Fusion proteins containing PD-L1 protein and uses thereof

Abstract

The present invention relates to a fusion protein comprising a PD-L1 protein and a modified immunoglobulin Fc region and uses thereof. Compared to existing fusion proteins, the fusion protein has significantly higher purity and production yield, has high binding affinity to PD-1, and is effective in reducing the proliferation of activated T cells, suppressing the production of cytokines produced by activated T cells, and suppressing the infiltration of T cells or macrophages into tissues, making it useful for treating immune diseases.

Description

[Technical field]

[0001] The present invention relates to a fusion protein comprising a PD-L1 protein and a modified immunoglobulin Fc region, and uses thereof. [Background technology]

[0002] Human PD-L1 (programmed cell death-ligand 1) is a ligand for PD-1 (programmed death-1) and is a single-pass transmembrane protein expressed not only in hematopoietic cells such as T lymphocytes, B lymphocytes, dendritic cells, and macrophages, but also in non-hematopoietic cells such as keratinocytes, pancreatic islet cells, and hepatocytes. Meanwhile, in order for T cells to be activated, a secondary signal stimulus (co-stimulation) is required in addition to the primary signal stimulus of the T cell receptor and antigen. Here, if neither of the two signals is present, the T cell will be in an inactivated state. PD-1 (programmed death-1) is a secondary signal stimulator (immune check point or immune modulator) that regulates the secondary signal activity of T cells. By binding to PD-L1 (programmed cell death ligand 1) or B7.1 (CD80) expressed on the cell surface of activated T cells (CD8 and / or CD4) and dendritic cells, it can suppress T cell proliferation and cytokine expression, thereby suppressing T cell function.

[0003] PD-1:PD-L1 binding is known to induce the activity of regulatory T cells (Immunol Rev.2010 Jul;236:219-42), and it has been observed that when PD-L1 protein fused with IgG1 Fc (PD-L1-Ig) was injected into a collagen-induced arthritis (CIA) mouse model, the symptoms of arthritis were alleviated (Rheumatol Int.2011 Apr;31(4):513-9). Since PD-1 is expressed in activated T cells, it is predicted that PD-L1 protein can be used as a therapeutic agent that specifically targets activated immune cells to induce immune tolerance not only in autoimmune diseases but also in organ transplantation.

[0004] To date, therapeutic agents for the PD-1 / PD-L1 cell signaling system have been developed as inhibitors that break immune tolerance and increase T cell activity. However, no immunotherapeutic agents based on inducing T cell immune tolerance using agonists have been developed to date. This is because, while PD-1 / PD-L1 inhibitors can be easily developed using antibody fusion technology, PD-1 / PD-L1 signal agonists must be developed as soluble proteins, making it technically difficult to develop.

[0005] Immunoglobulin (Ig) Fc fusion technology is one of the technologies to increase the half-life of protein therapeutics in vivo. However, in the case of IgG1 used in the existing Ig fusion technology, it causes antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) in the body, so Ig fusion proteins as therapeutic agents for autoimmune diseases or immune tolerance inducers in organ transplantation do not suppress inflammatory reactions but rather aggravate inflammation. Therefore, there is a need to develop technologies that can enhance the therapeutic efficacy of PD-L1 as an immune inhibitor by preventing it from inducing ADCC and CDC while maintaining the half-life of PD-L1 similar to that of existing Ig fusion protein therapeutics. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Immunol Rev.2010 Jul;236:219-42 [Non-Patent Document 2] Rheumatol Int.2011 Apr;31(4):513-9 Summary of the Invention [Problem to be solved by the invention]

[0007] An object of the present invention is to provide a fusion protein comprising a programmed cell death-ligand 1 (PD-L1) protein and a modified immunoglobulin Fc region.

[0008] It is another object of the present invention to provide a nucleic acid molecule encoding said fusion protein.

[0009] It is yet another object of the present invention to provide an expression vector comprising the nucleic acid molecule.

[0010] It is still another object of the present invention to provide a host cell containing the expression vector.

[0011] It is yet another object of the present invention to provide a pharmaceutical composition for the prevention or treatment of immune diseases, which comprises as an active ingredient a fusion protein comprising a PD-L1 protein and a modified immunoglobulin Fc region.

[0012] It is yet another object of the present invention to provide a use of a fusion protein comprising a PD-L1 protein and a modified immunoglobulin Fc region for producing a pharmaceutical preparation having the effect of preventing or treating an immune disease.

[0013] It is yet another object of the present invention to provide a method for preventing or treating an immune disease, comprising the step of administering to an individual a fusion protein comprising a PD-L1 protein and an engineered immunoglobulin Fc region, and a pharma- ceutical acceptable carrier. [Means for solving the problem]

[0014] To achieve the above objectives, the present invention provides a fusion protein comprising a PD-L1 protein and a modified immunoglobulin Fc region.

[0015] The present invention also provides a nucleic acid molecule encoding the fusion protein.

[0016] The present invention also provides an expression vector comprising the nucleic acid molecule.

[0017] The present invention also provides a host cell comprising the expression vector.

[0018] The present invention also provides a pharmaceutical composition for preventing or treating an immune disease, which comprises as an active ingredient a fusion protein comprising a PD-L1 protein and a modified immunoglobulin Fc region.

[0019] The present invention also provides use of a fusion protein comprising a PD-L1 protein and a modified immunoglobulin Fc region for producing a pharmaceutical preparation having an effect of preventing or treating an immune disease.

[0020] The present invention also provides a method for preventing or treating an immune disease, comprising the step of administering to an individual a fusion protein comprising a PD-L1 protein and a modified immunoglobulin Fc region, and a pharma- ceutical acceptable carrier. Effect of the Invention

[0021] The fusion protein according to the present invention is a fusion protein in which a PD-L1 protein and a modified immunoglobulin Fc region are linked by a sequence consisting of a GS sequence and an IgG1 hinge, and is characterized in that it is prepared so as to be capable of inducing a dimer while maintaining flexibility by being linked by a sequence consisting of the GS sequence and the IgG1 hinge.In addition, the fusion protein according to the present invention has a significantly higher purity and production yield than existing fusion proteins, has a high binding ability to PD-1, and has the effects of reducing the proliferation of activated T cells, suppressing the production of cytokines produced by activated T cells, and suppressing the infiltration of T cells or macrophages into tissues, and can be useful in the treatment of immune diseases. [Brief description of the drawings]

[0022] [Figure 1] Figure 1 shows the structure of a fusion protein comprising a PD-L1 protein and a modified immunoglobulin Fc region (PD-L1-hyFc21 fusion protein). [Figure 2A]Figure 2a shows the analysis result of the cell concentration over time of cells expressing PD-L1-hyFc21 fusion protein, and Figure 2b shows the analysis result of the purity of the target protein in the cell culture medium of cells expressing PD-L1-hyFc21 fusion protein by SE-HPLC. [Figure 2B] Same as above. [Figure 3A] Figure 3a shows the analysis result of the cell concentration over time of cells expressing PD-L1-hyFc5 fusion protein, and Figure 3b shows the analysis result of the purity of the target protein in the cell culture medium of cells expressing PD-L1-hyFc5 fusion protein by SE-HPLC. [Figure 3B] Same as above. [Figure 4] Figure 4 shows SDS-PAGE analysis of purified PD-L1-hyFc21 or PD-L1-hyFc5 fusion proteins. [Figure 5A] Figure 5a shows the SE-HPLC analysis of purified PD-L1-hyFc21 fusion protein, and Figure 5b shows the SE-HPLC analysis of purified PD-L1-hyFc5 protein. [Figure 5B] Same as above. [Figure 6] Figure 6 shows the results of Gel IEF analysis of purified PD-L1-hyFc21 or PD-L1-hyFc5 fusion proteins. [Figure 7] Figure 7 shows the differential scanning fluorometry (DSF) analysis of purified PD-L1-hyFc21 or PD-L1-hyFc5 fusion proteins. [Figure 8] Figure 8 compares the binding affinity of PD-L1-hyFc21 and PD-L1-hyFc5 fusion proteins to PD-1. [Figure 9] Figure 9 compares the ability of PD-L1-hyFc21 or PD-L1-hyFc5 fusion proteins to suppress mixed lymphocyte reaction. [Figure 10A]Figure 10a compares the ability of PD-L1-hyFc21 or PD-L1-hyFc5 fusion proteins to inhibit the proliferation of activated human CD4 T cells, and Figure 10b compares their ability to inhibit cytokine expression in activated human CD4 T cells. [Figure 10B] Same as above. [Figure 11] Figure 11 compares the ability of PD-L1-hyFc21 or PD-L1-hyFc5 fusion proteins to suppress cytokine expression in activated mouse CD4 T cells. [Figure 12A] Figure 12a shows the measurement of mouse ear thickness after subcutaneous administration of PD-L1-hyFc21 fusion protein into an IMQ-induced psoriasis mouse model, and Figure 12b shows the measurement of mouse ear thickness after intravenous administration of PD-L1-hyFc21 fusion protein into an IMQ-induced psoriasis mouse model. [Figure 12B] Same as above. [Figure 13A] Figure 13a shows the changes in skin epithelial tissue observed by H&E staining after subcutaneous administration of PD-L1-hyFc21 fusion protein in a rtTA-Peli1 psoriasis mouse model, and Figure 13b shows the measurement of the thickness of the skin epithelial layer. [Figure 13B] Same as above. [Figure 14] Figure 14 shows the results of immunofluorescence analysis showing the degree of infiltration of T cells and macrophages in a control group (rtTA) in which psoriasis was not induced or in the rtTA-Peli1 psoriasis mouse model in which psoriasis was induced (left panel: rtTA; and right panel: rtTA-Peli1). [Figure 15] Figure 15 shows the results of measuring the numbers of T cells and macrophages infiltrating into skin tissue after subcutaneous administration of PD-L1-hyFc21 fusion protein in the rtTA-Peli1 psoriasis mouse model. [Figure 16] Figure 16 shows the number of K14+ keratinocytes in skin tissue after subcutaneous administration of PD-L1-hyFc21 fusion protein in the rtTA-Peli1 psoriasis mouse model. [Figure 17]Figure 17 shows the results of analyzing the changes in the skin epithelium layer using a score index after intravenous administration of PD-L1-hyFc21 fusion protein to the rtTA-Peli1 psoriasis mouse model. [Figure 18] Figure 18 shows the results of measuring abdominal skin thickness after intravenous administration of PD-L1-hyFc21 fusion protein in the rtTA-Peli1 psoriasis mouse model. [Figure 19A] Figure 19a shows the changes in skin epithelial tissue observed by H&E staining after intravenous administration of PD-L1-hyFc21 fusion protein to a rtTA-Peli1 psoriasis mouse model, and Figure 19b shows the measurement of the thickness of the skin epithelial layer. [Figure 19B] Same as above. [Figure 20] Figure 20 shows the results of qRT-PCR analysis of changes in mRNA expression of Th17 cell-related genes (IL-17A and IL-22) and innate immune cell-related genes (IL-1β and IL-24) after intravenous administration of PD-L1-hyFc21 fusion protein to the rtTA-Peli1 psoriasis mouse model. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] The present invention will be described in detail below.

