Genetically modified cells containing nucleic acids encoding CD40L binding agents and uses thereof

Abstract

The present invention relates to genetically engineered cells in which a nucleic acid encoding a CD40L binder (e.g., a Stefin A protein variant that specifically binds to CD40L or a fusion protein containing the same) has been introduced into a host cell, and uses thereof. The present invention provides genetically engineered cells in which a CD40L binder is expressed and secreted, expressed on the cell membrane, and / or localized intracellularly, thereby reducing or inhibiting CD40L activity. The genetically engineered cells of the present invention exhibit the ability to suppress T cell activity and B cell activity, thereby having an immunosuppressive effect, and are therefore useful for the prevention or treatment of immune diseases such as autoimmune diseases or inflammatory diseases.

Description

[Technical Field]

[0001] cross reference This application claims priority to Korean Patent Application Publication No. 10-2022-0188961, filed on December 29, 2022, the contents of which are incorporated herein by reference.

[0002] The present invention relates to genetically modified cells in which a nucleic acid encoding a CD40L-binding protein (e.g., a Stefin A protein variant that specifically binds to CD40L) or a fusion protein containing the same has been introduced into cells (e.g., stem cells, e.g., human mesenchymal stem cells), and uses thereof. [Background technology]

[0003] The immune system is a biological network composed of various cells and organs that protects the body from external invasion. It operates based on the specific functions of the organs and cells that make up the immune system, as well as the signaling and interactions between each cell. The immune system maintains immunological homeostasis by maintaining an appropriate balance between immune tolerance, which suppresses and regulates immunity, and immune response, which promotes immunity. Various causes can lead to an imbalance between immune tolerance and immune response, which can lead to various diseases. For example, a strong immune tolerance mechanism can facilitate the development of cancer or the invasion of external infections, resulting in cancer, infectious diseases, and other conditions. On the other hand, a strong immune response mechanism can lead to inflammatory diseases such as autoimmune diseases and allergic diseases.

[0004] In recent years, active research has been conducted on the immune synapse, a signaling system between immune cells that make up the immune system or between target cells and immune cells. The immune synapse is composed of various cytokines and signaling substances secreted by cells, as well as various costimulatory molecules and receptors expressed on the cell surface. As various factors involved in maintaining immune system homeostasis have been reported, interest in immunotherapeutics that target these factors to regulate immune responses has increased. Most immunotherapeutics developed to date are antibody drugs against cytokines or cell surface molecules, but their use is severely limited due to insufficient efficacy or side effects. Recently, cell therapies utilizing differentiated immune cells such as T cells and NK cells or stem cells that can differentiate into various immune cells have been developed as more effective treatments for immune diseases. Cell therapy agents based on differentiated immune cells, such as CAR-T or CAR-NK, have limitations such as high cost and limited targeting due to the use of autologous cells, while mesenchymal stem cells have limitations such as low therapeutic efficacy compared to the cost of treatment and difficulty in explaining a clear mechanism of action (Blood Cancer J.11,69(2021);World J Stem Cells.2019;11(4):212-221).

[0005] On the other hand, CD40 ligand (CD40L, also named CD154) is a protein that binds to CD40 on antigen-presenting cells and exhibits diverse effects depending on the target cell (The Journal of Experimental Medicine. 175(4):1091-101). Representative CD40L binding partners include CD40, α5β1 integrin, and αIIbβ3. CD40L acts as a costimulatory molecule and has been reported to be particularly important for a subset of T cells known as T follicular helper cells (TFH cells) (Journal of Immunology. 149(12):3817-26). CD40L is primarily expressed on activated CD4+ T cells, but it has also been found in a soluble form. It has been reported to be expressed not only on T cells but also on platelets, mast cells, macrophages, basophils, NK cells, B cells, and non-hematopoietic cells such as endothelial and epithelial cells (Cellular and Molecular Life Sciences. 58(1):4-43). CD40L is classified as a member of the tumor necrosis factor (TNF) superfamily, and CD40 / CD40L signaling as a costimulator plays an important role in T cell activity and T cell-mediated B cell differentiation and activation. Furthermore, CD40 / CD40L signaling plays an important role in regulating the expression and signaling mechanism of OX40 / OX40L, which is also a costimulator, and is therefore involved in T cell survival and memory T cell development.

[0006] CD40L has been reported to play an important role in the interaction between antigen-presenting cells (APCs) and T cells in various immune homeostasis-related diseases. In particular, CD40L / CD40 interaction acts as a pathogenic factor in autoimmune or inflammatory diseases in which T cell and B cell activation play a major role in the pathology. Specifically, CD40L is a pathogenic factor in various diseases, such as type 1 diabetes, thyroiditis, psoriasis, lupus (systemic lupus erythematosus), rheumatoid arthritis (RA), and multiple sclerosis (MS). Various compounds or antibodies targeting CD40L have been developed for the treatment of such diseases (Semin Immunol. 2009;21(5):293-300; Advanced Drug Delivery Reviews Volume 141, 15 February 2019, Pages 92-103). Under these circumstances, the present inventors have made efforts to develop a new type of cell therapy agent that expresses a CD40L binding agent. Summary of the Invention

[0007] The present disclosure provides, at least in part, genetically engineered cells expressing CD40L-binding agents. In one embodiment, the provided cells exhibit potent immunomodulatory and therapeutic effects against immune-related diseases.

[0008] In one embodiment, a population of genetically modified cells is provided, wherein the cells comprise an exogenous nucleic acid containing a coding sequence encoding a CD40L binding agent, the CD40L binding agent comprising one or more binding domains derived from an antibody or antibody mimetic.

[0009] In one embodiment, the genetically modified cells and populations thereof are eukaryotic cells, hi one embodiment, the genetically modified cells and populations thereof are mammalian cells, hi one embodiment, the genetically modified cells and populations thereof are human cells.

[0010] In one embodiment, a population of genetically modified immune cells is provided, wherein the immune cells comprise an exogenous nucleic acid containing a coding sequence encoding a CD40L-binding agent, the CD40L-binding agent comprising one or more binding domains derived from an antibody or antibody mimetic. In one embodiment, the immune cells are selected from T cells, B cells, NK cells, monocytes, macrophages, Treg cells, and antigen-presenting cells (e.g., dendritic cells).

[0011] In one embodiment, a population of genetically modified stem cells is provided, wherein the stem cells comprise an exogenous nucleic acid containing a coding sequence encoding a CD40L binding agent, wherein the CD40L binding agent comprises one or more binding domains derived from an antibody or antibody mimetic.

[0012] In one embodiment, the genetically modified cells and populations thereof are mammalian stem cells. In one embodiment, the genetically modified cells and populations thereof are human stem cells. In one embodiment, the human stem cells are pluripotent stem cells. In one embodiment, the human stem cells are multipotent stem cells.

[0013] In one embodiment, genetically modified mesenchymal stromal cells (MSCs) are provided that contain a nucleic acid encoding a CD40L-binding agent. In one embodiment, the CD40L-binding agent is a CD40L-binding protein. In one embodiment, the CD40L-binding protein is an antibody, an antibody fragment, a stefin A protein variant, or a fusion comprising any of these.

[0014] In one embodiment, a population of genetically modified MSCs is provided, the MSCs comprising an exogenous nucleic acid comprising a coding sequence encoding a CD40L binding agent, the CD40L binding agent comprising one or more binding domains derived from an antibody or antibody mimetic.

[0015] In one embodiment, a method for producing a population of genetically modified MSCs is provided, wherein the MSCs comprise an exogenous nucleic acid containing a coding sequence encoding a CD40L-binding agent, the CD40L-binding agent comprising one or more binding domains derived from an antibody or antibody mimetic. In one embodiment, the method includes contacting a population of MSCs with a lentiviral vector containing an exogenous nucleic acid containing a coding sequence encoding a CD40L-binding agent and culturing the population of MSCs. In one embodiment, the population of MSCs is separated at the time of initiation of contact. In one embodiment, the method includes selecting MSCs that express a selection gene.

[0016] In one embodiment, the CD40L binding agent is a Stefin A protein variant, Fab, Fab', F(ab')2, Fv, Fd, scFv, sdFv, VL, VH, Camel Ig, V-NAR, VHH, trispecific (Fab3), bispecific (Fab2), diabody ((VL-VH)2 or (VH-VL)2), triabody (trimer), tetrabody (tetrabody), minibody ((scFv-CH3)2), bispecific single-chain Fv (Bis-scFv), VNAR (a shark heavy-chain-only antibody), microprotein (cysteine ​​knot protein, knottin), affibody, aptamer, avimer, nanobody, unibody, single domain antibody, antibody, affilin, affitin, adnectin, atrimer, evasin, DARPin, anticalin, fynomer, versabody, repebody, or duocalin.

[0017] In one embodiment, the CD40L binding agent is a CD40L binding protein, which may be a Stefin A protein variant, Fab, Fab', F(ab')2, Fv, Fd, scFv, sdFv, VL, VH, camel Ig, V-NAR, VHH, trispecific (Fab3), bispecific (Fab2), diabody ((VL-VH)2 or (VH-VL)2), triabody (tribody), tetrabody (tetrabody), minibody ((scFv-CH3)2), Bis-scFv (bispecific single-chain Fv), VNAR (a shark heavy-chain-only antibody), microprotein (cysteine ​​knot protein, knottin), affibody, avimer, nanobody, unibody, single domain antibody, affilin, affitin, adnectin, atrimer, evasin, DARPin, anticalin, finomer, versabody, lipibody or duocalin.

[0018] In one embodiment, the CD40L-binding agent is based on Stefin A protein variant technology. In particular, the present disclosure has developed genetically engineered cells that secrete or express anti-CD40L-binding Stefin A protein variants on the cell membrane by introducing a gene encoding an anti-CD40L Stefin A protein variant into immune cells, stem cells, or somatic cells. The genetically engineered cells not only exhibit significantly enhanced immunomodulatory capabilities compared to host cells, but also reduce immune rejection following allogeneic cell transplantation. Furthermore, the use of the genetically engineered cells has been shown to exhibit excellent therapeutic effects against immune-related diseases, such as inflammatory diseases and autoimmune diseases, leading to the completion of the present invention. In one embodiment, the CD40L-binding agent is or includes a Stefin A protein variant.

[0019] In one embodiment, the exogenous nucleic acid comprises a transcriptional regulatory sequence operably linked to a coding sequence. In one embodiment, the transcriptional regulatory sequence is a promoter selected from the group consisting of a CMV promoter, an EFS promoter, a CBh promoter, an MSCV promoter, an SFFV promoter, and an E1FA promoter. In one embodiment, the exogenous nucleic acid comprises a selection gene. In one embodiment, the exogenous nucleic acid sequentially comprises a promoter, a coding sequence encoding a CD40L binding agent (e.g., a CD40L binding protein, e.g., a CD40L antibody, a CD40L antibody fragment, a CD40L-binding Stefin A protein variant, or a fusion thereof), an IRES or 2A sequence, and an antibiotic selection gene.

[0020] In one embodiment, the mesenchymal stem cells (MSCs) are derived from pluripotent stem cells. In one embodiment, the mesenchymal stem cells are derived from or differentiated from induced pluripotent stem cells. In one embodiment, the mesenchymal stem cells are derived from or differentiated from embryonic stem cells.

[0021] In some embodiments, the MSCs express at least one cell surface marker selected from CD29, CD44, CD73, CD90, and CD105. In one embodiment, the MSCs express at least two cell surface markers selected from CD29, CD44, CD73, CD90, and CD105. In one embodiment, the MSCs express at least three cell surface markers selected from CD29, CD44, CD73, CD90, and CD105. In one embodiment, the MSCs express all of the following cell surface markers: CD29, CD44, CD73, CD90, and CD105.

[0022] In one embodiment, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, or at least 98% of the MSCs express at least one cell surface marker selected from CD29, CD44, CD73, CD90, and CD 105. In one embodiment, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, or at least 98% of the MSCs express at least two cell surface markers selected from CD29, CD44, CD73, CD90, and CD105. In one embodiment, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, or at least 98% of the MSCs express at least three cell surface markers selected from CD29, CD44, CD73, CD90, and CD 105. In one embodiment, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, or at least 98% of the MSCs express all of the following cell surface markers: CD29, CD44, CD73, CD90, and CD105.

[0023] In one embodiment, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, or at least 98% of the MSCs express CD90.

[0024] In one embodiment, expression of the cell surface markers is maintained at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, or at least 98% in the MSC population after a minimum of 15 passages.

[0025] In one embodiment, the MSCs do not express, or less than 1% of the MSCs in the population express, at least one cell surface marker selected from CD11b, CD14, CD34, CD45, CD79, HLA-DR, TRA-1-60, and TRA-1-81. In one embodiment, the MSCs do not express, or less than 1% of the MSCs in the population express, at least two cell surface markers selected from CD11b, CD14, CD34, CD45, CD79, HLA-DR, TRA-1-60, and TRA-1-81. In one embodiment, the MSCs do not express, or less than 1% of the MSCs in the population express, at least three cell surface markers selected from CD11b, CD14, CD34, CD45, CD79, HLA-DR, TRA-1-60, and TRA-1-81. In one embodiment, the MSCs do not express, or less than 1% of the MSCs in the population express, four or more cell surface markers selected from CD11b, CD14, CD34, CD45, CD79, HLA-DR, TRA-1-60, and TRA-1-81. In another embodiment, the MSCs do not express, or less than 1% of the MSCs in the population express, any one of CD34, CD45, HLA-DR, TRA-1-60, and TRA-1-81.

[0026] In one embodiment, at least 95% of the MSCs are CD73+ and CD105+, and less than 1% express CD45, SSEA-3, TRA-1-60, TRA-1-81, and HLA-DR.

[0027] In one embodiment, the CD40L-binding agent is present in a concentration of 1×10 -6 M, 1 x 10 -7 M, 1 x 10 -8 M, 1 x 10 -9 The Kd value is M or less.

[0028] In one embodiment, the CD40L-binding agent is present in a concentration of 1×10 -6 M, 1 x 10 -7 M, 1 x 10 -8 M, 1 x 10 -9A stefin A protein mutant exhibiting a Kd value of M or less. In one embodiment, the CD40L binding agent is a Stefin A protein variant, wherein the Stefin A protein variant comprises the amino acid sequence: MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVV-(Xaa)n-GTNYYIKVRAGDNKYMHLKVFKSL-(Xaa)m-EDLVLTGYQVDKNKDDELTGF (SEQ ID NO: 742); wherein Xaa is an amino acid residue, and n and m are each independently an integer from 3 to 20. In one embodiment, (Xaa)n comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 6 to 125. In one embodiment, (Xaa)m comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 126 to 245.

[0029] In one embodiment, the CD40L-binding agent is a Stefin A protein variant, wherein the Stefin A protein variant comprises or consists of a sequence at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence selected from the group consisting of SEQ ID NOs: 246 to 365. In another embodiment, the CD40L-binding agent is a Stefin A protein variant, wherein the Stefin A protein variant comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 246 to 365.

[0030] In one embodiment, the CD40L binding agent is a Stefin A protein variant, which further comprises a signal peptide.

[0031] In one embodiment, the CD40L binding agent comprises a trimer or tetramer of a Stefin A protein variant.

[0032] In one embodiment, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, or at least 98% of the mesenchymal stem cells (MSCs) of the population comprise the exogenous nucleic acid.

[0033] In one embodiment, the CD40L-binding agent is expressed on the surface of mesenchymal stem cells, hi one embodiment, the CD40L-binding agent is secreted extracellularly, hi one embodiment, the CD40L-binding agent is expressed intracellularly.

[0034] In one embodiment, when the CD40L-binding agent is secreted extracellularly, the population of cells express and secrete the CD40L-binding agent at an average level of 200 fg / cell / day or more. In one embodiment, when the CD40L-binding agent is secreted extracellularly, the population of cells express and secrete the CD40L-binding agent at an average level of 300 fg / cell / day or more. In one embodiment, when the CD40L-binding agent is secreted extracellularly, the population of cells express and secrete the CD40L-binding agent at an average level of 400 fg / cell / day or more.

[0035] In one embodiment, when the CD40L-binding agent is secreted extracellularly, the population of cells expresses and secretes the CD40L-binding agent at an average level of 200-1500 fg / cell / day. In another embodiment, when the CD40L-binding agent is secreted extracellularly, the population of cells expresses and secretes the CD40L-binding agent at a level of 300-1000 fg / cell / day. In another embodiment, when the CD40L-binding agent is secreted extracellularly, the population of cells expresses and secretes the CD40L-binding agent at a level of 400-800 fg / cell / day.

[0036] In one embodiment, the genetically modified MSCs of the present invention are used to prevent or treat immune diseases.

[0037] In one embodiment, there is provided a pharmaceutical composition comprising the genetically modified MSCs of the present invention, which is useful for preventing or treating immune diseases. In one embodiment, there is provided a method for treating immune disorders using the genetically modified MSCs of the present invention and / or a pharmaceutical composition comprising the same.

[0038] In one embodiment, the immune disease is selected from the group consisting of lupus (SLE), lupus nephritis (e.g., drug-induced lupus nephritis), immune thrombocytopenia (ITP), rheumatoid arthritis (RA), multiple sclerosis (MS), inflammatory bowel disease (IBD) (e.g., Crohn's disease and colitis / ulcerative colitis), graft-versus-host disease (GVHD) or allograft rejection, transplant / solid organ transplant (SOT), primary biliary cholangitis (PBC), psoriasis, psoriatic arthritis, collagen-induced arthritis, oophoritis, allergic rhinitis, asthma, Sjogren's syndrome, atopic eczema, myasthenia gravis, Graves' disease, and glomerulosclerosis.

[0039] In one embodiment, a lentiviral vector suitable for use in producing and / or transfecting MSCs of the present invention is provided. In one embodiment, a lentiviral vector is provided, comprising a nucleic acid comprising a transcriptional regulatory sequence operably linked to a sequence encoding a CD40L-binding agent of the present invention, an IRES or 2A sequence, and a selection gene. In one embodiment, the transcriptional regulatory sequence is a promoter selected from the group consisting of a CMV promoter, an EFS promoter, a CBh promoter, an MSCV promoter, an SFFV promoter, and an E1FA promoter. In one embodiment, the nucleic acid sequentially comprises a promoter, a sequence encoding a CD40L-binding agent (e.g., a CD40L-binding protein, e.g., a CD40L antibody, a CD40L antibody fragment, a CD40L-binding Stefin A protein variant, or any fusion thereof), an IRES or 2A sequence, and an antibiotic selection gene.

[0040] In one embodiment, a method is provided for genetically modifying cells to contain a nucleic acid encoding a CD40L binding agent (e.g., a Stefin A protein variant that specifically binds to CD40L and / or a fusion protein comprising the same).

[0041] In one embodiment, a cell therapy agent is provided, comprising genetically engineered cells containing a nucleic acid encoding a CD40L binding agent and / or a culture medium thereof. In one embodiment, the CD40L binding agent is an anti-CD40L antibody, an antibody fragment, a Stefin A protein variant, or any fusion thereof. In one embodiment, the genetically engineered cells are MSCs (e.g., human MSCs).

[0042] In one embodiment, the present invention provides a use of the genetically modified cells (e.g., genetically modified MSCs) and / or their culture medium for the production of a cell therapy agent.

[0043] In one embodiment, the present invention provides a pharmaceutical composition for preventing or treating an immune disease, comprising the genetically modified cells (e.g., genetically modified MSCs) and / or a culture medium thereof.

[0044] In one embodiment, the present invention provides a use of the genetically modified cells (e.g., genetically modified MSCs) and / or their culture medium for the prevention or treatment of immune diseases.

[0045] In one embodiment, the present invention provides a use of the genetically modified cells (e.g., genetically modified MSCs) and / or their culture medium for the preparation of a pharmaceutical composition for the prevention or treatment of an immune disease.

[0046] In one embodiment, the present invention provides a method for preventing or treating an immune disease, comprising administering the genetically modified cells (e.g., genetically modified MSCs) and / or a culture medium thereof to a subject.

[0047] In one embodiment, the present invention provides a composition for drug delivery comprising the genetically modified cells (e.g., genetically modified MSCs) and / or a culture medium thereof.

[0048] In one embodiment, the present invention provides a use of genetically modified cells (e.g., genetically modified MSCs) and / or their culture medium for drug delivery.

[0049] In one embodiment, the present invention provides a drug delivery method using genetically modified cells (e.g., genetically modified MSCs) and / or their culture medium. [Brief explanation of the drawings]

[0050] These and other embodiments, features, and other advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which: The drawings contained herein are for illustration purposes only and not for limitation. [Figure 1] FIG. 1 shows the results of direct binding ELISA of monomer clones to human CD40L. [Figure 2] FIG. 1 shows flow cytometry analysis of human CD40L expression in HEK293 cells through binding of monomeric clones. [Figure 3] FIG. 1 shows the results of confirming hCD40L expression in hCD40L-HEK293 cells (upper panel) compared with control HEK 293 cells (lower panel). [Figure 4] FIG. 1 shows the binding of clone 230 (SEQ ID NO: 249) to hCD40L-HEK293 cells at different concentrations. [Figure 5] FIG. 1 shows the dose effect of the Stefin A protein mutants of the present invention that specifically bind to CD40L on blocking the binding between hCD40L and CD40, as determined by CD40L HEK-BLUE reporter cell assay. [Figure 6] Figure 1 shows various in-line fusions (ILFs) used to increase avidity for CD40L. Concentrations of 1 μg (left) and 5 μg (right) of each format were tested. Each format is labeled on the gel (e.g., monomer, dimer, or trimer). [Figure 7] FIG. 1 shows binding of different clones containing ILF dimeric and trimeric structures to hCD40L using a BIACORE™ assay. [Figure 8] FIG. 1 shows the binding of different clones containing ILF dimeric and trimeric structures to hCD40L by flow cytometry. [Figure 9] FIG. 1 shows the dose effect of monomeric, dimeric, and trimeric Stefin A protein mutants or their ILFs on blocking the binding of hCD40L to CD40, as determined by a CD40L HEK-BLUE reporter cell assay. [Figure 10] FIG. 1 shows the results of competitive ELISA for the binding of clones of various formats (DT, trimer with a rigid linker; XT75, trimer with a rigid linker and an HSA-binding Stefin A protein mutant; XT76, tetramer with a rigid linker and an HSA-binding Stefin A protein mutant). [Figure 11] This figure shows the dose effect of various formats (DT, trimer with a rigid linker; XT75, trimer with a rigid linker and an HSA-binding Stefin A protein mutant; XT76, tetramer with a rigid linker and an HSA-binding Stefin A protein mutant) on blocking the binding of hCD40L to CD40, using a CD40L HEK-BLUE reporter cell assay. [Figure 12]Figure 12 shows the results of hCD40L / HSA bridging ELISAs using clone-230 XT75 (an ILF protein containing three clone 230 monomers and an HSA-binding stefin A protein variant (HSA affimer)) and clone-230 XT76 (an ILF protein containing four clone 230 monomers and an HSA-binding stefin A protein variant). 3t0 Gly XT58 is an ILF protein containing a non-hCD40L-targeting protein trimer and an HSA-binding stefin A protein variant (HSA affimer) linked to each other via a rigid linker. 3t0 Gly XT59 is an ILF protein containing two non-hCD40L-targeting proteins and an HSA-binding stefin A protein variant (HSA affimer) linked to each other via a rigid linker. Figure 12A shows the stefin A protein variants binding to bound hCD40L (measured with an anti-cystatin antibody). Figure 12B shows Stefin A protein variants binding to bound hCD40L in the presence of HSA (as measured with an anti-cystatin antibody), and Figure 12C shows Stefin A protein variants binding to bound hCD40L and HSA (as measured with an anti-HSA antibody). [Figure 13] This figure shows the results of comparing the gene transfer efficiency of transduction enhancers when mesenchymal stem cells were transformed with a lentiviral vector (SEQ ID NO: 729) containing the eGFP gene. After treatment with Polybrene (2, 4, 8 μg / mL), protamine sulfate (5, 10, 20 μg / mL), and Lentiboost (1:500, 1:100, 1:20), the eGFP gene transfer efficiency was confirmed by measuring the GFP fluorescence wavelength. [Figure 14] FIG. 1 shows the results of confirming the gene transfer efficiency using GFP fluorescence wavelength for a method in which adherent mesenchymal stem cells are treated with a lentiviral vector containing the eGFP gene, and a method in which non-adherent mesenchymal stem cells are treated with a lentiviral vector (reverse transduction). [Figure 15A]

[0033] Figure 1 shows the results of evaluating G418 concentration conditions for selecting gene-transfected cells. Naive MSC cells were treated with 500 μg / mL, 250 μg / mL, 125 μg / mL, 62.5 μg / mL, 31.25 μg / mL, 15.625 μg / mL, and 7.8125 μg / mL of G418, and cell death was confirmed using a CCK-8 assay at intervals of 1 day, 3 days, 5 days, and 7 days. [Figure 15B] FIG. 1 shows the results of FACS analysis of cells expressing the eGFP gene (SEQ ID NO: 729) selected using G418 before (left) and after (right) selection. [Figure 16A] This figure shows the results of comparing gene transfer efficiency using a fluorescence microscope after mesenchymal stem cells were transformed with the Lentivirus Promoter Blast™ kit (Applied Biological Materials Inc.) to evaluate the promoter-driven GFP gene expression efficiency. [Figure 16B] FIG. 1 shows the results of comparing gene transfer efficiency by promoter using the GFP gene by FACS analysis (CMV SEQ ID NO: 729). [Figure 17A] This figure shows the results of evaluating combinations of promoters and gene-linked peptides and comparing the expression of anti-CD40L Stefin A protein variants. Lentiviral vectors expressing anti-CD40L Stefin A protein variants were constructed using the CMV, EFS, CBh, MSCV, SFFV, and EF1A promoters, and these were then introduced into mesenchymal stem cells to generate cell lines. The anti-CD40L Stefin A protein variants secreted by the constructed cell lines were quantified using an ELISA kit, and the difference in expression was expressed as a fold difference. [Figure 17B] FIG. 1 shows the results of analyzing the secretion amount of anti-CD40L Stefin A protein variants while continuously subculturing cells after constructing a cell line in which the expression of anti-CD40L Stefin A protein variants is regulated by EF1A (SEQ ID NO: 733) and the CBh promoter (SEQ ID NO: 737). [Figures 18A-18C] Figure 18 shows the results of examining the passage stability of a cell line in which expression of the anti-CD40L Stefin A protein variant is controlled by EF1A (SEQ ID NO: 733) and the CBh promoter (SEQ ID NO: 737). Figure 18A shows the results of a comparative analysis of cell size during continuous subculture from PN9 to PN19, Figure 18B shows the results of the cell proliferation time (PDT), and Figure 18C shows the results of a comparative analysis of the cell proliferation rate (PDL). [Figure 19] This figure shows the results of binding ELISA analysis of the CD40L-binding ability of anti-CD40L Stefin A protein variants secreted outside the cells by promoter. *5C8: anti-CD40L monoclonal antibody; *eMSC (XT75, w / EF1A), eMSC (XT75, w / CBh). [Figure 20] This figure shows the results of a cell-based assay (HEK-Blue assay) analyzing the ability of promoter-specific anti-CD40L Stefin A protein variants secreted to the outside of cells to inhibit the binding between CD40L and CD40. *5C8: anti-CD40L monoclonal antibody; *eMSC (XT75, w / EF1A), eMSC (XT75, w / CBh). [Figure 21] This figure shows the results of an investigation into whether cells secreting anti-CD40L Stefin A protein variants have an inhibitory effect on PBMC activation (PBMC clustering assay). Naive MSCs, XT73 gene (SEQ ID NO: 692)-transfected cell lines, and XT75 gene (SEQ ID NO: 694)-transfected cell lines were co-cultured with PBMCs at different ratios (1:20 to 1:1), and the inhibitory effect on clustering of activated PBMCs was examined. *eMSCs (XT73, SEQ ID NO: 692) SEQ ID NO: 692, eMSCs (XT75, SEQ ID NO: 694). [Figure 22]This figure shows the results of comparing whether cells secreting anti-CD40L Stefin A protein mutants suppress the expansion of CD3+ T cells through FACS analysis. *eMSCs (XT73, SEQ ID NO: 692), eMSCs (XT75, SEQ ID NO: 694). [Figure 23] FIG. 1 shows the results of an analysis of whether a protein secreted by cells into which the anti-CD40L Stefin A protein mutant gene (SEQ ID NO: 694) has been introduced exhibits the activity of suppressing B cell clustering. [Figure 24] FIG. 1 shows the results of an analysis using the CD86 surface marker to determine whether a protein secreted by cells into which the anti-CD40L Stefin A protein mutant gene (SEQ ID NO: 694) has been introduced suppresses B cell activation. [Figure 25] FIG. 1 shows the results of a Western blot comparison of the expression of immunoregulatory factors between naive MSCs and MSCs transfected with the anti-CD40L Stefin A protein mutant gene (SEQ ID NO: 694). [Figure 26] FIG. 1 shows the results of FACS analysis of the purity of cells into which the anti-CD40L Stefin A protein mutant gene (SEQ ID NO: 694) was introduced. [Figure 27] Schematic diagram of the xenograft GVHD animal model (left) and experimental group design (right). [Figure 28] This figure shows the efficacy of cells into which the anti-CD40L Stefin A protein mutant gene (SEQ ID NO: 694) was introduced using a xenograft GVHD mouse model, expressed as clinical scores. *G1: normal control; *G2: CS10; *G3: 5c8; *G4: naive MSC; *G5: XT75 (eMSC). [Figure 29] FIG. 1 is a schematic diagram showing a haploidentical GVHD mouse model. [Figure 30]FIG. 1 shows the number of doses and intervals of administration of anti-CD40L Stefin A protein mutants (XT54, SEQ ID NO: 739, XT55, SEQ ID NO: 740) in a haploidentical GVHD mouse model. [Figure 31] FIG. 1 shows the efficacy of anti-CD40L Stefin A protein mutants (XT54 SEQ ID NO: 739, XT55 SEQ ID NO: 740) in a haploidentical GVHD mouse model, as measured by changes in body weight (top panel) and GVHD clinical score (bottom panel). DETAILED DESCRIPTION OF THE INVENTION

[0051] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which this invention belongs. Generally, the nomenclature used herein is that which is well known and commonly used in the art.

