Fusion protein containing programmed cell death ligand 1 protein and its uses

Abstract

The present invention relates to a fusion protein comprising a programmed cell death ligand 1 (PD-L1) protein and the Fc region of a modified immunoglobulin, and its use. The fusion protein has the following effects: compared with existing fusion proteins, the purity and production yield are significantly higher, the binding ability to programmed cell death receptor 1 is high, the proliferation of activated T cells is reduced, the production of cytokines produced by activated T cells is inhibited, and the infiltration of T cells or macrophages into tissues is inhibited, thereby being useful for treating immune diseases.

Description

Technical Field

[0001] The present invention relates to a fusion protein comprising a programmed cell death ligand 1 (PD-L1) protein and the Fc region of a modified immunoglobulin and uses thereof. Background Art

[0002] Human programmed cell death ligand 1 (PD-L1), a ligand for programmed death receptor 1 (PD-1), is a type 1 transmembrane protein expressed in hematopoietic cells such as T lymphocytes, B lymphocytes, dendritic cells, and macrophages, as well as non-hematopoietic cells such as keratinocytes, pancreatic islet cells, and hepatocytes. On the other hand, in order to activate T cells, in addition to the primary stimulation of the T cell receptor and antigen, a secondary stimulation signal (co-stimulation) is also required. In this case, without either of these two signals, the T cell remains in an anergy state. Programmed cell death receptor 1 (PD-1) is a second signal stimulator (immune checkpoint or immune modulator) that regulates the second signal activity of T cells. It can inhibit T cell function by binding to programmed cell death ligand 1 or B7.1 (CD80) expressed on the cell surface of activated T cells (CD8 and / or CD4) or dendritic cells, thereby inhibiting T cell proliferation and reducing cytokine expression.

[0003] It is known that the binding between programmed cell death receptor 1 and programmed cell death ligand 1 induces the activity of regulatory T cells (Immunol Rev. 2010 Jul; 236: 219-42). When the immune tolerance-inducing function of programmed cell death ligand 1 is utilized, when programmed cell death ligand 1 protein fused to the Fc of immunoglobulin G1 (PD-L1-Ig) is injected into a mouse model of collagen-induced arthritis (CIA), arthritis symptoms are alleviated (Rheumatol Int. 2011 Apr; 31(4): 513-9). Since programmed cell death receptor 1 is expressed in activated T cells, it is expected that PD-L1 protein will induce immune tolerance not only in autoimmune diseases but also in organ transplantation, and will be useful as a therapeutic agent that specifically targets activated immune cells.

[0004] To date, therapeutic agents for the programmed death receptor 1 / programmed cell death ligand 1 cell signaling system have been developed towards the direction of increasing T cell activity by acting as antagonists by hindering immune tolerance (tolerance breaking). However, to date, immunotherapeutic agents based on inducing T cell immune tolerance using agonists have not been developed. This is because, in the case of programmed death receptor 1 / programmed cell death ligand 1 antagonists, they can be easily developed using antibody fusion technology, but it is technically difficult to develop agonists of programmed death receptor 1 / programmed cell death ligand 1 signaling, which require the development of proteins in soluble form.

[0005] Immunoglobulin (Ig) Fc fusion technology is one technique used to increase the in vivo half-life of protein therapeutics. However, in the case of immunoglobulin G1, which is currently used in Ig fusion technology, it induces antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) in vivo. Consequently, Ig fusion proteins used as therapeutics for autoimmune diseases or as agents for inducing immune tolerance in organ transplantation fail to suppress inflammatory responses and instead exacerbate inflammation.

[0006] Therefore, there is a need to develop a technology that maintains the half-life of programmed cell death ligand 1 to be similar to that of existing immunoglobulin fusion protein therapeutics and does not induce antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity, thereby improving the therapeutic efficacy of programmed cell death ligand 1 as an immunosuppressant. Summary of the Invention

[0007] Problems to be solved by the invention

[0008] The object of the present invention is to provide a fusion protein comprising a programmed cell death ligand 1 protein and the Fc region of a modified immunoglobulin.

[0009] Furthermore, another object of the present invention is to provide a nucleic acid molecule that encodes the fusion protein.

[0010] Furthermore, another object of the present invention is to provide an expression vector comprising the nucleic acid molecule.

[0011] Furthermore, another object of the present invention is to provide a host cell comprising the expression vector.

[0012] Furthermore, another object of the present invention is to provide a pharmaceutical composition for preventing or treating immune diseases, comprising a fusion protein as an active ingredient, wherein the fusion protein comprises programmed cell death ligand 1 protein and the Fc region of a modified immunoglobulin.

[0013] Furthermore, another object of the present invention is to provide a use of a fusion protein comprising a programmed cell death ligand 1 protein and a modified immunoglobulin Fc region for producing a pharmaceutical preparation having a preventive or therapeutic effect on immune diseases.

[0014] Furthermore, another object of the present invention is to provide a method for preventing or treating immune diseases, comprising the step of administering a fusion protein comprising programmed cell death ligand 1 protein and the Fc region of a modified immunoglobulin and a pharmaceutically acceptable carrier to an individual.

[0015] Means of solving the problem

[0016] To achieve the above objectives, the present invention provides a fusion protein comprising a programmed cell death ligand 1 (PD-L1) protein and a modified immunoglobulin Fc region.

[0017] Furthermore, the present invention provides a nucleic acid molecule encoding the fusion protein.

[0018] Furthermore, the present invention provides an expression vector comprising the nucleic acid molecule.

[0019] Furthermore, the present invention provides a host cell comprising the expression vector.

[0020] The present invention provides a pharmaceutical composition for preventing or treating immune diseases, comprising a fusion protein as an active ingredient, wherein the fusion protein comprises a programmed cell death ligand 1 (PD-L1) protein and the Fc region of a modified immunoglobulin.

[0021] Furthermore, the present invention provides a use of a fusion protein comprising a programmed cell death ligand 1 protein and a modified immunoglobulin Fc region for producing a pharmaceutical preparation having a preventive or therapeutic effect on immune diseases.

[0022] Furthermore, the present invention provides a method for preventing or treating immune diseases, comprising the step of administering a fusion protein comprising programmed cell death ligand 1 protein and the Fc region of a modified immunoglobulin and a pharmaceutically acceptable carrier to an individual.

[0023] Effects of the Invention

[0024] The fusion protein according to the present invention comprises a programmed cell death ligand 1 protein and a modified immunoglobulin Fc region linked via a sequence consisting of an immunoglobulin G1 (IgG1) hinge and a GS sequence. The fusion protein is characterized by being prepared in a manner that induces dimerization while maintaining flexibility by linking the sequence consisting of the immunoglobulin G1 hinge and the GS sequence. Furthermore, the fusion protein according to the present invention exhibits significantly higher purity and production yield than existing fusion proteins, exhibits enhanced binding ability to programmed cell death receptor 1, reduces the proliferation of activated T cells, inhibits the production of cytokines produced by activated T cells, and suppresses tissue infiltration by T cells or macrophages, making it useful for treating immune diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The structure of a fusion protein comprising a programmed cell death ligand 1 protein and a modified immunoglobulin Fc region (programmed cell death ligand 1-hyFc21 (PD-L1-hyFc21) fusion protein) is shown.

[0026] Figure 2a The results are from analyzing the cell concentration of cells expressing programmed cell death ligand 1-hyFc21 fusion protein at different times. Figure 2b The results are obtained by analyzing the purity of the target protein by size exclusion high performance liquid chromatography (SE-HPLC) in the cell culture medium of cells expressing the programmed cell death ligand 1-hyFc21 fusion protein.

[0027] Figure 3a The results are from analyzing the cell concentration at different times of cells expressing programmed cell death ligand 1-hyFc5 (PD-L1-hyFc5) fusion protein. Figure 3b The results are obtained by analyzing the purity of the target protein by size exclusion high performance liquid chromatography in the cell culture medium of cells expressing the programmed cell death ligand 1-hyFc5 fusion protein.

[0028] Figure 4 This is the result of polyacrylamide gel electrophoresis (SDS-PAGE) analysis of purified programmed cell death ligand 1-hyFc21 or programmed cell death ligand 1-hyFc5 fusion protein.

[0029] Figure 5a This is the result of size exclusion high performance liquid chromatography analysis of the purified programmed cell death ligand 1-hyFc21 fusion protein. Figure 5b The results of size exclusion high performance liquid chromatography analysis of the purified programmed cell death ligand 1-hyFc5 protein.

[0030] Figure 6The results are obtained by gel isoelectric focusing (Gel IEF) analysis of purified programmed cell death ligand 1-hyFc21 or programmed cell death ligand 1-hyFc5 fusion proteins.

[0031] Figure 7 The results are differential scanning fluorimetry (DSF) analysis of purified programmed cell death ligand 1-hyFc21 or programmed cell death ligand 1-hyFc5 fusion proteins.

[0032] Figure 8 The results are a comparison of the binding affinity of programmed cell death ligand 1-hyFc21 or programmed cell death ligand 1-hyFc5 fusion proteins to programmed cell death receptor 1.

[0033] Figure 9 The results are a comparison of the mixed lymphocyte reaction inhibitory ability of programmed cell death ligand 1-hyFc21 or programmed cell death ligand 1-hyFc5 fusion proteins.

[0034] Figure 10a The results are a comparison of the ability to suppress proliferation of human CD4 T cells activated by programmed cell death ligand 1-hyFc21 or programmed cell death ligand 1-hyFc5 fusion proteins. Figure 10b The results are a comparison of the ability of activated human CD4 T cells to suppress cytokine expression.

[0035] Figure 11 The results show a comparison of the cytokine expression suppressive ability of mouse CD4 T cells activated by programmed cell death ligand 1-hyFc21 or programmed cell death ligand 1-hyFc5 fusion proteins.

[0036] Figure 12a The results are from measuring ear thickness of mice after subcutaneous administration of programmed cell death ligand 1-hyFc21 fusion protein to an imiquimod (IMQ)-induced psoriasis mouse model. Figure 12b The results are from measuring ear thickness of mice after intravenous administration of programmed cell death ligand 1-hyFc21 fusion protein to an imiquimod-induced psoriasis mouse model.

[0037] Figure 13a The results are based on the results of subcutaneous administration of programmed cell death ligand 1-hyFc21 fusion protein to the rtTA-Peli1 psoriasis mouse model, and the changes in skin epithelial tissue were confirmed by hematoxylin-eosin (H&E) staining. Figure 13b It is a measurement of the thickness of the epidermal layer of the skin.

[0038] Figure 14The results show the infiltration levels of T cells and macrophages by immunofluorescence analysis in a psoriasis-uninduced control group (rtTA) or a psoriasis-induced rtTA-Peli1 psoriasis mouse model (left panel: rtTA; and right panel: rtTA-Peli1).

[0039] Figure 15 The results are from measuring the number of T cells and macrophages infiltrating into skin tissue after subcutaneous administration of programmed cell death ligand 1-hyFc21 fusion protein to the rtTA-Peli1 psoriasis mouse model.

[0040] Figure 16 After subcutaneous administration of programmed cell death ligand 1-hyFc21 fusion protein to the rtTA-Peli1 psoriasis mouse model, K14 expression in skin tissue was measured. + The result is the number of keratin-producing cells.

[0041] Figure 17 The results are based on a scoring index analysis of changes in the skin epithelium following intravenous administration of programmed cell death ligand 1-hyFc21 fusion protein to the rtTA-Peli1 psoriasis mouse model.

[0042] Figure 18 The results show that abdominal skin thickness was measured after intravenous administration of programmed cell death ligand 1-hyFc21 fusion protein to the rtTA-Peli1 psoriasis mouse model.

[0043] Figure 19a The results are based on the results of intravenous administration of programmed cell death ligand 1-hyFc21 fusion protein to the rtTA-Peli1 psoriasis mouse model, and the changes in skin epithelial tissue were confirmed by hematoxylin-eosin staining. Figure 19b It is a measurement of the thickness of the epidermal layer of the skin.

[0044] Figure 20 The results are the results of confirming changes in messenger RNA (mRNA) expression of Th17 cell-related genes (interleukin-17A (IL-17A) and interleukin-22 (IL-22)) and innate immune cell-related genes (interleukin-1β (IL-1β) and interleukin-24 (IL-24)) by qRT-PCR after intravenous administration of programmed cell death ligand 1-hyFc21 fusion protein to the rtTA-Peli1 psoriasis mouse model. DETAILED DESCRIPTION

[0045] Hereinafter, the present invention will be described in detail.

[0046] The present invention provides a fusion protein comprising a programmed cell death ligand 1 protein and the Fc region of a modified immunoglobulin.

[0047] The programmed cell death ligand 1 protein may be the extracellular domain of the programmed cell death ligand 1 protein or a fragment thereof. The extracellular domain of the programmed cell death ligand 1 protein may be a polypeptide comprising the immunoglobulin V (Ig V)-like domain of programmed cell death ligand 1 and the immunoglobulin C (Ig C)-like domain of programmed cell death ligand 1.

[0048] Specifically, the extracellular domain of the programmed cell death ligand 1 protein is the protein portion exposed outside the cell membrane, and can be a polypeptide consisting of amino acids 19 to 238 of SEQ ID NO: 1 or a polypeptide consisting of amino acids 19 to 239 of SEQ ID NO: 1.

[0049] In this case, the extracellular domain of the programmed cell death ligand 1 protein contains an immunoglobulin V-like (Ig V, Ig V like) sequence, which is a conserved sequence similar to the amino acid sequence of immunoglobulins. The highly conserved immunoglobulin V-like sequence is the amino acid sequence of positions 68 to 114 of SEQ ID NO: 1. In addition, it contains an immunoglobulin C-like (Ig C, Ig C like) sequence, and the highly conserved sequence site is the amino acid sequence of positions 153 to 210 of SEQ ID NO: 1. In addition, the fragment of the extracellular domain of the programmed cell death ligand 1 protein may contain all or part of the immunoglobulin V-like domain, and the immunoglobulin V-like domain contains the immunoglobulin V-like sequence of programmed cell death ligand 1.

