantified via Coomassie blue staining and the BCA assay (Thermo Scientific) (Figure 5).

[0210] To confirm the binding affinity of purified antibody 1G1 to normal PD-1 protein expressed on the cell surface, a cell-based ELISA was performed.Keytruda (MSD) and Opdivo (BMS) were used as anti-hPD-1 antibody controls.

[0211] Briefly, for ELISA analysis, 10,000 CHO-S cells expressing human PD-1 or mouse PD-1 were coated onto collagen-coated 96-well plates (Thermo Scientific) overnight in a 37°C incubator. The coated cells were fixed with 8% paraformaldehyde for 15 min at room temperature. After blocking and washing, Keytruda, Opdivo, and purified hybridoma anti-PD-1 antibodies were added to the coated plates and incubated at room temperature for 2 h.

[0212] After washing, secondary antibodies were added and incubated overnight at 4°C. Mouse anti-human IgG Fc HRP (GenScript) was used for the wells containing Keytruda or Opdivo, and goat anti-mouse IgG Fc HRP (Thermo Fisher) was used for the wells containing purified hybridoma anti-PD-1 antibody. After washing, TMB substrate (Abcam) was added and the color reaction was stopped with STOP solution (Abcam). The absorbance at 450 / 650 nm was confirmed using a microplate reader (Thermo Fisher).

[0213] Figure 6 shows the results of a binding test of purified hybridoma 1G1 antibody to human PD-1 or mouse PD-1 on the cell surface using ELISA. The experimental results show that the EC of purified mouse 1G1 antibody and the control group (Keytruda, Opdivo) against the antigens. 50 was calculated.

[0214] The purified hybridoma anti-PD-1 antibody (1G1) showed approximately 5-fold improved binding to human PD-1 antigen (28.13 pM) compared to Keytruda (153.52 pM) and Opdivo (157.27 pM).In addition, it was confirmed that the purified hybridoma anti-PD-1 antibody (1G1) also showed similar binding to mouse PD-1 antigen (31.72 pM) as to human PD-1 antigen.

[0215] [Table 1] ND: not detected

[0216] Example 7. Cross-reactivity to human immune checkpoints To confirm whether the single clone cell (hybridoma) 1G1 prepared in Example 3 specifically binds to PD-1 present on the surface of human T cells, a cross-reactivity test against other immune checkpoints was performed using a protein-based ELISA assay. Keytruda (Invivogen) was used as the anti-hPD-1 antibody control group.

[0217] Human PD-1, CD28, CTLA-4, ICOS or BTLA protein was coated on 96-well plates (Nunc) overnight at 4°C. After blocking and washing, Keytruda culture supernatant or hybridoma culture supernatant was added to the coated plate and incubated at 37°C for 1 hour. After washing, secondary antibodies were added and incubated at 37°C for 2 hours. The secondary antibodies used were mouse anti-human IgG Fc HRP (Genscript) for Keytruda-added wells and goat anti-mouse IgG Fc HRP (Thermo Fisher) for hybridoma culture supernatant-added wells. After washing, TMB substrate (Abcam) was added and the color reaction was stopped with STOP solution (Abcam). The absorbance at 450 / 650 nm was confirmed using a microplate reader (Thermo Fisher).

[0218] Figure 7 shows the results of an ELISA test to determine whether the 1G1 hybridoma antibody binds to human T cell surface immune checkpoints. Similar to the control group, the 1G1 hybridoma antibody specifically bound only to human PD-1.

[0219] Example 8. Antibody Hybridoma Cell Sequencing RNA was extracted with Trizol reagent from the 1G1 hybridoma cells prepared in Example 3. cDNA was synthesized from the RNA using reverse transcriptase, and the VH and VL sequences of the antibody were amplified from the synthesized cDNA as follows.

[0220] The primers for amplifying the sequence are as follows:

[0221] [Table 2]

[0222] The PCR reaction was carried out as follows.

[0223] [Table 3]

[0224] The resulting PCR product (10 μL) was ligated with pCMV3 vector, and the VH and VL sequences of the hybridoma antibody were confirmed by sequence analysis using T7 (5'-TAATACGACTCACTATAGGG-3') and pCMV3_F (5'-CGAGGAGGATTTGATATTCAC-3') primers.

[0225] The confirmed VH and VL sequences are as follows, and the FR1-4 and CDR1-3 sequences of both VH and VL were confirmed by the Kabat system.