[0052] In most cases, the amino acids and amino acid sequences described herein may be naturally occurring amino acids found in proteins or anabolic or catabolic products of amino acids containing amino and carboxyl groups, and isomers thereof (e.g., D- or L-isomers).

[0053] The specific amino acid residues described herein include not only naturally occurring amino acids but also their analogs, derivatives, and congeners. For example, when the Stefin A protein variants of the present invention are produced by chemical synthesis, they may contain amino acid analogs such as, but not limited to, cyanoalanine, canavanine, djenkolic acid, norleucine, 3-phosphoserine, homoserine, dihydroxyphenylalanine, 5-hydroxytryptophan, 1-methylhistidine, 3-methylhistidine, diaminopimelic acid, ornithine, or diaminobutyric acid.

[0054] In the amino acid or nucleic acid sequences described herein, identity or percent identity refers to sequences that are identical or have a specified percentage of identity. When comparing and aligning two sequences for maximum correspondence, conservative amino acid substitutions may not be considered as part of the sequence identity.

[0055] The proteins or polypeptides described herein, e.g., binding proteins, e.g., Stefin A protein variants and / or fusion proteins, may include not only the amino acid sequences described in this context, but also proteins or polypeptides in which portions of the amino acid sequences have been substituted through conservative substitutions.

[0056] As used herein, "conservative substitution" refers to a modification of a polypeptide that involves replacing one or more amino acids with amino acids having similar biochemical properties, without causing loss of biological or biochemical function of the polypeptide.

[0057] A conservative amino acid substitution is a substitution of an amino acid residue with an amino acid residue having a similar side chain. Classes of amino acid residues with similar side chains have been defined and are well known in the art. These classes include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Generally, conservative substitutions in the sequences of the polypeptides and proteins described herein do not result in loss of their function. For example, Stefin A variants that specifically bind to CD40L do not abolish binding to CD40L due to their conservative substitutions. Methods for identifying such conservative substitutions are well known in the art. Avacta has previously screened anti-CD40L Stefin A protein variants (anti-CD40L AFFIMER® proteins) using the Affimer® platform technology and filed a patent application (U.S. Patent Application No. 63 / 308629, Avacta Life Sciences Limited, unpublished). In this patent application, the authors confirmed that the anti-CD40L-binding Stefin A protein variants can bind to CD40L with excellent affinity and specificity and can be used as antagonists by inhibiting the activity of CD40L.

[0058] Therefore, the following inventions are claimed in U.S. Patent Application No. 63 / 308,629 filed by Avacta Life Sciences Limited or in an application claiming priority thereto, and are excluded from the scope of the present invention:

[0059] (i) a genetically engineered cell for use in manufacturing the Stefin A protein variant that specifically binds to CD40L or a fusion protein containing the same; (ii) Use solely for the production of a Stefin A protein variant that specifically binds to CD40L of the genetically engineered cells, or a fusion protein, conjugate, etc. containing the same; and (iii) A method for producing a Stefin A protein mutant that specifically binds to CD40L, or a fusion protein, conjugate, etc. containing the same, using the genetically engineered cells.

[0060] The term "production" in the present invention means "production for producing" the Stefin A protein variant that specifically binds to CD40L or a fusion protein containing the same.

[0061] In one embodiment, the present invention provides a cell with superior immunoregulatory ability through genetic engineering. In one embodiment, the present invention provides a genetically engineered cell that stably secretes, expresses at the cell membrane, and / or intracellularly a Stefin A protein variant that specifically binds to CD40L. In one embodiment, the cell can be produced by introducing a gene encoding the Stefin A protein variant that specifically binds to CD40L into the cell.

[0062] In the examples of the present invention, it was confirmed that the genetically modified cells can specifically bind to target cells through the cell membrane expression of a Stefin A protein variant that specifically binds to CD40L or a fusion protein containing the same, and that the passage stability and immunomodulatory effect of the host cells are maintained despite gene introduction. Furthermore, when the genetically modified cells are used, they exhibit significantly superior immunomodulatory effect compared to the host cells.

[0063] Therefore, in one aspect, the present invention relates to a genetically modified cell in which a nucleic acid encoding a Stefin A protein variant that specifically binds to CD40L or a fusion protein comprising the same has been introduced into a host cell.

[0064] CD40L The term "CD40L" or "CD40 ligand" according to the present invention refers to a protein that binds to its receptor, CD40, also named "CD154". CD40L may be used interchangeably with other terms, including TNF-related activation protein, TRAP, tumor necrosis factor (ligand) superfamily member, TB cell-activating molecule, CD40 antigen ligand, T-cell antigen Gp39, TNFSF5, HCD40L, CD154, Gp39, tumor necrosis factor (ligand) superfamily, member 5 (hyper-IgM syndrome), tumor necrosis factor (ligand) superfamily, hyper-IgM syndrome, CD154, CD40LG, HIGM1, T-BAM, IMD3, IGM, and CD40-L, to mean substantially the same thing.

[0065] CD40L (Cluster of Differentiation 40 ligand) is a protein encoded by the CD40L gene in humans. CD40L acts as a ligand for CD40 / TNFRSF5 and costimulates T cell proliferation and cytokine production. Cross-linking to T cells, along with TCR / CD3 ligation and CD28 costimulation, generates costimulatory signals that enhance IL4 and IL10 production. CD40L induces activation of NK-kappa-B, activates the kinases MAPK8 and PAK2 in T cells, and induces tyrosine phosphorylation of CD28 isoform 3. CD40L not only produces IGE in the presence of IL4 but also mediates B-cell proliferation in the absence of costimulation. CD40L is also involved in immunoglobulin class switching. Human CD40L amino acid and nucleic acid sequences can be found in public databases such as GenBank, UniProt, and Swiss-Prot. For example, the amino acid sequence of human CD40L can be found under UniProt / Swiss-Prot. Accession No. P29965, and the nucleic acid sequence encoding it can be found under Accession No. NM_000074.2.

[0066] The CD40L includes any native CD40L and mature CD40L produced in cells from a CD40L precursor protein. Unless otherwise specified, the CD40L includes CD40L obtainable from any organism, preferably an animal, including, but not limited to, CD40L obtainable from mammals such as primates (e.g., humans and cynomolgus monkeys) and rodents (e.g., mice and rats).

[0067] The CD40L includes, but is not limited to, all CD40L proteins containing mutations (e.g., point mutations, fragments, insertions, deletions, and splice variants of full-length wild-type CD40L).The CD40L includes, but is not limited to, all CD40L proteins containing mutations (e.g., point mutations, fragments, insertions, deletions, and splice variants of full-length wild-type CD40L).

[0068] CD40L binding agents The present disclosure provides, inter alia, cells comprising an exogenous nucleic acid encoding a CD40L-binding agent.

[0069] In one embodiment, the CD40L binding agent is a recombinant protein comprising one or more binding domains from an antibody or antibody mimetic. In one embodiment, the CD40L binding agent is a recombinant protein comprising one or more binding domains from an antibody or antibody mimetic. In one embodiment, the CD40L binding agent is a recombinant protein comprising one or more binding domains from an antibody or antibody mimetic. In one embodiment, the CD40L binding agent is a recombinant protein comprising one or more binding domains from an antibody or antibody mimetic. In one embodiment, the recombinant protein is a Stefin A protein variant, Fab, Fab', F(ab')2, Fv, Fd, scFv, sdFv, VL, VH, camel Ig, V-NAR, VHH, trispecific (Fab3), bispecific (Fab2), diabody ((VL-VH)2 or (VH-VL)2), triabody (trivalent), tetrabody (tetravalent), minibody ((scFv-CH3)2), bispecific single chain Fv (Bis-scFv), shark heavy chain single antibody (VNAR), microprotein (cysteine ​​knot), or the like. protein, knottin, affibody, aptamer, avimer, nanobody, unibody, single domain antibody, affilin, affitin, adnectin, atrimer, evasin, DARPin, anticalin, finomer, versabody, lipibody or duocalin.

[0070] In one embodiment, the CD40L-binding domain may be characterized as being selected from the group consisting of, but not limited to, a Stefin A protein variant, an antibody or fragment thereof, an antibody mimetic, an antigen-binding peptide, a ligand-binding site of a receptor (e.g., a receptor-trap polypeptide), a receptor-binding ligand (e.g., a cytokine or growth factor), an engineered T-cell receptor, and an enzyme or catalytic fragment thereof.

[0071] In one embodiment, the CD40L-binding agent is an anti-CD40L antibody. The term "antibody" as used herein includes not only intact antibodies that specifically bind to a target (antigen) but also antigen-binding fragments of antibody molecules. An intact antibody has a structure comprising two full-length light chains and two full-length heavy chains, with each light chain linked to a heavy chain via a disulfide bond. The term "heavy chain" as used herein refers to a full-length heavy chain and fragments thereof, comprising a variable region domain VH containing an amino acid sequence with sufficient variable region sequence to confer specificity to the antigen, and three constant region domains CH1, CH2, and CH3. The term "light chain" as used herein refers to a full-length light chain and fragments thereof, comprising a variable region domain VL containing an amino acid sequence with sufficient variable region sequence to confer specificity to the antigen, and a constant region domain CL. Full-length antibodies include subclasses IgA, IgD, IgE, IgM, and IgG. Specifically, IgG includes IgG1, IgG2, IgG3, and IgG4. The heavy chain constant region has gamma (γ), mu (μ), alpha (α), delta (δ), and epsilon (ε) types, which are divided into subclasses such as gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3), gamma 4 (γ4), alpha 1 (α1), and alpha 2 (α2). The light chain constant region has kappa (κ) and lambda (λ) types.

[0072] In one embodiment, the CD40L binder is an anti-CD40L antigen-binding antibody fragment. An antigen-binding fragment of an antibody refers to a fragment that has antigen-binding ability and includes Fab, F(ab'), F(ab')2, and Fv. Among antibody fragments, Fab has a structure comprising light and heavy chain variable regions, a light chain constant region, and the first heavy chain constant region (CH1) and has one antigen-binding site. Fab' differs from Fab in that it has a hinge region containing at least one cysteine ​​residue at the C-terminus of the heavy chain CH1 domain. F(ab')2 is formed by a disulfide bond between cysteine ​​residues in the hinge region of Fab'.

[0073] Fv is the minimum antibody fragment containing only the heavy-chain variable region and the light-chain variable region. A two-chain Fv is a fragment in which the heavy-chain variable region and the light-chain variable region are non-covalently linked, while a single-chain Fv (scFv) is a fragment in which the heavy-chain variable region and the light-chain variable region are covalently linked, generally via a peptide linker, or directly linked at the C-terminus, to form a dimeric structure like a two-chain Fv. These antibody fragments can be obtained using proteases (e.g., Fab fragments can be obtained by digesting whole antibodies with papain and F(ab')2 fragments can be obtained by digesting whole antibodies with pepsin) or can be produced through genetic recombination techniques.

[0074] An "Fv" fragment is an antibody fragment that contains a complete antigen recognition and binding site. This region is a dimer of one heavy-chain variable domain and one light-chain variable domain.

[0075] The "Fab" fragment contains the variable and constant domains of the light chain and the variable and first constant domain (CH1) of the heavy chain. F(ab')2 antibody fragments generally contain a pair of Fab' fragments covalently linked by hinge region cysteines located at the C-terminus of the Fab' fragments.

[0076] "Single-chain Fv (scFv)" antibody fragments are constructs composed of a single polypeptide chain comprising the VH and VL domains of an antibody. scFvs can further comprise a polypeptide linker between the VH and VL domains to form the desired structure for antigen binding.

[0077] Examples of antibodies of the present invention may include, but are not limited to, monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, scFv, Fab fragments, F(ab')2 fragments, disulfide-linked Fv (sdFv), anti-idiotype antibodies, epitope-binding fragments of such antibodies, and the like.

[0078] The heavy chain constant region can be selected from isotypes such as gamma (γ), mu (μ), alpha (α), delta (δ), and epsilon (ε). For example, the constant region can be gamma 1 (IgG1), gamma 2 (IgG2), gamma 3 (IgG3), or gamma 4 (IgG4). The light chain constant region can be kappa or lambda type.

[0079] A monoclonal antibody is an antibody obtained from a substantially homogeneous antibody population, in which the individual antibodies comprising the population are identical except for minor mutations that may naturally occur. Monoclonal antibodies are highly specific and are directed against a single epitope of an antigen. Unlike typical (polyclonal) antibodies, which generally contain many antibodies directed against different determinants (epitopes), each monoclonal antibody targets a single determinant of the antigen.

[0080] Examples of CD40L antibodies and antigen-binding antibody fragments are described in the art. In humans, at least two distinct anti-CD40L mAb clones have been used in clinical trials for the treatment of various autoimmune diseases. Maribel et al., Mol. Immunol., 45:937-944 (2008). Examples of anti-CD40L antibodies are described, for example, in WO2006 / 030220A1 (which describes antibody polypeptides that monovalently bind to CD40L; incorporated herein by reference), WO2013 / 056068A1 (which describes antibody polypeptides that specifically bind to human CD40L; incorporated herein by reference), and WO2016 / 040571A1 (which describes therapeutic methods using anti-CD40L antibodies; incorporated herein by reference).

[0081] In one embodiment, the CD40L-binding agent comprises a ligand-binding domain of a receptor that acts as an inhibitor / antagonist of the bound ligand (i.e., the target), e.g., an "inhibitory receptor trap" or "decoy receptor." Inhibitory receptor traps bind to and / or sequester the target, essentially preventing the target from performing a function that could contribute to the disease and / or disorder being treated.

[0082] In one embodiment, the CD40L binding agent is or comprises the binding domain of the CD40 receptor, hi one embodiment, the CD40L binding agent is or comprises a fusion protein comprising the binding domain of the CD40 receptor.

[0083] Stefin A protein variants specifically binding to CD40L The term "Stefin A protein variant" according to the present invention refers to a sequence which is a scaffold of the Stefin A protein and which is derived from a Stefin A protein, for example a mammalian Stefin A protein, for example a human Stefin A protein.

[0084] In one embodiment, the term "Stefin A protein variant" may be used interchangeably with "Stefin A protein variant" or "AFFIMER® protein" to mean substantially the same thing. In one embodiment, the term "Stefin A protein variant" may be used interchangeably with "Stefin A polypeptide variant" or "AFFIMER® protein" to mean substantially the same thing.

[0085] Affimer®, developed by Avacta, is a small, stable protein molecule engineered based on the biological protein stefin A. Affimer® contains two short peptide sequences with random sequences and an N-terminal sequence, which allows it to bind to target substances with high affinity and specificity in a manner similar to that of a monoclonal antibody. Affimer® exhibits significantly improved binding affinity and specificity compared to free peptide libraries, and is significantly smaller and more stable than antibodies. Therefore, it has attracted considerable attention as a next-generation alternative pharmaceutical platform to replace antibodies (e.g., U.S. Patent Nos. 9,447,170 and 8,853,131, which are incorporated herein by reference).

[0086] Avacta has used its Affimer® platform technology to screen anti-CD40L Stefin A protein variants (anti-CD40L AFFIMER® proteins) and filed a patent application (U.S. Patent Application No. 63 / 308629, Avacta Life Sciences Limited, unpublished). In this patent application, the company confirmed that the anti-CD40L-binding Stefin A protein variants can bind to CD40L with excellent affinity and specificity, and can be used as antagonists by inhibiting the activity of CD40L.

[0087] In one embodiment, the Stefin A protein variant of the present invention may have two randomly arranged peptide loops and an N-terminal sequence, which allows it to bind to a target protein with high affinity and specificity in a manner similar to that of an antibody. Target protein-specific binding platforms utilizing such Stefin A protein variants are disclosed in detail in U.S. Patent Nos. 9,447,170 and 8,853,131.

[0088] In one embodiment, the Stefin A protein variant is characterized by being able to bind to the target protein CD40L with high affinity and specificity through manipulation of the Stefin A protein.

[0089] In one embodiment, the Stefin A protein variant that specifically binds to CD40L can be characterized by specifically binding to CD40L and reducing or inhibiting the activity of CD40L.

[0090] In one embodiment, the Stefin A protein variant may contain one or more solvent accessible loops from the wild-type Stefin A protein that binds to CD40L.

[0091] In one embodiment, the Stefin A protein variant has a 10 -6 It can be characterized as binding with a Kd value of M or less.

[0092] In one embodiment, the Stefin A protein variant may be derived from a Stefin A protein having a backbone sequence in which one or more of loop 2 (corresponding to (Xaa)n) and loop 4 (corresponding to (Xaa)m) are replaced with alternative loop sequences, (Xaa)n and (Xaa)m.

[0093] In one embodiment, the Stefin A protein variant may be characterized by having an amino acid sequence represented by Formula I: [Number 1] FR1-(Xaa)n-FR2-(Xaa)m-FR3, wherein the FR1 is a polypeptide sequence comprising the amino acid sequence represented by MIPGGLSEAK PATPEIQEIV DKVKPQLEEK TGETYGKLEA VQYKTQVX (SEQ ID NO: 1) and MIPGGLSEAK PATPEIQEIV DKVKPQLEEK TNETYGKLEA VQYKTQVLA (SEQ ID NO: 741), or a polypeptide sequence having at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%) identity to the amino acid sequence of SEQ ID NO: 1, wherein X can be V or D; FR2 is a polypeptide sequence comprising the amino acid sequence represented by GTNYYIKVRA GDNKYMHLKV FKSL (SEQ ID NO: 2) or a polypeptide sequence having at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%) identity to the amino acid sequence of SEQ ID NO: 2; FR3 is a polypeptide sequence comprising the amino acid sequence represented by EDLVLTGYQV DKNKDDELTG F (SEQ ID NO: 3) or a polypeptide sequence having at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%) identity to the amino acid sequence of SEQ ID NO: 3; and Xaa, in each occurrence, is individually any amino acid residue; n and m are each independently an integer of 3 to 20.

[0094] In one embodiment, (Xaa)n and (Xaa)m are each independently any sequence of 3 to 20 amino acids.

[0095] In one embodiment, the FR1 may have a polypeptide sequence having at least 80%, 85%, 90%, 95%, or 98% homology to SEQ ID NO: 1. In one embodiment, the FR1 may have a polypeptide sequence having at least 80%, 85%, 90%, 95%, or 98% identity to SEQ ID NO: 1.

[0096] In one embodiment, FR2 may have a polypeptide sequence having at least 80%, 85%, 90%, 95%, or 98% homology to SEQ ID NO: 2. In one embodiment, FR2 may have a polypeptide sequence having at least 80%, 85%, 90%, 95%, or 98% identity to SEQ ID NO: 2.

[0097] In one embodiment, the FR3 may have a polypeptide sequence having at least 80%, 85%, 90%, 95%, or 98% identity to SEQ ID NO: 3. In one embodiment, the FR3 may have a polypeptide sequence having at least 80%, 85%, 90%, 95%, or 98% identity to SEQ ID NO: 3.

[0098] In one embodiment, the Stefin A protein variant may comprise an amino acid sequence represented by Formula II: [Mathematics II] MIP-Xaa1-GLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQV-Xaa2-(Xaa)n-Xaa3-TNYYI KVRAGDNKYMHLKVF-Xaa4-Xaa5-Xaa6-(Xaa)m-Xaa7-D-Xaa8-VLTGYQVDKNKDDELTGF (SEQ ID NO: 4), Here, Xaa is, in each case, independently any number of amino acid residues, more preferably 3 or less (preferably 1 or 2) independently selected amino acids; and n and m are each independently an integer of 3 to 20.

[0099] In one embodiment, the Stefin A protein variant can comprise an amino acid sequence having at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identity to the following amino acid sequence: MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVV-(Xaa)n-GTNYYIKVRAGDNKYMHLKVFKSL-(Xaa)m-EDLVLTGYQVDKNKDDELTGF (SEQ ID NO: 742); MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVD-(Xaa)n-GTNYYIKVRAGDNKYMHLKVFKSL-(Xaa)m-EDLVLTGYQVDKNKDDELTGF (SEQ ID NO: 5); or MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVLA-(Xaa)n-GTNYYIKVRAGDNKYMHLKVFKSL-(Xaa)m-EDLVLTGYQVDKNKDDELTGF (SEQ ID NO: 743) Here, Xaa is, in each case, individually any amino acid residue; n and m are each independently an integer of 3 to 20.

[0100] In one embodiment, Xaa1 may be Gly, Ala, Val, Arg, Lys, Asp, or Glu, preferably Gly, Ala, Arg, or Lys, more preferably Gly or Arg.

[0101] In one embodiment, Xaa2 can be Val, Asp, or 'Leu-Ala'.

[0102] In one embodiment, Xaa3 may be Gly, Ala, Val, Ser or Thr, preferably Gly or Ser.

[0103] In one embodiment, Xaa4 may be Arg, Lys, Asn, Gln, Ser or Thr, preferably Arg, Lys, Asn or Gln, more preferably Lys or Asn.

[0104] In one embodiment, Xaa5 may be Gly, Ala, Val, Ser or Thr, preferably Gly or Ser.

[0105] In one embodiment, Xaa6 may be Ala, Val, Ile, Leu, Gly or Pro, preferably Ile, Leu or Pro, more preferably Leu or Pro.

[0106] In one embodiment, Xaa7 may be Gly, Ala, Val, Asp or Glu, preferably Ala, Val, Asp or Glu, more preferably Ala or Glu.

[0107] In one embodiment, Xaa8 may be Ala, Val, Ile, Leu, Arg, or Lys, preferably Ile, Leu, or Arg, more preferably Leu or Arg.

[0108] In one embodiment, n can be 3 to 15, 3 to 12, 3 to 9, 3 to 7, 5 to 7, 5 to 9, 5 to 12, 5 to 15, 7 to 12, or 7 to 9.

[0109] In one embodiment, m can be 3 to 15, 3 to 12, 3 to 9, 3 to 7, 5 to 7, 5 to 9, 5 to 12, 5 to 15, 7 to 12, or 7 to 9.

[0110] In one embodiment, the Xaa's can each independently be any amino acid that can be added to a polypeptide when expressed in a prokaryotic or eukaryotic cell, preferably one of the 20 naturally occurring amino acids.

[0111] In the sequences and formulas of the present invention, the (Xaa)n may be an amino acid sequence selected from SEQ ID NOs: 6 to 125 or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 98% identity to a sequence selected from SEQ ID NOs: 6 to 125. In one embodiment, the (Xaa)n is an amino acid sequence having at least 80%, 85%, 90%, 95%, or 98% identity to an amino acid sequence selected from SEQ ID NOs: 6 to 125.

[0112] In one embodiment of the sequences and formulas of the present invention, the (Xaa)n may be an amino acid sequence selected from SEQ ID NOs: 6 to 125, or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 98% identity to a sequence selected from SEQ ID NOs: 6 to 125. In one embodiment, the (Xaa)n may be an amino acid sequence having at least 80%, 85%, 90%, 95%, or 98% identity to a sequence selected from SEQ ID NOs: 6 to 125.

[0113] [Table 1] JPEG2026504813000002.jpg237128JPEG2026504813000003.jpg237128JPEG2026504813000004.jpg109128

[0114] In one embodiment of the sequences and formulas of the present invention, the (Xaa)m may be an amino acid sequence selected from SEQ ID NOs: 126 to 245 or a sequence having at least 80%, 85%, 90%, 95%, or 98% homology to an amino acid sequence selected from SEQ ID NOs: 126 to 245. In one embodiment, the (Xaa)m may be a sequence having at least 80%, 85%, 90%, 95%, or 98% identity to an amino acid sequence selected from SEQ ID NOs: 126 to 245.

[0115] [Table 2] JPEG2026504813000006.jpg238128JPEG2026504813000007.jpg238128JPEG2026504813000008.jpg91128

[0116] In one embodiment, the Stefin A protein variant comprises an amino acid sequence selected from SEQ ID NOs: 246-365.

[0117] In one embodiment, the Stefin A protein variant comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 98% identity to an amino acid sequence selected from SEQ ID NOs: 246 to 365.

[0118] [Table 3] JPEG2026504813000010.jpg223152JPEG2026504813000011.jpg225153JPEG2026504813000012.jpg224153 JPEG2026504813000013.jpg222151JPEG2026504813000014.jpg224151JPEG2026504813000015.jpg202152

[0119] In one embodiment, the Stefin A protein variant may include small deletions or additions to the Stefin A or Stefin A-derived sequences disclosed herein, in addition to the above-described insertions in Loop 2 and Loop 4. For example, up to 10 amino acid additions or deletions may be made relative to the Stefin A protein or Stefin A protein variant.

[0120] Nucleic Acid Encoding a CD40L-Binding Protein The term "nucleic acid" as used herein refers to a polynucleotide of any length, including DNA, RNA, or a combination of DNA and RNA. In one embodiment, the nucleic acid may be deoxyribonucleotides and ribonucleotides, as well as modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated by DNA or RNA polymerase.

[0121] The term "encoding nucleic acid" as used herein refers to a nucleic acid sequence that encodes a specific protein or polypeptide. If the sequence of a specific protein or polypeptide is published, methods for designing or deriving a nucleic acid that encodes it are well known in the art.

[0122] Thus, a nucleic acid encoding a CD40L-binding agent of the present invention (e.g., a Stefin A protein variant that specifically binds to CD40L) can be readily understood from the above description (e.g., "a Stefin A protein variant that specifically binds to CD40L").

[0123] In one embodiment, the Stefin A protein variant that specifically binds to CD40L has an amino acid sequence encoded by a nucleic acid comprising a coding sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 98% identity to a nucleic acid sequence selected from SEQ ID NOs: 366 to 485. In another embodiment, the Stefin A protein variant that specifically binds to CD40L has an amino acid sequence encoded by a nucleic acid having a coding sequence that hybridizes to a sequence selected from SEQ ID NOs: 366 to 485 under stringent conditions (e.g., hybridization in the presence of 6× sodium chloride / sodium citrate (SSC) at 45°C, followed by washing with 0.2× SSC at 65°C).

[0124] [Table 4] JPEG2026504813000017.jpg228153JPEG2026504813000018.jpg231152JPEG2026504813000019.jpg231153JPEG2026504813000020.jpg232153 JPEG2026504813000021.jpg233153JPEG2026504813000022.jpg225152JPEG2026504813000023.jpg233153JPEG2026504813000024.jpg228151 JPEG2026504813000025.jpg223149JPEG2026504813000026.jpg230152JPEG2026504813000027.jpg231153JPEG2026504813000028.jpg226153 JPEG2026504813000029.jpg232154JPEG2026504813000030.jpg231153JPEG2026504813000031.jpg226152JPEG2026504813000032.jpg125146

[0125] Properties of CD40L-binding agents In one embodiment, the CD40L binding agent (e.g., a Stefin A protein variant) can bind to human CD40L as a monomer with a dissociation constant (KD) of about 1 μM or less, about 100 nM or less, about 40 nM or less, about 20 nM or less, about 10 nM or less, about 1 nM or less, or about 0.1 nM or less.

[0126] In one embodiment, the CD40L binding agent (e.g., a Stefin A protein variant) is TM When measured by assay or other means, approximately 10 3 s -1 (Example: 1 / second unit) or less, 10 4 s -1 Below, 10 5 s -1 Below, 10 6 s -1 It can bind to human CD40L with a Koff (off-rate constant) value equal to or slower than this.

[0127] In one embodiment, the CD40L binding agent (e.g., a Stefin A protein variant) is TM at least about 10 for human CD40L, as measured by assay or other means 3 M -1 s -1 , at least about 10 4 M -1 s -1 , at least about 10 5 M -1 s -1 , at least about 10 6 M -1 s -1 Alternatively, it can bind to human CD40L with a faster Kon (association constant) value.

[0128] In one embodiment, the CD40L binding agent (e.g., a Stefin A protein variant) can bind to human CD40L in a competitive binding assay with an IC50 value of about 1 μM or less, about 100 nM or less, about 40 nM or less, about 20 nM or less, about 10 nM or less, about 1 nM or less, or about 0.1 nM or less.

[0129] In one embodiment, a CD40L binding agent (e.g., a Stefin A protein variant) can have a melting temperature (Tm, the temperature at which the folded and unfolded states are identical) of about 65° C. or higher, about 70° C. or higher, about 75° C. or higher, about 80° C. or higher, or about 85° C. or higher. The relative proportion of folded and unfolded protein can be measured by various methods known in the art (see, "Measuring the conformational stability of a protein" in Protein structure: A practical approach 2:299-321).

[0130] In one embodiment, a CD40L-binding agent (eg, a Stefin A protein variant that specifically binds to CD40L) can be expressed in a recombinant cell and secreted and / or membrane-anchored and displayed on the cell surface.

[0131] In one embodiment, a CD40L-binding agent (eg, a Stefin A protein variant that specifically binds to CD40L) can be expressed in a genetically engineered cell and localized to a particular organ or location within the cell.

[0132] In one embodiment, if the CD40L binding agent (e.g., a Stefin A protein variant that specifically binds to CD40L) expressed by the recombinant cell is secreted, a signal sequence for transport and secretion of the Stefin A protein variant can be included.

[0133] In one embodiment, the signal sequence (also referred to as a signal peptide or leader sequence) may be located at the N-terminus of the CD40L binding agent (e.g., a Stefin A protein variant). The signal sequence functions to target the CD40L binding agent (e.g., a Stefin A protein variant) to the endoplasmic reticulum for secretion. Generally, the signal sequence can be cleaved by a peptidase after transport into the endoplasmic reticulum. Cleavage of the signal sequence from the polypeptide generally occurs at a specific site in the amino acid sequence and may vary depending on the amino acid residue in the signal sequence, but is not limited thereto.

[0134] In one embodiment, the signal sequence is about 5 to 40 amino acids in length (e.g., about 5 to about 7, about 7 to about 10, about 10 to about 15, about 15 to about 20, about 20 to about 25, about 25 to about 30, about 30 to about 35, or about 35 to about 40 amino acids in length).

[0135] In one embodiment, the signal sequence is a native signal peptide, for example, derived from a human protein. In another embodiment, the signal sequence is a non-native signal peptide. For example, in one embodiment, the non-native signal peptide may include one or more substitutions, insertions, and / or deletions in a signal peptide derived from a corresponding native secreted human protein.