[0050] Furthermore, in the extracellular domain of the programmed cell death ligand 1 protein, the immunoglobulin V-like domain, as a site that can interact with the programmed cell death receptor 1, can be a polypeptide consisting of the amino acid sequence of positions 19 to 239 of SEQ ID NO: 1 (SEQ ID NO: 3), or a polypeptide consisting of the amino acid sequence of positions 21 to 239 of SEQ ID NO: 1. Furthermore, it can be a polypeptide consisting of the amino acid sequence of positions 19 to 133 of SEQ ID NO: 1 (SEQ ID NO: 4), or a polypeptide consisting of the amino acid sequence of positions 21 to 133 of SEQ ID NO: 1. Furthermore, it can be a polypeptide consisting of the amino acid sequence of positions 21 to 114 of SEQ ID NO: 1, or a polypeptide consisting of the amino acid sequence of positions 19 to 114 of SEQ ID NO: 1. Furthermore, it can be a polypeptide consisting of the amino acid sequence of positions 21 to 120 of SEQ ID NO: 1, or a polypeptide consisting of the amino acid sequence of positions 19 to 120 of SEQ ID NO: 1. Furthermore, the polypeptide may be a polypeptide consisting of the amino acid sequence from positions 19 to 127 of SEQ ID NO: 1 (SEQ ID NO: 5), or a polypeptide consisting of the amino acid sequence from positions 21 to 127 of SEQ ID NO: 1 (SEQ ID NO: 6). Furthermore, the polypeptide may be a polypeptide consisting of the amino acid sequence from positions 21 to 130 of SEQ ID NO: 1, or a polypeptide consisting of the amino acid sequence from positions 19 to 130 of SEQ ID NO: 1. Furthermore, the polypeptide may be a polypeptide consisting of the amino acid sequence from positions 21 to 131 of SEQ ID NO: 1, or a polypeptide consisting of the amino acid sequence from positions 19 to 131 of SEQ ID NO: 1.

[0051] Furthermore, when the fragment of the extracellular domain of the programmed cell death ligand 1 protein comprises an immunoglobulin V-like domain or a fragment thereof, the fragment of the extracellular domain of the programmed cell death ligand 1 protein may further comprise an immunoglobulin C-like domain of the extracellular domain of the programmed cell death ligand 1 protein. The immunoglobulin C-like domain may be a polypeptide consisting of the amino acid sequence from positions 133 to 225 of SEQ ID NO: 1 or a polypeptide consisting of the amino acid sequence from positions 134 to 225 of SEQ ID NO: 1.

[0052] Furthermore, when the fragment of the extracellular domain of the programmed cell death ligand 1 protein comprises an immunoglobulin V-like domain or a fragment thereof, the fragment of the extracellular domain of the programmed cell death ligand 1 protein may further comprise a polypeptide or a fragment thereof, wherein the polypeptide comprises an immunoglobulin C-like domain of the extracellular domain of the programmed cell death ligand 1 protein. The polypeptide comprising the immunoglobulin C-like domain refers to the extracellular domain of the programmed cell death ligand 1 protein excluding the immunoglobulin V domain, and may be a polypeptide having amino acids 134 to 239 of SEQ ID NO: 1 (SEQ ID NO: 7) or a polypeptide having amino acids 134 to 238 of SEQ ID NO: 1 (SEQ ID NO: 8).

[0053] Furthermore, the extracellular domain of the programmed cell death ligand 1 protein or a fragment thereof may be derived from human or mouse.

[0054] The extracellular domain of the human programmed cell death ligand 1 protein is a polypeptide (SEQ ID NO: 3) consisting of the amino acid sequence from positions 19 to 239 of SEQ ID NO: 1, and the extracellular domain of the mouse programmed cell death ligand 1 protein is a polypeptide consisting of the amino acid sequence from positions 19 to 239 of SEQ ID NO: 2. Furthermore, the extracellular domain of the programmed cell death ligand 1 protein may have about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more homology with the polypeptide sequence consisting of the amino acid sequence from positions 19 to 239 of SEQ ID NO: 1.

[0055] Specifically, the human programmed cell death ligand 1 protein has 290 amino acid residues and comprises the amino acid sequence of SEQ ID NO: 1 (Accession Number: Q9NZQ7). In the amino acid sequence of SEQ ID NO: 1, the amino acid residues 1 to 18 at the N-terminus are a signal sequence, and the mature human programmed cell death ligand 1 protein comprises the amino acid sequence of SEQ ID NO: 1 from positions 19 to 290. The extracellular domain of the human programmed cell death ligand 1 protein comprises the amino acid sequence of SEQ ID NO: 1 from positions 19 to 238 or from positions 19 to 239.

[0056] The human programmed cell death ligand 1 protein comprises an immunoglobulin V-like domain as amino acids 19 to 127 of SEQ ID NO: 1 and an immunoglobulin C-like domain as amino acids 134 to 226 of SEQ ID NO: 1.

[0057] The mouse programmed cell death ligand 1 protein reportedly comprises 290 amino acids and includes the amino acid sequence of SEQ ID NO: 2 (Accession Number: Q9EP73). Amino acid residues 1 to 18 of SEQ ID NO: 2 are a signal sequence, and the mature mouse programmed cell death ligand 1 protein includes the amino acid sequence of SEQ ID NO: 2 from 19 to 290. The extracellular domain of the mouse programmed cell death ligand 1 protein includes the amino acid sequence of SEQ ID NO: 2 from 19 to 239. The mouse programmed cell death ligand 1 protein includes an immunoglobulin V-like protein as amino acids 19 to 127 of SEQ ID NO: 2 and an immunoglobulin C-like domain as amino acids 133 to 224 of SEQ ID NO: 2.

[0058] The extracellular domain of the programmed cell death ligand 1 protein may include the entire immunoglobulin V-like domain or a fragment thereof. Furthermore, the fragment of the extracellular domain of the programmed cell death ligand 1 protein may also include a polypeptide (the extracellular domain of programmed cell death ligand 1 excluding the immunoglobulin V-like domain), wherein the polypeptide further includes an immunoglobulin C-like domain or an immunoglobulin C-like domain.

[0059] The extracellular domain of the programmed cell death ligand 1 protein or a fragment thereof may comprise a variety of modified proteins or peptides. The modification may be performed by substituting, deleting, or adding one or more proteins to the wild-type programmed cell death ligand 1 protein, as long as the function of the programmed cell death ligand 1 is not modified. Such multiple proteins or peptides may have 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology to the wild-type protein.

[0060] The term "extracellular domain of programmed cell death ligand 1 protein" in the present invention is also used as a concept encompassing "extracellular domain of programmed cell death ligand 1 protein and fragments thereof".

[0061] Unless otherwise specified, the terms "protein," "polypeptide," and "peptide" in the present invention are used interchangeably.

[0062] The terms "programmed cell death ligand 1 fusion protein" and "programmed cell death ligand 1-modified immunoglobulin Fc region fusion protein" of the present invention refer to a fusion protein in which programmed cell death ligand 1 protein, the extracellular domain of programmed cell death ligand 1 protein or a fragment thereof is combined with the Fc region of a modified immunoglobulin.

[0063] In the present invention, the programmed cell death ligand 1 protein can be fused to the N-terminus or C-terminus of the Fc region of a modified immunoglobulin. Preferably, the programmed cell death ligand 1 protein can be fused to the N-terminus of the Fc region of a modified immunoglobulin. The programmed cell death ligand 1 protein can be linked to the immunoglobulin Fc region via a linker peptide. The connecting peptide may include GGGSGGS (SEQ ID NO: 10), AAGSGGGGGSGGGGSGGGGS (SEQ ID NO: 17), GGSGG (SEQ ID NO: 18), GGSGGSGGS (SEQ ID NO: 19), GGGSGG (SEQ ID NO: 20), (G4S)n (n is an integer from 1 to 10), (GGS)n (n is an integer from 1 to 10), (GS)n (n is an integer from 1 to 10), (GSSGGS)n (n is an integer from 1 to 10), KESGSVSSEQLAQFRSLD (SEQ ID NO: 21), EGKSSGSGSESKST (SEQ ID NO: 22), GSAGSAAGSGEF (SEQ ID NO: 23), (EAAAK)n (n is an integer from 1 to 10), CRRRRREAEAC (SEQ ID NO: 24), A(EAAAK)4ALEA(EAAAK)4A, GGGGGGGG (SEQ ID NO: 25), ID NO: 25), GGGGGG (SEQ ID NO: 26), AEAAAAKEAAAAKA (SEQ ID NO: 27), PAPAP (SEQ ID NO: 28), (Ala-Pro) n (n is an integer from 1 to 10), VSQTSKLTRAETVFPDV (SEQ ID NO: 29), PLGLWA (SEQ ID NO: 30), TRHRQPRGWE (SEQ ID NO: 31), AGNRVRRRSVG (SEQ ID NO: 32), RRRRRRRR (SEQ ID NO: 33), GFLG (SEQ ID NO: 34), and GSSGGSGSSGGSGGGDEADGSRGSQKAGVDE (SEQ ID NO: 35), etc. Preferably, programmed cell death ligand 1 and the immunoglobulin Fc region can be linked via a linker peptide consisting of the amino acid sequence of GGGSGGS (SEQ ID NO: 10). When the programmed cell death ligand 1 protein and the Fc region of an immunoglobulin are linked using the linker peptide, the activity, stability, and productivity of the fusion protein can be optimized.

[0064] Furthermore, the fusion protein can exist as a dimer. The fusion proteins constituting the dimer can be bound together by disulfide bonds between cysteines present in the connecting peptide. The fusion proteins constituting the dimer are identical. That is, the dimer can be a homodimer. In this case, the fusion protein can be soluble, in particular, soluble in purified water or physiological saline.

[0065] The Fc region of the modified immunoglobulin can be any one of the Fc regions of immunoglobulin G1, immunoglobulin G2, immunoglobulin G3, immunoglobulin D, and immunoglobulin G4, or a combination thereof. The Fc region is modified so as not to bind to Fc receptors and / or complement. In particular, the Fc region of the modified immunoglobulin comprises a hinge region, a CH2 domain, and a CH3 domain along the direction from N-terminus to C-terminus, wherein the hinge region comprises the human immunoglobulin G1 hinge region (SEQ ID NO: 16), the CH2 domain comprises the amino acid residue portion of the CH2 domain of human immunoglobulin D and human immunoglobulin G4, and the CH3 domain may comprise the amino acid residue portion of the CH3 domain of human immunoglobulin G4.

[0066] The terms "Fc region," "Fc fragment," or "Fc" herein refer to a protein comprising the heavy chain constant region 2 (CH2) and heavy chain constant region 3 (CH3) of an immunoglobulin, and excluding the variable regions of the heavy and light chains and the light chain constant region 1 (CL1) of an immunoglobulin. It may also include the hinge region of the heavy chain constant region. In the present invention, hybrid Fc or hybrid Fc fragments are also referred to as "hFc" or "hyFc."

[0067] Furthermore, the Fc fragment of the present invention may be in the form of native sugar chains, in the form of increased sugar chains compared to the native form, or in the form of reduced sugar chains compared to the native form, or in the form of deglycosylated form. Immunoglobulin Fc sugar chains can be modified by conventional methods such as chemical methods, enzymatic methods, and genetic engineering methods using microorganisms. When sugar chains are removed from the Fc fragment, the binding affinity of the first complement component C1 to C1q is sharply reduced, and antibody-dependent cell-mediated cytotoxicity or complement-dependent cytotoxicity is reduced or lost, thereby not inducing unnecessary immune responses in the body. In this regard, deglycosylated or aglycosylated forms of immunoglobulin Fc fragments may be more suitable for the purposes of the present invention as drug carriers. As used herein, the term "deglycosylation" refers to the enzymatic removal of sugars from the Fc fragment. Furthermore, the term "aglycosylation" refers to an Fc fragment produced in an unglycosylated form by prokaryotes, preferably by E. coli.

[0068] In one embodiment of the present invention, the Fc region of the modified immunoglobulin may consist of the amino acid sequence of SEQ ID NO: 11 (hereinafter referred to as "hyFc").

[0069] In one embodiment of the present invention, the fusion protein can be represented by the following structural formula I.

[0070] N'-XLY-C' (Structural Formula I)

[0071] in,

[0072] N' is the N-terminus of the fusion protein, and C' is the C-terminus of the fusion protein;

[0073] The X is programmed cell death ligand 1 protein, the extracellular domain of programmed cell death ligand 1 protein or a fragment thereof;

[0074] L is a connecting peptide;

[0075] Y is the immunoglobulin Fc region.

[0076] Preferably, the fusion protein may consist of the amino acid sequence of SEQ ID NO: 12 or SEQ ID NO: 13. Furthermore, the fusion protein of the present invention may have about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more homology with the amino acid sequence of SEQ ID NO: 12.

[0077] Furthermore, the present invention provides a nucleic acid molecule encoding the fusion protein.

[0078] Preferably, the nucleic acid molecule may be a nucleic acid molecule encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 12 or SEQ ID NO: 13. Furthermore, the nucleic acid molecule may further comprise a signal sequence (or signal peptide) or a leader sequence.

[0079] The term "signal sequence (or signal peptide)" of the present invention refers to a short peptide present at the N-terminus of a newly synthesized protein classified as a secretory pathway. Signal sequences useful in the present invention include antibody light chain signal sequences such as antibody 1418 (Gillies et al., J Immunol Meth 1989 125: 191-202), antibody heavy chain signal sequences such as MOPC141 antibody heavy chain signal sequences (Sakano et al, Nature 1980 286: 676-683), and other signal sequences well known in the art (see, e.g., Watson et al, Nucleic Acid Research 1984 12: 5145-5164).