[0226] [Table 4]

[0227] Example 9. Generation, expression and purification of chimeric antibodies A chimeric antibody was prepared by replacing the constant region of a mouse anti-PD-1 antibody produced by a hybridoma with the constant region of a human antibody. The signal peptide and the VH or VL sequence of the antibody were linked by overlapping PCR, and ligated with the pTRIOZ-hIgG4 vector (Invivogen) expressing the human IgG4 constant region (S228P) (SEQ ID NOs: 38 to 41).

[0228] The pTRIOZ-hIgG4 vector containing the VH and VL sequences of the antibody was TM expression The antibody was expressed in the ExpiCHO-S cell line by transfection with the pTRIOZ-hIgG4 vector. TM The cells were cultured in Expression Medium and observed for cell viability. After 7-10 days after transfection, the cells with a viability of 70% or more were centrifuged and filtered through a 0.22 μm filter (Millipore) to collect the cell culture medium. The cell culture medium was loaded onto a chromatography column (Bio Rad) containing Protein A beads (Thermo Scientific) and eluted with IgG Elution Buffer (Thermo Scientific). To minimize damage to the protein due to the low pH of the IgG Elution Buffer (pH 2.5-3.0), the antibody was eluted into a tube containing 1M Tris-HCL solution (pH 8.0), and the pH of the neutralized protein was measured using a pH indicator strip. The purified antibodies were concentrated using an Amicon 50K centrifugal filter (Merck), and the proteins were confirmed and quantified via Coomassie blue staining and BCA assay (Thermo Scientific). Figure 5 shows the results of SDS-PAGE confirming the purified mouse 1G1 antibody (1G1 parental) and chimeric 1G1 antibody (Chimeric 1G1).

[0229] Example 10. Binding test of chimeric 1G1 antibody to PD-1 (SPR) The binding kinetics of the Chimeric 1G1 antibody prepared in Example 9 to human PD-1 was measured by SPR (surface plasma resonance) analysis using Biacore 8K (Cytiva). Anti-human IgG antibody was immobilized on a CM5 chip (Cytiva) via amine coupling. Purified antibodies (Keytruda, Opdivo, Chimeric 1G1 antibody) were flowed over the sensor chip and captured by anti-human IgG antibody. Human PD-1 and running buffer at concentrations of 0 to 100 nM (0, 6.25, 12.5, 25, 50, 100 nM) were flowed over the sensor chip at a flow rate of 30 μl / min for 120 s of the association phase, followed by dissociation for 900 s. The chip was regenerated with glycine at pH 1.5 after each experiment. Binding and dissociation curves were plotted using Cytiva evaluation software to determine kinetics and affinity values.

[0230] The table below shows the results of the binding affinity of the chimeric 1G1 antibody to human PD-1, as tested by SPR analysis.

[0231] [Table 5]

[0232] Chimeric anti-PD-1 antibody (1G1) binds human PD-1 with the same activity as Keytruda (6.33e -9 ) and Opdivo (1.05e -8 ) and has a bond strength (KD) of 3.35e -9 ) that the 1G1 chimeric anti-PD-1 antibody had a binding (K on ) of 2.23e +5 ) in terms of Keytruda (4.96e +5 ) and Opdivo (2.04e +5 ), but the dissociation (Koff) is similar (0.9-2.2 times) to -4 In terms of , Keytruda (3.14e -3) and Opdivo (2.14e -3 ) This is because it has improved binding properties with 2.9 to 4.2 times less dissociation compared to the control.

[0233] Example 11. Epitope mapping of 1G1 antibody Epitope mapping (alanine scanning) of the 1G1-chimeric (hIgG4(S228P)) antibody on human PD-1 antigen (SEQ ID NO: 62) was performed to confirm the epitope of the human PD-1 antigen recognized by the antibody. Amino acid residues of PD-1 were mutated to alanine using PCR mutagenesis. The mutated proteins were expressed and analyzed for binding to the 1G1 chimeric antibody by high-throughput flow cytometry.

[0234] As a result, three epitopes (P130, L128, and I126) were identified whose binding affinity to the mutant antigen was reduced to 50% or less compared to the binding affinity to the wild-type antigen. All of these amino acids are also conserved in mouse PD-1, supporting the phenomenon that the 1G1 antibody has cross-reactivity to human PD-1 and mouse PD-1.