[0136] In one embodiment, the signal sequence may be, but is not limited to, a signal peptide derived from a non-IgSF protein family or a variant thereof, such as an immunoglobulin (e.g., IgG heavy chain or IgG-kappa light chain), a cytokine (e.g., IL-2 or CD33), plasma albumin, a human azurocidin preprotein signal sequence, luciferase, trypsinogen (e.g., chymotrypsinogen or trypsinogen), or other signal peptide that enables efficient secretion of proteins from cells.

[0137] Table 5 below lists examples of signal peptides that can be used for secretion of the CD40L-binding agent (eg, a Stefin A protein variant) or a fusion protein thereof, but is not limited thereto.

[0138] [Table 5]

[0139] In one embodiment, when the CD40L-binding agent (e.g., a Stefin A protein variant) is membrane-anchored or expressed on the cell surface, the CD40L-binding agent (e.g., a Stefin A protein variant) may be directly or indirectly linked to a transmembrane domain and expressed as a fusion protein containing the transmembrane domain. Membrane-anchored or cell-surface-expressed CD40L-binding agents (e.g., Stefin A protein variants) are described in detail in the fusion protein section below.

[0140] In one embodiment, the CD40L binding agent is capable of forming multimers. In one embodiment, the CD40L binding agent is capable of forming multimers. In one embodiment, the CD40L binding agent is a multimeric CD40L binding agent. In one embodiment, the multimeric CD40L protein is a dimer, trimer, tetramer, pentamer, or higher multimer. In one embodiment, the CD40L binding agent is a multimeric Stefin A protein variant.

[0141] In some embodiments, the Stefin A protein variants can form multimers.

[0142] In one embodiment, the Stefin A protein variant can form a duplex, triplex, tetraplex, quinplex or higher multimer.

[0143] In one embodiment, the multimer may be formed by covalent or non-covalent binding via interactions between amino acid residues of the Stefin A protein variants.

[0144] In one embodiment, the multimer may be formed through the fusion domain of a fusion protein fused to a Stefin A protein variant.

[0145] In one embodiment, the multimer may be formed by in-line fusion of Stefin A protein variants, and such multimer-form in-line fusion proteins are described in detail below in the section on fusion proteins.

[0146] fusion proteins In one embodiment, the genetically modified cell may be characterized by having introduced therein a nucleic acid encoding a fusion protein comprising a CD40L-binding agent (e.g., a Stefin A protein variant that specifically binds to CD40L). In one embodiment, the fusion protein further comprises an additional insertion, substitution, and / or deletion that modulates or confers additional biological activity to the Stefin A protein variant. For example, the CD40L-binding agent (e.g., a Stefin A protein variant) may be fused to another polypeptide to bind to or inhibit CD40L, or to modulate circulating half-life, therapeutic half-life, stability of the AFFIMER® polypeptide, protease cleavage, dosage, release, or bioavailability, facilitate purification, reduce deamidation, extend shelf life, or improve or change the route of administration.

[0147] For example, the fusion protein may further comprise, but is not limited to, a protease cleavage sequence, a reactive group, an antibody binding domain, a fusion domain for protein expression and purification (e.g., FLAG, poly-His, GST, c-myc, etc.), a binding molecule (e.g., biotin, etc.), or even other therapeutic peptides or proteins.

[0148] In one embodiment, the fusion protein may comprise an additional peptide sequence (fusion domain) fused to one and / or the other end of the CD40L binding agent (eg, a Stefin A protein variant).

[0149] In one embodiment, for example, the fusion domain can be fused to confer expression characteristics such as secretion from a cell, anchoring to the cell surface, or localization within a cell; adding substrate or other recognition sequences for post-translational modification; generating multimeric structures that aggregate through protein-protein interactions; altering (generally extending) serum half-life; or altering tissue localization, tissue clearance, or other ADME properties, as well as adding proteins or peptides with other functions.

[0150] In one embodiment, the fusion domain may further comprise a therapeutic peptide or protein.

[0151] In other embodiments, the fusion domain may be fused for isolation and / or purification of the fusion protein. Well-known examples of fusion domains that facilitate expression or purification include affinity tags such as polyhistidine tags (e.g., His6 tags), Strep II tags, streptavidin-binding peptide (SBP) tags, calmodulin-binding peptide (CBP) tags, glutathione S-transferase (GST) tags, maltose-binding protein (MBP) tags, S-tags, HA tags, c-Myc tags, thioredoxin, protein A, and protein G.

[0152] In one embodiment, the fusion protein may include at least one linker separating the Stefin A protein variant and the fusion domain. In one embodiment, the linker may be inserted between the first polypeptide (e.g., the Stefin A protein variant that specifically binds to CD40L) and the second polypeptide (e.g., another Stefin A protein variant or the fusion domain) of the fusion protein of the invention.

[0153] In one embodiment, the linker can be generally classified into three categories based on its structure: flexible linker, rigid linker, and in vivo cleavable linker.

[0154] In one embodiment, in addition to its basic role of linking the fusion domains, the linker may provide various advantages in the production of fusion proteins, such as improved biological activity, increased expression yield, and favorable pharmacokinetic profile.

[0155] In one embodiment, the linker preferably does not negatively affect the expression, secretion, or functional activity of each domain of the fusion protein, and preferably is not antigenic and does not induce an immune response.

[0156] In one embodiment, the linker is preferably, but not limited to, a GS linker (gly-ser linker) containing glycine and serine residues, and may contain, but is not limited to, threonine and alanine.

[0157] In one embodiment, the length of the linker may vary, for example, from about 1 to 50 amino acids, 1 to 22 amino acids, 1 to 10 amino acids, 1 to 5 amino acids, or 1 to 3 amino acids. In some embodiments, the linker may include a cleavage site.

[0158] In one embodiment, the linker may be characterized as flexible. Flexible linkers are generally preferred, but not limited to, when the fusion domains linked to the Stefin A protein variants of the present invention require some degree of movement or interaction.

[0159] Examples of flexible linkers are described in, but not limited to, Argos P. (1990) "An investigation of oligopeptides linking domains in protein tertiary structures and possible candidates for general gene fusion," J. Mol. Biol. 211:943-958. In one embodiment, the flexible linker can allow flexibility of the fusion protein and mobility of the linked fusion domain. The inclusion of serine or threonine can maintain the stability of the linker in aqueous solution and reduce adverse interactions between the linker and the protein moiety. The most commonly used flexible linker has a sequence consisting primarily of an extension of Gly and Ser residues ("GS" linker). An example of the most widely used flexible linker has the sequence (Gly-Gly-Gly-Gly-Ser)n (SEQ ID NO: 508), but is not limited thereto. In SEQ ID NO: 508, n is an integer of 1 or more, and can be optimized for appropriate distance and interaction of the fusion domain by adjusting the number of Gly-Gly-Gly-Gly-Ser repeats. The flexible linker may further contain amino acids such as Thr, Ala, Lys, and Glu in addition to Gly and Ser.

[0160] In one embodiment, the linker may be rigid. Flexible linkers have the advantage of connecting functional fusion domains and allowing a certain degree of movement, but the lack of rigidity of such linkers can be a limitation in certain fusion protein embodiments, such as in expression yield or biological activity. In this case, rigid linkers have been successfully used to maintain a fixed distance between each domain of the fusion protein and maintain independent function.

[0161] Most natural linkers exhibit an α-helical structure, which forms a rigid and stable structure due to intrasegmental hydrogen bonds and a tightly packed backbone. Therefore, stiff α-helical linkers can be used as rigid spacers between protein domains (George et al. (2002) "An analysis of protein domain linkers: their classification and role in protein folding" Protein Eng. 15(11):871-9).

[0162] Generally, rigid linkers adopt an α-helical structure or contain multiple Pro residues, exhibiting a relatively rigid structure. In many cases, rigid linkers can separate functional fusion domains more efficiently than flexible linkers. The length of the linker can be easily optimized by adjusting the number of repeats to achieve an optimal distance between each domain. Consequently, rigid linkers can be used when spatial separation of domains is important for preserving the stability or biological activity of the fusion protein. In this regard, an α-helix-forming linker having the sequence (EAAAK)n (SEQ ID NO: 509) is commonly used in the construction of many fusion proteins. Other examples of rigid linkers include, but are not limited to, the Pro-rich sequence (XP)n (where X is any amino acid, preferably Ala, Lys, or Glu).

[0163] In one embodiment, Table 6 below lists examples of linkers that can be used for secreting the CD40L binding agent (e.g., Stefin A protein variant) of the present invention or its fusion protein, but is not limited thereto.

[0164] [Table 6]

[0165] Further examples of linkers that can be used in the fusion proteins include, but are not limited to: SerGly, GGSG (SEQ ID NO: 519), GSGS (SEQ ID NO: 520), GGGS (SEQ ID NO: 521), S(GGS)n (SEQ ID NO: 522) (n is 1 to 7), GRA, poly(Gly), poly(Ala), GGGSGGG (SEQ ID NO: 523), ESGGGGVT (SEQ ID NO: 524), LESGGGGVT (SEQ ID NO: 525), GRAQVT (SEQ ID NO: 526), ​​WRAQVT (SEQ ID NO: 527), and ARGRAQVT (SEQ ID NO: 528). In one embodiment, when an Fc domain is fused to the Stefin A protein variant, the hinge region may also be considered as a linker.

[0166] In one embodiment, the fusion protein may include one or more linker domains selected from the following: SGTTSGTTRLLSGHTCFTLTGLLGTLVTMGLLT (SEQ ID NO: 529) SGTSPGLSAGATVGIMIGVLVGVALI (SEQ ID NO: 530) SAPVLSAVATVGITIGVLARVALI (SEQ ID NO: 531) SSPDLSAGTAVSIMIGVLAGMALI (SEQ ID NO: 532) TLGGNSASYTFVSLLFSAVTLLLLC (SEQ ID NO: 533) SGTSPGLSAGATVGIMIGVLVGVALI (SEQ ID NO: 534)

[0167] In one embodiment, yet another modification that may be made to the polypeptide sequence of the CD40L binding agent (e.g., an AFFIMER® polypeptide sequence) or a flanking polypeptide moiety provided as part of a fusion protein is the inclusion of one or more sequences that serve as sites for enzymatic post-translational modifications, including, but not limited to, glycosylation, acetylation, acylation, lipid-modification, palmitoylation, palmitate addition, phosphorylation, glycolipid-linkage, and the like.

[0168] In one embodiment, a nucleic acid encoding the fusion protein is introduced into a genetically modified cell, which expresses the fusion protein, which can be secreted extracellularly and / or anchored to the cell membrane and expressed on the cell surface.

[0169] In one embodiment, the fusion protein can be expressed in a recombinant cell and localized to a specific organ or location within the cell.

[0170] In one embodiment, the fusion protein is a secreted fusion protein and / or a membrane-anchored fusion protein.

[0171] In one embodiment, when the fusion protein is a secretory fusion protein, it may contain a signal sequence, which, unless otherwise specified, may have the same characteristics as those described in the description of the Stefin A protein variant.

[0172] In one embodiment, the fusion protein may further comprise one or more fusion domains.

[0173] The term "fusion domain" according to the present invention refers to an additional domain or moiety that can be fused to a CD40L binding agent of the present invention (eg, a Stefin A protein variant that specifically binds to CD40L).

[0174] In one embodiment, the fusion protein may comprise a fusion domain selected from the group consisting of, but not limited to, an antigen-binding protein (domain), a cytokine, a half-life extension domain, a growth factor, an enzyme, and a cell-penetrating domain.

[0175] In one embodiment, the fusion protein may further comprise a therapeutic peptide or protein.

[0176] In one embodiment, the term "therapeutic peptide or protein" refers to any peptide or protein that has a preventive or therapeutic effect against a specific disease. In one embodiment, the therapeutic peptide or protein may be a Stefin A protein variant (which may be the same as or different from the Stefin A protein variant that specifically binds to CD40L of the present invention), and includes, but is not limited to, peptides and proteins reported in the art to have a preventive or therapeutic effect against a specific disease.

[0177] In one embodiment, the fusion protein may comprise a binding domain.

[0178] In one embodiment, when the fusion protein contains a binding domain, it can have multispecificity, binding to more than one target molecule in addition to CD40L.

[0179] In one embodiment, the binding domain may be characterized as being selected from the group consisting of, but not limited to, a Stefin A protein variant (which may be the same as or different from the Stefin A protein variant of the present invention that specifically binds to CD40L), an antibody or a fragment thereof, an antibody analog, an antigen-binding peptide, a ligand-binding site of a receptor (e.g., a receptor-trap polypeptide), a receptor-binding ligand (e.g., a cytokine or growth factor), an engineered T-cell receptor, and an enzyme or a catalytic fragment thereof.

[0180] In one embodiment, further examples of the binding domain include, but are not limited to, adnectins / monobodies, affilins, affibodies, affitins, anticalins, atrimers, avimers, bicyclic peptides, C7 peptide, centyrin, carbohydrate-binding modules (CBMs), Cys-knots, darpins, El-tandems, fynomers, knottins, Kunitz domains, O-bodies, pronectin, ScFv, Sac7d, Sso7d, and Tn3.

[0181] In one embodiment, the fusion domain fused to the Stefin A protein variant may be the same or a different Stefin A protein variant that specifically binds to CD40L and / or a Stefin A protein variant that specifically binds to another target.

[0182] In one embodiment, when the fusion domain is a Stefin A protein variant that specifically binds to CD40L, the two Stefin A protein variants contained in the fusion protein of the present invention can bind to the same or different sites on CD40L. In one embodiment, the fusion protein can bind to two sites (biparatopic) or two or more sites (multiparatopic) on CD40L.

[0183] In one embodiment, the fusion protein may comprise the Fc portion of an immunoglobulin. For example, if the fusion protein comprises an Fc portion, it may bind to an Fc receptor and activate Fc receptor-positive cells, thereby initiating or increasing the expression of cytokines and / or costimulatory antigens, and inducing antibody-dependent cellular cytotoxicity (ADCC).

[0184] In one embodiment, the fusion domain may be, for example, but is not limited to, an immune checkpoint protein, an immune co-stimulatory receptor, a receptor (or receptor agonist), a cytokine, a growth factor, or a tumor-associated antigen.

[0185] In one embodiment, the cytokine is used as a collective term for secreted proteins that play an important role in signal transduction between cells, including, but not limited to, chemokines, interferons, lymphokines, interleukins, tumor necrosis factors, etc.

[0186] In one embodiment, the growth factor refers to a naturally occurring substance or variant thereof that can stimulate cell proliferation, wound healing, and / or cell differentiation, including, but not limited to, GH, EGF, VEGF, FGF, bFGF, HGF, BMPs, M-CSF, G-CSF, GM-CSF, EPO, GDNF, IGF, KGF, BDNF, NGF, PDGF, TPO, and TGF.

[0187] In one embodiment, the term "enzyme" refers collectively to proteins that catalyze biological reactions, including, but not limited to, α-chymotrypsin, lysozyme, urate oxidase, acetylcholinesterase, Thermomyces lanuginosa lipase, glucose oxidase, superoxide dismutase, caspase, β-glucosidase, Trametes versicolor laccase, alcohol oxidase, Cas9, Cas12, Cas13, Cas14, zinc finger nuclease, TALLEN, dimethyl sulfoxide, uricase, agalsidase beta, agalsidase alpha, imiglucerase, taliglucerase alpha, velaglucerase alpha, alglucerase, sebelipase alpha, laronidase, idursulfase, elosulfase alpha, galsulfase, and alglucosidase alpha.

[0188] In one embodiment, the cell-penetrating peptide refers to a short peptide that promotes cellular uptake and absorption of various molecules. In one embodiment, when the fusion protein includes a cell-penetrating peptide, the Stefin A protein variant can be absorbed into cells. Examples of cell-penetrating peptides include, but are not limited to, Tat, penetratin, transportant, Pept1, Pept2, pVEC, DPV3, DPV6, R8, R9, MPG, MAP, Bip4, C105Y, and melittin.

[0189] In one embodiment, the fusion protein can be anchored to the cell membrane or expressed on the cell surface of the genetically engineered cell.

[0190] In one embodiment, when the fusion protein is anchored to a cell membrane or expressed on the cell surface, it may further comprise a transmembrane domain. The term "transmembrane domain" as used herein refers to a protein domain that spans the cell membrane. In one embodiment, the transmembrane domain preferably has an α-helical structure, but is not limited thereto.

[0191] In one embodiment, the transmembrane domain may be, for example, a transmembrane domain derived from CD3, CD4, CD5, CD8, CD28, CD99, PDGFR, PTGFRN, etc., or a variant thereof, but is not limited thereto.

[0192] In one embodiment, the fusion protein may further comprise a hinge domain in addition to the transmembrane domain. The term "hinge domain" as used herein refers to a series of amino acid sequences located between the transmembrane domain and the extracellular domain of a membrane-anchored protein. In one embodiment, the hinge domain may be located between the CD40L binder (e.g., a Stefin A protein variant that specifically binds to CD40L) and the transmembrane domain.

[0193] In one embodiment, the hinge domain may be, but is not limited to, a hinge domain derived from an immunoglobulin (e.g., IgG1, IgG4, IgD, etc.), PDGFR, PTGFRN, etc., or a variant thereof. In some embodiments, the CD40L-binding fusion protein comprises a hinge domain derived from an immunoglobulin (e.g., IgG1, IgG4, IgD, etc.).

[0194] In one embodiment, the fusion protein may further comprise a coiled-coil domain. The term "coiled-coil domain" as used herein refers to a structural motif of a protein in which two to seven alpha helices are wound like a rope strand. Preferably, the coiled-coil domain is characterized by having two or three alpha helices wound around it.

[0195] In one embodiment, the coiled-coil domain may be, but is not limited to, a coiled-coil domain derived from leucine zipper, foldon, cardiac phospholamban, a water-soluble analogue of a membrane phospholamban, cartilage oligomeric matrix protein (COMP), thrombospondin 3, thrombospondin 4, or vasodilator-stimulated phosphoprotein (VASP), or a variant thereof.

[0196] In one embodiment, the coiled-coil domain may be located between the Stefin A protein variant that specifically binds to CD40L and the transmembrane domain.

[0197] In one embodiment, the fusion protein may further comprise a virus-derived peptide or protein, such as syncytin-1, syncytin-2, VSVG (vesicular stomatitis virus glycoprotein), Nipah virus F and G proteins, measles virus F and H proteins, tupaia paramyxovirus F and H proteins, paramyxovirus F and G proteins, F and H proteins, or F and HN proteins, Hendra virus F and G proteins, Henipavirus F and G proteins, Morbilivirus F and H proteins, respirovirus F and HN proteins, Sendai virus F and HN proteins, or the like. The F and HN proteins may include, but are not limited to, the F and HN proteins of a rubulavirus, the F and HN proteins of a rubulavirus, or the F and HN proteins of an avulavirus, or variants thereof, or combinations thereof.

[0198] In one embodiment, the fusion protein may further comprise an immunomodulatory domain or an intracellular signaling domain.

[0199] In one embodiment, the immunomodulatory domain or intracellular signaling domain refers to a domain located in the cytoplasmic direction of a membrane-anchored protein, which activates or suppresses an immune response when a target antigen binds to the extracellular domain.

[0200] In one embodiment, the immunomodulatory domain or intracellular signaling domain may be, but is not limited to, an immunomodulatory domain derived from CD3, CD28, CD40L, ICOS, OX40, 4-1BB, TNFR2, etc.

[0201] In one embodiment, the fusion protein may be a chimeric antigen receptor (CAR). In one embodiment, when the fusion protein is a chimeric antigen receptor, the CD40L binder (e.g., a Stefin A protein variant that specifically binds to CD40L) may function as an extracellular binding domain.

[0202] In one embodiment, when the fusion protein is a chimeric antigen receptor, it may further comprise the transmembrane domain, hinge domain, and intracellular signaling domain, but is not limited thereto. The CD40L binder of the present invention (e.g., a Stefin A protein variant that specifically binds to CD40L) can be easily designed and prepared by modifying the extracellular antigen-binding domain of various chimeric antigen receptors or analogs thereof known in the art.

[0203] In one embodiment, the fusion protein may further comprise a localization domain. In one embodiment, when the fusion protein is expressed intracellularly, it is preferable that the fusion protein further comprises a localization domain. The term "localization domain" of the present invention refers to a peptide or protein sequence that functions to localize a protein to a specific organ or location within a cell. In one embodiment, the localization domain may be an organ-specific localization domain or an intracellular protein localization domain.

[0204] In one embodiment, the localization domain may be, for example, a nucleus-specific localization domain derived from VACM-1 / CUL5, CXCR4, VP1, 53BP1, ING4, IER5, ERK5, Hrp1, UL79, EWS, PTHrP, Pho4, and rpL23a; a mitochondrial-specific localization domain derived from ATP synthase F1b, cytochrome c oxidase polypeptide VIII, SOD2, citrate synthase, Tu translation elongation factor, and the like; or a peroxisomal localization domain derived from PTS1, PTS2, and the like, but is not limited thereto.

[0205] In one embodiment, the fusion protein may further comprise a half-life extension domain. The half-life extension domain refers to a domain or moiety fused to the Stefin A protein variant of the present invention for the purpose of extending its half-life. For example, the half-life extension domain may include, but is not limited to, an Fc domain, an Fc-binding protein or peptide, albumin (e.g., HSA), an albumin-binding protein or peptide, transferrin, or a transferrin-binding protein or peptide.

[0206] Pharmacokinetic and ADME (Absorption, Distribution, Metabolism, Excretion) properties of engineered fusion proteins The term "half-life" as used herein means the time it takes for an active ingredient, such as a Stefin A protein variant or fusion protein of the present invention, to lose half of its pharmacological or physiological activity or concentration. Biological half-life can be affected by the elimination, excretion, degradation (e.g., enzymatic degradation) of the substance, or absorption and concentration in a particular organ or tissue of the body.

[0207] In one embodiment, biological half-life can be assessed by determining the time it takes for the plasma concentration of a substance to reach half of its normal level ("plasma half-life").

[0208] In one embodiment, the CD40L binding agent (e.g., a Stefin A protein variant) or a fusion protein containing the same may not have an adequate half-life and / or pharmacokinetic profile (PK profile), and may further comprise a half-life extending domain (or half-life extending moiety) to further improve the half-life or PK profile.

[0209] The term "half-life prolonging domain" of the present invention refers to a pharmaceutically acceptable moiety, domain, or molecule that is conjugated or fused to the Stefin A protein mutant of the present invention directly or indirectly via a linker or the like.

[0210] In one embodiment, the half-life extending domain can improve or alter the pharmacokinetic or biophysical properties of a CD40L binding agent (e.g., a Stefin A protein mutant), including, but not limited to, preventing or mitigating in vivo proteolysis or reduced activity or modification of the CD40L binding agent (e.g., a Stefin A protein mutant), extending half-life, increasing absorption rate, reducing toxicity, improving solubility, reducing protein aggregation, increasing biological activity and / or target selectivity of the CD40L binding agent (e.g., a modified AFFIMER® polypeptide), increasing productivity, and / or reducing immunogenicity.

[0211] In one embodiment, the half-life prolonging domain may include, but is not limited to, a water-soluble polymer such as polyethylene glycol (PEG) or discrete PEG, hydroxyethyl starch (HES), lipid, branched or unbranched acyl group, branched or unbranched C8-C30 acyl group, branched or unbranched alkyl group, or branched or unbranched C8-C30 alkyl group; and a non-proteinaceous half-life prolonging moiety such as serum albumin, transferrin, Adnectin (e.g., albumin-binding Adnectin or pharmacokinetically extended Adnectin (PKE adnectin)), Fc domain, unstructured polypeptide (e.g., amino acids Pro, Ala, and / or Ser) such as XTEN and PAS polypeptide, and fragments thereof. In one embodiment, the half-life prolonging domain can prolong the half-life of the Stefin A protein variant of the present invention circulating in the serum of a subject, compared to the half-life of a fusion protein not comprising the half-life prolonging domain.

[0212] In one embodiment, the half-life may be extended by, but is not limited to, about 1.2-fold, 1.5-fold, 2.0-fold, 3.0-fold, 4.0-fold, 5.0-fold, or 6.0-fold or more. In another embodiment, the half-life may be extended by, but is not limited to, at least 6 hours, at least 12 hours, at least 24 hours, at least 72 hours, at least 96 hours, or at least 1 week after administration in vivo, compared to a protein without the half-life extending moiety.

[0213] In one embodiment, a preparation example of a fusion protein containing the half-life prolonging domain is as follows, but is not limited thereto:

[0214] - Genetic fusions of a CD40L binding agent (e.g., a stefin A protein variant) sequence with a naturally long half-life protein or protein domain (e.g., an Fc domain fusion, a transferrin (Tf) fusion, or an albumin fusion). For example, Beck et al. (2011) “Therapeutic Fc-fusion proteins and peptides as successful alternatives to antibodies. MAbs.3:1-2; Czajkowsky et al. (2012) “Fc-fusion proteins: new developments and future perspectives. EMBO Mol Med.4:1015-28; Huang et al. technology”Curr Opin Biotechnol.2009;20:692-9;Keefe et al.(2013) “Transferrin fusion protein therapies:acetylcholine receptor-transferrin fusion protein as a model.In:Schmidt S,editor.Fusion protein technologies for biopharmaceuticals:applications and challenges.Hoboken:Wiley;p.345-56;Weimer et al. al. (2013) "Recombinant albumin fusion proteins. In: Schmidt S, editor. Fusion protein technologies for biopharmaceuticals: applications and challenges. Hoboken: Wiley; 2013. p. 297-323; Walker et al. (2013) "Albumin-binding fusion proteins in the development of novel long-acting therapeutics. In: Schmidt S, editor.Fusion protein technologies for biopharmaceuticals: applications and challenges. Hoboken: Wiley; 2013. pp. 325-43.

[0215] - a genetic fusion of a CD40L binding agent (e.g., a Stefin A protein variant) with an inert polypeptide (e.g., XTEN (also known as recombinant PEG or rPEG), HAP (homoamino acid polymer, HAPylation), proline-alanine-serine polymer (PAS; PASylation), or elastin-like peptide (ELP; ELPylation)).For example, see Schellenberger et al. (2009) "A recombinant polypeptide extends the in vivo half-life of peptides and proteins in a tunable manner. Nat Biotechnol. 2009;27:1186-90; Schlapschy et al. Fusion of a recombinant antibody fragment with a homo-amino-acid polymer: effects on biophysical properties and prolonged plasma half-life. Protein Eng Des Sel. 2007;20:273-84; Schlapschy (2013) PASylation: a biological alternative to PEGylation for extending the plasma half-life of pharmaceutically active proteins. Protein Eng Des Sel. 26:489-501. Floss et al. (2012) "Elastin-like polypeptides revolutionize recombinant protein expression and their biomedical application. Trends Biotechnol. 28:37-45. Floss et al. "ELP-fusion technology for biopharmaceuticals. In: Schmidt S, editor. Fusion protein technologies for biopharmaceuticals: application and challenges. Hoboken: Wiley; 2013. p. 372-98. etc.

[0216] - Increase in hydrodynamic radius by chemical conjugation of pharmacologically active peptides or proteins to repeating chemical moieties such as PEG (PEGylation) or hyaluronic acid. For example, (Caliceti et al. (2003) “Pharmacokinetic and biodistribution properties of poly(ethylene glycol)-protein conjugates” Adv Drug Delivery Rev. 55:1261-77; Jevsevar et al. (2010) PEGylation of therapeutic proteins. Biotechnol J 5:113-28; Kontermann (2009) “Strategies to extend plasma half-lives of See "recombinant antibodies" BioDrugs.23:93-109; Kang et al. (2009) "Emerging PEGylated drugs" Expert Opin Emerg Drugs.14:363-80; and Mero et al. (2013) "Conjugation of hyaluronan to proteins" Carb Polymers.92:2163-70.

[0217] - Significantly increasing the negative charge of the fusion of pharmacologically active peptides or proteins by polysialylation; or alternatively, (b) the fusion of negatively charged, highly sialylated peptides (e.g., carboxy-terminal peptide [CTP; of chorionic gonadotropin (CG) b-chain], a well-known method for extending the half-life of natural proteins such as the human CG b-subunit). For example, (Gregoriadis et al. (2005) “Improving the therapeutic efficacy of peptides and proteins: a role for polysialic acids” Int J Pharm.2005;300:125-30; Duijkers et al. “Single pharmacokinetics and effects on follicular growth and hormonal serums of a long-acting recombinant FSH preparation (FSHCTP) in healthy pituitary-suppressed "Design of a longacting follitropin agonist by fusing the C-terminal sequence of the chorionic gonadotropin beta subunit to the follitropin beta subunit" (1992) Proc Natl Acad Sci USA.89:4304-8.35; See O-glycosylation. etc.

[0218] - Non-covalent binding to biologically active proteins through peptide or protein-binding domains, typically to proteins with long half-lives such as HSA, IgG, transferrin or fibronectin. For example, Andersen et al. (2011) “Extending half-life by indirect targeting of the neonatal Fc receptor(FcRn) using a minimal albumin binding domain” J Biol Chem.286:5234-41; O’Connor-Semmes et al. (2014) “GSK2374697, a novel albumin-binding domain antibody(albudAb), extends systemic exposure of extendin-4:first See "study in humans-PK / PD and safety" Clin Pharmacol Ther.2014;96:704-12.Sockolosky et al. (2014) "Fusion of a short peptide that binds immunoglobulin G to a recombinant protein substantially increases its plasma half-life in mice" PLoS One.2014;9:e102566.

[0219] Classical genetic fusion to long-lived serum proteins offers an alternative method for half-life extension, distinct from chemical conjugation to PEG or lipids. Antibody Fc domains and human serum albumin have traditionally been used as fusion domains for half-life extension. Fc fusion involves fusing a peptide, protein, or receptor exodomain to the Fc portion of an antibody. Fc-albumin fusion proteins not only increase the size and half-life of peptide drugs, but also utilize the body's natural recycling mechanism, the neonatal Fc receptor (FcRn). The pH-dependent binding of these proteins to FcRn prevents fusion protein degradation in the endosome. Fusion proteins fused with such proteins can have half-lives of approximately 3 to 16 days, which is significantly longer than the half-lives of typical PEGylated or lipidated peptides. Fusion of antibody Fc domains can improve the solubility and stability of peptide or protein drugs. One example of a peptide-Fc fusion is dulaglutide, a GLP-1 receptor agonist currently in late-stage clinical trials. Human serum albumin, the same protein used by fatty-acylated peptides, is another popular fusion partner used for half-life extension. Albiglutide is a GLP-1 receptor agonist based on this platform. The key difference between the Fc domain and albumin is that Fc is typically used dimerized, while HSA is a monomeric structure, and the fusion peptide can appear as a dimer or monomer depending on the choice of fusion partner. The dimeric nature of antibodies and / or AFFIMER®-Fc fusions can generate avidity effects, which may or may not be desirable depending on the target, if the antibody and / or AFFIMER® target are spaced sufficiently closely or are themselves dimeric.

