Anti-PD-1 antibody and its applications

Abstract

FIELD: biotechnology.SUBSTANCE: antibody and its antigen-binding fragment that specifically bind to PD-1 are proposed. Also a nucleic acid molecule encoding said antibody, an expression vector and a host cell for producing the antibody, a method for producing the antibody or its antigen-binding fragment, a transgenic animal, a bispecific antigen-binding molecule, an immunoconjugate, pharmaceutical compositions and a method for preventing or treating cancer associated with PD-1 are provided.EFFECT: providing a new cross-reactive antibody and its antigen-binding fragment that bind to both human and mouse PD-1, exhibiting high antitumor activity, and enabling the effective treatment of pathological conditions associated with PD-1.21 cl, 19 dwg, 7 tbl, 19 ex

Description

[0001] Technical field

[0002] The present invention relates to antibodies that bind to programmed cell death protein 1 (PD-1), antigen-binding fragments thereof, and uses thereof.

[0003] Technology Level

[0004] The description in this section merely provides information on the state of the art and does not constitute prior art.

[0005] Programmed cell death protein 1 (programmed death protein 1; PD-1; also known as CD279) is a cell surface protein commonly found on immune cells such as T cells, B cells, monocytes, natural killer (NK) cells, and dendritic cells. It negatively regulates the immune system by modulating the immune response and promotes self-tolerance by suppressing the inflammatory activity of T cells. Although this may prevent the development of autoimmune diseases, it may negatively affect the immune system's ability to kill tumor cells (Syn et al., 2017 Lancet Oncol., 18(12): e731-e741).

[0006] In recent years, immuno-oncology drugs targeting the immune system to restore and stimulate immunity have been actively developed. One of the immune checkpoint proteins, the PD-1 / PD-L1 pathway, has been clinically confirmed as a target for cancer immunotherapy (Patsoukis et al., 2020 Sci. Adv., 6: eabd2712). Consequently, PD-1 inhibitors are being developed to block PD-1 and activate the immune system to attack tumors and treat certain types of cancer.

[0007] Furthermore, upregulation of the PD-1 signaling pathway is also relevant to viral infection and viral expansion in humans. The viruses that cause infectious hepatitis, HBV and HCV, induce overexpression of PD-1 ligand in hepatocytes and activate the PD-1 signaling pathway in effector T cells, leading to T cell exhaustion and resistance to viral infection (Golden-Mason et al., 2008 J. Immunol., 180: 3637–3641). Similarly, the causative agent of HIV infection, HIV often evades the human immune system through similar mechanisms. It has been reported that therapeutic modulation of PD-1 signaling pathway by antagonist molecules can lead to recovery of immune cells from tolerance and elimination of cancer and chronic viral infection through reactivation of immune cells (Okazaki et al., 2007 Int. Immunol., 19:813- 824).

[0008] Meanwhile, agonistic antibodies against PD-1 are being developed to treat autoimmune diseases such as rheumatoid arthritis and to reduce the rejection of transplanted cells / tissues (Grebinoski and Vignali, 2020 Curr. Opin. Immunol., 67:1-9).

[0009] Furthermore, it has been suggested that IFNγ-dependent systemic immune response is useful for the treatment of Alzheimer's disease and other central nervous system pathologies that share common neuroinflammatory components, and International Application Publication WO 2015 / 136541 discloses the use of an anti-PD-1 antibody for the treatment of Alzheimer's disease. International application publication WO 2017 / 220990 discloses that blockade of the inhibitory immune checkpoints of the PD-1 / PD-L1 pathway results in increased secretion of IFNγ by IFNγ-producing cells, and that increased IFNγ activity can ensure selective recruitment of leukocytes to the choroid plexus of the brain and infiltration of T cells and monocytes into the injured CNS, targeting these immune cells to sites of neurodegenerative pathology and neuroinflammation, and can modulate the environment to become less toxic and more favorable for the removal of toxic agents, neuronal rescue, regeneration, and repair.

[0010] Despite the active development of therapeutic agents based on anti-PD-1 antibodies, there is still a pressing need to develop various anti-PD-1 antibodies with more diverse indications and characteristics. Furthermore, to effectively develop therapeutic agents based on anti-PD-1 antibodies or combinations containing them, it is important that the antibody binds not only to human PD-1 but also to murine PD-1 so that the antibody's efficacy, pharmacokinetic properties, and toxicity can be assessed in mice during preclinical studies. However, most commercially available antibodies bind only to human PD-1. Therefore, the development of a new anti-PD-1 antibody with cross-reactivity is necessary.

[0011] Disclosure of invention

[0012] Technical task

[0013] One object of the present invention is to provide a novel PD-1-binding agent that binds to PD-1.

[0014] One object of the present invention is to provide a novel anti-PD-1 antibody and an antigen-binding fragment thereof that bind to PD-1.

[0015] Another object of the present invention is to provide a novel cross-reactive protein binding agent, an antibody and an antigen-binding fragment thereof that bind to both human PD-1 and mouse PD-1.

[0016] Another object of the present invention is to provide a method for producing a novel PD-1-binding agent, an anti-PD-1 antibody and an antigen-binding fragment thereof.

[0017] Another object of the present invention is to provide a use of a novel PD-1-binding agent, an anti-PD-1 antibody and an antigen-binding fragment thereof.

[0018] However, the problems to be solved by the present invention are not limited to the above problems, and other problems not mentioned above will be clearly understood by those skilled in the art from the following description.

[0019] Solving a technical problem

[0020] In order to solve the above problems, the inventors of the present invention conducted numerous experiments and developed a new PD-1-binding agent, an anti-PD-1 antibody and an antigen-binding fragment thereof.

[0021] In one aspect, the present invention relates to an anti-PD-1 antibody and an antigen-binding fragment thereof that bind to PD-1, wherein the antibody and the antigen-binding fragment comprise a heavy chain variable region and / or a light chain variable region, wherein:

[0022] the heavy chain variable region comprises 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, and HCDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 5 and 68-82, or HCDR variant(s) with conservative amino acid substitution(s) or no more than three amino acid mutations compared to said sequences;

[0023] 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 LCDR variant(s) with conservative amino acid substitution(s) or no more than three amino acid mutations compared to the specified sequences.

[0024] In one aspect, the present invention relates to an anti-PD-1 antibody and an antigen-binding fragment thereof that bind to PD-1, wherein the antibody comprises a heavy chain variable region and / or a light chain variable region, wherein the heavy chain variable region comprises a sequence shown in SEQ ID NO: 13, 54, 56 or 58, or a variant thereof with 1-10 or less amino acid mutations compared to said sequences; and the light chain variable region comprises a sequence shown in SEQ ID NO: 15, 55, 57 or 59, or a variant with 1-10 or less amino acid mutations compared to said sequences.

[0025] In one aspect, the present invention relates to an anti-PD-1 antibody or antigen-binding fragment thereof that specifically binds to a PD-1 epitope comprising P130, L128, and I126 of human PD-1 (SEQ ID NO: 62). The anti-PD-1 antibody or antigen-binding fragment thereof can specifically bind to another epitope comprising at least one selected from the group consisting of N66, Y68, K78, A129, and A132 of SEQ ID NO: 62.

[0026] In one aspect, the present invention relates to 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, HCDR2 comprising the amino acid sequence of SEQ ID NO: 3, 63, 64, 65, 66 or 67, and HCDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 5 and 68-82, or HCDR variant(s) with conservative amino acid substitution(s) or no more than three amino acid mutations compared to said sequences.

[0027] In one aspect, the present invention relates to 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 LCDR variant(s) with conservative amino acid substitution(s) or no more than three amino acid mutations compared to said sequences.

[0028] In one aspect, the present invention relates to an isolated immunoglobulin heavy chain variable region polypeptide that binds to PD-1, comprising a sequence shown in SEQ ID NO: 13, 54, 56 or 58, or a variant with 1-10 or less amino acid mutations compared to said sequences.

[0029] In one aspect, the present invention relates to an isolated immunoglobulin light chain variable region polypeptide that binds to PD-1, comprising a sequence shown in SEQ ID NO: 15, 55, 57 or 59, or a variant with 1-10 or less amino acid mutations compared to said sequences.

[0030] In one aspect, the present invention relates to an isolated immunoglobulin heavy chain variable region polypeptide that binds to PD-1, having an amino acid sequence with at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 13, 54, 56 or 58.

[0031] In one aspect, the present invention relates to an isolated immunoglobulin light chain variable region polypeptide that binds to PD-1, having an amino acid sequence with at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 15, 55, 57 or 59.

[0032] In one aspect, the present invention relates to a heavy chain variable region polypeptide that binds to PD-1, having an amino acid sequence comprising an addition, deletion, or conservative substitution, or any combination thereof, of 1-10 amino acids compared to the amino acid sequence shown in SEQ ID NO: 13, 54, 56, or 58.

[0033] In one aspect, the present invention relates to a light chain variable region polypeptide that binds to PD-1, having an amino acid sequence comprising an addition, deletion, or conservative substitution, or any combination thereof, of 1-10 amino acids compared to the amino acid sequence shown in SEQ ID NO: 15, 55, 57, or 59.

[0034] In one aspect, the present invention relates to 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, HCDR2 comprising the amino acid sequence of SEQ ID NO: 3, 63, 64, 65, 66 or 67, and HCDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 5 and 68-82, or HCDR variant(s) with conservative amino acid substitution(s) or no more than three amino acid mutations compared to said sequences, and further comprising framework regions and constant regions.

[0035] In one aspect, the present invention relates to 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 LCDR variant(s) with conservative amino acid substitution(s) or no more than three amino acid mutations compared to said sequences, and further comprising framework regions and constant regions.

[0036] In one aspect, the present invention relates to an isolated immunoglobulin heavy chain polypeptide comprising a sequence shown in SEQ ID NO: 13, 54, 56 or 58, or a variant with 1-10 or less amino acid mutations compared to said sequences.

[0037] In one aspect, the present invention relates to an isolated immunoglobulin light chain polypeptide comprising a sequence shown in SEQ ID NO: 15, 55, 57 or 59, or a variant with 1-10 or less amino acid mutations compared to said sequences.

[0038] In one aspect, the present invention relates to an isolated immunoglobulin heavy chain polypeptide having an amino acid sequence with at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 13, 54, 56, or 58.

[0039] In one aspect, the present invention relates to an isolated immunoglobulin light chain polypeptide having an amino acid sequence with at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 15, 55, 57 or 59.

[0040] In one aspect, the present invention relates to an immunoglobulin heavy chain polypeptide that binds to PD-1, having an amino acid sequence comprising an addition, deletion, or conservative substitution, or any combination thereof, of 1-10 amino acids compared to the amino acid sequence of SEQ ID NO: 13, 54, 56, or 58.

[0041] In one aspect, the present invention relates to an immunoglobulin light chain polypeptide that binds to PD-1, having an amino acid sequence comprising an addition, deletion, or conservative substitution, or any combination thereof, of 1-10 amino acids compared to the amino acid sequence of SEQ ID NO: 15, 55, 57, or 59.

[0042] In one aspect, the present invention relates to a PD-1-binding agent comprising an anti-PD-1 antibody or an antigen-binding fragment thereof, an antibody conjugate, an immunoglobulin heavy chain polypeptide and / or an immunoglobulin light chain polypeptide, or an immunoglobulin heavy chain variable region polypeptide and / or an immunoglobulin light chain variable region polypeptide, as described above.

[0043] In one aspect, the present invention relates to a PD-1 binding agent, which 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.

[0044] In one aspect, the present invention relates to an isolated or purified polynucleotide molecule comprising a polynucleotide sequence encoding any PD-1-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, as described above.

[0045] In another aspect, the present invention relates to an isolated or purified polynucleotide molecule comprising the polynucleotide sequence of SEQ ID NO: 14, which encodes an immunoglobulin heavy chain variable region polypeptide according to the present invention.

[0046] In one aspect, the present invention relates to 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 invention.

[0047] In one aspect, the present invention relates to a vector comprising a polynucleotide molecule comprising a polynucleotide sequence encoding any PD-1-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 invention.

[0048] In one aspect, the present invention relates to an isolated host cell comprising a vector comprising a polynucleotide molecule comprising a polynucleotide sequence encoding any PD-1-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 invention.

[0049] In one aspect, the present invention relates to a transgenic animal engineered to express any PD-1-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 invention.

[0050] In one aspect, the present invention relates to a method for expressing any PD-1-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 invention, comprising culturing a host cell containing a vector containing an isolated polynucleotide encoding said PD-1-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 under conditions in which said isolated polynucleotide is expressed.

[0051] In one aspect, the present invention relates to a method for screening a PD-1-like compound, comprising reacting any PD-1-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 invention with a test compound and measuring the binding activity.

[0052] In one aspect, the present invention relates to a PD-1-like compound screened by a PD-1-like compound screening method comprising reacting any PD-1-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 invention with a test compound and measuring the binding activity.

[0053] In one aspect, the present invention relates to a method for producing an anti-PD-1 antibody, comprising immunizing PD-1 knockout mice with a PD-1 antigen, isolating B lymphocytes from the spleen isolated from the mice, and selecting a hybridoma that produces an antibody that interacts with the PD-1 antigen from hybridoma cells obtained by fusing myeloma cells with B lymphocytes.

[0054] In one aspect, the present invention relates to a hybridoma that produces an antibody interacting with the PD-1 antigen, selected by the method for producing antibodies according to the present invention.

[0055] In one aspect, the present invention relates to a multispecific antigen-binding molecule, immunoconjugate, chimeric antigen receptor, engineered T cell receptor, or oncolytic virus comprising any PD-1-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 invention.

[0056] In one aspect, the present invention relates to a pharmaceutical composition comprising at least one selected from the group consisting of any PD-1-binding agent, an anti-PD-1 antibody or an antigen-binding fragment thereof, an immunoglobulin heavy chain variable region polypeptide, an immunoglobulin light chain variable region polypeptide, an immunoglobulin heavy chain polypeptide, an immunoglobulin light chain polypeptide, and a multispecific antigen-binding molecule, an immunoconjugate, a chimeric antigen receptor, an engineered T cell receptor, or an oncolytic virus containing the above-mentioned agent, antibody, fragment, or polypeptide according to the present invention, and a pharmaceutically acceptable excipient or carrier.

[0057] The said pharmaceutical composition may be a pharmaceutical composition for the prevention, alleviation or treatment of a tumor, cancer, metastatic tumor, metastatic cancer, autoimmune disease, neurological disease, neurodegenerative disease or infectious disease.

[0058] In one aspect, the present invention relates to a pharmaceutical composition further comprising a second therapeutic agent in the above pharmaceutical composition.

[0059] In one aspect, the present invention relates to a kit for therapeutic, diagnostic or detective use, comprising at least one selected from the group consisting of any PD-1-binding agent, an anti-PD-1 antibody or an antigen-binding fragment thereof, an immunoglobulin heavy chain variable region polypeptide, an immunoglobulin light chain variable region polypeptide, an immunoglobulin heavy chain polypeptide, an immunoglobulin light chain polypeptide, and a multispecific antigen-binding molecule, an immunoconjugate, a chimeric antigen receptor, an engineered T cell receptor or an oncolytic virus containing the above-mentioned agent, antibody, fragment or polypeptide according to the present invention.

[0060] In one aspect, the present invention relates to a method for the prevention or treatment of a tumor, cancer, metastatic tumor, metastatic cancer, autoimmune disease, neurological disease, neurodegenerative disease, or infectious disease, comprising the step of administering to a subject at least one selected from the group consisting of any PD-1-binding agent, an anti-PD-1 antibody or an antigen-binding fragment thereof, an immunoglobulin heavy chain variable region polypeptide, an immunoglobulin light chain variable region polypeptide, an immunoglobulin heavy chain polypeptide, an immunoglobulin light chain polypeptide, and a multispecific antigen-binding molecule, an immunoconjugate, a chimeric antigen receptor, an engineered T cell receptor, or an oncolytic virus comprising the above-mentioned agent, antibody, fragment, or polypeptide according to the present invention.

[0061] In one aspect, the present invention relates to a method for modulating an immune response in a subject, comprising administering to the subject at least one selected from the group consisting of any PD-1-binding agent, an anti-PD-1 antibody or an antigen-binding fragment thereof, an immunoglobulin heavy chain variable region polypeptide, an immunoglobulin light chain variable region polypeptide, an immunoglobulin heavy chain polypeptide, an immunoglobulin light chain polypeptide, and a multispecific antigen-binding molecule, an immunoconjugate, a chimeric antigen receptor, an engineered T cell receptor, or an oncolytic virus comprising the above-mentioned agent, antibody, fragment, or polypeptide according to the present invention.

[0062] In one aspect, the present invention relates to the use of a PD-1-binding agent, an anti-PD-1 antibody or an antigen-binding fragment thereof, an immunoglobulin heavy chain variable region polypeptide, an immunoglobulin light chain variable region polypeptide, an immunoglobulin heavy chain polypeptide, an immunoglobulin light chain polypeptide, or a multispecific antigen-binding molecule, an immunoconjugate, a chimeric antigen receptor, an engineered T cell receptor, or an oncolytic virus comprising the above-mentioned agent, antibody, fragment, or polypeptide according to the present invention, in the manufacture of a medicament for the prevention, alleviation, or treatment of a tumor, cancer, metastatic tumor, metastatic cancer, autoimmune disease, neurological disease, neurodegenerative disease, or infectious disease.

[0063] In one aspect, the present invention also relates to a method for inhibiting the growth of tumor cells in a subject, comprising administering to the subject at least one selected from the group consisting of a PD-1-binding agent, an anti-PD-1 antibody or an antigen-binding fragment thereof, an immunoglobulin heavy chain variable region polypeptide, an immunoglobulin light chain variable region polypeptide, an immunoglobulin heavy chain polypeptide, an immunoglobulin light chain polypeptide, or a multispecific antigen-binding molecule, an immunoconjugate, a chimeric antigen receptor, an engineered T cell receptor, or an oncolytic virus containing the above-mentioned agent, antibody, fragment, or polypeptide according to the present invention in a therapeutically effective amount to inhibit the growth of tumor cells.

[0064] Other aspects will be apparent from the detailed description given herein and from general technical knowledge in the art.

[0065] Advantageous effects

[0066] A PD-1-binding agent, an anti-PD-1 antibody or an antigen-binding fragment thereof, an immunoglobulin heavy chain variable region polypeptide, an immunoglobulin light chain variable region polypeptide, an immunoglobulin heavy chain polypeptide, an immunoglobulin light chain polypeptide, or a multispecific antigen-binding molecule, an immunoconjugate, a chimeric antigen receptor, an engineered T cell receptor, or an oncolytic virus comprising the above-mentioned agent, antibody, fragment, or polypeptide according to the present invention can be used to modulate immune responses by binding to human PD-1. They are suitable, for example, for targeting T cells expressing PD-1 and for modulating PD-1 activity.For example, they can be used to prevent or treat a tumor, cancer, metastatic tumor, metastatic cancer, autoimmune disease, neurological disease, neurodegenerative disease, or infectious disease.

[0067] Furthermore, the PD-1-binding agent, the anti-PD-1 antibody or the antigen-binding fragment thereof, the immunoglobulin heavy chain variable region polypeptide, the immunoglobulin light chain variable region polypeptide, the immunoglobulin heavy chain polypeptide or the immunoglobulin light chain polypeptide according to the present invention can bind to mouse PD-1 as well as human PD-1, which allows for mouse testing to evaluate the efficacy, pharmacokinetic properties and toxicity of the antibody at the preclinical stage, which plays an important role in the efficient development of antibody-based drugs or combinations containing the above.

[0068] The effect of the present disclosure is not limited to such literal description, but includes effects that a person skilled in the art can infer from the present disclosure.

[0069] Brief description of figures

[0070] Fig. 1 shows graphs of the results of an ELISA analysis showing the binding of the hybridoma antibody 1G1 of the present invention to human PD-1 or mouse PD-1 on the cell surface.

[0071] Fig. 2 shows graphs of the results of analysis using flow cytometry, showing the binding of the hybridoma antibody 1G1 of the present invention to human PD-1 or mouse PD-1 on the cell surface.

[0072] Fig. 3 shows graphs of the results of an ELISA analysis showing the blocking evaluation of the binding of human PD-L1 or mouse PD-L1 and human or mouse PD-1 cell surface PD-1 by the hybridoma antibody 1G1 of the present invention.

[0073] Fig. 4 shows graphs of the results of analysis using flow cytometry, showing the evaluation of blocking the binding of human PD-L1 or mouse PD-L1 and human PD-1 or mouse PD-1 to the cell surface by the hybridoma antibody 1G1 of the present invention.

[0074] Fig. 5 shows the results of SDS-PAGE for the identification of purified hybridoma antibody 1G1 and chimeric antibody 1G1 (chimeric 1G1).

[0075] Fig. 6 shows graphs of the results of an ELISA analysis showing the binding of purified hybridoma antibody 1G1 to human PD-1 or mouse PD-1 on the cell surface.

[0076] Fig. 7 shows the results of an analysis using ELISA to confirm the extent to which the monoclonal cell (hybridoma) antibody 1G1 of the present invention binds to immune checkpoint proteins on the surface of human T cells.

[0077] Fig. 8 shows the amino acid sequence of the heavy chain of each of the three humanized 1G1 antibodies (humanized antibodies 1G1-h61, 1G1-h68, and 1G1-h70). The sequence is shown in the following order: leader sequence-VH / VL (in bold and underlined)-hIgG4CH / hIgkappaCL.

[0078] Figure 9 shows the amino acid sequence of the light chain of each of the three humanized 1G1 antibodies (humanized antibodies 1G1-h61, 1G1-h68, and 1G1-h70). The sequence is shown in the following order: leader sequence-VH / VL (in bold and underlined)-hIgG4CH / hIgkappaCL.

[0079] Fig. 10a shows the nucleic acid sequence of the heavy chain of humanized antibody 1G1-h61. The sequence is shown in the following order: leader sequence-VH / VL (in bold and underlined)-hIgG4CH / hIgkappaCL-stop codon (in italics).

[0080] Fig. 10b shows the nucleic acid sequence of the heavy chain of humanized antibody 1G1-h68. The sequence is shown in the following order: leader sequence-VH / VL (in bold and underlined)-hIgG4CH / hIgkappaCL-stop codon (in italics).

[0081] Fig. 10c shows the nucleic acid sequence of the heavy chain of humanized antibody 1G1-h70. The sequence is shown in the following order: leader sequence-VH / VL (in bold and underlined)-hIgG4CH / hIgkappaCL-stop codon (in italics).

[0082] Fig. 11 shows the nucleic acid sequences of the light chain of each of the three humanized 1G1 antibodies (humanized antibodies 1G1-h61, 1G1-h68, and 1G1-h70). The sequence is shown in the following order: leader sequence-VH / VL (in bold and underlined)-hIgG4CH / hIgkappaCL-stop codon (in italics).

[0083] Fig. 12a shows the binding kinetics of the humanized 1G1 antibodies of the present invention to human PD-1.

[0084] Fig. 12b shows the affinity of the humanized 1G1 antibody of the present invention for human PD-1, expressed by ka (Kon), kd (Koff), and KD values.

[0085] Fig. 13 shows the results of an ELISA assay to confirm that the 1G1 antibodies of the present invention (chimeric 1G1 antibody and humanized 1G1 antibodies) selectively bind (cross-reactivity) to the extracellular domain of PD-1 antigens of human, mouse, rabbit, cynomolgus macaque, and rat.

[0086] Fig. 14 shows the scheme of the animal experiment to evaluate the antitumor efficacy of the 1G1 antibody.

[0087] Fig. 15 is a graph showing the changes in tumor size over time after administration of the 1G1 antibody in a mouse melanoma model.

[0088] Fig. 16 is a graph showing the survival time after administration of the 1G1 antibody in a mouse melanoma model.

[0089] Fig. 17 shows the relative change in tumor size and the degree of tumor growth inhibition over time after administration of the 1G1 antibody in the syngeneic mouse model of MC38 colorectal cancer.

[0090] Figure 18 shows the binding regions of humanized antibody 1G1, Keytruda, and Opdivo to human PD-1.

[0091] Fig. 19a shows the binding kinetics of the humanized antibody 1G1 of the present invention to human PD-1 at pH 6.0.

[0092] Fig. 19b shows the affinity of the humanized antibody 1G1 of the present invention to human PD-1 at pH 6.0, expressed as ka (Kon), kd (Koff), and KD values.

[0093] Method according to the invention

[0094] It should be understood that since the specific methods and experimental conditions described herein may vary, the present invention is not limited to such methods and conditions. Since the scope of the invention will be limited only by the appended claims, the terminology used herein is for the purpose of describing particular aspects of the invention only and is not intended to limit the claims.

[0095] 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 invention belongs. Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents referenced herein are hereby incorporated by reference in their entirety.

[0096] The terms "programmed cell death protein 1," "PD-1," and "PD-1 protein" are used interchangeably herein and include variants, isoforms, species homologs of human PD-1, and analogs that share at least one epitope with PD-1. PD-1 is a T-cell coinhibitor, also known as CD279.

[0097] As used herein, the term "binding molecule" or "binding agent" includes antibodies, antigen-binding fragments thereof, and conjugates thereof with other molecules.