[0024] The present invention provides a fusion protein comprising a PD-L1 protein and a modified immunoglobulin Fc region.

[0025] The PD-L1 protein may be the extracellular domain of the PD-L1 protein or a fragment thereof. The extracellular domain of the PD-L1 protein may be a polypeptide comprising an Immunoglobulin V like domain of PD-L1 and an Immunoglobulin C like domain of PD-L1.

[0026] Specifically, the extracellular domain of the PD-L1 protein is a protein site that is exposed outside the cell membrane, and may 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.

[0027] Here, the extracellular domain of the PD-L1 protein contains an Ig V-like sequence, which is a conserved sequence similar to the amino acid sequence of immunoglobulin (Ig), and the highly conserved Ig V-like sequence is the amino acid sequence of positions 68 to 114 of SEQ ID NO: 1. Additionally, the extracellular domain of the PD-L1 protein contains an Ig C-like sequence, and the highly conserved sequence portion is the amino acid sequence of positions 153 to 210 of SEQ ID NO: 1. Additionally, the fragment of the extracellular domain of the PD-L1 protein may contain the entire or a part of the Ig V-like domain including the Ig V-like sequence of PD-L1.

[0028] In addition, the Ig V-like domain in the extracellular domain of the PD-L1 protein is a site capable of interacting with PD-1, and may be a polypeptide consisting of the amino acid sequence of positions 19 to 239 of SEQ ID NO: 1 (SEQ ID NO: 3), or may be a polypeptide consisting of the amino acid sequence of positions 21 to 239 of SEQ ID NO: 1. Also, it may be a polypeptide consisting of the amino acid sequence of positions 19 to 133 of SEQ ID NO: 1 (SEQ ID NO: 4), or may be a polypeptide consisting of the amino acid sequence of positions 21 to 133 of SEQ ID NO: 1. Also, it may be a polypeptide consisting of the amino acid sequence of positions 21 to 114 of SEQ ID NO: 1, or may be a polypeptide consisting of the amino acid sequence of positions 19 to 114 of SEQ ID NO: 1. Also, it may be a polypeptide consisting of the amino acid sequence of positions 21 to 120 of SEQ ID NO: 1, or may be a polypeptide consisting of the amino acid sequence of positions 19 to 120 of SEQ ID NO: 1. Also, it may be a polypeptide consisting of the amino acid sequence of positions 19 to 127 of SEQ ID NO: 1 (SEQ ID NO: 5), or may be a polypeptide consisting of the amino acid sequence of positions 21 to 127 of SEQ ID NO: 1 (SEQ ID NO: 6). Moreover, it may be a polypeptide consisting of the amino acid sequence of positions 21 to 130 of SEQ ID NO: 1, or it may be a polypeptide consisting of the amino acid sequence of positions 19 to 130 of SEQ ID NO: 1. Moreover, it may be a polypeptide consisting of the amino acid sequence of positions 21 to 131 of SEQ ID NO: 1, or it may be a polypeptide consisting of the amino acid sequence of positions 19 to 131 of SEQ ID NO: 1.

[0029] Furthermore, when the fragment of the extracellular domain of PD-L1 protein comprises an Ig V-like domain or a fragment thereof, it may further comprise an Ig C-like domain (Immunoglobulin C like domain) of the extracellular domain of PD-L1 protein. The Ig C-like domain may be a polypeptide consisting of the amino acid sequence of positions 133 to 225 of SEQ ID NO:1, or a polypeptide consisting of the amino acid sequence of positions 134 to 225 of SEQ ID NO:1.

[0030] Furthermore, when the fragment of the extracellular domain of PD-L1 protein comprises an Ig V-like domain or a fragment thereof, it may further comprise a polypeptide comprising an Ig C-like domain of the extracellular domain of PD-L1 protein or a fragment thereof. The polypeptide comprising an Ig C-like domain refers to the extracellular domain of PD-L1 protein excluding the Ig V domain, and may be a polypeptide comprising amino acids 134 to 239 of SEQ ID NO: 1 (SEQ ID NO: 7) or a polypeptide comprising amino acids 134 to 238 of SEQ ID NO: 1 (SEQ ID NO: 8).

[0031] Additionally, the extracellular domain of the PD-L1 protein or a fragment thereof may be of human or mouse origin.

[0032] The extracellular domain of the human PD-L1 protein is a polypeptide consisting of the amino acid sequence of amino acids 19 to 239 of SEQ ID NO: 1 (SEQ ID NO: 3), and the extracellular domain of the mouse PD-L1 protein is a polypeptide consisting of the amino acid sequence of amino acids 19 to 239 of SEQ ID NO: 2. The extracellular domain of the PD-L1 protein may have approximately 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more homology to the polypeptide sequence consisting of the amino acid sequence of amino acids 19 to 239 of SEQ ID NO: 1.

[0033] Specifically, human PD-L1 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, amino acid residues 1 to 18 at the N-terminus are a signal sequence, and mature human PD-L1 protein comprises the amino acid sequence of SEQ ID NO: 1 from amino acid residues 19 to 290. The extracellular domain of human PD-L1 protein comprises the amino acid sequence of SEQ ID NO: 1 from amino acid residues 19 to 238 or amino acid sequence of SEQ ID NO: 1 from amino acid residues 19 to 239.

[0034] Human PD-L1 protein contains an Ig V-like domain, which is amino acids 19 to 127 of SEQ ID NO:1, and an Ig C-like domain, which is amino acids 134 to 226 of SEQ ID NO:1.

[0035] Mouse PD-L1 protein is reported to contain 290 amino acids, which comprises 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 PD-L1 protein comprises the amino acid sequence of SEQ ID NO:2 from 19 to 290. The extracellular domain of mouse PD-L1 protein comprises the amino acid sequence of SEQ ID NO:2 from 19 to 239. Mouse PD-L1 protein comprises an Ig V-like domain, which is amino acids 19 to 127 of SEQ ID NO:2, and an Ig C-like domain, which is amino acids 133 to 224 of SEQ ID NO:2.

[0036] The extracellular domain of the PD-L1 protein may comprise the entire Ig V-like domain or a fragment thereof, and the fragment of the extracellular domain of the PD-L1 protein may additionally comprise an Ig C-like domain or a polypeptide containing an Ig C-like domain (the extracellular domain of PD-L1 excluding the Ig V-like domain).

[0037] The extracellular domain of the PD-L1 protein or fragments thereof may comprise variously modified proteins or peptides, which may be made by substituting, deleting or adding one or more proteins to the wild-type PD-L1 protein, provided that the modifications do not alter the function of PD-L1. Such modified 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.

[0038] In the present invention, the term "extracellular domain of PD-L1 protein" can be used in a conceptual sense to include "the extracellular domain of PD-L1 protein and fragments thereof".

[0039] As used herein, the terms "protein", "polypeptide" and "peptide" can be used interchangeably unless otherwise specified.

[0040] As used herein, the terms “PD-L1 fusion protein” and “PD-L1-modified immunoglobulin Fc region fusion protein” refer to a fusion protein in which PD-L1 protein, the extracellular domain of PD-L1 protein, or a fragment thereof, is linked to a modified immunoglobulin Fc region.

[0041] In the present invention, the PD-L1 protein can be fused to the N-terminus or C-terminus of the modified immunoglobulin Fc domain, preferably, the PD-L1 protein can be fused to the N-terminus of the modified immunoglobulin Fc domain, and the PD-L1 protein can be linked to the immunoglobulin Fc domain via a linker peptide. The linker is selected from the group consisting of 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), (GS)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 ( Examples of PD-L1 include SEQ ID NO: 24), A(EAAAK)4ALEA(EAAAK)4A, GGGGGGGG (SEQ ID NO: 25), GGGGGG (SEQ ID NO: 26), AEAAAKEAAAAKA (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), AGNRVRRSVG (SEQ ID NO: 32), RRRRRRRR (SEQ ID NO: 33), GFLG (SEQ ID NO: 34), and GSSGGSGSSGGSGGGDEADGSRGSQKAGVDE (SEQ ID NO: 35). Preferably, PD-L1 and the immunoglobulin Fc region may be linked by a linker peptide consisting of the amino acid sequence of GGGSGGS (SEQ ID NO: 10). When the linker peptide is used to link the PD-L1 protein and the immunoglobulin Fc region, the activity, stability, and productivity of the fusion protein can be optimized.

[0042] The fusion protein may also exist in a dimeric form. The bond between the fusion proteins constituting the dimer may be a disulfide bond formed by a cysteine ​​present in the linker. The fusion proteins constituting the dimer are the same. That is, the dimer may be a homodimer. Here, the fusion protein may be soluble, particularly, soluble in purified water or physiological saline.

[0043] The modified immunoglobulin Fc region may be any one of IgG1, IgG2, IgG3, IgD, and IgG4 Fc regions, or a combination thereof. The Fc region is modified so as not to bind to an Fc receptor and / or not to bind complement. In particular, the modified immunoglobulin Fc region may comprise, from the N-terminus to the C-terminus, a hinge region, a CH2 domain, and a CH3 domain, the hinge region comprising a human IgG1 hinge region (SEQ ID NO: 16), the CH2 domain comprising a portion of amino acid residues of the CH2 domains of human IgD and human IgG4, and the CH3 domain comprising a portion of amino acid residues of the CH3 domain of human IgG4.

[0044] The term "Fc region", "Fc fragment" or "Fc" of the present invention refers to a protein that includes the heavy chain constant region 2 (CH2) and the heavy chain constant region 3 (CH3) of an immunoglobulin, but does not include the variable regions of the heavy and light chains of the immunoglobulin and the light chain constant region 1 (CL1). It may further include the hinge region of the heavy chain constant region. A hybrid Fc or a hybrid Fc fragment is also referred to as "hFc" or "hyFc" in the present invention.

[0045] The Fc fragment of the present invention may have a native glycan, an increased glycan compared to the native glycan, a decreased glycan compared to the native glycan, or a form in which the glycan has been removed. The immunoglobulin Fc glycan can be modified by conventional methods such as chemical methods, enzymatic methods, and genetic engineering methods using microorganisms. Removal of the glycan from the Fc fragment sharply reduces the binding affinity of the primary complement component C1 to C1q, resulting in a reduction or absence of ADCC or CDC, and therefore does not induce unnecessary immune responses in vivo. From this perspective, immunoglobulin Fc fragments in a deglycosylated or aglycosylated form are more suitable for the purpose of the present invention as a drug carrier. The term "deglycosylation" as used herein means that sugar is enzymatically removed from the Fc fragment. Additionally, the term "aglycosylation" means that the Fc fragment is produced in an unglycosylated form by a prokaryote, preferably E. coli.

[0046] In one embodiment of the present invention, the modified immunoglobulin Fc region may consist of the amino acid sequence of SEQ ID NO: 11 (hereinafter referred to as "hyFc").

