WO2022098186A1 - Pharmaceutical composition for treating transplant rejection, comprising fusion protein of pd-l1 and il-10 as active ingredient - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for treating transplant rejection, comprising a fusion protein of PD-L1 and IL-10 as an active ingredient. A fusion protein of PD-L1 and IL-10, according to the present invention, does not cause antibody dependent cell-mediated cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC), has increased in vivo half-life, and has remarkably increased immunosuppressive ability due to the fusion of monomeric IL-10 variants of which immunostimulating activity is removed and aggregation is inhibited, and thus can be effectively used in the prevention or treatment of transplant rejection.

Description

A pharmaceutical composition for the treatment of transplant rejection reaction comprising a fusion protein of PD-L1 and IL-10 as an active ingredient

The present invention relates to a pharmaceutical composition for the treatment of transplant rejection reaction comprising a fusion protein of PD-L1 and IL-10 as an active ingredient.

Human programmed cell death-ligand 1 (PD-L1) is a ligand for programmed death-1 (PD-1), in addition to hematopoietic cells such as T lymphocytes, B lymphocytes, dendritic cells, or macrophages, It is a type 1 transmembrane protein that is also expressed in non-hematopoietic cells such as keratinocytes, islet cells, hepatocytes, and the like. On the other hand, in order to activate T cells, in addition to the primary signal stimulation of the T cell receptor and antigen, secondary signal stimulation (co-stimulation) is simultaneously required. At this time, if there is no signal of either one, the T cell is in an inactive (anergy) state. PD-1 (programmed death-1) is a secondary signaling factor (immune check point or immune modulator) that regulates secondary signaling activity of T cells, activated T cells (CD8 and/or CD4) or dendritic cells ( dendritic cells), it binds to PD-L1 (programmed cell death ligand 1) or B7.1 (CD80) expressed on the cell surface, thereby inhibiting T cell proliferation and reducing cytokine expression. It can act to inhibit cell function.

Binding between PD-1:PD-L1 is known to induce regulatory T cell activity, and by using the immune tolerance inducing function of PD-L1, the PD-L1 protein (PD-L1-Ig ) when injected into a CIA (collagen induced arthritis) mouse model, it has been observed that arthritis symptoms are alleviated. Since PD-1 is expressed in activated T cells, PD-L1 protein is expected to be usefully used as a therapeutic agent that specifically targets active immune cells in autoimmune diseases as well as immune tolerance induction in organ transplantation. .

To date, therapeutic agents for the PD-1/PD-L1 cell signaling system have been developed in the direction of increasing T cell activity by inhibiting immune tolerance as an antagonist. However, an immunotherapeutic agent based on induction of T cell immune tolerance using an agonist has not been developed so far. In the case of a PD-1/PDL1 antagonist, it can be easily developed using antibody fusion technology, but the PD-1/PD-L1 signal agonist, which should be developed as a soluble protein, is technically Because development is not easy.

The Fc fusion technology of immunoglobulin (Ig) is one of the technologies for increasing the half-life of protein therapeutics. 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. The Ig fusion protein as an immune tolerance inducer does not play a role in suppressing the inflammatory response, but rather exacerbates inflammation.

Accordingly, there is a need to develop a technology to increase the therapeutic efficacy of PD-L1 as an immunosuppressant by not inducing ADCC and CDC while maintaining the half-life of PD-L1 similar to that of existing Ig fusion protein therapeutics.

Interleukin 10 (hereinafter referred to as "IL-10") is an anti-inflammatory cytokine expressed as a non-covalently bound homodimer of about 37 kDa called cytokine synthesis inhibitory factor (CSIF). IL-10 plays an important role in the induction and maintenance of immune tolerance, and its predominant anti-inflammatory properties have been known for a long time. IL-10 inhibits secretion of pro-inflammatory cytokines such as TNFα, IL-1, IL-6, and IL-12, as well as Th1 cytokines such as IL-2 and INFγ; It is known to regulate the differentiation and proliferation of macrophages, B cells and T cells. In addition, it is reported to be a potent inhibitor of antigen presentation, inhibiting MHC II expression as well as upregulation of co-stimulatory factors CD80 and CD86. Because of these properties, studies have been conducted to use IL-10 as a therapeutic agent for autoimmune diseases such as inflammatory bowel disease or psoriasis.

However, IL-10 is known to have the dual characteristic of having the opposite action of immunostimulatory activity. Specifically, IL-10 may stimulate B cell activation, prolong the survival of B cells, and contribute to class switching of B cells. In addition, it can stimulate NK cell proliferation and cytokine production, and can act as a growth factor promoting the proliferation of a specific subpopulation of CD8+ T cells. Importantly, it has been reported that high doses (20 and 25 μg/kg, respectively) of IL-10 in humans can increase the production of INFγ.

Accordingly, it has been reported that isoleucine, the 87th amino acid of IL-10, is involved in immune activation, and that if it is substituted with alanine, the immune activation action may be suppressed. However, in the case of the mutant IL-10, it is confirmed that the affinity with IL-10R1 is very weak compared to that of the wild type, so there is a disadvantage that a high dose is required for equivalent immunosuppressive activity.

On the other hand, when IL-10 is produced as a recombinant protein due to its structural characteristics, a large amount of insoluble aggregates are generated, which causes a great problem in productivity. Accordingly, in the secondary structure of IL-10 53-2, monomeric IL-10 that does not form a dimer has been developed by introducing a linker peptide of 6 a.a. between the fourth and fifth alpha helices, but it has a short half-life in the body and Because of its very low activity, it is an obstacle to its use as a therapeutic agent.