[0098] The term "antibody" as used herein includes whole antibodies and any antigen-binding fragment (i.e., "antigen-binding site") or individual chains thereof. "Antibody" refers to a protein comprising at least two heavy chains and two light chains linked together by disulfide bonds, or its antigen-binding site. Each heavy chain consists of a heavy chain variable region and a heavy chain constant region. The heavy chain constant region consists of three domains: CH1, CH2, and CH3. Each light chain consists of a light chain variable region and a light chain constant region. The light chain constant region consists of one domain, CL. The variable region of the heavy chain (VH) and the variable region of the light chain (VL) can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs).Each VH and VL consists of three CDRs and four FRs, arranged from amino terminus to carboxyl 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 the antigen.

[0099] The term "antibody" as used herein refers to an immunoglobulin, a fragment or derivative thereof, and includes any polypeptide comprising an antigen-binding site, regardless of whether it is produced in vitro or in vivo. The term includes, but is not limited to, polyclonal, monoclonal, monospecific, polyspecific, non-specific, humanized, single-chain, chimeric, synthetic, recombinant, hybrid, mutated, and grafted antibodies. The term "antibody" also includes an antibody fragment, such as Fab, Fab', F(ab')2, Fv, scFv, BsFv, dsFv, (dsFv)2, dsFv-dsFv', Fd, dAb, and other antibody fragments that retain the antigen-binding ability, i.e., the ability to specifically bind to PD-1. Typically, such a fragment may include antigen-binding fragments.

[0100] For the purposes of the present invention, the terms "antigen-binding fragment," "antigen-binding domain," and "binding fragment" refer to the region of an antibody molecule containing amino acids that mediate specific binding between the antibody and the antigen. For example, if the antigen is a large molecule, the antigen-binding fragment can only bind to a portion of the antigen. The region of the antigen molecule that mediates specific interaction with the antigen-binding fragment is called an "epitope" or "antigenic determinant."

[0101] An antigen-binding fragment may contain a light chain variable region (VL) and a heavy chain variable region (VH), but not necessarily both. For example, the so-called Fd fragment of an antibody consists only of the VH region but still retains some of the antigen-binding function of the intact antibody.

[0102] The term "epitope" defines an antigenic determinant that is specifically bound / identified by a binding moiety, as defined above. The binding moiety may specifically bind / interact with conformational or discontinuous epitopes that are unique to the target structure, such as human PD-1 and rodent PD-1. A conformational or discontinuous epitope is characteristic of polypeptide antigens with two or more discrete amino acid residues that are separated in the primary sequence but come together on the surface of the molecule when the polypeptide folds into the native protein / antigen. The two or more discrete amino acid residues that make up the epitope are present at separate sites on one or more polypeptide chains. These residues come together on the surface of the molecule when the polypeptide chain(s) fold into a three-dimensional structure, forming the epitope.In contrast, a continuous or linear epitope consists of two or more contiguous amino acid residues that are in the same linear segment of the polypeptide chain.

[0103] The expression "binds to a PD-1 epitope" indicates that the antibody has specific binding to a specific PD-1 epitope, which may be determined by the linear amino acid sequence or tertiary, i.e., three-dimensional, conformation, within a region of the PD-1 polypeptide. Specific binding means that the antibody's affinity for the PD-1 region is significantly higher than its affinity for other related polypeptides.

[0104] The term "higher affinity" means that there is a measurable increase in affinity for the PD-1 region compared to the affinity for other related polypeptides. Preferably, the affinity is at least 1.5 times, 2 times, 5 times, 10 times, 100 times, 10 2 times, 10 3 times, 10 4 times, 10 5 once or 10 6times higher for a specific region of PD-1 than for other proteins. Binding affinity can be determined using enzyme-linked immunosorbent assay (ELISA), fluorescence-activated cell sorting (FACS), or surface plasmon resonance (SPR).

[0105] The term "cross-reactivity" as used herein refers to the binding of the antigen-binding fragment described herein to the same target molecule in humans and rodents (mice or rats). Therefore, "cross-reactivity" should be understood as interspecies reactivity to the same molecule X expressed in different species, rather than to a molecule different from X. For example, the cross-species specificity of a monoclonal antibody that recognizes both human PD-1 and rodent PD-1 (mouse or rats) can be determined, for example, by FACS analysis.

[0106] In the context of the present invention, "individual" or "subject" means a subject in need of treatment of a disease, more specifically, a mammal such as a human or a non-human primate, a rat, a mouse, a dog, a cat, a horse, and a cow.

[0107] For the purposes of the present invention, "treatment" refers to any action in which the symptoms of a disease are alleviated or beneficially modified by administering a pharmaceutical composition according to the present invention. Treatment also includes prophylaxis. Subjects in need of treatment include those who already have a particular disease, as well as those who eventually develop the disease.

[0108] "Relieving" in the present invention means any action that at least reduces a parameter associated with the pathological condition being treated, such as the severity of a symptom.

[0109] Next, the details of the present invention will be described in detail with reference to specific examples and the accompanying figures. Items that do not differ from the prior art and are not necessary for understanding the technical concept of the present disclosure are excluded from the description.

[0110] Examples of anti-PD-1 antibodies and PD-1-binding agents, etc.

[0111] The present invention in one aspect relates to an anti-PD-1 antibody or antigen-binding fragment thereof that binds to PD-1, wherein said antibody or antigen-binding fragment comprises a heavy chain variable region and / or a light chain variable region, wherein:

[0112] the heavy chain variable region comprises 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, and HCDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 5 and 68-82, or HCDR variant(s) with conservative amino acid substitution(s) or no more than three amino acid mutations compared to said sequences;

[0113] 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 LCDR variant(s) with conservative amino acid substitution(s) or no more than three amino acid mutations compared to said sequences.

[0114] In some embodiments, the phrase "no more than three amino acid mutations" means mutation(s) of 3, 2, 1, or 0 amino acids.

[0115] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof of the present invention may have an equivalent level of binding affinity to human PD-1 and mouse PD-1.

[0116] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof binds to PD-1 with a KD value of 10 -7 M or lower; in some embodiments, they bind to PD-1 with a KD value of 10 -8 , 10 -9 , 10 -10 or 10 -11 M or below.

[0117] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof binds to PD-1 even in a low pH environment with a KD of 10 -9 M or lower, preferably with KD 10 -10 M or lower, preferably with KD 10 -11 M or lower. In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof binds to PD-1 with a KD of 9×10 -10 M or below at pH 6.0.

[0118] The present invention relates, in one aspect, to an anti-PD-1 antibody and an antigen-binding fragment thereof that bind to PD-1, wherein the antibody comprises a heavy chain variable region and / or a light chain variable region, wherein the heavy chain variable region comprises a sequence shown in SEQ ID NO: 13, 54, 56 or 58, or a variant thereof with 1-10 or less amino acid mutations compared to said sequences; the light chain variable region comprises a sequence shown in SEQ ID NO: 15, 55, 57 or 59, or a variant with 1-10 or less amino acid mutations compared to said sequences.

[0119] In one aspect, the present invention relates to 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, HCDR2 comprising the amino acid sequence of SEQ ID NO: 3, 63, 64, 65, 66 or 67, and HCDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 5 and 68-82, or HCDR variant(s) with conservative amino acid substitution(s) or no more than three amino acid mutations compared to said sequences.

[0120] In one aspect, the present invention relates to 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 LCDR variant(s) with conservative amino acid substitution(s) or no more than three amino acid mutations compared to said sequences.

[0121] In one aspect, the present invention relates to an isolated immunoglobulin heavy chain variable region polypeptide that binds to PD-1, comprising a sequence shown in SEQ ID NO: 13, 54, 56 or 58, or a variant with 1-10 or less amino acid mutations compared to said sequences.

[0122] In one aspect, the present invention relates to an isolated immunoglobulin light chain variable region polypeptide that binds to PD-1, comprising a sequence shown in SEQ ID NO: 15, 55, 57 or 59, or a variant with 1-10 or less amino acid mutations compared to said sequences.

[0123] “Isolated” in the present invention refers to a compound that has been separated from its natural environment.

[0124] In one aspect, the present invention relates to an isolated immunoglobulin heavy chain variable region polypeptide that binds to PD-1, having an amino acid sequence with at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 13, 54, 56 or 58.

[0125] In one aspect, the present invention relates to an isolated immunoglobulin light chain variable region polypeptide that binds to PD-1, having an amino acid sequence with at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 15, 55, 57 or 59.

[0126] Sequence similarity for polypeptides is typically measured using sequence analysis software. Protein analysis software compares similar sequences using similarity scores assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. For example, GCG software includes programs such as GAP and BESTFIT, which can be used with default parameters to determine sequence similarity or sequence identity between two closely related polypeptides, such as homologous polypeptides from different species, or between a wild-type protein and its mutein. See, for example, GCG Version 6.1. Polypeptide sequences can also be compared using FASTA with default or recommended parameters; a program included with GCG Version 6.1.FASTA (e.g., FASTA2 and FASTA3) provides alignments and percent sequence identity for the regions of best overlap between a given and a target sequence. Another preferred algorithm for comparing a sequence of the invention with sequences from a database containing a large number of sequences from different organisms is the BLAST computer program, in particular BLASTP or TBLASTN, using default parameters. See, e.g., Altschul et al. (1990) J. Mol. Biol., 215: 403–410 and (1997) Nucleic Acids Res., 25: 3389, 402, each of which is incorporated herein by reference.

[0127] Residues that are not identical may differ, for example by conservative amino acid substitutions.

[0128] In one aspect, the present invention relates to a heavy chain variable region polypeptide that binds to PD-1, having an amino acid sequence comprising an addition, deletion, or conservative substitution, or any combination thereof, of 1-10 amino acids compared to the amino acid sequence of SEQ ID NO: 13, 54, 56, or 58.

[0129] In one aspect, the present invention relates to a light chain variable region polypeptide that binds to PD-1, having an amino acid sequence comprising an addition, deletion, or conservative substitution, or any combination thereof, of 1-10 amino acids compared to the amino acid sequence of SEQ ID NO: 15, 55, 57, or 59.

[0130] A "conservative amino acid substitution" is a substitution in which an amino acid residue is replaced by another amino acid residue whose side chain (R group) has similar chemical properties (e.g., charge or hydrophobicity). Generally, a conservative amino acid substitution does not substantially change the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percentage or degree of similarity can be increased by adjusting for the conservative nature of the substitution. Methods for making such adjustments are well known to those skilled in the art. See, e.g., Pearson (1994) Methods Mol. Biol., 24: 307–331.Examples of groups of amino acids that contain 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: aspartate and glutamate; and 7) sulfur-containing side chains: cysteine ​​and methionine. Preferred groups of conservative amino acid substitutions are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine. Alternatively, a conservative substitution is any change that has a positive value on the log-likelihood matrix PAM250 described in Gonnet et al. (1992) Science, 256: 1443–45.A "moderately conservative" substitution is any change that does not have a negative value in the PAM250 log-likelihood matrix.

[0131] In one aspect, the present invention relates to 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, HCDR2 comprising the amino acid sequence of SEQ ID NO: 3, 63, 64, 65, 66 or 67, and HCDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 5 and 68-82, or HCDR variant(s) with conservative amino acid substitution(s) or no more than three amino acid mutations compared to said sequences, and further comprising framework regions and constant regions.

[0132] In one aspect, the present invention relates to 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 LCDR variant(s) with conservative amino acid substitution(s) or no more than three amino acid mutations compared to said sequences, and further comprising framework regions and constant regions.

[0133] In one aspect, the present invention relates to an isolated immunoglobulin heavy chain polypeptide comprising a sequence shown in SEQ ID NO: 13, 54, 56 or 58, or a variant with 1-10 or less amino acid mutations compared to said sequences.

[0134] In one aspect, the present invention relates to an isolated immunoglobulin light chain polypeptide comprising a sequence shown in SEQ ID NO: 15, 55, 57 or 59, or a variant with 1-10 or less amino acid mutations compared to said sequences.

[0135] In one aspect, the present invention relates to an isolated immunoglobulin heavy chain polypeptide having an amino acid sequence with at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 13, 54, 56, or 58.

[0136] In one aspect, the present invention relates to an isolated immunoglobulin light chain polypeptide having an amino acid sequence with at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 15, 55, 57, or 59.

[0137] In one aspect, the present invention relates to an immunoglobulin heavy chain polypeptide that binds to PD-1, having an amino acid sequence comprising an addition, deletion, or conservative substitution, or any combination thereof, of 1-10 amino acids compared to the amino acid sequence of SEQ ID NO: 13, 54, 56, or 58.

[0138] In one aspect, the present invention relates to an immunoglobulin light chain polypeptide that binds to PD-1, having an amino acid sequence comprising an addition, deletion, or conservative substitution, or any combination thereof, of 1-10 amino acids compared to the amino acid sequence of SEQ ID NO: 15, 55, 57, or 59.

[0139] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof may be a recombinant antibody, preferably a murine antibody, a chimeric antibody, or a humanized antibody.

[0140] In some embodiments, the heavy chain constant region of the chimeric or humanized anti-PD-1 antibody may be derived from human IgG1, IgG2, IgG3, IgG4, or mutant sequence(s) thereof, and the light chain constant region may be derived from human kappa, lambda chain, or mutant sequence(s) thereof.

[0141] In some embodiments of the anti-PD-1 antibody or antigen-binding fragment thereof of the present invention, the antibody is a chimeric antibody, and the constant region is derived from a constant region of a human antibody or a mutant thereof.

[0142] In some embodiments of the anti-PD-1 antibody or antigen-binding fragment thereof of the present invention, the antibody is a humanized antibody, and the light chain framework region (FR) and the heavy chain framework region of the antibody are derived from a human germline light chain and heavy chain, respectively, or mutant sequence(s) thereof.

[0143] In one aspect, the present invention relates to a PD-1-binding agent comprising an anti-PD-1 antibody or an antigen-binding fragment thereof, an antibody conjugate, an immunoglobulin heavy chain polypeptide and / or an immunoglobulin light chain polypeptide, or an immunoglobulin heavy chain variable region polypeptide and / or an immunoglobulin light chain variable region polypeptide according to the present invention.

[0144] The PD-1-binding agent may be, for example, an antibody, an antibody conjugate, or an antigen-binding fragment thereof, but is not limited thereto.

[0145] In some embodiments, the present invention relates to an isolated PD-1-binding agent, anti-PD-1 antibody, or antigen-binding fragment thereof that competes with any PD-1-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 described above for binding to PD-1, or binds to the same epitope of PD-1 as the PD-1-binding agent, anti-PD-1 antibody, or antigen-binding fragment thereof.

[0146] Antigen-binding fragment

[0147] In one aspect of the present invention, the PD-1 binding agent of the present invention may be, but is not limited to, 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.

[0148] Unless otherwise specifically stated, the term "antibody" as used herein encompasses antibody molecules comprising two immunoglobulin heavy chains and two immunoglobulin light chains (i.e., "complete antibody molecules"), as well as antigen-binding fragments thereof. The terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, and the like as used herein include any naturally occurring, enzymatically produced, synthetic, or engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. The term "antigen-binding fragment" or "antibody fragment" of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to PD-1. The antibody fragment may include a Fab fragment, an F(ab')2 fragment, an Fv fragment, a dAb fragment, a fragment containing a CDR, or an isolated CDR.In some embodiments, the term "antigen-binding fragment" refers to a polypeptide fragment of a multispecific antigen-binding molecule. Antigen-binding fragments of an antibody can be obtained, for example, from complete antibody molecules using any suitable standard methods, such as proteolytic cleavage or recombinant genetic engineering techniques involving the processing and expression of DNA encoding the variable and (optionally) constant domains of the antibody. Such DNA is known and / or readily available, for example, from commercial sources and DNA libraries (including, for example, antibody libraries in phage display format), or can be synthesized.DNA can be sequenced and manipulated chemically or using molecular biology techniques, for example, to arrange one or more variable and / or constant domains in a suitable configuration or to introduce codons, create cysteine ​​residues, modify, add or delete amino acids, etc.

[0149] Non-limiting examples of antigen-binding fragments include Fab, Fab', F(ab')2, Fv, scFv, BsFv, dsFv, (dsFv)2, dsFv-dsFv', Fd, dAb, and minimal recognition units consisting of amino acid residues that mimic a hypervariable region of an antibody (e.g., an isolated complementarity determining region (CDR), such as a CDR3 peptide), or a FR3-CDR3-FR4 delimited peptide. Other engineered molecules such as domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, tribodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark IgNAR variable domains are also encompassed by the term "antigen-binding moiety" as used herein.

[0150] An antigen-binding fragment of an antibody typically comprises at least one variable domain. The variable domain can be of any size or amino acid composition and will typically contain at least one CDR that is adjacent to one or more framework sequences or is in frame. In antigen-binding fragments having a VH domain linked to a VL domain, the VH and VL domains can be positioned relative to each other in any suitable arrangement. For example, the variable region can be dimeric and comprise VH-VH, VH-VL, or VL-VL dimers. Alternatively, the antigen-binding fragment of an antibody can comprise a monomeric VH or VL domain.

[0151] In some embodiments, the antigen-binding fragment of an antibody may comprise at least one variable domain covalently linked to at least one constant domain. Non-limiting examples of variable and constant domain configurations that may be found in the antigen-binding fragment of an antibody of the present invention 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 given above, the variable and constant domains may either be directly linked to each other or may be linked through a full or partial hinge or linker region.The hinge region may be composed of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids, resulting in a flexible or semi-flexible linkage between adjacent variable and / or constant domains in a single polypeptide molecule. Furthermore, the antigen-binding fragment of an antibody of the present invention may comprise a homodimer or heterodimer (or other multimer) of any of the above configurations of variable and constant domains in a non-covalent association or a covalent association (e.g., disulfide bond(s)) with each other and / or with one or more monomeric VH or VL domains. As with complete antibody molecules, antigen-binding fragments may be monospecific or multispecific (e.g., bispecific).A multispecific antigen-binding antibody fragment typically comprises at least two different variable domains, where each variable domain is capable of specifically binding to a separate antigen or to a different epitope 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 the antigen-binding antibody fragment of the present invention using standard techniques available in the art.

[0152] Nucleic acids encoding exemplary anti-PD-1 antibodies and binding agents, etc.

[0153] In one aspect, the present invention relates to an isolated or purified polynucleotide molecule comprising a polynucleotide sequence that encodes any PD-1-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 invention.

[0154] In another aspect, the present invention relates to an isolated or purified polynucleotide molecule comprising the polynucleotide sequence of SEQ ID NO: 14, which encodes an immunoglobulin heavy chain variable region polypeptide according to the present invention.

[0155] The present invention relates, in one aspect, to 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 invention.

[0156] Production of exemplary anti-PD-1 antibodies and binding agents, etc.

[0157] In one aspect, the present invention relates to a method for producing an antibody, comprising immunizing PD-1 knockout mice with a PD-1 antigen, isolating B lymphocytes from the spleen isolated from the mice, and selecting a hybridoma producing an antibody that reacts with the human PD-1 antigen from hybridoma cells obtained by fusing myeloma cells with B lymphocytes.

[0158] In one aspect, the present invention relates to a hybridoma obtained by the above method for producing an antibody.

[0159] In one aspect, the present invention relates to a vector comprising a polynucleotide molecule comprising a polynucleotide sequence encoding any PD-1-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 invention.

[0160] In one aspect, the present invention relates to an isolated host cell comprising a vector comprising a polynucleotide molecule comprising a polynucleotide sequence encoding any PD-1-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 invention.

[0161] In one embodiment, the host cell comprises: (1) a vector comprising a polynucleotide encoding an amino acid sequence comprising a VL antibody according to the present invention and an amino acid sequence comprising a VH antibody, or (2) a first vector comprising a polynucleotide encoding an amino acid sequence comprising a VL antibody according to the present invention and a second vector comprising a polynucleotide encoding an amino acid sequence comprising a VH antibody (e.g., the host cell is transformed with these vectors).

[0162] In one embodiment, the present invention relates to a method for producing an anti-PD1 antibody, comprising culturing a host cell containing a polynucleotide encoding an antibody that binds to PD-1 under conditions suitable for expressing the antibody, and selectively isolating the antibody from the host cell (or a cell culture medium of the host cells).

[0163] Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells. For example, antibodies can be produced by bacteria, particularly when glycosylation and Fc effector function are not required. For expression of antibody fragments and polypeptides in bacteria, see, for example, U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523. See also Charlton, K. A., In: Methods in Molecular Biology, Vol. 248, Lo B. K. C. (ed.), Humana Press, Totowa, NJ (2003), pp. 245–254, which describes expression of antibody fragments in E. coli. After expression, the antibody can be isolated from the bacterial cell mass as a soluble fraction and can be further purified.In addition to prokaryotes, suitable hosts for cloning or expression of antibody-encoding vectors include eukaryotic microorganisms such as filamentous fungi or yeast, including fungal and yeast strains whose glycosylation pathways have been "humanized," resulting in the production of antibodies with partially or fully human glycosylation patterns. See Gerngross TU, Nat. Biotech., 22 (2004) 1409–1414 and Li H. et al., Nat. Biotech., 24 (2006) 210–215.

[0164] Suitable host cells for expression of glycosylated antibodies are also obtained from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculovirus strains have been identified that can be used in combination with insect cells, particularly for transfection of Spodoptera frugiperda cells. Plant cell cultures can also be used as hosts (see, e.g., U.S. Patents 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (which describe PLANTIBODIES™ technology for producing antibodies in transgenic plants)).

[0165] Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted to growth in suspension can be used. Other examples of suitable mammalian host cell lines are the SV40-transformed monkey kidney line CV1 (COS-7); the human embryonic kidney cell line (293 or 293 cells, as described, for example, by Graham FL et al., J. Gen Virol., 36 (1977) 59-74); baby hamster kidney cells (BHK); mouse Sertoli cells (TM4 cells, as described, for example, by Mather JP, Biol. Reprod., 23 (1980) 243-252); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK); buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor cells (MMT 060562); TRI cells, as described, for example, in Mather JP et al., Annals NYAcad. Sci., 383 (1982) 44–68; MRC5 cells; and FS4 cells. Other suitable 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 some mammalian host cell lines suitable for antibody production, see, for example, Yazaki P. and Wu A. M., Methods in Molecular Biology, Vol. 248, Lo B. K. S. (ed.), Humana Press, Totowa, NJ (2004), pp. 255–268.

[0166] In one aspect, the present invention relates to a transgenic animal engineered to express any PD-1-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 invention. The animal can be, for example, a rodent such as a mouse, a rat, etc.

[0167] In one aspect, the present invention relates to a method of expressing any PD-1-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 invention, comprising culturing a host cell containing a vector containing an isolated polynucleotide encoding said PD-1-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, under conditions in which said polynucleotide is expressed.

[0168] In one aspect, the present invention relates to a method for screening a PD-1-like compound, comprising reacting any PD-1-binding agent, an anti-PD-1 antibody or an antigen-binding fragment thereof, an immunoglobulin heavy chain variable region polypeptide, an immunoglobulin light chain variable region polypeptide, an immunoglobulin heavy chain polypeptide or an immunoglobulin light chain polypeptide, with a test compound and measuring the binding activity.

[0169] In one aspect, the present invention relates to a PD-1-like compound screened by a method for screening a PD-1-like compound comprising reacting any PD-1-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 invention with a test compound and measuring the binding activity.

[0170] Multispecific antigen-binding molecule, immunoconjugate

[0171] In one aspect, the present invention relates to a multispecific antigen-binding molecule, immunoconjugate, chimeric antigen receptor, engineered T cell receptor, or oncolytic virus comprising any PD-1-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 invention.

[0172] A multispecific (e.g., bispecific) antigen-binding fragment of an antibody typically comprises at least two different variable domains, where each variable domain is capable of specifically binding 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 the antigen-binding fragment of the antibody of the present invention using conventional techniques available in the art.

[0173] In one aspect, the present invention includes multispecific antigen-binding molecules or antigen-binding fragments thereof, wherein one immunoglobulin specificity is specific for the extracellular domain of PD-1 or a fragment thereof, and another immunoglobulin specificity is specific for binding outside the extracellular domain of PD-1, which is specific for a second therapeutic target or is conjugated to a therapeutic agent. The other immunoglobulin specificity may be specific for a second target antigen. The second target antigen may be in the same cell as PD-1 or in a different cell. In one embodiment, the second target cell is in a population of an immune cell other than a T cell, such as a B cell, an antigen-presenting cell, a monocyte, a macrophage, or a dendritic cell.In some embodiments, the second target antigen may be on a tumor cell or a tissue cell in an autoimmune disease, or on a cell infected with a virus.

[0174] In another aspect, the present invention relates to multispecific antigen-binding molecules or antigen-binding fragments thereof, having a first antigen-binding specificity that provides binding to PD-1, and a second antigen-binding specificity that provides binding to a T-cell receptor, a B-cell receptor, or an Fc receptor. In a related aspect, the present invention relates to multispecific antigen-binding molecules or antigen-binding fragments thereof, having a first antigen-binding specificity that provides binding to PD-1, and a second antigen-binding specificity that provides binding to another T-cell coinhibitor, such as LAG-3, CTLA-4, BTLA, CD-28, 2B4, LY108, TIGIT, TIM3, LAIR1, ICOS, and CD160.

[0175] In another aspect, the present invention relates to multispecific antigen-binding molecules or antigen-binding fragments thereof, having a first antigen-binding specificity that provides binding to PD-1, and a second antigen-binding specificity that provides binding to an autoimmune tissue-specific antigen. In some embodiments, the antibodies can be activating or agonistic antibodies.