[0047] In one embodiment of the present invention, the fusion protein may be represented by the following structural formula I:

[0048] N'-XLY-C' (Structural formula I)

[0049] In the above formula, N' is the N-terminus of the fusion protein and C' is the C-terminus of the fusion protein; X is a PD-L1 protein, an extracellular domain of a PD-L1 protein, or a fragment thereof; L is a linker; Y is an immunoglobulin Fc region.

[0050] Preferably, the fusion protein may consist of the amino acid sequence of SEQ ID NO: 12 or SEQ ID NO: 13. The fusion protein of the present invention may have about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more homology to the amino acid sequence of SEQ ID NO: 12.

[0051] The present invention also provides a nucleic acid molecule encoding the fusion protein.

[0052] 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. The nucleic acid molecule may further include a signal sequence (or signal peptide) or a leader sequence.

[0053] The term "signal sequence (or signal peptide)" as used herein refers to a short peptide present at the N-terminus of a newly synthesized protein sorted into the 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 the MOPC141 antibody heavy chain signal sequence (Sakano et al., Nature 1980 286:676-683), and other signal sequences known in the art (see, for example, Watson et al., Nucleic Acid Research 1984 12:5145-5164).

[0054] The characteristics of the signal peptide are well known in the art and are generally known to contain 16-30 amino acid residues, although they may contain more or fewer amino acid residues. A typical signal peptide is composed of three regions: a basic N-terminal region, a central hydrophobic region, and a more polar C-terminal region.

[0055] The central hydrophobic region contains 4-12 hydrophobic residues that anchor the signal sequence through the membrane lipid bilayer during translocation of the immature polypeptide. After initiation, the signal sequence is cleaved in the lumen of the ER by cellular enzymes generally known as signal peptidases. Here, the signal sequence may be a secretory signal sequence of tPa (tissue plasminogen activation), HSV gDs, or growth hormone. Preferably, a secretory signal sequence used in higher eukaryotic cells including mammals can be used, more preferably a tPa sequence or the 1st to 18th amino acid sequence of SEQ ID NO: 1 can be used. The signal sequence of the present invention can also be used by substituting a codon that is frequently expressed in a host cell.

[0056] The present invention also provides an expression vector comprising the nucleic acid molecule.

[0057] The term "vector" in the present invention is understood as a nucleic acid means comprising a nucleotide sequence that can be introduced into a host cell to recombine and insert into the host cell genome or can replicate autonomously as an episome. Said vectors include linear nucleic acids, plasmids, phagemids, cosmids, RNA vectors, viral vectors and their analogs. Examples of viral vectors include, but are not limited to, retroviruses, adenoviruses and adeno-associated viruses.

[0058] In the present invention, a useful expression vector may be RcCMV (Invitrogen, Carlsbad) or a variant thereof. A useful expression vector may contain a human CMV (cytomegalovirus) promoter to drive continuous transcription of a gene of interest in mammalian cells, and a bovine growth hormone polyadenylation signal sequence to increase steady-state levels of post-transcriptional RNA. In one embodiment of the present invention, the expression vector is pAD15, a modified vector of RcCMV.

[0059] As used herein, the term "host cell" refers to prokaryotic and eukaryotic cells into which a recombinant expression vector can be introduced.

[0060] In the present invention, suitable host cells can be transformed or transfected with the DNA sequence of the present invention and used for expression and / or secretion of the protein of interest. Currently preferred host cells that can be used in the present invention include immortal hybridoma cells, NS / 0 myeloma cells, 293 cells, Chinese hamster ovary cells (CHO cells), HeLa cells, CapT cells (human amniotic fluid derived cells) and COS cells.

[0061] As used herein, the terms "transformation" and "transfection" refer to the introduction of a nucleic acid (eg, a vector) into a cell by various techniques known in the art.

[0062] The present invention also provides a composition for preventing or treating an immune disease, which comprises as an active ingredient a fusion protein comprising a PD-L1 protein and a modified immunoglobulin Fc region.

[0063] The immune disease may be a disease selected from the group consisting of an autoimmune disease, an inflammatory disease, and a cell, tissue, or organ transplantation rejection disease.

[0064] The autoimmune diseases include 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, arthritis chronica progrediente, deformans arthritis, primary chronic polyarthritis, polyarthritis chronica primaria, reactive arthritis, and ankylosing spondylitis), inflammatory hyperproliferative skin diseases, plaque psoriasis, guttate psoriasis, pustular psoriasis, and psoriasis of the genital tract, such as psoriasis of the rectum, ... psoriasis such as psoriasis of the nails, dermatitis including contact dermatitis, chronic contact dermatitis, allergic dermatitis, allergic contact dermatitis, dermatitis herpetiformis, and atopic dermatitis, urticaria such as chronic idiopathic urticaria, including X-linked hyper IgM syndrome, chronic allergic urticaria, and chronic autoimmune urticaria, polymyositis / dermatomyositis, juvenile dermatomyositis, toxic epidermal necrolysis, scleroderma (including systemic sclerosis), systemic sclerosis, spino-optical MS, Multiple sclerosis (MS), such as primary progressive MS (PPMS), and relapsing remitting MS (RRMS),MS, sclerosis including progressive systemic sclerosis, atherosclerosis, arteriosclerosis, sclerosis disseminata, and ataxic sclerosis, 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, neonatal necrotizing enterocolitis, and transmural colitis), and autoimmune 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, neonatal necrotizing enterocolitis, and transmural colitis). disease), pyoderma gangrenosum, erythema nodosum, and primary sclerosing cholangitis, episcleritis in adults or acute respiratory distress syndrome;Respiratory distress syndrome including ARDS, meningitis, inflammation of the uvea or of part of it, iritis, choroiditis, autoimmune hematological disorders, rheumatoid spondylitis, sudden deafness, IgE-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 uveitis, nongranulomatous uveitis, uveitis such as uveitis, phacoantigenic uveitis, posterior uveitis, or autoimmune uveitis; nephrotic syndrome such as chronic or acute glomerulonephritis such as primary GN; glomerulonephritis (GN) with or without concomitant disease, immune-mediated GN, membranous GN, membranous nephropathy, idiopathic membranous nephropathy, or idiopathic membranous GN; membranous- or membranous proliferative GN, including types I and II;MPGN, and rapidly progressive GN; allergic diseases, allergic reactions, eczema including allergic or atopic eczema; asthma such as asthma bronchiale, bronchial asthma and autoimmune asthma; diseases associated with T cell infiltration and chronic inflammatory reactions; chronic inflammatory pulmonary 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 disseminatus, lupus (including nephritis, cerebritis, pediatric, non-renal, extra-renal, discoid, and alopecia), pediatric insulin-dependent diabetes mellitus;Juvenile onset (Type I) diabetes mellitus, including IDDM, adult onset diabetes mellitus (Type II), autoimmune diabetes, idiopathic diabetes insipidus, immune responses associated with acute and delayed hypersensitivity mediated by T lymphocytes and cytokines, tuberculosis, sarcoidosis, granulomatosis, including lymphomatoid granulomatosis, Wegener's granulomatosis, agranulocytosis, vasculitis, including giant cell vasculitis (polymyalgia rheumatica and giant cell (Takayasu's) arteritis), Kawasaki disease, polyarteritis nodosa vasculitides including medium-sized vasculitis including vascular endothelial ulcers (VA) (including vascular endothelial ulcers), microscopic polyarteritis, CNS arthritis, necrotizing, cutaneous, or hypersensitivity vasculitis, systemic necrotizing vasculitis, vasculitis including Churg-Strauss vasculitis or ANCA-associated vasculitis such as CSS (Antibody-associated vasculitis), temporal arteritis, aplastic anemia, autoimmune aplastic anemia, Coombs anemia, Diamond Blackfan anemia, hemolytic anemia or immune hemolytic anemia including autoimmune hemolytic anemia (AIHA), pernicious anemia (anemia perniciosa), Addison's disease pure red cell anemia aplasia;PRCA, factor VHI deficiency, hemophilia A, autoimmune neutropenia, pancytopenia, leukopenia, diseases associated with leukocyte diapedesis, CNS inflammatory diseases, septicemia, multiple organ damage secondary to trauma, hemorrhage, antigen-antibody complex-mediated diseases, antiglomerular basement membrane disease, antiphospholipid syndrome, allergic neuritis, Bechet's or Behcet's disease, Castleman's syndrome, Goodpasture's syndrome, Raynaud's syndrome, Sjogren's syndrome, Stevens-Johnson syndrome, pemphigoid, such as pemphigoid bullous and skin pemphigoid, pemphigus (including pemphigus vulgaris, pemphigus foliaceus, pemphigus mucous-membrane pemphigoid, and pemphigus erythematosus); autoimmune polyendocrinopathies, Reiter's disease or syndrome, chronic neuropathy such as immune complex nephritis, antibody-mediated nephritis, neuromyelitis optica, polyneuropathies, IgM polyneuropathy or IgM-mediated neuropathy, thrombocytopenia (e.g. as occurs in patients with myocardial infarction), including autoimmune or immune-mediated thrombocytopenia such as thrombotic thrombocytopenic purpura (TTP) and idiopathic thrombocytopenia (ITP), including chronic or acute ITP, autoimmune diseases of the testis and ovary, including autoimmune orchitis and oophoritis, primary hypothyroidism, hypothyroidism, hypothyroidism including thyroiditis such as autoimmune thyroiditis, autoimmune endocrine disorders, Hashimoto's disease, chronic thyroiditis; or subacute thyroiditis, autoimmune thyroid disorders, idiopathic hypothyroidism, polyglandular syndromes such as Graves' disease, autoimmune polyglandular syndromes (polyglandular endocrinopathy syndromes), paraneoplastic syndromes including paraneoplastic neurological syndromes such as Lambert-Eaton myasthenic syndrome or Eaton-Lambert syndrome, stiff-man or stiff-person syndrome, allergic encephalomyelitis (or encephalomyelitis allergica) and experimental allergic encephalomyelitis (AE).Encephalomyelitis such as encephalomyelitis (EAE), myasthenia gravis such as thymoma-associated myasthenia gravis, cerebellar degeneration, neuromyotonia, opsoclonus or opsoclonus myoclonus syndrome (OMS), and sensory neuropathy, multifocal motor neuropathy, Sheehan's syndrome, autoimmune hepatitis, chronic hepatitis, lupoid hepatitis, giant cell hepatitis, chronic active hepatitis or autoimmune chronic active hepatitis, lymphoid interstitial pneumonia, pneumonitis, bronchiolitis obliteran (non-transplant) vs. NSIP, Guillain-Barré syndrome, Berger's disease (IgA nephropathy), idiopathic IgA nephropathy, linear IgA dermatosis, primary biliary cirrhosis, pneumonocirrhosis, autoimmune enteropathy syndrome, celiac disease, coeliac disease, celiac sprue, gluten enteropathy, refractory sprue, idiopathic sprue, cryoglobulinemia, amyotrophic lateral sclerosis (ALS; Lou Gehrig's coronary artery diseaseAutoimmune ear disease, autoimmune inner ear disease (AGED), autoimmune hearing loss, opsoclonus myoclonus syndrome (OMS), polychondritis (refractory or relapsing polychondritis), pulmonary alveolar proteinosis, amyloidosis, scleritis, non-cancerous lymphocytosis, primary lymphocytosis including monoclonal B-cell lymphocytosis (e.g., benign monoclonal gammopathy and monoclonal gammopathy of undetermined significance (MGUS)), peripheral neuropathy, neuropathy, paraneoplastic syndromes, epilepsy, migraine, arrhythmias, muscle diseases, deafness, blindness, periodic paralysis, and channelopathies such as CNS channelopathies, autism, inflammatory myopathy, focal segmental glomerulosclerosis (FSGS), endocrine ophthalmopathy, uveoretinitis, chorioretinitis, autoimmune hepatological disorder, fibromyalgia, multiple endocrine failure, Schmidt's syndrome,Demyelinating diseases such as pulmonary circulation disorders, pulmonary edema, pulmonary edema, pulmonary malformation ... multiforme, post-cardiotomy syndrome, Cushing's syndrome, bird-fancier's lung, allergic granulomatous angiitis, benign lymphocytic angiitis, Alport's syndrome, alveolitis such as allergic alveolitis and fibrosing alveolitis, interstitial lung disease, transfusion reactions, leprosy, malaria, leishmaniasis, trypanosomiasis, schistosomiasis, ascariasis, aspergillosis, Samter's syndrome, Caplan's syndromesyndrome, dengue, endocarditis, endomyocardial fibrosis, diffuse interstitial pulmonary fibrosis, interstitial lung fibrosis, idiopathic pulmonary fibrosis, cystic fibrosis, endophthalmitis, erythema elevatum et diutinum, erythroblastosis fetalis, eosinophilic fasciitis, Shulman's syndrome, Felty's syndrome syndrome, filariasis, chronic cyclitis, metachronous cyclitis, iridocyclitis or Fuch's cyclitis, Henoch-Schonlein purpura, human immunodeficiency virus (HIV) infection, echovirus 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 dysfunction, failure), Sydenham's chorea, poststreptococcal nephritis, and thromboangiitis obliterans.obliterans), thyrotoxicosis, tabes dorsalis, chorioiditis, giant cell polymyalgia, endocrine ophthalmopathy, chronic hypersensitivity pneumonitis, keratoconjunctivitis sicca, epidemic keratoconjunctivitis, idiopathic nephritic syndrome, minimal change nephrosis nephropathy), benign familial and ischemia-reperfusion injury, retinal autoimmunity, joint inflammation, bronchitis, chronic obstructive airway disease, silicosis, aphthae, aphthous stomatitis 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, rheumatic fever, Hamman-Rich's disease, acute sensorineural hearing loss, haemoglobinuria paroxysmatica, hypogonadism, regional ileitisregionalis, leucopenia, mononucleosis infectiosa, traverse myelitis, primary idiopathic myxedema, nephrosis, ophthalmia symphatica, orchitis granulomatosa), pancreatitis, polyradiculitis acuta, pyoderma gangrenosum, Quervain's thyreoiditis, acquired spenic atrophy, 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 Leishmania, toxic-shock syndrome, food poisoning, diseases associated with T cell infiltration, leukocyte adhesion deficiency, immune responses mediated by cytokines and T cells in acute and delayed hypersensitivity, diseases associated with leukocyte leakage, multiple organ injury syndrome, antigen-antibody complex-mediated diseases, antiglomerular basement membrane disease, allergic neuritis, autoimmune polyendocrinopathies, oophoritis, primary myxedema, autoimmune atrophic gastritis,gastritis), sympathetic ophthalmia, rheumatic diseases, mixed connective tissue diseases, nephrotic syndrome, insulitis, polyendocrine failure, peripheral neuropathy, autoimmune polyglandular syndrome type I I), adult-onset idiopathic hypoparathyroidism (AOIH), alopecia totalis, dilated cardiomyopathy, epidermolisis bullosa acquisita (EBA), hemochromatosis, myocarditis, nephrotic syndrome, primary sclerosing cholangitis sclerosing cholangitis, suppurative or non-suppurative sinusitis, acute or chronic sinusitis, ethmoid, frontal, maxillary, or sphenoid sinusitis, eosinophil disorders such as eosinophilia, pulmonary infiltration eosinophilia, eosinophilia-myalgia syndrome, Loffler's syndrome, chronic eosinophilic pneumonia, tropical pulmonary eosinophilia, bronchopulmonary aspergillosis, aspergillosis, aspergilloma, or eosinophilic granulomas, anaphylaxis, seronegative spondyloarthropathyspondyloarthritides, polyendocrine autoimmune disease, sclerosing cholangitis, sclera, episclerosis, chronic mucocutaneous candidiasis, Bruton's syndrome, transient hypogammaglobulinemia of infancy, Wiskott-Aldrich syndrome, ataxia telangiectasia, autoimmune disorders associated with collagen diseases, rheumatism, neurological diseases, ischemia-reperfusion disease, hypotensive response, vascular insufficiency, angiectasis, tissue injury, cardiovascular ischemia, hyperalgesia, cerebral ischemia ischemia, and diseases involving vascularization, allergic hypersensitivity diseases, glomerulonephritides, reperfusion injury, myocardial or other tissue reperfusion injury, dermatoses with an acute inflammatory component, acute purulent meningitis or other central nervous system inflammatory diseases, ocular and orbital inflammatory diseases, granulocyte transfusion-associated syndromes, cytokine-induced toxicity, acute serious inflammation, chronic intractable inflammation, pyelitis, pneumonocirrhosis, diabetic retinopathy, diabetic large-artery disorder, endarterial hyperplasia,The disease may be selected from the group consisting of gastrointestinal hyperplasia, peptic ulcer, valvulitis and endometriosis.