[0235] Example 12. Humanization of 1G1 antibody To eliminate the immunogenicity of mouse-derived 1G1 antibody and ensure stable antibody efficacy in the human body, we used the back mutation library method to humanize the 1G1 antibody. Three types of 1G1 humanized antibodies (humanized antibodies 1G1-h61, 1G1-h68, and 1G1-h70) were produced by replacing the framework (FR) sequence, excluding the complementarity determining region (CDR) sequence, with human antibody sequences, and purified by protein A affinity chromatography.

[0236] The binding affinity of the three 1G1 humanized antibodies (humanized antibodies 1G1-h61, 1G1-h68, and 1G1-h70) to human PD-1 antigen was analyzed by surface plasma resonance (SPR) analysis using Biacore 8K (Cytiva). All three humanized 1G1 antibodies were confirmed to have antigen binding strengths similar to those of the chimeric 1G1 antibody.

[0237] The sequences of each of the three humanized 1G1 antibodies obtained were analyzed and are illustrated in Figures 8 to 11. Specifically, the heavy chain variable region (VH) and light chain variable region (VL) of humanized antibody 1G1-h61 have the amino acid sequences of SEQ ID NOs: 54 and 55, respectively. The heavy chain variable region (VH) and light chain variable region (VL) of humanized antibody 1G1-h68 have the amino acid sequences of SEQ ID NOs: 56 and 57, respectively. The heavy chain variable region (VH) and light chain variable region (VL) of humanized antibody 1G1-h70 have the amino acid sequences of SEQ ID NOs: 58 and 59, respectively.

[0238] Example 13. Comparative evaluation of binding kinetics of humanized 1G1 antibody to PD-1 antigen The binding kinetics of each antibody to human PD-1 was measured using surface plasma resonance (SPR) analysis with Biacore 8K (Cytiva), and the binding strength of the antibodies to the antigen was compared.

[0239] The anti-human PD-1 antibody controls, Keytruda (MSD (Lot #T020031)) and Opdivo (BMS (Lot #043FB)), were purchased from Shinwon Pharmacy Co., Ltd. as human pharmaceuticals.

[0240] Control antibodies (Keytruda, Opdivo) and 1G1 antibodies (chimeric antibody 1G1-chimeric, humanized antibodies 1G1-h61, 1G1-h68, 1G1-h70) were flowed and captured on a Protein A chip (Cytiva). Seven concentrations of human PD-1 (0-100M) were flowed over the sensor chip at a flow rate of 30 μL / min for 120 s of the association phase, followed by 1,800 s of dissociation. The chip was regenerated with glycine at pH 1.5 after each experiment. Binding and dissociation curves were plotted using Cytiva evaluation software to measure kinetics and affinity values. The significance of the antibody kinetics and affinity values ​​was determined using one-way ANOVA with Tukey test in the GraphPad Prism program (****, P<0.0001).

[0241] Figures 12A and 12B show the binding kinetics and affinity for human PD-1 through K (ka), K (kd), and KD values. In conclusion, the humanized anti-PD-1 antibody (1G1) binds, on average, to human PD-1 in a manner similar to that of Keytruda (7.06e -9 ) and Opdivo (7.54e -9 ) and similar levels of binding strength (KD) (7.26e -9 It was confirmed that the 1G1 humanized anti-PD-1 antibody has a binding (K on ) of 5.45e +4 In terms of , Keytruda (4.18e +5 ) and Opdivo (1.41e +5 ) compared to , it binds 2.6 to 7.7 times more slowly, but dissociates (Koff) (3.87e -4 ) in terms of Keytruda (2.95e -3 ) and Opdivo (1.06 -3 ) compared to human 1G1, which has 2.7-7.6 fold improved non-dissociated binding properties. Considering the mechanism of action of anti-PD-1 antibodies, the 1G1 humanized anti-PD-1 antibody exhibits improved anti-cancer activity due to its non-dissociated binding properties.