In order to overcome this low stability in the body, an attempt was made to express the Fc domain of IgA in the form of a fusion protein, but recovery of IL-10 activity was limited.

Meanwhile, in previous studies, there have been no reports of a fusion protein in which PD-L1 protein and IL-10 are used in combination or a fusion protein in which PD-L1 protein and IL-10 are fused. Accordingly, the present inventors completed the present invention by developing a fusion protein in which PD-L1 protein and IL-10, which can be usefully used in the treatment of immune diseases, are fused.

It is an object of the present invention to provide a pharmaceutical composition for treatment or prevention of transplant rejection reaction comprising a fusion protein in which a programmed cell death-ligand 1 (PD-L1) protein and an IL-10 variant are fused as an active ingredient.

Another object of the present invention is to provide a method for preventing or treating transplant rejection, comprising administering to an individual the pharmaceutical composition for treatment or prevention of transplant rejection.

In order to achieve the above object, the present invention provides a pharmaceutical composition for the treatment or prevention of transplant rejection reaction comprising a fusion protein in which a PD-L1 protein and an IL-10 variant are fused as an active ingredient.

In addition, the present invention provides a method for preventing or treating transplant rejection, comprising administering to an individual the pharmaceutical composition for the treatment or prevention of transplant rejection.

The fusion protein of PD-L1 and IL-10 according to the present invention does not induce ADCC (antibody dependent cell-mediated cytotoxicity) and CDC (complement dependent cytotoxicity), has an effect of increasing half-life in the body, and has the effect of eliminating immune activity Monomeric IL-10 variants with suppressed aggregation are fused and have the effect of remarkably increased immunosuppressive ability, which can be usefully used for the prevention or treatment of transplant rejection.

1 shows the structure of a fusion protein (referred to as “PD-L1-hyFc-IL10m”) fused to a modified immunoglobulin Fc with a PD-L1 protein and a monomeric IL-10 variant.

Figure 2 shows the analysis results of polyacrylamide gel electrophoresis (sodium dodecyl sulfate polyacrylamide gel electrophoresis, SDS-PAGE) for the PD-L1-hyFc-IL10m fusion protein (M: size marker, 3 μL; Lane 1: HCCF (harvest cell culture fluid), 3 μg; Lane 2: Capture Elute, 3 μg; Lane 3: Q FF Input, 3 μg; Lane 4: Q FF Elute, 3 μg; Lane 5: PD-L1-hyFc-IL10m Fusion protein, 3 μg; Lane 6: PD-L1-hyFc-IL10m fusion protein, 3 μg; and Lane 7: flow through sample after 1st step purification, 20 μg).

3 shows the analysis results of size-exclusion chromatography (SE-HPLC) for the PD-L1-hyFc-IL10m fusion protein.

4 shows the results of isoelectric focusing (IEF) analysis on the PD-L1-hyFc-IL10m fusion protein (M: pH 3-10 IEF marker; Lane 1: PD-L1-hyFc-IL10m fusion) protein; and Lane 2: PD-L1-hyFc-IL10m fusion protein).

FIG. 5 shows that hyFc (control), PD-L1-hyFc fusion protein or PD-L1-hyFc-IL10m fusion protein was simultaneously stimulated with allogeneic human-derived peripheral blood mononuclear cells (PBMC), which are normal recipient cells (responder). After treatment with 0.1 μM and 0.5 μM, respectively, and culturing for 6 days, the results of FACS analysis are shown after staining with a FACS antibody capable of identifying CD4 and CD8 T cells.

The present invention provides a pharmaceutical composition for the treatment or prevention of transplant rejection reaction comprising a fusion protein in which a programmed cell death-ligand 1 (PD-L1) protein and an IL-10 variant are fused as an active ingredient.

The fusion protein may be expressed by the following formula.

X1 - IgFc - L1 - X2 (I)

In the above, X1 is a polypeptide comprising a PD-L1 protein or a fragment thereof; IgFc is the Fc region of a modified immunoglobulin; L1 is a linker; X2 is an IL-10 variant.

The PD-L1 protein may be an extracellular domain of PD-L1 or a fragment thereof, and the extracellular domain of PD-L1 is an Ig V-like domain of PD-L1 (Immunoglobulin V like domain) and Ig C It may be a polypeptide comprising a similar domain (Immunoglobulin C likedomain).

The PD-L1 protein may consist of the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4. Specifically, the extracellular domain of PD-L1 is a protein region exposed outside the cell membrane, and is a polypeptide consisting of amino acids 19 to 238 of SEQ ID NO: 3 or a polypeptide consisting of amino acids 19 to 239 of SEQ ID NO: 3, or SEQ ID NO: It may be a polypeptide consisting of the amino acid sequence of 4. In addition, the extracellular domain of PD-L1 is about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 of the polypeptide sequence. %, 98%, or 99% or more.