[0220] Classical genetic fusion to long-lived serum proteins offers an alternative method for half-life extension, distinct from chemical conjugation to PEG or lipids. Antibody Fc domains and human serum albumin have traditionally been used as fusion domains for half-life extension. Fc fusion involves fusing a peptide, protein, or receptor exodomain to the Fc portion of an antibody. Fc-albumin fusion proteins not only increase the size and half-life of peptide drugs, but also utilize the body's natural recycling mechanism, the neonatal Fc receptor (FcRn). The pH-dependent binding of these proteins to FcRn prevents fusion protein degradation in endosomes. Fusion proteins incorporating such proteins can have half-lives of approximately 3 to 16 days, which is significantly longer than the half-lives of typical PEGylated or lipidated peptides. Fusion of antibody Fc domains can improve the solubility and stability of peptide or protein drugs. An example of a peptide-Fc fusion is dulaglutide, a GLP-1 receptor agonist currently in late-stage clinical trials. Human serum albumin, the same protein used by fatty acylated peptides, is another popular fusion partner used for half-life extension.

[0221] The main difference between the Fc domain and albumin is that the Fc domain is generally used for dimerization, while the HSA domain is used in a monomeric structure. The fusion protein of the Stefin A protein variant and the Fc domain of the present invention can be used as a dimer, but is not limited thereto.

[0222] Fc fusion In one embodiment, the fusion protein can include an immunoglobulin Fc domain (Fc domain) or a fragment or variant thereof, e.g., a functional Fc region. In one embodiment, the Fc region is an FcγR null-binding Fc region. In one embodiment, the fusion protein can include at least one CD40L-binding Stefin A protein variant linked directly or indirectly via a peptide backbone to an immunoglobulin Fc region. In one embodiment, the fusion protein can include the Fc region of antibody 62 / 179 (to enhance effector function and pharmacokinetic properties) and a CD40L-binding agent (e.g., a Stefin A protein variant that specifically binds to CD40L) as part of the same peptide. Alternatively, the immunoglobulin Fc region can be indirectly linked to at least one CD40L-binding Stefin A protein variant via a linker. A variety of linkers for use in the fusion proteins of the present invention are known in the art. In one embodiment, the fusion protein comprising an Fc domain may be used in a dimerized form, and may be used in a homodimerized or heterodimerized form.

[0223] In one embodiment, when the fusion protein includes an Fc domain, stability can be improved and antibody-like properties conferred by the Fc region can be utilized. In one embodiment, when the fusion protein includes an Fc domain, the Fc domain can participate in the salvage neonatal FcRn receptor pathway, which involves FcRn-mediated recycling of the fusion protein to the cell surface after intracellular entry, thereby avoiding lysosomal degradation and releasing it into the bloodstream, thereby extending its half-life. In addition to extending its half-life, fusion of the Fc domain can also be useful for the isolation and purification of the fusion protein of the present invention.

[0224] The Fc domain can include the constant region of an antibody excluding the first constant region immunoglobulin domain. Thus, the Fc domain refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, the last three constant region immunoglobulin domains of IgE and IgM, and the flexible hinge N-terminal to these domains. In the case of IgA and IgM, the Fc domain can include the J chain. In the case of IgG, the Fc domain can include immunoglobulin domains Cγ2 and Cγ3 and the hinge between Cγ1 and Cγ2. While the boundaries of the Fc domain can vary, the human IgG heavy chain Fc region is generally defined to include the carboxyl-terminal residues C226 or P230. Fc can refer to this region alone or in the context of a whole antibody, antibody fragment, or Fc fusion protein. Polymorphisms are observed at various Fc positions, and these are also encompassed by the Fc domains used in the present invention.

[0225] In one embodiment, the Fc domain is a "functional Fc region," which refers to an Fc domain or a fragment thereof that maintains the ability to bind to FcRn. The functional Fc region binds to FcRn but does not have an effector function. The ability of the Fc region or a fragment thereof to bind to FcRn can be determined by standard binding assays known in the art. The "effector function" can include, for example, C1q binding; complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down-regulation of cell surface receptors (e.g., B cell receptors), etc. The effector function can be evaluated using various assays known in the art.

[0226] In one embodiment, the Fc domain may be derived from IgG1, IgG2, IgG3, or IgG4.

[0227] In one embodiment, the Fc domain is derived from an IgG1 subclass, for example, the Fc domain may comprise the following sequence: DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 535).

[0228] In one embodiment, the Fc region included in the fusion protein can include a hinge region. For example, the hinge region can include core hinge residues located at positions 1-16 of the human IgG1 Fc domain sequence (e.g., DKTHTCPPCPAPELLG (SEQ ID NO: 536)). In one embodiment, the fusion protein can have a multimeric structure (e.g., a dimer) due in part to cysteine ​​residues at positions 6 and 9 within the hinge region of the human IgG1 immunoglobulin Fc domain sequence. In one embodiment, the hinge region can further include residues derived from the CH1 and CH2 regions flanking the core hinge sequence of the IgG immunoglobulin Fc domain sequence. In one embodiment, the hinge sequence can include or consist of GSTHTCPPCPAPELLG (SEQ ID NO: 537) or EPKSCDKTHTCPPCPAPELLG (SEQ ID NO: 538).

[0229] In one embodiment, the hinge region may include one or more substitutions that preferably confer pharmacokinetic, biophysical, and / or biological properties, for example, the hinge region may include or consist of the following sequence: EPKSCDKTHTCPPCPAPELLGGPS (SEQ ID NO: 539); EPKSSDKTHTCPPCPAPELLGGPS (SEQ ID NO: 540); EPKSSDKTHTCPPCPAPELLGGSS (SEQ ID NO: 541); EPKSSGSTHTCPPCPAPELLGGSS (SEQ ID NO: 542); DKTHTCPPCPAPELLGGPS (SEQ ID NO: 543); and DKTHTCPPCPAPELLGGSS (SEQ ID NO: 544).

[0230] In one embodiment, the 18th residue P of the human IgG1 immunoglobulin Fc domain sequence may be substituted with S to eliminate Fc effector function; an example of such a substitution is as follows: EPKSSDKTHTCPPCPAPELLGGSS (SEQ ID NO: 541); EPKSSGSTHTCPPCPAPELLGGSS (SEQ ID NO: 542); and DKTHTCPPCPAPELLGGSS (SEQ ID NO: 544).

[0231] In one embodiment, DK at positions 1-2 of the human IgG1 immunoglobulin Fc domain may be substituted with GS to remove a potential clip site, and an example of such a substitution is as follows: EPKSSGSTHTCPPCPAPELLGGSS (SEQ ID NO: 542).

[0232] In one embodiment, residue 103, C, of ​​the heavy chain constant region (e.g., CH1-CH3) of human IgG can be substituted with S to prevent inappropriate cysteine ​​bond formation in the absence of a light chain. An example of such a substitution is as follows: EPKSSDKTHTCPPCPAPELLGGPS (SEQ ID NO: 540), EPKSSDKTHTCPPCPAPELLGGSS (SEQ ID NO: 541), and EPKSSGSTHTCPPCPAPELLGGSS (SEQ ID NO: 542).

[0233] In one embodiment, the Fc domain may be an Fc domain derived from a mammal, preferably a human Fc domain. In one embodiment, the Fc domain may be an Fc domain derived from IgG1, IgG2, IgG3, or IgG4. In one embodiment, the Fc domain may have about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity with a native Fc domain and / or the Fc region of a parent polypeptide. In one embodiment, the Fc domain may have 90% or more sequence identity with a native Fc domain and / or the Fc domain of a parent polypeptide.

[0234] In one embodiment, the Fc domain may comprise an amino acid sequence selected from SEQ ID NOs: 545 to 558 or an Fc domain of SEQ ID NOs: 545 to 558 described in the Examples. In one embodiment, a C-terminal lysine of the Fc domain should be understood as an optional configuration for a fusion protein comprising an Fc domain. In one embodiment, the Fc domain comprises an amino acid sequence selected from SEQ ID NOs: 545 to 558, and the C-terminal lysine is omitted. In one embodiment, the Fc domain comprises an amino acid sequence selected from SEQ ID NOs: 545 to 558. In one embodiment, the Fc domain comprises an amino acid sequence selected from SEQ ID NOs: 545 to 558, and the C-terminal lysine is omitted.

[0235] [Table 7] JPEG2026504813000036.jpg170154

[0236] As used herein, the term "antibody-dependent cellular cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which cytotoxic effector cells of secretory immunoglobulins bound to Fc receptors (FcRs) present on specific cytotoxic cells specifically bind to antigen-bearing targets, thereby exerting cytotoxicity and killing the cells.

[0237] In one embodiment, the fusion protein may comprise an Fc domain that lacks or has reduced ADCC and / or complement activation or effector function. For example, the Fc domain may comprise a naturally inactivated constant region of an IgG2 or IgG4 isotype or a mutated IgG1 constant region. Such modified Fc domains are described in, for example, EP 0307434. For example, the Fc domain may comprise, but is not limited to, substitutions of alanine residues at positions 235 and 237 (EU index numbering).

[0238] In one embodiment, the fusion protein comprises an Fc domain and retains some or all of the Fc function. For example, but not limited to, the fusion protein comprises the Fc domain of human IgG1 or IgG3. The level of effector function of the Fc domain of the present invention can be adjusted by known methods. For example, mutations in the CH2 domain, such as one or more mutations selected from positions 239, 332, and 330 of the CH2 of IgG1, such as S239D, I332E, and A330L, can be included to improve the effector function of the antibody and / or alter the glycosylation profile, e.g., to reduce fucosylation in the Fc region.

[0239] Albumin fusion In one embodiment, the fusion protein can include an albumin sequence or a fragment thereof. In one embodiment, the albumin sequence or a fragment thereof can be fused to a polypeptide sequence containing a Stefin A protein variant through conjugation or chemical linkage. In one embodiment, the albumin, albumin variant, or albumin fragment can be human serum albumin (HSA), a variant, or a fragment thereof. In one embodiment, albumin serum proteins other than HSA can be derived from, for example, cynomolgus monkeys, cows, dogs, rabbits, and mice. Among non-human species, BSA is structurally most similar to HSA (Kosa et al., (2007) J Pharm Sci. 96(11):3117-24). In one embodiment, the albumin can be a non-human serum albumin, such as, but not limited to, canine serum albumin or bovine serum albumin.

[0240] Mature HSA, a 585 amino acid polypeptide (approximately 67 kDa) with a serum half-life of approximately 20 days, is primarily responsible for maintaining colloid osmotic pressure, blood pH, and the transport and distribution of numerous endogenous and exogenous ligands. Serum albumin proteins have three structurally homologous domains (domains I, II, and III), exhibit a mostly α-helical conformation, and are highly stabilized by 17 disulfide bonds. In one embodiment, an antibody and / or AFFIMER® formulation may be an albumin fusion protein comprising at least one antibody and / or AFFIMER® polypeptide sequence and mature human serum albumin (e.g., SEQ ID NO: 559) or a variant or fragment thereof, which can maintain the PK and / or biodistribution properties of mature albumin at a desired level in the fusion protein.

[0241] (SEQ ID NO: 559)

[0242] In one embodiment, the albumin sequence may be separated from other flanking sequences of the Stefin A protein variant or fusion protein by a linker sequence.

[0243] Unless otherwise specified, in the present invention, albumin or mature albumin refers to human serum albumin (HSA). However, full-length HSA may include an 18-amino acid signal peptide (MKWVTFISLLFLFSSAYS (SEQ ID NO: 486)) followed by a 6-amino acid prodomain sequence (RGVFRR (SEQ ID NO: 560)), and these 24 amino acids may be designated the preprodomain. In one embodiment, the fusion protein may be expressed and secreted via the HSA preprodomain. In one embodiment, a fusion protein comprising a Stefin A protein variant and HSA may include the signal sequence described for the Stefin A protein variant and may be expressed and secreted via the signal sequence.

[0244] In one embodiment, the serum albumin may be covalently coupled to the Stefin A protein variant or a fusion protein containing the same by a bond other than an amide bond, such as by chemical conjugation through an amino acid side chain of the Stefin A protein variant or a fusion protein containing the same.

[0245] Serum Binding Domains In one embodiment, the fusion protein can include a serum-binding domain (or portion), which can be included as part of the antibody and / or AFFIMER® polypeptide sequence and the fusion protein sequence, or can be chemically conjugated through another site that is not part of a contiguous polypeptide chain.

[0246] In one embodiment, the serum-binding domain may be an albumin-binding domain. Albumin contains multiple hydrophobic binding pockets and can serve as a carrier for various ligands, such as fatty acids and steroids, as well as drugs. Albumin also has a negatively charged surface and is highly water-soluble.

[0247] The term "albumin-binding domain" of the present invention refers to any chemical group that binds to albumin, and can refer to, for example, a compound that has albumin-binding affinity. Albumin can not only bind to endogenous ligands such as fatty acids, but also interact with exogenous ligands such as warfarin, penicillin, and diazepam. The binding of such drugs to albumin is reversible, and albumin-drug complexes can serve as drug reservoirs or drug delivery vehicles that can improve the biodistribution and bioavailability of drugs. Methods have been used to enhance albumin binding and increase drug efficacy by adding components that mimic endogenous albumin-binding ligands, such as fatty acids.

[0248] In one embodiment, a chemical modification that can be applied to the fusion proteins of the present invention to increase protein half-life is lipidation, which covalently attaches a fatty acid to a peptide side chain. This method is well known in the art as a method for extending the half-life of insulin and shares the same half-life extension mechanism as PEGylation: increasing the hydrodynamic radius and reducing renal filtration. However, the lipid moiety itself is relatively small, and the effect is indirectly mediated by non-covalent binding to circulating albumin. While lipidation can reduce the water solubility of the peptide, this can be controlled by manipulating the linker between the peptide and the fatty acid, for example, by using glutamic acid or mini-PEG within the linker. Linker manipulation and lipid modification can affect self-aggregation, delay biodistribution independently of albumin, and contribute to increased half-life.

[0249] In one embodiment, the albumin-binding domain is an albumin-binding adnectin (albumin-binding (PKE2) adnectin; see WO2011140086 "Serum Albumin Binding Molecules," WO2015143199 "Serum albumin-binding fibronectin type III domains," and WO2017053617 "Fast-off rate serum albumin-binding fibronectin type III domains"), albumin-binding domain 3 (ABD3) of protein G from Streptococcus G148, an albumin-binding domain antibody (e.g., GSK2374697, AlbudAb), or an albumin-binding nanobody (e.g., ATN103, Ozolralizumab).

[0250] AFFIMER® XT In one embodiment, the fusion protein may comprise a Stefin A protein variant that binds to a serum protein.

[0251] In one embodiment, at least one of the solvent-accessible loops of the HSA-binding Stefin A protein variant (HSA AFFIMER®) can be derived from the wild-type Stefin A protein tract, allowing it to bind to HSA. In one embodiment, the HSA-binding Stefin A protein variant can be 10 -6 It can bind to HSA with a Kd of less than M

[0252] In one embodiment, the Stefin A protein variant that binds to HSA has a binding affinity of 1×10 at pH 7.4-7.6. -9 M~1×10 -6 In one embodiment, the HSA-binding Stefin A protein variant can bind to HSA with a Kd of 10 M at pH 7.4-7.6. -6In one embodiment, the HSA-binding Stefin A protein variant can bind to HSA with a Kd of 10 M or less at pH 7.4 to 7.6. -7 In one embodiment, the HSA-binding Stefin A protein variant can bind to HSA with a Kd of 10 M or less at pH 7.4 to 7.6. -8 In one embodiment, the HSA-binding Stefin A protein variant can bind to HSA with a Kd of 10 M or less at pH 7.4 to 7.6. -9 In one embodiment, the HSA-binding Stefin A protein variant can bind to HSA with a Kd of 10 M or less at pH 7.4 to 7.6. -10 In one embodiment, the HSA-binding Stefin A protein variant can bind to HSA with a Kd of 10 M or less at pH 7.4 to 7.6. -11 In one embodiment, the HSA-binding Stefin A protein variant can bind to HSA with a Kd of 1×10 M or less at pH 7.4. -9 M~1×10 -6 In one embodiment, the HSA-binding Stefin A protein variant can bind to HSA with a Kd of 10 M at pH 7.4. -6 In one embodiment, the HSA-binding Stefin A protein variant can bind to HSA with a Kd of 10 M or less at pH 7.4. -7 In one embodiment, the HSA-binding Stefin A protein variant can bind to HSA with a Kd of 10 M or less at pH 7.4. -8 In one embodiment, the HSA-binding Stefin A protein variant can bind to HSA with a Kd of 10 M or less at pH 7.4. -9 In one embodiment, the HSA-binding Stefin A protein variant can bind to HSA with a Kd of 10 M or less at pH 7.4. -10 In one embodiment, the HSA-binding Stefin A protein variant can bind to HSA with a Kd of 10 M or less at pH 7.4.-11 It can bind to HSA with a Kd of less than M

[0253] In one embodiment, the HSA-binding Stefin A protein variant may be derived from a wild-type human Stefin A protein having a backbone sequence in which one or more of loop 2 (corresponding to (Xaa)n) and loop 4 (corresponding to (Xaa)m) are replaced with alternative loop sequences, (Xaa)n and (Xaa)m.

[0254] In one embodiment, the Stefin A protein variant may comprise an amino acid sequence represented by Formula I: [Number I] FR1-(Xaa)n-FR2-(Xaa)m-FR3(I), wherein the FR1 is an amino acid sequence having at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%) identity to the amino acid sequence represented by MIPGGLSEAK PATPEIQEIV DKVKPQLEEK TNETYGKLEA VQYKTQVLA (SEQ ID NO: 741); FR2 is an amino acid sequence represented by GTNYYIKVRA GDNKYMHLKV FKSL (SEQ ID NO: 2) or an amino acid sequence having at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%) identity to the amino acid sequence of SEQ ID NO: 2; FR3 is an amino acid sequence represented by EDLVLTGYQV DKNKDDELTGF (SEQ ID NO: 3) or an amino acid sequence having at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%) identity to the amino acid sequence of SEQ ID NO: 3; and Xaa, in each occurrence, is any amino acid residue; n is an integer of 3 to 20, and m is an integer of 3 to 20.

[0255] In one embodiment, FR1 is a polypeptide sequence having at least 80% to 98%, 82% to 98%, 84% to 98%, 86% to 98%, 88% to 98%, 90% to 98%, 92% to 98%, 94% to 98%, or 96% to 98% identity to SEQ ID NO: 741. In one embodiment, FR1 is a polypeptide sequence having at least 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, or 98% identity to SEQ ID NO: 741. In one embodiment, FR1 is the polypeptide sequence of SEQ ID NO: 741. In one embodiment, FR2 is a polypeptide sequence having at least 80% to 96%, 84% to 96%, 88% to 96%, or 92% to 96% identity to SEQ ID NO: 2. In one embodiment, FR2 is a polypeptide sequence having at least 80%, 84%, 88%, 92%, or 96% identity to SEQ ID NO: 2. In one embodiment, FR2 is a polypeptide sequence having at least 80%, 85%, 90%, 95%, or 98% identity to SEQ ID NO: 2. In one embodiment, FR3 is a polypeptide sequence having at least 80%-95%, 85%-95%, or 90%-95% identity to SEQ ID NO: 3. In one embodiment, FR3 is a polypeptide sequence having at least 80%, 85%, 90%, or 95% identity to SEQ ID NO: 3. In one embodiment, FR3 is the polypeptide sequence of SEQ ID NO: 3. In one embodiment, the HSA-binding Stefin A protein variant may comprise an amino acid sequence represented by Formula II (SEQ ID NO: 4): [Mathematics II] MIP-Xaa1-GLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQV-Xaa2-(Xaa)n-Xaa3-TNYYI KVRAGDNKYMHLKVF-Xaa4-Xaa5-Xaa6-(Xaa)m-Xaa7-D-Xaa8-VLTGYQVDKNKDDELTGF (SEQ ID NO: 4), wherein Xaa is, in each case, any number of amino acid residues, preferably three or less (preferably one or two) independently selected amino acids; and n and m are each independently an integer of 3 to 20.

[0256] In one embodiment, the HSA-binding Stefin A protein variant may comprise the amino acid sequence shown below: MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVV-(Xaa)n-GTNYYIKVRAGDNKYMHLKVFKSL-(Xaa)m-EDLVLTGYQVDKNKDDELTGF (SEQ ID NO: 742); MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVD-(Xaa)n-GTNYYIKVRAGDNKYMHLKVFKSL-(Xaa)m-EDLVLTGYQVDKNKDDELTGF (SEQ ID NO: 5); or MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVLA-(Xaa)n-GTNYYIKVRAGDNKYMHLKVFKSL-(Xaa)m-EDLVLTGYQVDKNKDDELTGF (SEQ ID NO: 743) Here, Xaa is, in each case, individually any amino acid residue; n and m are each independently an integer of 3 to 20.

[0257] In one embodiment, Xaa1 can be Gly, Ala, Val, Arg, Lys, Asp, or Glu; Xaa2 can be Val, Asp, or 'Leu-Ala'; Xaa3 can be Gly, Ala, Val, Ser, or Thr; Xaa4 can be Arg, Lys, Asn, Gln, Ser, or Thr; Xaa5 can be Gly, Ala, Val, Ser, or Thr; Xaa6 can be Ala, Val, Ile, Leu, Gly, or Pro; Xaa7 can be Gly, Ala, Val, Asp, or Glu; and Xaa8 can be Ala, Val, Ile, Leu, Arg, or Lys.

[0258] In one embodiment, Xaa1 is Gly, Ala, Arg, or Lys. In one embodiment, Xaa1 is Gly or Arg. In one embodiment, Xaa2 is Val, Asp, or "Leu-Ala." In one embodiment, Xaa3 is Gly, Ala, Val, Ser, or Thr. In one embodiment, Xaa3 is Gly or Ser. In one embodiment, Xaa4 is Arg, Lys, Asn, Gln, Ser, or Thr. In one embodiment, Xaa4 is Arg, Lys, Asn, or Gln. In one embodiment, Xaa4 is Lys or Asn. In one embodiment, Xaa5 is Gly, Ala, Val, Ser, or Thr. In one embodiment, Xaa5 is Gly or Ser. In one embodiment, Xaa6 is Ala, Val, Ile, Leu, Gly, or Pro. In one embodiment, Xaa6 is Ile, Leu, or Pro. In one embodiment, Xaa6 is Leu or Pro. In one embodiment, Xaa7 is Gly, Ala, Val, Asp, or Glu. In one embodiment, Xaa7 is Ala, Val, Asp, or Glu. In one embodiment, Xaa7 is Ala or Glu. In one embodiment, Xaa8 is Ala, Val, Ile, Leu, Arg, or Lys. In one embodiment, Xaa8 is Ile, Leu, or Arg. In one embodiment, Xaa8 is Leu or Arg.

[0259] In one embodiment, n is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In one embodiment, n can be 8-10, 7-11, 6-12, 5-13, 4-14, or 3-15. In one embodiment, m can be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In one embodiment, m can be 8-10, 7-11, 6-12, 5-13, 4-14, or 3-15. In one embodiment, (Xaa)n is represented by Formula IV: [Mathematics IV] aa1-aa2-aa3-aa4-aa5-aa6-aa7-aa8-aa9, aa3 is an amino acid with a neutral non-polar hydrophobic side chain; aa4 is an amino acid with a neutral polar hydrophilic side chain; aa5 is an amino acid with a positively charged polar hydrophilic side chain; aa6 is an amino acid with a positively charged polar hydrophilic side chain; aa7 is an amino acid with a neutral non-polar hydrophobic side chain; aa8 is an amino acid with a neutral non-polar hydrophobic side chain; and aa9 is an amino acid with a neutral non-polar hydrophobic side chain.

[0260] In one embodiment, (Xaa)m is represented by Formula V: [Several V] aa1-aa2-aa3-aa4-aa5-aa6-aa7-aa8-aa9(V), aa2 is an amino acid with a positively charged polar hydrophilic side chain; aa3 is an amino acid with a neutral nonpolar hydrophobic side chain; aa4 is an amino acid with a positively charged polar hydrophilic side chain; aa5 is an amino acid with a neutral polar hydrophilic side chain; aa6 is an amino acid with a neutral polar hydrophilic side chain; aa7 is an amino acid with a negatively charged polar hydrophilic side chain; aa8 is an amino acid with a positively charged polar hydrophilic side chain; and aa9 is an amino acid with a neutral nonpolar hydrophilic side chain.

[0261] Examples of amino acids having neutral non-polar hydrophilic side chains include cysteine ​​(Cys) and glycine (Gly). In one embodiment, the amino acid having a neutral non-polar hydrophilic side chain is Cys. In one embodiment, the amino acid having a neutral non-polar hydrophilic side chain is Gly.

[0262] Examples of amino acids having a neutral nonpolar hydrophobic side chain include alanine (Ala), isoleucine (Ile), leucine (Leu), methionine (Met), phenylalanine (Phe), proline (Pro), tryptophan (Trp), and valine (Val). In one embodiment, the amino acid having a neutral nonpolar hydrophobic side chain is Ala. In one embodiment, the amino acid having a neutral nonpolar hydrophobic side chain is Ile. In one embodiment, the amino acid having a neutral nonpolar hydrophobic side chain is Leu. In one embodiment, the amino acid having a neutral nonpolar hydrophobic side chain is Met. In one embodiment, the amino acid having a neutral nonpolar hydrophobic side chain is Phe. In one embodiment, the amino acid having a neutral nonpolar hydrophobic side chain is Pro. In one embodiment, the amino acid having a neutral nonpolar hydrophobic side chain is Trp. In one embodiment, the amino acid having a neutral nonpolar hydrophobic side chain is Val.

[0263] Examples of amino acids having a neutral polar hydrophilic side chain include asparagine (Asn), glutamine (Gln), serine (Ser), threonine (Thr), and tyrosine (Tyr). In one embodiment, the amino acid having a neutral polar hydrophilic side chain is Asn. In one embodiment, the amino acid having a neutral polar hydrophilic side chain is Gln. In one embodiment, the amino acid having a neutral polar hydrophilic side chain is Ser. In one embodiment, the amino acid having a neutral polar hydrophilic side chain is Thr. In one embodiment, the amino acid having a neutral polar hydrophilic side chain is Tyr.

[0264] Examples of amino acids having a positively charged polar hydrophilic side chain include arginine (Arg), histidine (His), and lysine (Lys). In one embodiment, the amino acid having a positively charged polar hydrophilic side chain is Arg. In one embodiment, the amino acid having a positively charged polar hydrophilic side chain is His. In one embodiment, the amino acid having a positively charged polar hydrophilic side chain is Lys.

[0265] Examples of amino acids having negatively charged polar hydrophilic side chains include aspartic acid (Asp) and glutamic acid (Glu). In one embodiment, the amino acid having a negatively charged polar hydrophilic side chain is Asp. In one embodiment, the amino acid having a negatively charged polar hydrophilic side chain is Glu. In one embodiment, (Xaa)n is represented by Formula IV: [Mathematics IV] aa1-aa2-aa3-aa4-aa5-aa6-aa7-aa8-aa9(IV), wherein aa1 is an amino acid selected from Asp, Gly, Asn, and Val; aa2 is an amino acid selected from Trp, Tyr, His, and Phe; aa3 is an amino acid selected from Trp, Tyr, Gly, Trp, and Phe; aa4 is an amino acid selected from Gln, Ala, and Pro; aa5 is an amino acid selected from Ala, Gln, Glu, Arg, and Ser; aa6 is an amino acid selected from Lys, Arg, and Tyr; aa7 is an amino acid selected from Trp and Gln; aa8 is an amino acid selected from Pro and His; and / or aa9 is an amino acid selected from His, Gly, and Gln. In one embodiment, aa1 is Asp. In one embodiment, aa1 is Gly. In one embodiment, aa1 is Asn. In one embodiment, aa2 is Trp. In one embodiment, aa2 is Tyr. In one embodiment, aa2 is His. In one embodiment, aa2 is Phe. In one embodiment, aa3 is Trp. In one embodiment, aa3 is Tyr. In one embodiment, aa3 is Gly. In one embodiment, aa3 is Trp. In one embodiment, aa3 is Phe. In one embodiment, aa4 is Gln. In one embodiment, aa4 is Ala. In one embodiment, aa4 is Pro. In one embodiment, aa5 is Ala. In one embodiment, aa5 is Gln. In one embodiment, aa5 is Glu. In one embodiment, aa5 is Arg. In one embodiment, aa5 is Ser. In one embodiment, aa6 is Lys. In one embodiment, aa6 is Arg. In one embodiment, aa6 is Tyr. In one embodiment, aa7 is Trp. In one embodiment, aa7 is Gln. In one embodiment, aa8 is Pro. In one embodiment, aa8 is His. In one embodiment, aa9 is His. In one embodiment, aa9 is Gly. In one embodiment, aa9 is Gln.

[0266] In one embodiment, (Xaa)m is represented by Formula IV: [Mathematics IV] aa1-aa2-aa3-aa4-aa5-aa6-aa7-aa8-aa9(IV), aa3 is an amino acid selected from Val, Asn, Gly, Gln, Ala, and Phe; aa4 is an amino acid selected from His, Thr, Lys, Trp, Lys, Val, and Arg; aa5 is an amino acid selected from Gln, Ser, Gly, Pro, and Asn; aa6 is an amino acid selected from Ser, Tyr, Glu, Leu, Lys, and Thr; aa7 is an amino acid selected from Ser, Asp, Val, and Lys; aa8 is an amino acid selected from Gly, Leu, Ser, Pro, His, Asp, and Arg; and / or aa9 is an amino acid selected from Gly, Gln, Glu, and Ala.