[0080] The characteristics of the signal peptide are well known in the art and are known to typically comprise 16 to 30 amino acid residues, and may comprise more or fewer amino acid residues. Conventional signal peptides consist of three regions: a basic N-terminal region, a central hydrophobic region, and a more polar C-terminal region.

[0081] The central hydrophobic region comprises 4 to 12 hydrophobic residues, and the hydrophobic residues fix the signal sequence by the membrane lipid bilayer during the translocation of the immature polypeptide. After startup, the signal sequence is usually cut by the cellular enzyme known as signal peptidase (signal peptidases) in the lumen (lumen) of the endoplasmic reticulum (ER). Now, the signal sequence can be the secretion signal sequence of tissue plasminogen activator (tPa, tissue Plasminogen Activation), herpes simplex virus glycoprotein D (HSV gDs) or growth hormone. Preferably, the secretion signal sequence used in the higher eukaryotic cells including mammals etc. can be used, more preferably, the amino acid sequence of the 1st to 18th of tissue plasminogen activator sequence or SEQ ID NO:1 can be used. In addition, the signal sequence of the present invention can be used by replacing the codon with the high expression frequency in the host cell.

[0082] Furthermore, the present invention provides an expression vector comprising the nucleic acid molecule.

[0083] The term "vector" of the present invention is understood to mean a nucleic acid unit comprising a nucleotide sequence that can be introduced into a host cell for recombination, insertion into the host cell genome, or capable of autonomous replication as an episome. Said vectors include linear nucleic acids, plasmids, phagemids, cosmids, RNA vectors, viral vectors and the like. Examples of viral vectors include, but are not limited to, retroviruses, adenoviruses, and adeno-associated viruses.

[0084] In the present invention, a useful expression vector can be RcCMV (Invitrogen, Carlsbad) or a variant thereof. Useful expression vectors can include a human cytomegalovirus (CMV) promoter for promoting continuous transcription of the target gene in mammalian cells and a bovine growth hormone (BOH) polyadenylation signal sequence for increasing steady-state levels of post-transcriptional RNA. In one embodiment of the present invention, the expression vector is pAD15, which is a modified vector of RcCMV.

[0085] The term "host cell" of the present invention refers to prokaryotic and eukaryotic cells into which a recombinant expression vector can be introduced.

[0086] In the present invention, suitable host cells can be transformed or transfected with the deoxyribonucleic acid (DNA) sequences of the present invention for expression and / or secretion of the target protein. Currently preferred host cells for use in the present invention include immortal hybridoma cells, NS / 0 myeloma cells, 293 cells, Chinese hamster ovary cells (CHO cells), HeLa cells, CapT cells (cells derived from human amniotic fluid), and COS cells.

[0087] The terms "transformation" and "transfection" of the present invention refer to the introduction of nucleic acid (eg, vector) into cells by various techniques well known in the art.

[0088] Furthermore, the present invention provides a pharmaceutical composition for preventing or treating immune diseases, comprising a fusion protein as an active ingredient, wherein the fusion protein comprises a programmed cell death ligand 1 protein and an Fc region of a modified immunoglobulin.

[0089] The immune disease may be a disease selected from the group consisting of autoimmune diseases, inflammatory diseases, and cell, tissue, or organ transplant rejection diseases.

[0090] The autoimmune disease may be selected from arthritis [acute arthritis, chronic rheumatoid arthritis, gouty arthritis, acute gouty arthritis, chronic inflammatory arthritis, degenerative arthritis, infectious arthritis, Lyme arthritis, proliferative arthritis, psoriatic arthritis, spondyloarthritis and rheumatoid arthritis such as juvenile-onset rhematoid arthritis, osteoarthritis, chronic progressive arthritis, deformans arthritis, primary chronic polyarthritis, reactive arthritis and ankylosing spondylitis], inflammatory proliferative skin diseases, psoriasis such as plaque psoriasis, guttate psoriasis, pustular psoriasis and psoriasis of the nails. nails), dermatitis, including contact dermatitis, chronic contact dermatitis, allergic dermatitis, allergic contact dermatitis, herpetiformis dermatitis, and atopic dermatitis, X-linked hyperimmunoglobulinemia M syndrome; urticaria, such as chronic idiopathic urticaria, including chronic allergic urticaria and chronic autoimmune urticaria, polymyositis / dermatomyositis, juvenile dermatomyositis, toxic epidermal necrolysis, scleroderma (including systemic scleroderma), systemic sclerosis, sclerosis, including, for example, spino-optical multiple sclerosis. Multiple sclerosis (MS), primary progressive MS (PPMS), and relapsing remitting MS (RRMS), progressive systemic sclerosis (MS), andsystemic sclerosis), atherosclerosis, arteriosclerosis, sclerosis disseminata, and ataxia, inflammatory bowel disease (IBD) [e.g., Crohn's disease, autoimmune-mediated gastrointestinal diseases, colitis such as ulcerative colitis, colitis ulcerosa, microscopic colitis, collagenous colitis, colitis polyposa, necrotizing enterocolitis, and transmural colitis, and autoimmune inflammatory bowel disease], pyoderma gangrenosum, erythema nodosum, primary sclerosing cholangitis, cholangitis, episcleritis, respiratory distress syndrome including adult or acute respiratory distress syndrome (ARDS), meningitis, inflammation of all or part of the uvea, iritis, choroiditis, autoimmune hematological disorders, rheumatoid spondylitis, sudden hearing loss, immunoglobulin E-mediated diseases such as anaphylaxis and allergic and atopic rhinitis, encephalitis such as Rasmussen's encephalitis and limbic and / or brainstem encephalitis, anterior uveitis, acute anterior uveitis, granulomatous uveitisuveitis, nongranulomatous uveitis, phacoantigenic uveitis, posterior uveitis, or autoimmune uveitis, glomerulonephritis (GN) with or without nephrotic syndrome such as primary GN, chronic or acute glomerulonephritis, immune-mediated glomerulonephritis, membranous nephropathy, idiopathic membranous nephropathy, or idiopathic membranous GN, including types I and II, membranous or membranous proliferative glomerulonephritis. GN (MPGN) and rapidly progressive glomerulonephritis, allergic diseases, allergic reactions, eczema including allergic or atopic eczema, asthma such as bronchiole, bronchial asthma, and autoimmune asthma, diseases associated with T cell invasion and chronic inflammatory response, chronic pulmonary inflammatory disease, autoimmune myocarditis, leukocyte adhesion deficiency, systemic lupus erythematosus such as systemic lupus erythematosus (SLE) or cutaneous SLE, subacute cutaneous lupus erythematosus, neonatal lupus syndrome (NLE), disseminated lupus erythematosus (DLE), disseminatus), lupus (including nephritis, cerebritis, pediatric, non-renal, extra-renal, discoid, alopecia), including pediatric insulin-dependent diabetes mellitusmellitus; IDDM), juvenile onset (Type I) diabetes mellitus, adult-onset diabetes mellitus (Type II), autoimmune diabetes mellitus, idiopathic diabetes insipidus, immune responses related to cytokines and T lymphocytes and mediated acute and delayed hypersensitivity reactions, tuberculosis, sarcoidosis, granulomatosis including lymphomatoid granulomatosis, Wegener's granulomatosis, agranulocytosis, vasculitis (including large vessel vasculitis (polymyalgia rheumatica and giant cell (Takayasu's) arteritis)), medium-vessel vasculitis including Kawasaki disease and polyarteritis nodosa, microscopic polyarteritis polyarteritis, central nervous system (CNS) arthritis, including necrotizing, cutaneous, or allergic vasculitis, systemic necrotizing vasculitis, vasculitis including ANCA-associated vasculitis such as Churg-Strauss vasculitis or Churg-Strauss syndrome (CSS), temporal arteritis, aplastic anemia, autoimmune aplastic anemia, Coombs-positive anemia, Diamond Blackfan anemia, immune hemolytic anemia including hemolytic anemia or autoimmune hemolytic anemia (AIHA), pernicious anemia (anemia perniciosa), Addison's disease, pure red cell aplasia aplasia; PRCA), VHI factor deficiency, hemophilia A, and autoimmune neutropenia (autoimmuneneutropenia), pancytopenia, leukopenia, diseases associated with leukocyte diapedesis, central nervous system inflammatory disorders such as septicemia, trauma, multi-organ damage secondary to hemorrhage, antigen-antibody complex-mediated diseases, anti-glomerular basement membrane disease, antiphospholipid antibody syndrome, allergic neuritis, Behcet's disease, Castleman's syndrome, Goodpasture's syndrome, Raynaud's syndrome, Sjögren's syndrome, Stevens-Johnson syndrome, such as pemphigoid bullous and skin pemphigoid Pemphigoid, pemphigus (including pemphigus vulgaris, pemphigus foliaceus, pemphigus mucous-membrane pemphigoid, and pemphigus erythematosus), autoimmune polyendocrinopathies, Reiter's disease or syndrome, immune complex nephritis, antibody-mediated nephritis, neuromyelitis optica, polyneuropathies, such as immunoglobulin M polyneuropathy or immunoglobulin M-mediated neuropathy, chronic neuropathy including thrombotic thrombocytopenic purpura,purpura; TTP) and autoimmune or immune-mediated thrombocytopenia (e.g., occurring in patients with myocardial infarction) such as idiopathic thrombocytopenic purpura (ITP) (including chronic or acute idiopathic thrombocytopenia), autoimmune diseases of the testicles and ovaries including autoimmune orchitis and oophoritis, primary hypothyroidism, hypothyroidism including thyroiditis such as autoimmune thyroiditis, autoimmune endocrine diseases, Hashimoto's disease, chronic thyroiditis or subacute thyroiditis, autoimmune thyroid diseases, idiopathic hypothyroidism, Graves' disease, polyglandular syndromes such as autoimmune polyglandular syndromes (or polyglandular endocrinopathy syndromes), syndrome, paraneoplastic syndromes including paraneoplastic syndromes of the nervous system such as Lambert-Eaton myasthenic syndrome or Eaton-Lambert syndrome, stiff-man or stiff-person syndrome, encephalomyelitis such as allergic encephalomyelitis (encephalomyelitis allergica) and experimental allergic encephalomyelitis (EAE), myasthenia gravis such as thymoma-associated myasthenia gravis, cerebellar degeneration, neuromyotonia, and opsoclonus or opsoclonus myoclonus syndrome. syndrome; OMS) and sensory neuropathy, multifocal motor neuropathymotor neuropathy), Sheehan's syndrome, autoimmune hepatitis, chronic hepatitis, lupus hepatitis, giant cell hepatitis, chronic active hepatitis or autoimmune chronic active hepatitis, lymphoid interstitial pneumonitis, bronchiolitis obliteran (non-transplant) and nonspecific interstitial pneumonia (NSIP), Guillain-Barré syndrome, Berger's disease (IgA nephritis), idiopathic IgA nephropathy, linear IgA dermopathy, primary biliary cirrhosis, pneumonocirrhosis, autoimmune enteropathy syndrome, Celiac disease, and celiac disease. disease), celiac sprue (gluten enteropathy), refractory sprue, idiopathic sprue, cryoglobulinemia, amyotrophic lateral sclerosis (ALS; Lou Gehrig's disease), coronary artery disease, autoimmune ear disease such as autoimmune inner ear disease (AGED), autoimmune hearing loss, opsoclonus myoclonus syndrome (OMS), polychondritis such as refractory or relapsing polychondritis, pulmonary alveolar proteinosis, alveolar proteinosis), amyloidosis, scleritis, non-cancerouslymphocytosis, primary lymphocytosis including monoclonal B-cell lymphocytosis (e.g., benign monoclonal gammopathy and monoclonal gammopathy of undetermined significance (MGUS)), peripheral neuropathy, paraneoplastic syndrome, epilepsy, migraine, arrhythmia, muscle disease, deafness, blindness, periodic paralysis, and channelopathies such as central nervous system channelopathies, autism, inflammatory myopathy, focal segmental glomerulosclerosis (FSGS), endocrine ophthalmopathy, ophthalmopathy, uveoretinitis, chorioretinitis, autoimmune hepatological disorder, fibromyalgia, multiple endocrine failure, Schmidt's syndrome, adrenalitis, gastric atrophy, presenile dementia, demyelinating diseases such as autoimmune demyelinating diseases, diabetic nephropathy, Dressler's syndrome, alopecia areata, CREST syndrome (calcinosis, Raynaud's phenomenon), esophageal motility disorderdysmotility), sclerodactyl and telangiectasia, male and female infertility, mixed connective tissue disease, Chagas' disease, rheumatic fever, recurrent abortion, farmer's lung, erythema multiforme, post-cardiotomy syndrome, Cushing's syndrome, bird-fancier's lung, allergic granulomatousangiitis, benign lymphocytic angiitis, Alport's syndrome, alveolitis such as allergic alveolitis and fibrosing periostitis, interstitial lung disease disease, transfusion disease, leprosy, malaria, leishmaniasis, trypanosomiasis, schistosomiasis, ascariasis, aspergillosis, Samter's syndrome, Caplan's syndrome, dengue fever, endocarditis, endomyocardial fibrosis, diffuse interstitial pulmonary fibrosis, interstitial lung fibrosis, idiopathic pulmonary fibrosis, cystic fibrosis, endophthalmitis, erythema elevatum et diutinum, erythroblastosis fetalis fetalis), eosinophilic fasciitis, Shulman'ssyndrome, Felty's syndrome, fliasis, cyclitis such as chronic cyclitis, heterochromic chronic cyclitis, iridocyclitis, or Fuch's cyclitis, Henoch-Schonlein purpura, human immunodeficiency virus (HIV) infection, ECHO virus infection, cardiomyopathy, Alzheimer's disease, parvovirus infection, rubella virus infection, post-vaccination syndromes, congenital rubella infection, Epstein-Barr virus infection, mumps, Evan's syndrome, autoimmune gonadal failure failure), Sydenham's chorea, poststreptococcal nephritis, thromboangiitis obliterans, thyrotoxicosis, tabes dorsalis, chorioiditis, giant cell polymyalgia, endocrine ophthalmopathy, chronic hypersensitivity pneumonitis, keratoconjunctivitis sicca, epidemic keratoconjunctivitis, idiopathic nephritic syndrome, minimal change nephropathy, benign familial and ischemia-reperfusion injury, retinal autoimmunity, joint arthritis,inflammation), bronchitis, chronic obstructive airway disease, silicosis, aphthae, aphthous stomatitis, arteriosclerotic disorders, aspermatogenesis, autoimmune hemolysis, Boeck's disease, cryoglobulinemia, Dupuytren's contracture, endophthalmia phacoanaphylactica, enteritis allergica, erythema nodosum leprosum, idiopathic facial paralysis, chronic fatigue syndrome, febrisrheumatica, Hamman-Rich syndrome disease, sensorineural hearing loss, hemoglobinuria paroxysmatica, hypogonadism, ileitis regionalis, leucopenia, mononucleosis infectiosa, transverse myelitis, primary idiopathic myxedema, nephrosis, ophthalmia symphatica, orchitis granulomatosa, pancreatitis, polyradiculitis acuta, pyoderma gangrenosum, Quervain's thyreoiditis, acquired spenicatrophy, infertility due to antispermatozoan antobodies, non-malignant thymoma, vitiligo, SCID and Epstein-Barr virus-associated diseases, acquired immune deficiency syndrome (AIDS), parasitic diseases such as leishmaniasis, toxic-shock syndrome, food poisoning, diseases involving T-cell infiltration, leukocyte adhesion defects, immune responses related to cytokine- and T-lymphocyte-mediated acute and delayed hypersensitivity, diseases associated with leukocytic infiltration, multiple organ injury syndrome, diseases mediated by antigen-antibody complexes, antiglomerular basement membrane disease, allergic neuritis, autoimmune polyendocrine disease, polyendocrinopathies, oophoritis, primary myxedema, autoimmune atrophic gastritis, sympathetic ophthalmia, rheumatic diseases, mixed connective tissue disease, nephrotic syndrome, insulitis, polyendocrine failure, peripheral neuropathy, autoimmune polyglandular syndrome type I, adult-onset idiopathic hypoparathyroidism (AOIH), alopecia totalis, dilated cardiomyopathy, epidermolysis bullosabullosaacquisita (EBA), hemochromatosis, myocarditis, nephrotic syndrome, primary sclerosing cholangitis, suppurative or non-suppurative sinusitis, acute or chronic sinusitis, ethmoid, frontal, maxillary, or sphenoid sinusitis, eosinophilic disorders such as eosinophilia, pulmonary infiltration eosinophilia, eosinophilia-myalgia syndrome, Loffler's syndrome, chronic eosinophilic pneumonia, tropical pulmonary eosinophilia, eosinophilia, bronchopneumonicaspergillosis, aspergilloma or granulomas containing eosinophils, anaphylaxis, seronegative spondyloarthritides, polyendocrine autoimmune disease, sclerosing cholangitis, sclera, episclera, chronic mucocutaneous candidiasis, Bruton's syndrome, transient hypogammaglobulinemia of infancy, Wiskott-Aldrich syndrome, ataxia telangiectasiatelangiectasia), autoimmune diseases associated with collagen disorders, rheumatic diseases, neurological diseases, ischemia-reperfusion injury, hypotensive response, vascular dysfunction, angiectasis, tissue damage, cardiovascular ischemia, hyperalgesia, cerebral ischemia and diseases associated with vascularization, allergic hypersensitivity disorders, glomerulonephritides, reperfusion injury, myocardial or other tissue reperfusion injury, dermatoses with acute inflammation, acute purulent meningitis or other central nervous system inflammatory conditions, ocular and orbital inflammatory diseases, granulocyte transfusion-associated syndromes, cytokine-induced toxicity, acute serious inflammation, chronic refractory inflammation The group consists of diabetic retinopathy, diabetic large-artery disorder, intractable inflammation, pyelitis, pneumonocirrhosis, endarterial hyperplasia, peptic ulcer, valvulitis, and endometriosis.