The Ig V-like domain of the PD-L1 extracellular domain is a site capable of interacting with PD-1, and may be a polypeptide having amino acids 21 to 114 of SEQ ID NO: 3, and numbers 19 to 114 of SEQ ID NO: 3 It may be a polypeptide having amino acids. In addition, it may be a polypeptide having amino acids 21 to 120 of SEQ ID NO: 3, and may be a polypeptide having amino acids 19 to 120 of SEQ ID NO: 3. In addition, it may be a polypeptide having amino acids 21 to 127 of SEQ ID NO: 3, and may be a polypeptide having amino acids 19 to 127 of SEQ ID NO: 3. In addition, it may be a polypeptide having amino acids 21 to 130 of SEQ ID NO: 3, and may be a polypeptide having amino acids 19 to 130 of SEQ ID NO: 3. In addition, it may be a polypeptide having amino acids 21 to 131 of SEQ ID NO: 3, and may be a polypeptide having amino acids 19 to 131 of SEQ ID NO: 3. In addition, it may be a polypeptide having amino acids 21 to 133 of SEQ ID NO: 3, and may be a polypeptide having amino acids 19 to 133 of SEQ ID NO: 3. In addition, it may be a polypeptide having amino acids 21 to 239 of SEQ ID NO: 3, and may be a polypeptide having amino acids 19 to 239 of SEQ ID NO: 3.

When the fragment of the PD-L1 extracellular domain includes an Ig V-like domain or a fragment thereof, it may further include an Ig C-like domain of the PD-L1 extracellular domain (Immunoglobulin C like domain). The Ig C-like domain may be a polypeptide having amino acids 133 to 225 of SEQ ID NO: 3, or a polypeptide having amino acids 134 to 225 of SEQ ID NO: 3.

In addition, when the fragment of the PD-L1 extracellular domain includes an Ig V-like domain or a fragment thereof, it may further include a polypeptide or a fragment thereof comprising an Ig C-like domain of the PD-L1 extracellular domain. The polypeptide comprising the Ig C-like domain refers to the extracellular domain of PD-L1 excluding the Ig V domain, and the polypeptide having amino acids 134 to 239 of SEQ ID NO: 3 or 134 to 238 of SEQ ID NO: 3 It may be a polypeptide having amino acids.

The extracellular domain of PD-L1 or a fragment thereof may be derived from a human.

Human PD-L1 protein has 290 amino acid residues, and may consist of the amino acid sequence of SEQ ID NO: 3 (Accession Number: Q9NZQ7). In the amino acid sequence of SEQ ID NO: 3, the N-terminal amino acid residues 1 to 18 are the signal sequence, and mature human PD-L1 is a protein composed of amino acids 19 to 290 of SEQ ID NO: 3. The extracellular domain of human PD-L1 includes amino acids 19 to 238 of SEQ ID NO: 3 or 19 to 239 of SEQ ID NO: 3.

The human PD-L1 protein includes an Ig V-like domain that is amino acids 19 to 127 of SEQ ID NO: 3 and an Ig C-like domain that is amino acids 134 to 226 of SEQ ID NO: 3.

The extracellular domain of human PD-L1 protein or fragment thereof may include variously modified proteins or peptides. The modification may be performed by substituting, deleting or adding one or more proteins to wild-type PD-L1 as long as the function of PD-L1 is not altered. These various proteins or peptides can be combined with wild-type proteins by 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99 % homology.

Typically, substitution of wild-type amino acid residues is alanine, but can be accomplished by conservative amino acid substitutions that do not affect or weaken the charge, polarity or hydrophobicity of the entire protein.

Additional conservative substitutions include "homologs" of amino acids. In this case, the "homolog" refers to an amino acid in which a methylene group (CH2) is inserted into the side chain at the beta position of the side chain of the amino acid. Examples of such "homologs" include, but are not limited to, homophenylalanine, homoarginine, homoserine, and the like.

As used herein, the terms “protein”, “polypeptide” and “peptide” may be used interchangeably unless otherwise specified.

The IL-10 mutant is alanine with isoleucine (I), the 87th amino acid of wild-type human IL-10 protein, in order to remove immune-stimulatory functions from human IL-10 protein (Accession number: NM_000572.3). (A) introduced a mutation (I87A) to substitute, and in order to prevent aggregation of IL-10, between asparagine (N134), the 134th amino acid of the wild-type IL-10 protein, and lysine (K135), the 135th amino acid The GGSGGSGGS (9 aa) sequence was introduced as a linker peptide to improve productivity and purity. Specifically, the IL-10 variant may consist of the amino acid sequence of SEQ ID NO: 5.

As used herein, the term “fusion protein” refers to a recombinant protein in which two or more proteins or domains responsible for a specific function in the protein are linked so that each protein or domain performs its original function. The two or more proteins or domains may be directly coupled or coupled through a linker peptide having a flexible structure.

The linker peptide is AAGSGGGGGSGGGGSGGGGS, GGSGG, GGSGGSGGS, GGGSGG, (G4S)n (n is an integer from 1 to 10), (GGS)n (n is an integer from 1 to 10), (GS)n (n is 1 to 10) of), (GSSGGS)n (n is an integer from 1 to 10), KESGSVSSEQLAQFRSLD, EGKSSGSGSESKST, GSAGSAAGSGEF, (EAAAK)n (n is an integer from 1 to 10), CRRRRRREAEAC, A(EAAAK)4ALEA(EAAAK)4A, GGGGGGGG, GGGGGG, AEAAAKEAAAAKA, PAPAP, (Ala-Pro)n (n is an integer from 1 to 10), VSQTSKLTRAETVFPDV, PLGLWA, TRHRQPRGWE, AGNRVRRSVG, RRRRRRRR, GFLG, and GSSGGSGSSGGSGGGDEADGSRGSQKSGGGDEADGSRGSQ, and the like may be included. It may consist of an amino acid sequence of

The fusion protein may include one or more fusion partner proteins responsible for different functions, such as an antibody that specifically binds to a target protein, an antigen-binding fragment of the antibody, and the target protein. It may be an antibody analog that specifically binds to, an Fc region of an antibody, an antibody Fc region receptor, the antibody Fc region receptor, or an Fc region binding extracellular domain, dimerization domain, cytokine, or immunoregulatory peptide thereof.