[0176] Any of the multispecific antigen-binding molecules of the present invention or variants thereof can be constructed using standard molecular biological techniques (e.g., recombinant DNA and protein expression technology) known to those skilled in the art.

[0177] In some embodiments, PD-1-specific antibodies are generated in a bispecific format (“bispecific antibodies”), in which variable regions that bind to different domains of PD-1 are linked to one another to impart dual-domain specificity within a single binding molecule. Appropriately designed bispecific constructs can enhance the overall inhibitory efficacy of PD-1 by increasing both binding specificity and avidity. Variable regions with specificity for individual domains (e.g., segments of the N-terminal domain) or that can bind to different regions within a single domain are paired in a structural scaffold, allowing each region to simultaneously bind to distinct epitopes or to different regions within a single domain.

[0178] In one example of bispecific constructs, heavy chain variable regions (VH) from a binding compound with specificity for one domain are recombined with light chain variable regions (VL) from a number of binding compounds with specificity for a second domain to identify unrelated VL partners that can be combined with the original VHs without compromising the original specificity for that VH. Thus, a single VL segment (e.g., VL1) can be combined with two different VH domains (e.g., VH1 and VH2) to create a bispecific construct consisting of two binding arms (VH1-VL1 and VH2-VL1). The use of a single VL segment reduces the complexity of the system and thereby increases the efficiency of the cloning, expression, and purification processes used to produce the bispecific antibody (see, e.g., US 13 / 022759 and US 2010 / 0331527).

[0179] Alternatively, antibodies that bind to one or more domains and to a second target, such as, but not limited to, a second antibody different from anti-PD-1, can be produced in a bispecific format using the methods described herein or other methods known to those skilled in the art. The variable regions of the antibody that bind to different regions can be linked together with variable regions that bind to corresponding sites, such as the extracellular domain of PD-1, to impart dual antigen specificity within a single binding molecule. Appropriately designed bispecific antibodies of this type perform dual functions.Variable regions with specificity for the extracellular domain are combined with a variable region with specificity outside the extracellular domain and paired in a structural scaffold that allows each variable region to bind to distinct antigens.

[0180] An exemplary format of a bispecific antibody that can be used in the context of the present invention includes the use of a first CH3 domain of an immunoglobulin (Ig) and a second CH3 domain of an Ig, wherein the first and second CH3 domains of an Ig differ from each other by at least one amino acid, and wherein at least the difference by one amino acid reduces the binding of the bispecific antibody to protein A compared to a bispecific antibody that does not have a difference in the amino acid.

[0181] In one embodiment, the first Ig CH3 domain binds to protein A, and the second Ig CH3 domain comprises a mutation that reduces or abolishes binding to protein A, such as an H95R modification (according to the IMGT exon numbering system; H435R according to the EU numbering system). The second CH3 may further comprise a Y96F modification (according to the IMGT numbering system; Y436F according to the EU numbering system). Additional modifications that can be found in the second CH3 include: D16E, L18M, N44S, K52N, V57M, and V82I (according to the IMGT numbering system; D356E, L358M, N384S, K392N, V397M, and V422I according to the EU numbering system) in the case of IgG1 antibodies; N44S, K52N and V82I (according to the IMGT numbering system; N384S, K392N and V422I according to the EU numbering system) in the case of IgG2 antibodies; and Q15R, N44S, K52N, V57M, R69K, E79Q and V82I (according to the IMGT numbering system; Q355R, N384S, K392N, V397M, R409K, E419Q and V422I according to the EU numbering system) in the case of IgG4 antibodies.Variations of the bispecific antibody format described above are contemplated within the scope of the present invention.

[0182] Other exemplary bispecific formats that can be used in the context of the present invention include, for example, scFv or diabody-based bispecific formats, IgG-scFv fusion constructs, dual variable domain (DVD)-Ig, Quadroma, knob-in-hole, common light chain (e.g., common light chain according to knob-in-hole technology, etc.), CrossMab, CrossFab, (SEED)body, leucine zipper, DuoBody, IgG1 / IgG2, dual-acting Fab (DAF)-IgG, and Mab2 bispecific formats (see, for example, Klein et al. 2012, mAbs, 4:6, 1-11, and references cited therein for a review of the aforementioned formats).Bispecific antibodies can also be constructed using peptide / nucleic acid conjugation, for example, in which unnatural amino acids with orthogonal chemical reactivity are used to generate site-specific antibody-oligonucleotide conjugates that then self-assemble into multimeric complexes with defined composition, valence, and geometry (see, e.g., Kazane et al., J. Am. Chem. Soc. [Epub: Dec. 4 2012]).

[0183] The present invention relates to a human monoclonal anti-PD-1 antibody conjugated to a therapeutic moiety ("immunoconjugate"), such as a cytotoxin or chemotherapeutic agent, for the treatment of cancer. Within the framework of the present invention, the term "immunoconjugate" refers to an antibody that is chemically or biologically linked to a cytotoxin, radioactive agent, cytokine, interferon, target or reporter molecule, enzyme, toxin, peptide or protein, or therapeutic agent. The antibody can be linked to a cytotoxin, radioactive agent, cytokine, interferon, target or reporter molecule, enzyme, toxin, peptide, or therapeutic agent at any site on the molecule, provided that it is capable of binding to its target. Examples of immunoconjugates include antibody-drug conjugates and antibody-toxin fusion proteins.

[0184] When determining the type of therapeutic molecule that can be conjugated to an anti-PD-1 antibody, the pathological condition to be treated and the desired therapeutic effect to be achieved will be taken into account. Examples of suitable agents for forming immunoconjugates are known in the art; see, for example, WO 05 / 103081.

[0185] Therapeutic administration and formulations

[0186] In one aspect, the present invention relates to a pharmaceutical composition comprising at least one selected from the group consisting of any PD-1-binding agent, an anti-PD-1 antibody or an antigen-binding fragment thereof, an immunoglobulin heavy chain variable region polypeptide, an immunoglobulin light chain variable region polypeptide, an immunoglobulin heavy chain polypeptide, an immunoglobulin light chain polypeptide, and a multispecific antigen-binding molecule, an immunoconjugate, a chimeric antigen receptor, an engineered T cell receptor, or an oncolytic virus containing the above-mentioned agent, antibody, fragment, or polypeptide according to the present invention, and a pharmaceutically acceptable excipient or carrier.

[0187] The said pharmaceutical composition may be a pharmaceutical composition for the prevention, alleviation or treatment of a tumor, cancer, metastatic tumor, metastatic cancer, autoimmune disease, neurological disease, neurodegenerative disease or infectious disease.

[0188] In one aspect, the present invention relates to a pharmaceutical composition further comprising a second therapeutic agent in said pharmaceutical composition.

[0189] In one aspect, the present invention relates to a method for the prevention, amelioration and / or treatment of a tumor, cancer, metastatic tumor, metastatic cancer, autoimmune disease, neurological disease, neurodegenerative disease or infectious disease, comprising the step of administering to a subject at least one selected from the group consisting of any PD-1-binding agent, an anti-PD-1 antibody or antigen-binding fragment thereof, an immunoglobulin heavy chain variable region polypeptide, an immunoglobulin light chain variable region polypeptide, an immunoglobulin heavy chain polypeptide, an immunoglobulin light chain polypeptide, and a multispecific antigen-binding molecule, an immunoconjugate, a chimeric antigen receptor, an engineered T cell receptor or an oncolytic virus comprising the above-mentioned agent, antibody, fragment or polypeptide according to the present invention,and a pharmaceutically acceptable excipient or carrier.

[0190] A PD-1-binding agent, an anti-PD-1 antibody or an antigen-binding fragment thereof, an immunoglobulin heavy chain variable region polypeptide, an immunoglobulin light chain variable region polypeptide, an immunoglobulin heavy chain polypeptide, an immunoglobulin light chain polypeptide, and a multispecific antigen-binding molecule, an immunoconjugate, a chimeric antigen receptor, an engineered T cell receptor, or an oncolytic virus comprising the above-mentioned agent, antibody, fragment, or polypeptide according to the present invention are useful, among other things, for the treatment, prevention, and / or amelioration of any disease, disorder, or pathological condition associated with or mediated by the expression, signaling pathway, or activity of PD-1, or treatable by blocking the interaction of PD-1 and a PD-1 ligand (e.g., PD-L1 or PD-L2), or otherwise inhibiting the activity and / or signaling pathway PD-1.

[0191] The pharmaceutical composition of the present invention can be used for the prevention, treatment or alleviation of pathological conditions associated with PD-1.

[0192] Pathological conditions associated with PD-1 may be, but are not limited to, a tumor, cancer, metastatic tumor, metastatic cancer, autoimmune disease, neurological disease, neurodegenerative disease, or infectious disease.

[0193] PD-1-associated pathological conditions may include, but are not limited to, 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 cancer, 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 and classical Hodgkin lymphoma (CHL), primary mediastinal large B-cell lymphoma, T-cell / histiocyte-rich B-cell lymphoma, Epstein-Barr virus (EBV)-positive and Epstein-Barr virus (EBV)-negative post-transplant lymphoproliferative disorder (PTLD) or EBV-associated diffuse large B-cell lymphoma (DLBCL), plasmablastic lymphoma,extranodal NK / T-cell lymphoma, nasopharyngeal carcinoma or primary effusion lymphoma associated with human herpes virus 8 (HHV8), or other hematological malignancies including Hodgkin's lymphoma, neoplasms of the central nervous system including primary central nervous system (CNS) lymphoma, spinal cord tumor and brainstem glioma.

[0194] The pharmaceutical composition of the present invention can be used to treat the symptoms of early or late stage cancer. In one aspect, the antibody or antigen-binding fragment thereof can be used to treat metastatic cancer. The pharmaceutical composition of the present invention is useful for suppressing, inhibiting, or reducing solid tumors and blood cancer. In some embodiments, treatment with the pharmaceutical composition causes at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% tumor regression in a subject. In some embodiments, the pharmaceutical composition can be used to prevent tumor recurrence. In some embodiments, the pharmaceutical composition is useful for increasing the overall survival of a subject with cancer.In some embodiments, the pharmaceutical composition is useful for reducing toxicity caused by chemotherapy or radiation therapy while maintaining long-term survival in cancer patients.

[0195] PD-1-associated autoimmune disease may be, for example, but not limited to, lupus, systemic lupus erythematosus, Sjogren's syndrome, arthritis, rheumatoid arthritis, asthma, COPD, pelvic inflammatory disease, Alzheimer's disease, inflammatory bowel disease, Crohn's disease, ulcerative colitis, Peyronie's disease, celiac disease, gallbladder disease, pilonidal disease, peritonitis, psoriasis, psoriatic arthritis, vasculitis, adhesions after surgery, stroke, type 1 diabetes mellitus, Lyme disease, meningoencephalitis, autoimmune uveitis, multiple sclerosis, Guillain-Barré syndrome, atopic dermatitis, autoimmune hepatitis, fibrosing alveolitis, Graves' 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, graft rejection, cardiac disease including ischemic diseases such as myocardial infarction and atherosclerosis, intravascular coagulation, bone resorption, osteoporosis, osteoarthritis, periodontitis and hypochlorhydria, infertility due to maternal-fetal intolerance, vitiligo, myasthenia gravis or systemic sclerosis.

[0196] It has been suggested that IFNγ-dependent systemic immune response is beneficial for the treatment of Alzheimer's disease and other central nervous system pathologies that share common neuroinflammatory components, and the publication of international application WO 2015 / 136541 discloses the use of anti-PD-1 antibodies for the treatment of Alzheimer's disease. International application publication WO 2017 / 220990 discloses that blockade of the PD-1 / PD-L1 immune checkpoint pathway results in increased secretion of IFNγ by IFNγ-producing cells, and that increased IFNγ activity can selectively recruit leukocytes to the choroid plexus of the brain and infiltrate T cells and monocytes into the injured CNS, targeting these immune cells to sites of neurodegenerative pathology and neuroinflammation, and can modulate the environment to become less toxic and more favorable for the removal of toxic agents, neuronal rescue, regeneration, and repair.

[0197] PD-1 is associated not only with cognitive function, learning, and memory in the central nervous system, but also with other central nervous system disorders such as brain tumor, Alzheimer's disease, stroke, spinal cord injury, multiple sclerosis, glioblastoma, melanoma, and pain (Zunli Zhao et al., “Emerging role of PD-1 in the central nervous system and brain diseases,” Neurosci. Bull., 2021.04.20, published online). In addition, PD-1 has been reported to be associated with retinal ganglion cells, which are known to degenerate in neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, multiple sclerosis, amyotrophic lateral sclerosis (ALS), etc. (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).Anti-PD-1 antibody is known to have potential in neurodegenerative diseases, as shown by attenuating cognitive impairment and pathological characteristics in the 5XFAD mouse model of Alzheimer's disease and a mouse model of dementia (Michal Schwartz et al., “Potential Immunotherapy for Alzheimer's Disease and Age-Related Dementia,” Dialogues in Clinical Neuroscience, 21(1), 21, 2019), and 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)).

[0198] In one aspect of the present invention, the pharmaceutical composition of the present invention can be used for the prevention, alleviation and treatment of neurological diseases and neurodegenerative diseases associated with PD-1, including, but not limited to, cognitive impairment, brain tumor, Alzheimer's disease, dementia, stroke, spinal cord injury, amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, multiple sclerosis, glioblastoma, melanoma, pain and memory loss.

[0199] In one aspect of the present invention, the pharmaceutical composition of the present invention is suitable for treating a subject suffering from a chronic viral infection. In some embodiments, it is suitable for reducing viral titer and / or restoring exhausted T cells in a host in accordance with the present invention.

[0200] PD-1-associated infectious disease may be a chronic viral infection, including viral infection such as hepatitis B, hepatitis C, herpes virus, Epstein-Barr virus, HIV, cytomegalovirus, herpes simplex virus type 1, herpes simplex virus type 2, human papillomavirus, adenovirus, Kaposi-West sarcoma-associated with herpes virus epidemics, ring virus (torketenovirus), lymphocytic choriomeningitis virus (LCMV), JC virus or BK virus.

[0201] In one aspect, the pharmaceutical composition of the present invention can be used to treat a viral infection such as that caused by simian immunodeficiency virus (SIV) in a monkey subject such as a cynomolgus macaque.

[0202] In one aspect, the pharmaceutical composition of the present invention can be administered to alleviate or prevent the severity of one or more symptoms or conditions of a disease or disorder. This document also contemplates the prophylactic use of the pharmaceutical composition of the present invention for patients at risk of developing a disease or disorder, such as cancer, autoimmune disease, and chronic viral infection.

[0203] In another embodiment of the present invention, the pharmaceutical composition of the present invention can be used as an adjuvant therapy with any other agent or any other therapy known to those skilled in the art suitable for the treatment of cancer, an autoimmune disease or a viral infection.

[0204] The pharmaceutical composition of the present invention can be administered with suitable carriers, excipients and other agents that are included in the formulations to provide improved transport, delivery, tolerability, etc. Many suitable formulations can be found in a pharmaceutical formulary known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid-containing vesicles (cationic or anionic) (such as LIPOFECTIN™), DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, carbowax emulsions (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax (see Powell et al., “Compendium of excipients for parentheral products,” PDA (1998) J. Pharm. Sci. Technol., 52:238-311).

[0205] The antibody dosage may vary depending on the age and size of the subject to be administered, the target disease, the general health condition, the route of administration, etc. When the antibody of the present invention is used to treat a disease or disorder in an adult patient or to prevent such a disease, it is preferable to administer the antibody of the present invention generally at a single dose of about 0.1 to about 60 mg / kg body weight, more preferably about 5 to about 60, about 10 to about 50, or about 20 to about 50 mg / kg body weight. The frequency and duration of treatment can be adjusted depending on the severity of the pathological condition.In some embodiments, an antibody or antigen-binding fragment thereof of the present invention can be administered as an initial dose of at least about 0.1 mg to about 800 mg, about 1 mg to about 500 mg, about 5 mg to about 300 mg, or about 10 mg to about 200 mg, up to about 100 mg, or up to about 50 mg.In some embodiments, the initial dose may be followed by administration of a second or multiple subsequent doses of the antibody or antigen-binding fragment thereof in an amount that may be about the same as or less than the initial dose, wherein the subsequent doses are separated by an interval of at least 1-3 days; at least one 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.

[0206] The pharmaceutical composition of the present invention can be administered using various delivery systems, such as encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, and receptor-mediated endocytosis (see, for example, Wu et al. (1987) J. Biol. Chem., 262:4429-4432). The routes of administration include, but are not limited to, intradermal, transdermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural and oral routes. The composition can be administered by any conventional route, such as by infusion or bolus injection, or by absorption through epithelial or mucocutaneous membranes (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and can be administered together with other biologically active agents. Administration can be systemic or local.

[0207] The pharmaceutical composition can also be delivered in a vesicle, in particular in a liposome (see, for example, Langer (1990) Science, 249:1527-1533). The use of nanoparticles for delivering the antibodies of the present invention is also contemplated herein. Nanoparticles conjugated with antibodies can be used for both therapeutic and diagnostic purposes. Nanoparticles conjugated with antibodies and methods for their preparation and use 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 designed and conjugated with antibodies contained in pharmaceutical compositions to target tumor cells, autoimmune disease tissue cells, or virus-infected cells.Nanoparticles for drug delivery have also been described, for example, in U.S. Patent Nos. 8,257,740 or 8,246,995, each of which is herein incorporated in its entirety.

[0208] In certain situations, the pharmaceutical composition may be delivered in a controlled-release system. In one embodiment, a pump may be used. In another embodiment, polymeric materials may be used. In yet another embodiment, the controlled-release system may be placed in close proximity to the target of the composition, thereby requiring only a portion of the systemic dose. Injectable preparations may include dosage forms for intravenous, subcutaneous, intradermal, intracranial, intraperitoneal, and intramuscular injections, drip infusions, etc. These injectable preparations can be prepared by generally known methods. For example, the injectable preparations can be prepared by dissolving, suspending, or emulsifying the antibody or salt thereof described above in a sterile aqueous medium or oil medium commonly used for injection.As an aqueous injection medium, there are used, for example, physiological saline, isotonic solution containing glucose and other auxiliary agents, which can be used in combination with an appropriate solubilizing agent such as an alcohol (e.g., ethanol), a polyhydric alcohol (e.g., propylene glycol, polyethylene glycol), a nonionic surfactant [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)], etc. As an oil medium, there are used sesame oil, soybean oil, etc., which can be used in combination with a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc. The injection preparation thus prepared is preferably placed in an appropriate ampoule.

[0209] The pharmaceutical composition of the present invention can be administered subcutaneously or intravenously using a standard needle and syringe. Furthermore, for subcutaneous delivery, a syringe pen is readily used to deliver the pharmaceutical composition of the present invention. Such a syringe pen can be reusable or disposable. A reusable syringe pen typically uses a replaceable cartridge containing the pharmaceutical composition. Once the pharmaceutical composition in the cartridge has been administered and the cartridge is empty, the empty cartridge can simply be discarded and replaced with a new cartridge containing the pharmaceutical composition. The syringe pen can then be reused. A disposable syringe pen does not have a replaceable cartridge.In contrast, a single-use injection pen delivery device is supplied pre-filled with the pharmaceutical composition contained in a reservoir within the device. Once the reservoir is emptied of the pharmaceutical composition, the entire device is discarded. Numerous reusable injection pens and autoinjectors are used for subcutaneous delivery of the pharmaceutical composition of the present invention. Examples include, but are not limited to, AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ pen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75 / 25™ pen, HUMALOG™ pen, HUMALIN 70 / 30™ pen (Eli Lilly and Co., Indianapolis, India), NOVOPEN™ I, II, and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), BD™ pen (Becton Dickinson, Franklin Lakes, NJ), OPTIPEN™, OPTIPEN PRO™, OPTIPEN STARLET™, and OPTICLIK™ (Sanofi-Aventis, Frankfurt, Germany).Examples of disposable injection pens used for subcutaneous delivery of the pharmaceutical composition of the present invention include, but are not limited to, the SOLOSTAR™ injection pen (Sanofi-Aventis), FLEXPEN™ (Novo Nordisk), KWIKPEN™ (Eli Lilly), SURECLICK™ autoinjector (Amgen, Thousand Oaks, CA), PENLET™ (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, LP), and the HUMIRA™ injection pen (Abbott Labs, Abbott Park, IL).

[0210] Preferably, the pharmaceutical compositions for oral or parenteral use described above are prepared in the form of dosage forms in a single dose corresponding to the dose of the active ingredients. Such dosage forms in a single dose include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. The amount of the antibody contained is usually about 5 to about 500 mg per dosage form in a single dose; in particular, in the form of an injection, it is preferable that the antibody be contained from about 5 to about 100 mg, and from about 10 to about 250 mg for other dosage forms.

[0211] In one aspect, the present invention relates to a method for modulating an immune response in a subject, comprising administering to the subject at least one selected from the group consisting of any PD-1-binding agent, an anti-PD-1 antibody or antigen-binding fragment thereof, an immunoglobulin heavy chain variable region polypeptide, an immunoglobulin light chain variable region polypeptide, an immunoglobulin heavy chain polypeptide, an immunoglobulin light chain polypeptide, and a multispecific antigen-binding molecule, an immunoconjugate, a chimeric antigen receptor, an engineered T cell receptor, or an oncolytic virus comprising the above-mentioned agent, antibody, fragment, or polypeptide according to the present invention.

[0212] In one aspect, the present invention relates to the use of a PD-1-binding agent, an anti-PD-1 antibody or an antigen-binding fragment thereof, an immunoglobulin heavy chain variable region polypeptide, an immunoglobulin light chain variable region polypeptide, an immunoglobulin heavy chain polypeptide, an immunoglobulin light chain polypeptide, and a multispecific antigen-binding molecule, an immunoconjugate, a chimeric antigen receptor, an engineered T cell receptor, or an oncolytic virus comprising the above-mentioned agent, antibody, fragment, or polypeptide according to the present invention, in the manufacture of a medicament for the prevention, alleviation, or treatment of a tumor, cancer, metastatic tumor, metastatic cancer, autoimmune disease, neurological disease, neurodegenerative disease, or infectious disease.

[0213] In one aspect, the present invention also relates to a method for inhibiting the growth of tumor cells in a subject, comprising administering to the subject at least one selected from the group consisting of a PD-1-binding agent, an anti-PD-1 antibody or an antigen-binding fragment thereof, an immunoglobulin heavy chain variable region polypeptide, an immunoglobulin light chain variable region polypeptide, an immunoglobulin heavy chain polypeptide, an immunoglobulin light chain polypeptide, and a multispecific antigen-binding molecule, an immunoconjugate, a chimeric antigen receptor, an engineered T cell receptor, or an oncolytic virus comprising the above-mentioned agent, antibody, fragment, or polypeptide according to the present invention, in a therapeutically effective amount to inhibit the growth of tumor cells.

[0214] Combination administration

[0215] In one aspect of the present invention, the pharmaceutical composition of the present invention provides a pharmaceutical composition further comprising a second therapeutic agent.

[0216] In various embodiments, the second combination therapeutic agent may be an anti-PD-L1 antibody, a second anti-PD-1 antibody (e.g., nivolumab), a LAG-3 inhibitor, a CTLA-4 inhibitor (e.g., ipilimumab), a TIM3 inhibitor, a BTLA inhibitor, a TIGIT inhibitor, a CD47 inhibitor, an antagonist of another T cell coinhibitor or ligand (e.g., an anti-CD-28, 2B4, LY108, LAIR1, ICOS, CD160, or VISTA antibody), an indoleamine 2,3-dioxygenase (IDO) inhibitor, a vascular endothelial growth factor (VEGF) antagonist [e.g., a “VEGF trap” such as aflibercept or another VEGF inhibitory fusion protein as described in U.S. Patent No. 7,087,411, or an anti-T-cell antibody] VEGF or its antigen-binding fragment (eg, bevacizumab, ranibizumab), or a small molecule inhibitor of VEGF receptor kinase (eg, sunitinib, sorafenib, or pazopanib)], an Ang2 inhibitor (eg, nesvacumab), an inhibitor of transforming growth factor beta (TGFβ),an epidermal growth factor receptor (EGFR) inhibitor (e.g., erlotinib, cetuximab), a costimulatory receptor agonist (e.g., a glucocorticoid-induced TNFR-related protein agonist), an antibody to a tumor-specific antigen (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), a vaccine (e.g., Bacillus Calmette-Guerin tumor vaccine), an adjuvant to enhance antigen presentation (e.g., granulocyte-macrophage colony-stimulating factor), a bispecific antibody (e.g., CD3×CD20 bispecific antibody, PSMA×CD3 bispecific antibody), cytotoxin, chemotherapeutic agent (e.g., dacarbazine, temozolomide, cyclophosphamide, docetaxel, doxorubicin, daunorubicin, cisplatin, carboplatin, gemcitabine, methotrexate, mitoxantrone, oxaliplatin, paclitaxel, and vincristine), cyclophosphamide,radiation therapy, an IL-6R inhibitor (e.g., sarilumab), an IL-4R inhibitor (e.g., dupilumab), an IL-10 inhibitor, a cytokine such as IL-2, IL-7, IL-21, and IL-4. 15, an antibody-drug conjugate (ADC) (e.g., anti-CD19-DM4 ADC, anti-DS6-DM4 ADC), an anti-inflammatory drug (e.g., corticosteroids, non-steroidal anti-inflammatory drugs), a dietary supplement such as antioxidants, or a palliative in the treatment of cancer.