[0091] Preferably, the autoimmune disease can be selected from type 1 diabetes, alopecia areata, antiphospholipid antibody syndrome, rheumatoid arthritis, psoriasis or psoriatic arthritis, multiple sclerosis, systemic lupus erythematosus, inflammatory bowel disease, Addison's disease, Graves' disease, Sjogren's syndrome, Guillain-Barre syndrome, Hashimoto's thyroiditis, myasthenia gravis, inflammatory myophathy, autoimmune vasculitis, autoimmune hepatitis, hemolytic anemia, idiopathic thrombocytopenic purpura, The group consisting of primary thrombocytopenic purpura, primary biliary cirrhosis, scleroderma, vitiligo, pernicious anemia and celiac disease.

[0092] The inflammatory disease may be selected from the group consisting of arthritis, ankylosing spondylitis, reactive arthritis, Reiter's syndrome, crystal arthropathies, Lyme disease, polymyalgia rheumatica, systemic sclerosis, polymyositis, dermatomyositis, polyarteritis nodosa, Wegener's granulomatosis, Churg-Strauss syndrome, sarcoidosis, atherosclerotic vascular disease, atherosclerosis, ischemic heart disease, myocardial infarction, and the like. The group consisting of eye infarction, stroke, peripheral vascular disease, uveitis, corneal disease, iritis, iridocyclitis and cataracts.

[0093] The transplant rejection disease may be a tissue or organ transplant rejection reaction, which may be selected from the group consisting of bone marrow transplantation, heart transplantation, corneal transplantation, intestinal transplantation, liver transplantation, lung transplantation, pancreas transplantation, kidney transplantation, and skin transplantation rejection reactions.

[0094] The term "inflammatory skin disease" in the present invention refers to a disease that occurs in the skin through an inflammatory response. Skin keratinocytes, as a component of the majority of epidermal cells, form keratin and produce a variety of cytokines that participate in various inflammatory and immune responses. If skin keratinocytes are exposed to environmental or physiological stress, an inflammatory response is induced, thereby secreting various inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), and chemokine (CC motif) ligands (CCL). These cytokines reduce the proliferation rate of skin keratinocytes, thereby hindering the formation of the dermal matrix, thereby reducing the healing rate of damaged skin. Therefore, the proliferation of skin keratinocytes plays an important role in inflammatory skin diseases and is closely related to inflammatory skin diseases such as skin aging, atopic dermatitis, and psoriasis. Therefore, the inflammatory skin disease may be psoriasis or atopic dermatitis, preferably, it may be psoriasis.

[0095] The term "psoriasis" in this application refers to a skin disease characterized by silvery-white scales with distinct borders, forming erythematous papules and plaques of varying sizes. Histologically, it is characterized by epithelial hyperproliferation and exhibits a variety of clinical manifestations. It is a chronic inflammatory skin disease with recurrent exacerbations and remissions.

[0096] The preferred dosage of the pharmaceutical composition varies according to the patient's condition and body weight, degree of illness, pharmaceutical form, route of administration and time, but can be appropriately selected by those skilled in the art. In the pharmaceutical composition for treating or preventing psoriasis according to the present invention, as long as its active ingredient shows psoriasis therapeutic activity, any amount (effective amount) can be included according to the purpose, dosage form, formulation purpose, etc., and the conventional effective amount can be determined in the range of 0.001 weight % to 20.0 weight % based on the gross weight of the composition. Wherein, "effective amount" refers to the amount of the active ingredient that can guide the effect of psoriasis treatment (treatment). This effective amount can be determined by experiment within the conventional capabilities of those skilled in the art.

[0097] The term "treatment" herein may be used to encompass both therapeutic and preventative treatments. In this context, prevention may be used to refer to alleviating or reducing a pathological condition or disease in an individual. In one embodiment, the term "treatment" encompasses application or any form of administration for treating a disease in mammals, including humans. Furthermore, the term includes: inhibiting or slowing the progression of a disease or illness; partially or completely alleviating a disease by restoring or repairing damaged or missing functions; stimulating inefficient processes; and alleviating severe disease.

[0098] Here, "therapeutically effective amount" or "pharmaceutically effective amount" refers to the amount of a compound or composition effective for preventing or treating a target disease, meaning an amount that is sufficient to treat the disease at a reasonable benefit / risk ratio without causing side effects, as applicable to medical treatment. The level of the effective amount can be determined based on the patient's health status, the type and severity of the disease, the drug's activity, sensitivity to the drug, the method of administration, the time of administration, the route of administration, factors including excretion rate, treatment duration, formulated or concomitant medications, and other factors well known in the medical field. In one embodiment, a therapeutically effective amount refers to an amount of a drug used to effectively treat psoriasis.

[0099] The composition of the present invention may contain a pharmaceutically acceptable carrier, and in addition to the carrier, may further contain a pharmaceutically acceptable adjuvant, excipient or diluent.

[0100] Term of the present invention " pharmaceutically acceptable " refers to and can be accepted physiologically and does not cause allergic reaction for example gastrointestinal disease or the composition of dizziness or similar reaction when being applied to the people usually.As the example of described carrier, excipient and diluent, can give example lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, polyvinylpyrrolidone, water, methylparaben, propylparaben, talc, magnesium stearate and mineral oil.And, also can comprise filler, anticaking agent, lubricant, wetting agent, spices, emulsifying agent and preservative etc.

[0101] The pharmaceutical compositions of the present invention can be formulated using methods known in the art so as to provide rapid, sustained, or delayed release of the active ingredient when administered to a mammal. Dosage forms include powders, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatin capsules, sterile injectable solutions, and sterile powder forms.

[0102] The composition of the present invention can be formulated with a pharmaceutically acceptable carrier in an appropriate formulation. Examples of pharmaceutically acceptable carriers include carriers for parenteral administration, such as water, suitable oils, saline, water-soluble glucose, and ethylene glycol, and may also include stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium bisulfite, sodium sulfite, or ascorbic acid. Suitable preservatives include benzalkonium chloride, methylparaben, propylparaben, and chlorobutanol. Furthermore, depending on the method of administration or dosage form, the composition of the present invention may, if necessary, appropriately include a suspending agent, a cosolvent, a stabilizer, an isotonic agent, a preservative, an adsorption inhibitor, a surfactant, a diluent, an excipient, a pH adjuster, an analgesic, a buffer, an antioxidant, and the like. Pharmaceutically acceptable carriers and formulations suitable for the present invention, including the above examples, are described in detail in the document "Remington's Pharmaceutical Sciences (latest edition)".

[0103] The compositions of the present invention can be sterilized by conventional, well-known sterilization techniques. The compositions may include pharmaceutically acceptable auxiliary substances and adjuvants, toxicity-adjusting agents, and the like required for regulating physiological conditions, such as pH, such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, and the like. In such dosage forms, the concentration of the fusion protein may vary, for example, by weight, and may be approximately less than or equal to 0.5%, typically or at least about 1% to 15% or 20%, and may be selected based on the specific method of administration selected, preferably based on body fluid volume, viscosity, and the like.

[0104] Depending on the patient's condition, weight, sex, age, severity of the disease, and route of administration, the preferred dosage of the composition of the present invention may be in the range of 0.01 μg / kg to 10 g / kg per day or in the range of 0.01 mg / kg to 1 g / kg per day. Administration may be once a day or divided into multiple doses. In no respect should this dosage be construed as limiting the scope of this application.

[0105] The composition of the present invention can be applied (prescribed) to mammals and humans, particularly, preferably humans. Depending on the patient's condition and the presence or absence of side effects, the fusion protein or fusion protein dimer can be administered to the target subject using a variety of methods and dosages. Those skilled in the art can select the optimal administration method, dosage, and frequency within an appropriate range.

[0106] The compositions of the present invention can be administered by any route. The compositions of the present invention can be administered to an animal directly (e.g., topically, by injection, implantation, or topical administration to a tissue site) or systemically (e.g., parenterally or orally) by any suitable means. When the compositions of the present invention are administered parenterally, such as intravenously, subcutaneously, ophthalmically, intraperitoneally, intramuscularly, orally, rectally, intraorbitally, intracerebrally, intracranially, intraspinally, intraventricularly, intrathecally, intracisternally, intracapsularly, intranasally, or by aerosol, the compositions preferably comprise a portion of an aqueous or physiologically acceptable body fluid suspension or solution. Thus, the carrier or vehicle is physiologically acceptable and can be added to the composition for delivery to a patient without adversely affecting the patient's electrolyte and / or volume balance. Therefore, as a body fluid medium used for the preparation, physiological saline may generally be included.

[0107] A DNA construct (or gene construct) comprising a nucleic acid encoding a fusion protein of the present invention can be used as part of a gene therapy regimen carrying a nucleic acid encoding the fusion protein construct.

[0108] In the present invention, an expression vector that infects and expresses the fusion protein in a specific cell type is administered together with any biologically effective vector to reconstruct or supplement the desired function of the fusion protein. For example, it can be any dosage form or composition that can effectively transport a gene encoding a fusion protein required by cells in an organism or its fusion protein.

[0109] The target gene can be inserted into viral vectors including recombinant retrovirus, adenovirus, adeno-associated virus and herpes simplex virus-1, or recombinant viral plasmids or recombinant eukaryotic plasmids for gene therapy using nucleic acids encoding the fusion protein.

[0110] The dosage of the nucleic acid encoding the fusion protein of the present invention for human administration is in the range of 0.1 mg to 100 mg, preferably 1 mg to 10 mg, more preferably 2 mg to 10 mg. The optimal dosage and administration form can be determined by routine experiments within the skill of the art.