[0242] Example 14. Comparative evaluation of cross-reactivity to PD-1 antigen Protein-based ELISA was performed to confirm the cross-reactivity of antibodies to PD-1 antigen. Briefly, 96-well plates (Thermo Scientific) were coated with 10 ng of human, mouse, rabbit, cynomolgus, and rat extracellular domain (ECD) PD-1 proteins overnight at 4°C. After blocking and washing, control antibodies (Keytruda, Opdivo) and 1G1 antibody (Chimeric antibody, humanized antibody) at 1 μg / ml were added to the coated plates and incubated at 37°C for 2 hours. After washing, secondary antibodies (anti-human IgG antibodies) were added and incubated at 37°C for 2 hours. After washing, TMB substrate (Abcam) was added and the color reaction was stopped with STOP solution (Abcam). The absorbance at 450 / 650 nm was measured using a microplate reader (Thermo Fisher).

[0243] Figure 13 shows the results of measuring the cross-reactivity of antibodies to PD-1 antigen. All of Keytruda, Opdivo, and 1G1 antibodies have cross-reactivity to human PD-1 and cynomolgus PD-1 antigens. It was confirmed that 1G1 antibody also shows cross-reactivity to mouse PD-1 antigen.

[0244] Example 15. Evaluation of in vivo anticancer efficacy The anticancer efficacy evaluation experiment of the 1G1 antibody using a mouse melanoma model was carried out as shown in FIG. 14. Mouse melanoma cells (B16F11 / OT.EGFP) were cultured at 3×10 6 The cells were subcutaneously transplanted into the right dorsal skin of 8-week-old C57BL / 6 female mice. Six days after tumor cell transplantation, the size of the cancer nodules was measured and the mice were divided into 5 groups. Starting from the 7th day after transplantation, 1G1-parental antibody was intraperitoneally administered 5 times at 3-day intervals, and the tumor size was measured at 3-day intervals. The tumor size was calculated using the following formula: LxW 2 / 2 Here, L is the major axis length and W is the minor axis length.

[0245] Survival rate was 1.0% for tumors with a volume of 1,000 mm 3 The day when the tumor growth inhibition rate exceeded 700 mm or when tumor ulceration or mouse death occurred was recorded. The significance between tumor growth inhibition rates was determined using two-way ANOVA with Bonferroni test in the GraphPad Prism program, and the significance between survival rate values ​​was determined using the log-rank test (Mantel-Cox test) (*: P<0.05, ***: P<0.001, survival rate analysis endpoint: 700 mm 3 ).

[0246] FIG. 15 shows tumor growth over time and tumor growth inhibition depending on the administration concentration of 1G1-parental antibody. The 1G1 1 mg / kg administration group showed tumor growth similar to that of the isotype control group, while the 1G1 2.5-10 mg / kg administration group showed tumor growth inhibition rates of about 90-100% on day 19, 94-112% on day 22, and 86-94% on day 25 (day 22 in FIG. 15), and an increase in survival rate of about 20% was observed (FIG. 16). Based on these results, it was confirmed that the 1G1-parental antibody exhibits excellent anticancer effects.

[0247] Example 16. Evaluation of in vivo anticancer efficacy in an experimental colon cancer model The anti-cancer efficacy of 1G1 antibody was evaluated in the MC38 colon cancer syngeneic model. 7-8 week-old C57BL / 6 female mice were inoculated with 5 × 10 5 MC38 mouse colon cancer cells were subcutaneously injected. After injection, the average tumor size was 50 to 150 mm 3When the tumor volume reached 100 mg / kg, the animals were divided into 4 groups (n=13 each) based on tumor volume, and human hIgG4, rat rIgG2a, 1G1-h70 antibody, and RMP1-14 (mouse PD-1) antibody were administered at 10 mg / kg each, 3 days apart, for a total of 5 doses. For each group, the animal's weight and tumor size were measured and evaluated 3 times a week. Final weight and tumor size were measured on the day the study reached its endpoint, which was when the average tumor size in the control group reached 1,500 mm 3 Percent tumor growth inhibition (% TGI) was determined using the initial (i) and final (f) tumor measurements for the treated (T) vs. control (C) groups using the following equation: %TGI=1-(Tf-Ti) / (Cf-Ci)*100

[0248] The relative tumor size change over time and tumor growth inhibition rate for each group are shown in Figure 17. The 1G1-h70 antibody showed significantly better tumor growth inhibition than the mouse PD-1 antibody RMP1-14 antibody. The MC38 colon cancer syngeneic model is known to not respond well to anti-PD-1 therapy. Therefore, the excellent tumor growth inhibition effect of the 1G1-h70 antibody was not expected. There was no statistically significant change in the body weight of the mice treated with each group (data not shown).