[0065] Preferably, the autoimmune disease is selected from the group consisting of 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, and the like. The anemia may be selected from the group consisting of idiopathic thrombocytopenic purpura, primary biliary cirrhosis, scleroderma, vitiligo, pernicious anemia and celiac disease.

[0066] The inflammatory diseases include 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, and the like. The present invention may be directed to a disease selected from the group consisting of arteriosclerosis, ischemic heart disease, myocardial infarction, stroke, peripheral vascular disease, uveitis, corneal disease, iritis, iridocyclitis, and cataracts.

[0067] The transplant rejection disease may be a tissue or organ transplant rejection, and the tissue or organ transplant rejection may be selected from bone marrow transplant, heart transplant, cornea transplant, intestine transplant, liver transplant, lung transplant, pancreas transplant, kidney transplant and skin transplant rejection.

[0068] The term "inflammatory skin disease" of the present invention means a disease that occurs in the skin due to an inflammatory reaction. Skin keratinocytes are a component that constitutes the majority of epidermal cells, form keratin, and produce various cytokines to participate in various inflammatory and immune reactions. When skin keratinocytes are exposed to environmental and physiological stress, an inflammatory reaction occurs, and therefore various types of inflammatory cytokines such as TNF-α (tumor necrosis factor-α), IL-1β (interleukin-1β), IL-6, and CCL (chemokine (CC motif) ligand) are secreted. These cytokines reduce the proliferation rate of skin keratinocytes and impede the organization of the dermis layer, thereby slowing down 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, and preferably may be psoriasis.

[0069] The term "psoriasis" as used herein refers to a disease that forms erythematous papules and plaques of various sizes, with clear borders, covered with silvery white scales on the skin. It is a chronic inflammatory skin disease that is histologically characterized by epithelial hyperproliferation, shows various clinical manifestations, and repeats exacerbations and improvements.

[0070] The preferred dosage of the pharmaceutical composition varies depending on the condition and weight of the patient, the degree of the disease, the drug form, the route of administration and the period of administration, and can be appropriately selected by those skilled in the art. In the pharmaceutical composition for treating or preventing psoriasis of the present invention, the active ingredient can be contained in any amount (effective amount) depending on the application, dosage form, purpose of formulation, etc., as long as it can show psoriasis treatment activity, and the effective amount is usually determined within the range of 0.001% by weight to 20.0% by weight based on the total weight of the composition. Here, the "effective amount" refers to the amount of the active ingredient that can induce a psoriasis treatment effect. Such an effective amount can be experimentally determined within the normal ability of those skilled in the art.

[0071] The term "treatment" as used herein includes both therapeutic and prophylactic treatments. Here, prophylaxis can be used to alleviate or reduce a pathological condition or disease in an individual. In one embodiment, the term "treatment" includes any form of medication or application for treating a disease in a mammal, including a human. The term also includes inhibiting or slowing the progression of a disease or disease; restoring or repairing a damaged or defective function to partially or completely alleviate a disease; or stimulating an inefficient process; and alleviating serious diseases.

[0072] Here, the term "therapeutically effective amount" or "pharmaceutical effective amount" refers to an amount of a compound or composition effective to prevent or treat a target disease, and refers to an amount sufficient to treat the disease with a reasonable benefit / risk ratio applicable to medical treatment and without causing side effects. The level of the effective amount can be determined by factors including the patient's health condition, type and severity of the disease, drug activity, sensitivity to the drug, administration method, administration time, administration route and excretion rate, treatment period, co-administered or co-used drugs, and other factors well known in the medical field. In one embodiment, the therapeutically effective amount refers to an amount of a drug effective to treat psoriasis.

[0073] The composition of the present invention may contain a pharma- ceutically acceptable carrier, and may further contain a pharma- ceutically acceptable adjuvant, excipient, or diluent in addition to the carrier.

[0074] The term "pharmaceutical acceptable" in the present invention refers to a composition that is physiologically acceptable and does not usually cause gastrointestinal disorders, allergic reactions such as dizziness, or similar reactions when administered to humans. Examples of the carrier, excipient, and diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition, fillers, anti-agglomerating agents, lubricants, wetting agents, flavorings, emulsifiers, and preservatives may be further included.

[0075] The pharmaceutical composition of the present invention can be formulated by a method known in the art to enable immediate release, sustained or delayed release of the active ingredient when administered to a mammal, and includes powder, granule, tablet, emulsion, syrup, aerosol, soft or hard gelatin capsule, sterile injection solution, and sterile powder form.

[0076] The composition of the present invention can be formulated in a suitable form together with a pharma- ceutically acceptable carrier. The pharma- ceutically acceptable carrier may be, for example, a parenteral carrier such as water, a suitable oil, saline, aqueous glucose, and glycol, and may further include a stabilizer and a preservative. Suitable stabilizers include antioxidants such as sodium bisulfite, sodium sulfite, or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben, and chlorobutanol. The composition of the present invention may also include, as necessary, a suspending agent, a solubilizing agent, a stabilizer, an isotonic agent, a preservative, an adsorption inhibitor, a surfactant, a diluent, an excipient, a pH adjuster, a soothing agent, a buffer, an antioxidant, and the like, depending on the administration method and dosage form. The pharma- ceutically acceptable carriers and formulations suitable for the present invention, including those exemplified above, are described in detail in the literature [Remington's Pharmaceutical Sciences, latest edition].