[0111] The human unit dose of fusion protein of the present invention is 0.1mg / kg to 1500mg / kg, preferably 1mg / kg to 100mg / kg, more preferably 5mg / kg to 20mg / kg. Unit dose can be different according to the presence or absence of the disease of the treatment target object and side effect. However, the optimal dose can be determined by routine experiments. The use of fusion protein can be carried out by regular rapid injection (periodic bolus injections) or from the outside supply source (external reservoir) (for example, intravenous bag (intravenous bag)) or the persistence of inside (for example, biodegradable implant (bioerodable implant)) intravenous, subcutaneous or intraperitoneal administration.

[0112] The composition of the present invention can be administered in combination with other drugs or physiologically active substances having a preventive or therapeutic effect on the disease to be prevented or treated, or can be formulated in the form of a combination formulation with such other drugs.

[0113] The method of preventing or treating a disease using the fusion protein or composition of the present invention may also include administering the fusion protein or composition of the present invention in combination with other drugs or physiologically active substances that have a preventive or therapeutic effect on the disease. The route, time and amount of combined administration can be determined based on the type of disease, the patient's disease state, the purpose of treatment or prevention, and the other drugs or physiologically active substances in combination.

[0114] Furthermore, the present invention provides the use of a fusion protein comprising a programmed cell death ligand 1 protein and a modified immunoglobulin Fc region for producing a pharmaceutical preparation having a preventive or therapeutic effect on immune diseases.

[0115] Furthermore, the present invention provides a method for preventing or treating immune diseases, comprising the step of administering a fusion protein comprising a programmed cell death ligand 1 protein and an Fc region of a modified immunoglobulin and a pharmaceutically acceptable carrier to an individual.

[0116] Preferably, the therapeutically effective amount is applied differently depending on various factors, including the type and extent of the response to be achieved, including whether other agents are used in different situations, the specific composition, the individual's age, weight, general health, sex, and diet, the time of administration, the route of administration, the secretion rate of the composition, the duration of treatment, various factors including drugs used together or concurrently with the specific composition, and similar factors known in the medical field. Therefore, preferably, the effective amount of the composition suitable for the purpose of the present invention should be determined taking into account the above matters.

[0117] The subject can be applied to any mammal, including not only humans and primates but also livestock such as cattle, pigs, sheep, horses, dogs and cats.

[0118] Embodiments of the invention

[0119] Hereinafter, the present invention will be described in more detail by way of examples. These examples are only used to describe the present invention in more detail, and the scope of the present invention is not limited to these examples.

[0120] Example 1. Preparation of a gene construct for producing a fusion protein comprising programmed cell death ligand 1 protein and the Fc region of a modified immunoglobulin

[0121] A gene construct for producing a fusion protein of a human programmed cell death ligand 1 protein and a modified immunoglobulin Fc domain was prepared. Specifically, the human programmed cell death ligand 1 gene was prepared at TOP Gene Technologies (Canada, Quebec) using a known amino acid sequence (Accession number: Q9NZQ7) to produce a gene construct comprising the extracellular domain (19-239aa) of the programmed cell death ligand 1 protein. The human programmed cell death ligand 1 protein was prepared to be fused to the N-terminus of the modified immunoglobulin Fc domain. The modified immunoglobulin Fc domain (SEQ ID NO: 11) is a hybrid of the Fc of human immunoglobulin D and the Fc of human immunoglobulin G4, characterized in that it comprises an immunoglobulin G1 hinge region (SEQ ID NO: 16) consisting of 8 amino acids.

[0122] The fusion protein of the present invention is prepared so that the programmed cell death ligand 1 protein is connected to the Fc region of the modified immunoglobulin through a connecting peptide. In the present invention, in order to induce dimerization and maintain flexibility, a fusion protein comprising a human programmed cell death ligand 1 protein and a modified immunoglobulin Fc region connected by a GS connecting peptide consisting of a 7-amino acid sequence (SEQ ID NO: 10) was prepared (hereinafter, "programmed cell death ligand 1-hyFc21" or "programmed cell death ligand 1-hyFc21 fusion protein", SEQ ID NO: 12 or SEQ ID NO: 13). As a control group, a fusion protein comprising an immunoglobulin Fc region (hereinafter, "programmed cell death ligand 1-hyFc5" or "programmed cell death ligand 1-hyFc5 fusion protein", SEQ ID NO: 15) was used, wherein the immunoglobulin Fc region comprises the immunoglobulin D hinge region of SEQ ID NO: 14.

[0123] A recombinant expression vector was prepared using a gene construct comprising nucleotides encoding the fusion protein. The prepared recombinant expression vector was transformed using the following gene introduction method (NeonTM kit, 10 μL, Invitrogen Cat. MPK1096), that is, a deoxyribonucleic acid (DNA) solution was suspended in the CHO DG44 cell line and a DC high voltage pulse was passed through to introduce deoxyribonucleic acid into the cell. Afterwards, HT selection (HT selection) (HT supplement (HTsupplement), Invitrogen, 11067-030) and methotrexate (MTX) (Methotrexate, Sigma, M8407) amplification steps were performed to perform cell passage to screen only cells showing high expression rates. The cells were cultured every 3 days at a rate of 0.4×10 6The amount of cells / mL is passaged, and the cell number and survival rate are determined using a cell counting device (Vi-cell, Beckman coulter). HT selection is a screening method in which HT is removed from the culture medium to allow only the transformed cells to survive, and methotrexate amplification is a method in which methotrexate is added to the passage medium at a screened concentration to amplify the gene. The screened cell pool (pool) utilizes a limiting dilution cloning (Limiting Dilution cloning) method to perform single cell cloning (Single CellCloning). In short, cells are seeded in a 96-well plate (well plate) to 1 cell / well. On day 0, day 7, and day 14, cell images are stored using a clone selection imager (Molecular Devices), and clones derived from one cell are reversely searched. The productivity of the selected single-cell cell lines was confirmed using an Fc ELISA (Human IgG ELISA Quantitation Set, Bethyl, E80-104). Five to six clones were finally screened for batch culture and long-term stability evaluation. The clones with confirmed stability were established as a Research Cell Bank (RCB).

[0124] Example 2. Ensuring the expression of programmed cell death ligand 1-hyFc21 or programmed cell death ligand 1-hyFc5 fusion protein

[0125] In order to mass-produce the PD-1-hyFc21 or PD-1-hyFc5 fusion protein, the target protein was isolated and purified from the PD-1-hyFc5 obtained in Example 1 or from the cell culture fluid produced using the PD-1-hyFc21 suspension cell line.

[0126] To obtain large quantities of the programmed cell death ligand 1-hyFc21 or programmed cell death ligand 1-hyFc5 fusion protein, cell lines expressing the programmed cell death ligand 1-hyFc21 or programmed cell death ligand 1-hyFc5 fusion protein were cultured in a 15L glass bioreactor for 20 days using the same fed-batch culture method to produce the programmed cell death ligand 1-hyFc21 or programmed cell death ligand 1-hyFc5 fusion protein. During the fed-batch culture, the cell viability of the cell lines expressing the fusion protein was measured, and size exclusion high-performance liquid chromatography (SE-HPLC) was performed to confirm the final expression level and purity of the target protein.

[0127] The results of confirming the maximum cell concentration of the expression cell line at different times and the final expression level of the fusion protein showed that the maximum cell concentration of the expression cell line of programmed cell death ligand 1-hyFc21 fusion protein at different times was 13.9×10 6 cells / mL( Figure 2a ), the expression level of the fusion protein finally recovered was 5.6 g / L ( Figure 2b In contrast, it was confirmed that the maximum cell concentration of the programmed cell death ligand 1-hyFc5 fusion protein-expressing cell line at different times was 16.7×10 6 cells / mL( Figure 3a ), the expression level of the fusion protein finally recovered was 2.2 g / L ( Figure 3b ).

[0128] Furthermore, the purity of the fusion protein was confirmed to be 79.5% for the programmed cell death ligand 1-hyFc21 fusion protein, including 7.7% high molecular weight impurities (HMW) and 9.8% low molecular weight impurities (LMW) (Table 1). In contrast, the purity of the programmed cell death ligand 1-hyFc5 fusion protein was confirmed to be 47.8%, including 31.7% high molecular weight impurities and 20.5% low molecular weight impurities (Table 1).

[0129]

Table 1

[0130]

[0131] *HMW: High Molecular Weight Impurity; Main: Target protein; and LMW: Low Molecular Weight Impurity.

[0132] Subsequently, to obtain highly pure programmed cell death ligand 1-hyFc21 or programmed cell death ligand 1-hyFc5 fusion protein, the following three-step purification process was performed: culture medium => Protein A affinity chromatography => Anion exchange chromatography 1 => Anion exchange chromatography 2 => Ultrafiltration / diafiltration.

[0133] The purity and yield of the obtained target protein were confirmed by size exclusion high-performance liquid chromatography (SE-HPLC). The programmed cell death ligand 1-hyFc21 fusion protein had a purity of 97.3% and a yield of 30.2%. In contrast, the programmed cell death ligand 1-hyFc5 fusion protein had a purity of 93.2% and a yield of 5.1% (Table 2).

[0134]

Table 2

[0135]

[0136] These results confirmed that the programmed cell death ligand 1-hyFc21 fusion protein exhibited approximately 2.5-fold higher production yield than the programmed cell death ligand 1-hyFc5 fusion protein, and also showed high protein purity.

[0137] Example 3. Characterization Analysis of Programmed Cell Death Ligand 1-hyFc21 or Programmed Cell Death Ligand 1-hyFc5 Fusion Proteins

[0138] 3.1. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)

[0139] To confirm the molecular weight of the purified programmed cell death ligand 1-hyFc21 or programmed cell death ligand 1-hyFc5 fusion protein, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed. Briefly, the fusion protein was diluted with deionized water and subjected to NuPAGE. TMThe sample was mixed with LDS sample buffer (Thermo Fisher Scientific) and loaded onto a 4-12% Bis-Tris gel (Invitrogen) for electrophoresis at 3 μg / well. The gel after electrophoresis was stained with Coomassie staining.

[0140] As a result, under non-reducing conditions, the purified programmed cell death ligand 1-hyFc21 and programmed cell death ligand 1-hyFc5 fusion proteins were confirmed to be located near the size marker 98 kDa. Figure 4 However, the purified programmed cell death ligand 1-hyFc5 fusion protein contains low-molecular-weight impurities in the cleaved form.

[0141] 3.2. Size-exclusion chromatography (SE-HPLC)

[0142] Size-exclusion high-performance liquid chromatography (SE-HPLC) was performed to analyze impurity peaks, such as the main peak and abnormal peptides in dimers, multimers, or cleaved forms, in the purified programmed cell death ligand 1-hyFc21 or programmed cell death ligand 1-hyFc5 fusion protein. Briefly, the fusion protein was diluted to 1.0 mg / mL with formulation buffer, and the area ratio (% Area) of the main peak of the fusion protein in the separated peaks was analyzed using a gel filtration column (TOSOH TSK-GEL G3000SWxL column, 7.8 mm × 300 mm).

[0143] As a result, it was confirmed that the purity of programmed cell death ligand 1-hyFc21 fusion protein was 97.3% ( Figure 5a ), compared with the purity of programmed cell death ligand 1-hyFc5 fusion protein was 93.2% ( Figure 5b ).

[0144] 3.3. Isoelectric focusing (IEF)

[0145] In order to confirm the charge variants and distribution of the purified programmed cell death ligand 1-hyFc5 and programmed cell death ligand 1-hyFc21 proteins, isoelectric focusing electrophoresis was performed. Isoelectric focusing electrophoresis is an electrophoresis method that analyzes separated proteins using the pI value of the protein. The pI value is the pH value at which a charged protein becomes neutrally charged, which is called the isoelectric point. In isoelectric electrophoresis, proteins that reach the isoelectric point no longer move, remain on the gel, and are separated. In short, in the present invention, isoelectric gels with a pH of 3 to 10 (Invitrogen) were used to separate the fusion proteins according to their pI values. After the gel was fixed with a 12% trichloroacetic acid (TCA) solution, it was stained using Coomassie staining.

[0146] As a result, it was confirmed that the pI values of programmed cell death ligand 1-hyFc21 and programmed cell death ligand 1-hyFc5 fusion proteins were both in the range of 5.2 to 6.0 ( Figure 6 ).

[0147] 3.4. Differential Scanning Fluorimetry (DSF)

[0148] To analyze the thermal stability of purified programmed cell death ligand 1-hyFc21 or programmed cell death ligand 1-hyFc5 fusion proteins, the The experiment was performed using a thermal shift stability assay kit. The assay kit contains a fluorescent dye for detecting protein aggregation, allowing determination of the temperature at which large amounts of protein aggregate under heat stress. The aggregation temperature (Tagg) serves as an indicator of protein stability, confirming the stability of the protein structure.

[0149] As a result, it was confirmed that the aggregation temperature of the programmed cell death ligand 1-hyFc21 fusion protein was 55.8°C, while the aggregation temperature of the programmed cell death ligand 1-hyFc5 fusion protein was 50.7°C ( Figure 7 ). This confirmed that, structurally, the programmed cell death ligand 1-hyFc21 fusion protein has superior thermal stability compared to the programmed cell death ligand 1-hyFc5.

[0150] Example 4. In vitro activity analysis of programmed cell death ligand 1-hyFc21 or programmed cell death ligand 1-hyFc5 fusion proteins

[0151] 4.1. Analysis of programmed death receptor 1 binding ability using Jurkat (SHP-1) cell line expressing programmed death receptor 1

[0152] The binding ability of programmed cell death ligand 1-hyFc21 or programmed cell death ligand 1-hyFc5 fusion protein to programmed cell death receptor 1 was analyzed using Jurkat (SHP-1) cell line expressing programmed cell death receptor 1. Briefly, Jurkat cells ( Jurkat PD-1 (SHP-1, DiscoverX) was stabilized in a 37°C 5% CO2 incubator for 2 hours. Then, programmed cell death ligand 1-hyFc21 or programmed cell death ligand 1-hyFc5 fusion protein was added to 75nM and 600nM, respectively, and then reacted in a 37°C 5% CO2 incubator for 1 hour. After the reaction, a reagent for detecting SHP-1 expression through the programmed cell death receptor 1: programmed cell death ligand 1 signaling response ( Bioassay Detection Kit, DiscoverX) was used to measure the luminescence of SHP-1.