[0249] Example 17. Epitope mapping of 1G1 antibody using crystallography Epitope mapping (X-ray crystallography) of the 1G1-h70 humanized antibody on human PD-1 antigen (SEQ ID NO: 62) was performed to confirm the epitope of the human PD-1 antigen recognized by the antibody. The bound complex of the Fab of the 1G1-h70 antibody and PD-1 was crystallized at 20°C using a crystallization solution by the hanging drop vapor diffusion method (drop volume = protein 0.8 μl + reservoir 0.8 μl, reservoir volume 400 μl). X-ray diffraction experiments were performed on the generated single crystals, and data with a resolution of 2.30 Å was obtained. Table 4 below summarizes information related to X-ray diffraction data collection and structure refinement.

[0250] [Table 6]

[0251] In the interaction between 1G1-h70 Fab and PD-1, it was confirmed that the 1G1-h70 antibody forms hydrogen bonds with N66, Y68, K78, A129, P130, and A132 residues on the PD-1 protein antigen (sequence 62). Among them, the side chains of Y68, K78, and N66 are involved in hydrogen bonds.

[0252] In addition, it was observed that the 1G1-h70 antibody formed hydrophobic bonds with the I126, L128, A129, P130, and A132 residues on the PD-1 protein antigen (sequence 62) through the interaction between 1G1-h70 Fab and PD-1. As confirmed through the Ala scanning experiment in Example 11, the three amino acid residues (P130, L128, and I126) that were found to play the most important role in binding to the 1G1 antibody are located in the FG loop region of PD-1, and it is believed that the hydrophobic interactions between them exert synergy and contribute greatly to the binding affinity.

[0253] In fact, it is known that the loop region of the PD-1 molecule is very flexible and adopts a conformation appropriate for binding depending on the binding partner. Figure 18 shows the structural differences in the FG loop, which plays an important role in binding to the 1G1 antibody, the C'D loop, which is important for binding to Keytruda, and the N-terminal region, which is important for binding to Opdivo, in PD-1. Such differences in structural changes give rise to differences in the binding patterns with the PD-1 interactome, and the anti-cancer immune patterns of each antibody may differ.

[0254] Example 18. Binding strength of 1G1 antibody depending on pH It is known that there is a pH difference between blood (pH 7.4) and the tumor microenvironment (pH 5.0-7.0). Therefore, the therapeutic efficacy of an immune anti-cancer antibody is affected by the pH dependency of the antibody binding strength. Generally, the residue most sensitive to binding strength due to pH difference is histidine, but in the case of the conventional PD-1 antibodies Keytruda and Opdivo, there is no histidine in the residues involved in binding. In contrast, the 1G1 antibody has a histidine residue involved in hydrogen bonding or hydrophobic bonding with the PD-1 protein antigen in the CDR region (Kabat system), such as the H52 residue of the heavy chain CDR2, and is therefore expected to contribute to binding with PD-1 even in the tumor microenvironment with a low pH. To this end, we investigated whether the PD-1 binding strength of the 1G1-h70 antibody is pH dependent, together with Keytruda and Opdivo.

[0255] The binding kinetics of each antibody to human PD-1 was measured by SPR (surface plasma resonance) analysis using Biacore 8K (Cytiva), and the binding strength to the antigen between the antibodies was compared. Keytruda (MSD (Lot #T020031)) and Opdivo (BMS (Lot #043FB)), which are anti-human PD-1 antibody controls, were purchased from Shinwon Pharmaceutical as human pharmaceuticals. In addition, the 2E5 antibody was produced from the 2E5 clone from WO 2018 / 053709 of CStone Pharmaceuticals. The 2E5 antibody binds to both human PD-1 and mouse PD-1, and the epitope analysis results have been disclosed to be located on the FG ring of PD-1.

[0256] Control antibodies (Keytruda, Opdivo), 2E5 antibody, and 1G1 antibody (1G1-h70) were flowed onto a Protein A chip (Cytiva) and captured. Seven concentrations (0-100M) of human PD-1 were flowed onto the sensor chip at a flow rate of 30 μL / min for 120 s of the association phase, followed by 1,800 s of dissociation. Capture, association, and dissociation were performed in HBS-EP+ buffer at pH 6.0. The chip was regenerated with glycine at pH 1.5 after each experiment. Binding and dissociation curves were plotted using Cytiva evaluation software to measure kinetics and affinity values. One-way ANOVA with Tukey test in GraphPad Prism program was used to determine the significance of the antibody kinetics and affinity values ​​(****, P<0.0001).