[0267] In one embodiment, aa1 is Tyr. In one embodiment, aa1 is Phe. In one embodiment, aa1 is Trp. In one embodiment, aa1 is Asn. In one embodiment, aa2 is Lys. In one embodiment, aa2 is Pro. In one embodiment, aa2 is His. In one embodiment, aa2 is Ala. In one embodiment, aa2 is Thr. In one embodiment, aa3 is Val. In one embodiment, aa3 is Asn. In one embodiment, aa3 is Gly. In one embodiment, aa3 is Gln. In one embodiment, aa3 is Ala. In one embodiment, aa3 is Phe. In one embodiment, aa4 is His. In one embodiment, aa4 is Thr. In one embodiment, aa4 is Lys. In one embodiment, aa4 is Trp. In one embodiment, aa4 is Lys. In one embodiment, aa4 is Val. In one embodiment, aa4 is Arg. In one embodiment, aa5 is Gln. In one embodiment, aa5 is Ser. In one embodiment, aa5 is Gly. In one embodiment, aa5 is Pro. In one embodiment, aa5 is Asn. In one embodiment, aa6 is Ser. In one embodiment, aa6 is Tyr. In one embodiment, aa6 is Glu. In one embodiment, aa6 is Leu. In one embodiment, aa6 is Lys. In one embodiment, aa6 is Thr. In one embodiment, aa7 is Ser. In one embodiment, aa7 is Asp. In one embodiment, aa7 is Val. In one embodiment, aa7 is Lys. In one embodiment, aa8 is Gly. In one embodiment, aa8 is Leu. In one embodiment, aa8 is Ser. In one embodiment, aa8 is Pro. In one embodiment, aa8 is His. In one embodiment, aa8 is Asp. In one embodiment, aa8 is Arg. In one embodiment, aa9 is Gly. In one embodiment, aa9 is Gln. In one embodiment, aa9 is Glu. In one embodiment, aa9 is Ala.

[0268] In one embodiment, (Xaa)n is represented by Formula V: [Several V] Asn-aa1-aa2-Gln-Gln-Arg-Arg-Trp-Pro-Gly(V), wherein aa1 is an amino acid selected from Trp and Phe; and aa2 is an amino acid selected from Tyr and Phe. In one embodiment, aa1 is Trp. In one embodiment, aa1 is Phe. In one embodiment, aa2 is Tyr. In one embodiment, aa2 is Phe.

[0269] In one embodiment, (Xaa)n is represented by Formula VI: [Mathematics VI] aa1-aa2-Trp-aa3-aa4-Lys-Trp-Pro-aa5(VI), wherein aa1 is an amino acid selected from Asp and Gly; aa2 is an amino acid selected from Trp, Tyr, and Phe; aa3 is an amino acid selected from Gln and Ala; aa4 is an amino acid selected from Ala and Ser; and aa5 is an amino acid selected from His and Gly. In one embodiment, aa1 is Asp. In one embodiment, aa1 is Gly. In one embodiment, aa2 is Trp. In one embodiment, aa2 is Tyr. In one embodiment, aa2 is Phe. In one embodiment, aa3 is Gln. In one embodiment, aa3 is Ala. In one embodiment, aa4 is Ala. In one embodiment, aa4 is Ser. In one embodiment, aa5 is His. In one embodiment, aa5 is Gly.

[0270] In one embodiment, (Xaa)n is represented by Formula VII: [Mathematics VII] aa1-aa2-aa3-aa4-aa5-aa6-Trp-Pro-Gly(VII), wherein aa1 is an amino acid selected from Gly and Asn; aa2 is an amino acid selected from Tyr, Phe, Trp, and His; aa3 is an amino acid selected from Trp, Tyr, and Phe; aa4 is an amino acid selected from Ala and Gln; aa5 is an amino acid selected from Ala, Ser, Gln, and Arg; and aa6 is an amino acid selected from Lys, Arg, and Tyr. In one embodiment, aa1 is Gly. In one embodiment, aa1 is Asn. In one embodiment, aa2 is Tyr. In one embodiment, aa2 is Phe. In one embodiment, aa2 is Trp. In one embodiment, aa2 is His. In one embodiment, aa3 is Trp. In one embodiment, aa3 is Tyr. In one embodiment, aa3 is Phe. In one embodiment, aa4 is Ala. In one embodiment, aa4 is Gln. In one embodiment, aa5 is Ala. In one embodiment, aa5 is Ser. In one embodiment, aa5 is Gln. In one embodiment, aa5 is Arg. In one embodiment, aa6 is Lys. In one embodiment, aa6 is Arg. In one embodiment, aa6 is Tyr.

[0271] In one embodiment, (Xaa)n is represented by Formula VIII: [Mathematics VIII] Gly-aa1-aa2-Ala-aa3-aa4-Trp-Pro-Gly(VIII) (SEQ ID NO: 561), wherein aa1 is an amino acid selected from Tyr, Phe, and His; aa2 is an amino acid selected from Trp and Tyr; aa3 is an amino acid selected from Ala, Ser, and Arg; and aa4 is an amino acid selected from Lys and Tyr. In one embodiment, aa1 is Tyr. In one embodiment, aa1 is Phe His. In one embodiment, aa1 is His. In one embodiment, aa2 is Trp. In one embodiment, aa2 is Tyr. In one embodiment, aa3 is Ala. In one embodiment, aa3 is Ser. In one embodiment, aa3 is Arg. In one embodiment, aa4 is Lys. In one embodiment, aa4 is Tyr.

[0272] In one embodiment, (Xaa)n is represented by Formula IX: [Number IX] aa1-aa2-aa3-Gln-aa4-aa5-Trp-Pro-aa6(IX), wherein aa1 is an amino acid selected from Asp and Asn; aa2 is an amino acid selected from Trp and Phe; aa3 is an amino acid selected from Trp, Tyr, and Phe; aa4 is an amino acid selected from Ala, Gln, and Arg; aa5 is an amino acid selected from Lys and Arg; and aa6 is an amino acid selected from His and Gly. In one embodiment, aa1 is Asp. In one embodiment, aa1 is Asn. In one embodiment, aa2 is Trp. In one embodiment, aa2 is Phe. In one embodiment, aa3 is Trp. In one embodiment, aa3 is Tyr. In one embodiment, aa3 is Phe. In one embodiment, aa4 is Ala. In one embodiment, aa4 is Gln. In one embodiment, aa4 is Arg. In one embodiment, aa5 is Lys. In one embodiment, aa5 is Arg. In one embodiment, aa6 is His. In one embodiment, aa6 is Gly.

[0273] In one embodiment, the HSA-binding Stefin A protein variant comprises a loop 2 amino acid selected from the amino acids of SEQ ID NOs: 562 to 614 (Table 8). In another embodiment, the HSA-binding Stefin A protein variant comprises a loop 4 amino acid selected from the amino acids of SEQ ID NOs: 615 to 667 (Table 8).

[0274] [Table 8] JPEG2026504813000038.jpg197145

[0275] In one embodiment, the (Xaa)n comprises an amino acid sequence having at least 80% or at least 90% identity to the amino acid sequence of any one of SEQ ID NOs: 562 to 614. In one embodiment, the (Xaa)n comprises an amino acid sequence having 80% to 90% identity to the amino acid sequence of any one of SEQ ID NOs: 562 to 614. In one embodiment, the (Xaa)n comprises an amino acid sequence of any one of SEQ ID NOs: 562 to 614.

[0276] In one embodiment, the (Xaa)m comprises an amino acid sequence having at least 80% or at least 90% identity to the amino acid sequence of any one of SEQ ID NOs: 615 to 667. In one embodiment, the (Xaa)m comprises an amino acid sequence having 80% to 90% identity to the amino acid sequence of any one of SEQ ID NOs: 615 to 667. In one embodiment, the (Xaa)m comprises an amino acid sequence of any one of 615 to 667.

[0277] In one embodiment, the HSA-binding Stefin A protein variant comprises any one of the amino acid sequences selected from SEQ ID NOs: 668 to 674 (Table 9).

[0278] [Table 9]

[0279] In one embodiment, the HSA-binding Stefin A protein variant comprises an amino acid sequence having at least 80% or at least 90% identity to any one of the amino acid sequences of SEQ ID NOs: 668-674.

[0280] In one embodiment, the HSA-binding Stefin A protein variant comprises an amino acid sequence having 80% to 90% identity to any one of the amino acid sequences of SEQ ID NOs: 668 to 674.

[0281] In one embodiment, the HSA-binding Stefin A protein variant can be linked to another molecule (e.g., a therapeutic polypeptide or a Stefin A protein variant of the present invention that specifically binds to CD40L) to extend the half-life of the molecule. In one embodiment, the HSA-binding Stefin A protein variant can have a variety of binding affinities that cross-react with other species, such as mouse and cynomolgus monkey. Herein, the HSA-binding Stefin A protein variant is AFFIMER XT. TM In one embodiment, the HSA-binding Stefin A protein variant can extend the half-life of other Stefin A protein variants or fusion proteins thereof conjugated by single gene fusion in a controlled manner through in vivo pharmacokinetic (PK) studies. In one embodiment, the HSA-binding Stefin A protein variant (AFFIMER XT TM ) can be used to extend the half-life of other peptide or protein therapeutics.

[0282] In one embodiment, the HSA-binding Stefin A protein variant can increase the serum half-life of a molecule, e.g., a therapeutic protein, such as a Stefin A protein variant of the present invention that specifically binds to CD40L or a fusion protein comprising the same, in vivo. For example, the HSA-binding Stefin A protein variant can increase the serum half-life of a molecule, e.g., a therapeutic protein, such as a Stefin A protein variant of the present invention that specifically binds to CD40L or a fusion protein comprising the same, by at least two-fold compared to a molecule to which the HSA-binding Stefin A protein variant is not linked.

[0283] In one embodiment, when the fusion protein comprises the HSA-binding Stefin A protein variant, the serum half-life can be increased by at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 20-fold, or at least 30-fold compared to a molecule to which the HSA-binding Stefin A protein variant is not linked. In one embodiment, when the fusion protein comprises the HSA-binding Stefin A protein variant, the serum half-life can be increased by 2-fold to 5-fold, 2-fold to 10-fold, 3-fold to 5-fold, 3-fold to 10-fold, 15-fold to 5-fold, 4-fold to 10-fold, or 5-fold to 10-fold compared to a molecule to which the HSA-binding Stefin A protein variant is not linked. In one embodiment, when the fusion protein contains the Stefin A protein variant that binds to HSA, the serum half-life can be increased by at least 6 hours, at least 12 hours, at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, e.g., at least 1 week after administration in vivo, compared to a molecule to which the Stefin A protein variant that binds to HSA is not linked.

[0284] In one embodiment, the Stefin A protein variant that specifically binds to CD40L has an increased serum half-life and comprises an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, or 98% identical to the sequence of SEQ ID NOs: 667-702 (Table 10). In one embodiment, the Stefin A protein variant that specifically binds to CD40L has an increased serum half-life and comprises an amino acid sequence encoded by a nucleic acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, or 98% identical to the sequence of SEQ ID NOs: 667-702 (Table 10) and SEQ ID NOs: 703-728 (Table 11).

[0285] PEGylated, XTEN, PAS and other polymers A wide variety of macromolecular polymers and other molecules can be linked to the CD40L-binding agents of the present invention (e.g., Stefin A protein variants that specifically bind to CD40L) to modulate and / or provide new biological properties.

[0286] In the present invention, the polymer can be linked to a CD40L binding agent of the present invention (e.g., a Stefin A protein variant that specifically binds to CD40L) through a naturally encoded amino acid, a non-naturally encoded amino acid, or any functional substituent of a natural or non-natural amino acid, or any substituent or functional group added to a natural or non-natural amino acid.

[0287] In one embodiment, the molecular weight of the high molecular weight polymer may be, but is not limited to, about 100 Da to about 100,000 Da, and may be within a wide range, such as 100,000 Da, 95,000 Da, 90,000 Da, 85,000 Da, 80,000 Da, 75,000 Da, 70,000 Da, 65,000 Da, 60,000 Da, 55,000 Da, 50,000 Da, 45,000 Da, 40,000 Da, 35,000 Da, 30,000 Da, 25,000 Da, 20,000 Da, and 15,000 Da. The molecular weight may be from about 100 Da to about 100,000 Da, including, but not limited to, 0 Da, 10,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000 Da, 5,000 Da, 4,000 Da, 3,000 Da, 2,000 Da, 1,000 Da, 900 Da, 800 Da, 700 Da, 600 Da, 500 Da, 400 Da, 300 Da, 200 Da, and 100 Da. In one embodiment, the molecular weight of the polymer is about 100 Da to about 50,000 Da, about 100 Da to about 40,000 Da, about 1,000 Da to about 40,000 Da, about 5,000 Da to about 40,000 Da, or about 10,000 Da to about 40,000 Da.

[0288] For this purpose, methods have been developed that involve PEGylation, polysialylation, HESylation, glycosylation, or recombinant PEG analogs fused to flexible hydrophilic amino acid chains (500-600 amino acids) (see, e.g., Chapman, (2002) Adv Drug Deliv Rev. 54, 531-545; Schlapschy et al., (2007) Prot Eng Des Sel. 20, 273-283; Contermann (2011) Curr Op Biotechnol. 22, 868-876; Jevsevar et al., (2012) Methods Mol Biol. 901, 233-246).

[0289] In one embodiment, the polymer may be, for example, polyalkyl ethers and their alkoxy-capped analogs (e.g., polyoxyethylene glycol, polyoxyethylene / propylene glycol and its methoxy- or ethoxy-capped analogs, particularly polyoxyethylene glycol, the latter also referred to as polyethylene glycol); discrete PEG (dPEG); polyvinylpyrrolidones; polyvinylalkyl ethers; polyoxazolines, polyalkyloxazolines, and polyhydroxyalkyloxazolines; polyacrylamides, polyalkyl acrylamides, and polyhydroxyalkyl acrylamides (e.g., polyhydroxypropylmethacrylamide and its derivatives); polyhydroxyalkyl acrylates; polysialic acid acids and their analogues; hydrophilic peptide sequences; dextran and dextran derivatives (e.g., carboxymethyldextran polysaccharides and their derivatives), polysaccharides and their derivatives, including amide dextran; cellulose and its derivatives (e.g., carboxymethyl cellulose, hydroxyalkyl celluloses);Chitin and its derivatives (e.g., chitosan, succinyl chitosan, carboxymethylchitin, carboxymethylchitosan); hyaluronic acid and its derivatives; starches; alginates; chondroitin sulfate; albumin; pullulan and carboxymethyl pullulan; polyaminoacids and derivatives thereof (e.g., polyglutamic acids, polylysines, polyaspartic acids, polyaspartamides); maleic anhydride copolymers (e.g., styrene maleic anhydride copolymers) Examples of suitable vinyl copolymers include, but are not limited to, divinylethyl ether maleic anhydride copolymer, divinylethyl ether maleic anhydride copolymer, polyvinyl alcohols, copolymers thereof, terpolymers thereof, mixtures thereof, and derivatives thereof.

[0290] In one embodiment, the polymer may be a water-soluble polymer so that the fusion protein does not precipitate in an aqueous environment, such as a physiological environment. In one embodiment, the water-soluble polymer may have any structure, including, but not limited to, linear, forked, or branched. Generally, the water-soluble polymer may be a polyalkylene glycol, such as polyethylene glycol (PEG), but may also be other water-soluble polymers, including, but not limited to, PEG. For example, PEG is used in some embodiments of the present invention. In one embodiment, the polymer may be pharmaceutically acceptable.

[0291] The term "polyethylene glycol (PEG)" used herein is broadly used to encompass any polyethylene glycol molecule, regardless of its size or whether or not the PEG terminus is modified, and can be characterized by being linked to the Stefin A protein variant of the present invention as shown in the following chemical formula: XO-(CH2CH2O)n-CH2CH2-, or XO-(CH2CH2O)n-, where n is 2 to 10,000, and X is H or a non-limiting example, C 1-4 Terminal modifications such as alkyl, protecting groups, or terminal functional groups.

[0292] In one embodiment, the PEG may be terminated at one end with hydroxy or methoxy, for example, X may be H or CH3 (methoxy PEG).

[0293] In the formula, the other end of the PEG, designated by the terminal "-", can be attached to the Stefin A protein variant of the present invention through a naturally occurring or non-naturally encoded amino acid. For example, the attachment can be to an amine group (e.g., the epsilon amine of lysine, or the N-terminus, including, but not limited to, the N-terminus) through an amide, carbamate, or urea linkage. Alternatively, the polymer can be linked to a thiol group (e.g., the thiol group of cysteine, including, but not limited to, a maleimide bond). When the polymer is conjugated to a fusion protein in this manner, it is necessary to change the residue of the Stefin A protein variant of the present invention to cysteine.

[0294] The number of water-soluble polymers (e.g., including the degree of PEGylation or glycosylation) linked to a CD40L binding agent (e.g., a Stefin A protein variant) of the invention or a fusion protein thereof can be adjusted to provide altered (including, but not limited to, increased or decreased) pharmacological, pharmacokinetic, or pharmacodynamic properties, such as in vivo half-life. In one embodiment, the half-life of a CD40L binding agent (e.g., a Stefin A protein variant) of the invention or a fusion protein thereof comprising such a polymer is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 2-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 50-fold, or at least 100-fold compared to the unaltered polypeptide.

[0295] Yet another variation of the polymer system useful for modifying the PK or biological properties of the CD40L binding agents (e.g., Stefin A protein variants or fusion proteins) of the invention is the use of unstructured hydrophilic amino acid polymers that are functional analogs of PEG. For example, hydrophilic amino acid polymers that are functional analogs of PEG may be useful as part of a fusion protein with a Stefin A protein variant. The inherent biodegradability of polypeptides has highlighted them as potentially safer alternatives to PEG. Yet another advantage is the precise molecular structure of hydrophilic amino acid polymers, in contrast to the polydispersity of PEG.

[0296] Unlike the fusion of HSA and Fc domains, where the tertiary folding of the fusion domain must be maintained, most fusion proteins containing unstructured domains can be subjected to harsh conditions such as higher temperatures or HPLC purification.

[0297] One such advanced form of polypeptide is XTEN (Amunix), which is 864 amino acids long and consists of six amino acids (A, E, G, P, S, and T). See Schellenberger et al., "A recombinant polypeptide extends the in vivo half-life of peptides and proteins in a tunable manner," 2009 Nat Biotechnol. 27(12):1186-90. The biodegradable polymer's properties are far greater than those of the commonly used 40 kDa PEG, providing an even greater half-life extension effect. When fused to a CD40L-binding fusion protein containing XTEN (e.g., a polypeptide containing AFFIMER®), the half-life can be extended 60 to 130 times compared to the unmodified polypeptide.

[0298] A second polymer based on a similar concept is PAS (XL-Protein GmbH). See Schlapschy et al., "PASYlation: a biological alternative to PEGylation for extending the plasma half-life of pharmaceutically active proteins," 2013 Protein Eng Des Sel. 26(8):489-501. This random coil polymer is composed of a limited set of uncharged amino acids: proline, alanine, and serine. Similar to Fc domains, HSA, and XTEN, PAS variants can be produced as in-line fusion proteins by being genetically encoded and expressed with a CD40L binding agent of the invention (e.g., a stefin A protein variant).

[0299] In-line fusion protein In one embodiment, the fusion protein may be selected from any one of the polypeptides in Table 10 below, or may be encoded by one of the polynucleotides provided in Table 11.

[0300] [Table 10] JPEG2026504813000041.jpg228152JPEG2026504813000042.jpg229153JPEG20265048130 00043.jpg231154JPEG2026504813000044.jpg225153JPEG2026504813000045.jpg107153

[0301] In one embodiment, the nucleic acid encoding the fusion protein may be characterized by being selected from, but not limited to, the nucleic acids provided in Table 11. It is obvious that a nucleic acid sequence encoding a fusion protein can be designed and predicted based on the amino acid sequence of the Stefin A protein of the present invention that specifically binds to CD40L and the nucleic acid sequence encoding the same, and the polypeptide sequence of the fusion domain contained in the fusion protein of the present invention and the nucleic acid sequence encoding the same.

[0302] [Table 11] JPEG2026504813000047.jpg228151JPEG2026504813000048.jpg228152JPEG2026504813000049.jpg227151JPEG202 6504813000050.jpg228152JPEG2026504813000051.jpg228152JPEG2026504813000052.jpg228153JPEG20265048130 00053.jpg228152JPEG2026504813000054.jpg228151JPEG2026504813000055.jpg227150JPEG2026504813000056.j pg227150JPEG2026504813000057.jpg228151JPEG2026504813000058.jpg227150JPEG2026504813000059.jpg184151

[0303] Host cells and genetically modified cells In one embodiment, a nucleic acid encoding the above-described CD40L-binding agent (eg, a Stefin A protein variant that specifically binds to CD40L) and / or a fusion protein comprising the same is introduced into a host cell.

[0304] The term "host cell" according to the present invention refers to a cell into which a nucleic acid encoding a CD40L-binding agent (e.g., a Stefin A protein variant that specifically binds to CD40L) and / or a fusion protein comprising the same described in the present invention is introduced prior to introduction.

[0305] The term "genetically modified cell" as used herein refers to a cell that has been modified to contain an exogenous nucleic acid. The genetically modified cell includes cells containing an exogenous nucleic acid, regardless of whether the exogenous nucleic acid is integrated into a host. In one embodiment, the genetically modified cell is modified to express the CD40L binding agent (e.g., a cell expressing a Stefin A protein variant and / or a fusion protein comprising the same). In this embodiment, the genetically modified cell is produced by introducing a nucleic acid encoding a CD40L binding agent of the invention (e.g., a Stefin A protein variant that specifically binds to CD40L) and / or a fusion protein comprising the same.

[0306] However, the present invention excludes from its scope genetically modified cells for use in producing Stefin A protein variants that specifically bind to CD40L and / or fusion proteins containing the same.

[0307] In one embodiment, the genetically modified cells may contain additional genetic modifications in addition to the introduction of a nucleic acid encoding a CD40L binding agent of the present invention (e.g., a Stefin A protein variant that specifically binds to CD40L) and / or a fusion protein comprising the same.

[0308] In one embodiment, host cells can be genetically modified to contain nucleic acids encoding the CD40L binding agents of the present invention (e.g., Stefin A protein variants that specifically bind to CD40L) and / or fusion proteins comprising the same, thereby producing genetically modified cells that express the CD40L binding agents and / or fusion proteins comprising the same.

[0309] The genetically modified cells of the present invention may be any cell, including eukaryotic cells and prokaryotic cells, including, but not limited to, bacterial cells such as Escherichia coli, Streptomyces, and Salmonella typhimurium; yeast cells; fungal cells such as Pichia pastoris; insect cells such as Drosophila, Spodoptera, and Sf9 cells; animal cells such as CHO, COS, NSO, 293, and bow melanoma cells; and plant cells.

[0310] In one embodiment, the host cells may be selected from the group consisting of stem cells, immune cells, and somatic cells.

[0311] In one embodiment, the host cell can be naturally occurring, e.g., derived from an animal, preferably a mammal, more preferably a human; or a cell engineered through cell or genetic engineering.

[0312] The term "stem cell" as used herein refers to cells that can differentiate into various cells that constitute biological tissues, and collectively refers to undifferentiated cells that can be obtained from embryonic, fetal, and adult tissues before differentiation. Stem cells differentiate into specific cells in response to differentiation stimuli (environments), and unlike cells that have completed differentiation and ceased cell division, stem cells have the property of self-renewal through cell division, allowing them to proliferate (expansion), and are characterized by their plasticity in differentiation, as they can differentiate into other cells in response to different environments or different differentiation stimuli.

[0313] In one embodiment, the stem cells may include, but are not limited to, pluripotent stem cells, multipotent stem cells, and unipotent stem cells depending on their differentiation potential.

[0314] In one embodiment, the pluripotent stem cells (PS cells) refer to stem cells that can differentiate into the three germ layers that constitute a living body, and include, but are not limited to, embryonic stem cells (ES cells) or induced pluripotent stem cells (iPS cells).

[0315] In one embodiment, the present invention provides transgenic tolipotent stem cells expressing a CD40L-binding agent and / or a fusion thereof.

[0316] In one embodiment, the omnipotent stem cells comprise a nucleic acid encoding a CD40L-binding agent and / or a fusion thereof. In one embodiment, the genetically modified PS cells are iPS cells. In some embodiments, the genetically modified PS cells provided are ES cells.

[0317] In one embodiment, genetically modified PS cells expressing a CD40L binding agent (e.g., a Stefin A protein variant) or a fusion protein thereof are produced by introducing a nucleic acid encoding the CD40L binding agent or a fusion protein comprising the same into PS cells (e.g., iPS cells or ES cells).

[0318] In one embodiment, the multipotent stem cells refer to cells that have the potential to differentiate from progenitor cells into cells belonging to a specific type family, including, but not limited to, hematopoietic stem cells, mesenchymal stem cells, and neural stem cells.

[0319] In one embodiment, the stem cells may be mesenchymal stem cells. The terms "mesenchymal stem cells" or "mesenchymal stromal cells" as used herein refer to cells that can differentiate into osteoblasts, adipocytes, chondrocytes, etc., derived from the mesoderm, one of the three germ layers of embryonic tissue. Mesenchymal stem cells may be extracted from bone marrow, adipose tissue, umbilical cord blood, synovial membrane, trabecular bone, infrapatellar fat pad, etc. Mesenchymal stem cells are known to be suitable for allotransplantation and xenotransplantation because they possess the immunomodulatory ability to 1) suppress the activity and proliferation of T lymphocytes and B lymphocytes, 2) suppress the activity of natural killer cells (NK cells), and 3) regulate the function of dendritic cells and macrophages.

[0320] In one embodiment, the present invention provides genetically modified mesenchymal stem cells (MSCs) that express a CD40L-binding agent and / or a fusion thereof, wherein the MSCs comprise a nucleic acid encoding the CD40L-binding agent and / or a fusion thereof.

[0321] In one embodiment, genetically modified mesenchymal stem cells expressing a CD40L binding agent (e.g., a Stefin A protein variant) or a fusion protein thereof are produced by introducing a nucleic acid encoding the CD40L binding agent or a fusion protein comprising the same into mesenchymal stem cells.

[0322] The Stefin A protein variants of the present invention that specifically bind to CD40L exhibit CD40L antagonistic effects and thus exhibit immune suppressive effects, such as suppression of T cell and / or B cell activity. Thus, in one embodiment of the present invention, a nucleic acid encoding the Stefin A protein variant was introduced into mesenchymal stem cells (MSCs) as host cells. It was confirmed that the Stefin A protein variants acquired novel immune-modulating activity through the CD40L antagonism while maintaining the immune-modulating effects of mesenchymal stem cells (e.g., suppression of T cell activity). This resulted in highly effective immune suppression, and further demonstrated significantly superior therapeutic effects in immune-related diseases such as GVHD or autoimmune diseases.

[0323] In one embodiment, the mesenchymal stem cells may be derived from pluripotent stem cells. In one embodiment, the mesenchymal stem cells may be capable of long-term subculture. Methods for producing mesenchymal stem cells from pluripotent stem cells and long-term subculture methods are well known in the art. For example, Korean Patent Application Publication No. 10-2021-0072734 and Korean Patent No. 10-1135636 disclose in detail methods for maintaining undifferentiated potential and marker expression characteristics even after several dozen subcultures.

[0324] In one embodiment, the mesenchymal stem cells may express any one or more of CD29, CD44, CD73, CD90, and CD105. In another embodiment, the mesenchymal stem cells may not express at least one cell surface marker selected from c-kit, CD11b, CD19, CD14, CD34, CD45, CD14, CD79, HLA-DR, TRA-1-60, and TRA-1-81. In another embodiment, the mesenchymal stem cells may not express at least one cell surface marker selected from CD14, CD19, CD34, CD45, HLA-DR, SSEA-3, TRA-1-60, TRA-1-81, Nanog, and Oct3 / 4.

[0325] In one embodiment, the mesenchymal stem cells may express one or more cell surface markers selected from CD29, CD44, CD73, and CD105.

[0326] In one embodiment, the mesenchymal stem cells (MSCs) express CD90, ie, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, or at least 98% of the MSCs express CD90.

[0327] In one embodiment, the mesenchymal stem cells may maintain cell surface marker expression of at least 70% or more, at least 75% or more, at least 80% or more, at least 85% or more, at least 90% or more, or at least 95% or more even after at least 10 passages, at least 11 passages, at least 12 passages, at least 13 passages, at least 14 passages, at least 15 passages, at least 16 passages, at least 17 passages, at least 18 passages, at least 19 passages, or at least 20 passages.

[0328] In one embodiment, the passaging may be based on a specific fold increase in the number of cells. For example, one passaging may be defined as an increase in the number of cells by at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, or 8-fold compared to the start of the previous passaging, but is not limited thereto.

[0329] In one embodiment, the mesenchymal stem cells may not express one or more cell surface markers selected from CD14, CD19, CD34, CD45, HLA-DR, SSEA-3, TRA-1-60, TRA-1-81, Nanog, and Oct3 / 4. In another embodiment, the mesenchymal stem cells may not express one or more cell surface markers selected from CD11b, CD14, CD34, CD45, CD79, HLA-DR, TRA-1-60, and TRA-1-81. In some embodiments, the mesenchymal stem cells may not express one or more cell surface markers selected from CD34, CD45, HLA-DR, TRA-1-60, and TRA-1-81.

[0330] In one embodiment, the host cell may be an immune cell.

[0331] The term "immune cells" as used herein comprehensively refers to all types of cells that constitute the immune system. Cellular therapy utilizing the immunoregulatory ability of immune cells has been used to treat various diseases such as cancer and autoimmune diseases. However, due to their non-specific effects, immune cell therapy agents engineered to enable target-specific immunoregulation, such as chimeric antigen receptors, have attracted much attention.

[0332] The CD40L binding agents of the present invention (e.g., Stefin A protein variants) target CD40L, which is involved in immunoregulation, and in particular have the ability to antagonize CD40L. Therefore, when a nucleic acid encoding a CD40L binding agent (e.g., Stefin A protein variants that specifically bind to CD40L) and / or a fusion protein containing the same is introduced into immune cells, a significantly superior immunoregulatory effect can be exhibited.