[0077] The compositions of the present invention can be sterilized by conventional sterilization techniques. The compositions may contain pharma- ceutically acceptable auxiliary substances and adjuvants required to regulate physiological conditions, such as pH regulation, toxicity control agents and the like, such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, and the like. The concentration of the fusion protein in such formulations can vary widely, for example, up to about 0.5% by weight, and generally can range from about 1% to 15% or 20%, and can be selected based on the body fluid volume, viscosities, and the like, depending on the particular method of administration selected.

[0078] The preferred dosage for the composition of the present invention may be in the range of 0.01 μg / kg to 10 g / kg or 0.01 mg / kg to 1 g / kg per day depending on the patient's condition, body weight, sex, age, severity of the patient, and route of administration. Administration may be once or several times a day. Such dosage should not be construed as limiting the scope of the present invention in any aspect.

[0079] The subject to which the composition of the present invention can be applied (formulated) is a mammal or a human, and preferably a human. The administration route, dosage and number of administrations of the fusion protein or fusion protein dimer can be administered to the subject in various ways and amounts depending on the condition of the patient and the presence or absence of side effects, and the optimal administration method, dosage and number of administrations can be selected within an appropriate range by a person skilled in the art.

[0080] The composition of the present invention can be administered by any route. The composition of the present invention can be provided to animals by any suitable means, directly (e.g., locally, by injection, implantation or local administration to tissue site) or systemically (e.g., parenterally or orally). When the composition of the present invention is provided parenterally, such as intravenously, subcutaneously, ophthalmic, intraperitoneally, intramuscularly, buccal, rectal, intraorbital, intracerebral, intracranial, intraspinal, intraventricular, intrathecal, intracisternal, intracapsular, intranasal or aerosol administration, the composition is preferably aqueous or contains a portion of a physiologically compatible body fluid suspension or solution. This allows the carrier or vehicle to be added to the composition and delivered to the patient so that it does not adversely affect the patient's electrolyte and / or volume balance, and thus may typically include physiologic saline as the body fluid medium for the formulation.

[0081] A DNA construct (or gene construct) containing a nucleic acid encoding a fusion protein of the present invention can be used as part of a gene therapy protocol to deliver the nucleic acid encoding said fusion protein construct.

[0082] The present invention allows the administration of an expression vector that infects and expresses the fusion protein in a particular cell type in vivo to reconstitute or supplement the function of the desired fusion protein in combination with any biologically effective carrier, such as any dosage form or composition that can efficiently deliver a gene encoding the desired fusion protein or the fusion protein to an in vivo cell.

[0083] For gene therapy using a nucleic acid encoding the fusion protein, the gene of interest can be inserted into a viral vector, including recombinant retrovirus, adenovirus, adeno-associated virus, and herpes simplex virus-1, or a recombinant bacterial or eukaryotic plasmid.

[0084] The dosage of the nucleic acid encoding the fusion protein of the present invention is in the range of 0.1 mg to 100 mg in humans, preferably 1 mg to 10 mg, and more preferably 2 mg to 10 mg. The optimal amount and dosage form can be determined by routine experiments within the level of skill in the art.

[0085] The unit dosage of the fusion protein of the present invention is 0.1 mg / kg to 1,500 mg / kg in humans, preferably 1 mg / kg to 100 mg / kg, more preferably 5 mg / kg to 20 mg / kg. The unit dosage may vary depending on the disease to be treated and the presence or absence of side effects. However, the optimal dosage can be determined using routine experimentation. The administration of the fusion protein may be by periodic bolus injections, or by continuous intravenous, subcutaneous, or intraperitoneal administration from an external reservoir (e.g., an intravenous bag) or internal (e.g., a bioerodable implant).

[0086] The compositions of the present invention can be administered in combination with other drugs or physiologically active substances that have 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.

[0087] A method for preventing or treating a disease using the fusion protein or composition of the present invention may also include administering, in combination with the fusion protein or composition of the present invention, other drugs or physiologically active substances having a preventive or therapeutic effect against the disease, but the route, timing, and dosage of the combined administration can be determined depending on the type of disease, the disease state of the patient, the purpose of treatment or prevention, and the other drugs or physiologically active substances to be used in combination.

[0088] The present invention also provides use of a fusion protein comprising a PD-L1 protein and a modified immunoglobulin Fc region for producing a pharmaceutical preparation having an effect of preventing or treating an immune disease.

[0089] The present invention also provides a method for preventing or treating an immune disease, comprising the step of administering to an individual a fusion protein comprising a PD-L1 protein and a modified immunoglobulin Fc region, and a pharma- ceutical acceptable carrier.

[0090] The therapeutically effective amount is preferably applied differently depending on various factors including the type and degree of the reaction to be achieved, the specific composition including whether or not other preparations are used, the age, weight, general health condition, sex and diet of the individual, administration time, administration route and secretion rate of the composition, treatment period, drugs used together with the specific composition or used simultaneously, and similar factors well known in the pharmaceutical field. Therefore, the effective amount of the composition suitable for the purpose of the present invention is preferably determined in consideration of the above-mentioned matters.

[0091] The individual may be any mammal, including humans and primates, as well as domestic animals such as cows, pigs, sheep, horses, dogs and cats. EXAMPLES

[0092] The present invention will be described in more detail below with reference to examples. These examples are intended to more specifically explain the present invention, and the scope of the present invention is not limited to these examples.

[0093] Example 1. Preparation of a gene construct for producing a fusion protein containing a PD-L1 protein and a modified immunoglobulin Fc region A gene construct was prepared to produce a fusion protein in which human PD-L1 (programmed cell death-ligand 1) protein and a modified immunoglobulin Fc domain were fused. Specifically, the human PD-L1 gene had a known amino acid sequence (Accession number: Q9NZQ7), and a gene construct containing the extracellular domain (19-239 aa) of the PD-L1 protein was prepared by TOP Gene Technologies (Quebec, Canada). The human PD-L1 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 human IgD Fc and human IgG4 Fc, and is characterized by including an IgG1 hinge region (SEQ ID NO: 16) consisting of eight amino acids.

[0094] The fusion proteins of the present invention were prepared in which the PD-L1 protein was linked to a modified immunoglobulin Fc domain via a peptide linker. In the present invention, a fusion protein (hereinafter "PD-L1-hyFc21" or "PD-L1-hyFc21 fusion protein", SEQ ID NO: 12 or 13) was prepared that contains human PD-L1 protein and a modified immunoglobulin Fc domain linked by a GS linker (SEQ ID NO: 10) consisting of a 7 amino acid sequence in order to maintain flexibility while inducing dimerization. As a control, a fusion protein (hereinafter "PD-L1-hyFc5" or "PD-L1-hyFc5 fusion protein", SEQ ID NO: 15) containing an immunoglobulin Fc domain containing the IgD hinge region of SEQ ID NO: 14 was used.

[0095] A recombinant expression vector was constructed using a gene construct containing nucleotides encoding the fusion protein. The constructed recombinant expression vector was transformed into CHO DG44 cell line using a gene transfer method (NeonTM kit, 10μL, Invitrogen Cat.MPK1096) in which DNA is introduced into the cells by suspending the DNA solution and passing a high-voltage DC pulse. Then, HT selection (HT supplement, Invitrogen, 11067-030) and MTX (Methotrexate, Sigma, M8407) amplification steps were performed, and the cells were passaged to select only cells showing high expression rates. The cells were cultured at 0.4×10 every 3 days. 6 The cells were subcultured at 1000 cells / mL, and the cell count and viability were measured using a cell counting device (Vi-cell, Beckman Coulter). HT selection is a selection method in which HT is removed from the media to allow only transformed cells to survive, and MTX amplification is a method in which MTX is added to the subculture medium at a selected concentration to amplify genes. The selected cell pool was subjected to single cell cloning using the limiting dilution cloning method. Briefly, cells were dispensed into a 96-well plate at 1 cell / well, and cell images were saved using a CSI device (clone selection imager, Molecular Devices) on days 0, 7, and 14 to trace back clones derived from a single cell. The productivity of the selected single cell-derived cell lines was confirmed using Fc ELISA (Human IgG ELISA Quantitation Set, Bethyl, E80-104). The final 5-6 selected clones were subjected to batch culture and long-term stability evaluation, and clones that were confirmed to be stable were produced at the Research Cell Bank (RCB).

[0096] Example 2. Obtaining PD-L1-hyFc21 or PD-L1-hyFc5 fusion proteins To mass-produce PD-L1-hyFc21 or PD-L1-hyFc5 fusion proteins, the target proteins were isolated and purified from the cell culture medium produced by the PD-L1-hyFc5 or PD-L1-hyFc21 suspension cell lines obtained in Example 1 above.

[0097] To obtain large quantities of PD-L1-hyFc21 or PD-L1-hyFc5 fusion proteins, the cell lines expressing PD-L1-hyFc21 or PD-L1-hyFc5 fusion proteins were cultured in a 15L Glass bioreactor for 20 days using the same fed-batch culture method to produce PD-L1-hyFc21 or PD-L1-hyFc5 fusion proteins. During fed-batch culture, the cell viability of the cell lines expressing the fusion proteins was measured, and size exclusion chromatography (SE-HPLC) was performed to confirm the final expression level and purity of the target proteins.

[0098] The maximum cell concentration over time of the expressing cell line and the final expression level of the fusion protein were confirmed. The maximum cell concentration over time of the PD-L1-hyFc21 fusion protein expressing cell line was 13.9×10 6 The expression level of the fusion protein was confirmed to be 5.6 g / L (Fig. 2b). In contrast, the maximum cell concentration over time for the PD-L1-hyFc5 fusion protein expressing cell line was 16.7 × 10 6 The expression level of the finally recovered fusion protein was confirmed to be 2.2 g / L (FIG. 3b).

[0099] Additionally, the purity of the fusion proteins was determined, and the purity of the PD-L1-hyFc21 fusion protein was 79.5%, with 7.7% high molecular weight impurities (HMW) and 9.8% low molecular weight impurities (LMW) (Table 1). In contrast, the purity of the PD-L1-hyFc5 fusion protein was 47.8%, with 31.7% high molecular weight impurities (HMW) and 20.5% low molecular weight impurities (LMW) (Table 1).

[0100] [Table 1] *HMW: High Molecular Weight Impurity; Main: Target protein; and LMW: Low Molecular Weight Impurity.

[0101] Then, to ensure high purity PD-L1-hyFc21 or PD-L1-hyFc5 fusion protein, a three-step purification process was performed: culture medium → Protein A affinity chromatography → anion exchange chromatography 1 → anion exchange chromatography 2 → ultrafiltration / diafiltration.

[0102] The purity and yield of the obtained target protein were confirmed by size exclusion chromatography (SE-HPLC). The purity of PD-L1-hyFc21 fusion protein was confirmed to be 97.3%, and the yield was 30.2%. In contrast, the purity of PD-L1-hyFc5 fusion protein was confirmed to be 93.2%, and the yield was 5.1% (Table 2).

[0103] [Table 2]

[0104] The results showed that PD-L1-hyFc21 fusion protein was produced in approximately 2.5-fold higher yield than PD-L1-hyFc5 fusion protein, and the protein purity was also higher.