[0257] Figure 19A and Figure 19B show the binding kinetics and affinity for human PD-1 through Kon (ka value), Koff (kd value), and KD value. At pH 7.4 (blood), 1G1-h70 antibody (KD = 5.4nM) shows a similar level of binding affinity for human PD-1 compared to Keytruda (KD = 6.6nM), Opdivo (KD = 7.1nM), and 2E5 antibody (KD = 12.6nM). However, at pH 6.0 (tumor microenvironment), 1G1-h70 antibody (KD = 0.9nM) was confirmed to show a much stronger binding affinity for human PD-1 compared to Keytruda (KD = 4.2nM), Opdivo (KD = 3.1nM), and 2E5 antibody (KD = 4.5nM). This is because the 1G1-h70 antibody has improved binding properties, such as no dissociation in terms of dissociation (Koff), even at pH 6.0, compared to the KEYTRUDA, OPDIVO, and 2E5 antibodies. The anti-cancer efficacy of anti-cancer drugs, including immune anti-cancer antibodies, against tumors is affected by the low pH of the tumor microenvironment, and the 1G1-h70 antibody, which exhibits strong human PD-1 binding affinity even at the low pH of the tumor microenvironment, is the basis for demonstrating significantly superior anti-cancer activity in the in vivo tumor microenvironment.

[0258] Example 19. Affinity maturation In order to increase the affinity of the 1G1-h70 antibody for human PD-1, affinity maturation was carried out. Each amino acid residue in the CDR region of the 1G1-h70 antibody was mutated to another 19 amino acids using the optimal codon for E. coli. A DNA oligonucleotide library was synthesized on a microarray, clones were selected, and expressed in E. coli.

[0259] Crude proteins secreted into the medium were analyzed by ELISA against BSA and human PD-1 protein to analyze expression and binding affinity. Clones with improved values ​​were selected and sequence analyzed. Beneficial mutants were identified by affinity ranking by SPR. Off-rate screening was performed on a Biacore T200. The running buffer was HBS-EP (10 mM HEPES, 500 mM NaCl, 3 mM EDTA, 0.05% Tween 20) (pH 7.4). Fab-SASA of selected clones secreted into the culture medium was captured on a SASA capture biosensor. After equilibration, antigen was injected for 120 seconds (association phase), followed by running buffer for 420 seconds (dissociation phase). Before injecting other selected clones, the surface was regenerated and this process was repeated until all samples were analyzed. The off-rates of Fab-SASA clones were obtained by fitting the experimental data locally to a 1:1 interaction model using the Biacore T200 evaluation software. Selected mutations were ranked by their off-rates (kd).

[0260] Once "good mutations" were identified, combinatorial libraries consisting of random combinations of those mutations were constructed by PCR. Combination clones were analyzed by ELISA, and DNA sequence analysis and affinity ranking were performed. The top combinations of "good mutations" that caused the highest affinity increase without reducing expression were finally selected, and antibody affinity was measured.

[0261] After affinity maturation, a total of 13 humanized PD-1 antibodies (AHF16556, AHF16557, AHF16558, AHF16559, AHF16560, AHF16561, AHF16563, AHF16564, AHF16565, AHF16566, AHF16568, AHF16569 and AHF16570) were obtained, which have amino acid substitutions in three CDR regions (VHCDR2, VHCDR3, VLCDR1) compared to the 1G1-h70 parent antibody (WT) as shown in Table 5 below.

[0262] [Table 7]

[0263] The binding kinetics data of these antibodies to human PD-1 are shown in Table 6 below.

[0264] [Table 8]

[0265] Array List [Table 9-1] [Table 9-2] [Table 9-3] [Table 9-4] [Table 9-5] [Table 9-6] [Table 9-7] [Table 9-8] [Table 9-9] [Table 9-10] [Table 9-11]

[0266] Although the present disclosure has been described above, the present disclosure is not limited to the disclosed embodiments and the accompanying drawings, and may be modified in various ways by those skilled in the art within the scope of the technical ideas of the present disclosure. In addition, the technical ideas described in the embodiments of the present disclosure may be implemented independently or in combination with each other.

Claims

1. An antibody or antigen-binding fragment thereof that binds to an epitope of programmed cell death 1 (PD-1) protein comprising amino acids P130, L128, and I126 of SEQ ID NO:

62.