[0333] In one embodiment, the immune cells may be characterized as being selected from the group consisting of T cells, B cells, natural killer (NK) cells, nkT cells, and dendritic cells, but are not limited thereto.

[0334] In one embodiment, the immune cells may be characterized as being isolated from the human body, blood, or peripheral blood mononuclear cells (PBMCs); or differentiated from stem cells (preferably pluripotent stem cells), but are not limited thereto.

[0335] In one embodiment, when a nucleic acid encoding a fusion protein (e.g., a chimeric antigen receptor) comprising a CD40L-binding agent (e.g., a Stefin A protein variant that specifically binds to CD40L) as an extracellular binding domain is introduced into the immune cells, it can be used as a cell therapy agent such as CAR-T or CAR-NK.

[0336] In one embodiment, the host cell may be a somatic cell.

[0337] The term "somatic cells" as used herein refers to general cells, excluding germ cells, that constitute the body of an animal, preferably a human. Examples of cell therapy agents using somatic cells are well known in the art. For example, epidermal cells such as keratinocytes, fibroblasts, and mucous membrane cells can be used for the treatment of skin burns, scars, and cosmetic purposes, while chondrocytes, adipocytes, pancreatic islet cells, and skeletal myoblasts can be used to treat diseases such as degenerative arthritis and subcutaneous fat deficiency, but are not limited thereto.

[0338] When the host cells of the present invention are used as somatic cells and a gene encoding a CD40L binding agent (e.g., a Stefin A protein variant that specifically binds to CD40L) and / or a fusion protein containing the same is introduced, an immunomodulatory effect can be exhibited through the suppression of CD40L activity.

[0339] In one embodiment, the genetically modified cells are capable of secreting, expressing at the cell membrane, and / or localizing to a specific site within the cell the CD40L-binding agent (e.g., a Stefin A protein variant that specifically binds to CD40L) and / or a fusion protein comprising the same.

[0340] Method for introducing nucleic acid into host cells The term "introduction" as used herein means causing a host cell to harbor an exogenous gene (nucleic acid) that the host cell does not possess.

[0341] In one embodiment, a nucleic acid encoding the CD40L binding agent (e.g., a Stefin A protein variant that specifically binds to CD40L) or a fusion protein containing the same can be introduced into a host cell using a vector containing the same.

[0342] The term "vector" of the present invention refers to a means for expressing a gene of interest in a host cell, and includes, for example, adenovirus vectors, retrovirus vectors, adeno-associated virus vectors, vaccinia virus (Puhlmann M. et al., Human Gene Therapy, 10:649-657 (1999); Ridgeway, 467-492 (1988); Baichwal and Sugden, In: Kucherlapati R, ed. Gene transfer. New York: Plenum Press, 117-148 (1986) and Coupar et al., Gene, 68:1-10 (1988)), lentivirus (Wang G. et al., J. Clin. Invest., 104(11):R55-62 (1999)), herpes simplex virus (Chamber R., et al., Proc. Natl. Acad. Sci USA, 92:1411-1415(1995)), fox virus (GCE, NJL, Krupa M, Esteban M., Curr Gene Ther 8(2):97-120(2008)), reovirus, measles virus, Semliki Forest virus, and poliovirus-derived vectors, as well as non-viral vectors such as plasmid vectors (Sambrook et al., 1989) and minicircle vectors (Yew et al. 2000 Mol Ther 1(3),255-62).

[0343] Generally, a vector may include one or more components selected from, but not limited to, a signal sequence, an origin of replication, one or more antibiotic resistance marker genes, an enhancer element, a promoter, and a transcription termination sequence. The nucleic acid encoding the Stefin A protein variant of the present invention or a fusion protein containing the same may be operably linked to a promoter, a transcription termination sequence, etc.

[0344] "Operably linked" refers to a functional connection between a nucleic acid expression control sequence (e.g., a promoter, signal sequence, or an array of transcriptional regulator binding sites) and another nucleic acid sequence, such that the control sequence controls the transcription and / or translation of the other nucleic acid sequence.

[0345] When a prokaryotic cell is used as the host, it generally contains a strong promoter capable of driving transcription (e.g., tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pLλ promoter, pRλ promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter, T7 promoter, etc.), a ribosome binding site for initiating translation, and a transcription / translation termination sequence. Furthermore, for example, when a eukaryotic cell is used as the host, promoters derived from the genome of a mammalian cell (e.g., metallothionine promoter, β-actin promoter, human hemoglobin promoter, and human muscle creatine promoter) or promoters derived from mammalian viruses (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus (CMV) promoter, HSV tk promoter, mouse mammary tumor virus (MMTV) promoter, HIV LTR promoter, Moloney virus promoter, Epstein-Barr virus (EBV) promoter, and Rous sarcoma virus (RSV) promoter) can be used, and a polyadenylation sequence is generally used as a transcription termination sequence.

[0346] In one embodiment, the promoter may be a eukaryotic promoter, preferably selected from the group consisting of a CMV promoter, a PGK promoter, an EF1α promoter, an EFS promoter, a CBh promoter, an MSCV promoter, an SFFV promoter, and an UbC promoter, and most preferably selected from the group consisting of a cytomegalovirus (CMV) promoter, an EF1α promoter, and a CBh promoter, but is not limited thereto.

[0347] In one embodiment, the promoter may further comprise an enhancer sequence, but is not limited thereto.

[0348] In some cases, the vector may be fused with other sequences to facilitate purification of the antibody expressed therefrom, such as glutathione S-transferase (Pharmacia, USA), maltose-binding protein (NEB, USA), FLAG (IBI, USA), and 6x His (hexahistidine; Quiagen, USA).

[0349] The vector may contain an antibiotic resistance gene commonly used in the art as a selection marker, including, but not limited to, resistance genes to ampicillin, gentamycin, carbenicillin, chloramphenicol, streptomycin, kanamycin, puromycin, blasticidin, hygromycin, geneticin, neomycin, and tetracycline.

[0350] In one embodiment, a nucleic acid encoding the Stefin A protein variant that specifically binds to CD40L or a fusion protein containing the same can be incorporated (integrated) and introduced into the gene of a host cell.

[0351] In one embodiment, a nucleic acid encoding the CD40L binding agent (e.g., a Stefin A protein variant that specifically binds to CD40L) or a fusion protein containing the same can be produced by chemical synthesis using an oligonucleotide synthesizer. Oligonucleotides can be designed based on the amino acid sequence of the desired polypeptide and the codons preferred by the host cell in which the recombinant polypeptide of interest will be produced. Polynucleotide sequences encoding isolated polypeptides of interest can be synthesized using standard methods. For example, a reverse-translated gene can be constructed using the complete amino acid sequence. Alternatively, DNA oligomers containing nucleoside sequences encoding specific isolated polypeptides can be synthesized. For example, multiple small oligonucleotides encoding portions of the desired polypeptide can be synthesized and then ligated. Individual oligonucleotides typically contain 5' or 3' overhangs for complementary assembly.

[0352] In one embodiment, once a nucleic acid sequence encoding the CD40L binding agent (e.g., a Stefin A protein variant that specifically binds to CD40L) or a fusion protein containing the same is obtained, a vector containing the same can be produced by recombinant DNA technology using techniques widely known in the art. Expression vectors containing a sequence encoding a CD40L binding agent (e.g., a Stefin A protein variant) of the present invention or a fusion protein containing the same and appropriate transcriptional and translational control signals can be constructed using methods well known to those skilled in the art. Such methods include, for example, in vitro recombinant DNA technology, synthetic techniques, and in vivo genetic recombination (e.g., Sambrook et al., 1990, MOLECULAR CLONING, A LABORATORY MANUAL, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, and Ausubel et al. eds., 1998, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, NY).

[0353] In one embodiment, a nucleic acid encoding the CD40L binding agent (e.g., a Stefin A protein variant that specifically binds to CD40L) or a fusion protein containing the same, or a non-viral expression vector containing the same, can be delivered to host cells by conventional techniques (e.g., electroporation, liposome transfection, and calcium phosphate precipitation).

[0354] The vector can be introduced into a host cell by methods such as transformation or transfection. As used herein, the term "transformation" refers to introducing DNA into a host cell so that the DNA becomes replicable, either as an extrachromosomal element or by chromosomal integration. As used herein, the term "transfection" refers to the host cell harboring an expression vector, regardless of whether any coding sequences are actually expressed. To introduce the vector, a wide variety of techniques commonly used to introduce exogenous nucleic acid (DNA or RNA) into prokaryotic or eukaryotic host cells can be used, including, but not limited to, electrophoresis, calcium phosphate precipitation, DEAE-dextran transfection, and lipofection.

[0355] Of course, it should be understood that not all vectors and expression control sequences function equally well in expressing the DNA sequences of the present invention. Similarly, not all hosts function equally well in the same expression system. However, one of ordinary skill in the art can select from a wide variety of vectors, expression control sequences, and hosts without undue experimental burden and without departing from the scope of the present invention. For example, when selecting a vector, the host must be considered because the vector must replicate within it. The vector's copy number, the ability to control that copy number, and the expression of other proteins encoded by the vector, such as antibiotic markers, must also be considered. When selecting an expression control sequence, various factors must also be considered, such as the relative strength, regulatability, and compatibility with the DNA sequences of the present invention, particularly in relation to potential secondary structures. A unicellular host must be selected taking into account factors such as the toxicity of the selected vector, the product encoded by the DNA sequences of the present invention, secretion characteristics, the ability to correctly fold the protein, culture and fermentation requirements, and the ease with which the product encoded by the DNA sequences of the present invention can be purified from the host. Within these variables, one skilled in the art can select various combinations of vectors / expression regulatory sequences / hosts that can express the DNA sequences of the present invention in fermentation or large-scale animal culture. Screening methods for cloning cDNA by expression cloning include binding, panning, and film emulsion. In one embodiment, a nucleic acid encoding a CD40L-binding agent or a fusion protein comprising the same is introduced into a host cell using a lentivirus.

[0356] In one embodiment, a nucleic acid encoding a Stefin A protein variant that specifically binds to CD40L or a fusion protein comprising the same is introduced into a host cell using a lentivirus.

[0357] In a specific embodiment, when a gene is introduced using a lentivirus, a transformation enhancer may be used.

[0358] In some embodiments, the transformation enhancer can be selected from, for example, but not limited to, polybrene, protamine sulfate, and Sirion's LentiBOOST, most preferably polybrene.

[0359] In one embodiment, the method may be characterized by simultaneously infecting adherent or non-adherent host cells with a transformation enhancing agent and a lentivirus, preferably by infecting the cells before attachment, but is not limited thereto.

[0360] In another aspect, the present invention relates to genetically engineered cells expressing a CD40L-binding agent (eg, a Stefin A protein variant that specifically binds to CD40L) and / or a fusion protein comprising the same.

[0361] Method for producing genetically modified cells and culture fluid for genetically modified cells In another aspect, the present invention relates to a method for producing a genetically modified cell into which a CD40L binding agent (a Stefin A protein variant that specifically binds to CD40L and / or a fusion protein containing the same) has been introduced, the method comprising the steps of: (a) introducing into a host cell a nucleic acid encoding a Stefin A protein variant that specifically binds to CD40L and / or a fusion protein comprising the same; and (b) selecting and harvesting host cells into which a nucleic acid encoding the CD40L binding agent (e.g., a Stefin A protein variant that specifically binds to CD40L) and / or a fusion protein containing the same has been introduced;

[0362] In one embodiment, step (a) can be performed through various means known in the art.

[0363] In one embodiment, step (a) may be performed using a lentivirus containing nucleic acid encoding a CD40L binding agent (e.g., a Stefin A protein variant that specifically binds to CD40L) and / or a fusion protein containing the same.

[0364] In one embodiment, the lentivirus may be characterized by having a vector containing a nucleic acid encoding a CD40L binding agent (e.g., a Stefin A protein variant that specifically binds to CD40L) and / or a fusion protein containing the same.

[0365] In one embodiment, the vector may further include one or more of a signal sequence, an origin of replication, one or more antibiotic resistance marker genes, an enhancer element, a promoter, and a transcription termination sequence.

[0366] In one embodiment, the promoter may be a eukaryotic promoter, preferably selected from the group consisting of a CMV promoter, a PGK promoter, an EF1α promoter, an EFS promoter, a CBh promoter, an MSCV promoter, an SFFV promoter, and an UbC promoter, and most preferably selected from the group consisting of a cytomegalovirus (CMV) promoter, an EF1α promoter, and a CBh promoter, but is not limited thereto.

[0367] In one embodiment, the promoter may further comprise an enhancer sequence, but is not limited thereto.

[0368] In one embodiment, the enhancer is a short DNA region of about 50 bp to 1500 bp in length that can be bound to a transcriptional regulatory protein. The enhancer can be located at the transcription initiation site or upstream or downstream of the promoter. Enhancers for various promoters are well known in the art, and those of ordinary skill in the art can select and apply enhancers without limitation.

[0369] In one embodiment, step (a) may include infecting host cells with the lentivirus to transform the host cells.

[0370] In one embodiment, the step of infecting a host cell with the lentivirus and transforming the host cell may be performed in the presence of a transformation enhancer.

[0371] In one embodiment, the transformation enhancer is preferably a cationic polymer, which allows the negatively charged nucleic acid or gene to be easily introduced into the host cell.

[0372] In one embodiment, the transformation enhancer may be a cationic polymer, such as, but not limited to, polybrene, protamine sulfate, and Sirion's Lentiboost, most preferably polybrene.

[0373] In one embodiment, the step of infecting and transforming the host cells with the lentivirus can be carried out by treating the lentivirus after attaching the host cells, or by treating and infecting the host cells with the lentivirus before attaching the host cells.

[0374] In one embodiment, the method of treating the host cells with a lentivirus before attachment and infecting the host cells during the attachment process is also known as reverse transduction.

[0375] In one embodiment, the step of infecting and transforming the host cells with the lentivirus preferably involves treating and infecting the host cells with the lentivirus before attaching the host cells, but is not limited thereto.

[0376] In one embodiment, step (b) may involve selecting host cells into which a nucleic acid encoding the CD40L binding agent (e.g., a Stefin A protein variant that specifically binds to CD40L) and / or a fusion protein containing the same has been introduced using an antibiotic and its resistance gene.

[0377] Methods for selecting transformed cells into which genes have been introduced using vectors containing antibiotics and their resistance genes are well known in the art.

[0378] In one embodiment, step (b) may involve treating the cells with an aminoglycoside antibiotic and selecting the transfected genetically modified cells.

[0379] In one embodiment, the antibiotic may be, for example, ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, puromycin, blasticidin, hygromycin, geneticin, neomycin, and tetracycline, but is not limited thereto.

[0380] In one embodiment, the genetically modified cell may be characterized in that a neomycin resistance gene has been additionally introduced into the cell in addition to the nucleic acid encoding the CD40L binding agent (e.g., a Stefin A protein variant that specifically binds to CD40L) and / or a fusion protein containing the same.

[0381] In one embodiment, for example, the antibiotic may be administered at a concentration of 250 μg / mL to 500 μg / mL for 3 to 7 days, at a concentration of about 125 μg / mL for 5 days, or at a concentration of about 62.5 μg / mL for 7 days, but is not limited thereto.

[0382] In another aspect, the present invention relates to a culture medium for the genetically modified cells. In one embodiment, the culture medium can be prepared by culturing the genetically modified cells using appropriate conditions and medium depending on the type of host cell.

[0383] In one embodiment, the genetically modified cells can express and secrete a CD40L-binding agent (e.g., a Stefin A protein variant that specifically binds to CD40L) and / or a fusion protein containing the same. In this case, the culture medium of the genetically modified cells can contain the CD40L-binding agent (e.g., a Stefin A protein variant that specifically binds to CD40L) and / or a fusion protein containing the same secreted together with the genetically modified cells.

[0384] In one embodiment, genetically modified mesenchymal stem cells (MSCs) are provided that contain a nucleic acid encoding a CD40L-binding agent as described herein. In one embodiment, genetically modified mesenchymal stem cells expressing the CD40L-binding agent are provided, wherein the mesenchymal stem cells are derived from induced pluripotent stem cells (iPS cells). In one embodiment, genetically modified mesenchymal stem cells expressing the CD40L-binding agent are provided, wherein the mesenchymal stem cells have the pluripotency to differentiate into cells selected from the group consisting of adipocytes, osteocytes, chondrocytes, muscle cells, neurons, and cardiomyocytes. In one embodiment, genetically modified mesenchymal stem cells expressing the CD40L-binding agent are provided, wherein the MSCs can be stored for a long period of time and / or repeatedly passaged. In one embodiment, genetically modified mesenchymal stem cells expressing the CD40L-binding agent are provided, wherein the expression of mesenchymal stem cell surface markers is maintained at 90% or more in the mesenchymal stem cells after 20 or more passages.

[0385] In one embodiment, when the CD40L-binding agent is secreted extracellularly, the cell population expresses and secretes the CD40L-binding agent at an average level of 200 fg / cell / day or greater. In one embodiment, when the CD40L-binding agent is secreted extracellularly, the cell population expresses and secretes the CD40L-binding agent at an average level of 300 fg / cell / day or greater. In some embodiments, when the CD40L-binding agent is secreted extracellularly, the cell population expresses and secretes the CD40L-binding agent at an average level of 400 fg / cell / day or greater.

[0386] In one embodiment, when the CD40L-binding agent is secreted extracellularly, the cell population expresses and secretes the CD40L-binding agent at an average level of 200-1500 fg / cell / day. In another embodiment, when the CD40L-binding agent is secreted extracellularly, the cell population expresses and secretes the CD40L-binding agent at an average level of 300-1000 fg / cell / day. In another embodiment, when the CD40L-binding agent is secreted extracellularly, the cell population expresses and secretes the CD40L-binding agent at an average level of 400-800 fg / cell / day.

[0387] In one embodiment, when the host cells are mesenchymal stem cells, methods for long-term subculture thereof are well known in the art. For example, Korean Patent Application Publication No. 10-2021-0072734 and Korean Patent No. 10-1135636 disclose in detail methods for maintaining undifferentiated potential and marker expression characteristics even after several dozen subcultures.

[0388] Purpose The present invention excludes from its scope uses for producing Stefin A protein variants and / or fusion proteins containing the same that specifically bind to CD40L in genetically modified cells.

[0389] The uses of the recombinant cells of the present invention include, without limitation, all uses except for the production of Stefin A protein variants and / or fusion proteins containing the same, including, but not limited to, medical, pharmaceutical, and clinical uses.

[0390] Use - Cellular therapeutic agent or pharmaceutical composition In particular, it is well known in the art that activation of T cells and B cells through CD40L / CD40 interaction acts as a pathogenic factor in autoimmune or inflammatory diseases, which plays a major role in pathology. Specifically, CD40L is a pathogenic factor in various diseases, such as type 1 diabetes, thyroiditis, psoriasis, lupus (systemic lupus erythematosus; SLE), rheumatoid arthritis (RA), and multiple sclerosis (MS). Various compounds or antibodies targeting CD40L have been developed for the treatment of such diseases (Semin Immunol. 2009;21(5):293-300; Advanced Drug Delivery Reviews Volume 141, 15 February 2019, Pages 92-103).

[0391] In the examples of the present invention, it was confirmed that the genetically modified cells of the present invention have significantly superior suppressive abilities to T cell activity and B cell activity.

[0392] In another example of the present invention, it was confirmed that administration of the CD40L binding agent (e.g., a Stefin A protein variant that specifically binds to CD40L) in an animal model of graft-versus-host disease (GVHD) showed a significant therapeutic effect.

[0393] In another aspect, the present invention provides a cell therapy agent comprising the genetically modified cells.

[0394] In yet another aspect, the present invention provides a pharmaceutical composition for preventing or treating immune diseases, which comprises the recombinant cell or a culture medium thereof.

[0395] In one embodiment, a composition is provided comprising genetically modified cells (e.g., mesenchymal stem cells) that contain or express a CD40L-binding agent. In one embodiment, a pharmaceutical composition is provided comprising the genetically modified mesenchymal stem cells.

[0396] In one embodiment, when the genetically modified cells contain a CD40L binding agent (e.g., a Stefin A protein variant that specifically binds to CD40L) and / or a fusion protein containing the same, the genetically modified cells may be included in the pharmaceutical composition in the form of a culture medium containing not only the genetically modified cells but also the CD40L binding agent (e.g., a Stefin A protein variant that specifically binds to CD40L) secreted therethrough and / or a fusion protein containing the same.

[0397] The term "prevention" as used herein means any action of administering the pharmaceutical composition provided by the present invention to an individual who is expected to develop an immune disease, thereby suppressing or delaying the onset of the immune disease.

[0398] The term "treatment" as used herein refers to any clinical intervention aimed at altering the natural processes of the individual or cells being treated, and may be performed during or to prevent the progression of a clinical pathological condition. The intended therapeutic effects include preventing the onset or recurrence of the disease, alleviating symptoms, inhibiting all direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, alleviating or temporarily alleviating the disease state, and improving prognosis. For purposes of the present invention, the term "treatment" may be interpreted as including, but is not limited to, any action of administering the pharmaceutical composition of the present invention to a patient suffering from an autoimmune disease, such as psoriasis, to improve the course of the autoimmune disease.

[0399] In one embodiment, the pharmaceutical composition may further comprise one or more pharmaceutically acceptable carriers.

[0400] Pharmaceutically acceptable carriers contained in the composition of the present invention include those commonly used in formulation, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginic acid, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil, but are not limited thereto. In addition to the above ingredients, the composition of the present invention may further contain lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc.

[0401] The pharmaceutical composition of the present invention can be administered orally or parenterally. Parenteral administration includes intravenous injection, subcutaneous injection, intramuscular injection, peritoneal injection, intradermal administration, topical administration, intranasal administration, intrapulmonary administration, and intrarectal administration.

[0402] Since proteins or peptides are digested during oral administration, oral compositions must be formulated to coat or protect the active agent from degradation in the stomach. Additionally, pharmaceutical compositions can be administered by any device that allows the active agent to be delivered to target cells.

[0403] The appropriate dosage of the composition of the present invention varies depending on various factors such as formulation method, administration method, age, weight, sex, pathological condition, diet of the patient, administration time, administration route, excretion rate, and reaction sensitivity, and a skilled physician can generally easily determine and prescribe an effective dosage for the desired treatment or prevention. As used herein, the term "pharmaceutically effective amount" means an amount sufficient for the prevention or treatment of an immune disease.

[0404] The pharmaceutical compositions of the present invention can be prepared in unit dose form or in multi-dose containers by formulating them with pharmaceutically acceptable carriers and / or excipients in a manner easily understood by those skilled in the art, and the dosage form may be in the form of a solution, suspension, or emulsion in an oily or aqueous medium, or in the form of an extract, powder, suppository, powder, granule, tablet, or capsule, and may further contain a dispersing agent or stabilizer.

[0405] The pharmaceutical composition of the present invention can be administered in combination with known pharmaceuticals or pharmaceutical compositions that have the effect of preventing, ameliorating, or treating the symptoms of immune diseases, and can be administered in combination with, for example, one or more other immunotherapeutic agents, chemotherapeutic agents, antibody therapeutic agents, etc.

[0406] Specifically, the pharmaceutical composition of the present invention may be used in combination with a drug selected from the group consisting of disease modifying antirheumatic drugs (DMARDs); nonsteroidal anti-inflammatory drugs (NSAIDs); corticosteroids; Janus kinase inhibitors; calcineurin inhibitors; mTOR inhibitors; IMDH inhibitors; and biological agents, but is not limited thereto.

[0407] In one embodiment, the disease modifying antirheumatic drugs (DMARDs) may include, but are not limited to, Actarit, Auranofin, Azathioprine, Bucillamine, Cyclophophamide, D-Penicillamine, Leflunomide, Lobenzarit disodium, Methotrexate, Minocycline hydrochloride, Mizoribine, and Salazosulfapyridine.

[0408] In one embodiment, the nonsteroidal anti-inflammatory drug (NSAID) may include, but is not limited to, celecoxib, diclofenac sodium, ibuprofen, ketoprofen, meloxicam, naproxen, piroxicam, etc.

[0409] In one embodiment, the corticosteroids may include, but are not limited to, prednisone (Deltasone, Orasone), budesonide (Entocort EC), prednisolone (Millipred), methylprednisolone, etc.

[0410] In one embodiment, the Janus kinase inhibitors include, for example, approved drugs such as tofacitinib, abrocitinib, baricitinib, delgocitinib, fedratinib, filgotinib, oclacitinib, peficitinib, and ruxolitinib. Drugs currently in clinical trials include, but are not limited to, cerdulatinib, gandotinib, lestaurtinib, momelotinib, pacritinib, deucravacitinib, and the like.

[0411] In one embodiment, the calcineurin inhibitor may include, but is not limited to, cyclosporine, tacrolimus, etc.

[0412] In one embodiment, the mTOR inhibitor may include, but is not limited to, sirolimus (Rapamune), everolimus (Afinitor, Zortress), etc.

[0413] In one embodiment, the IMDH inhibitor may include, but is not limited to, azathioprine (Azasan, Imuran), mycophenolate (CellCept, Myfortic), etc.

[0414] In one embodiment, examples of the biological agent include, but are not limited to, abatacept, adalimumab, anakinra, certolizumab, etanercept, golimumab, infliximab, ixekizumab, natalizumab, rituximab, secukinumab, tocilizumab, ustekinumab, vedolizumab, basiliximab, and daclizumab.

[0415] In one embodiment, the immune disease may be an autoimmune disease or an inflammatory disease.

[0416] In one embodiment, the immune disease may be characterized as being selected from the group consisting of, but not limited to, lupus (SLE), lupus nephritis (e.g., drug-induced lupus nephritis), immune thrombocytopenia (ITP), rheumatoid arthritis (RA), multiple sclerosis (MS), inflammatory bowel disease (IBD) (e.g., Crohn's disease and colitis / ulcerative colitis), graft-versus-host disease (GvHD) or allograft rejection, transplant / solid organ transplant (SOT), primary biliary cholangitis (PBC), psoriasis, psoriatic arthritis, collagen-induced arthritis, experimental allergic encephalomyelitis (EAE), oophoritis, allergic rhinitis, asthma, Sjogren's syndrome, atopic eczema, myasthenia gravis, Graves' disease, and / or glomerulosclerosis.

[0417] Lupus Lupus, also known as systemic lupus erythematosus (SLE), is a chronic autoimmune disease that can cause swelling (inflammation) and pain throughout the body. There are different types of lupus, with systemic lupus erythematosus being the most common. Other types of lupus include: Cutaneous lupus erythematosus: This type of lupus affects the skin. "Cutaneous" is the term that means skin. People with cutaneous lupus erythematosus may experience skin problems such as sun sensitivity and rashes. Hair loss can also be a symptom of the disease.

[0418] Drug-induced lupus: This type of lupus is caused by certain drugs. People with drug-induced lupus may have many of the same symptoms as systemic lupus erythematosus, but it is generally temporary.

[0419] Neonatal lupus: A rare form of lupus, neonatal lupus is a disease that is detected in infants at birth. Babies born with neonatal lupus inherit antibodies from their mothers, who either had lupus during pregnancy or are at risk of developing the disease later in life. Not all babies born to mothers with lupus will develop lupus.

[0420] Therapies that may be used in combination with the pharmaceutical compositions of the present invention may include, but are not limited to, for example, steroids (including corticosteroids, prednisone); hydroxychloroquine (Plaquenil®); azathioprine (Imuran®); methotrexate (Rheumatrex®); cyclophosphamide (Cytoxan®) and mycophenolate mofetil (CellCept®); belimumab (Benlysta®); and / or rituximab (Rituxan®).

[0421] Lupus nephritis Lupus nephritis occurs as a complication of lupus. It occurs when lupus autoantibodies affect the kidney's mechanisms that filter waste. This causes kidney inflammation, which can lead to blood in the urine, protein in the urine, high blood pressure, kidney dysfunction, or kidney failure. Lupus nephritis occurs in about half of adults with systemic lupus. Systemic lupus causes immune system proteins to damage the kidneys, impairing their ability to filter waste.

[0422] Rheumatoid arthritis Rheumatoid arthritis is a type of chronic (ongoing) arthritis that affects both joints of the body, such as the hands, wrists, and knees. The short-term goal of rheumatoid arthritis medications is to reduce joint pain and swelling and improve joint function. The long-term goal is to slow or stop the progression of the disease, particularly joint damage.

[0423] Arthritis is a general term describing inflammation of the joints. Rheumatoid arthritis is a type of chronic (ongoing) arthritis (causing pain and swelling) that generally occurs symmetrically in joints (on both sides of the body, such as the hands, wrists, and knees). This involvement of many joints helps distinguish rheumatoid arthritis from other types of arthritis.

[0424] Besides affecting the joints, rheumatoid arthritis can sometimes affect the skin, eyes, lungs, heart, blood, nerves, or kidneys.

[0425] Therapies that may be used in combination with the pharmaceutical compositions of the present invention to treat rheumatoid arthritis include, for example:

[0426] Analgesics and anti-inflammatory drugs: These products include nonsteroidal anti-inflammatory drugs (NSAIDs), such as ibuprofen (MOTRIN®), naproxen (ALEVE®), celecoxib, diclofenac sodium, ketoprofen, meloxicam, and piroxicam. Another class of drugs, COX-2 inhibitors, also falls into this category and are used to relieve the signs and symptoms of rheumatoid arthritis. One COX-2 inhibitor, celecoxib (CELEBREX®), is available in the United States. COX-2 inhibitors are designed to reduce gastrointestinal bleeding side effects.

[0427] Disease-modifying antirheumatic drugs (DMARDs): Unlike other NSAIDs, DMARDs can actually modify the immune system and slow disease progression. Previous DMARDs included methotrexate (TREXALL®), gold salts, penicillamine (CUPRIMINE®), hydroxychloroquine (PLAQUENIL®), sulfasalazine (AZULFIDINE®), cyclosporine (SANDIMMUNE®), cyclophosphamide (CYTOXAN®), and leflunomide (ARAVA®). Currently, methotrexate, leflunomide, hydroxychloroquine, and sulfasalazine are most frequently used (cyclosporine, cyclophosphamide, gold salts, and penicillamine are no longer commonly used).