[0217] In some embodiments, the second therapeutic agent may include antitumor vaccines, including dendritic cell-based vaccines, oncolytic viruses, tumor cell-based vaccines, etc., to enhance the antitumor response. Examples of antitumor vaccines may include the MAGE3 vaccine for melanoma and bladder cancer, the MUC1 vaccine for breast cancer, EGFRv3 (e.g., rindopepimut) for brain cancer (including glioblastoma multiforme), or ALVAC-CEA (for CEA+ cancer).

[0218] Methods and kits for diagnosis and detection

[0219] In one aspect, the present invention relates to a kit for treating, diagnosing or detecting a disease, comprising at least one selected from the group consisting of any PD-1-binding agent, an anti-PD-1 antibody or an antigen-binding fragment thereof, an immunoglobulin heavy chain variable region polypeptide, an immunoglobulin light chain variable region polypeptide, an immunoglobulin heavy chain polypeptide, an immunoglobulin light chain polypeptide, and a multispecific antigen-binding molecule, an immunoconjugate, a chimeric antigen receptor, an engineered T cell receptor or an oncolytic virus containing the above-mentioned agent, antibody, fragment or polypeptide according to the present invention.

[0220] The anti-PD-1 antibodies of the present invention can be used to detect and / or quantify PD-1 in a sample, for example, for diagnostic purposes. In some embodiments, the use of one or more antibodies of the present invention in assays for detecting a disease or disorder, such as cancer, an autoimmune disease, or a chronic viral infection, is contemplated. Examples of diagnostic assays for PD-1 can include, for example, contacting a sample obtained from a patient with an anti-PD-1 antibody of the present invention, wherein the anti-PD-1 antibody comprises a detectable label or reporter molecule, or is used as a capture ligand for the selective isolation of PD-1 from patient samples.

[0221] Alternatively, an unlabeled anti-PD-1 antibody can be used for diagnostic purposes in combination with a secondary antibody that is itself detectably labeled. The detectable label or reporter molecule can be a radioisotope such as 3H, 14C, 32P, 35S, or 125I; a fluorescent or chemiluminescent molecule such as fluorescein isothiocyanate or rhodamine; or an enzyme such as alkaline phosphatase, β-galactosidase, horseradish peroxidase, or luciferase.

[0222] Specific illustrative assays that can be used to detect or quantify PD-1 in a sample include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence-activated cell sorting (FACS).

[0223] Samples that can be used in the PD-1 diagnostic assays of the present invention include any tissue or fluid sample obtained from a patient that contains detectable amounts of either the PD-1 protein or its fragments under normal or pathological conditions. Typically, PD-1 levels in a given sample obtained from a healthy subject (e.g., a subject not suffering from cancer or an autoimmune disease) will be measured to initially establish a baseline or standard PD-1 level. This baseline PD-1 level can then be compared with PD-1 levels measured in samples obtained from subjects suspected of having a cancer-related condition or symptoms associated with such a condition.

[0224] The polypeptide, PD-1 binding agent, antibody, etc. specific for PD-1 of the present invention may not contain additional labels or groups, or may contain an N-terminal or C-terminal label or group. In one embodiment, the label or group is biotin. In a binding assay, the location of the label (if present) can be determined by the orientation of the peptide relative to the surface to which the peptide is bound. For example, if the surface is coated with avidin, the peptide containing the N-terminal biotin will be oriented such that the C-terminal region of the peptide is away from the surface.

[0225] One aspect of the present invention relates to the use of the disclosed antibodies as markers for predicting the prognosis of cancer or autoimmune disease in patients. The polypeptide, PD-1-binding agent, antibody, and the like of the present invention can be used in diagnostic assays to assess the prognosis of cancer in a patient and predict survival.

[0226] Next, a method for producing an antibody according to specific embodiments of the present invention will be described in more detail. However, this is provided as an example of the invention and does not limit the scope of the invention. Those skilled in the art will appreciate that various modifications to the embodiments are possible within the scope of the invention.

[0227] Examples

[0228] Example 1. Obtaining an immunogen and creating cell lines

[0229] Protein antigen

[0230] The extracellular domain of human PD-1 was synthesized from the pCMV3-C-FLAG (Sino) vector containing the cDNA of human PD-1 by PCR and inserted into the pEM.CMV-SF-IRES-EGFP vector. The constructed vector was transfected into the CHO-S cell line, and cells producing the human PD-1 extracellular domain were selected based on EGFP expression using flow cytometry. Human PD-1 was purified and quantified from cell culture media using FLAG-tagged affinity chromatography.

[0231] Cell line antigen

[0232] The pCMV3-C-FLAG vector (Sino) containing human PD-1 cDNA was transfected into the CT26 cell line, derived from a malignant colon tumor in BALB / c mice. Cells expressing human PD-1 were selected by flow cytometry using the anti-human PD-1 antibody APC-cy7. Single clones were obtained by limiting dilution in a 96-well plate.

[0233] Obtaining control groups (Keytruda, Opdivo)

[0234] Keytruda, a product of InvivoGen (human IgG4 isotype (S228P), catalog number hpd1pe-mab14) or a clinical product from MSD was purchased and used as a control antibody. Opdivo, a clinical product from BMS, was purchased and used.

[0235] Creation of cell lines

[0236] The pCMV3-C-FLAG vector (Sino) containing human or mouse PD-1 cDNA was transfected into the CHO-S cell line. Cells expressing human or mouse PD-1 were selected by flow cytometry using the anti-human PD-1 antibody APC-cy7 or the anti-mouse PD-1 antibody APC-cy7. Single clones were obtained by limiting dilution in a 96-well plate.

[0237] Example 2. Construction of PD-1 knockout mice PD-1 knockout mice were constructed using the CRISPR-CAS system in mice (C57BL / 6N). After obtaining guide RNA specific for the mouse PD-1 gene sequence and testing the ability of mouse PD-1 DNA to be cleaved in vitro, the guide RNA and Cas9 protein were microinjected into the zygote. Surviving zygotes injected with guide RNA were selected and transplanted into the oviducts of surrogate mother mice. After transplantation, the tails of 2-week-old mice were clipped to isolate genomic DNA, and deletion of the mouse PD-1 gene was confirmed by PCR.

[0239] Example 3. Obtaining hybridomas for antibody production

[0240] Immunization of mice

[0241] Eight-week-old female PD-1 knockout mice were immunized with human PD-1 antigen to induce antibody production. The antigens used for immunization were purified human PD-1 antigen and CT26 cell line-derived human PD-1 antigen.

[0242] For immunization with protein antigen, 50 μg of protein antigen per mouse was mixed with 50 μg of Titermax gold adjuvant and administered subcutaneously (s.c.) to the left and right hindquarters of mice. For immunization with cell line antigen, X-ray irradiation of the cell line antigen was performed 1 day before immunization to suppress cell growth, and 1×10 was administered intraperitoneally (i.p.). 6 cells per mouse. Immunizations were administered at 3-week intervals, and blood was collected from each mouse via submandibular blood sampling 10 days after immunization. Antibody titers in serum obtained from whole blood were measured using ELISA.

[0243] Cell fusion and cell generation (hybridomas) to produce monoclonal antibodies

[0244] B lymphocytes were isolated from the spleens of mice immunized with human PD-1 and then fused with cultured myeloma cells (sp2 / 0). The fused cells were cultured in a medium supplemented with hypoxanthine, aminopterin, and thymidine (HAT medium), and only hybridomas representing the fusion of myeloma and B lymphocytes were selectively selected and cultured.

[0245] Among the resulting hybridoma cells, a hybridoma producing an antibody that reacts with the human PD-1 antigen was identified using protein-based ELISA. The hybridoma interacting with human PD-1 was cloned multiple times using the limiting dilution method, yielding cells (hybridomas) producing a monoclonal antibody (1G1) reactive with the human PD-1 antigen.

[0246] Example 4. Binding analysis of hybridoma 1G1 with PD-1

[0247] To confirm the binding of the monoclonal antibody-producing cell (hybridoma) (1G1) obtained in Example 3 to the cell-surface expressed PD-1 conformational protein, a cell-based ELISA and flow cytometry assay were performed. Keytruda (Invivogen) was used as a control anti-hPD-1 antibody.

[0248] ELISA assay summary: 10,000 CHO-S cells expressing human or mouse PD-1 were coated onto collagen-coated 96-well plates (ThermoFisher) and incubated overnight at 37°C. Coated cells were fixed with 8% paraformaldehyde at room temperature for 15 min. After blocking and washing, Keytruda or hybridoma supernatant was added to the coated plate and incubated for 2 h at room temperature. After washing, secondary antibody was added and the plate was incubated overnight at 4°C. HRP-conjugated mouse anti-human IgG Fc (GenScript) was used as the secondary antibody in wells containing Keytruda, and HRP-conjugated goat anti-mouse IgG Fc (ThermoFisher) was used in wells containing the hybridoma supernatant. After washing, TMB substrate (abcam) was added, and the color reaction was stopped with STOP solution (abcam).Absorbance at 450 / 650 nm was measured using a microplate reader (ThermoFisher).

[0249] Flow cytometry analysis summary: CHO-S cells expressing human or mouse PD-1 were loaded into a 96-well V-bottom plate (Corning) at a density of 1×10 6 cells / well, Keytruda or hybridoma supernatant was added and incubated at 4°C for 1 h. After washing with 1X PBS / 2% BSA, secondary antibody was added and the cells were incubated at 4°C for 1 h. PE-conjugated anti-human IgG Fc (Biolegend) was used as the secondary antibody in wells with Keytruda, and AF647 goat anti-mouse IgG (H+L) (ThermoFisher) was used in wells with hybridoma supernatant. Cells were then washed and resuspended in 1X PBS / 2% BSA and analyzed using flow cytometry (BD) and FlowJo software.

[0250] Figure 1 shows graphs showing the results of the binding analysis of the hybridoma antibody 1G1 obtained in Example 3 to human PD-1 or mouse PD-1 on the cell surface using ELISA. Figure 2 shows graphs showing the results of the binding analysis of the hybridoma antibody obtained in Example 3 to human PD-1 or mouse PD-1 on the cell surface using flow cytometry. Antibody 1G1 bound to human PD-1 and mouse PD-1 with high affinity.

[0251] On the other hand, Keytruda, used as a control antibody, bound only to human cell surface PD-1, while the hybridoma antibody 1G1 obtained in Example 3 bound to both human PD-1 and mouse cell surface PD-1, showing cross-reactivity.

[0252] Example 5. Ligand blocking assay of hybridoma antibody 1G1

[0253] To determine the extent to which the cell (hybridoma) producing the monoclonal antibody (1G1) obtained in Example 3 blocks PD-L1 binding to cell surface PD-1, cell-based ELISA and flow cytometry were performed. Keytruda (Invivogen) was used as a control anti-hPD-1 antibody.

[0254] Summary of the ELISA assay: 10,000 CHO-S cells expressing human or mouse PD-1 were coated onto collagen-coated 96-well plates and incubated overnight at 37°C. The coated cells were fixed with 8% paraformaldehyde at room temperature for 15 min. After blocking and washing, human llama Fcβ PD-L1 protein was added to the wells coated with human PD-1-expressing CHO-S cells, and mouse Fcβ PD-L1 protein was added to the wells coated with mouse PD-1-expressing CHO-S cells; the plates were then incubated at room temperature for 20 min. Keytruda or hybridoma supernatant was added and incubated for 1 h and 20 min at room temperature.

[0255] After washing, the secondary antibody was added and incubated overnight at 4°C. HRP-conjugated mouse anti-llama IgG2 / IgG3 (antibody online) was used as the secondary antibody in the wells to which llama Fc PD-L1 protein was added, and HRP-conjugated mouse anti-human IgG Fc was added to the wells to which mouse Fc PD-L1 protein was added. After washing, TMB substrate (abcam) was added, and the color reaction was stopped with STOP solution (abcam). Absorbance at 450 / 650 nm was measured using a microplate reader (ThermoFisher).

[0256] Flow cytometry analysis summary: CHO-S cells expressing human or mouse PD-1 were loaded into a 96-well V-bottom plate (Corning) at a density of 1×10 6cells / well and a mixture of PD-L1 protein and Keytruda or hybridoma supernatant was added and incubated at 4°C for 1 h. A mixture of llama Fc PD-L1 protein and antibodies was added to wells loaded with CHO-S cells producing human PD-1, and a mixture of mouse Fc PD-L1 protein and antibodies was added to wells loaded with CHO-S cells producing mouse PD-1. After washing with 1X PBS / 2% BSA, secondary antibody was added and incubated at 4°C for 1 h.

[0257] FITC-conjugated goat anti-llama IgG (abcam) was used as the secondary antibody in wells containing a mixture of llama Fc, human PD-L1 protein, and antibodies, and PE-conjugated anti-human IgG Fc (Biolegend) was used in wells containing a mixture of mouse Fc, human PD-L1 protein, and antibodies. After washing, the cells were resuspended in 1X PBS / 2% BSA and analyzed using flow cytometry (BD) and FlowJo software.

[0258] Fig. 3 is a graph showing the results of an ELISA assay to determine how much the monoclonal cell (hybridoma) antibody 1G1 obtained in Example 3 blocks the binding of human PD-L1 and mouse PD-L1 to human PD-1 and mouse PD-1 on the cell surface, respectively.

[0259] Fig. 4 shows graphs showing the results of an analysis using flow cytometry regarding the extent to which the monoclonal cell (hybridoma) 1G1 antibody obtained in Example 3 blocks the binding of human PD-L1 and mouse PD-L1 to human PD-1 and mouse PD-L1 on the cell surface, respectively.

[0260] The control antibody, Keytruda, blocked only the binding of human PD-1 and cell surface PD-L1, whereas the hybridoma antibody 1G1 blocked the binding of both human PD-1 / PD-L1 and mouse PD-1 / PD-L1.

[0261] Example 6. Binding assay of purified hybridoma antibody 1G1 to PD-1 (ELISA)

[0262] To produce antibodies in the hybridoma, the hybridoma was cultured in DMEM (HyClone, Cytiva) containing 3% FBS with low IgG content (Gibco) for 1 week, centrifuged, and then filtered through a 0.22 μm filter (Millipore) to isolate the cell culture medium.

[0263] 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 particles (Cytiva) and eluted with IgG elution buffer (Thermo Scientific). To minimize protein damage due to the low pH of the IgG elution buffer (pH 2.5-3.0), the antibody was eluted into a tube containing 1 M Tris-HCl (pH 8.0), and the pH of the neutralized protein was measured using a pH indicator strip.

[0264] The purified antibody was concentrated using an Amicon 100K centrifugal filter (Merck), and protein identification and quantification were performed by Coomassie blue staining and bicinchoninic acid BCA assay (Thermo Scientific) (Fig. 5).

[0265] To assess 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 control anti-hPD-1 antibodies.

[0266] ELISA assay summary: 10,000 CHO-S cells expressing either human PD-1 or mouse PD-1 were coated onto a 96-well collagen-coated plate (Thermo Scientific) and incubated overnight at 37°C. Coated cells were fixed with 8% paraformaldehyde at room temperature for 15 min. After blocking and washing, Keytruda, Opdivo, and purified anti-PD-1 hybridoma antibody were added to the coated plate and incubated at room temperature for 2 h.

[0267] After washing, the secondary antibody was added and incubated overnight at 4°C. HRP-conjugated mouse anti-human IgG Fc (GenScript) was used as the secondary antibody for wells containing Keytruda or Opdivo, and HRP-conjugated goat anti-mouse IgG Fc (ThermoFisher) was used for wells containing purified anti-PD-1 hybridoma antibody. After washing, TMB substrate (abcam) was added, and the color reaction was stopped with STOP solution (abcam). Absorbance at 450 / 650 nm was measured using a microplate reader (ThermoFisher).

[0268] Figure 6 shows graphs showing the results of the binding assay using ELISA to measure the binding activity of purified hybridoma antibody 1G1 to human or mouse PD-1 cell surfaces. Based on the test results, the EC50 values ​​of purified mouse antibody 1G1 and control antibodies (Keytruda, Opdivo) for the antigen were calculated.

[0269] The purified anti-PD-1 hybridoma antibody (1G1) exhibited approximately 5-fold higher binding activity (28.13 pM) to the human PD-1 antigen compared with Keytruda (153.52 pM) and Opdivo (157.27 pM). In addition, the purified anti-PD-1 hybridoma antibody (1G1) bound to the mouse PD-1 antigen with a binding activity (31.72 pM) similar to that of human PD-1.

[0270] Keytruda Opdivo Purified hybridoma antibody 1G1 EC50: hPD-1 (pM) 153,52 157,27 28,13 EC50: mPD-1 (pM) not defined not defined 31,72

[0271] Example 7. Cross-reactivity to human immune checkpoints

[0272] To determine the specificity of the cell (hybridoma) producing monoclonal antibody 1G1 obtained in Example 3 to bind to PD-1 on the surface of human T cells, cross-reactivity with other immune checkpoints was analyzed using a protein-based ELISA. Keytruda (Invivogen) was used as a control antibody against hPD-1.

[0273] Human PD-1, CD28, CTLA-4, ICOS, and BTLA proteins were coated onto 96-well plates (Nunc) and incubated overnight at 4°C. After blocking and washing, Keytruda or hybridoma culture supernatant was added to the coated plates and incubated at 37°C for 1 h. After washing, secondary antibody was added and incubated at 37°C for 2 h. HRP-conjugated mouse anti-human IgG Fc (Genscript) was used as the secondary antibody in wells with Keytruda, and HRP-conjugated goat anti-mouse IgG Fc (ThermoFisher) was used in wells with hybridoma supernatant. After washing, TMB substrate (abcam) was added, and the color reaction was stopped with STOP solution (abcam). Absorbance at 450 / 650 nm was measured using a microplate reader (ThermoFisher).

[0274] Figure 7 shows the results of an ELISA assay measuring binding to human T cell surface immune checkpoints. The hybridoma antibody 1G1 specifically bound only to human PD-1, as did the control antibody (Keytruda).

[0275] Example 8. Sequencing of antibody produced by hybridoma cells

[0276] RNA was extracted from 1G1 hybridoma cells obtained in Example 3 using Triazole reagent. cDNA was synthesized from RNA using reverse transcriptase, and the VH and VL sequences of the antibody were amplified from the synthesized cDNA as follows:

[0277] The primers for sequence amplification were as follows:

[0278] Table 1

[0279] The PCR reaction was carried out as follows:

[0280] Table 2 Reaction system (50 µl) Reaction conditions cDNA 1 µl 95°C 5 min 1 cycle 10× Taq buffer 5 µl 95°C 1 min 30 cycles dNTP (2.5 mM) 4 µl 45°C 1 min Forward primer (100 pmol / µl) 1 µl 72°C 1 min Reverse primer (100 pmol / µl) 1 µl 72°C 5 min 1 cycle Taq (5 U / µl) 0.25 µl TDW 37.75 µl

[0281] The resulting PCR product (10 μl) was ligated with pCMV3 vector, and the VH and VL sequences of the hybridoma antibody were identified by sequencing using T7 primers (5'-TAATACGACTCACTATAGGG-3') and pCMV3_F (5'-CGAGGAGGATTTGATATTCAC-3').

[0282] The identified VH and VL sequences were as follows, and FR1-4 and CDR1-3 sequences were identified in both VH and VL according to the numbering system of Kabat.

[0283] Table 3

[0284] Example 9. Construction, expression, and purification of chimeric antibodies

[0285] A chimeric antibody was generated in which the constant region of a mouse anti-PD-1 antibody produced in a hybridoma was replaced with the constant region of a human antibody. The signal peptide and VH or VL sequences of the antibody were linked using overlapping PCR and ligated with the pTRIOZ-hIgG4 vector (Invivogen) expressing the constant region of human IgG4 (S228P) (SEQ ID NOs: 38-41).

[0286] The pTRIOZ-hIgG4 vector containing the VH and VL sequences of the antibody was introduced into the ExpiCHO expression system. TM (Thermo Scientific) for antibody expression. After transfection of the pTRIOZ-hIgG4 vector into ExpiCHO-S cells, the cells were cultured in ExpiCHO expression medium. TM, which did not contain FBS, and cell viability was monitored. Seven to ten days after transfection, cell cultures with at least 70% viability were centrifuged and filtered using a 0.22 μm filter (Millipore) to obtain cell culture medium. The cell culture medium was applied to a chromatography column (Bio Rad) containing Protein A particles (Thermo Scientific) and eluted with IgG elution buffer (Thermo Scientific). To minimize protein damage due to the low pH (pH 2.5-3.0) of the IgG elution buffer, the antibody was eluted into a tube containing 1 M Tris-HCl (pH 8.0), and the pH of the neutralized protein was measured using a pH indicator strip. The purified antibody was concentrated using an Amicon 50K centrifugal filter (Merck), and protein identification and quantification were performed by Coomassie blue staining and bicinchoninic acid BCA assay (Thermo Scientific).5 shows the SDS-PAGE results identifying purified mouse antibody 1G1 (parent 1G1) and chimeric antibody 1G1 (chimeric 1G1).

[0287] Example 10. Chimeric antibody 1G1 binding assay for PD-1 (SPR)

[0288] The binding kinetics of the chimeric antibody 1G1 obtained in Example 9 to human PD-1 was measured by SPR (surface plasmon resonance) analysis using Biacore 8K (Cytiva). Anti-human IgG antibody was immobilized on a CM5 chip (Cytiva) via an amine coupling reaction. Purified antibodies (Keytruda, Opdivo, chimeric antibody 1G1) were passed through the sensor chip, where they were captured by the anti-human IgG antibody. Human PD-1 and running buffer at a concentration of 0-100 nM (0, 6.25, 12.5, 25, 50, 100 nM) were passed through the sensor chip at a flow rate of 30 μl / min for an association phase of 120 s, followed by dissociation for 900 s. The chip was regenerated with glycine at pH 1.5 after each run. Association and dissociation curves were constructed using Cytiva evaluation software, and kinetics and affinity parameters were determined.

[0289] The table below shows the binding affinity of chimeric antibody 1G1 to human PD-1 determined by SPR analysis.

[0290] Keytruda Opdivo Chimeric antibody 1G1 Kon (1 / Ms) 4.96e+5 2.04e+5 2.23e+5 Koff (1 / s) 3.14e-3 2.14e-3 7.49e-4 KD (M) 6.33e-9 1.05e-8 3.35e-9

[0291] The chimeric anti-PD-1 antibody (1G1) had approximately 2-3-fold higher binding affinity (KD, 3.35e-9) for human PD-1 compared with Keytruda (6.33e-9) and Opdivo (1.05e-8). This was due to the improved binding properties of the chimeric anti-PD-1 antibody 1G1, with an association speed (Kon, 2.23e+5) that was similar (0.9-2.2-fold) to Keytruda (4.96e+5) and Opdivo (2.04e+5), but dissociation was 2.9-4.2-fold slower (Koff, 7.49e-4) compared with Keytruda (3.14e-3) and Opdivo (2.14e-3).

[0292] Example 11. Epitope mapping of 1G1 antibody

[0293] Epitope mapping (alanine scanning) of the chimeric antibody 1G1 (hIgG4(S228P)) to the human PD-1 antigen (SEQ ID NO: 62) was performed to determine the epitope of the human PD-1 antigen recognized by the antibody. Selected amino acid residues of PD-1 were mutated to alanine using PCR-based mutagenesis. The mutated proteins were expressed and analyzed for binding to the chimeric antibody 1G1 using high-throughput flow cytometry.

[0294] As a result, three epitopes (P130, L128, and I126) were identified, the mutation of which resulted in a decrease in binding activity to 50% or less of that of the wild-type antigen. All of these amino acids are conserved in mouse PD-1, supporting the cross-reactivity of the 1G1 antibody with human and mouse PD-1.

[0295] Example 12. Humanization of antibody 1G1

[0296] To ​​eliminate the immunogenicity of murine antibody 1G1 and ensure stable antibody efficacy in humans, humanization of antibody 1G1 was performed using the reverse mutation library method. The framework (FR) sequences of antibody 1G1, excluding the complementarity-determining region (CDR) sequences, were replaced with human antibody sequences, yielding three humanized antibodies 1G1 (humanized antibodies 1G1-h61, 1G1-h68, and 1G1-h70), which were purified by protein A affinity chromatography.

[0297] The binding affinity of the resulting three humanized 1G1 antibodies (humanized antibodies 1G1-h61, 1G1-h68, and 1G1-h70) to the human PD-1 antigen was analyzed by SPR (surface plasmon resonance) analysis using Biacore 8K (Cytiva). All three humanized 1G1 antibodies had antigen-binding affinity similar to that of the chimeric antibody 1G1.

[0298] The sequences of each of the obtained three humanized 1G1 antibodies were analyzed and are shown in Figs. 8 to 11. Specifically, the heavy chain variable region (VH) and the light chain variable region (VL) of the humanized antibody 1G1-h61 have the amino acid sequences of SEQ ID NOs: 54 and 55, respectively. The heavy chain variable region (VH) and the light chain variable region (VL) of the humanized antibody 1G1-h68 have the amino acid sequences of SEQ ID NOs: 56 and 57, respectively. The heavy chain variable region (VH) and the light chain variable region (VL) of the humanized antibody 1G1-h70 have the amino acid sequences of SEQ ID NOs: 58 and 59, respectively.