[0105] Example 3. Characterization of PD-L1-hyFc21 or PD-L1-hyFc5 fusion proteins 3.1. Polyacrylamide gel electrophoresis (sodium dodecyl sulfate-polyacrylamide gel electrophoresis, SDS-PAGE) To confirm the molecular weight of the purified PD-L1-hyFc21 or PD-L1-hyFc5 fusion proteins, polyacrylamide gel electrophoresis (sodium dodecyl sulfate polyacrylamide gel electrophoresis, SDS-PAGE) was performed. Briefly, the fusion proteins were diluted with deionized water and subjected to NuPAGE. TM The mixture was mixed with LDS Sample Buffer (Thermo Fisher Scientific) and loaded onto a 4-12% Bis-Tris gel (Invitrogen) at 3 μg / well for electrophoresis. After electrophoresis, the gel was stained with Coomassie staining.

[0106] As a result, the purified PD-L1-hyFc21 and PD-L1-hyFc5 fusion proteins were both confirmed to be around the size marker of 98 kDa under non-reducing conditions (Figure 4), however, the purified PD-L1-hyFc5 fusion protein contained cleaved forms of low molecular weight impurities.

[0107] 3.2. Size-exclusion chromatography (SE-HPLC) Size-exclusion chromatography (SE-HPLC) was performed to analyze the main peak and impurity peaks such as dimers, multimers, or abnormal peptides in the form of truncated forms of the purified PD-L1-hyFc21 or PD-L1-hyFc5 fusion proteins. Briefly, the fusion proteins were diluted with formulation buffer to a concentration of 1.0 mg / mL, and then a gel filtration chromatography column (TOSOH TSK-GEL G3000SWxL column, 7.8 mm x 300 mm) was used to separate the peaks, and the area ratio (%Area) of the main peak of the fusion protein was analyzed.

[0108] As a result, the purity of the PD-L1-hyFc21 fusion protein was confirmed to be 97.3% (Fig. 5a), while the purity of the PD-L1-hyFc5 fusion protein was confirmed to be 93.2% (Fig. 5b).

[0109] 3.3. Isoelectric focusing (IEF) Isoelectric focusing (IEF) was performed to confirm the charge variants and distribution of purified PD-L1-hyFc5 and PD-L1-hyFc21 proteins. Isoelectric focusing is an electrophoretic method that analyzes separated proteins using their pI value, where pI refers to the pH value at which a charged protein becomes electrically neutral, which is called the isoelectric point. In isoelectric focusing, proteins that reach the isoelectric point do not move any further and remain on the gel for separation. Briefly, in the present invention, fusion proteins were separated according to pI value using a pH 3-10 isoelectric focusing gel (Invitrogen), and the gel after electrophoresis was fixed with a 12% TCA (trichloroacetic acid) solution and stained with Coomassie staining.

[0110] As a result, it was confirmed that the pI values ​​of both the PD-L1-hyFc21 and PD-L1-hyFc5 fusion proteins were within the range of 5.2 to 6.0 (Figure 6).

[0111] 3.4. Differential Scanning Fluorimetry (DSF) To analyze the thermal stability of purified PD-L1-hyFc21 or PD-L1-hyFc5 fusion proteins, experiments were performed using the PROTEOSTAT® Thermal Transfer Stability Assay Kit. The assay kit contains a fluorescent dye that detects protein aggregation, and can determine the temperature at which a large amount of protein aggregates under heat stress conditions. The aggregation temperature (Tagg) is an indicator of protein stability and can determine the stability of the protein structure.

[0112] As a result, the aggregation temperature of the PD-L1-hyFc21 fusion protein was determined to be 55.8°C, while the aggregation temperature of the PD-L1-hyFc5 fusion protein was determined to be 50.7°C (Figure 7). This confirmed that the PD-L1-hyFc21 fusion protein has a more superior thermal stability structure than PD-L1-hyFc5.

[0113] Example 4. In vitro activity analysis of PD-L1-hyFc21 or PD-L1-hyFc5 fusion proteins 4.1. PD-1 binding analysis using PD-1 expressing Jurkat (SHP-1) cell line The binding ability of PD-L1-hyFc21 or PD-L1-hyFc5 fusion protein to PD-1 was analyzed using a PD-1 expressing Jurkat (SHP-1) cell line. Briefly, Jurkat cells (PathHunter® Jurkat PD-1 (SHP-1), DiscoverX) were dispensed into a 96-well plate and stabilized in a 37°C 5% CO2 incubator for 2 hours. Then, PD-L1-hyFc21 or PD-L1-hyFc5 fusion protein was added to the plate at 75 and 600 nM, respectively, and incubated in a 37°C 5% CO2 incubator for 1 hour. After the reaction, a reagent (PathHunter® Bioassay Detection Kit, DiscoverX) that detects SHP-1 expressed in response to PD-1:PD-L1 signaling was added to measure the luminescence of SHP-1.

[0114] The results showed that the PD-L1-hyFc21 fusion protein group had higher SHP-1 emission levels at both 75 and 600 nM concentrations compared to the PD-L1-hyFc5 fusion protein group (Figure 8). This confirmed that the PD-L1-hyFc21 fusion protein has a higher binding ability to PD-1 compared to the PD-L1-hyFc5 fusion protein, and in particular, the binding ability to PD-1 increased as the concentration increased.

[0115] 4.2. Analysis of the inhibitory ability of mixed lymphocyte reaction We analyzed the ability of PD-L1-hyFc21 or PD-L1-hyFc5 fusion proteins to suppress mixed lymphocyte reaction. Briefly, peripheral blood mononuclear cells (PBMCs) from donors and recipients were prepared and analyzed by CellTrace CT (CTV) to detect intracellular proteins. TM Violet) and CTR (CellTrace TMThe donor and recipient PBMCs were mixed in a 1:1 ratio as responder and stimulator cells and placed in a 96-well U-bottom plate. PD-L1-hyFc21 or PD-L1-hyFc5 fusion protein was added at 0.5μM and incubated at 37℃ in a 5% CO2 incubator for 5 days. The CTV (CellTrace Far Red) staining of the responder cells was performed after the reaction was completed. TM The degree to which PD-L1-hyFc21 or PD-L1-hyFc5 fusion proteins suppressed the mixed lymphocyte reaction was compared by analyzing the degree to which PD-L1-hyFc21 or PD-L1-hyFc5 fusion proteins reduced fluorescence (P < 0.05).

[0116] As a result, it was confirmed that the proliferation of CD4 T cells and CD8 T cells in activated responder cells by mixed lymphocyte reaction was reduced in the groups treated with PD-L1-hyFc21 or PD-L1-hyFc5 fusion protein compared to the control group (Allo,hyFc treatment group) (Figure 9). In particular, it was confirmed that the proliferation of CD4 T cells and CD8 T cells in activated responder cells under the same concentration conditions was more significantly reduced in the group treated with PD-L1-hyFc21 fusion protein compared to the group treated with PD-L1-hyFc5 fusion protein (Figure 9).

[0117] 4.3. Analysis of proliferation inhibition and cytokine production inhibition of activated human T cells We compared the ability of PD-L1-hyFc21 or PD-L1-hyFc5 fusion proteins to inhibit human T cell proliferation under T cell activation conditions using human PBMCs. Briefly, PD-L1-hyFc21 or PD-L1-hyFc5 fusion proteins were coated onto a 96-well plate together with an anti-CD3 antibody at 4°C for 1 day. CD4 T cells were isolated from normal human PBMCs using microbeads (MACS), and the isolated CD4 T cells were stained with CellTrace (CTV) to stain intracellular proteins. TMAfter the coating, the plate was washed with PBS, and the CD4 T cells stained with CTV were placed in the test plate and cultured for 3 days in a 37℃ 5% CO2 incubator. After 4 days, the cells in the test plate were harvested and analyzed for the degree of decrease in CTV fluorescence using flow cytometry.

[0118] As a result, it was confirmed that the proliferation of activated human T cells was reduced in the groups treated with PD-L1-hyFc21 or PD-L1-hyFc5 fusion protein compared to the control groups (no treatment or hyFc treatment group) (Figure 10a). In particular, it was confirmed that the proliferation of activated human T cells was significantly suppressed by approximately two-fold in the group treated with PD-L1-hyFc21 fusion protein compared to the group treated with PD-L1-hyFc5 fusion protein (Figure 10a).

[0119] We also compared the ability of PD-L1-hyFc21 or PD-L1-hyFc5 fusion proteins to suppress cytokine expression in human T cells under T cell activation conditions using human PBMCs. Briefly, PD-L1-hyFc21 or PD-L1-hyFc5 fusion proteins were diluted with anti-CD3 antibody to be treated at 0.5 μM and coated on a 48-well plate at 4°C for 1 day. CD4 T cells were isolated from normal human PBMCs using microbeads (Microbead, Miltenyi Biotech). CD4 T cells were isolated from normal human PBMCs using microbeads (Microbead, MACS). After coating, the plate was washed with PBS, and the isolated CD4 T cells were placed in the test plate and cultured in a 37°C 5% CO2 incubator for 3 days. Anti-IFN-γ antibody (DuoSet Human IFN-gamma ELISA set, R&D system) was placed in the test plate and coated at room temperature. After coating, the test plate was washed three times with PBS containing 0.05% Tween-20 (PBST), and then PBS containing 1% BSA was added to block the test plate at room temperature for 2 hours. CD4 cell culture medium was transferred to the test plate and incubated at room temperature for 2 hours. The plate was washed five times with PBST, and the detection antibody was added and incubated at room temperature for 2 hours, after which the plate was washed five times with PBST. Streptavidin-HRP was added to the washed test plate and incubated at room temperature for 20 minutes. The plate was then washed five times with PBST, and TMB substrate was added, followed by the addition of 2N sulfuric acid (H2SO4), and the absorbance was measured at 450 nm. The measured absorbance values ​​were used to analyze the ability of PD-L1-hyFc21 or PD-L1-hyFc5 fusion protein to inhibit IFN-γ production produced by activated CD4 T cells.

[0120] As a result, it was confirmed that the amount of IFN-γ produced by activated human CD4 T cells was significantly reduced in the PD-L1-hyFc21 or PD-L1-hyFc5 fusion protein treatment groups compared to the control groups (no treatment or hyFc treatment groups) (Figure 10b). In particular, it was confirmed that the amount of IFN-γ produced by activated human CD4 T cells was significantly suppressed by about three-fold in the PD-L1-hyFc21 fusion protein treatment group compared to the PD-L1-hyFc5 fusion protein treatment group (Figure 10b).