In the fusion protein, the antigen-binding fragment of the antibody may be Fab, F(ab')2, Fab', scFv, diabody, triabody, sdAb (single domain antibody), VNAR or VHH, and the antibody analogue may be an affibody , affilin, affimer, affitin, alphabody, anticalin, avimer, DARPin, Fynomer, Kunitz domain peptide, monobody, repebody, VLR, or nanoCLAMP. The dimerization domain may be a hinge domain of an antibody, a LIM/double zinc-finger motif, a RAG1 domain, a HAT dimerization domain, a TRFH dimerization domain, a Stat3 dimerization, or a LFB1/HNF1 dimerization domain, but is not limited thereto.

As used herein, the term "antibody" is an immunoglobulin molecule, a heterotetrameric protein produced by combining two identical heavy chains and two identical light chains, and the variable region (VL) of the light chain and the variable region of the heavy chain An antigen-specific binding occurs through an antigen-binding site constituted by the region (VH), thereby inducing an antigen-specific humoral immune response.

As used herein, the term "antigen-binding fragment of antibody" refers to a fragment having antigen-binding ability derived from an antibody, and not only a fragment produced by cleaving an antibody with a protease, but also a recombinant method. All of the resulting single chain fragments are included, including Fab, F(ab')2, scFv, diabody, triabody, sdAb, and VHH.

As used herein, the term "Fab" refers to a fragment produced by cleaving an antibody molecule with papain, a protease, as an antigen-binding antibody fragment, and a dimer of two peptides, VH-CH1 and VL-CL. and other fragments produced by papain are referred to as fragment crystallizable (Fc).

As used herein, the term "F(ab')2" refers to a fragment containing an antigen-binding site among fragments produced by cleaving an antibody with pepsin, a proteolytic enzyme, and refers to a tetrameric form in which the two Fabs are linked by a disulfide bond. . Another fragment produced by pepsin is referred to as pFc'.

As used herein, the term "Fab'" refers to a molecule having a structure similar to that of Fab produced by isolating the F(ab')2 under mild reducing conditions.

As used herein, the term "scFv (single chain variable fragment)" is not a fragment of an actual antibody, but is prepared by linking the heavy chain variable region (VH) and light chain variable region (VL) of an antibody with a linker peptide having a size of about 25 aa. Although it is not a unique antibody fragment as a kind of fusion protein, it is known to have antigen-binding ability.

As used herein, the terms “diabody” and “triabody” refer to antibody fragments in which two and three scFvs are linked by a linker, respectively.

As used herein, the term "sdAb (single domain antibody)" is an antibody fragment composed of a single variable region fragment of an antibody, also called a nanobody. An sdAb derived from a heavy chain is mainly used, but it has been reported that a single variable region fragment derived from a light chain also binds specifically to an antigen. Unlike conventional antibodies composed of heavy and light chains, VNAR composed of a variable region fragment of a shark antibody composed only of a single-chain dimer and VHH composed of a variable region fragment of a camelid antibody are also included in the sdAb.

As used herein, the term "antibody mimetic" refers to a monobody, variable lymphocyte receptor (VLR), unlike a conventional full-length antibody in which two heavy chains and two light chains form a quaternary structure of a heterotetramer and exert their functions. ) is a concept including a protein having a function similar to that of an antibody prepared from a non-antibody-derived protein scaffold, that is, an antigen-binding ability. Such antibody analogs include Affibody derived from the Z domain of protein A, Affilin derived from Gamma-B crystallin or Ubiquitin, Affimer derived from Cystatin, Affitin derived from Sac7d, Alphabody derived from triple helix coiled coil protein, Anticalin derived from lipocalin, and various membranes. Avimer derived from receptor domain, DARPin derived from ankyrin repeat motif, Fynomer derived from SH3 domain of Fyn protein, Kunitz domain peptide derived from Kunitz domain of various protein inhibitors, monobody derived from the 10th type 3 domain of fibronectin, carbohydrate binding module 32-2-derived nanoCLAMP, hagfish-derived variable lymphocyte receptor (VLR), and a repebody engineered to enhance antigen affinity based on the VLR are included.

In the present invention, the PD-L1 protein or monomeric IL-10 variant may be fused to the N-terminus or C-terminus of the Fc region of a modified immunoglobulin, and specifically, the PD-L1 protein is a modified immunoglobulin. is fused to the N-terminus of the Fc region of , and the monomeric IL-10 variant may be fused to the C-terminus of the Fc region of the modified immunoglobulin. In addition, in the case of a monomeric IL-10 variant, it may be linked with a linker peptide consisting of the amino acid sequence of AAGSGGGGGSGGGGSGGGGS (SEQ ID NO: 6).

The Fc region of the modified immunoglobulin may be any one or a combination of Fc regions of IgG1, IgG2, IgG3, IgD and IgG4. The Fc region is modified so that binding to the Fc receptor and/or complement (complement) does not occur. In particular, the Fc region of the modified immunoglobulin comprises in an N-terminal to C-terminal direction a hinge region, a CH2 domain and a CH3 domain, wherein the hinge region comprises a human IgG1 hinge region, and wherein the CH2 domain is a human IgD and a portion of an amino acid residue of a CH2 domain of human IgG4, wherein the CH3 domain may include a portion of an amino acid residue of a CH3 domain of human IgG4.