[0153] As a result, compared with the programmed cell death ligand 1-hyFc5 fusion protein treatment group, in the programmed cell death ligand 1-hyFc21 fusion protein treatment group, the luminescence levels of all SHP-1 were high at concentrations of 75 nM and 600 nM ( Figure 8 ). This confirmed that the programmed cell death ligand 1-hyFc21 fusion protein has a higher binding ability to programmed cell death receptor 1 than the programmed cell death ligand 1-hyFc5 fusion protein. In particular, the binding ability to programmed cell death receptor 1 further increases with increasing concentration.

[0154] 4.2. Analysis of the inhibitory ability of mixed lymphocyte reaction

[0155] The ability to suppress mixed lymphocyte reactions by programmed cell death ligand 1-hyFc21 or programmed cell death ligand 1-hyFc5 fusion proteins was analyzed. Briefly, peripheral blood mononuclear cells (PBMCs) from donors and recipients were prepared and stained with CTV (CellTrace™ Violet) and CTR (CellTrace™ Far Red), which stain intracellular proteins. Peripheral blood mononuclear cells from each donor and recipient were mixed at a 1:1 ratio as reacting and stimulating cells and added to a 96-well U-bottom plate. 0.5 μM of programmed cell death ligand 1-hyFc21 or programmed cell death ligand 1-hyFc5 fusion protein was added and the cells were reacted in a 37°C, 5% CO2 incubator for 5 days. In the cells after the reaction, the degree of fluorescence reduction of CTV (CellTrace™ Violet) stained on the reacting cells was analyzed to compare the degree of inhibition of the mixed lymphocyte reaction by programmed cell death ligand 1-hyFc21 or programmed cell death ligand 1-hyFc5 fusion protein.

[0156] As a result, it was confirmed that the proliferation of CD4 T cells and CD8 T cells activated by mixed lymphocyte reaction was reduced in the programmed cell death ligand 1-hyFc21 or programmed cell death ligand 1-hyFc5 fusion protein-treated groups compared with the control group (Allo, hyFc-treated groups). Figure 9 In particular, it was confirmed that, under the same concentration conditions, the proliferation of activated responder cells, CD4 T cells and CD8 T cells, was further significantly reduced in the programmed cell death ligand 1-hyFc5 fusion protein-treated group ( Figure 9 ).

[0157] 4.3. Analysis of Proliferation Inhibition and Cytokine Production Inhibition of Activated Human T Cells

[0158] Under T cell activation conditions, the ability of human T cell proliferation to be inhibited by either the programmed cell death ligand 1-hyFc21 or programmed cell death ligand 1-hyFc5 fusion proteins was compared using human peripheral blood mononuclear cells. Briefly, 96-well plates were treated with either the programmed cell death ligand 1-hyFc21 or programmed cell death ligand 1-hyFc5 fusion proteins and anti-CD3 antibodies and coated at 4°C for one day. CD4 T cells were isolated from normal human peripheral blood mononuclear cells using microbeads (MACS), and the isolated CD4 T cells were stained with CTV (CellTrace™ Violet, 2.5μM), an intracellular protein stain. The coated plates were washed with phosphate-buffered saline (PBS), and the CTV-stained CD4 T cells were placed in the test plates and cultured in a 37°C, 5% CO2 incubator for three days. After 4 days, the cells in the test plate were recovered and the degree of CTV fluorescence reduction was analyzed by flow cytometry.

[0159] As a result, it was confirmed that the proliferation of activated human T cells was reduced in the programmed cell death ligand 1-hyFc21 or programmed cell death ligand 1-hyFc5 fusion protein-treated groups compared to the control group (no treatment or hyFc-treated group). Figure 10a In particular, it was confirmed that the proliferation of activated human T cells was significantly suppressed by about 2 times in the programmed cell death ligand 1-hyFc21 fusion protein-treated group compared to the programmed cell death ligand 1-hyFc5 fusion protein-treated group ( Figure 10a ).

[0160] Furthermore, the ability of human T cells to suppress cytokine expression by programmed cell death ligand 1-hyFc21 or programmed cell death ligand 1-hyFc5 fusion proteins was compared using human peripheral blood mononuclear cells under T cell activation conditions. Briefly, programmed cell death ligand 1-hyFc21 or programmed cell death ligand 1-hyFc5 fusion proteins were diluted to 0.5 μM and coated in 48-well plates at 4°C for one day. CD4 T cells were isolated from normal human peripheral blood mononuclear cells using microbeads (Miltenyi Biotech). CD4 T cells were isolated from normal human peripheral blood mononuclear cells using microbeads (MACS). The coated plates were washed with phosphate-buffered saline, and the isolated CD4 T cells were placed in the test plates and cultured in a 37°C incubator with 5% CO2 for three days. Anti-interferon-γ (IFN-γ) antibody (DuoSet Human IFN-gamma ELISA set, R&D system) was placed in the test plate and coated at room temperature. After washing the coated test plate three times with phosphate-buffered saline (PBST) supplemented with 0.05% Tween-20, 1% bovine serum albumin (BSA) in phosphate-buffered saline was added and the test plate was blocked at room temperature for 2 hours. The culture medium was transferred to the CD4 cells on the test plate and reacted at room temperature for 2 hours. After washing five times with phosphate buffered saline, the detection antibody was added and reacted at room temperature for 2 hours, and then washed again with phosphate buffered saline five times. After adding streptavidin-HRP to the washed test plate, the plate was reacted at room temperature for 20 minutes. After that, the plate was washed five times with phosphate buffered saline, substrate solution (TMB substrate) was added, and then 2N sulfuric acid (H2SO4) was added and the absorbance was measured at 450nm. The measured absorbance values were used to analyze the ability of CD4 T cells activated by the programmed cell death ligand 1-hyFc21 or programmed cell death ligand 1-hyFc5 fusion protein to suppress interferon-γ production.

[0161] As a result, it was confirmed that the amount of interferon-γ produced by activated human CD4 T cells was significantly reduced in the programmed cell death ligand 1-hyFc21 or programmed cell death ligand 1-hyFc5 fusion protein-treated groups compared to the control group (no treatment or hyFc-treated group). Figure 10b In particular, it was confirmed that the amount of interferon-γ produced by activated human CD4 T cells was significantly suppressed by about 3 times in the programmed cell death ligand 1-hyFc5 fusion protein-treated group ( Figure 10b ).

[0162] 4.4. Analysis of Cytokine Production Inhibition Ability of Activated Mouse T Cells

[0163] The ability of programmed cell death ligand 1-hyFc21 or programmed cell death ligand 1-hyFc5 fusion proteins to suppress cytokine expression in mouse T cells was compared under mouse T cell activation conditions. Briefly, 0.5 μM of programmed cell death ligand 1-hyFc21 or 1-hyFc5 fusion proteins were placed in test plates along with 5 μg / mL of anti-CD3 antibody and coated at 4°C for one day. CD4 T cells were isolated from lymphocytes isolated from mouse lymph nodes using microbeads (Miltenyi Biotech). After washing the coated plates with phosphate-buffered saline, the isolated CD4 T cells were placed in the plates and cultured in a 37°C, 5% CO2 incubator for three days. Anti-interleukin-2 (IL-2) antibody (Mouse IL-2 ELISA MAX™ Deluxe, Biolegend) and anti-interferon-γ antibody (Mouse IFNgamma ELISA MAX™ Deluxe, Biolegend) were placed in a new test plate and coated at room temperature. The test plate, which had been coated the previous day, was washed four times with wash buffer, and assay diluent A was added and blocked at room temperature for one hour. The test plate was washed again four times with wash buffer and then incubated with CD4 T cell culture medium, incubated at room temperature for two hours. After washing four times with wash buffer, detection antibody was added and the plate was shaken at room temperature for one hour, followed by washing four times with wash buffer. Streptavidin-HRP was added to the washed plate and incubated at room temperature for 30 minutes. After washing five times with wash buffer, substrate solution (TMB substrate) was added and the plate was incubated at room temperature with light blocked for 30 minutes. The reaction was terminated with the addition of a stop solution, and the absorbance was measured at 450 nm over 15 minutes. Interleukin-2 and interferon-γ concentrations were calculated using the measured absorbance values, and the ability to inhibit interleukin-2 and interferon-γ production by CD4 T cells activated with either the programmed cell death ligand 1-hyFc21 or programmed cell death ligand 1-hyFc5 fusion proteins was analyzed.

[0164] As a result, it was confirmed that the amounts of interleukin-2 and interferon-γ produced by activated mouse CD4 T cells were significantly reduced in the programmed cell death ligand 1-hyFc21 or programmed cell death ligand 1-hyFc5 fusion protein-treated groups compared to the control group (no treatment or hyFc-treated group). Figure 11 In particular, it was confirmed that the amount of interleukin-2 produced by activated human CD4 T cells in the programmed cell death ligand 1-hyFc5 fusion protein-treated group was significantly suppressed by about 1.8 times, and the amount of interferon-γ was significantly suppressed by about 4 times, compared to the programmed cell death ligand 1-hyFc5 fusion protein-treated group ( Figure 11 ).

[0165] Example 5. In vivo activity analysis of programmed cell death ligand 1-hyFc21 fusion protein

[0166] 5.1. Efficacy Evaluation of Programmed Cell Death Ligand 1-hyFc21 Fusion Protein in Imiquimod (IMQ)-Induced Psoriasis Mouse Model

[0167] During the onset of psoriasis, dendritic cells are activated through Toll-like receptors 7 / 8 (TLR7 / 8). Imiquimod (IMQ), a ligand for both TLR7 and TLR8, is known to induce psoriasis-like inflammatory responses when repeatedly applied to mouse skin. Imiquimod activates not only dendritic cells but also T cells, which are adaptive immune cells. Among these, gamma delta T cells (γδT17), which secrete interleukin-17, are particularly involved in psoriatic inflammation. Therefore, the imiquimod-induced psoriasis mouse model has a phenotype similar to that of psoriasis patients and can be used as a suitable model for the development of psoriasis therapeutics.

[0168] Briefly, to induce psoriasis in a mouse model, 20 mg / mouse / day of imiquimod cream was applied to both ears of the mice daily for 6 days. From day 0, the day of imiquimod application, until day 6, the end of the study, the ear thickness of each group of mice was measured daily with a caliper for 7 days. The ear thickness of mice not treated with imiquimod cream (no treatment) was used as a benchmark for comparison. Programmed cell death ligand 1-hyFc21 fusion protein was administered subcutaneously or intravenously on days 0, 1, 3, and 5. Alternatively, an anti-IL12p40 antibody was administered intravenously (IV) once on day 2 of the study. The control and experimental groups are shown in Table 3 below.

[0169]

Table 3

[0170]

[0171] As a result, it was confirmed that the ear thickness increased by about 68% ( 0.05 %) in the imiquimod-treated group (IMG-vehicle) on the sixth day after the end of the experiment, compared with the imiquimod-untreated control group (No treatment-vehicle). Figure 12a However, in the groups to which 3 mg / kg, 10 mg / kg, and 30 mg / kg of programmed cell death ligand 1-hyFc21 fusion protein were subcutaneously administered, differences in mouse ear thickness were observed starting from day 4 ( Figure 12a In particular, compared to the imiquimod-treated group (IMG-vehicle), in the group that subcutaneously administered the lowest dose of 3 mg / kg of programmed cell death ligand 1-hyFc21 fusion protein, ear thickness was suppressed by approximately 51% on day 6 after the end of the experiment. Compared to the imiquimod-treated group (IMG-vehicle), in the groups that subcutaneously administered the medium and high doses of 10 mg / kg and 30 mg / kg of programmed cell death ligand 1-hyFc21 fusion protein, inhibition rates of 67% and 66%, respectively, were shown. Figure 12a ). Also, significant differences were shown according to the dose between the group administered with 10 mg / kg or 30 mg / kg and the group administered with a low volume of 3 mg / kg, but no significant difference was shown between the group administered with 10 mg / kg and the group administered with 30 mg / kg.

[0172] Furthermore, compared to the imiquimod-treated group (IMG-vehicle), the groups that received intravenous administration of 3 mg / kg, 10 mg / kg, and 30 mg / kg of programmed cell death ligand 1-hyFc21 fusion protein all showed the following effect: the thickness of the mouse ears was significantly suppressed by about 87-92% (6 days after the end of the experiment). Figure 12b ).

[0173] 5.2. Efficacy Evaluation of Subcutaneous Administration of Programmed Cell Death Ligand 1-hyFc21 Fusion Protein in Doxycycline-Inducible Peli1 Gene Transformation Psoriasis Mouse Model

[0174] To evaluate the efficacy of the programmed cell death ligand 1-hyFc21 fusion protein in a psoriasis mouse model of chronic inflammation, the present inventors utilized, in addition to the imiquimod (IMQ)-induced psoriasis mouse model, a psoriasis mouse model in which long-term chronic psoriasis symptoms are induced by overexpression of the Peli1 (Pellino homolog 1) gene through administration of doxycycline (hereinafter referred to as the "rtTA-Peli1 psoriasis mouse model"). The rtTA-Peli1 psoriasis mouse model induces the development of psoriatic lesions in the epidermis and increases in inflammatory responses, such as increased production of inflammatory cytokines, an increase in the epithelial cell layer, and increased infiltration of macrophages, dendritic cells, and Th17 cells in the dermis, as psoriasis progresses.