[0299] Example 13. Comparative evaluation of the binding kinetics of humanized 1G1 antibodies to the PD-1 antigen

[0300] The binding kinetics of each antibody to human PD-1 were measured by SPR (surface plasmon resonance) analysis using Biacore 8K (Cytiva), and their binding affinities to the antigen were compared.

[0301] The anti-human PD-1 control antibodies, Keytruda (MSD (Lot No. T020031)) and Opdivo (BMS (Lot No. 043 FB)), were products for human clinical use obtained from Shinwon Pharmacy Co., Ltd.

[0302] Control antibodies (Keytruda, Opdivo) and 1G1 antibodies (chimeric 1G1 antibody-chimeric, humanized 1G1-h61, 1G1-h68, and 1G1-h70 antibodies) were passed through a Protein A chip (Cytiva), where they were captured. Seven concentrations (0-100 M) of human PD-1 were passed through the sensor chip at a flow rate of 30 μl / min for an association phase of 120 s, followed by dissociation for 1800 s. The chip was regenerated with glycine at pH 1.5 after each run. Association and dissociation curves were generated using Cytiva evaluation software, and kinetics and affinity values ​​were measured. One-way analysis of variance with Tukey's test in GraphPad Prism was performed to determine whether there was a significant difference in kinetics and affinity between antibodies (****P < 0.0001).

[0303] Figures 12a and 12b show the binding kinetics and affinity values ​​for human PD-1, expressed as Kon (ka value), Koff (kd value), and KD values. In conclusion, the humanized anti-PD-1 antibodies (1G1) had, on average, a binding affinity for human PD-1 (KD, 7.26e-9) similar to that of Keytruda (7.06e-9) and Opdivo (7.54e-9). This indicates that humanized anti-PD-1 antibodies 1G1 have improved binding properties, dissociating 2.7-7.6 times slower (Koff, 3.87e-4) than Keytruda (2.95e-3) and Opdivo (1.06e-3), while in terms of association rate, they bind 2.6-7.7 times slower (Kon, 5.45e+4) than Keytruda (4.18e+5) and Opdivo (1.41e+5). Considering the mechanism of action of anti-PD-1 antibodies, humanized anti-PD-1 antibodies 1G1 exhibit enhanced antitumor activity due to their slow-dissociation binding property.

[0304] Example 14. Comparative evaluation of cross-reactivity to PD-1 antigens

[0305] To determine the cross-reactivity of the antibody to PD-1 antigens, a protein-based ELISA was performed. Brief description of the ELISA assay: A 96-well plate (Thermo Scientific) was coated with 10 ng of the extracellular domain (ECD) proteins of human, mouse, rabbit, cynomolgus macaque, and rat PD-1 and incubated overnight at 4°C. After blocking and washing, 1 μg / mL of control antibodies (Keytruda and Opdivo) and 1G1 antibodies (chimeric antibody and humanized antibodies) were added to the coated plate and incubated at 37°C for 2 h. After washing, secondary antibody (anti-human IgG antibody) was added and incubated at 37°C for 2 h. After washing, TMB substrate (abcam) was added, and the color reaction was stopped with STOP solution (abcam). Absorbance at 450 / 650 nm was measured using a microplate reader (ThermoFisher).

[0306] Figure 13 shows the results of the antibody cross-reactivity to PD-1 antigens. Keytruda, Opdivo, and 1G1 antibodies exhibit cross-reactivity with human PD-1 and cynomolgus monkey PD-1 antigens. 1G1 antibodies also exhibited cross-reactivity with the mouse PD-1 antigen.

[0307] Example 15. Evaluation of antitumor efficacy in vivo

[0308] An experiment to evaluate the antitumor efficacy of antibody 1G1 was performed using a mouse melanoma model as shown in Fig. 14. Mouse melanoma cells (B16F11 / OT.EGFP) were cultured and 3×10 6The cells were inoculated subcutaneously into the right hindlimb of 8-week-old female C57BL / 6 mice. Six days after tumor cell inoculation, the tumor nodule size was measured, and the mice were divided into five groups. Beginning on day seven after inoculation, the parent antibody 1G1 was administered intraperitoneally five times at three-day intervals, and tumor size was measured at three-day intervals. Tumor size was calculated using the following formula:

[0309]

[0310] Survival was recorded as the day when tumor volume exceeded 1000 mm 3, or tumor ulceration or mouse death occurred. Two-way analysis of variance with Bonferroni correction in GraphPad Prism was used to determine significant differences between tumor growth inhibition rates, and the log-rank (Mantel-Cox) test was used to determine the significance of differences between survival rates (*P < 0.05; ***P < 0.001, endpoint of survival analysis: 700 mm 3 ).

[0311] Fig. 15 shows the tumor growth and tumor growth inhibition over time according to the dose of parent antibody 1G1. In the 1 mg / kg antibody 1G1 group, tumor growth similar to that of the isotype control group was observed; in the 2.5 mg / kg to 10 mg / kg antibody 1G1 groups, tumor growth inhibition was approximately 90-100% on day 19, 94-112% on day 22, and 86-94% on day 25 (Fig. 15; day 22), and the survival rate was increased by approximately 20% (Fig. 16). The results confirmed that the parent antibody 1G1 exhibited high antitumor effect.

[0312] Example 16. Evaluation of in vivo antitumor efficacy in a colorectal cancer model

[0313] The antitumor efficacy of antibody 1G1 was evaluated in a syngeneic colorectal cancer model MC38. 5×10 5MC38 mouse colorectal carcinoma cells were inoculated subcutaneously into the left flank of 7-8-week-old female C57BL / 6 mice. After injection, when the average tumor size reached 50 to 150 mm 3 Mice were divided into 4 groups (n=13 each) based on tumor volume and administered human hIgG4, rat rIgG2a, 1G1-h70 antibody, and RMP1-14 (murine PD-1) antibody at 3-day intervals at a dose of 10 mg / kg each for a total of 5 doses. Animal body weight and tumor size were measured and assessed three times a week in each group. Final body weight and tumor size were measured on the day the study reached the endpoint. The endpoint was determined when the average tumor size in the control group reached 1500 mm 3 The tumor growth inhibition rate (%TGI) was determined for each treatment group (T) compared to the control group (C) using the initial (i) and final (f) tumor measurements according to the formula below:

[0314] %TGI=1 - (Tf - Ti) / (Cf - Ci)×100

[0315] The relative change in tumor size and tumor growth inhibition rate over time for each group is shown in Fig. 17. The 1G1-h70 antibody demonstrated significantly higher tumor growth inhibition compared with the anti-mouse PD-1 antibody, RMP1-14. The syngeneic colorectal cancer model MC38 is known to respond poorly to anti-PD-1 therapy. Thus, the high tumor growth inhibitory effect of the 1G1-h70 antibody was unexpected. However, no statistically significant change in mouse body weight was observed as a result of treatment in each group (data not shown).

[0316] Example 17. Epitope mapping of antibody 1G1 using crystallography

[0317] Epitope mapping (X-ray crystallography method) of humanized antibody 1G1-h70 to human PD-1 antigen (SEQ ID NO: 62) was performed to determine the epitope on the human PD-1 antigen recognized by the antibody. The binding complex of antibody 1G1-h70 Fab and PD-1 was crystallized at 20°C using a crystallization solution by the hanging drop vapor diffusion method (drop volume = 0.8 μL protein + 0.8 μL reservoir, 400 μL reservoir volume). X-ray diffraction experiment was performed on the obtained single crystal to obtain a data resolution of 2.30 Å. Table 4 below summarizes the information of X-ray diffraction data collection and structure refinement.

[0318] Table 4

[0319] Analysis of the interaction of 1G1-h70 Fab with PD-1 revealed that the 1G1-h70 antibody formed hydrogen bonds with residues N66, Y68, K78, A129, P130, and A132 on the PD-1 antigen (SEQ ID NO: 62). Of these, Y68, K78, and N66 have side chains that are involved in hydrogen bond formation.

[0320] In addition, the interaction analysis of 1G1-h70 Fab and PD-1 showed that the 1G1-h70 antibody formed hydrophobic bonds with residues I126, L128, A129, P130, and A132 on the PD-1 antigen (SEQ ID NO: 62). As also confirmed by the alanine scanning experiment results in Example 11, three amino acid residues (P130, L128, I126) that play the most important roles in binding to the 1G1 antibody are located in the FG loop region of PD-1, and the hydrophobic interactions formed by the residues were found to exhibit synergism, significantly contributing to the binding affinity.

[0321] The loop regions of the PD-1 molecule are known to be highly flexible and adopt a conformation suitable 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. Differences in these structures result in differences in the binding pattern of the PD-1 interactome and in the antitumor immunity of each antibody.

[0322] Example 18. pH-dependent binding of antibody 1G1

[0323] 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 immuno-oncology antibody may be affected by its pH-dependent binding activity. In general, histidine is the most sensitive residue for pH-dependent binding activity. Keytruda and Opdivo, which are conventional anti-PD-1 antibodies, do not contain histidine among the residues involved in binding. In contrast, since the 1G1 antibody has a histidine residue, such as residue H52 of the heavy chain CDR2 in the CDR region (Kabat numbering system), which is involved in the formation of hydrogen bonds or hydrophobic bonds with the PD-1 antigen, it is expected that H52 contributes to the binding of PD-1 in the tumor microenvironment with a low pH. Therefore, the pH dependence of 1G1-h70 binding to PD-1 was examined compared with Keytruda and Opdivo.

[0324] The binding kinetics of each antibody to human PD-1 were measured by SPR (surface plasmon resonance) analysis using Biacore 8K (Cytiva), and the binding affinities of the antibodies to the antigen were compared with each other. The control anti-human PD-1 antibodies, Keytruda (MSD (lot No. T020031)) and Opdivo (BMS (lot No. 043 FB)), were products for clinical use in humans obtained from Shinwon Pharmacy Co., Ltd. In addition, the 2E5 antibody was obtained according to the 2E5 clone described in WO 2018 / 053709 of CStone Pharmaceuticals. The 2E5 antibody was found to be able to bind to both human and mouse PD-1, and its epitopes are located on the FG loop of PD-1.

[0325] Control antibodies (Keytruda, Opdivo), 2E5 antibody, and 1G1 antibody (1G1-h70) were passed through a protein A chip (Cytiva), where they were captured. Seven concentrations (0-100 M) of human PD-1 were passed through the sensor chip at a flow rate of 30 μl / min for an association phase of 120 s, followed by dissociation for 1800 s. 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 run. Association and dissociation curves were generated using Cytiva evaluation software, and kinetics and affinity parameters were determined. The significance of differences between the kinetics and affinity parameters of antibodies was determined using one-way ANOVA with the Tukey test of the GraphPad Prism program (****, P <0.0001).

[0326] Fig. 19a and 19b show the binding kinetics and affinity to human PD-1 expressed as Kon (ka value), Koff (kd value), and KD values. At pH 7.4 (blood), the 1G1-h70 antibody (KD=5.4 nM) had a binding affinity to human PD-1 comparable to that of Keytruda (KD=6.6 nM), Opdivo (KD=7.1 nM), and 2E5 antibody (KD=12.6 nM). However, at pH 6.0 (tumor microenvironment), antibody 1G1-h70 (KD=0.9 nM) demonstrated significantly higher binding affinity to human PD-1 than Keytruda (KD=4.2 nM), Opdivo (KD=3.1 nM), and antibody 2E5 (KD=4.5 nM). This is due to the fact that antibody 1G1-h70 has improved binding capacity in terms of dissociation (Koff), dissociating more slowly compared to antibodies Keytruda, Opdivo, and 2E5 at pH 6.0.Since the antitumor efficacy of antitumor drugs, including immuno-oncology antibodies, is affected by the low pH of the tumor microenvironment, the high binding affinity of the 1G1-h70 antibody to human PD-1 even at a low pH of the tumor microenvironment provides the basis for ensuring high antitumor activity in the tumor microenvironment in vivo.

[0327] Example 19. Affinity Maturation

[0328] Affinity maturation was performed to increase the affinity of antibody 1G1-h70 to human PD-1. Each amino acid residue in the CDR region of antibody 1G1-h70 was mutated to another 19 amino acids using codons optimized for E. coli. DNA oligonucleotide libraries were synthesized on microarrays, and clones were selected for expression in E. coli.

[0329] The crude protein secreted into the medium was analyzed by ELISA against BSA and human PD-1 to assess expression and binding affinity. Clones with improved values ​​were selected for sequencing. “Useful mutants” were confirmed by affinity ranking using SPR. Screening for outliers 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 the selected clones secreted into the culture medium was captured by SASA capture biosensors. After equilibration, antigen was injected for 120 s (association phase), followed by injecting the running buffer for 420 s (dissociation phase). The surface was regenerated before introducing other selected clones. The process was repeated until all samples had been analyzed.Dissociation rates of Fab-SASA clones were obtained by locally fitting experimental data to a 1:1 interaction model using Biacore T200 evaluation software. Selected mutants were ranked by their dissociation rate constants (kd).

[0330] Once the "beneficial mutants" were identified, a combinatorial library with random combinations of these mutations was constructed using PCR. The combinatorial clones were analyzed by ELISA and subjected to DNA sequencing and affinity ranking. Finally, the best combinations of "beneficial mutants" that maximized affinity without compromising expression were selected for antibody affinity measurements.

[0331] After affinity maturation, a total of 13 humanized anti-PD-1 antibodies (AHF16556, AHF16557, AHF16558, AHF16559, AHF16560, AHF16561, AHF16563, AHF16564, AHF16565, AHF16566, AHF16568, AHF16569, and AHF16570) were obtained. These antibodies had amino acid substitutions in three CDR regions (VH CDR2, VH CDR3, VL CDR1) compared with the parent antibody 1G1-h70 (WT), as shown in Table 5 below.

[0332] Table 5

[0333] The binding kinetics data of antibodies to human PD-1 are shown in Table 6 below.

[0334] Table 6

[0335] Sequence List

[0336] Table 7 Sequence number Sequences 1 HCDR1 amino acids GYWMH 2 HCDR1 nucleotides GGCTACTGGATGCAC 3 HCDR2 amino acids MIHPNSDTTTYNEKFKN 4 HCDR2 nucleotides ATGATTCATCCTAACAGTGATACTACTACCTACAATGAGAAGTTCAAAAAC 5 HCDR3 amino acids TDQAAWFAF 6 HCDR3 nucleotides ACAGATCAGGCCGCCTGGTTTGCTTTC 7 LCDR1 amino acids RSSQNIVHSNGDTYLE 8 LCDR1 nucleotides agatctagtcagaacattgtacatagtaatggagacacctatttagaa 9 LCDR2 amino acids KVSKRFS 10 LCDR2 nucleotides aaagtttccaagcgattttct 11 LCDR3 amino acids FQGSHVPWT 12 LCDR3 nucleotides tttcaaggttcacatgttccgtggacg 13 аминокислоты HCVR EVQLQQSGAELVKPGASVKLSCKASGYTFTGYWMHWVKQRPGQGLEWIGMIHPNSDTTTYNEKFKNRATLTVDKSSGTAYMQLSSLTSEDSAVYYCTGTDQAAWFAFWGQGTLVTVSA 14 нуклеотиды HCVR GAGGTCCAGCTGCAGCAGTCTGGGGCTGAGCTGGTTAAGCCTGGGGCTTCAGTGAAGCTGTCCTGCAAGGCTTCTGGCTACACTTTCACCGGCTACTGGATGCACTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGAATGATTCATCCTAACAGTGATACTACTACCTACAATGAGAAGTTCAAAAACAGGGCCACACTGACTGTAGACAAATCCTCCGGCACAGCCTACATGCAACTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTACAGGGACAGATCAGGCCGCCTGGTTTGCTTTC TGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA 15 аминокислоты LCVR DIVLTQTPLSLPVSLGDQASISCRSSQNIVHSNGDTYLEWYLQKPGQSPKLLIYKVSKRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPWTFGGGTKLEIK 16 нуклеотиды LCVR GATATTGTGCTGACACAAACTCCActctccctgcctgtcagtcttggagatcaagcctccatctcttgcagatctagtcagaacattgtacatagtaatggagacacctatttagaatggtacctgcagaaaccaggccagtctccaaagctcctgatctacaaagtttccaagcgattttctggggtcccagacaggttcagtggcagtggatcagggacagatttcacactcaagatcagcagagtggaggctgaggatctgggagtttattactgctttcaaggttcacatgttccgtggacgttcggtggaggcaccaagctggaaatcaaa 17 HFR1 amino acids EVQLQQSGAELVKPGASVKLSCKASGYTFT 18 HFR1 nucleotides GAGGTCCAGCTGCAGCAGTCTGGGGCTGAGCTGGTTAAGCCTGGGGCTTCAGTGAAGCTGTCCTGCAAGGCTTCTGGCTACACTTTCACC 19 HFR2 amino acids WVKQRPGQGLEWIG 20 HFR2 nucleotides TGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGA 21 HFR3 amino acids RATLTVDKSSGTAYMQLSSLTSEDSAVYYCTG 22 HFR3 nucleotides AGGGCCACACTGACTGTAGACAAATCCTCCGGCACAGCCTACATGCAACTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTACAGGG 23 HFR4 amino acids WGQGTLVTVSA 24 HFR4 nucleotides TGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA 25 LFR1 amino acids DIVLTQTPLSLPVSLGDQASISC 26 LFR1 nucleotides GATATTGTGCTGACACAAACTCAcctctccctgcctgtcagtcttggagatcaagcctccatctcttgc 27 LFR2 amino acids WYLQKPGQSPKLLIY 28 LFR2 nucleotides tggtacctgcagaaaccaggccagtctccaaagctcctgatctac 29 LFR3 amino acids GVPDRFSGSGSGTDFTLKISRVEAEDLGVYYC 30 LFR3 nucleotides ggggtcccagacaggttcagtggcagtggatcagggacagatttcacactcaagatcagcagagtggaggctgaggatctgggagtttattactgc 31 LFR4 amino acids FGGGTKLEIK 32 LFR4 nucleotides ttcggtggaggcaccaagctggaaatcaaa 33 primer MH1 AATTTGCTAGCSARGTNMAGCTGSAGSAGTC 34 primer MH2 AATTTGCTAGCSARGTNMAGCTGSAGSAGTCWGG 35 IgG1 primer AATTTGGATCCATAGACAGATGGGGGTGTCGTTTTGGC 36 primer MK AATTGGATCCAGGGGCCAGTGGATAGACTGATGG 37 CK primer AATTTGCGGCCGCGGATACAGTTGGTGCAGCATC 38 nucleotides of the constant region of the heavy chain of human IgG4 (S228P) GCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCCGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGTCCCCCATGCCCACCATGCCCAGCACCTGAGTTCCTGGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCCGGGTAAA 39 amino acids of the constant region of the heavy chain of human IgG4 (S228P) ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVE VHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPGK 40 nucleotides of the constant region of the kappa light chain of human IgG4 (S228P) CGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGG AGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT 41 amino acids of the constant region of the kappa light chain of human IgG4 (S228P) RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 42 heavy chain nucleotides of the humanized antibody 1G1-h61 CAAGTTCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCTGGAGCCAGCGTGAAAGTGTCCTGCAAGGCCTCTGGCTACACCTTTACAGGCTACTGGATGCACTGGGTGCGGCAGGCCCCTGGACAGGGCCTGGAATGGATGGGCATGATCCACCCCAACAGCGACACCACAACCTACAACGAGAAGTTCAAGAATAGAGTGACCATGACAAGAGATACCAGCATCAGCACCGCCTACATGGAACTGAGCAGACTGCGGTCCGATGACACAGCTGTGTACTATTGTGCCGGCACCGACCAGGCCGCTTGGTTCGCCTTCTGGGGGCAGGGCACCACAGTCACCGTGAGCTCTGCCAGCACCAAGGGCCCTTCCGTGTTTCCCCTGGCCCCTTGCTCCCGGTCCACATCTGAGAGCACCGCCGCCCTGGGCTGTCTGGTGAAGGACTACTTCCCAGAGCCCGTGACCGTGAGCTGGAACAGCGGCGCCCTGACAAGCGGCGTGCACACATTTCCCGCCGTGCTGCAGAGCTCCGGCCTGTACTCCCTGTCTAGCGTGGTGACAGTGCCTTCCTCTAGCCTGGGCACCAAGACATATACCTGTAACGTGGACCACAAGCCAAGCAATACCAAGGTGGATAAGCGGGTGGAGTCTAAGTACGGCCCTCCTTGCCCTAGCTGTCCTGCTCCAGAGTTTCTGGGCGGCCCTTCCGTGTTCCTGTTTCCACCCAAACCAAAGGACACACTGATGATCTCTAGAACACCAGAGGTGACCTGCGTGGTGGTGGACGTGAGCCAGGAGGATCCCGAGGTGCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCACAATGCCAAGACCAAGCCAAGAGAGGAGCAGTTTAACTCTACATACAGGGTGGTGAGCGTGCTGACCGTGCTGCACCAGGATTGGCTCAACGGCAAGGAGTATAAGTGCAAGGTGTCCAATAAGGGCCTGCCCTCCTCTATCGAGAAGACAATCTCTAAGGCTAAGGGCCAGCCAAGAGAGCCTCAGGTGTACACCCTGCCTCCAAGCCAGGAGGAGATGACAAAGAACCAGGTGTCCCTGACATGTCTGGTGAAGGGCTTCTATCCCTCCGACATCGCCGTGGAGTGGGAGTCTAATGGCCAGCCTGAGAACAATTACAAGACCACACCCCCTGTGCTGGACTCTGATGGCAGCTTCTTTCTGTATTCCAGGCTGACCGTGGATAAGTCTCGGTGGCAGGAGGGCAACGTGTTCAGCTGCTCTGTGATGCACGAAGCCCTGCATAATCACTATACTCAGAAAAGTCTGTCACTGTCACTGGGAAAGTGATAA 43 amino acids of the heavy chain of the humanized antibody 1G1-h61 QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYWMHWVRQAPGQGLEWMGMIHPNSDTTTYNEKFKNRVTMTRDTSISTAYMELSRLRSDDTAVYYCAGTDQAAWFAFWGQG TTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGP PCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKT ISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 44 heavy chain nucleotides of the humanized antibody 1G1-h68 CAAGTTCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCTGGAGCCAGCGTGAAAGTGTCCTGCAAGGCCTCTGGCTACACCTTTACAGGCTACTGGATGCACTGGGTGCGGCAGGCCCCTGGACAGGGCCTGGAATGGATCGGCATGATCCACCCCAACAGCGACACCACAACCTACAACGAGAAGTTCAAGAATAGAGTGACCATGACAAGAGATACCAGCATCAGCACCGCCTACATGGAACTGAGCAGACTGCGGTCCGATGACACAGCTGTGTACTATTGTACCGGCACCGACCAGGCCGCTTGGTTCGCCTTCTGGGGGCAGGGCACCACAGTCACCGTGAGCTCTGCCAGCACCAAGGGCCCTTCCGTGTTTCCCCTGGCCCCTTGCTCCCGGTCCACATCTGAGAGCACCGCCGCCCTGGGCTGTCTGGTGAAGGACTACTTCCCAGAGCCCGTGACCGTGAGCTGGAACAGCGGCGCCCTGACAAGCGGCGTGCACACATTTCCCGCCGTGCTGCAGAGCTCCGGCCTGTACTCCCTGTCTAGCGTGGTGACAGTGCCTTCCTCTAGCCTGGGCACCAAGACATATACCTGTAACGTGGACCACAAGCCAAGCAATACCAAGGTGGATAAGCGGGTGGAGTCTAAGTACGGCCCTCCTTGCCCTAGCTGTCCTGCTCCAGAGTTTCTGGGCGGCCCTTCCGTGTTCCTGTTTCCACCCAAACCAAAGGACACACTGATGATCTCTAGAACACCAGAGGTGACCTGCGTGGTGGTGGACGTGAGCCAGGAGGATCCCGAGGTGCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCACAATGCCAAGACCAAGCCAAGAGAGGAGCAGTTTAACTCTACATACAGGGTGGTGAGCGTGCTGACCGTGCTGCACCAGGATTGGCTCAACGGCAAGGAGTATAAGTGCAAGGTGTCCAATAAGGGCCTGCCCTCCTCTATCGAGAAGACAATCTCTAAGGCTAAGGGCCAGCCAAGAGAGCCTCAGGTGTACACCCTGCCTCCAAGCCAGGAGGAGATGACAAAGAACCAGGTGTCCCTGACATGTCTGGTGAAGGGCTTCTATCCCTCCGACATCGCCGTGGAGTGGGAGTCTAATGGCCAGCCTGAGAACAATTACAAGACCACACCCCCTGTGCTGGACTCTGATGGCAGCTTCTTTCTGTATTCCAGGCTGACCGTGGATAAGTCTCGGTGGCAGGAGGGCAACGTGTTCAGCTGCTCTGTGATGCACGAAGCCCTGCATAATCACTATACTCAGAAAAGTCTGTCACTGTCACTGGGAAAGTGATAA 45 amino acids of the heavy chain of the humanized antibody 1G1-h68 QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYWMHWVRQAPGQGLEWIGMIHPNSDTTTYNEKFKNRVTMTRDTSISTAYMELSRLRSDDTAVYYCTGTDQAAWFAFWGQG TTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGP PCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKT ISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 46 heavy chain nucleotides of the humanized antibody 1G1-h70 CAAGTTCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCTGGAGCCAGCGTGAAAGTGTCCTGCAAGGCCTCTGGCTACACCTTTACAGGCTACTGGATGCACTGGGTGCGGCAGGCCCCTGGACAGGGCCTGGAATGGATCGGCATGATCCACCCCAACAGCGACACCACAACCTACAACGAGAAGTTCAAGAATAGAGTGACCATGACAAGAGATACCAGCATCAGCACCGCCTACATGGAACTGAGCAGACTGCGGTCCGATGACACAGCTGTGTACTATTGTGCCGGCACCGACCAGGCCGCTTGGTTCGCCTTCTGGGGGCAGGGCACCACAGTCACCGTGAGCTCTGCCAGCACCAAGGGCCCTTCCGTGTTTCCCCTGGCCCCTTGCTCCCGGTCCACATCTGAGAGCACCGCCGCCCTGGGCTGTCTGGTGAAGGACTACTTCCCAGAGCCCGTGACCGTGAGCTGGAACAGCGGCGCCCTGACAAGCGGCGTGCACACATTTCCCGCCGTGCTGCAGAGCTCCGGCCTGTACTCCCTGTCTAGCGTGGTGACAGTGCCTTCCTCTAGCCTGGGCACCAAGACATATACCTGTAACGTGGACCACAAGCCAAGCAATACCAAGGTGGATAAGCGGGTGGAGTCTAAGTACGGCCCTCCTTGCCCTAGCTGTCCTGCTCCAGAGTTTCTGGGCGGCCCTTCCGTGTTCCTGTTTCCACCCAAACCAAAGGACACACTGATGATCTCTAGAACACCAGAGGTGACCTGCGTGGTGGTGGACGTGAGCCAGGAGGATCCCGAGGTGCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCACAATGCCAAGACCAAGCCAAGAGAGGAGCAGTTTAACTCTACATACAGGGTGGTGAGCGTGCTGACCGTGCTGCACCAGGATTGGCTCAACGGCAAGGAGTATAAGTGCAAGGTGTCCAATAAGGGCCTGCCCTCCTCTATCGAGAAGACAATCTCTAAGGCTAAGGGCCAGCCAAGAGAGCCTCAGGTGTACACCCTGCCTCCAAGCCAGGAGGAGATGACAAAGAACCAGGTGTCCCTGACATGTCTGGTGAAGGGCTTCTATCCCTCCGACATCGCCGTGGAGTGGGAGTCTAATGGCCAGCCTGAGAACAATTACAAGACCACACCCCCTGTGCTGGACTCTGATGGCAGCTTCTTTCTGTATTCCAGGCTGACCGTGGATAAGTCTCGGTGGCAGGAGGGCAACGTGTTCAGCTGCTCTGTGATGCACGAAGCCCTGCATAATCACTATACTCAGAAAAGTCTGTCACTGTCACTGGGAAAGTGATAA 47 amino acids of the heavy chain of the humanized antibody 1G1-h70 QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYWMHWVRQAPGQGLEWIGMIHPNSDTTTYNEKFKNRVTMTRDTSISTAYMELSRLRSDDTAVYYCAGTDQAAWFAFWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGP PCPSCPAPEFLFGGPSVFLFPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 48 nucleotides of the humanized chain of antibody 1G1-h61 GATATCGTTATGACCCAGACCCCTCTGAGCCTGTCCGTGACCCCAGGCCAACCTGCCTCTATCAGCTGTAGAAGCAGCCAGAACATCGTGCACAGCAACGGCGACACCTACCTGGAATGGTATCTGCAGAAACCTGGACAGAGCCCCAAGCTGCTGATCTACAAGGTGTCCAAGCGGTTTTCCGGCGTGCCTGATAGATTCAGCGGATCTGGCAGCGGCACAGACTTCACCCTGAAGATTTCTAGAGTGGAGGCCGAGGACGTGGGCGTCTACTACTGCTTCCAGGGCAGCCACGTGCCCTGGACATTCGGCGGCGGAACAAAGGTGGAAATCAAGAGGACAGTGGCCGCCCCAAGCGTGTTCATCTTTCCCCCTTCCGACGAGCAGCTGAAGTCTGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCTCGGGAGGCCAAGGTCCAGTGGAAGGTGGATAACGCCCTGCAGTCTGGCAATAGCCAGGAGTCCGTGACCGAGCAGGACTCTAAGGATAGCACATATTCCCTGTCTAGCACCCTGACACTGAGCAAGGCCGATTACGAGAAGCACAAGGTGTATGCCTGTGAAGTCACCCATCAGGGGCTGTCATCACCCGTCACTAAGTCATTCAATCGCGGAGAATGCTGATAA 49 аминокислоты легкой цепи гуманизированного антитела 1G1-h61 DIVMTQTPLSLSVTPGQPASISCRSSQNIVHSNGDTYLEWYLQKPGQSPKLLIYKVSKRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 50 нуклеотиды легкой цепи гуманизированного антитела 1G1-h68 GATATCGTTATGACCCAGACCCCTCTGAGCCTGTCCGTGACCCCAGGCCAACCTGCCTCTATCAGCTGTAGAAGCAGCCAGAACATCGTGCACAGCAACGGCGACACCTACCTGGAATGGTATCTGCAGAAACCTGGACAGAGCCCCCAGCTGCTGATCTACAAGGTGTCCAAGCGGTTTTCCGGCGTGCCTGATAGATTCAGCGGATCTGGCAGCGGCACAGACTTCACCCTGAAGATTTCTAGAGTGGAGGCCGAGGACGTGGGCGTCTACTACTGCTTCCAGGGCAGCCACGTGCCCTGGACATTCGGCGGCGGAACAAAGGTGGAAATCAAGAGGACAGTGGCCGCCCCAAGCGTGTTCATCTTTCCCCCTTCCGACGAGCAGCTGAAGTCTGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCTCGGGAGGCCAAGGTCCAGTGGAAGGTGGATAACGCCCTGCAGTCTGGCAATAGCCAGGAGTCCGTGACCGAGCAGGACTCTAAGGATAGCACATATTCCCTGTCTAGCACCCTGACACTGAGCAAGGCCGATTACGAGAAGCACAAGGTGTATGCCTGTGAAGTCACCCATCAGGGGCTGTCATCACCCGTCACTAAGTCATTCAATCGCGGAGAATGCTGATAA 51 аминокислоты легкой цепи гуманизированного антитела 1G1-h68 DIVMTQTPLSLSVTPGQPASISCRSSQNIVHSNGDTYLEWYLQKPGQSPQLLIYKVSKRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 52 нуклеотиды легкой цепи гуманизированного антитела 1G1-h70 GATATCGTTATGACCCAGACCCCCTGAGCCTGTCCGTGACCCCAGGCCAACCTGCCTCTATCAGCTGTAGAAGCAGCCAGAACATCGTGCACAGCCAGGGCGACACCTACCTGGAATGGTATCTGCAGAAACCTGGACAGAGCCCCCAGCTGCTGATCTACAAG GTGTCCAAGCGGTTTTCCGGCGTGCCTGATAGATTCAGCGGATCTGGCAGCGGCACAGACTTCACCCTGAAGATTTCTAGAGTGGAGGCCGAGGACGTGGGCGTCTACTACTGCTTCCAGGGCAGCCACGTGCCCTGGACATTCGGCGGCGAACAAAGGTGGAAA TCAAGAGGACAGTGGCCGCCCCAAGCGTGTTCATCTTTCCCCCTTCCGACGAGCAGCTGAAGTCTGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCTCGGGAGGCCAAGGTCCAGTGGAAGGTGGATAACGCCCTGCAGTCTGGCAATAGCCAGGA GTCCGTGACCGAGCAGGACTCTAAGGATAGCACATATTCCCTGTCTAGCACCCTGACACTGAGCAAGGCCGATTACGAGAAGCAAGGTGTATGCCTGTGAAGTCACCCATCAGGGGCTGTCATCACCCGTCACTAAGTCATTCAATCGCGGAGAATGCTGATAA 53 amino acids of the light chain of the humanized antibody 1G1-h70 DIVMTQTPLSSVTPGQPASISCRSSQNIVHSQGDTYLEWYLQKPGQSPQLLIYKVSKRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPWTFGGGGTKV EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 54 amino acids of the VH of the humanized antibody 1G1-h61 QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYWMHWVRQAPGQGLEWMGMIHPNSDTTTYNEKFKNRVTMTRDTSISTAYMELSRLRSDDTAVYYCAGTDQAAWFAFWGQGTTVTVSS 55 amino acids VL of humanized antibody 1G1-h61 DIVMTQTPLSLSVTPGQPASISCRSSQNIVHSNGDTYLEWYLQKPGQSPKLLIYKVSKRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPWTFGGGTKVEIK 56 VH amino acids of humanized antibody 1G1-h68 QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYWMHWVRQAPGQGLEWIGMIHPNSDTTTYNEKFKNRVTMTRDTSISTAYMELSRLRSDDTAVYYCTGTDQAAWFAFWGQGTTVTVSS 57 amino acids VL of humanized antibody 1G1-h68 DIVMTQTPLSLSVTPGQPASISCRSSQNIVHSNGDTYLEWYLQKPGQSPQLLIYKVSKRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPWTFGGGTKVEIK 58 VH amino acids of humanized antibody 1G1-h70 QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYWMHWVRQAPGQGLEWIGMIHPNSDTTTYNEKFKNRVTMTRDTSISTAYMELSRLRSDDTAVYYCAGTDQAAWFAFWGQGTTVTVSS 59 amino acids VL of humanized antibody 1G1-h70 DIVMTQTPLSLSVTPGQPASISCRSSQNIVHSQGDTYLEWYLQKPGQSPQLLIYKVSKRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPWTFGGGTKVEIK 60 LCDR1 amino acids RSSQNIVHSQGDTYLE 61 LCDR1 nucleotides agaagcagccagaacatcgtgcacagccagggcgacacctacctggaa 62 The complete amino acid sequence of the human PD-1 protein MQIPQAPWPVVWAVLQLGWRPGW FLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL Signal sequence is in italics. ECD (extracellular domain) is underlined. 63 HCDR2 amino acids MIHPNSDTTVYNEKFKN 64 HCDR2 amino acids MIHPNSDTTIYNEKFKN 65 HCDR2 amino acids MIHPNSDTTTYNWKFKN 66 HCDR2 amino acids MIHPNSDTTVYNWKFKN 67 HCDR2 amino acids MIHPNSDTTIYNWKFKN 68 HCDR3 amino acids TDQEAWFAF 69 HCDR3 amino acids TDQAAWAAF 70 HCDR3 amino acids TDQAAWMAF 71 HCDR3 amino acids TDQAAWHAF 72 HCDR3 amino acids TDQAAWFGF 73 HCDR3 amino acids TDQEAWAAF 74 HCDR3 amino acids TDQEAWMAF 75 HCDR3 amino acids TDQEAWHAF 76 HCDR3 amino acids TDQEAWFGF 77 HCDR3 amino acids TDQAAWAGF 78 HCDR3 amino acids TDQAAWMGF 79 HCDR3 amino acids TDQAAWHGF 80 HCDR3 amino acids TDQEAWAGF 81 HCDR3 amino acids TDQEAWMGF 82 HCDR3 amino acids TDQEAWHGF 83 LCDR1 amino acids RSSQNIVRSNGDTYLE 84 LCDR1 amino acids RSSQNIVRSQGDTYLE