[0121] 4.4. Cytokine production inhibition analysis of activated mouse T cells We compared the ability of PD-L1-hyFc21 or PD-L1-hyFc5 fusion proteins to suppress cytokine expression in mouse T cells under mouse T cell activation conditions. Briefly, PD-L1-hyFc21 or PD-L1-hyFc5 fusion proteins (0.5 μM) were added to test plates together with 5 μg / mL anti-CD3 antibody and coated at 4°C for 1 day. CD4 T cells were isolated from lymphocytes isolated from mouse lymph nodes using microbeads (microbead, Miltenyi Biotech). After washing the coated plates with PBS, the isolated CD4 T cells were added and cultured in a 37°C 5% CO2 incubator for 3 days. Anti-IL-2 antibody (Mouse IL-2 ELISA MAX TM Deluxe, Biolegend) and anti-IFN-γ antibody (Mouse IFNgamma ELISA MAX TMDeluxe, Biolegend) was placed in a new test plate and coated at room temperature. The test plate coated the day before was washed 4 times with washing buffer, and assay diluent A was added and blocked at room temperature for 1 hour. The test plate was washed 4 times with washing buffer, and CD4 T cell culture medium was transferred and stored, and reacted at room temperature for 2 hours. After washing 4 times with washing buffer, the detection antibody was added and reacted with shaking at room temperature for 1 hour, and washed 4 times with washing buffer. After washing, streptavidin-HRP was added to the test plate and reacted at room temperature for 30 minutes. After washing 5 times with washing buffer, TMB substrate was added and reacted at room temperature for 30 minutes in the dark. The stop solution was added and the absorbance was measured at 450 nm within 15 minutes. The measured absorbance values ​​were used to calculate the concentrations of IL-2 and IFN-γ, and the ability of PD-L1-hyFc21 or PD-L1-hyFc5 fusion proteins to inhibit the production of IL-2 and IFN-γ produced by activated CD4 T cells was analyzed.

[0122] As a result, it was confirmed that the amount of IL-2 and IFN-γ produced by activated mouse CD4 T cells was significantly reduced in the groups treated with PD-L1-hyFc21 or PD-L1-hyFc5 fusion protein compared to the control groups (no treatment or hyFc treatment) (Figure 11). In particular, it was confirmed that the amount of IL-2 produced by activated human CD4 T cells was significantly suppressed by about 1.8-fold in the group treated with PD-L1-hyFc21 fusion protein compared to the group treated with PD-L1-hyFc5 fusion protein, and the amount of IFN-γ was significantly suppressed by about 4-fold (Figure 11).

[0123] Example 5. In vivo activity analysis of PD-L1-hyFc21 fusion protein 5.1. Efficacy evaluation of PD-L1-hyFc21 fusion protein in imiquimod (IMQ)-induced psoriasis mouse model When psoriasis develops, dendritic cells are activated by Toll-like receptor 7 / 8 (TLR7 / 8). Imiquimod (IMQ), a ligand for TLR7 and TLR8, is known to induce psoriasis-like inflammatory reactions when repeatedly applied to mouse skin. Imiquimod (IMQ) is involved not only in dendritic cells but also in T cells, which are adaptive immune cells. Among these, gamma delta T cells (γδT17), which secrete interleukin-17 (IL-17), are preferentially involved in psoriatic inflammation. Therefore, the IMQ-induced psoriasis mouse model has a phenotype similar to that of psoriasis patients and can be used as a model suitable for the development of psoriasis therapeutics.

[0124] Briefly, to induce psoriasis in a mouse animal model, IMQ cream was applied to both ears of mice at 20 mg / mouse / day every day for 6 days. The ear thickness of mice in each group was measured using a caliper every day for 7 days from day 0, when IMQ cream was applied, to day 6, which was the end of the study, and the changes in ear thickness in mice in each group were compared with the ear thickness of mice not treated with IMQ cream (no treatment). PD-L1-hyFc21 fusion protein was administered subcutaneously or intravenously on days 0, 1, 3, and 5, or 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.

[0125] [Table 3]

[0126] As a result, the IMQ-treated group (IMG-vehicle) showed a 68% increase in ear thickness compared to the non-IMQ-treated control group (No treatment-vehicle) on Day 6, the final day of the study (Figure 12a). In contrast, differences in ear thickness were observed from Day 4 in the groups subcutaneously administered with 3, 10, and 30 mg / kg PD-L1-hyFc21 fusion protein (Figure 12a). In particular, the group subcutaneously administered with the lowest dose of 3 mg / kg PD-L1-hyFc21 fusion protein showed a 51% inhibition of ear thickness on Day 6, the final day of the study, compared to the IMQ-treated group (IMG-vehicle), and the groups subcutaneously administered with the medium and high doses of 10 mg / kg and 30 mg / kg PD-L1-hyFc21 fusion protein showed 67% and 66%, respectively, inhibition rates compared to the IMQ-treated group (IMG-vehicle) (Figure 12a). Additionally, there was a significant difference in dose between the groups administered 10 mg / kg or 30 mg / kg and the group administered the low dose of 3 mg / kg, but no significant difference was observed between the groups administered 10 mg / kg and 30 mg / kg.

[0127] Additionally, in all groups that received 3, 10, and 30 mg / kg of PD-L1-hyFc21 fusion protein intravenously, ear thickness was significantly suppressed by approximately 87-92% compared to the IMQ-treated group (IMG-vehicle) until the end of the study on the 6th (Figure 12b).

[0128] 5.2. Efficacy evaluation of subcutaneous administration of PD-L1-hyFc21 fusion protein in a doxycycline-inducible Peli1 gene-transfected psoriasis mouse model To evaluate the efficacy of PD-L1-hyFc21 fusion protein in a mouse model of chronic inflammation, the inventors used an imiquimod (IMQ)-induced psoriasis mouse model, as well as a mouse model in which the Pellino homolog 1 (Peli1) gene was overexpressed by administration of doxycycline, in order to induce long-term chronic psoriasis symptoms (hereinafter referred to as the "rtTA-Peli1 psoriasis mouse model"). As psoriasis is induced in the rtTA-Peli1 psoriasis mouse model, the mouse model induces the development of psoriatic lesions in the epidermis, increased inflammatory responses such as increased inflammatory cytokines, increased epithelial cell layers, and increased infiltration of phagocytes, dendritic cells, and Th17 cells into the dermis.

[0129] Briefly, to induce psoriasis in mice, 4-week-old mice were given drinking water containing 5% sucrose and 2 mg / mL doxycycline for 6 months. Saline containing doxycycline was continuously provided during the study period. Mice that were not transformed with the Peli gene (hereinafter referred to as "rtTA") were used as the control group. PD-L1-hyFc21 fusion protein was administered subcutaneously once a week for 8 weeks.

[0130] First, PD-L1-hyFc21 fusion protein was subcutaneously administered to rtTA-Peli1 psoriasis mouse model, and then H&E staining was used to measure changes in skin epithelial tissue and the thickness of the skin epithelial layer. H&E staining results were observed at 200X magnification, and six areas of each photograph were randomly selected for measurement. As a result, the average thickness of the epithelial layer was 8.74μm in the control group (rtTA) where psoriasis was not induced, and the average thickness of the epithelial layer was 48.56μm in the rtTA-Peli1 group where psoriasis was induced (rtTA-Peli1 Vehicle), and the epithelial layer thickness was approximately 5.6-fold increased in the rtTA-Peli1 group where psoriasis was induced (rtTA-Peli1 Vehicle) compared to the control group (Figure 13a and Figure 13b). In contrast, in the groups subcutaneously administered with 3, 10, and 30 mg / kg PD-L1-hyFc21 fusion protein, the average epithelial layer thickness was 38.29 μm, 40.18 μm, and 31.74 μm, respectively, confirming the effect of reducing the epithelial layer thickness compared to the rtTA-Peli1 group in which psoriasis was induced (rtTA-Peli1 Vehicle) (Figure 13a and Figure 13b), although no dose-dependent effect was observed.

[0131] In addition, after subcutaneous administration of PD-L1-hyFc21 fusion protein to rtTA-Peli1 psoriasis mouse model, an experiment was conducted to compare the infiltration of T cells and macrophages into the dermis layer using immunofluorescence staining. Anti-CD3 antibody was used to detect T cells, and anti-F4 / 80 antibody was used to detect macrophages. The results were observed under an immunofluorescence microscope at 400X magnification, and the number of cells in the same area was calculated.

[0132] As a result, in the control group (rtTA) where psoriasis was not induced, it was confirmed that almost no T cells and macrophages had infiltrated into the skin tissue, while in the rtTA-Peli1 group (rtTA-Peli1 Vehicle) where psoriasis was induced, the number of T cells and macrophages infiltrating into the skin tissue was increased (Figure 14). In particular, in the rtTA-Peli1 group (rtTA-Peli1 Vehicle) where psoriasis was induced, the number of T cells infiltrating into the skin tissue was approximately 47 (Figure 15). In contrast, in the groups subcutaneously administered with 3, 10, and 30 mg / kg PD-L1-hyFc21 fusion protein, 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).

[0133] In addition, after subcutaneous administration of PD-L1-hyFc21 fusion protein to the rtTA-Peli1 psoriasis mouse model, changes in keratinocytes in skin tissue were analyzed using immunofluorescence staining with anti-K14 antibody. K14 (Keratin 14) expression appears in the basal layer, which is responsible for proliferation, and this increased expression can be interpreted as an increase in the cells responsible for proliferation among keratinocytes. The results were compared by measuring the number of cells in the same area. As a result, in the control group (rtTA) where psoriasis was not induced, the number of K14 + The number of keratinocytes in the psoriasis-induced rtTA-Peli1 group was 59, while the number of keratinocytes in the psoriasis-induced rtTA-Peli1 group (rtTA-Peli1 Vehicle) was 197, indicating that the number of keratinocytes in the psoriasis-induced rtTA-Peli1 group was increased by approximately 3.3-fold compared to the control group (Figure 16). In contrast, in the groups subcutaneously administered with 3, 10, and 30 mg / kg of PD-L1-hyFc21 fusion protein, the number of keratinocytes was approximately 179, 150, and 98, respectively, indicating a dose-dependent decrease in the number of keratinocytes, with a particularly significant decrease in the number of keratinocytes at the 30 mg / kg dose (Figure 16).

[0134] 5.3. Efficacy evaluation of intravenous administration of PD-L1-hyFc21 fusion protein in a doxycycline-inducible Peli1 gene-transfected psoriasis mouse model After intravenously administering PD-L1-hyFc21 fusion protein to the rtTA-Peli1 psoriasis mouse model once a week for 5 weeks, the changes in the skin epithelium were analyzed by score index and the thickness of the abdominal skin was measured using a caliper. The score index was evaluated as follows: 0=normal skin; 1=keratoplasia appear; 2=keratoplasia appear on half of back skin, or skin lesions slightly overtop the normal skin; 3=thickness appear on whole back skin, or skin lesions significantly overtop the normal skin; 4=skin lesions sclerosis (Biomedicine & Pharmacotherapy, Volume 110, February 2019, Pages 265-274).

[0135] As a result, the score index of the rtTA-Peli1 group (rtTA-Peli1 Vehicle) with psoriasis induction increased by nearly 3 compared to the control group (rtTA), whereas the score index of the group administered PD-L1-hyFc21 fusion protein intravenously decreased compared to the rtTA-Peli1 group (Figure 17). In addition, similar to the score index, the abdominal skin thickness of the rtTA-Peli1 group (rtTA-Peli1 Vehicle) with psoriasis induction increased by approximately 100 μm compared to the control group (rtTA), whereas the skin thickness of the group administered PD-L1-hyFc21 fusion protein intravenously decreased to a similar level to the control group (rtTA) (Figure 18). However, no dose-dependent effect was observed in the above results.