[0175] Briefly, to induce psoriasis in mice, four-week-old mice were given drinking water containing 5% sucrose and 2 mg / mL of doxycycline for six months. During the experimental period, saline containing doxycycline was also continuously supplied. Mice not transformed with the Peli gene (hereinafter, "rtTA") served as a control group. The programmed cell death ligand 1-hyFc21 fusion protein was administered subcutaneously once weekly for eight weeks.

[0176] First, after subcutaneous administration of programmed cell death ligand 1-hyFc21 fusion protein to the rtTA-Peli1 psoriasis mouse model, an experiment was performed to measure the changes in skin epithelial tissue and the thickness of the skin epithelial layer by hematoxylin-eosin staining. The hematoxylin-eosin staining results were observed at a magnification of 200×, and 6 parts of each photo were arbitrarily selected and measured. As a result, it was confirmed that in the control group (rtTA) where psoriasis was not induced, the average thickness of the epithelial layer was 8.74μm, and in the rtTA-Peli1 group (rtTA-Peli1 Vehicle) where psoriasis was induced, the average thickness of the epithelial layer was 48.56μm. Compared with the control group, the thickness of the epithelial layer in the rtTA-Peli1 group (rtTA-Peli1 Vehicle) where psoriasis was induced increased by about 5.6 times ( Figure 13a and Figure 13b However, it was confirmed that in the groups where 3 mg / kg, 10 mg / kg, and 30 mg / kg of programmed cell death ligand 1-hyFc21 fusion protein were subcutaneously administered, the average thickness of the epithelial layer was 38.29 μm, 40.18 μm, and 31.74 μm, respectively, which showed an effect of reducing the thickness of the epithelial layer compared to the rtTA-Peli1 group (rtTA-Peli1 Vehicle) that induced psoriasis ( Figure 13a and Figure 13b ). However, no dose-dependent effect was shown.

[0177] Furthermore, after subcutaneous administration of a programmed cell death ligand 1-hyFc21 fusion protein to the rtTA-Peli1 psoriasis mouse model, an experiment was conducted to compare the extent of T cell and macrophage infiltration into the dermis using immunofluorescence staining. Anti-CD3 antibodies were used to detect T cells, and anti-F4 / 80 antibodies were used to detect macrophages. Results were observed using an immunofluorescence microscope at 400× magnification, and the number of cells within the same area was counted.

[0178] As a result, it was confirmed that in the control group (rtTA) where psoriasis was not induced, T cells and macrophages hardly infiltrated into the skin tissue, whereas in the rtTA-Peli1 group (rtTA-Peli1 Vehicle) where psoriasis was induced, the number of T cells and macrophages infiltrating into the skin tissue increased ( Figure 14 In particular, it was confirmed that the number of T cells infiltrating into the skin tissue in the rtTA-Peli1 group (rtTA-Peli1 Vehicle) that induced psoriasis was approximately 47 ( Figure 15 However, it was confirmed that in the groups where 3 mg / kg, 10 mg / kg, and 30 mg / kg of programmed cell death ligand 1-hyFc21 fusion protein was subcutaneously administered, the number of T cells infiltrating into the skin tissue was approximately 32, 14, and 7, respectively, and the number of infiltrating T cells was significantly reduced in a dose-dependent manner ( Figure 15 ).

[0179] Furthermore, after subcutaneous administration of programmed cell death ligand 1-hyFc21 fusion protein to the rtTA-Peli1 psoriasis mouse model, changes in keratin-producing cells in the skin tissue were analyzed by immunofluorescence staining using anti-keratin 14 (K14) antibodies. The expression of keratin (K14, Keratin 14) was shown in the basal layer responsible for proliferation, and their increased expression can be interpreted as an increase in cells responsible for proliferation among keratin-producing cells. The number of cells in the same area was measured and the results were compared. As a result, in the control group (rtTA) where psoriasis was not induced, K14+ The number of keratin-producing cells was 59. In the psoriasis-induced rtTA-Peli1 group (rtTA-Peli1 Vehicle), the number of keratin-producing cells was 197. Compared with the control group, the number of keratin-producing cells in the psoriasis-induced rtTA-Peli1 group increased by about 3.3 times ( Figure 16 However, in the groups to which 3 mg / kg, 10 mg / kg, and 30 mg / kg of programmed cell death ligand 1-hyFc21 fusion protein was subcutaneously administered, the number of keratinocytes was approximately 179, 150, and 98, respectively, and the number of keratinocytes was reduced in a dose-dependent manner. In particular, it was confirmed that the number of keratinocytes was significantly reduced at a dose of 30 mg / kg ( Figure 16 ).

[0180] 5.3. Efficacy Evaluation of Intravenous Administration of Programmed Cell Death Ligand 1-hyFc21 Fusion Protein in a Doxycycline-Inducible Peli1 Gene Transformed Psoriasis Mouse Model

[0181] After weekly intravenous administration of programmed cell death ligand 1-hyFc21 fusion protein to the rtTA-Peli1 psoriasis mouse model for 5 weeks, changes in the skin epithelium were analyzed as a score index and the skin thickness of the abdomen was measured using a caliper. The score index was evaluated as follows: 0 = normal skin; 1 = keratoplasia appeared; 2 = keratoplasia appeared on half of the back skin, or skin lesions slightly overtop the normal skin; 3 = thickness appeared on the whole back skin, or skin lesions significantly overtop the normal skin; 4 = skin lesions sclerosis (Biomedicine & Pharmacotherapy, Volume 110, February 2019, Pages 265-274).

[0182] As a result, it was confirmed that the score index increased by nearly 3 in the psoriasis-induced rtTA-Peli1 group (rtTA-Peli1 Vehicle) compared to the control group (rtTA), whereas the score index decreased in the group intravenously administered with programmed cell death ligand 1-hyFc21 fusion protein compared to the rtTA-Peli1 group ( Figure 17 ). Furthermore, similar to the scoring index, it was confirmed that the skin thickness of the abdomen increased by approximately 100 μm in the psoriasis-induced rtTA-Peli1 group (rtTA-Peli1 Vehicle) compared to the control group (rtTA), whereas the skin thickness decreased at a level similar to that of the control group (rtTA) in the group receiving intravenous administration of programmed cell death ligand 1-hyFc21 fusion protein ( Figure 18 ). However, no dose-dependent effect was confirmed in the above results.

[0183] In addition, after intravenous administration of programmed cell death ligand 1-hyFc21 fusion protein to the rtTA-Peli1 psoriasis mouse model, an experiment was performed to measure the changes in skin epithelial tissue and the thickness of the skin epithelial layer by hematoxylin-eosin staining. The results of hematoxylin-eosin staining were observed at 400× magnification under a microscope, and 6 parts of the epithelial layer were randomly selected to measure the thickness of the skin epithelial layer using the Image J program. As a result, it was confirmed that in the control group (rtTA) where psoriasis was not induced, the average thickness of the epithelial layer was 23.8μm, and in the rtTA-Peli1 group (rtTA-Peli1 Vehicle) where psoriasis was induced, the average thickness of the epithelial layer was 104.5μm. Compared with the control group, the thickness of the epithelial layer in the rtTA-Peli1 group (rtTA-Peli1 Vehicle) where psoriasis was induced increased by about 4.4 times ( Figure 19a and Figure 19b However, it was confirmed that in the groups where 1 mg / kg, 3 mg / kg, and 10 mg / kg of programmed cell death ligand 1-hyFc21 fusion protein was intravenously administered, the average thickness of the epithelial layer was approximately 97.9 μm, 86.3 μm, and 73.3 μm, respectively, which showed a dose-dependent effect of reducing the thickness of the epithelial layer compared to the psoriasis-inducing rtTA-Peli1 group (rtTA-Peli1 Vehicle) ( Figure 19a and 19b ).

[0184] Furthermore, after intravenous administration of programmed cell death ligand 1-hyFc21 fusion protein to the rtTA-Peli1 psoriasis mouse model, qRT-PCR was performed to confirm changes in messenger RNA expression of Th17 cell-associated genes such as interleukin 17A (IL-17A) and interleukin-22 (IL-22) and innate immune cell-associated genes such as interleukin-1β (IL-1β) and interleukin-24 (IL-24). As a result, it was confirmed that the expression of messenger RNA of Th17 cell-related genes such as interleukin 17A (IL-17A) and interleukin-22 (IL-22) was increased in the rtTA-Peli1 group (rtTA-Peli1 Vehicle) in which psoriasis was induced, compared with the control group (rtTA), whereas the expression of messenger RNA of Th17 cell-related genes was decreased in the group to which programmed cell death ligand 1-hyFc21 fusion protein was intravenously administered ( Figure 20 Similarly, in the psoriasis-induced rtTA-Peli1 group (rtTA-Peli1 Vehicle), the expression of innate immune cell-related genes, such as interleukin-1β (IL-1β) and interleukin-24 (IL-24), increased compared to the control group (rtTA). However, in the group to which programmed cell death ligand 1-hyFc21 fusion protein was intravenously administered, the expression of Th17 cell-related genes showed a tendency to decrease ( Figure 20 In particular, in the group to which 10 mg / kg of programmed cell death ligand 1-hyFc21 fusion protein was intravenously administered, the expression of interleukin-24 messenger RNA was significantly reduced ( Figure 20 ).