[0428] Biologics: In addition to these "traditional" DMARDs, newer drugs have been approved. Currently, there are seven classes of drugs, each with its own distinct classes, depending on the case (some of which have been used since 2000 as anti-TNF drugs). Collectively, these DMARDs are known by other names: biologics (or biological agents). Compared to traditional DMARDs, these products target molecules that cause inflammation in rheumatoid arthritis. Inflammatory cells in the joints are involved in the pathogenesis of rheumatoid arthritis itself. Biologics reduce the inflammatory process that ultimately leads to the joint damage seen in rheumatoid arthritis. By attacking cells at a more specific level than inflammation itself, biologics are considered more effective and more specifically targeted. Biologics include etanercept (ENBREL®), infliximab (REMICADE®), adalimumab (HUMIRA®), anakinra (KINARET®), abatacept (ORENCIA®), rituximab (RITUXAN®), certolizumab pegol (CIMZIA®), golimumab (SYMPON®), tocilizumab (ACTEMRA®), and tofacitinib (XELJANJ®). Some biologics are used in combination with traditional DMARDs, particularly methotrexate.

[0429] Multiple sclerosis Multiple sclerosis (MS) is an autoimmune disease. In this condition, the immune system mistakenly attacks healthy cells. In people with multiple sclerosis, the immune system attacks myelin cells, the protective membrane that surrounds nerves in the brain and spinal cord. When myelin is damaged, nerve signals from the brain to the rest of the body are blocked. The damage can trigger symptoms that affect the brain, spinal cord, and eyes.

[0430] There are four types of multiple sclerosis. Clinically isolated syndrome (CIS): When the first symptoms of MS occur, healthcare providers typically classify this as CIS. Not everyone with CIS progresses to multiple sclerosis. Relapse-remitting MS (RRMS): This is the most frequent form of multiple sclerosis. People with RRMS have flares (also called relapses or exacerbations) of new or worsening symptoms. Periods of remission follow (when symptoms stabilize or disappear). Primary-progressive MS (PPMS): People diagnosed with PPMS have recurring or gradually worsening symptoms that do not go away. Secondary-progressive MS (SPMS): In most cases, people originally diagnosed with RRMS eventually progress to SPMS. With secondary-progressive multiple sclerosis, nerve damage continues to accumulate. Symptoms gradually worsen. You may experience some relapses or flares (when symptoms increase), but these are followed by no further periods of relief (when symptoms stabilize or disappear).

[0431] Treatments that may be used in combination with the pharmaceutical compositions of the present invention include, for example: Disease-modifying therapy (DMT): Many medications have received FDA approval for long-term MS treatment. These medications help reduce relapses (also called flares or attacks). They slow the progression of the disease and can prevent new lesions from forming in the brain and spinal cord. Drug treatment for relapse management: If you have severe attacks, your neurologist may recommend high doses of corticosteroids. These drugs can quickly reduce inflammation and slow damage to the myelin sheath that surrounds nerve cells. Physical rehabilitation therapy: Multiple sclerosis can affect your physical function. Staying physically healthy and strong helps you maintain mobility. Mental health consultation: Coping with a chronic illness can be emotionally difficult. MS can sometimes affect mood and memory. Working with a neuropsychologist or other emotional support is an essential part of managing the disease.

[0432] inflammatory bowel disease Inflammatory bowel diseases (IBD) are a group of disorders that cause chronic inflammation (pain and edema) of the intestine. Crohn's disease and ulcerative colitis are the main types of IBD. The types are: Crohn's disease causes pain and swelling in the digestive tract. It can affect any part of the digestive tract, from the mouth to the anus. It most frequently affects the small intestine and upper part of the large intestine. Ulcerative colitis causes swelling and sores (ulcers) in the large intestine (colon and rectum). Microcolitis induces intestinal inflammation that can only be detected under a microscope.

[0433] Treatments that may be used in combination with the pharmaceutical compositions of the present invention include, for example: Aminosalicylates (anti-inflammatory drugs such as sulfasalazine, mesalamine, or balsalazide) minimize irritation to internal organs; antibiotics treat infections and abscesses; biologics block immune system signals that trigger inflammation; corticosteroids such as prednisone suppress the immune system and manage flares; immunomodulators calm an overactive immune system; antidiarrheals; nonsteroidal anti-inflammatory drugs (NSAIDs); and supplements such as vitamins and probiotics.

[0434] Graft-versus-host disease (GvHD) Graft-versus-host disease (GvHD) is a condition that can occur after allogeneic transplantation. In GvHD, the donated bone marrow and peripheral blood stem cells perceive the recipient's body as foreign and attack the donated cells / bone marrow. GvHD can include acute graft-versus-host disease (aGvHD); and chronic graft-versus-host disease (cGvHD).

[0435] Psoriasis Psoriasis is a chronic skin disorder that refers to a skin condition that does not go away. People with psoriasis have thick pink or red patches of skin covered with white or silvery scales. The thick, scaly patches are called plaques. Psoriasis typically begins early in adulthood, but can also begin later. In addition to the red, scaly patches, symptoms of psoriasis include itchy, cracked, dry skin, a scaly scalp, sore skin, indented nails, cracked or brittle nails, and joint pain.

[0436] Treatments that may be used in combination with the pharmaceutical compositions of the present invention include, but are not limited to, steroid creams, moisturizers for dry skin, anthralin (a drug that slows the production of skin cells), medicated lotions to improve scalp psoriasis, shampoos and bath detergents, vitamin D3 ointment, vitamin A or retinoid creams, phototherapy, PUVA (a treatment that combines the drug psoralen with a special form of ultraviolet light exposure), methotrexate, retinoids, cyclosporine, and / or immunotherapy.

[0437] Sjogren's syndrome Sjögren's syndrome is a lifelong autoimmune disease that reduces the amount of moisture produced by the sweat glands in the eyes and mouth. It is named after the Swedish ophthalmologist Henrik Sjögren, who first described the disease. Dry mouth and dry eyes are the main symptoms, but most people with these problems do not have Sjögren's syndrome. Dry mouth is also called xerostomia.

[0438] Sjogren's syndrome comes in two forms: primary Sjogren's syndrome (occurring on its own and not due to another health condition), and secondary Sjogren's syndrome (occurring in addition to other autoimmune diseases such as rheumatoid arthritis, lupus, and psoriatic arthritis).

[0439] Treatments that may be used in combination with the pharmaceutical compositions of the present invention include, for example, dry eye treatments (e.g., artificial tears, prescription eye drops, punctal plugs, surgery, autologous serum eye drops), dry mouth treatments (e.g., saliva-generating agents), and treatments for joint or organ problems (e.g., painkillers, antirheumatic drugs, immunosuppressants, steroids, antifungal agents, and agents for treating vaginal dryness).

[0440] Myasthenia gravis Myasthenia gravis (MG) is an autoimmune disease in which the body's immune system mistakenly attacks its own body. MG affects the transmission of signals between nerves and muscles (neuromuscular junctions).

[0441] Patients with myasthenia gravis lose the ability to voluntarily control their muscles. They experience muscle weakness and fatigue of varying severity. They may be unable to move muscles in the eyes, face, neck, and limbs. MG is a lifelong neuromuscular disorder.

[0442] Myasthenia gravis affects approximately 20 out of 100,000 people. Experts estimate that 36,000 to 60,000 Americans suffer from this neuromuscular disease. The actual number of people affected may be even higher, as some mildly affected individuals may not know they have the disease. MG primarily affects women between the ages of 20 and 40 and men between the ages of 50 and 80. Approximately 1 in 10 cases of MG occurs in teenagers (adolescent MG). The disease can affect people of all ages, but it rarely occurs in children. Autoimmune MG is the most frequent form of this neuromuscular disease. Autoimmune MG is as follows:

[0443] Ocular MG: The muscles that move the eyes and eyelids become weak. The eyelids may droop or the eyes may not be able to open. Some people have double vision. Vision loss is often the first sign of MG. Nearly half of people with ocular MG develop the systemic form within two years of the initial symptoms.

[0444] Systemic MG: Muscle weakness affects other parts of the body, such as the eyes, face, neck, arms, legs, and neck. It can make it difficult to speak or drink, lift your arms above your head, stand from a seated position, walk long distances, or climb stairs.

[0445] Therapies that may be used in combination with the pharmaceutical compositions of the present invention include, for example, drugs, monoclonal antibodies, IV immunoglobulin (IVIG), plasma exchange (plasmapheresis), and / or surgery.

[0446] In yet another aspect, the present invention relates to a method for preventing or treating an immune disease, which comprises the step of administering the genetically modified cells or a culture medium thereof.

[0447] In yet another aspect, the present invention relates to use of the genetically modified cells or their culture medium for preventing or treating immune diseases.

[0448] In yet another aspect, the present invention relates to the use of the genetically modified cells or their culture medium for producing a pharmaceutical composition for preventing or treating immune diseases.

[0449] Uses - Drug delivery compositions In yet another aspect, the present invention relates to a composition for drug delivery comprising the genetically modified cells.

[0450] In one embodiment of the present invention, the genetically modified cells may contain at least one drug carried or attached to the cell surface.

[0451] In one embodiment of the present invention, the drug may further include one or more drugs selected from the group consisting of genes, viruses, and small molecule compounds. In one embodiment of the present invention, the drug may be characterized by having immunomodulatory activity, and more preferably, may be a drug having immunosuppressive activity.

[0452] In one embodiment of the present invention, the genetically modified cells of the present invention can express a CD40L binding agent (e.g., a Stefin A protein variant that specifically binds to CD40L), thereby inhibiting the adhesion and activity of immune cells to CD40L targets.

[0453] In one embodiment of the present invention, the genetically modified cells may be mesenchymal stem cells. When the host cells of the present invention are mesenchymal stem cells, the mesenchymal stem cells have a homing function that biologically searches for damaged or infected sites in the body and have excellent targeting ability, so that drugs can be delivered effectively and accurately to the desired site in the body.

[0454] In one embodiment of the present invention, the drug may be internally loaded into the mesenchymal stem cells, attached to their surface, or both, but is not limited thereto. The drug may be directly internally loaded / loaded into the mesenchymal stem cells or attached to their surface, but is not limited thereto. The drug may be loaded into a nanostructure, or attached to a specific molecule, and then internally loaded / loaded into the mesenchymal stem cells or attached to their surface, but is not limited thereto. In one embodiment of the present invention, the nanostructure may include inorganic nanoparticles, polymeric nanoparticles, proteins, or liposomes. The inorganic nanoparticles may include, but are not limited to, iron oxide nanoparticles, quantum dot nanoparticles, metal oxide nanoparticles, etc. The nanostructure may include, but is not limited to, a porous nanostructure. For example, the drug may be loaded into the mesenchymal stem cells by being loaded in the pores of the porous nanostructure or attached to the surface of the nanostructure, but is not limited thereto. The polymeric nanoparticles are nanoparticles mainly used for drug delivery and are made mainly of polymers and lipids.

[0455] In one embodiment of the present invention, the genetically modified cells can release a drug from a target site. In one embodiment of the present invention, a drug loaded within the genetically modified cells can be released. In one embodiment of the present invention, a drug attached to the surface of the genetically modified cells can be detached from the surface and released by the target environment. In one embodiment of the present invention, when a drug is loaded onto the nanostructure, the drug can be released by temperature-specific and pH-specific structural changes of the nanostructure.

[0456] Numbered Examples Example 1. A recombinant cell in which a nucleic acid encoding a Stefin A protein variant that specifically binds to CD40L or a fusion protein containing the Stefin A protein variant is introduced into a host cell. Embodiment 2: In embodiment 1, the Stefin A protein mutant is 1×10 -6 Genetically modified cells exhibiting Kd values ​​below M. Embodiment 3. The recombinant cell of embodiment 1, wherein the Stefin A protein variant comprises an amino acid sequence represented by the following: MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVV-(Xaa)n-GTNYYIKVRAGDNKYMHLKVFKSL-(Xaa)m-EDLVLTGYQVDKNKDDELTGF; or MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVLA-(Xaa)n-GTNYYIKVRAGDNKYMHLKVFKSL-(Xaa)m-EDLVLTGYQVDKNKDDELTGF Here, Xaa is an amino acid residue, and n and m are each independently an integer of 3 to 20. Embodiment 4: The recombinant cell of embodiment 1, wherein the Stefin A protein variant comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 246 to 365. Embodiment 5: The recombinant cell of embodiment 3, wherein (Xaa)n comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 6 to SEQ ID NO: 125. Embodiment 6: The recombinant cell of embodiment 3, wherein (Xaa)m comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 126 to SEQ ID NO: 245. Embodiment 7: The recombinant cell of embodiment 1, wherein the Stefin A protein variant or the fusion protein comprising the Stefin A protein variant additionally comprises a signal peptide. Embodiment 8: The recombinant cell of embodiment 1, wherein the fusion protein comprises a trimer or tetramer of a Stefin A protein variant. Embodiment 9: The recombinant cell of embodiment 8, wherein the fusion protein comprises the amino acid sequence of SEQ ID NO: 681, 691, 694, or 695. Embodiment 10: The genetically modified cell of embodiment 8, wherein the triplet or quadruplet is a Stefin A protein mutant linked by a linker. Embodiment 11. The genetically modified cell of embodiment 10, wherein the linker is a rigid linker or a flexible linker. Embodiment 12: The recombinant cell of embodiment 10, wherein the linker is selected from the group consisting of SEQ ID NO: 508 to SEQ ID NO: 534. Embodiment 13. The genetically modified cell of embodiment 1, wherein the fusion protein further comprises one or more selected from the group consisting of a binding domain, a cytokine, a half-life extension domain, a growth factor, an enzyme, and a cell-penetrating domain. Embodiment 14: The recombinant cell of embodiment 1, wherein the fusion protein further comprises one or more domains selected from the group consisting of a transmembrane domain, a hinge domain, a coiled-coil domain, a virus-derived domain, an intracellular signaling domain, and a localization domain. Embodiment 15: The recombinant cell of embodiment 1, wherein the fusion protein further comprises a therapeutic peptide or protein. Embodiment 16: In embodiment 1, the host cell is a genetically modified cell selected from the group consisting of stem cells, immune cells, and somatic cells. Embodiment 17. As in embodiment 16, the stem cells are genetically modified cells selected from the group consisting of omnipotent stem cells, multipotent stem cells, and unipotent stem cells. Embodiment 18: The method of embodiment 16, wherein the host cell is a genetically modified cell that is a mesenchymal stem cell. Embodiment 19: In embodiment 18, the mesenchymal stem cells are genetically modified cells differentiated from human pluripotent stem cells. Embodiment 20: As in embodiment 18, the mesenchymal stem cells are genetically modified cells that express at least one cell surface marker selected from the group consisting of CD29, CD44, CD73, and CD105. Embodiment 21: In embodiment 20, the mesenchymal stem cells are genetically modified cells in which the expression of the cell surface marker is maintained at 90% or more even after at least 15 passages. Embodiment 22: In embodiment 18, the mesenchymal stem cells are genetically modified cells that do not express at least one cell surface marker selected from the group consisting of CD34, CD45, HLA-DR, TRA-1-60, and TRA-1-81. Embodiment 23: The recombinant cell of embodiment 1, wherein the Stefin A protein variant or a fusion protein containing the Stefin A protein variant is expressed intracellularly. Embodiment 24: The recombinant cell of embodiment 1, wherein the Stefin A protein variant or a fusion protein containing the Stefin A protein variant is expressed on the cell surface. Embodiment 25: A recombinant cell according to any one of Embodiments 1 to 22, wherein the Stefin A mutant or a fusion protein containing the Stefin A mutant is secreted extracellularly. Embodiment 26. A culture medium of the genetically modified cells of embodiment 25. Embodiment 27. The culture medium of embodiment 26, wherein the culture medium contains a Stefin A protein variant secreted by a genetically engineered cell or a fusion protein containing the Stefin A protein variant. Embodiment 28: A cell therapy agent for preventing or treating immune diseases, comprising any one of the genetically modified cells of Embodiments 1 to 24. Embodiment 29: A pharmaceutical composition for preventing or treating immune diseases, comprising the culture medium of embodiment 26. Embodiment 30. In Embodiment 29, the immune disease is selected from the group consisting of lupus (SLE), lupus nephritis (e.g., drug-induced lupus nephritis), immune thrombocytopenia (ITP), rheumatoid arthritis (RA), multiple sclerosis (MS), inflammatory bowel disease (IBD) (e.g., Crohn's disease and ulcerative colitis), graft-versus-host disease (GvHD) or allograft rejection, transplantation / solid organ transplantation (SOT), primary biliary cholangitis (PBC), psoriasis, and psoriatic arthritis. Arthritis, Collagen-Induced Arthritis, Oophoritis, Allergic Rhinitis, Asthma, Sjogren's Syndrome, Atopic Eczema, Myasthenia Gravis, Graves' Disease, and Glomerulosclerosis. Embodiment 31: A composition for drug delivery comprising any one of the genetically modified cells of Embodiments 1 to 24. Embodiment 32: The composition of embodiment 31, wherein the genetically modified cells are configured to be additionally loaded with a gene, a virus, or a low molecular weight compound. [Example]

[0457] The present invention will be described in more detail below through examples. It will be obvious to those skilled in the art that these examples are merely for the purpose of illustrating the present invention and should not be construed as limiting the scope of the present invention.

[0458] Example 1: Selection of CD40L-binding Stefin A protein variants in a phage display library Candidate clone identification The CD40L-binding stefin A protein variants (hereinafter, "anti-CD40L stefin A protein variants") of the present disclosure were identified by selection from a stefin A protein variant library containing two random loop sequences, each loop approximately 9 amino acids long and displayed on a fixed stefin A protein variant framework based on the amino acid sequence of stefin A. Such selection procedures are well known in the art (see Tiede et al. Protein Eng Des Sel. 2014.27(5):145-155 and Hughes et al. Sci Signal. 2018.10(505):eaaj2005). According to such procedures, a suspension of phage expressing a stefin A protein variant (Affimer®) was cultured with human CD40L (ACROBiosystems, CDL-H82Q8) or mouse CD40L (ACROBiosystems, CDL-M5248), as appropriate. Otherwise, CD40L was biotinylated and alternately captured on streptavidin and neutravidin beads; otherwise, CD40L was passively absorbed onto the surface. Unbound phage particles were then washed, and bound phage were eluted after washing. Bound phage were eluted by incubating the antigen in a low pH solution followed by exposure to a high pH solution and trypsin. The eluted phage particles were then used to infect Escherichia coli (E. coli), and the infected bacteria were cultured under conditions favorable for bacteriophage replication. After bacteriophage particles were released from these infected bacteria, they were allowed to bind to the target antigen, and the bound phage particles were eluted. The eluted phage particles were propagated in bacteria, and the cycle of isolating the released phage particles from the infected bacteria was repeated to enrich the bacteriophage population for phage particles displaying proteins that bind to the target antigen. In this cycle, certain conditions were modified to select for phage particle-displayed proteins that bind more strongly or specifically to the target antigen by increasing the number of washing steps, decreasing the amount of available antigen, or adding blocking agents.

[0459] After multiple rounds of phage display library selection and amplification, the proteins expressed by the phage were expressed and screened via enzyme-linked immunosorbent assay (ELISA). Specifically, Stefin A protein variants were overexpressed from phagemid vectors, bacterial cells were lysed, and the lysates were used as substrates in ELISA. In ELISA, human CD40L was immobilized on a plate, lysates were added, and the amount of CD40L-binding Stefin A protein variants on each plate was measured using a detection antibody specific for the 6xMyc tag expressed on the candidate Stefin A protein variants. Phagemid vectors encoding the Stefin A protein variants with the best human CD40L-binding activity were sequenced to confirm the DNA sequences of candidate clones for further development. The amino acid sequences of loop 2 and loop 4 of each of these candidate clones are listed in Tables 1 and 2, respectively.

[0460] Example 2: Screening of anti-CD40L Stefin A protein variants by direct ELISA To measure the affinity of different monomeric DAW01 clones for hCD40L, a binding ELISA was performed. Specifically, plates were coated with 5 μg / ml hCD40L antigen and incubated overnight at 4°C. The plates were washed twice with 150 μl of wash buffer (PBS, Tween 20 0.1%) using a plate washer and saturated with 5% casein (Sigma) in PBS for 90 minutes at room temperature (25 ± 1°C). For binding, one-third of each DAW01 clone and one-third of rhCD40 Fc were added to the plate at 1 μM and 300 nM, respectively. The plates were washed three times as described above. Next, biotinylated polyclonal anti-human CD40 antibody was added to the plate and incubated for 90 minutes. Next, a biotinylated polyclonal antibody to cystatin A (BAF1407) was diluted in dilution buffer (PBS, 1% casein, 0.01% Tween 20) to a concentration of 0.05 μg / ml, and the plate was incubated at room temperature (25±1°C) for 90 minutes. Next, poly-HRP-streptavidin was diluted in dilution buffer, and the plate was incubated at room temperature (25±1°C) for 90 minutes. Next, the plate was washed three times as described above, and substrate (TMB, Pierce Thermo-Scientific) was added to the plate for 10 minutes. The reaction was stopped with an acidic solution, and the plate was read at 450 nm and 630 nm. The results are shown in Figure 1, with EC50 values ​​ranging from 0.67 nM to 60 nM.

[0461] Example 3: Screening of anti-CD40L Stefin A protein variants in hCD40L-HEK293 cells by flow cytometry To investigate the binding capacity of the DAW01 monomeric stefin A protein variants to cell surface-expressed hCD40L, a flow cytometry cell binding assay was performed. Briefly, hCD40L-HEK-293 cells (Crown Biosciences, C2041) were collected by centrifugation at 300 rpm for 5 minutes. The cells were resuspended in PBS and aliquoted at 200,000 cells per well of a round-bottom 96-well plate. The cells were then washed with PBS. The stefin A protein variants and controls were diluted in duplicate in a staining buffer containing 1% BSA, 0.01% sodium azide (NaN3), and 2 mM EDTA in DPBS and added to the cells for approximately 60 minutes at 4 ± 1°C. The cells were washed and stained with anti-cystatin A (R&D, AF1407) secondary antibody diluted at 0.2 mg / ml in staining buffer for approximately 45 minutes at 4 ± 1°C. The cells were washed again and stained with A488 anti-goat (ThermoFisher, A21467) detection antibody diluted at 1:500 in staining buffer for approximately 30 minutes at 4 ± 1°C. Finally, the cells were washed and stained for live and dead cells with L / D stain Zombie Yellow (Biolegend, 423103) diluted in staining buffer for 10 minutes at 4 ± 1°C. The cells were washed again, and fixation buffer (R&D) was added to each well for 10 minutes at 4 ± 1°C. PBS containing EDTA (Lonza) was then added, and the plate was read on a flow cytometer (Guava 12 HT, Millipore). Dead cells were excluded and the green fluorescence channel (488 nm / 525 / 30) was acquired. Results were analyzed using Incyte, and data were displayed as a dot plot using GraphPad. An example of the results at 1 μM is shown in Figure 2. Stefin A protein variants were confirmed to specifically bind to hCD40L-HEK293 cells (dark gray). HEK-293 negative cells were also used in the experiment to assess nonspecific binding (light gray). HuCD40L HEK293 cells and control HEK293 cells were assessed for hCD40L expression using BV711 mAb (clone 24-31).The results are shown in Figure 3. The binding of clone 230 (SEQ ID NO: 249) to hCD40L-HEK293 cells was evaluated at different doses ranging from 0.7 to 500 nM. As shown in Figure 4, the binding of clone 230 was dose-dependent between 0.7 nM and 55 nM, and reached saturation above 55 nM.

[0462] Example 4: Screening of anti-CD40L Stefin A protein variants in the CD40-HEK Blue reporter assay HEK-Blue CD40-expressing cells (Invivogen) can detect bioactive CD40L through NF-κB activation after CD40 stimulation. Activation of the NF-κB pathway can be determined by measuring the levels of secreted embryonic alkaline phosphatase (SEAP) in the cell culture medium. The assay was performed according to the manufacturer's instructions.

[0463] Briefly, cells were plated in test medium (DMEM high glucose containing Blasticin and Zeocin) and aliquoted at 20,000 cells per well of a 96-well flat-bottom tissue culture plate and incubated overnight at 37°C and 5% CO2. Next, 50 μl of test medium was removed from the cells, and 50 μl of a 4X dilution of the test Stefin A protein variant or control containing hCD40L (final concentration 0.8 nM) was added to the cells. The plates were then incubated for 22 hours at 37°C and 5% CO2. The following day, the supernatant was collected, and 30 μl of each well was mixed with 200 μl of HEK-Blue detection reagent (Invivogen) to detect SEAP activity. The mixture was incubated at 37°C, and any color change was monitored periodically. The absorbance (640 nm) was measured over 3 hours using a Pherastar plate reader. The data were displayed as a dot plot. The IC50 was then calculated using an interpolated non-linear four-parameter curve, where OD = f (log concentration). The positive control for this assay was the clinical-grade monoclonal anti-hCD40L antibody 5C8. The calculated IC50 ranged from 11.6 nM to 100 nM (e.g., clone 230 (SEQ ID NO: 249) and clone 248 (SEQ ID NO: 267)). An example of the results is shown in Figure 5.

[0464] Example 5: Formatting of Stefin A protein mutants into dimers or trimers for increased binding to hCD40L Stefin A protein variants can be designed to assemble into stable multimeric oligomers to increase binding avidity. The resulting Stefin A protein variants are ILF (In Line Fusion) proteins, and can be dimers or trimers with various linkers (rigid linkers, e.g., SEQ ID NOS: 508, 510-514; or flexible linkers, e.g., SEQ ID NOS: 509, 515-518) as shown in Figure 6.

[0465] The binding of different Stefin A protein variants (monomer, dimer, and trimer) to hCD40L was investigated as described above. The results, shown in Figure 7, confirmed that the ILF proteins have binding affinities for their target ligands that are equal to or greater than those of the monomeric form. Further analysis of this binding by flow cytometry (Figure 8) demonstrated that the Stefin A protein variants (monomer, dimer, and trimer forms) bound to hCD40L at levels similar to those of anti-CD40L antibodies.

[0466] Alternative formats were also screened using the HEK-Blue cell-based assay as described above, and the results are shown in Figure 9, showing that the clone 230 DJ format (trimer) was the best with an EC50 of 5.99 nM.

[0467] Example 6: Characterization of Trimeric or Tetrameric In-Line Fusion (ILF) Stefin A Protein Variants Using HSA-Binding Stefin A Protein Variants In additional experiments, Stefin A protein variants were engineered to assemble into stable multimeric oligomers. The Stefin A protein variants generated were ILF (In Line Fusion) proteins, which could be trimers or tetramers with various linkers (ridged linkers, see e.g., SEQ ID NOS: 508, 510-514; or flexible linkers, see e.g., SEQ ID NOS: 509, 515-518). Four Stefin A protein variants were tested: Clone-230 DT (trimer with a rigid linker), Clone-230 XT75 (trimer with a rigid linker and an HSA Stefin A protein variant), Clone-230 DS (tetramer with a rigid linker), and Clone-230 XT76 (tetramer with a rigid linker and an HSA Stefin A protein variant).

[0468] To evaluate the binding capacity of various formats, a CD40L competitive ELISA was performed. Briefly, plates were coated with 1 μg / ml rhCD40 Fc and incubated overnight at 4°C. The plates were washed twice with 150 μl of wash buffer (PBS, Tween 200.1%) using a plate washer and saturated with 5% casein (Sigma) in PBS for 90 minutes at room temperature (25±1°C). Human CD40L at 2× EC80 (1 nM) was combined with anti-hCD40L monoclonal antibodies or each test ILF clone starting at 10 nM. The resulting solutions were then mixed and added to the plates. The plates were washed three times as described above. Biotinylated anti-hCD40L polyclonal antibodies were then diluted in dilution buffer and added to the plates. The plates were incubated for 90 minutes at room temperature (25±1°C). The plates were then washed. Next, poly-HRP-streptavidin was diluted in dilution buffer and added to the plate. The plate was incubated for an additional 90 minutes at room temperature (25±1°C). The plate was washed three times as described above, and substrate (TMB, Pierce Thermo-Scientific) was added to the plate for 10 minutes. The reaction was stopped with an acidic solution, and the plate was read at 450 nm to 630 nm. The final percentage of inhibition was calculated. The results are shown in Figure 10.

[0469] The CD40L inhibitory activity of the ILF (In Line Fusion) fusion proteins containing the Stefin A protein mutants was evaluated by HEK-Blue assay using the same method as described in Example 4. The results are shown in Figure 11.

[0470] To demonstrate that distinct ILF proteins can simultaneously bind two targets (human CD40L and HSA), a bridging ELISA was performed. In this assay, hCD40L was used to capture the bispecific Stefin A variants, and anti-HSA antibodies were used to detect the Stefin A variants, i.e., HSA-binding Stefin A variants. Briefly, a 96-well plate was coated with 0.5 mg / ml human CD40L in carbonate buffer. After saturation with 5% casein / PBS buffer, the plate was washed, and dilutions of Stefin A variants or controls were incubated with HSA at a final concentration of 10 μM for 90 minutes. The plate was then washed, and a biotinylated polyclonal antibody to HSA (HRP-conjugated) (Abcam) was added for 90 minutes. After the final wash step, TMB was added for experimental development, and the plate was read at 450 nm. EC50 values ​​were then calculated using an interpolated nonlinear four-parameter standard curve (Figure 12). In control experiments, an anti-cystatin antibody bound to the stefin A framework of the stefin A protein variant was added instead of the anti-HSA antibody in the absence (Figure 12A) or presence (Figure 12B) of HSA, demonstrating that the stefin A protein variant bound to hCD40L under both conditions. Bridging ELISA data showed that the degree of HSA incorporation did not affect CD40L binding in either of the two stefin A protein variant formats tested.