[0136] In addition, after intravenous administration of PD-L1-hyFc21 fusion protein to rtTA-Peli1 psoriasis mouse model, we performed an experiment to measure changes in skin epithelial tissue and the thickness of the skin epithelial layer through H&E staining. The H&E staining results were observed under a microscope at 400X magnification, and 6 areas of the epithelial layer were randomly selected and the thickness of the skin epithelial layer was measured using the Image J program. As a result, the average thickness of the epithelial layer was 23.8μm in the control group (rtTA) where psoriasis was not induced, and the average thickness of the epithelial layer was 104.5μm in the rtTA-Peli1 group where psoriasis was induced (rtTA-Peli1 Vehicle), and it was confirmed that the epithelial layer thickness of the rtTA-Peli1 group where psoriasis was induced (rtTA-Peli1 Vehicle) was approximately 4.4 times higher than the control group (Figure 19a and Figure 19b). In contrast, in the groups that received 1, 3, and 10 mg / kg of PD-L1-hyFc21 fusion protein intravenously, the average thickness of the epithelial layer was approximately 97.9 μm, 86.3 μm, and 73.3 μm, respectively, demonstrating a dose-dependent reduction in the thickness of the epithelial layer compared to the psoriasis-induced rtTA-Peli1 group (rtTA-Peli1 Vehicle) (Figures 19a and 19b).

[0137] In addition, qRT-PCR was performed to confirm changes in mRNA expression of Th17 cell-associated genes such as IL-17A and IL-22, and innate immune cell-associated genes such as IL-1β and IL-24 after intravenous administration of PD-L1-hyFc21 fusion protein to the rtTA-Peli1 psoriasis mouse model. The results showed that the rtTA-Peli1 group with psoriasis (rtTA-Peli1 Vehicle) showed increased mRNA expression of Th17 cell-associated genes such as IL-17A and IL-22 compared to the control group (rtTA), whereas the group administered PD-L1-hyFc21 fusion protein intravenously showed decreased mRNA expression of Th17 cell-associated genes (Figure 20). Similarly, the rtTA-Peli1 group (rtTA-Peli1 Vehicle) in which psoriasis was induced showed increased mRNA expression of innate immune cell-related genes such as IL-1β and IL-24 compared to the control group (rtTA), whereas the group administered PD-L1-hyFc21 fusion protein intravenously showed a tendency to decrease mRNA expression of Th17 cell-related genes (Figure 20). In particular, the group administered 10 mg / kg PD-L1-hyFc21 fusion protein intravenously showed a significant decrease in IL-24 mRNA expression (Figure 20).

[0138] These results suggest that the PD-L1-hyFc21 fusion protein, when administered subcutaneously or intravenously, can suppress the proliferation of CD4 T cells, which are important factors in the pathogenesis of autoimmune diseases such as psoriasis, and suppress the expression of inflammatory cytokines associated with the lesions, and therefore can be used as a treatment for immune diseases such as psoriasis. The present invention provides, for example, the following items. (Item 1) A fusion protein comprising the programmed cell death-ligand 1 (PD-L1) protein and a modified immunoglobulin Fc region. (Item 2) 2. The fusion protein of item 1, wherein the PD-L1 protein is the extracellular domain of PD-L1 protein or a fragment thereof. (Item 3) 3. The fusion protein according to item 2, wherein the PD-L1 protein consists of an amino acid sequence selected from the group consisting of SEQ ID NO:1 to SEQ ID NO:6, or is a polypeptide having 70% or more homology to an amino acid sequence selected from the group consisting of SEQ ID NO:1 to SEQ ID NO:6. (Item 4) 2. The fusion protein of item 1, wherein the PD-L1 protein is fused to the N-terminus or C-terminus of the Fc region of an engineered immunoglobulin. (Item 5) 2. The fusion protein of item 1, wherein the PD-L1 protein is linked to an engineered immunoglobulin Fc region via a linker peptide. (Item 6) The linker peptide is selected from the group consisting of 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 of 1 to 10), (GGS)n (n is an integer of 1 to 10), (GS)n (n is an integer of 1 to 10), (GSSGGS)n (n is an integer of 1 to 10), KESGSVSSEQLAQFRSLD (SEQ ID NO: 21), EGKSSGSGSESKST (SEQ ID NO: 22), GSAGSAAGSGEF (SEQ ID NO: 23), (EAAAK)n (n is an integer of 1 to 10), CRRRRREAEAC (SEQ ID NO: 24), A(EAAAK)4 6. The fusion protein according to item 5, comprising one or more amino acid sequences selected from the group consisting of ALEA(EAAAK)4A, GGGGGGGG (SEQ ID NO: 25), GGGGGG (SEQ ID NO: 26), AEAAKEAAAAKA (SEQ ID NO: 27), PAPAP (SEQ ID NO: 28), (Ala-Pro)n (n is an integer of 1 to 10), VSQTSKLTRAETVFPDV (SEQ ID NO: 29), PLGLWA (SEQ ID NO: 30), TRHRQPRGWE (SEQ ID NO: 31), AGNRVRRSVG (SEQ ID NO: 32), RRRRRRRR (SEQ ID NO: 33), GFLG (SEQ ID NO: 34), and GSSGGSGSSGGSGGGDEADGSRGSQKAGVDE (SEQ ID NO: 35). (Item 7) 7. The fusion protein according to item 6, wherein the linker peptide consists of the amino acid sequence GGGSGGS (SEQ ID NO: 10). (Item 8) 2. The fusion protein according to item 1, wherein the modified immunoglobulin Fc region is any one of IgG1, IgG2, IgG3, IgD and IgG4 Fc regions or a combination thereof. (Item 9) the Fc region of the modified immunoglobulin comprises, from the N-terminus to the C-terminus, a hinge region, a CH2 domain, and a CH3 domain; the hinge region comprises a human IgG1 hinge region; the CH2 domain comprises a portion of amino acid residues of the CH2 domains of human IgD and human IgG4, The CH3 domain comprises a portion of amino acid residues of the CH3 domain of human IgG4. The fusion protein according to claim 1. (Item 10) 2. The fusion protein according to item 1, wherein the modified immunoglobulin Fc region consists of the amino acid sequence of SEQ ID NO: 11. (Item 11) 11. The fusion protein according to item 10, wherein the modified immunoglobulin Fc region comprises an IgG1 hinge region consisting of the amino acid sequence of SEQ ID NO: 16. (Item 12) The fusion protein according to item 1, wherein the fusion protein forms a dimer. (Item 13) 2. The fusion protein according to item 1, wherein the fusion protein consists of the amino acid sequence of SEQ ID NO: 12 or SEQ ID NO: 13. (Item 14) 14. A nucleic acid molecule encoding the fusion protein according to any one of items 1 to 13. (Item 15) 15. An expression vector comprising the nucleic acid molecule of item 14. (Item 16) A host cell comprising the expression vector according to item 15. (Item 17) 14. A pharmaceutical composition for preventing or treating an immune disease, comprising the fusion protein according to any one of items 1 to 13 as an active ingredient. (Item 18) 18. The pharmaceutical composition according to item 17, wherein the composition further comprises a pharma- ceutically acceptable carrier. (Item 19) 18. The pharmaceutical composition according to item 17, wherein the immune disease is selected from the group consisting of autoimmune diseases and inflammatory diseases. (Item 20) The autoimmune diseases include type 1 diabetes, alopecia areata, antiphospholipid antibody syndrome, rheumatoid arthritis, psoriasis, 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, and the like. 20. The pharmaceutical composition according to item 19, wherein the anemia is selected from the group consisting of idiopathic thrombocytopenic purpura, primary biliary cirrhosis, scleroderma, vitiligo, pernicious anemia and celiac disease. (Item 21) The inflammatory diseases include arthritis, ankylosing spondylitis, and the like. g 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, ischaemic heart disease, myocardial infarction 20. The pharmaceutical composition according to item 19, wherein the ocular disease is selected from the group consisting of ocular diseases, infarction, stroke, peripheral vascular disease, uveitis, corneal disease, iritis, iridocyclitis and cataracts.

Claims

1. A fusion protein comprising a programmed cell death-ligand 1 (PD-L1) protein and a modified immunoglobulin Fc region, the Fc region of the modified immunoglobulin comprises, from N-terminus to C-terminus, a hinge region, a CH2 domain, and a CH3 domain; The hinge region comprises a human IgG1 hinge region consisting of the amino acid sequence of SEQ ID NO: 16, the CH2 domain comprises a portion of amino acid residues of the CH2 domains of human IgD and human IgG4; the CH3 domain comprises a portion of amino acid residues of the CH3 domain of human IgG4, the PD-L1 protein is linked to the Fc region of the modified immunoglobulin via a linker peptide, the linker peptide having the amino acid sequence GGGSGGS (SEQ ID NO: 10); A fusion protein consisting of the amino acid sequence of SEQ ID NO:12 or SEQ ID NO:

13.

2. The fusion protein of claim 1 , wherein the fusion protein forms a dimerization.

3. A nucleic acid molecule encoding the fusion protein of claim 1.

4. An expression vector comprising the nucleic acid molecule of claim 3.

5. A host cell comprising the expression vector of claim 4.

6. A pharmaceutical composition for preventing or treating an immune disease, comprising the fusion protein according to claim 1 or 2 as an active ingredient.

7. The pharmaceutical composition of claim 6 , wherein the composition further comprises a pharma- ceutically acceptable carrier.

8. The pharmaceutical composition of claim 6, wherein the immune disease is selected from the group consisting of an autoimmune disease and an inflammatory disease.

9. The autoimmune diseases include type 1 diabetes, alopecia areata, antiphospholipid antibody syndrome, rheumatoid arthritis, psoriasis, psoriatic arthritis, multiple sclerosis, systemic lupus erythematosus, inflammatory bowel disease, Addison's disease, Graves' disease, and the like. disease), Shogren's syndrome, Guillain-Barre syndrome, Hashimoto's thyroiditis, Myasthenia gravis gravis), inflammatory myopathy, autoimmune vasculitis, autoimmune hepatitis, hemolytic anemia anemia), idiopathic thrombocytopenic purpura 9. The pharmaceutical composition of claim 8, wherein the liver disease is selected from the group consisting of primary biliary cirrhosis, scleroderma, vitiligo, pernicious anemia and celiac disease.

10. The inflammatory diseases include arthritis, ankylosing spondylitis, reactive arthritis, Reiter's syndrome, crystal arthropathies, Lyme disease, polymyalgia rheumatica, systemic sclerosis, polymyositis, dermatomyositis, polyarteritis nodosa, Wegener's granulomatosis, and inflammatory bowel disease. granulomatosis, Churg-Strauss syndrome, sarcoidosis, atherosclerotic vascular disease, ischemic heart disease, myocardial infarction, stroke, peripheral vascular disease, uveitis, corneal disease 9. The pharmaceutical composition of claim 8, wherein the inflammatory disease is selected from the group consisting of iritis, iridocyclitis and cataracts.

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