[0337] Although the present invention has been described above, the present invention is not limited to the disclosed examples and accompanying drawings, and can be variously modified by those skilled in the art within the scope without departing from the essence of the present invention. Furthermore, the technical ideas described in the examples of the present disclosure can be implemented independently, or two or more can be implemented in combination with each other.

[0338] --->

[0339] Sequence List

[0340] <110> GENUV Inc.

[0341] <120> ANTI-PD-1 ANTIBODY AND ITS APPLICATIONS

[0342] <130> GNUV201_P_0002

[0343] <150> KR 10-2021-0015937

[0344] <151> 2021-02-04

[0345] <150> KR 10-2021-0070443

[0346] <151> 2021-05-31

[0347] <150> KR 10-2021-0139442

[0348] <151> 2021-10-19

[0349] <160> 84

[0350] <170> KoPatentIn 3.0

[0351] <210> 1

[0352] <211> 5

[0353] <212> PROTEIN

[0354] <213> Artificial sequence

[0355] <220>

[0356] <223> HCDR1 aa

[0357] <400> 1

[0358] Gly Tyr Trp Met His

[0359] 1 5

[0360] <210> 2

[0361] <211> 15

[0362] <212> DNA

[0363] <213> Artificial sequence

[0364] <220>

[0365] <223> HCDR1 nucleotide

[0366] <400> 2

[0367] ggctactgga tgcac 15

[0368] <210> 3

[0369] <211> 17

[0370] <212> PROTEIN

[0371] <213> Artificial sequence

[0372] <220>

[0373] <223> HCDR2 aa

[0374] <400> 3

[0375] Met Ile His Pro Asn Ser Asp Thr Thr Thr Tyr Asn Glu Lys Phe Lys

[0376] 1 5 10 15

[0377] Asn

[0378] <210> 4

[0379] <211> 51

[0380] <212> DNA

[0381] <213> Artificial sequence

[0382] <220>

[0383] <223> HCDR2 nucleotide

[0384] <400> 4

[0385] atgattcatc ctaacagtga tactactacc tacaatgaga agttcaaaaa c 51

[0386] <210> 5

[0387] <211> 9

[0388] <212> PROTEIN

[0389] <213> Artificial sequence

[0390] <220>

[0391] <223> HCDR3 aa

[0392] <400> 5

[0393] Thr Asp Gln Ala Ala Trp Phe Ala Phe

[0394] 1 5

[0395] <210> 6

[0396] <211> 27

[0397] <212> DNA

[0398] <213> Artificial sequence

[0399] <220>

[0400] <223> HCDR3 nucleotide

[0401] <400> 6

[0402] acagatcagg ccgcctggtt tgctttc 27

[0403] <210> 7

[0404] <211> 16

[0405] <212> PROTEIN

[0406] <213> Artificial sequence

[0407] <220>

[0408] <223> LCDR1 aa

[0409] <400> 7

[0410] Arg Ser Ser Gln Asn Ile Val His Ser Asn Gly Asp Thr Tyr Leu Glu

[0411] 1 5 10 15

[0412] <210> 8

[0413] <211> 48

[0414] <212> DNA

[0415] <213> Artificial sequence

[0416] <220>

[0417] <223> LCDR1 nucleotide

[0418] <400> 8

[0419] agatctagtc agaacattgt acatagtaat ggagacacct atttagaa 48

[0420] <210> 9

[0421] <211> 7

[0422] <212> PROTEIN

[0423] <213> Artificial sequence

[0424] <220>

[0425] <223> LCDR2 aa

[0426] <400> 9

[0427] Lys Val Ser Lys Arg Phe Ser

[0428] 1 5

[0429] <210> 10

[0430] <211> 21

[0431] <212> DNA

[0432] <213> Artificial sequence

[0433] <220>

[0434] <223> LCDR2 nucleotide

[0435] <400> 10

[0436] aaagtttcca agcgattttc t 21

[0437] <210> 11

[0438] <211> 9

[0439] <212> PROTEIN

[0440] <213> Artificial sequence

[0441] <220>

[0442] <223> LCDR3 aa

[0443] <400> 11

[0444] Phe Gln Gly Ser His Val Pro Trp Thr

[0445] 1 5

[0446] <210> 12

[0447] <211> 27

[0448] <212> DNA

[0449] <213> Artificial sequence

[0450] <220>

[0451] <223> LCDR3 nucleotide

[0452] <400> 12

[0453] tttcaaggtt cacatgttcc gtggacg 27

[0454] <210> 13

[0455] <211> 118

[0456] <212> PROTEIN

[0457] <213> Artificial sequence

[0458] <220>

[0459] <223> HCVR aa

[0460] <400> 13

[0461] Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala

[0462] 1 5 10 15

[0463] Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr

[0464] 20 25 30

[0465] Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

[0466] 35 40 45

[0467] Gly Met Ile His Pro Asn Ser Asp Thr Thr Thr Tyr Asn Glu Lys Phe

[0468] 50 55 60

[0469] Lys Asn Arg Ala Thr Leu Thr Val Asp Lys Ser Ser Gly Thr Ala Tyr

[0470] 65 70 75 80

[0471] Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

[0472] 85 90 95

[0473] Thr Gly Thr Asp Gln Ala Ala Trp Phe Ala Phe Trp Gly Gln Gly Thr

[0474] 100 105 110

[0475] Leu Val Thr Val Ser Ala

[0476] 115

[0477] <210> 14

[0478] <211> 354

[0479] <212> DNA

[0480] <213> Artificial sequence

[0481] <220>

[0482] <223> HCVR nucleotide

[0483] <400> 14

[0484] gaggtccagc tgcagcagtc tggggctgag ctggttaagc ctggggcttc agtgaagctg 60

[0485] tcctgcaagg cttctggcta cactttcacc ggctactgga tgcactgggt gaagcagagg 120

[0486] cctggacaag gccttgagtg gattggaatg attcatccta acagtgatac tactacctac 180

[0487] aatgagaagt tcaaaaacag ggccacactg actgtagaca aatcctccgg cacagcctac 240

[0488] atgcaactca gcagcctgac atctgaggac tctgcggtct attactgtac agggacagat 300

[0489] caggccgcct ggtttgcttt ctggggccaa gggactctgg tcactgtctc tgca 354

[0490] <210> 15

[0491] <211> 112

[0492] <212> БЕЛОК

[0493] <213> Read more

[0494] <220>

[0495] <223> LCVR aa

[0496] <400> 15

[0497] Asp Ile Val Leu Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly

[0498] 1 5 10 15

[0499] Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Asn Ile Val His Ser

[0500] 20 25 30

[0501] Asn Gly Asp Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser

[0502] 35 40 45

[0503] Pro Lys Leu Leu Ile Tyr Lys Val Ser Lys Arg Phe Ser Gly Val Pro

[0504] 50 55 60

[0505] Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

[0506] 65 70 75 80

[0507] Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly

[0508] 85 90 95

[0509] Ser His Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

[0510] 100 105 110

[0511] <210> 16

[0512] <211> 336

[0513] <212> DNA

[0514] <213> Artificial sequence

[0515] <220>

[0516] <223> LCVR nucleotide

[0517] <400> 16

[0518] gatattgtgc tgacacaaac tccactctcc ctgcctgtca gtcttggaga tcaagcctcc 60

[0519] atctcttgca gatctagtca gaacattgta catagtaatg gagacaccta tttagaatgg 120

[0520] tacctgcaga aaccaggcca gtctccaaag ctcctgatct acaaagtttc caagcgattt 180

[0521] tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac actcaagatc 240

[0522] agcagagtgg aggctgagga tctgggagtt tattactgct ttcaaggttc acatgttccg 300

[0523] tggacgttcg gtggaggcac caagctggaa atcaaa 336

[0524] <210> 17

[0525] <211> 30

[0526] <212> PROTEIN

[0527] <213> Artificial sequence

[0528] <220>

[0529] <223> HFR1 aa

[0530] <400> 17

[0531] Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala

[0532] 1 5 10 15

[0533] Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr

[0534] 20 25 30

[0535] <210> 18

[0536] <211> 90

[0537] <212> DNA

[0538] <213> Artificial sequence

[0539] <220>

[0540] <223> HFR1 nucleotide

[0541] <400> 18

[0542] gaggtccagc tgcagcagtc tggggctgag ctggttaagc ctggggcttc agtgaagctg 60

[0543] tcctgcaagg cttctggcta cactttcacc 90

[0544] <210> 19

[0545] <211> 14

[0546] <212> PROTEIN

[0547] <213> Artificial sequence

[0548] <220>

[0549] <223> HFR2 aa

[0550] <400> 19

[0551] Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly

[0552] 1 5 10

[0553] <210> 20

[0554] <211> 42

[0555] <212> DNA

[0556] <213> Artificial sequence

[0557] <220>

[0558] <223> HFR2 nucleotide

[0559] <400> 20

[0560] tgggtgaagc agaggcctgg acaaggcctt gagtggattg ga 42

[0561] <210> 21

[0562] <211> 32

[0563] <212> PROTEIN

[0564] <213> Artificial sequence

[0565] <220>

[0566] <223> HFR3 aa

[0567] <400> 21

[0568] Arg Ala Thr Leu Thr Val Asp Lys Ser Ser Gly Thr Ala Tyr Met Gln

[0569] 1 5 10 15

[0570] Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Thr Gly

[0571] 20 25 30

[0572] <210> 22

[0573] <211> 96

[0574] <212> DNA

[0575] <213> Artificial sequence

[0576] <220>

[0577] <223> HFR3 nucleotide

[0578] <400> 22

[0579] agggccacac tgactgtaga caaatcctcc ggcacagcct acatgcaact cagcagcctg 60

[0580] acatctgagg actctgcggt ctattactgt acaggg 96

[0581] <210> 23

[0582] <211> 11

[0583] <212> PROTEIN

[0584] <213> Artificial sequence

[0585] <220>

[0586] <223> HFR4 aa

[0587] <400> 23

[0588] Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala

[0589] 1 5 10

[0590] <210> 24

[0591] <211> 33

[0592] <212> DNA

[0593] <213> Artificial sequence

[0594] <220>

[0595] <223> HFR4 nucleotide

[0596] <400> 24

[0597] tggggccaag ggactctggt cactgtctct gca 33

[0598] <210> 25

[0599] <211> 23

[0600] <212> PROTEIN

[0601] <213> Artificial sequence

[0602] <220>

[0603] <223> LFR1 aa

[0604] <400> 25

[0605] Asp Ile Val Leu Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly

[0606] 1 5 10 15

[0607] Asp Gln Ala Ser Ile Ser Cys

[0608] 20

[0609] <210> 26

[0610] <211> 69

[0611] <212> DNA

[0612] <213> Artificial sequence

[0613] <220>

[0614] <223> LFR1 nucleotide

[0615] <400> 26

[0616] gatattgtgc tgacacaaac tccactctcc ctgcctgtca gtcttggaga tcaagcctcc 60

[0617] atctcttgc 69

[0618] <210> 27

[0619] <211> 15

[0620] <212> PROTEIN

[0621] <213> Artificial sequence

[0622] <220>

[0623] <223> LFR2 aa

[0624] <400> 27

[0625] Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr

[0626] 1 5 10 15

[0627] <210> 28

[0628] <211> 45

[0629] <212> DNA

[0630] <213> Artificial sequence

[0631] <220>

[0632] <223> LFR2 nucleotide

[0633] <400> 28

[0634] tggtacctgc agaaaccagg ccagtctcca aagctcctga tctac 45

[0635] <210> 29

[0636] <211> 32

[0637] <212> PROTEIN

[0638] <213> Artificial sequence

[0639] <220>

[0640] <223> LFR3 aa

[0641] <400> 29

[0642] Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

[0643] 1 5 10 15

[0644] Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys

[0645] 20 25 30

[0646] <210> 30

[0647] <211> 96

[0648] <212> DNA

[0649] <213> Artificial sequence

[0650] <220>

[0651] <223> LFR3 nucleotide

[0652] <400> 30

[0653] ggggtcccag acaggttcag tggcagtgga tcagggacag atttcacact caagatcagc 60

[0654] agagtggagg ctgaggatct gggagtttat tactgc 96

[0655] <210> 31

[0656] <211> 10

[0657] <212> PROTEIN

[0658] <213> Artificial sequence

[0659] <220>

[0660] <223> LFR4 aa

[0661] <400> 31

[0662] Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

[0663] 1 5 10

[0664] <210> 32

[0665] <211> 30

[0666] <212> DNA

[0667] <213> Artificial sequence

[0668] <220>

[0669] <223> LFR4 nucleotide

[0670] <400> 32

[0671] ttcggtggag gcaccaagct ggaaatcaaa 30

[0672] <210> 33

[0673] <211> 31

[0674] <212> DNA

[0675] <213> Artificial sequence

[0676] <220>

[0677] <223> primer MH1

[0678] <400> 33

[0679] aatttgctag csargtnmag ctgsagsagt c 31

[0680] <210> 34

[0681] <211> 34

[0682] <212> DNA

[0683] <213> Artificial sequence

[0684] <220>

[0685] <223> primer MH2

[0686] <400> 34

[0687] aatttgctag csargtnmag ctgsagsagt cwgg 34

[0688] <210> 35

[0689] <211> 38

[0690] <212> DNA

[0691] <213> Artificial sequence

[0692] <220>

[0693] <223> IgG1 primer

[0694] <400> 35

[0695] aatttggatc catagacaga tggggggtgtc gttttggc 38

[0696] <210> 36

[0697] <211> 34

[0698] <212> DNA

[0699] <213> Artificial sequence

[0700] <220>

[0701] <223> primer MK

[0702] <400> 36

[0703] aattggatcc aggggccagt ggatagactg atgg

[0704] <210> 37

[0705] <211> 34

[0706] <212> ДНК

[0707] <213> Искусствнная послеватеность

[0708] <220>

[0709] <223> Flying CK

[0710] <400> 37

[0711] atttgcggc cgcggataca gttggtgcag catc

[0712] <210> 38

[0713] <211> 981

[0714] <212> ДНК

[0715] <213> Искусствнная послеватеность

[0716] <220>

[0717] <223> IgG4 CH liver

[0718] <400> 38

[0719] gctagcacca agggcccatc ggtcttcccc ctggcgccct gctccaggag cacctccgag

[0720] agcacagccg ccctgggctg cctggtcaag gactacttcc ccgaacccgt gacggtgtcg

[0721] tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct cacagtcctca

[0722] ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacgaagacc

[0723] tacacctgca acgtagca caagcccagc aacaccaagg tggacaagag agttgagtcc

[0724] aaatatggtc ccccatgccc accatgccca gcacctgagt tcctgggggg accatcagtc 360

[0725] ttcctgttcc ccccaaaacc caaggacact ctcatgatct cccggacccc tgaggtcacg 420

[0726] tgcgtggtgg tggacgtgag ccaggaagac cccgaggtcc agttcaactg gtacgtggat 480

[0727] ggcgtggagg tgcataatgc caacaaag ccgcgggagg agcagttcaa cagcacgtac 540

[0728] cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaacggcaa ggatcaag 600