As used herein, the term "Fc region", "Fc fragment" or "Fc" includes the heavy chain constant region 2 (CH2) and heavy chain constant region 3 (CH3) of an immunoglobulin, and includes the variable regions of the heavy and light chains of an immunoglobulin. and light chain constant region 1 (CL1). It may further comprise a hinge region of the heavy chain constant region. Hybrid Fc or hybrid Fc fragments are also referred to herein as “hFc” or “hyFc”. Also, in the present invention, the term "Fc region variant" refers to one prepared by substituting some amino acids in the Fc region or combining different types of Fc regions. The Fc region variant may be modified to prevent cleavage at the hinge region.

The Fc fragment of the present invention may be in the form of a native sugar chain, an increased sugar chain compared to the native form, a reduced sugar chain compared to the native form, or a form in which the sugar chain is removed. The increase, decrease or removal of immunoglobulin Fc sugar chains may be performed by conventional methods known in the art, such as chemical methods, enzymatic methods, and genetic engineering methods using microorganisms. Removal of sugar chains from the Fc fragment sharply reduces the binding affinity of the primary complement component C1 to Clq, resulting in reduction or elimination of antibodydependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC), thereby It does not induce unnecessary immune response in vivo. In this regard, the immunoglobulin Fc fragment in a deglycosylated or aglycosylated form may be more suitable for the purpose of the present invention as a drug carrier. As used herein, the term "deglycosylation" means that sugars are enzymatically removed from an Fc fragment, and the term "aglycosylation" means that the Fc fragment is in a prokaryotic organism, preferably E. coli. This means that it is produced in an unglycosylated form.

In the present invention, the Fc region of the modified immunoglobulin may consist of the amino acid sequence of SEQ ID NO: 7 or the amino acid sequence of SEQ ID NO: 8 into which a point mutation is introduced.

In the present invention, the fusion protein of PD-L1 and IL-10 may consist of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

The transplant rejection reaction may be a tissue or organ transplant rejection reaction, and the tissue or organ transplant rejection reaction includes bone marrow transplantation, heart transplantation, corneal transplantation, intestinal transplantation, liver transplantation, lung transplantation, pancreatic transplantation, kidney transplantation and It may be selected from among the rejection reactions of skin grafts.

The composition of the present invention may include a pharmaceutically acceptable carrier, and may additionally include a pharmaceutically acceptable adjuvant, excipient or diluent in addition to the carrier.

As used herein, the term "pharmaceutically acceptable" refers to a composition that is physiologically acceptable and does not normally cause gastrointestinal disorders, allergic reactions such as dizziness, or similar reactions when administered to humans. Examples of such carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In addition, fillers, anti-agglomeration agents, lubricants, wetting agents, flavoring agents, emulsifiers and preservatives and the like may be further included.

The pharmaceutical composition of the present invention may be formulated using methods known in the art to enable rapid, sustained or delayed release of the active ingredient upon administration to a mammal. Formulations include powders, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatin capsules, sterile injectable solutions, and sterile powder forms.

The composition of the present invention may be formulated in a suitable form together with a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include, for example, carriers for parenteral administration such as water, suitable oils, saline, aqueous glucose and glycol, and the like, and may further include stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium hydrogen sulfite, sodium sulfite or ascorbic acid. Suitable preservatives are benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol. In addition, the composition according to the present invention can be used as a suspending agent, solubilizer, stabilizer, isotonic agent, preservative, adsorption inhibitor, surfactant, diluent, excipient, pH adjuster, analgesic agent, buffer, if necessary depending on the administration method or formulation , antioxidants and the like may be included as appropriate.

The compositions of the present invention may be sterilized according to commonly known sterilization techniques. The composition may contain pharmaceutically acceptable auxiliary substances and adjuvants required to control physiological conditions such as pH control, toxicity control agents and analogs thereof, for example, sodium acetate, sodium chloride ( sodium chloride), potassium chloride, calcium chloride, sodium lactate, and the like. The fusion protein concentration in such formulations can vary widely, for example up to about 0.5% by weight, typically or at least about 1% to 15% or up to 20%, depending on the particular method of administration chosen. Accordingly, the selection may be preferentially based on body fluid volume, viscosities, and the like.

The composition of the present invention may be administered by any route. The composition of the present invention may be provided to an animal either directly (eg, by injection, implantation or topical administration at a tissue site, topically) or systemically (eg, parenterally or orally) by any suitable means. there is. The composition of the present invention can be administered intravenously, subcutaneously, ophthalmic, intraperitoneally, intramuscularly, oral, rectal, intraorbital, intracerebral, intracranial, intraspinal, ventricle. When given parenterally, such as intraventricular, intrathecal, intracistenal, intracapsular, intranasal or aerosol administration, the composition is preferably aqueous or It is preferred to include portions of physiologically applicable bodily fluid suspensions or solutions. Accordingly, the carrier or vehicle is physiologically acceptable and thus can be added to a composition and delivered to a patient without adversely affecting the patient's electrolytes and/or volumetric balance. Accordingly, as a body fluid medium for the preparation, it may generally contain physiological saline.

A DNA construct (or gene construct) comprising a nucleic acid encoding a fusion protein of the present invention can be used as part of a gene therapy protocol delivering a nucleic acid encoding the fusion protein construct.