2. The antibody or antigen-binding fragment thereof of claim 1 , wherein the antibody or antigen-binding fragment thereof binds to an additional epitope including one or more selected from the group consisting of N66, Y68, K78, A129 and A132 of SEQ ID NO:

62.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the antibody or antigen-binding fragment thereof has a binding affinity to human PD-1 and mouse PD-1 at a similar level.

4. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the antibody or antigen-binding fragment thereof binds to human PD-1 with a KD of 9E-10 M or less at pH 6.

0.

5. An antibody or antigen-binding fragment thereof that binds to PD-1 protein, the antibody or antigen-binding fragment comprising: A heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence of SEQ ID NO:1; HCDR2 comprising the amino acid sequence of SEQ ID NO: 3, 63, 64, 65, 66 or 67; HCDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:5 and SEQ ID NOs:68-82; A light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence of SEQ ID NO: 7, 60, 83 or 84; LCDR2 comprising the amino acid sequence of SEQ ID NO:9; and an LCDR3 comprising the amino acid sequence of SEQ ID NO:

11.

6. The antibody or antigen-binding fragment thereof according to claim 5 , wherein the antibody is a murine antibody, a chimeric antibody, or a humanized antibody.

7. An antibody or antigen-binding fragment thereof that binds to PD-1 protein, the antibody or antigen-binding fragment comprising: A heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 13, 54, 56 or 58; A light chain variable region comprising the amino acid sequence of SEQ ID NO: 15, 55, 57 or 59, or an antibody or antigen-binding fragment thereof.

8. An antibody or antigen-binding fragment thereof that binds to PD-1 protein, the antibody or antigen-binding fragment comprising: a heavy chain variable region comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 13, 54, 56 or 58; and a light chain variable region comprising an amino acid sequence that has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 15, 55, 57 or 59, or more.

9. A nucleic acid molecule encoding an antibody or antigen-binding fragment thereof according to any one of claims 1, 2 and 5 to 8.

10. A cloning vector or expression vector comprising the nucleic acid molecule of claim 9.

11. A host cell comprising the cloning vector or expression vector of claim 10.

12. A method for producing an antibody or antigen-binding fragment thereof described in any one of claims 1, 2, and claims 5 to 8, comprising the step of culturing a host cell containing a cloning vector or an expression vector containing a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof described in any one of claims 1, 2, and claims 5 to 8.

13. The antibody or antigen-binding fragment may be a camelized single domain antibody, a diabody, a F(ab') ２ 9. The antibody or antigen-binding fragment thereof according to any one of claims 1, 2, and claims 5 to 8, which is selected from the group consisting of Fab', Fab, Fv, scFv, scFV dimer, BsFv, dsFv, (dsFv)2, dsFv-dsFv', Fv fragment, ds diabody, nanobody, minibody, domain antibody, bivalent domain antibody, dAb, and single-chain binding polypeptide.

14. A transgenic animal engineered to express the antibody or antigen-binding fragment thereof of any one of claims 1, 2, and 5 to 8.

15. The transgenic animal of claim 14 , wherein the animal is a rodent.

16. A polyspecific antigen-binding molecule, an immunoconjugate, a chimeric antigen receptor, an engineered T cell receptor or a tumor-killing virus comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1, 2 and claims 5 to 8.

17. A pharmaceutical composition for preventing or treating a condition associated with PD-1, comprising the antibody or antigen-binding fragment thereof according to any one of claims 1, 2, and 5 to 8, or a multispecific antigen-binding molecule, immunoconjugate, chimeric antigen receptor, engineered T cell receptor, or tumor-killing virus comprising the antibody or antigen-binding fragment thereof.

18. The pharmaceutical composition of claim 17, wherein the condition associated with PD-1 is a tumor, a cancer, an autoimmune disease, a neurological disease, a neurodegenerative disease, or an infectious disease.

19. The tumor or cancer that is a condition associated with PD-1 is selected from the group consisting of non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, kidney cancer, liver cancer, bone cancer, skin cancer, colon cancer, rectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric carcinoma, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic carcinoma, leukemia, lymphoma, myeloma, mycosis fungoides, Merkel cell carcinoma, classical Hodgkin lymphoma (CHL), primary mediastinal large B cell lymphoma, T cell / histiocyte-rich B cell lymphoma, Epstein-Barr disease, and ...