[0729] tgcaaggtct ccaacaaagg cctcccgtcc tccatcgaga aaaccatctc caaagccaaa 660

[0730] gggcagcccc gagagccaca ggtgtacacc ctgcccccat cccaggagga gatgaccaag 720

[0731] aaccaggtca gcctgacctg cctggtcaaa ggcttctacc ccagcgacat cgccgtggag 780

[0732] tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 840

[0733] gacggctcct tcttctctca cagcaggcta accgtggaca agagcaggtg gcaggagggg 900

[0734] aatgtcttct catgctccgt gatgcatgag gctctgcaca accactacac acagaagagc 960

[0735] ctctccctgt ctccggggtaa a 981

[0736] <210> 39

[0737] <211> 327

[0738] <212> PROTEIN

[0739] <213> Artificial sequence

[0740] <220>

[0741] <223> Human IgG4 CH

[0742] <400> 39

[0743] Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

[0744] 1 5 10 15

[0745] Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

[0746] 20 25 30

[0747] Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

[0748] 35 40 45

[0749] Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

[0750] 50 55 60

[0751] Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

[0752] 65 70 75 80

[0753] Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

[0754] 85 90 95

[0755] Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro

[0756] 100 105 110

[0757] Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

[0758] 115 120 125

[0759] Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

[0760] 130 135 140

[0761] Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

[0762] 145 150 155 160

[0763] Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

[0764] 165 170 175

[0765] Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

[0766] 180 185 190

[0767] Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

[0768] 195 200 205

[0769] Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

[0770] 210 215 220

[0771] Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys

[0772] 225 230 235 240

[0773] Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

[0774] 245 250 255

[0775] Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

[0776] 260 265 270

[0777] Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

[0778] 275 280 285

[0779] Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

[0780] 290 295 300

[0781] Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

[0782] 305 310 315 320

[0783] Leu Ser Leu Ser Pro Gly Lys

[0784] 325

[0785] <210> 40

[0786] <211> 321

[0787] <212> DNA

[0788] <213> Artificial sequence

[0789] <220>

[0790] <223> Human IgG4 Ckappa

[0791] <400> 40

[0792] cgtacggtgg ctgcaccatc tgtcttcatc ttcccgccat ctgatgagca gttgaaatct 60

[0793] ggaactgcct ctgttgtgtg cctgctgaat aacttctatc ccagagaggc caaagtacag 120

[0794] tggaaggtgg ataacgccct ccaatcgggt aactcccagg agagtgtcac agagcaggac 180

[0795] agcaaggaca gcacctacag cctcagcagc accctgacgc tgagcaaagc agactacgag 240

[0796] aaacacaaag tctacgcctg cgaagtcacc catcagggcc tgagctcgcc cgtcacaaag 300

[0797] agcttcaaca ggggagagtg t 321

[0798] <210> 41

[0799] <211> 107

[0800] <212> PROTEIN

[0801] <213> Artificial sequence

[0802] <220>

[0803] <223> Human IgG4 Ckappa

[0804] <400> 41

[0805] Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

[0806] 1 5 10 15

[0807] Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

[0808] 20 25 30

[0809] Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

[0810] 35 40 45

[0811] Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

[0812] 50 55 60

[0813] Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

[0814] 65 70 75 80

[0815] Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

[0816] 85 90 95

[0817] Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

[0818] 100 105

[0819] <210> 42

[0820] <211> 1341

[0821] <212> DNA

[0822] <213> Artificial sequence

[0823] <220>

[0824] <223> Humanized antibody 1G1-h61 heavy chain nucleotides

[0825] <400> 42

[0826] caagttcagc tggtgcagag cggcgccgag gtgaagaagc ctggagccag cgtgaaagtg 60

[0827] tcctgcaagg cctctggcta cacctttaca ggctactgga tgcactgggt gcggcaggcc 120

[0828] cctggacagg gcctggaatg gatgggcatg atccacccca acagcgacac cacaacctac 180

[0829] aacgagaagt tcaagaatag agtgaccatg acaagagata ccagcatcag caccgcctac 240

[0830] atggaactga gcagactgcg gtccgatgac acagctgtgt actattgtgc cggcaccgac 300

[0831] caggccgctt ggttcgcctt ctgggggcag ggcaccacag tcaccgtgag ctctgccagc 360

[0832] accaagggcc cttccgtgtt tcccctggcc ccttgctccc ggtccacatc tgagagcacc 420

[0833] gccgccctgg gctgtctggt gaaggactac ttcccagagc ccgtgaccgt gagctggaac 480

[0834] agcggcgccc tgacaagcgg cgtgcacaca tttcccgccg tgctgcagag ctccggcctg 540

[0835] tactccctgt ctagcgtggt gacagtgcct tcctctagcc tgggcaccaa gacatatacc 600

[0836] tgtaacgtgg accacaagcc aagcaatacc aaggtggata agcgggtgga gtctaagtac 660

[0837] ggccctcctt gccctagctg tcctgctcca gagtttctgg gcggcccttc cgtgttcctg 720

[0838] tttccaccca aaccaaagga cacactgatg atctctagaa caccagaggt gacctgcgtg 780

[0839] gtggtggacg tgagccagga ggatcccgag gtgcagttca actggtacgt ggatggcgtg 840

[0840] gaggtgcaca atgccaagac caagccaaga gaggagcagt ttaactctac atacagggtg 900

[0841] gtgagcgtgc tgaccgtgct gcaccaggat tggctcaacg gcaaggagta taagtgcaag 960

[0842] gtgtccaata agggcctgcc ctcctctatc gagaagacaa tctctaaggc taagggccag 1020

[0843] ccaagagagc ctcaggtgta caccctgcct ccaagccagg aggagatgac aaagaaccag 1080

[0844] gtgtccctga catgtctggt gaagggcttc tatccctccg acatcgccgt ggagtgggag 1140

[0845] tctaatggcc agcctgagaa caattacaag accacacccc ctgtgctgga ctctgatggc 1200

[0846] agcttctttc tgtattccag gctgaccgtg gataagtctc ggtggcagga gggcaacgtg 1260

[0847] ttcagctgct ctgtgatgca cgaagccctg cataatcact atactcagaa aagtctgtca 1320

[0848] ctgtcactgg gaaagtgata a 1341

[0849] <210> 43

[0850] <211> 445

[0851] <212> PROTEIN

[0852] <213> Artificial sequence

[0853] <220>

[0854] <223> Humanized antibody 1G1-h61 amino acid heavy chain

[0855] <400> 43

[0856] Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

[0857] 1 5 10 15

[0858] Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr

[0859] 20 25 30

[0860] Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

[0861] 35 40 45

[0862] Gly Met Ile His Pro Asn Ser Asp Thr Thr Thr Tyr Asn Glu Lys Phe

[0863] 50 55 60

[0864] Lys Asn Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr

[0865] 65 70 75 80

[0866] Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

[0867] 85 90 95

[0868] Ala Gly Thr Asp Gln Ala Ala Trp Phe Ala Phe Trp Gly Gln Gly Thr

[0869] 100 105 110

[0870] Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

[0871] 115 120 125

[0872] Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly

[0873] 130 135 140

[0874] Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

[0875] 145 150 155 160

[0876] Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

[0877] 165 170 175

[0878] Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

[0879] 180 185 190

[0880] Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser

[0881] 195 200 205

[0882] Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys

[0883] 210 215 220

[0884] Pro Ser Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu

[0885] 225 230 235 240

[0886] Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

[0887] 245 250 255

[0888] Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln

[0889] 260 265 270

[0890] Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

[0891] 275 280 285

[0892] Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu

[0893] 290 295 300

[0894] Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

[0895] 305 310 315 320

[0896] Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys

[0897] 325 330 335

[0898] Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

[0899] 340 345 350

[0900] Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

[0901] 355 360 365

[0902] Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

[0903] 370 375 380

[0904] Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

[0905] 385 390 395 400

[0906] Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln

[0907] 405 410 415

[0908] Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

[0909] 420 425 430

[0910] His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

[0911] 435 440 445

[0912] <210> 44

[0913] <211> 1341

[0914] <212> DNA

[0915] <213> Artificial sequence

[0916] <220>

[0917] <223> Humanized antibody 1G1-h68 heavy chain nucleotides

[0918] <400> 44

[0919] caagttcagc tggtgcagag cggcgccgag gtgaagaagc ctggagccag cgtgaaagtg 60

[0920] tcctgcaagg cctctggcta cacctttaca ggctactgga tgcactgggt gcggcaggcc 120

[0921] cctggacagg gcctggaatg gatcggcatg atccacccca acagcgacac cacaacctac 180

[0922] aacgagaagt tcaagaatag agtgaccatg acaagagata ccagcatcag caccgcctac 240

[0923] atggaactga gcagactgcg gtccgatgac acagctgtgt actattgtac cggcaccgac 300

[0924] caggccgctt ggttcgcctt ctgggggcag ggcaccacag tcaccgtgag ctctgccagc 360

[0925] accaagggcc cttccgtgtt tcccctggcc ccttgctccc ggtccacatc tgagagcacc 420

[0926] gccgccctgg gctgtctggt gaaggactac ttcccagagc ccgtgaccgt gagctggaac 480

[0927] agcggcgccc tgacaagcgg cgtgcacaca tttcccgccg tgctgcagag ctccggcctg 540

[0928] tactccctgt ctagcgtggt gacagtgcct tcctctagcc tgggcaccaa gacatatacc 600

[0929] tgtaacgtgg accacaagcc aagcaatacc aaggtggata agcgggtgga gtctaagtac 660

[0930] ggccctcctt gccctagctg tcctgctcca gagtttctgg gcggcccttc cgtgttcctg 720

[0931] tttccaccca aaccaaagga cacactgatg atctctagaa caccagaggt gacctgcgtg 780

[0932] gtggtggacg tgagccagga ggatcccgag gtgcagttca actggtacgt ggatggcgtg 840

[0933] gaggtgcaca atgccaagac caagccaaga gaggagcagt ttaactctac atacagggtg 900

[0934] gtgagcgtgc tgaccgtgct gcaccaggat tggctcaacg gcaaggagta taagtgcaag 960

[0935] gtgtccaata agggcctgcc ctcctctatc gagaagacaa tctctaaggc taaggggccag 1020

[0936] ccaagagagc ctcaggtgta caccctgcct ccaagccagg aggagatgac aaagaaccag 1080

[0937] gtgtccctga catgtctggt gaagggcttc tatccctccg acatcgccgt ggagtgggag 1140

[0938] tctaatggcc agcctgagaa caattacaag accacacccc ctgtgctgga ctctgatggc 1200

[0939] agcttctttc tgtattccag gctgaccgtg gataagtctc ggtggcagga gggcaacgtg 1260

[0940] ttcagctgct ctgtgatgca cgaagccctg cataatcact atactcagaa aagtctgtca 1320

[0941] ctgtcactgg gaaagtgata a 1341

[0942] <210> 45

[0943] <211> 445

[0944] <212> PROTEIN

[0945] <213> Artificial sequence

[0946] <220>

[0947] <223> Humanized antibody 1G1-h68 amino acid heavy chain

[0948] <400> 45

[0949] Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

[0950] 1 5 10 15

[0951] Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr

[0952] 20 25 30

[0953] Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

[0954] 35 40 45

[0955] Gly Met Ile His Pro Asn Ser Asp Thr Thr Thr Tyr Asn Glu Lys Phe

[0956] 50 55 60

[0957] Lys Asn Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr

[0958] 65 70 75 80

[0959] Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

[0960] 85 90 95

[0961] Thr Gly Thr Asp Gln Ala Ala Trp Phe Ala Phe Trp Gly Gln Gly Thr

[0962] 100 105 110

[0963] Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

[0964] 115 120 125

[0965] Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly

[0966] 130 135 140

[0967] Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

[0968] 145 150 155 160

[0969] Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

[0970] 165 170 175

[0971] Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

[0972] 180 185 190

[0973] Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser

[0974] 195 200 205

[0975] Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys

[0976] 210 215 220

[0977] Pro Ser Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu

[0978] 225 230 235 240

[0979] Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

[0980] 245 250 255

[0981] Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln

[0982] 260 265 270

[0983] Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

[0984] 275 280 285

[0985] Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu

[0986] 290 295 300

[0987] Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

[0988] 305 310 315 320

[0989] Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys

[0990] 325 330 335

[0991] Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

[0992] 340 345 350

[0993] Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

[0994] 355 360 365

[0995] Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

[0996] 370 375 380

[0997] Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

[0998] 385 390 395 400

[0999] Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln

[1000] 405 410 415

[1001] Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

[1002] 420 425 430

[1003] His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

[1004] 435 440 445

[1005] <210> 46

[1006] <211> 1341

[1007] <212> DNA

[1008] <213> Artificial sequence

[1009] <220>

[1010] <223> Humanized 1G1-h70 heavy chain nucleotide antibody

[1011] <400> 46

[1012] caagttcagc tggtgcagag cggcgccgag gtgaagaagc ctggagccag cgtgaaagtg 60

[1013] tcctgcaagg cctctggcta cacctttaca ggctactgga tgcactgggt gcggcaggcc 120

[1014] cctggacagg gcctggaatg gatcggcatg atccacccca acagcgacac cacaacctac 180

[1015] aacgagaagt tcaagaatag agtgaccatg acaagagata ccagcatcag caccgcctac 240

[1016] atggaactga gcagactgcg gtccgatgac acagctgtgt actattgtgc cggcaccgac 300

[1017] caggccgctt ggttcgcctt ctgggggcag ggcaccacag tcaccgtgag ctctgccagc 360

[1018] accaagggcc cttccgtgtt tcccctggcc cttgctccc ggtccacatc tgagagcacc 420

[1019] gccgccctgg gctgtctggt gaaggactac ttcccagagc ccgtgaccgt gagctggaac 480

[1020] agcggcgccc tgacaagcgg cgtgcacaca tttcccgccg tgctgcagag ctccggcctg

[1021] tactccctgt ctagcgtggt gacagtgcct tcctctagcc tgggcaccaa gacatatacc

[1022] tgtaacgtgg accacaagcc aagcaatacc aaggtggata agcgggtgga gtctaagtac

[1023] ggccctcctt gccctagctg tcctgctcca gagtttctgg gcggcccttc cgtgttcctg 720

[1024] tttccaccca aaccaaagga cacactgatg atctctagaa caccagaggt gacctgcgtg

[1025] gtggtggacg tgagccagga ggatcccgag gtgcagttca actggtacgt ggatggcgtg

[1026] gaggtgcaca atgccaagac caagccaaga gaggagcagt ttaactctac atacagggtg

[1027] gtgagcgtgc tgaccgtgct gcaccaggat tggctcaacg gcaaggagta tagtgcaag

[1028] gtgtccaata agggcctgcc ctcctctatc gagagacaa tctctaaggc tagggccag

[1029] caagagagc ctcaggtgta caccctgcct ccaagccagg aggagatgac aaagaaccag

[1030] gtgtccctga catgtctggt gaagggcttc tatccctccg acatcgccgt ggagtgggag

[1031] tctaatggcc agcctgagaa caattacaag accacacccc ctgtgctgga ctctgatggc 1200

[1032] agcttctttc tgtattccag gctgaccgtg gataagtctc ggtggcagga gggcaacgtg 1260

[1033] ttcagctgct ctgtgatgca cgaagccctg cataatcact atactcagaa aagtctgtca 1320

[1034] ctgtcactgg gaaagtgata a 1341

[1035] <210> 47

[1036] <211> 445

[1037] <212> PROTEIN

[1038] <213> Artificial sequence

[1039] <220>

[1040] <223> Humanized antibody 1G1-h70 amino acid heavy chain

[1041] <400> 47

[1042] Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

[1043] 1 5 10 15

[1044] Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr

[1045] 20 25 30

[1046] Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

[1047] 35 40 45

[1048] Gly Met Ile His Pro Asn Ser Asp Thr Thr Thr Tyr Asn Glu Lys Phe

[1049] 50 55 60

[1050] Lys Asn Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr

[1051] 65 70 75 80

[1052] Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

[1053] 85 90 95

[1054] Ala Gly Thr Asp Gln Ala Ala Trp Phe Ala Phe Trp Gly Gln Gly Thr

[1055] 100 105 110

[1056] Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

[1057] 115 120 125

[1058] Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly

[1059] 130 135 140

[1060] Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

[1061] 145 150 155 160

[1062] Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

[1063] 165 170 175

[1064] Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

[1065] 180 185 190

[1066] Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser

[1067] 195 200 205

[1068] Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys

[1069] 210 215 220

[1070] Pro Ser Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu

[1071] 225 230 235 240

[1072] Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

[1073] 245 250 255

[1074] Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln

[1075] 260 265 270

[1076] Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

[1077] 275 280 285

[1078] Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu

[1079] 290 295 300

[1080] Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

[1081] 305 310 315 320

[1082] Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys

[1083] 325 330 335

[1084] Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

[1085] 340 345 350

[1086] Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

[1087] 355 360 365

[1088] Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

[1089] 370 375 380

[1090] Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

[1091] 385 390 395 400

[1092] Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln

[1093] 405 410 415

[1094] Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

[1095] 420 425 430

[1096] His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

[1097] 435 440 445

[1098] <210> 48

[1099] <211> 663

[1100] <212> DNA

[1101] <213> Artificial sequence

[1102] <220>

[1103] <223> Subscribe to the 1G1-h61 range of videos

[1104] <400> 48

[1105] gatatcgtta tgacccagac ccctctgagc ctgtccgtga ccccaggcca acctgcctct

[1106] atcagctgta gaagcagcca gaacatcgtg cacagcaacg gcgacaccta cctggaatgg

[1107] tatctgcaga aacctggaca gagccccag ctgctgatct acaaggtgtc caagcggttt

[1108] tccggcgtgc ctgatagatt cagcggatct ggcagcggca cagacttcac cctgaagatt

[1109] tctagagtgg aggccgagga cgtgggcgtc tactactgct tccagggcag ccacgtgccc 300

[1110] tggacattcg gcggcggac aaaggtgga atcaagagga cagtggccgc cccaagcgtg

[1111] ttcatctttc ccccttccga cgagcagctg aagtctggca ccgccagcgt ggtgtgcctg

[1112] ctgaacaact tctaccctcg ggaggccag gtccagtgga aggtggataa cgccctgcag

[1113] tctggcaata gccaggagtc cgtgaccgag caggactcta aggatagcac atattccctg

[1114] tctagcaccc tgacactgag caaggccgat tacgagaagc acaaggtgta tgcctgtgaa

[1115] gtcacccatc aggggctgtc atcacccgtc actaagtcat tcaatcgcgg agaatgctga 660

[1116] taa 663

[1117] <210> 49

[1118] <211> 219

[1119] <212> PROTEIN

[1120] <213> Artificial sequence

[1121] <220>

[1122] <223> Humanized antibody 1G1-h61 amino acid light chain

[1123] <400> 49

[1124] Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly

[1125] 1 5 10 15

[1126] Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Asn Ile Val His Ser

[1127] 20 25 30

[1128] Asn Gly Asp Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser

[1129] 35 40 45

[1130] Pro Lys Leu Leu Ile Tyr Lys Val Ser Lys Arg Phe Ser Gly Val Pro

[1131] 50 55 60

[1132] Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

[1133] 65 70 75 80

[1134] Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly

[1135] 85 90 95

[1136] Ser His Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

[1137] 100 105 110

[1138] Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

[1139] 115 120 125

[1140] Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

[1141] 130 135 140

[1142] Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

[1143] 145 150 155 160

[1144] Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

[1145] 165 170 175

[1146] Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

[1147] 180 185 190

[1148] Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

[1149] 195 200 205

[1150] Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

[1151] 210 215

[1152] <210> 50

[1153] <211> 663

[1154] <212> DNA

[1155] <213> Artificial sequence

[1156] <220>

[1157] <223> Humanized antibody 1G1-h68 light chain nucleotides

[1158] <400> 50

[1159] gatatcgtta tgacccagac cctctgagc ctgtccgtga cccaggcca acctgcctct 60

[1160] atcagctgta gaagcagcca gaacatcgtg cacagcaacg gcgacaccta cctggaatgg 120

[1161] tatctgcaga aacctggaca gagcccccag ctgctgatct acaaggtgtc caagcggttt 180

[1162] tccggcgtgc ctgatagatt cagcggatct ggcagcggca cagacttcac cctgaagatt 240

[1163] tctagagtgg aggccgagga cgtgggcgtc tactactgct tccagggcag ccacgtgccc 300

[1164] tggacattcg gcggcggaac aaaggtggaa atcaagagga cagtggccgc cccaagcgtg 360

[1165] ttcatctttc ccccttccga cgagcagctg aagtctggca ccgccagcgt ggtgtgcctg 420

[1166] ctgaacaact tctaccctcg ggaggccaag gtccagtgga aggtggataa cgccctgcag 480

[1167] tctggcaata gccaggagtc cgtgaccgag caggactcta aggatagcac atattccctg 540

[1168] tctagcaccc tgacactgag caaggccgat tacgagaagc acaaggtgta tgcctgtgaa 600

[1169] gtcacccatc aggggctgtc atcacccgtc actaagtcat tcaatcgcgg agaatgctga 660

[1170] taa 663

[1171] <210> 51

[1172] <211> 219

[1173] <212> PROTEIN

[1174] <213> Artificial sequence

[1175] <220>

[1176] <223> Humanized antibody 1G1-h68 amino acid light chain

[1177] <400> 51

[1178] Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly

[1179] 1 5 10 15

[1180] Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Asn Ile Val His Ser

[1181] 20 25 30

[1182] Asn Gly Asp Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser

[1183] 35 40 45

[1184] Pro Gln Leu Leu Ile Tyr Lys Val Ser Lys Arg Phe Ser Gly Val Pro

[1185] 50 55 60

[1186] Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

[1187] 65 70 75 80

[1188] Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly

[1189] 85 90 95

[1190] Ser His Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

[1191] 100 105 110

[1192] Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

[1193] 115 120 125

[1194] Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

[1195] 130 135 140

[1196] Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

[1197] 145 150 155 160

[1198] Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

[1199] 165 170 175

[1200] Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

[1201] 180 185 190

[1202] Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

[1203] 195 200 205

[1204] Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

[1205] 210 215

[1206] <210> 52

[1207] <211> 663

[1208] <212> DNA

[1209] <213> Artificial sequence

[1210] <220>

[1211] <223> Humanized 1G1-h70 nucleotide light chain antibody

[1212] <400> 52

[1213] gatatcgtta tgacccagac ccctctgagc ctgtccgtga cccaggcca acctgcctct 60

[1214] atcagctgta gaagcagcca gaacatcgtg cacagccagg gcgacaccta cctggaatgg 120

[1215] tatctgcaga aacctggaca gagcccccag ctgctgatct acaaggtgtc caagcggtttt 180

[1216] tccggcgtgc ctgatagatt cagcggatct ggcagcggca cagacttcac cctgaagatt 240

[1217] tctagagtgg aggccgagga cgtgggcgtc tactactgct tccagggcag ccacgtgccc 300

[1218] tggacattcg gcggcggaac aaaggtggaa atcaagagga cagtggccgc cccaagcgtg 360

[1219] ttcatctttc ccccttccga cgagcagctg aagtctggca ccgccagcgt ggtgtgcctg 420

[1220] ctgaacaact tctaccctcg ggaggccaag gtccagtgga aggtggataa cgccctgcag 480

[1221] tctggcaata gccaggagtc cgtgaccgag caggactcta aggatagcac atattccctg 540

[1222] tctagcaccc tgacactgag caaggccgat tacgagaagc acaaggtgta tgcctgtgaa 600

[1223] gtcacccatc aggggctgtc atcacccgtc actaagtcat tcaatcgcgg agaatgctga 660

[1224] taa 663

[1225] <210> 53

[1226] <211> 219

[1227] <212> PROTEIN

[1228] <213> Artificial sequence

[1229] <220>

[1230] <223> Humanized antibody 1G1-h70 amino acid light chain

[1231] <400> 53

[1232] Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly

[1233] 1 5 10 15

[1234] Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Asn Ile Val His Ser

[1235] 20 25 30

[1236] Gln Gly Asp Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser

[1237] 35 40 45

[1238] Pro Gln Leu Leu Ile Tyr Lys Val Ser Lys Arg Phe Ser Gly Val Pro

[1239] 50 55 60

[1240] Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

[1241] 65 70 75 80

[1242] Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly

[1243] 85 90 95

[1244] Ser His Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

[1245] 100 105 110

[1246] Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

[1247] 115 120 125

[1248] Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

[1249] 130 135 140

[1250] Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