[0185] These results confirm that the programmed cell death ligand 1-hyFc21 fusion protein has the following effects: inhibiting the proliferation of CD4 T cells, which are an important factor in the pathogenesis of autoimmune diseases such as psoriasis, and suppressing the expression of inflammatory cytokines associated with pathological changes, through subcutaneous or intravenous administration, and is therefore useful as a therapeutic agent for immune diseases such as psoriasis. <110> Gnasini Ltd. <120> Fusion protein containing programmed cell death ligand 1 protein and use thereof <130> PCT5040687 <150> KR 10-2020-0008991 <151> 2020-01-23 <160> 35 <170> KopatentIn 2.0 <210> 1 <211> 290 <212> PRT <213> Artificial sequence <220> <223> Amino acid sequence of human PD-L1 (Accession number: Q9NZQ7) <400> 1 Met Arg Ile Phe Ala Val Phe Ile Phe Met Thr Tyr Trp His Leu Leu 1 5 10 15 Asn Ala Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr 20 25 30 Gly Ser Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu 35 40 45 Asp Leu Ala Ala Leu Ile Val Tyr Trp Glu Met Glu Asp Lys Asn Ile 50 55 60 Ile Gln Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser 65 70 75 80 Tyr Arg Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn 85 90 95 Ala Ala Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr 100 105 110 Arg Cys Met Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Val 115 120 125 Lys Val Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val 130 135 140 Asp Pro Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr 145 150 155 160 Pro Lys Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser 165 170 175 Gly Lys Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn 180 185 190 Val Thr Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr 195 200 205 Cys Thr Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu 210 215 220 Val Ile Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg Thr His 225 230 235 240 Leu Val Ile Leu Gly Ala Ile Leu Leu Cys Leu Gly Val Ala Leu Thr 245 250 255 Phe Ile Phe Arg Leu Arg Lys Gly Arg Met Met Asp Val Lys Lys Cys 260 265 270 Gly Ile Gln Asp Thr Asn Ser Lys Lys Gln Ser Asp Thr His Leu Glu 275 280 285 Glu Thr 290 <210> 2 <211> 290 <212> PRT <213> Artificial sequence <220> <223> Mouse PD-L1 amino acid sequence (Accession number: Q9EP73) <400> 2 Met Arg Ile Phe Ala Gly Ile Ile Phe Thr Ala Cys Cys His Leu Leu 1 5 10 15 Arg Ala Phe Thr Ile Thr Ala Pro Lys Asp Leu Tyr Val Val Glu Tyr 20 25 30 Gly Ser Asn Val Thr Met Glu Cys Arg Phe Pro Val Glu Arg Glu Leu 35 40 45 Asp Leu Leu Ala Leu Val Val Tyr Trp Glu Lys Glu Asp Glu Gln Val 50 55 60 Ile Gln Phe Val Ala Gly Glu Glu Asp Leu Lys Pro Gln His Ser Asn 65 70 75 80 Phe Arg Gly Arg Ala Ser Leu Pro Lys Asp Gln Leu Leu Lys Gly Asn 85 90 95 Ala Ala Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr 100 105 110 Cys Cys Ile Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Leu 115 120 125 Lys Val Asn Ala Pro Tyr Arg Lys Ile Asn Gln Arg Ile Ser Val Asp 130 135 140 Pro Ala Thr Ser Glu His Glu Leu Ile Cys Gln Ala Glu Gly Tyr Pro 145 150 155 160 Glu Ala Glu Val Ile Trp Thr Asn Ser Asp His Gln Pro Val Ser Gly 165 170 175 Lys Arg Ser Val Thr Thr Ser Arg Thr Glu Gly Met Leu Leu Asn Val 180 185 190 Thr Ser Ser Leu Arg Val Asn Ala Thr Ala Asn Asp Val Phe Tyr Cys 195 200 205 Thr Phe Trp Arg Ser Gln Pro Gly Gln Asn His Thr Ala Glu Leu Ile 210 215 220 Ile Pro Glu Leu Pro Ala Thr His Pro Pro Gln Asn Arg Thr His Trp 225 230 235 240 Val Leu Leu Gly Ser Ile Leu Leu Phe Leu Ile Val Val Ser Thr Val 245 250 255 Leu Leu Phe Leu Arg Lys Gln Val Arg Met Leu Asp Val Glu Lys Cys 260 265 270 Gly Val Glu Asp Thr Ser Ser Lys Asn Arg Asn Asp Thr Gln Phe Glu 275 280 285 Glu Thr 290 <210> 3 <211> 221 <212> PRT <213> Artificial sequence <220> <223> Amino acid sequence of human PD-L1 VC-like domain (19-239) <400> 3 Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr Gly Ser 1 5 10 15 Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu Asp Leu 20 25 30 Ala Ala Leu Ile Val Tyr Trp Glu Met Glu Asp Lys Asn Ile Ile Gln 35 40 45 Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser Tyr Arg 50 55 60 Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn Ala Ala 65 70 75 80 Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr Arg Cys 85 90 95 Met Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Val Lys Val 100 105 110 Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val Asp Pro 115 120 125 Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr Pro Lys 130 135 140 Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser Gly Lys 145 150 155 160 Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn Val Thr 165 170 175 Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr Cys Thr 180 185 190 Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu Val Ile 195 200 205 Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg Thr 210 215 220 <210> 4 <211> 115 <212> PRT <213> Artificial sequence <220> <223> Amino acid sequence of human PD-L1 V-like domain (19-133) <400> 4 Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr Gly Ser 1 5 10 15 Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu Asp Leu 20 25 30 Ala Ala Leu Ile Val Tyr Trp Glu Met Glu Asp Lys Asn Ile Ile Gln 35 40 45 Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser Tyr Arg 50 55 60 Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn Ala Ala 65 70 75 80 Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr Arg Cys 85 90 95 Met Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Val Lys Val 100 105 110 Asn Ala Pro 115 <210> 5 <211> 109 <212> PRT <213> Artificial sequence <220> <223> Amino acid sequence of human PD-L1 V-like domain (19-127) <400> 5 Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr Gly Ser 1 5 10 15 Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu Asp Leu 20 25 30 Ala Ala Leu Ile Val Tyr Trp Glu Met Glu Asp Lys Asn Ile Ile Gln 35 40 45 Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser Tyr Arg 50 55 60 Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn Ala Ala 65 70 75 80 Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr Arg Cys 85 90 95 Met Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr 100 105 <210> 6 <211> 107 <212> PRT <213> Artificial sequence <220> <223> Amino acid sequence of human PD-L1 V-like domain (21-127) <400> 6 Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr Gly Ser Asn Met 1 5 10 15 Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu Asp Leu Ala Ala 20 25 30 Leu Ile Val Tyr Trp Glu Met Glu Asp Lys Asn Ile Ile Gln Phe Val 35 40 45 His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser Tyr Arg Gln Arg 50 55 60 Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn Ala Ala Leu Gln 65 70 75 80 Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr Arg Cys Met Ile 85 90 95 Ser Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr 100 105 <210> 7 <211> 106 <212> PRT <213> Artificial sequence <220> <223> Amino acid sequence containing the human PD-L1 C-like domain (134-239) <400> 7 Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val Asp Pro Val Thr Ser 1 5 10 15 Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr Pro Lys Ala Glu Val 20 25 30 Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser Gly Lys Thr Thr Thr 35 40 45 Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn Val Thr Ser Thr Leu 50 55 60 Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr Cys Thr Phe Arg Arg 65 70 75 80 Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu Val Ile Pro Glu Leu 85 90 95 Pro Leu Ala His Pro Pro Asn Glu Arg Thr 100 105 <210> 8 <211> 105 <212> PRT <213> Artificial sequence <220> <223> Amino acid sequence including the human PD-L1 C-like domain (134-238) <400> 8 Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val Asp Pro Val Thr Ser 1 5 10 15 Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr Pro Lys Ala Glu Val 20 25 30 Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser Gly Lys Thr Thr Thr 35 40 45 Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn Val Thr Ser Thr Leu 50 55 60 Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr Cys Thr Phe Arg Arg 65 70 75 80 Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu Val Ile Pro Glu Leu 85 90 95 Pro Leu Ala His Pro Pro Asn Glu Arg 100 105 <210> 9 <211> 107 <212> PRT <213> Artificial sequence <220> <223> Amino acid sequence of mouse PD-L1 V-like domain (21-127) <400> 9 Ile Thr Ala Pro Lys Asp Leu Tyr Val Val Glu Tyr Gly Ser Asn Val 1 5 10 15 Thr Met Glu Cys Arg Phe Pro Val Glu Arg Glu Leu Asp Leu Leu Ala 20 25 30 Leu Val Val Tyr Trp Glu Lys Glu Asp Glu Gln Val Ile Gln Phe Val 35 40 45 Ala Gly Glu Glu Asp Leu Lys Pro Gln His Ser Asn Phe Arg Gly Arg 50 55 60 Ala Ser Leu Pro Lys Asp Gln Leu Leu Lys Gly Asn Ala Ala Leu Gln 65 70 75 80 Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr Cys Cys Ile Ile 85 90 95 Ser Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr 100 105 <210> 10 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Linker between PD-L1 and Fc <400> 10 Gly Gly Gly Ser Gly Gly Ser 1 5 <210> 11 <211> 223 <212> PRT <213> Artificial Sequence <220> <223> Modified Fc Region <400> 11 Thr His Thr Cys Pro Pro Cys Pro Ser His Thr Gln Pro Leu Gly Val 1 5 10 15 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 20 25 30 Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu 35 40 45 Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 50 55 60 Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser 65 70 75 80 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 85 90 95 Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile 100 105 110 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 115 120 125 Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 130 135 140 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 145 150 155 160 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 165 170 175 Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg 180 185 190 Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 195 200 205 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 210 215 220 <210> 12 <211> 451 <212> PRT <213> Artificial Sequence <220> <~223> sPD-L1-hyFc <400> 12 [[ID=~38]]Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr Gly Ser 1 5 10 15 Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu Asp Leu 20 25 30 Note: There seems to be an issue with the tag <223> in the original text. I've translated it as <~223> to maintain the format consistency as it's not clear what the correct form should be. If this is a known or specific tag format, it might need to be adjusted accordingly.Ala Ala Leu Ile Val Tyr Trp Glu Met Glu Asp Lys Asn Ile Ile Gln 35 40 45 Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser Tyr Arg 50 55 60 Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn Ala Ala 65 70 75 80 Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr Arg Cys 85 90 95 Met Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Val Lys Val 100 105 110 Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val Asp Pro 115 120 125 Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr Pro Lys 130 135 140 Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser Gly Lys 145 150 155 160 Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn Val Thr 165 170 175 Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr Cys Thr 180 185 190 Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu Val Ile 195 200 205 Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg Thr Gly Gly Gly 210 215 220 Ser Gly Gly Ser Thr His Thr Cys Pro Pro Cys Pro Ser His Thr Gln 225 230 235 240 Pro Leu Gly Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln 260 265 270 Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr 290 295 300 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305 310 315 320 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile 325 330 335 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350 Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser 355 360 365 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val 405 410 415 Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445 Leu Gly Lys 450 <210> 13 <211> 345 <212> PRT <213> Artificial Sequence <220> <223> sPD-L1 Variant-hyFc (PD-L1-GS / IgG1 / hyFc) <400> 13 Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr Gly Ser 1 5 10 15 Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu Asp Leu 20 25 30 Ala Ala Leu Ile Val Tyr Trp Glu Met Glu Asp Lys Asn Ile Ile Gln 35 40 45 Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser Tyr Arg 50 55 60 Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn Ala Ala 65 70 75 80 Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr Arg Cys 85 90 95 Met Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Val Lys Val 100 105 110 Asn Ala Pro Gly Gly Gly Ser Gly Gly Ser Thr His Thr Cys Pro Pro 115 120 125 Cys Pro Ser His Thr Gln Pro Leu Gly Val Phe Leu Phe Pro Pro Lys 130 135 140 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 145 150 155 160 Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr 165 170 175 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 180 185 190 Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 195 200 205 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 210 215 220 Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 225 230 235 240 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met 245 250 255 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 260 265 270 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 275 280 285 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 290 295 300 Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val 305 310 315 320 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 325 330 335 Lys Ser Leu Ser Leu Ser Leu Gly Lys 340 345 <210> 14 <211> 30 <212> PRT <213> Artificial sequence <220> <223> IgD hinge (from hyFc5) <400> 14 Arg Asn Thr Gly Arg Gly Gly Glu Glu Lys Lys Lys Glu Lys Glu Lys 1 5 10 15 Glu Glu Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys Pro 20 25 30 <210> 15 <211> 466 <212> PRT <213> Artificial sequence <220> <223> sPD-L1 variant-hyFc (PD-L1-IgD / hyFc) <400> 15 Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr Gly Ser 1 5 10 15 Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu Asp Leu 20 25 30 Ala Ala Leu Ile Val Tyr Trp Glu Met Glu Asp Lys Asn Ile Ile Gln 35 40 45 Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser Tyr Arg 50 55 60 Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn Ala Ala 65 70 75 80 Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr Arg Cys 85 90 95 Met Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Val Lys Val 100 105 110 Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val Asp Pro 115 120 125 Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr Pro Lys 130 135 140 Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser Gly Lys 145 150 155 160 Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn Val Thr 165 170 175 Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr Cys Thr 180 185 190 Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu Val Ile 195 200 205 Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg Thr Arg Asn Thr 210 215 220 Gly Arg Gly Gly Glu Glu Lys Lys Lys Glu Lys Glu Lys Glu Glu Gln 225 230 235 240 Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys Pro Ser His Thr Gln Pro 245 250 255 Leu Gly Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 260 265 270 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu 275 280 285 Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 290 295 300 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg 305 310 315 320 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 325 330 335 Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu 340 345 350 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 355 360 365 Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu 370 375 380 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 385 390 395 400 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 405 410 415 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp 420 425 430 Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His 435 440 445 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu 450 455 460 Gly Lys 465 <210> 16 <211> 8 <212> PRT <213> Artificial sequence <220> <223> IgG1 hinge (from modified Fc region) <400> 16 Thr His Thr Cys Pro Pro Cys Pro 1 5 <210> 17 <211> 20 <212> PRT <213> Artificial sequence <220> <223> Linker between PD-L1 and Fc <400> 17 Ala Ala Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser 20 <210> 18 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Linker between PD-L1 and Fc <400> 18 Gly Gly Ser Gly Gly 1 5 <210> 19 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Linker between PD-L1 and Fc <400> 19 Gly Gly Ser Gly Gly Ser Gly Gly Ser 1 5 <210> 20 <211> 6 <212> PRT <213> Artificial sequence <220> <223> Linker between PD-L1 and Fc <400> 20 Gly Gly Gly Ser Gly Gly 1 5 <210> twenty one <211> 18 <212> PRT <213> Artificial sequence <220> <223> Linker between PD-L1 and Fc <400> twenty one Lys Glu Ser Gly Ser Val Ser Ser Glu Gln Leu Ala Gln Phe Arg Ser 1 5 10 15 Leu Asp <210> twenty two <211> 14 <212> PRT <213> Artificial sequence <220> <223> Linker between PD-L1 and Fc <400> twenty two Glu Gly Lys Ser Ser Gly Ser Gly Ser Glu Ser Lys Ser Thr 1 5 10 <210> twenty three <211> 12 <212> PRT <213> Artificial sequence <220> <223> Linker between PD-L1 and Fc <400> twenty three Gly Ser Ala Gly Ser Ala Ala Gly Ser Gly Glu Phe 1 5 10 <210> twenty four <211> 12 <212> PRT <213> Artificial sequence <220> <223> Linker between PD-L1 and Fc <400> twenty four Cys Arg Arg Arg Arg Arg Arg Glu Ala Glu Ala Cys 1 5 10 <210> 25 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Linker between PD-L1 and Fc <400> 25 Gly Gly Gly Gly Gly Gly Gly Gly 1 5 <210> 26 <211> 6 <212> PRT <213> Artificial sequence <220> <223> Linker between PD-L1 and Fc <400> 26 Gly Gly Gly Gly Gly Gly 1 5 <210> 27 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Linker between PD-L1 and Fc <400> 27 Ala Glu Ala Ala Ala Lys Glu Ala Ala Ala Ala Lys Ala 1 5 10 <210> 28 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Linker between PD-L1 and Fc <400> 28 Pro Ala Pro Ala Pro 1 5 <210> 29 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Linker between PD-L1 and Fc <400> 29 Val Ser Gln Thr Ser Lys Leu Thr Arg Ala Glu Thr Val Phe Pro Asp 1 5 10 15 Val <210> 30 <211> 6 <212> PRT <213> Artificial sequence <220> <223> Linker between PD-L1 and Fc <400> 30 Pro Leu Gly Leu Trp Ala 1 5 <210> 31 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Linker between PD-L1 and Fc <400> 31 Thr Arg His Arg Gln Pro Arg Gly Trp Glu 1 5 10 <210> 32 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Linker between PD-L1 and Fc <400> 32 Ala Gly Asn Arg Val Arg Arg Ser Val Gly 1 5 10 <210> 33 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Linker between PD-L1 and Fc <400> 33 Arg Arg Arg Arg Arg Arg Arg Arg 1 5 <210> 34 <211> 4 <212> PRT <213> Artificial sequence <220> <223> Linker between PD-L1 and Fc <400> 34 Gly Phe Leu Gly 1 <210> 35 <211> 31 <212> PRT <213> Artificial sequence <220> <223> Linker between PD-L1 and Fc <400> 35 Gly Ser Ser Gly Gly Ser Gly Ser Ser Gly Gly Ser Gly Gly Gly Asp 1 5 10 15 Glu Ala Asp Gly Ser Arg Gly Ser Gln Lys Ala Gly Val Asp Glu 20 25 30

Claims

1. A fusion protein, characterized in that The invention relates to a fusion protein comprising a programmed cell death ligand 1 (PD-L1) protein and an Fc region of a modified immunoglobulin, wherein the fusion protein consists of the amino acid sequence of SEQ ID NO: 12 or SEQ ID NO:

13.

2. A nucleic acid molecule, characterized in that Encode the fusion protein according to claim 1.

3. An expression vector, characterized in that Comprising the nucleic acid molecule according to claim 2.

4. A host cell, characterized in that Comprising the expression vector according to claim 3.

5. Use of the fusion protein according to claim 1 as an effective ingredient in the preparation of a drug for treating immune diseases, wherein: The immune disease is selected from the group consisting of psoriasis, inflammatory bowel disease, rheumatoid arthritis and systemic lupus erythematosus.

6. The use according to claim 5, characterized in that The drug further comprises a pharmaceutically acceptable carrier.

Images