19. The pharmaceutical composition of claim 18, wherein the cancer is selected from the group consisting of Epstein-Barr virus (EBV) positive and EBV negative post-transplant lymphoproliferative disorders (PTLD), EBV-associated persistent large B-cell lymphoma (DLBCL), plasmablastic lymphoma, extracellular NK / T cell lymphoma, non-pharyngeal carcinoma, human herpesvirus 8 (HHV8) associated primary effusion lymphoma, other hematological cancers including Hodgkin's lymphoma, primary central nervous system (CNS) lymphoma, spinal tumors, and neoplasms of the central nervous system including brainstem glioma.

20. Autoimmune diseases that are conditions associated with PD-1 include lupus, systemic lupus erythematosus, Sjogren's syndrome, arthritis, rheumatoid arthritis, asthma, chronic obstructive pulmonary disease (COPD), pelvic inflammatory disease, Alzheimer's disease, inflammatory bowel disease, Crohn's disease, ulcerative colitis, Peyronie's disease, coeliac disease, gallbladder disease, pilonidal disease, peritonitis, psoriasis, psoriatic arthritis, vasculitis, surgical adhesions, stroke, type 1 diabetes, Lyme disease, meningoencephalitis, autoimmune uveitis, multiple sclerosis, Guillain-Barre syndrome, and others.

19. The pharmaceutical composition of claim 18, wherein the therapeutic agent is selected from the group consisting of inflammatory bowel disease, inflammatory bowel disease, inflammatory bowel syndrome, atopic dermatitis, autoimmune hepatitis, fibrosing alveolitis, Grave's disease, IgA nephropathy, idiopathic thrombocytopenic purpura, Meniere's disease, pemphigus, primary biliary cirrhosis, sarcoidosis, scleroderma, Wegener's granulomatosis, other autoimmune diseases, pancreatitis, trauma (surgery), graft versus host disease, transplant rejection, heart disease including ischemic diseases such as myocardial infarction and atherosclerosis, intravascular coagulation, bone resorption, osteoporosis, osteoarthritis, periodontitis and hypochlorhydria, infertility related to lack of fetal-maternal tolerance, leukoplakia, myasthenia gravis and systemic sclerosis.

21. 19. The pharmaceutical composition of claim 18, wherein the nervous system disease or neurodegenerative disease that is a condition associated with PD-1 is selected from the group consisting of cognitive impairment, brain tumors, Alzheimer's disease, dementia, stroke, spinal cord injury, amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, multiple sclerosis, glioblastoma, melanoma, pain, and memory decline.

22. 19. The pharmaceutical composition of claim 18, wherein the infectious disease, which is a condition associated with PD-1, is selected from the group consisting of hepatitis B and hepatitis C viral infections, herpes viruses, Epstein-Barr virus, HIV, cytomegalovirus, herpes simplex virus type I, herpes simplex virus type II, human papilloma virus, adenovirus, Kaposi-West sarcoma associated with herpes virus transmission, thin ring virus (Torque teno virus), JC virus, or BK virus infection.

23. A method for preventing or treating a tumor, cancer, an autoimmune disease, a nervous system disease, a neurodegenerative disease or an infectious disease, comprising the step of administering to an individual one or more selected from the group consisting of an antibody or antigen-binding fragment thereof described in any one of claims 1, 2, and claims 5 to 8, and a polyspecific antigen-binding molecule, an immunoconjugate, a chimeric antigen receptor, an engineered T cell receptor and a tumor-killing virus comprising the antibody or antigen-binding fragment thereof.

24. A pharmaceutical composition for increasing an immune response in a cancer-bearing tumor, comprising an antibody or an antigen-binding fragment thereof described in any one of claims 1, 2, and claims 5 to 8, or a polyspecific antigen-binding molecule, immunoconjugate, chimeric antigen receptor, engineered T cell receptor, or tumor-killing virus comprising the same.

25. A method for producing the antibody or antigen-binding fragment according to any one of claims 1, 2, and claims 5 to 8, comprising immunizing a PD-1 knockout mouse with a PD-1 antigen, removing the spleen, isolating B lymphocytes, and fusing the B lymphocytes with myeloma cells to obtain hybridoma cells, and selecting a hybridoma that produces an antibody that reacts with the human PD-1 antigen.

26. A hybridoma produced by the method of claim 25.

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