In the present invention, an expression vector for infecting and expressing the fusion protein in vivo in a specific cell type in order to reconstitute or supplement the desired function of the fusion protein may be administered together with any biologically effective carrier, for example, an in vivo cell Any formulation or composition capable of efficiently delivering a gene encoding a desired fusion protein or a fusion protein thereof is the same.

For gene therapy using a nucleic acid encoding the fusion protein, a viral vector comprising a recombinant retrovirus, an adenovirus, an adeno-associated virus, and herpes simplex virus-1, or a recombinant bacterial plasmid Alternatively, the target gene may be inserted into a recombinant eukaryotic plasmid.

The dosage of the nucleic acid encoding the fusion protein of the present invention ranges from 0.1 mg to 100 mg for humans. In one embodiment, the preferred dosage of the nucleic acid encoding the fusion protein of the present invention is 1 mg to 10 mg for humans. In another embodiment, the preferred dosage of the nucleic acid encoding the fusion protein of the present invention is 2 mg to 10 mg for humans. The optimal amount and dosage form can be determined by routine experimentation within the level of skill in the art.

The unit dose of the fusion protein of the present invention is 0.1 mg/kg to 1,500 mg/kg in humans. In one embodiment, the unit dose of the fusion protein is 1 mg/kg to 100 mg/kg in humans. In another embodiment, the unit dose of the fusion protein is 5 mg/kg to 20 mg/kg in humans. 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. Administration of the fusion protein may be by periodic bolus injections, or from an external reservoir (eg, intravenous bag) or internal (eg, bioerodable implant). continuous intravenous, subcutaneous, or intraperitoneal administration of

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

In certain embodiments, the composition further comprises one or more immunosuppressive agents. As used herein, the terms "immunosuppressant" and "immunosuppressive agent" include compounds or compositions that inhibit an immune response or symptoms associated therewith. Immunosuppressants include, but are not limited to, purine analogs (eg, azathioprine), methotrexate, cyclosporine (eg, cyclosporine A), cyclophosphamide, leflunomide, mycophenolate (mycophenolate parent). fetil), steroids (e.g., glucocorticoids, corticosteroids), methylprednisone, prednisone, nonsteroidal anti-inflammatory drugs (NSAIDs), chloroquine, hydroxychloroquine, chlorambucil, CD20 antagonists (e.g., rituximab, ocrelizumab) , beltuzumab or ofatumumab), abatacept, TNF antagonists (eg, infliximab, adalimumab, etanercept), macrolides (eg, pimecrolimus, tacrolimus, and sirolimus), dihydroepiandrosterone, lenalidomide, CD40 antagonist or agonist, CD83 antagonist or agonist, CD45RB antagonist or agonist, avetimus sodium, BLys antagonist (eg anti-BLyS (eg belimumab)), dactinomycin , bucilamine, penicillamine, leflunomide, mercaptopurine, pyrimidine analogs (eg, cytosine arabinoside), mizoribine, alkylating agents (eg, nitrogen mustard, phenylalanine mustard, busulfan, and cyclophosphamide) ), folate antagonists (eg, aminopterin and methotrexate), antibiotics (eg, rapamycin, actinomycin D, mitomycin C, furamycin, and chloramphenicol), human IgG, anti-lymphocyte globulin (ALG), Antibodies (eg, anti-CD3 (OKT3), anti-CD4 (OKT4), anti-CD5, anti-CD7, anti-IL-2 receptors (eg, daclizumab and basiliximab), anti-alpha/beta antibodies to TCR, anti-ICAM-1, murononab-CD3, anti-IL-12, alemutuzumab and immunotoxins), 1-methyltryptophan, and derivatives and analogs thereof. In certain embodiments, the immunosuppressive agent is methotrexate, hydroxychloroquine, a CD20 antagonist (eg, rituximab, ocrelizumab, veltuzumab or ofatumumab), abatacept, a TNF antagonist (eg, infliximab, a dalimumab, etanercept), sirolimus, and a BLyS antagonist (eg, anti-BLyS (eg, belimumab)). In certain embodiments, the immunosuppressive agent is a CD20 antagonist, a TNF antagonist, or a BLyS antagonist.

In a preferred embodiment, the immunosuppressant may be administered simultaneously or sequentially with the fusion protein.

In addition, the present invention provides a method for preventing or treating transplant rejection, comprising administering to an individual the pharmaceutical composition for the treatment or prevention of transplant rejection.

The method for preventing or treating transplant rejection using the fusion protein or composition of the present invention comprises administering another drug or physiologically active substance having an effect of preventing or treating transplantation rejection in combination with the fusion protein or composition of the present invention. It may also include, and the route, administration timing, and dosage of the combined administration may be determined according to the type of disease, the disease state of the patient, the purpose of treatment or prevention, and other drugs or physiologically active substances used in combination.

Hereinafter, the present invention will be described in more detail through examples. These examples are for explaining the present invention in more detail, and the scope of the present invention is not limited to these examples.