[1251] 145 150 155 160

[1252] Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

[1253] 165 170 175

[1254] Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

[1255] 180 185 190

[1256] Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

[1257] 195 200 205

[1258] Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

[1259] 210 215

[1260] <210> 54

[1261] <211> 118

[1262] <212> PROTEIN

[1263] <213> Artificial sequence

[1264] <220>

[1265] <223> Humanized antibody 1G1-h61 VH amino acid

[1266] <400> 54

[1267] Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

[1268] 1 5 10 15

[1269] Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr

[1270] 20 25 30

[1271] Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

[1272] 35 40 45

[1273] Gly Met Ile His Pro Asn Ser Asp Thr Thr Thr Tyr Asn Glu Lys Phe

[1274] 50 55 60

[1275] Lys Asn Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr

[1276] 65 70 75 80

[1277] Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

[1278] 85 90 95

[1279] Ala Gly Thr Asp Gln Ala Ala Trp Phe Ala Phe Trp Gly Gln Gly Thr

[1280] 100 105 110

[1281] Thr Val Thr Val Ser Ser

[1282] 115

[1283] <210> 55

[1284] <211> 112

[1285] <212> PROTEIN

[1286] <213> Artificial sequence

[1287] <220>

[1288] <223> Humanized antibody 1G1-h61 VL amino acids

[1289] <400> 55

[1290] Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly

[1291] 1 5 10 15

[1292] Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Asn Ile Val His Ser

[1293] 20 25 30

[1294] Asn Gly Asp Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser

[1295] 35 40 45

[1296] Pro Lys Leu Leu Ile Tyr Lys Val Ser Lys Arg Phe Ser Gly Val Pro

[1297] 50 55 60

[1298] Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

[1299] 65 70 75 80

[1300] Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly

[1301] 85 90 95

[1302] Ser His Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

[1303] 100 105 110

[1304] <210> 56

[1305] <211> 118

[1306] <212> PROTEIN

[1307] <213> Artificial sequence

[1308] <220>

[1309] <223> Humanized antibody 1G1-h68 VH amino acid

[1310] <400> 56

[1311] Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

[1312] 1 5 10 15

[1313] Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr

[1314] 20 25 30

[1315] Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

[1316] 35 40 45

[1317] Gly Met Ile His Pro Asn Ser Asp Thr Thr Thr Tyr Asn Glu Lys Phe

[1318] 50 55 60

[1319] Lys Asn Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr

[1320] 65 70 75 80

[1321] Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

[1322] 85 90 95

[1323] Thr Gly Thr Asp Gln Ala Ala Trp Phe Ala Phe Trp Gly Gln Gly Thr

[1324] 100 105 110

[1325] Thr Val Thr Val Ser Ser

[1326] 115

[1327] <210> 57

[1328] <211> 112

[1329] <212> PROTEIN

[1330] <213> Artificial sequence

[1331] <220>

[1332] <223> Humanized antibody 1G1-h68 VL amino acids

[1333] <400> 57

[1334] Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly

[1335] 1 5 10 15

[1336] Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Asn Ile Val His Ser

[1337] 20 25 30

[1338] Asn Gly Asp Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser

[1339] 35 40 45

[1340] Pro Gln Leu Leu Ile Tyr Lys Val Ser Lys Arg Phe Ser Gly Val Pro

[1341] 50 55 60

[1342] Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

[1343] 65 70 75 80

[1344] Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly

[1345] 85 90 95

[1346] Ser His Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

[1347] 100 105 110

[1348] <210> 58

[1349] <211> 118

[1350] <212> PROTEIN

[1351] <213> Artificial sequence

[1352] <220>

[1353] <223> Humanized antibody 1G1-h70 VH amino acid

[1354] <400> 58

[1355] Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

[1356] 1 5 10 15

[1357] Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr

[1358] 20 25 30

[1359] Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

[1360] 35 40 45

[1361] Gly Met Ile His Pro Asn Ser Asp Thr Thr Thr Tyr Asn Glu Lys Phe

[1362] 50 55 60

[1363] Lys Asn Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr

[1364] 65 70 75 80

[1365] Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

[1366] 85 90 95

[1367] Ala Gly Thr Asp Gln Ala Ala Trp Phe Ala Phe Trp Gly Gln Gly Thr

[1368] 100 105 110

[1369] Thr Val Thr Val Ser Ser

[1370] 115

[1371] <210> 59

[1372] <211> 112

[1373] <212> PROTEIN

[1374] <213> Artificial sequence

[1375] <220>

[1376] <223> Humanized antibody 1G1-h70 VL amino acids

[1377] <400> 59

[1378] Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly

[1379] 1 5 10 15

[1380] Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Asn Ile Val His Ser

[1381] 20 25 30

[1382] Gln Gly Asp Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser

[1383] 35 40 45

[1384] Pro Gln Leu Leu Ile Tyr Lys Val Ser Lys Arg Phe Ser Gly Val Pro

[1385] 50 55 60

[1386] Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

[1387] 65 70 75 80

[1388] Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly

[1389] 85 90 95

[1390] Ser His Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

[1391] 100 105 110

[1392] <210> 60

[1393] <211> 16

[1394] <212> PROTEIN

[1395] <213> Artificial sequence

[1396] <220>

[1397] <223> LCDR1 amino acids

[1398] <400> 60

[1399] Arg Ser Ser Gln Asn Ile Val His Ser Gln Gly Asp Thr Tyr Leu Glu

[1400] 1 5 10 15

[1401] <210> 61

[1402] <211> 48

[1403] <212> DNA

[1404] <213> Artificial sequence

[1405] <220>

[1406] <223> LCDR1 nucleotides

[1407] <400> 61

[1408] agaagcagcc agaacatcgt gcacagccag ggcgacacct acctggaa 48

[1409] <210> 62

[1410] <211> 288

[1411] <212> PROTEIN

[1412] <213> Artificial sequence

[1413] <220>

[1414] <223> Human PD-1 protein full-length amino acid sequence

[1415] <400> 62

[1416] Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln

[1417] 1 5 10 15

[1418] Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp

[1419] 20 25 30

[1420] Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp

[1421] 35 40 45

[1422] Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val

[1423] 50 55 60

[1424] Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala

[1425] 65 70 75 80

[1426] Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg

[1427] 85 90 95

[1428] Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg

[1429] 100 105 110

[1430] Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu

[1431] 115 120 125

[1432] Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val

[1433] 130 135 140

[1434] Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro

[1435] 145 150 155 160

[1436] Arg Pro Ala Gly Gln Phe Gln Thr Leu Val Val Gly Val Val Gly Gly

[1437] 165 170 175

[1438] Leu Leu Gly Ser Leu Val Leu Leu Val Trp Val Leu Ala Val Ile Cys

[1439] 180 185 190

[1440] Ser Arg Ala Ala Arg Gly Thr Ile Gly Ala Arg Arg Thr Gly Gln Pro

[1441] 195 200 205

[1442] Leu Lys Glu Asp Pro Ser Ala Val Pro Val Phe Ser Val Asp Tyr Gly

[1443] 210 215 220

[1444] Glu Leu Asp Phe Gln Trp Arg Glu Lys Thr Pro Glu Pro Pro Val Pro

[1445] 225 230 235 240

[1446] Cys Val Pro Glu Gln Thr Glu Tyr Ala Thr Ile Val Phe Pro Ser Gly

[1447] 245 250 255

[1448] Met Gly Thr Ser Ser Pro Ala Arg Arg Gly Ser Ala Asp Gly Pro Arg

[1449] 260 265 270

[1450] Ser Ala Gln Pro Leu Arg Pro Glu Asp Gly His Cys Ser Trp Pro Leu

[1451] 275 280 285

[1452] <210> 63

[1453] <211> 17

[1454] <212> PROTEIN

[1455] <213> Artificial sequence

[1456] <220>

[1457] <223> HCDR2 aa

[1458] <400> 63

[1459] Met Ile His Pro Asn Ser Asp Thr Thr Val Tyr Asn Glu Lys Phe Lys

[1460] 1 5 10 15

[1461] Asn

[1462] <210> 64

[1463] <211> 17

[1464] <212> PROTEIN

[1465] <213> Artificial sequence

[1466] <220>

[1467] <223> HCDR2 aa

[1468] <400> 64

[1469] Met Ile His Pro Asn Ser Asp Thr Thr Ile Tyr Asn Glu Lys Phe Lys

[1470] 1 5 10 15

[1471] Asn

[1472] <210> 65

[1473] <211> 17

[1474] <212> PROTEIN

[1475] <213> Artificial sequence

[1476] <220>

[1477] <223> HCDR2 aa

[1478] <400> 65

[1479] Met Ile His Pro Asn Ser Asp Thr Thr Thr Tyr Asn Trp Lys Phe Lys

[1480] 1 5 10 15

[1481] Asn

[1482] <210> 66

[1483] <211> 17

[1484] <212> PROTEIN

[1485] <213> Artificial sequence

[1486] <220>

[1487] <223> HCDR2 aa

[1488] <400> 66

[1489] Met Ile His Pro Asn Ser Asp Thr Thr Val Tyr Asn Trp Lys Phe Lys

[1490] 1 5 10 15

[1491] Asn

[1492] <210> 67

[1493] <211> 17

[1494] <212> PROTEIN

[1495] <213> Artificial sequence

[1496] <220>

[1497] <223> HCDR2 aa

[1498] <400> 67

[1499] Met Ile His Pro Asn Ser Asp Thr Thr Ile Tyr Asn Trp Lys Phe Lys

[1500] 1 5 10 15

[1501] Asn

[1502] <210> 68

[1503] <211> 9

[1504] <212> PROTEIN

[1505] <213> Artificial sequence

[1506] <220>

[1507] <223> HCDR3 aa

[1508] <400> 68

[1509] Thr Asp Gln Glu Ala Trp Phe Ala Phe

[1510] 1 5

[1511] <210> 69

[1512] <211> 9

[1513] <212> PROTEIN

[1514] <213> Artificial sequence

[1515] <220>

[1516] <223> HCDR3 aa

[1517] <400> 69

[1518] Thr Asp Gln Ala Ala Trp Ala Ala Phe

[1519] 1 5

[1520] <210> 70

[1521] <211> 9

[1522] <212> PROTEIN

[1523] <213> Artificial sequence

[1524] <220>

[1525] <223> HCDR3 aa

[1526] <400> 70

[1527] Thr Asp Gln Ala Ala Trp Met Ala Phe

[1528] 1 5

[1529] <210> 71

[1530] <211> 9

[1531] <212> PROTEIN

[1532] <213> Artificial sequence

[1533] <220>

[1534] <223> HCDR3 aa

[1535] <400> 71

[1536] Thr Asp Gln Ala Ala Trp His Ala Phe

[1537] 1 5

[1538] <210> 72

[1539] <211> 9

[1540] <212> PROTEIN

[1541] <213> Artificial sequence

[1542] <220>

[1543] <223> HCDR3 aa

[1544] <400> 72

[1545] Thr Asp Gln Ala Ala Trp Phe Gly Phe

[1546] 1 5

[1547] <210> 73

[1548] <211> 9

[1549] <212> PROTEIN

[1550] <213> Artificial sequence

[1551] <220>

[1552] <223> HCDR3 aa

[1553] <400> 73

[1554] Thr Asp Gln Glu Ala Trp Ala Ala Phe

[1555] 1 5

[1556] <210> 74

[1557] <211> 9

[1558] <212> PROTEIN

[1559] <213> Artificial sequence

[1560] <220>

[1561] <223> HCDR3 aa

[1562] <400> 74

[1563] Thr Asp Gln Glu Ala Trp Met Ala Phe

[1564] 1 5

[1565] <210> 75

[1566] <211> 9

[1567] <212> PROTEIN

[1568] <213> Artificial sequence

[1569] <220>

[1570] <223> HCDR3 aa

[1571] <400> 75

[1572] Thr Asp Gln Glu Ala Trp His Ala Phe

[1573] 1 5

[1574] <210> 76

[1575] <211> 9

[1576] <212> PROTEIN

[1577] <213> Artificial sequence

[1578] <220>

[1579] <223> HCDR3 aa

[1580] <400> 76

[1581] Thr Asp Gln Glu Ala Trp Phe Gly Phe

[1582] 1 5

[1583] <210> 77

[1584] <211> 9

[1585] <212> PROTEIN

[1586] <213> Artificial sequence

[1587] <220>

[1588] <223> HCDR3 aa

[1589] <400> 77

[1590] Thr Asp Gln Ala Ala Trp Ala Gly Phe

[1591] 1 5

[1592] <210> 78

[1593] <211> 9

[1594] <212> PROTEIN

[1595] <213> Artificial sequence

[1596] <220>

[1597] <223> HCDR3 aa

[1598] <400> 78

[1599] Thr Asp Gln Ala Ala Trp Met Gly Phe

[1600] 1 5

[1601] <210> 79

[1602] <211> 9

[1603] <212> PROTEIN

[1604] <213> Artificial sequence

[1605] <220>

[1606] <223> HCDR3 aa

[1607] <400> 79

[1608] Thr Asp Gln Ala Ala Trp His Gly Phe

[1609] 1 5

[1610] <210> 80

[1611] <211> 9

[1612] <212> PROTEIN

[1613] <213> Artificial sequence

[1614] <220>

[1615] <223> HCDR3 aa

[1616] <400> 80

[1617] Thr Asp Gln Glu Ala Trp Ala Gly Phe

[1618] 1 5

[1619] <210> 81

[1620] <211> 9

[1621] <212> PROTEIN

[1622] <213> Artificial sequence

[1623] <220>

[1624] <223> HCDR3 aa

[1625] <400> 81

[1626] Thr Asp Gln Glu Ala Trp Met Gly Phe

[1627] 1 5

[1628] <210> 82

[1629] <211> 9

[1630] <212> PROTEIN

[1631] <213> Artificial sequence

[1632] <220>

[1633] <223> HCDR3 aa

[1634] <400> 82

[1635] Thr Asp Gln Glu Ala Trp His Gly Phe

[1636] 1 5

[1637] <210> 83

[1638] <211> 16

[1639] <212> PROTEIN

[1640] <213> Artificial sequence

[1641] <220>

[1642] <223> LCDR1 aa

[1643] <400> 83

[1644] Arg Ser Ser Gln Asn Ile Val Arg Ser Asn Gly Asp Thr Tyr Leu Glu

[1645] 1 5 10 15

[1646] <210> 84

[1647] <211> 16

[1648] <212> PROTEIN

[1649] <213> Artificial sequence

[1650] <220>

[1651] <223> LCDR1 aa

[1652] <400> 84

[1653] Arg Ser Ser Gln Asn Ile Val Arg Ser Gln Gly Asp Thr Tyr Leu Glu

[1654] 1 5 10 15

[1655] <---

Claims

1. An antibody or antigen-binding fragment thereof that binds to PD-1, wherein the antibody or antigen-binding fragment thereof comprises: (a) 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, HCDR3, comprising the amino acid sequence of SEQ ID NO: 5, light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence of SEQ ID NO: 7 or 60, LCDR2, comprising the amino acid sequence of SEQ ID NO: 9, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 11; or (b) the antibody or antigen-binding fragment comprises: HCDR1 comprising the amino acid sequence of SEQ ID NO: 1, LCDR2 comprising the amino acid sequence of SEQ ID NO: 9, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 11, and also comprises: (b-1) HCDR2 comprising the amino acid sequence of SEQ ID NO: 66, HCDR3 comprising the amino acid sequence of SEQ ID NO: 73, and LCDR1 comprising the amino acid sequence of SEQ ID NO: 83; or (b-2) HCDR2 comprising the amino acid sequence of SEQ ID NO: 66, HCDR3 comprising the amino acid sequence of SEQ ID NO: 77, and LCDR1 comprising the amino acid sequence of SEQ ID NO: 60; or (b-3) HCDR2 comprising the amino acid sequence of SEQ ID NO: 3, HCDR3 comprising the amino acid sequence of SEQ ID NO: 75, and LCDR1 comprising the amino acid sequence of SEQ ID NO: 83; or (b-4) HCDR2 comprising the amino acid sequence of SEQ ID NO: 63, HCDR3 comprising the amino acid sequence of SEQ ID NO: 71, and LCDR1 comprising the amino acid sequence of SEQ ID NO: 60; or (b-5) HCDR2 comprising the amino acid sequence of SEQ ID NO: 64, HCDR3 comprising the amino acid sequence of SEQ ID NO: 82, and LCDR1 comprising the amino acid sequence of SEQ ID NO: 83; or (b-6) HCDR2 comprising the amino acid sequence of SEQ ID NO: 64, HCDR3 comprising the amino acid sequence of SEQ ID NO: 77, and LCDR1 comprising the amino acid sequence of SEQ ID NO: 83; or (b-7) HCDR2 comprising the amino acid sequence of SEQ ID NO: 66, HCDR3 comprising the amino acid sequence of SEQ ID NO: 81, and LCDR1 comprising the amino acid sequence of SEQ ID NO: 60; or (b-8) HCDR2 comprising the amino acid sequence of SEQ ID NO: 3, HCDR3 comprising the amino acid sequence of SEQ ID NO: 80, and LCDR1 comprising the amino acid sequence of SEQ ID NO: 83; or (b-9) HCDR2 comprising the amino acid sequence of SEQ ID NO: 66, HCDR3 comprising the amino acid sequence of SEQ ID NO: 82, and LCDR1 comprising the amino acid sequence of SEQ ID NO: 83; or (b-10) HCDR2 comprising the amino acid sequence of SEQ ID NO: 66, HCDR3 comprising the amino acid sequence of SEQ ID NO: 69, and LCDR1 comprising the amino acid sequence of SEQ ID NO: 60; or (b-11) HCDR2 comprising the amino acid sequence of SEQ ID NO: 67, HCDR3 comprising the amino acid sequence of SEQ ID NO: 81, and LCDR1 comprising the amino acid sequence of SEQ ID NO: 83; or (b-12) HCDR2 comprising the amino acid sequence of SEQ ID NO: 67, HCDR3 comprising the amino acid sequence of SEQ ID NO: 73, and LCDR1 comprising the amino acid sequence of SEQ ID NO: 83; or (b-13) HCDR2 comprising the amino acid sequence of SEQ ID NO: 67, HCDR3 comprising the amino acid sequence of SEQ ID NO: 77, and LCDR1 comprising the amino acid sequence of SEQ ID NO:

83.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody is a murine antibody, a chimeric antibody, or a humanized antibody.

3. An antibody or antigen-binding fragment thereof that binds to PD-1, wherein said antibody or antigen-binding fragment thereof comprises: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 13, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 15; or (b) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 54, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 55; or (c) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 56, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 57; or (d) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 58, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:

59.

4. An antibody or antigen-binding fragment thereof that binds to PD-1, wherein the antibody or antigen-binding fragment thereof comprises: (a) a heavy chain variable region comprising an amino acid sequence that has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 13, wherein HCDR1 comprises SEQ ID NO: 1, HCDR2 comprises SEQ ID NO: 3, and HCDR3 comprises SEQ ID NO: 5; 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% identity to the amino acid sequence of SEQ ID NO: 15, wherein LCDR1 comprises SEQ ID NO: 7, LCDR2 comprises SEQ ID NO: 9, and LCDR3 comprises SEQ ID NO: 11; or (b) a heavy chain variable region comprising an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 54, wherein HCDR1 comprises SEQ ID NO: 1, HCDR2 comprises SEQ ID NO: 3, and HCDR3 comprises SEQ ID NO: 5; and a light chain variable region comprising an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 55, wherein LCDR1 comprises SEQ ID NO: 7, LCDR2 comprises SEQ ID NO: 9, and LCDR3 comprises SEQ ID NO: 11; or (c) a heavy chain variable region comprising an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 56, wherein HCDR1 comprises SEQ ID NO: 1, HCDR2 comprises SEQ ID NO: 3, and HCDR3 comprises SEQ ID NO: 5; and a light chain variable region comprising an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 57, wherein LCDR1 comprises SEQ ID NO: 7, LCDR2 comprises SEQ ID NO: 9, and LCDR3 comprises SEQ ID NO: 11; or (d) a heavy chain variable region comprising an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 58, wherein HCDR1 comprises SEQ ID NO: 1, HCDR2 comprises SEQ ID NO: 3, and HCDR3 comprises SEQ ID NO: 5; and a light chain variable region comprising an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 59, wherein LCDR1 comprises SEQ ID NO: 60, LCDR2 comprises SEQ ID NO: 9 and LCDR3 comprises SEQ ID NO:

11.

5. A nucleic acid molecule encoding an antibody or antigen-binding fragment thereof according to any one of claims 1-4.

6. An expression vector containing a nucleic acid molecule according to claim 5.

7. A host cell for producing an antibody or antigen-binding fragment thereof that binds to PD-1, containing the expression vector according to claim 6.

8. A method for producing an antibody or an antigen-binding fragment thereof according to any one of claims 1-4, comprising culturing a host cell containing an expression vector containing a nucleic acid molecule encoding the antibody or an antigen-binding fragment thereof according to any one of claims 1-4.

9. The antibody or antigen-binding fragment thereof according to any one of claims 1-4, wherein the antibody or antigen-binding fragment thereof is selected from a diabody, F(ab')2, Fab', Fab, Fv, scFv, scFv dimer, BsFv, dsFv, (dsFv)2, dsFv- dsFv', Fv fragment, ds diabody, minibody and single-chain binding polypeptide.

10. A transgenic animal engineered to express an antibody or antigen-binding fragment thereof according to any one of claims 1-4, comprising an expression vector according to claim 6.

11. The transgenic animal of claim 10, wherein the animal is a rodent.

12. A bispecific antigen-binding molecule that binds to PD-1 and a second target, comprising a PD-1 binding domain and a second target binding domain, characterized in that it comprises an antibody or antigen-binding fragment thereof according to any one of claims 1-4, wherein the second target is selected from BTLA, CTLA-4, ICOS, LAG-3, TIGIT and TIM-3.

13. An immunoconjugate that binds to PD-1, containing a cytotoxin or a chemotherapeutic agent, characterized in that it contains an antibody or an antigen-binding fragment thereof according to any one of claims 1-4.

14. A pharmaceutical composition for the prevention or treatment of a pathological condition associated with PD-1, comprising an antibody or antigen-binding fragment thereof according to any one of claims 1-4, or a bispecific antigen-binding molecule according to claim 12, or an immunoconjugate according to claim 13 and a pharmaceutically acceptable excipient or carrier.

15. The pharmaceutical composition of claim 14, wherein the pathological condition associated with PD-1 is a tumor, cancer, autoimmune disease, neurological disease, neurodegenerative disease, or infectious disease.

16. The pharmaceutical composition of claim 15, wherein the tumor or cancer associated with PD-1 is selected from non-small cell lung cancer, small cell lung cancer, renal cell cancer, kidney cancer, liver cancer, bone cancer, skin cancer, colon cancer, rectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymus carcinoma, leukemia, lymphoma, myeloma, mycosis fungoides, Merkel cell carcinoma and classical Hodgkin's lymphoma (CHL), primary mediastinal large B-cell lymphoma, T-cell / histiocyte-rich B-cell lymphoma, Epstein-Barr virus (EBV)-positive and Epstein-Barr virus (EBV)-negative post-transplant lymphoproliferative disease (PTLD) or EBV-associated diffuse large B-cell lymphoma (DLBCL),plasmablastic lymphoma, extranodal NK / T-cell lymphoma, nasopharyngeal carcinoma or primary effusion lymphoma associated with human herpes virus 8 (HHV8), other hematological malignancies including Hodgkin's lymphoma, neoplasms of the central nervous system including primary central nervous system (CNS) lymphoma, spinal cord tumor and brainstem glioma.

17. The pharmaceutical composition of claim 15, wherein the PD-1-associated autoimmune disease is selected from lupus, systemic lupus erythematosus, Sjogren's syndrome, arthritis, rheumatoid arthritis, asthma, COPD, pelvic inflammatory disease, Alzheimer's disease, inflammatory bowel disease, Crohn's disease, ulcerative colitis, Peyronie's disease, celiac disease, gallbladder disease, pilonidal disease, peritonitis, psoriasis, psoriatic arthritis, vasculitis, adhesions after surgery, stroke, type 1 diabetes mellitus, Lyme disease, meningoencephalitis, autoimmune uveitis, multiple sclerosis, Guillain-Barré syndrome, atopic dermatitis, autoimmune hepatitis, fibrosing alveolitis, Graves' 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 associated with maternal-fetal intolerance, vitiligo, myasthenia gravis or systemic sclerosis.

18. The pharmaceutical composition of claim 15, wherein the neurological disease or neurodegenerative disease associated with PD-1 is selected from cognitive impairment, brain tumor, Alzheimer's disease, dementia, stroke, spinal cord injury, amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, multiple sclerosis, glioblastoma, melanoma, pain and memory loss.

19. The pharmaceutical composition according to claim 15, wherein the infectious disease associated with PD-1 is selected from chronic viral infections, including a viral infection such as hepatitis B, hepatitis C caused by the herpes virus, Epstein-Barr virus, HIV, cytomegalovirus, herpes simplex virus type 1, herpes simplex virus type 2, human papillomavirus, adenovirus, Kaposi-West sarcoma associated with herpes virus epidemics, ring virus (torquetenovirus), JC virus or BK virus.

20. A method for preventing or treating PD-1-associated cancer, comprising administering to a subject at least one selected from the group consisting of an antibody or antigen-binding fragment thereof according to any one of claims 1-4, and a multispecific antigen-binding molecule according to claim 12, or an immunoconjugate according to claim 13.

21. A pharmaceutical composition for enhancing an immune response in a subject suffering from a PD-1-associated cancer, comprising an antibody or antigen-binding fragment thereof according to any one of claims 1-4, or a multispecific antigen-binding molecule according to claim 12, or an immunoconjugate according to claim 13 and a pharmaceutically acceptable excipient or carrier.

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