Example 1. Preparation of a gene construct for producing a fusion protein in which PD-L1 protein and monomeric IL-10 variants are fused

To obtain a fusion protein in which human programmed cell death-ligand 1 (PD-L1) protein and a monomeric IL-10 variant are fused, a gene encoding the fusion protein is inserted to construct a gene t was prepared. In the gene construct encoding the fusion protein, the genes encoding the human PD-L1 protein and the monomeric IL-10 variant, respectively, were fused to the gene encoding the Fc region of a modified immunoglobulin (Ig), whereby It did not induce ADCC (antibody dependent cell mediated cytotoxicity, fusion protein is antibody dependent cytotoxicity) and CDC (complement dependent cytotoxicity, complement dependent cytotoxicity), and was prepared to increase half-life in the body. Specifically, the gene encoding the N-terminus of the Fc region of the modified immunoglobulin was fused to the human PD-L1 gene, and the gene encoding the C-terminus of the Fc region of the modified immunoglobulin was a monomeric IL-10 variant. It was prepared by fusion to the gene. In particular, a linker peptide consisting of the amino acid sequence of SEQ ID NO: 6 was connected between the C-terminus of the Fc region of the modified immunoglobulin and the monomeric IL-10 variant ( FIG. 1 ).

In the above, a known amino acid sequence (Accession number: Q9NZQ7) was used for the human PD-L1 gene, and a known amino acid sequence (Accession number: NM_000572.3) was used for the human IL-10 gene.

In the above, as the gene encoding the Fc region of the modified immunoglobulin, a nucleotide encoding the amino acid sequence of SEQ ID NO: 7, which is a hybrid type of a human IgD gene and a human IgG4 gene (referred to as "hyFc gene") was used. Specifically, the hyFc gene was bound in the order of 5'-IgD-IgG4-3', and in the case of the hinge, the GS linker and the IgG1 hinge sequence were combined to maintain flexibility while inducing dimerization. connected and used.

In addition, as the gene encoding the Fc region of the modified immunoglobulin, a nucleotide encoding the amino acid sequence of SEQ ID NO: 8 mutated in the hyFc gene (referred to as "NTIG gene") was used. In the NTIG gene, a two-point mutation was introduced to increase the binding affinity of the neonatal Fc receptor (FcRn) based on the sequence of the hyFc gene.

As described above, a fusion protein consisting of the amino acid sequence of SEQ ID NO: 1 in which human PD-L1 protein and a monomeric IL-10 variant are fused to the hyFc gene (referred to as “PD-L1-hyFc-IL10m”) or NTIG gene The gene construct each encoding a fusion protein consisting of the amino acid sequence of SEQ ID NO: 2 (referred to as "PD-L1-NTIG-IL10m") in which a human PD-L1 protein and a monomeric IL-10 variant are fused is PD-L1 After synthesizing the protein, the Fc region of the modified immunoglobulin (hyFc or NTIG), and each gene fragment of the monomeric IL-10 mutant and preparing it as a sub-vector, the three synthesized gene segments are prepared as one gene segment For Golden Gateway assembly, an expression vector was obtained as a pBispec vector.

To develop a high-efficiency cell line, the expression vector was treated with restriction enzymes EcoR1/NheI, the vector size of 6.4 Kb was isolated, and the insert was cut by treating the same EcoR1/NheI in the pBispec vector, and then ligated with ligase to finalize the expression vector. The gene pAD15 PD-L1-hyFc-IL10m plasmid or pAD15 PD-L1-NTIG-IL10m plasmid was obtained.

Example 2. Securing of PD-L1-hyFc-IL10m or PD-L1-NTIG-IL10m fusion protein

In order to mass-produce the PD-L1-hyFc-IL10m or PD-L1-NTIG-IL10m fusion protein, the PD-L1-hyFc-IL10m or PD-L1-NTIG-IL10m fusion protein obtained in Example 1 is encoded. The plasmid vector containing the gene was transformed into a suspension cell line such as CHO cells, and the target protein produced in the high-efficiency cell line was isolated and purified from the cell culture medium. In order to purify a high-purity protein, the target protein was obtained through a purification process of anion exchange chromatography.

Example 3. Characterization of PD-L1-hyFc-IL10m fusion protein

3.1. Polyacrylamide gel electrophoresis (sodium dodecyl sulfatepolyacrylamide gel electrophoresis, SDS-PAGE)

To confirm the molecular weight of the PD-L1-hyFc-IL10m fusion protein, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed. Briefly, fusion proteins were diluted with deionized water, mixed with NuPAGE™ LDS Sample Buffer (Thermo Fisher Scientific), and loaded into 4-15% Mini-PROTEANⓡTGX™ (Bio-Rad) to 3 μg/well. ) to perform electrophoresis. After electrophoresis, the gel was stained by Coomassie staining method.

As a result, the PD-L1-hyFc-IL10m fusion protein was confirmed above 150 kD of the size marker in a nonreducing condition ( FIG. 2 ).

3.2. Size-exclusion chromatography (SE-HPLC)

Size-exclusion chromatography (SE-HPLC) to analyze the main peak and impurity peaks such as a dimer, multimer, or truncated abnormal peptide in the purified PD-L1-hyFc-IL10m fusion protein ) was performed. Briefly, the fusion protein was diluted with a formulation buffer to prepare 1.0 mg/mL, and then the main peak of the fusion protein was separated using a gel filtration chromatography column (TOSOH TSK-GEL G3000SWxL column, 7.8 mm x 300 mm). The area ratio (% Area) was analyzed.

As a result, it was confirmed that the purity of the PD-L1-hyFc-IL10m fusion protein was 97.38% ( FIG. 3 ).

3.3. isoelectric focusing (IEF)

In order to confirm the charge variants and distribution of the D-L1-hyFc-IL10m fusion protein, isoelectric focusing (IEF) was performed. Isoelectric point electrophoresis is an electrophoresis method that analyzes separated proteins using the pI value of the protein. The pI value refers to the pH value at which a charged protein becomes electrically neutral, which is called the isoelectric point. In isoelectric point electrophoresis, the protein that has reached the isoelectric point does not move any more and stays on the gel and is separated. Briefly, in the present invention, the fusion protein was separated according to the pI value using a pH 3-10 isoelectric point gel, and the gel after electrophoresis was fixed with a 12% trichloroacetic acid (TCA) solution and then stained by Coomassie staining. The conditions of the isoelectric point electrophoresis method are shown in Table 1 below.

As a result, the PD-L1-hyFc-IL10m fusion protein was confirmed to have a pI value of 5.2 to 6.9 ( FIG. 4 ).

test condition
Gel	pH 3-10 IEF Gel
sample loading	10μg or max/ 20μl/well
Loading condition	100V 1hr, 200V 1hr, 500V 1hr
size marker	IEF Marker 3-10 (10μL/well)

Example 4. In vitro cell division inhibitory efficacy through single administration of PD-L1-hyFc-IL10m fusion protein

Treatment of PD-L1-hyFc-IL10m fusion protein in allogenic mixed lymphocyte reaction (MLR) that can represent the transplantation situation using normal donor peripheral blood mononuclear cells (PBMC) An experiment was performed to confirm the cell division inhibitory efficacy. Briefly, normal human-derived peripheral blood mononuclear cells (PBMCs) 1 x 105 cells were stained with Cell TraceTM violet (2.5 mM) and then treated with allogeneic irradiated 3rd party PBMCs (1 x 105 PBMCs). Simultaneously with stimulation, hyFc (control), PD-L1-hyFc fusion protein or PD-L1-hyFc-IL10m fusion protein was treated with 0.1 μM and 0.5 μM, respectively, and cultured for 6 days. Then, after staining with a FACS antibody capable of identifying CD4 and CD8 T cells, FACS analysis was performed.

As a result, it was confirmed that the proliferation of CD4 and CD8 T cells was reduced when the PD-L1-hyFc fusion protein fused with PD-L1 and modified immunoglobulin Fc was treated compared to the control group. Moreover, in the case of the PD-L1-hyFc-IL10m fusion protein in which the PD-L1 protein and the monomeric IL-10 variant are fused to the Fc of the modified immunoglobulin, CD4 compared to the control and the PD-L1-hyFc fusion protein. And it was confirmed that the proliferation of CD8 T cells was significantly reduced (FIG. 5).

Accordingly, the PD-L1-hyFc-IL10m fusion protein in which the PD-L1 protein and the monomeric IL-10 variant are fused to the modified immunoglobulin Fc suppresses the cell division of CD4 and CD8 T cells, thereby suppressing the immune response following transplantation. It was confirmed that there is an inhibitory or reducing effect.

Claims (15)

1. A pharmaceutical composition for the treatment or prevention of transplantation rejection, comprising as an active ingredient a fusion protein in which a programmed cell death-ligand 1 (PD-L1) protein and an IL-10 variant are fused.
2. The pharmaceutical composition according to claim 1, wherein the fusion protein is expressed by the following formula:

   X1 - IgFc - L1 - X2 (I)

   In the above, X1 is a polypeptide comprising a PD-L1 protein or a fragment thereof;

   IgFc is the Fc region of a modified immunoglobulin;

   L1 is a linker;

   X2 is an IL-10 variant.
3. The pharmaceutical composition of claim 2, wherein the PD-L1 protein consists of the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4.
4. The pharmaceutical composition according to claim 2, wherein the IL-10 variant consists of the amino acid sequence of SEQ ID NO: 5.
5. The pharmaceutical composition according to claim 2, wherein the linker consists of the amino acid sequence of SEQ ID NO: 6.
6. The pharmaceutical composition according to claim 2, wherein the Fc region of the modified immunoglobulin is any one or a combination of Fc regions of IgG1, IgG2, IgG3, IgD and IgG4.
7. 7. The method of claim 6,

   The Fc region of the modified immunoglobulin comprises a hinge region, a CH2 domain and a CH3 domain in an N-terminal to C-terminal direction,

   wherein the hinge region comprises a human IgG1 hinge region,

   wherein the CH2 domain comprises a portion of the amino acid residues of the CH2 domain of human IgD and human IgG4,

   The CH3 domain comprises a portion of the amino acid residues of the CH3 domain of human IgG4.
8. The pharmaceutical composition according to claim 2, wherein the Fc region of the modified immunoglobulin consists of the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 8.
9. The pharmaceutical composition according to claim 1, wherein the fusion protein consists of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.
10. The pharmaceutical composition according to claim 1, wherein the transplant rejection reaction is a transplant rejection reaction of a tissue or organ.
11. The method of claim 10, wherein the tissue or organ transplant rejection is selected from rejection of bone marrow transplantation, heart transplantation, corneal transplantation, intestinal transplantation, liver transplantation, lung transplantation, pancreatic transplantation, kidney transplantation and skin transplantation, pharmaceutical composition.
12. The pharmaceutical composition according to claim 1, wherein the composition further comprises an immunosuppressant.
13. The pharmaceutical composition according to claim 12, wherein the immunosuppressant is administered simultaneously or sequentially with the fusion protein.
14. The pharmaceutical composition according to claim 12, wherein the immunosuppressant is at least one selected from the group consisting of tacrolimus, sirolimus, CD40 antagonist or agonist, CD83 antagonist or agonist, and CD45RB antagonist or agonist.
15. A method for preventing or treating transplant rejection, comprising administering the pharmaceutical composition according to any one of claims 1 to 14 to a subject.

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