[0471] Example 7: Introduction of anti-CD40L Stefin A protein mutant gene via lentivirus Example 7-1: Screening of transformation enhancers We selected a transformation enhancer to increase the transduction efficiency of lentivirus-mediated gene transfer into PSC-derived MSCs (obtained from the Institute of Population Medicine, Seoul National University Graduate School of Medicine). The transformation enhancer is a cationic polymer that promotes lentivirus-cell binding through ion neutralization, thereby increasing gene transfer efficiency. Representative transformation enhancers include polybrene and protamine sulfate. Because their efficiency and sensitivity vary depending on the cell characteristics, it is important to select an appropriate transformation enhancer. Therefore, to select a transformation enhancer suitable for PSC-derived MSCs, we treated them with three types of enhancers: polybrene (Sigma-Aldrich, TR-1003-G), protamine sulfate (Sigma-Aldrich, P3369), and Sirion's lentiboost (SIRION Biotech, SB-P-LV-101-02), at different concentrations, and compared the transduction efficiency. Lentivirus (SIRION Biotech, SEQ ID NO: 729) engineered with a vector containing the eGFP gene was treated at an MOI of 5. Polybrene was added at concentrations of 2 μg / mL, 4 μg / mL, and 8 μg / mL; protamine sulfate was added at concentrations of 5 μg / mL, 10 μg / mL, and 20 μg / mL; and Lentiboost was added at concentrations of 1:500, 1:100, and 1:20, as recommended by the manufacturer. Sixteen to 20 hours after lentivirus treatment, the lentivirus-containing culture medium was removed and replaced with fresh culture medium (FUJIFILM Irvine Scientific, 991333). The cells were then cultured for 48 hours. After 48 hours, the cells were harvested and the transduction efficiency was compared between the GFP-transduced cell populations using a flow cytometer. As a result, as shown in Figure 13, polybrene showed an efficiency of 66.27% when treated with 2 μg / mL, protamine sulfate showed an infusion efficiency of 63.90% at 20 μg / mL, and Lentiboost showed an infusion efficiency of 21.98% at a concentration of 1:500, confirming a tendency for cell death to increase with increasing treatment concentration.In summary, when transducing genes into PSC-derived MSCs using lentivirus, polybrene, which showed the highest transduction efficiency at a low concentration, showed significantly superior transduction efficiency.

[0472] The vectors used to construct the lentiviruses used in the examples of the present invention are shown in SEQ ID NOs: 729 to 738 in Table 12 below.

[0473] [Table 12] TIFF2026504813000061.tif254166TIFF2026504813000062.tif254166TIFF2026504813000063.tif254166TIFF2026504813000064.tif254166TIFF2026504813000065.tif254166TIFF2026504813000066.tif254166TIFF2026504813000067.tif254166TIFF2026504813000068.tif254166TIFF2026504813000069.tif254166TIFF2026504813000070.tif254166TIFF2026504813000071.tif254166TIFF2026504813000072.tif254166TIFF2026504813000073.tif254166TIFF2026504813000074.tif254166TIFF2026504813000075.tif254166TIFF2026504813000076.tif254166TIFF2026504813000077.tif254166TIFF2026504813000078.tif254166TIFF2026504813000079.tif254166TIFF2026504813000080.tif253166TIFF2026504813000081.tif253166TIFF2026504813000082.tif254166TIFF2026504813000083.tif254166TIFF2026504813000084.tif254166TIFF2026504813000085.tif254166TIFF2026504813000086.tif254166TIFF2026504813000087.tif254166TIFF2026504813000088.tif254166TIFF2026504813000089.tif254166TIFF2026504813000090.tif254166TIFF2026504813000091.tif254166TIFF2026504813000092.tif254166TIFF2026504813000093.tif254166TIFF2026504813000094.tif248166.

[0474] Example 7-2: Establishment of cell transfection conditions When using lentivirus to transduce genes into PSC-derived MSCs, it is important to select an appropriate transduction method to maximize transduction efficiency. A common method for lentivirus administration is to infect adherent cells with lentiviral particles mixed with a transformation enhancer. Another method is to treat cells with both lentiviral particles and a transformation enhancer before they attach, and then infect them with lentivirus during the attachment process (reverse transduction). To identify which of these two methods could further enhance the efficiency of lentiviral transduction into PSC-derived MSCs, we compared transduction efficiency using lentivirus engineered with an eGFP fluorescent vector (SIRION Biotech, SEQ ID NO: 729). Frozen PSC-derived MSCs were thawed, counted, and seeded. Cell attachment was confirmed the next day, and cells were treated with lentivirus at 1-5 MOI and polybrene at 28-8 μg / mL for 16-20 hours. Under other conditions, lentivirus at 1-5 MOI and polybrene at 2 μg / mL-8 μg / mL were mixed and plated. The same number of cells were then seeded and treated for 16-20 hours. In both conditions, 16-20 hours after lentivirus treatment, the lentivirus-containing culture medium was removed and replaced with fresh medium, followed by 48 hours of culture. After 48 hours, the cells were harvested and the transduction efficiency was compared using a flow cytometer for the GFP-transduced cell population. As shown in Figure 14, the transduction efficiency was 6.61% when lentivirus was administered to adherent cells, while it increased approximately 3.26-fold to 21.57% when lentivirus was administered simultaneously with cell seeding. These results confirm that lentivirus administration during the cell attachment process can enhance the efficiency of lentiviral gene transduction into PSC-derived MSCs.

[0475] Example 7-3: Selection of genetically modified cells into which genes have been introduced To select only transfected cells after gene transfer via lentivirus into PSC-derived MSCs, a neomycin resistance gene was engineered to be expressed downstream of the Stefin A mutant (AFFIMER®) gene to be transferred during lentivirus construction. When the Stefin A mutant (AFFIMER®) gene is transferred into cells, the neomycin resistance gene is also expressed, conferring antibiotic resistance. However, prolonged treatment with high concentrations of antibiotics can lead to genetic mutations within the cells, so the minimum concentration required to select only transfected cells must be selected. Since this varies depending on the characteristics of each cell, to determine the minimum G418 concentration that will kill PSC-derived MSCs, we treated the cells with various concentrations of G418 antibiotic and examined whether the cells died after culture. PSC-derived MSCs were uniformly seeded in a 96-well plate and treated the following day with 500 μg / mL, 250 μg / mL, 125 μg / mL, 62.5 μg / mL, 31.25 μg / mL, 15.625 μg / mL, or 7.8125 μg / mL G418. The G418-containing culture medium was replaced every two days, and cell death was confirmed 1, 3, 5, and 7 days after the initial treatment. To confirm cell viability, CCK-8 (Cell Counting Kit-8) was diluted 1:10 in culture medium, and 100 μL of each was added after removing the entire G418-containing culture medium. The cells were incubated at 37°C for 1 hour. After incubation, absorbance was measured at 460 nm using a multimode microplate reader. As shown in Figure 15A, when treated with 500 μg / mL, cell viability was 71.40% on day 1, while 1.34% of the cells had died by day 3. When treated with 250 μg / mL, cell viability was 86.29% on day 1, but 3.07% on day 3, indicating that most of the cells had died, similar to the 500 μg / mL treatment. When treated with 125 μg / mL, cell viability was 89.14% on day 1, 13.43% on day 3, and 5.82% on day 5.When treated with 62.5 μg / mL, cell viability was confirmed to be 90.38% on day 1, 38.51% on day 3, 11.52% on day 5, and 2.84% on day 7.

[0476] After determining the G418 concentration and treatment time, the ratio of transfected cells selected before and after G418 treatment was confirmed. To compare the suitability of G418 treatment conditions, a lentivirus (SEQ ID NO: 729) was constructed to express a neomycin resistance gene downstream of the eGFP fluorescent gene. A 5 MOI of lentivirus was mixed with 2 μg / mL of polybrene, and the mixture was applied to PSC-derived MSCs, which were then cultured for 16 to 20 hours. The culture medium containing the lentivirus was then completely removed and replaced with fresh culture medium, followed by 48 hours of culture. When the cell confluency reached 90% or more, the cells were harvested and 0.4 to 1.0 × 10 4 cells / cm 2 The cells were then seeded at a cell density of 100 μg / mL. After culturing for 18–24 hours, the culture medium was replaced with one containing 100 μg / mL or 250 μg / mL G418, and then replaced with the corresponding G418-containing culture medium at two-day intervals. The 100 μg / mL treatment was continued for 5 days, and the 250 μg / mL treatment was continued for 3 days, after which the culture medium was replaced with a G418-free medium. When the cell confluency reached 90% or greater, the cells were harvested and the proportion of EGFP transfected cells was measured using a flow cytometer. As shown in Figure 15B, the proportion of EGFP transfected cells was 22.00% before G418 treatment, 96.01% after 5 days of treatment with 100 μg / mL, and 93.47% after 3 days of treatment with 250 μg / mL.

[0477] Example 7-4: Promoter screening To select a promoter capable of maintaining stable and high expression when transducing stefin A mutants into PSC-derived MSCs, four promoters that induce constitutive expression were evaluated. Cell lines were generated using lentiviral particles containing a vector (Applied Biological Materials Inc. LV950) engineered to express eGFP fluorescent protein downstream of four promoters, including CMV, PGK, EF1A, and UbC. Cells were treated with lentivirus at 1-5 MOI and 2-8 μg / mL polybrene and cultured at 37°C and 5% CO2 for 16-20 hours. A positive control cell line was generated under the same conditions using a lentivirus (SIRION Biotech, Table 12) in which eGFP fluorescent protein was fused downstream of the CMV promoter. The culture medium containing the lentivirus was then completely removed and replaced with fresh medium. The cells were then cultured at 37°C and 5% CO2 for 48 hours. After 48 hours, cells were harvested and the transfection efficiency was compared using a flow cytometer to determine the percentage of GFP-transfected cells. As shown in Figures 16A and 16B, the positive control group showed a 26.9% EGFP transfected cell rate, while the CMV-IE promoter showed a 1.4-fold higher rate of 36.5%. The other promoters, PGK, EF1A, and UbC, showed very low levels of transfection, with rates of 1.6%, 1.5%, and 0.2%, respectively. Of the four promoters, the CMV promoter / enhancer sequence was found to induce protein expression with the highest efficiency.

[0478] Example 7-5: Confirmation of passage stability of MSCs transfected with anti-CD40L Stefin A protein mutant gene We constructed cell lines in which anti-CD40L stefin A protein variant expression was regulated by a vector (VB211001-1274, SEQ ID NO: 731) containing an enhancer-containing CMV promoter (CMV-IE), and compared their long-term subculture stability with that of untransfected cell lines (naive MSCs). Lentivirus was constructed using a vector containing the anti-CD40L stefin A protein variant gene and a neomycin resistance gene. After thawing frozen naive MSCs, lentivirus was mixed at 1 to 5 MOI with 2 to 8 μg / mL polybrene and inoculated into cell culture dishes. After culturing for 16 to 20 hours at 37°C and 5% CO2, the entire culture medium containing the lentivirus was removed and replaced with fresh culture medium. The cells were then cultured for 48 hours at 37°C and 5% CO2. The cells were then harvested and 0.4 to 1.0 × 10 4 cells / cm 2The cells were then seeded again at a cell density of 100 μg / mL and cultured for 18–24 hours at 37°C in 5% CO2. The culture medium was then replaced with 100 μg / mL G418, and cultured for 5 days, with the medium replaced with G418 every two days. When the cells reached 90% or greater confluency, they were harvested and frozen. Naive MSCs of the same passage number as the frozen transfected cell line were thawed and seeded into a T175 flask. After culturing for 18–24 hours, the culture medium was completely replaced with fresh medium every two days. When the cells reached 90% or greater confluency, they were harvested, seeded again into a T175 flask, and cultured at 37°C in 5% CO2. Continuous culture was continued until the PDL of the cells decreased to 3.0 or less. The remaining cells after each passage were frozen. After continuous culture, the frozen cells were thawed and analyzed for purity and immune markers using a flow cytometer. Expression of mesenchymal stem cell surface markers CD29, CD44, CD73, and CD105, as well as expression of hematopoietic stem cell-specific marker CD45, embryonic stem cell-specific markers SSEA-3, TRA-1-60, and TRA-1-81, and immune marker HLA-DR were analyzed from passages 10 to 18. As shown in Tables 13 and 14, regardless of gene transfection, the expression of mesenchymal stem cell surface markers CD29, CD44, CD73, and CD105 was maintained at over 95% up to PN18. Furthermore, the expression of CD45, SSEA-3, TRA-1-60, TRA-1-81, and HLA-DR was maintained at less than 1% up to PN18, demonstrating the successful maintenance of mesenchymal stem cell characteristics. These results confirmed that the important characteristics and long-term passage stability of mesenchymal stem cells were maintained even when the anti-CD40L Stefin A protein mutant gene was introduced.

[0479] [Table 13]

[0480] [Table 14]

[0481] Example 7-6: Additional screening of promoters for expression of anti-CD40L Stefin A protein variants To introduce the stefin A mutant gene into PSC-derived MSCs, eight lentiviral vectors with the characteristics shown in Figure 17A were constructed, and a promoter capable of inducing stable and high expression was selected.

[0482] Cell lines were constructed using lentiviral particles containing vectors expressing Stefin A variants (AFFIMER®) downstream of each promoter and antibiotic genes downstream of an IRES or T2A sequence. Cells were treated with lentivirus at 1-5 MOI and 2-8 μg / mL polybrene, and then cultured at 37°C and 5% CO2 for 16-20 hours. The culture medium containing the lentivirus was then completely removed and replaced with fresh medium. The cells were then cultured at 37°C and 5% CO2 for 48 hours. After 48 hours, cells were harvested and the expression efficiency of Stefin A variants was evaluated by ELISA. As shown in Figure 17A, two promoters (EF1A and CBh) were identified that increased Stefin A variant expression by more than 10-fold compared to the control (SEQ ID NO: 730).

[0483] Cell lines in which expression of the stefin A mutant protein (AFFIMER®) was controlled by the EF1A promoter (SEQ ID NO: 733) or the CBh promoter (SEQ ID NO: 737) were constructed, and the culture stability, expression level, and activity of the stefin A mutant protein (AFFIMER®) were evaluated by long-term subculture (binding ELISA, competition ELISA, and functional cell assay). As shown in Figure 17B, stable expression of the stefin A mutant protein was maintained from PN9 to PN17 in both cell lines, confirming the combination of expression cassette structures suitable for the expression of the stefin A mutant protein in PSC-derived MSCs.

[0484] To confirm the stable maintenance of expression of Stefin A mutants through long-term passage, transgenic cell lines in which expression of Stefin A mutants was controlled by the EF1A promoter (SEQ ID NO: 733) or the CBh promoter (SEQ ID NO: 737) were constructed using the method described in Example 7-5. The two transgenic cell lines constructed were thawed and inoculated into T175 flasks using culture medium. After culturing for 18-24 hours at 37°C and 5% CO2, the culture medium was completely replaced, and the culture medium was completely replaced with fresh medium every two days. When the cell confluency reached 70-80%, the cells were harvested and re-inoculated into T175 flasks for continuous subculture. Continuous culture was continued until the PDL of the cells decreased to 3.0 or less. After each subculture, the cell culture medium was harvested and frozen, and the remaining cells were frozen. After the continuous culture was completed, the frozen cell culture medium was completely thawed and diluted, and the Stefin A mutants in the cell culture medium were quantified using sandwich ELISA. Because secretion levels may vary depending on the cell number and the time spent in culture, the number of cells harvested during subculture was divided by the time it took to harvest the culture medium after medium change to calculate the daily secretion level of Stefin A mutants per cell. As shown in Table 15, the expression of Stefin A mutants was confirmed to be stable even during long-term subculture.

[0485] [Table 15]

[0486] Example 7-7: Confirmation of long-term passage stability when using an additional promoter After constructing anti-CD40L Stefin A protein mutant gene-transfected cell lines (eMSCs) containing two promoters, the long-term subculture stability was compared with that of naive MSCs using the same method as in Example 7-6.

[0487] During subculture, cell size, viability, total cell number, PDT, and PDL were monitored, and cell morphology was observed using a phase-contrast microscope. Morphological observations confirmed that both the two transfected cell lines and naive MSCs maintained a spindle shape from PN9 to PN19. Cell viability was maintained at over 95% from PN9 to PN19 for both transfected cell lines, and the secretion levels of stefin A mutant protein, PDT, and PDL were similar (Figures 18A-18C). Naive MSCs showed a gradual increase in PDT from 19.21 hours at PN9 to 37.23 hours at PN19, while the two transfected cell lines showed a tendency to increase to 21.78 and 22.86 hours at PN9 and 38.11 and 40.67 hours at PN19. In the case of PDL, both naive MSCs and the two gene-transfected cell lines maintained a PDL of 3.0 or higher until PN 18, then decreased to 3.0 or lower at PN 19. By confirming that the long-term passage stability of PSC-derived MSCs was maintained even when the Stefin A mutant gene was introduced, this technology was confirmed to be a suitable platform technology for the development of cell-based gene therapy agents (Table 16).

[0488] [Table 16]

[0489] Example 8: Analysis of binding and inhibitory activity of anti-CD40L Stefin A protein variants The Stefin A protein variants secreted from the constructed anti-CD40L Stefin A protein variant transfected cell lines were examined to determine whether they possessed the ability to bind to and inhibit CD40L. Anti-CD40L Stefin A protein variant transfected cell lines containing two promoters were constructed using lentiviruses as described in Example 7-6. The cell lines were cultured at 4.0 × 10 in culture medium. 3 cells / cm 2The cells were seeded into a T175 flask at a cell density of 7.0 × 10 cells / ml. After culturing for 18 to 24 hours at 37°C in 5% CO2, the culture medium was completely replaced with fresh medium every two days. After culturing for 3 days, the cells were harvested and seeded at a cell density of 7.0 × 10 cells / ml in one T175 flask. 6 After seeding, the cells were cultured for 18–24 hours at 37°C and 5% CO2. The culture medium was then completely removed and washed with 10 mL of MEM Alpha to completely remove residual medium. 30 mL of MEM Alpha medium was added to a T175 tube and cultured for 48 hours. After 48 hours, the cell culture medium was completely collected and centrifuged to remove residual cells and impurities. The cell culture medium was transferred to a Vivaspin 20 tube and centrifuged to concentrate the culture medium. The amount of stefin A protein variants was quantified by sandwich ELISA (LSBio, LS-F4620). For the binding ELISA, recombinant human CD40L (R&D Systems, 6420-CL-025 / CF) was first coated onto a 96-well plate, and two concentrated culture mediums were serially diluted 1 / 3 each and reacted with CD40L. After washing to remove unbound Stefin A mutants, the binding of Stefin A mutants to CD40L was measured by measuring the difference in absorbance observed after reaction with TMB substrate. The bELISA EC50 values ​​of the Stefin A mutants secreted by the two transfected cell lines were found to be 0.0039 nM and 0.0053 nM (Figure 19).

[0490] To confirm whether the Stefin A protein mutants secreted by the two transfected cell lines have inhibitory activity against CD40L, analysis was performed using the HEK-Blue CD40L cell line (InvivoGen, hkb-cd40). The HEK-Blue CD40L cell line was plated at 2.0 × 10 cells per well in a 96-well plate. 4After seeding at 1000 cells / well, the cells were cultured at 37°C and 5% CO2 for 18–24 hours. After confirming successful attachment of the HEK-Blue CD40L cell line, 50 μL of the medium was removed. The two concentrated culture media were serially diluted 1 / 3 and mixed with human Mega CD40L recombinant protein (Enzo Lifesciences, ALX-522-100-C010). 50 μL of each was added to the 96-well plate seeded with HEK-Blue CD40L cells and incubated at 37°C and 5% CO2 for 20–22 hours. The HEK-Blue CD40L culture media was then harvested and incubated with HEK-Blue solution for 3 hours at 37°C. The absorbance of each well was measured using a multiplate reader to analyze whether the secreted Stefin A protein mutants had CD40L binding inhibitory activity. The analysis showed that the IC50 for the benchmark molecule against hMega CD40L was 0.470 nM, while the IC50s for the Stefin A protein variants secreted by the two transfected cell lines were 0.303 nM and 0.400 nM, respectively, demonstrating similar activity. These results demonstrate that the Stefin A protein variants secreted by the transfected cell lines have CD40L binding and inhibitory abilities similar to those of the reference substance (Figure 20).

[0491] Example 9: Confirmation of immunosuppressive activity of MSCs transfected with anti-CD40L Stefin A protein mutant gene Example 9-1: Confirmation of the effect of suppressing T cell activity The inhibitory effect of the stefin A protein mutant secreted by the anti-CD40L stefin A protein mutant gene-transfected cell line on immune cell activity due to its antagonistic effect against CD40L was confirmed. The constructed stefin A protein mutant gene (SEQ ID NO: 692 or 694)-transfected cell line and naive MSCs were co-cultured with PBMCs (Stem Cell Technologies, 70025), and the activation rate of T cells within the PBMCs was compared. 5.0 × 10 PBMCs were placed in a 24-well plate.5 Two anti-CD40L Stefin A mutant transfected cell lines (SEQ ID NO: 692 or 694) and naive MSCs were mixed at ratios of 1:20, 1:10, 1:5, 1:2.5, and 1:1 and inoculated into wells. PBMCs were stained for CFSE to monitor cell proliferation, and then cultured for 7 days after addition of anti-CD3 and CD28 Dynabeads (Gibco, 11161D) and IL-2 (Gibco, PHC0023). On day 4 after inoculation, the medium was replaced with anti-CD3 and CD28 Dynabeads and IL-2. PBMC clustering, which can be used to assess T cell activation, was observed under a phase-contrast microscope. The most significant and clearest clustering was observed when PBMCs were activated with Dynabeads alone. We observed that the clustering of PBMCs tended to decrease as the ratio of naive MSCs to the two transfected cell lines increased, and that clustering was even less with the transfected cell lines than with naive MSCs (Figure 21).

[0492] PBMCs were collected and analyzed using a flow cytometer to compare the degree of T cell activity through marker analysis. The results showed that the number of CFSE low / CD3 positive cells, a proliferating T cell population, tended to decrease as the ratio of naive MSCs to the two transfected cell lines increased. Furthermore, we confirmed that co-culture with the two transfected cell lines exhibited a stronger suppressive effect on T cell activity than co-culture with naive MSCs (Figure 22).

[0493] Example 9-2: Confirmation of the effect of suppressing B cell activity The inhibitory effect of the Stefin A protein variant secreted by the anti-CD40L Stefin A protein variant gene-transduced cell line on T cell-mediated B cell activation was confirmed. The anti-CD40L Stefin A protein variant gene (SEQ ID NO: 694)-transduced cell line was constructed using lentivirus, and then 4.0 × 10 cells were cultured in culture medium. 3 cells / cm 2The cells were inoculated into a T175 flask at a cell density of 7.0 × 10 cells / ml and cultured. The culture medium was replaced with fresh medium every two days and cultured for three days, after which the cells were harvested. The harvested cells were placed in one T175 flask at a cell density of 7.0 × 10 cells / ml. 6 After seeding with 1000 cells, the cells were cultured at 37°C and 5% CO2 for 18 to 24 hours. The entire culture medium was then removed and washed with MEM-Alpha to completely remove any remaining medium. 30 mL of MEM-Alpha medium was added to a T175 flask and cultured for 48 hours. After 48 hours, the entire cell culture medium was collected and centrifuged to remove remaining cells and impurities. The cell culture medium was transferred to a Vivaspin 20 flask and concentrated by centrifugation, and the amount of stefin A protein variants was quantified by sandwich ELISA. Frozen B cells (Lonza, 4W-601) were thawed and stabilized overnight in a T75 flask. Then, 5.0 × 10 cells were placed in a 24-well plate. 5 B cells were seeded at 1000 cells / well. MEGACD40L (Enzo Lifesciences, ALX-522-110-C010), IgM (Jackson Immuno Research Laboratories, 109-006-129), and IL-21 (Peprotech, 200-21) were added to the culture medium to induce B cell activation. Concurrently, B cell clustering, which can be assessed for the level of B cell activation, was observed under a phase-contrast microscope after 30 hours of culture. Clear clustering was observed when MEGACD40L, IgM, or IL-21 was added alone. However, clustering was reduced when the cells were added with the enriched culture medium, indicating that B cell activation was suppressed (Figure 23).

[0494] B cells were harvested and analyzed using a flow cytometer to assess the level of B cell activation using cell surface markers. The population of CD19-positive / CD86-positive cells, which are markers of activated B cells, was examined. The population of B cells, which increased to approximately 80% upon activation induction, was significantly reduced when the enriched culture medium was added. Furthermore, the inhibitory effect decreased as the enriched culture medium was diluted, demonstrating concentration dependency. This result was not observed in the culture medium of naive MSCs that were not transfected with the stefin A mutant gene. This confirmed that the stefin A mutant secreted by the stefin A mutant gene-transfected cell line suppressed CD40L signaling, thereby suppressing B cell activation (Figure 24).

[0495] Example 9-3: Confirmation of immunoregulatory factor expression in MSCs (eMSCs) transfected with anti-CD40L Stefin A protein mutant gene To confirm whether the introduction of the stefin A mutant gene results in changes in the expression of immunoregulatory factors in an inflammatory environment, an inflammatory environment was induced in the stefin A mutant gene (SEQ ID NO: 694)-transfected cell line and naive MSCs. Naive MSCs at the same passage number as the frozen anti-CD40L stefin A mutant gene-transfected cell line were thawed and cultured at 4.0 × 10 in culture medium. 3 cells / cm 2 The two cell lines were seeded into a T175 flask at a cell density of 1.5 × 10 cells per 100 mm dish. The culture medium was replaced with fresh medium every 2 days and the cells were harvested after 3 days of culture. 6After seeding, cells were cultured for 18 to 24 hours. The culture medium was then completely removed and replaced with medium containing 20 ng / mL IFN-gamma (PEPROTECH, AF-300-02) and 10 ng / mL TNF-alpha (PEPROTECH, AF-300-01A). The cells were cultured for 48 hours to induce an inflammatory environment. After 48 hours, the culture medium was completely removed, the cells were washed with PBS, and then harvested using a scraper. The cell pellet was isolated and the entire protein was dissolved using RIPA buffer. Protein was quantified using BCA and then loaded in equal amounts onto SDS-PAGE. After transferring the cells to a membrane, they were reacted with antibodies to secreted factors induced in an inflammatory environment, such as TGF-beta1 (abcam, ab179695), IDO (abcam, ab76157), IL-10 (abcam, ab133575), MCP-1 (abcam, ab214819), and TSG-6 (abcam, ab267469), as well as cell surface-expressed factors, such as ICAM-1 (abcam, ab282575), VCAM-1 (abcam, ab174279), PD-L1 (abcam, ab243877), and PD-L2 (abcam, ab283344), to confirm their expression. As a result, when naive MSCs were treated with IFN-gamma and TNF-alpha to induce an inflammatory environment, the expression of secreted immunomodulatory factors such as TGF-beta1, IDO, and TSG-6 increased, as did the expression of cell surface expression factors such as ICAM-1, PD-L1, and PD-L2 (Figure 25). When an inflammatory environment was induced not only in naive MSCs but also in Stefin A mutant transgenic cell lines, the expression of immunomodulatory factors increased, and no difference in expression was observed between naive MSCs and Stefin A mutant transgenic cell lines (Figure 25).

[0496] Example 10: Purity verification of anti-CD40L Stefin A mutant gene-transfected MSCs (eMSCs) The purity of the constructed stefin A mutant gene-transfected mesenchymal stem cells was verified by analyzing the percentage of cells expressing the transfected stefin A mutant gene (SEQ ID NO: 694) (the percentage of unintended cells). When constructing the stefin A mutant gene-transfected cell line, a signal peptide sequence (SEQ ID NO: 503) was added to enable extracellular secretion of the stefin A mutant. Therefore, since the stefin A mutant gene-transfected cell line secretes all of the stefin A mutant extracellularly, it was necessary to inhibit secretory protein secretion to identify cells transfected with the stefin A mutant gene. Golgiplug (Brefeldin A; BD Pharmingen, 555029) was used to accumulate secretory proteins in the Golgi apparatus, an intracellular organelle, and the percentage of cells expressing the stefin A mutant was confirmed. After inhibiting secretory protein secretion through Golgiplug and allowing intracellular protein accumulation, the expression of the transfected stefin A mutant was confirmed. After thawing frozen naive MSCs at the same passage number as the anti-CD40L Stefin A mutant gene-transduced cell line, 3.5 x 10 cells were cultured in the culture medium. 4 cells / cm 2 The cells were seeded onto 100 mm dishes at a cell density of 100 μg / mL and cultured. The next day, the culture medium was removed and replaced with culture medium containing 0.1 μg / mL Golgiplug. The cells were incubated at 37°C and 5% CO for 4 hours. The cells were then harvested, permeabilized, and stained with an antibody (Novus Biologicals, NBP2-59470) that detects stefin A mutants. The percentage of cells expressing the stefin A mutant was determined using a flow cytometer. The percentage of stefin A mutant transfected cells was 96.6%. The percentage of stefin A mutant transfected cells was maintained at a very high level in the constructed stefin A mutant transfected cell line (Figure 26).

[0497] Example 11: Therapeutic effect of anti-CD40L Stefin A protein-secreting cell line on GVHD animal model Example 11-1: Experimental Method Five- to six-week-old NOD.Cg-Prkdcscid Il2rgtm1Wjl / SzJ (NSG) mice were purchased from Jackson Laboratory and JABio. After an acclimation period of one to two weeks, 7-week-old mice were used for the experiments.

[0498] To induce GVHD after xenotransplantation, four to five NSG mice were irradiated with 1.5 Gy of radiation, and then 2 × 10 human PBMCs (Lonza) were injected the next day. 6 The cells / head were administered intravenously to induce GVHD.

[0499] In the cell administration group, the drug was administered intravenously on D0 and D7, 24 hours after PBMC administration, while in the standard 5c8 antibody administration group, the drug was administered intravenously on D0 and D7, 24 hours after PBMC administration (Figure 27).

[0500] Each animal in this study was randomly assigned to the study based on individual body weight before irradiation, and an analysis of variance (ANOVA) was performed to determine homogeneity between groups.

[0501] The animals were observed daily for general symptoms, especially before and after cell and substance administration. If an animal showed severe pain or was moribund during the observation period, the symptoms were recorded and the animal was euthanized. The mice were weighed twice a week, and the GVHD clinical score was measured every three days starting from day 0 after drug administration and followed for 60 days.

[0502] Example 11-2: Experimental Results GVHD clinical scoring was performed on five items: weight loss, posture, activity, fur texture, and skin integrity. Each item was assigned a score of 0 to 2, and the combined scores are shown diagrammatically in Figure 28. The group treated with engineered MSCs expressing the XT75 gene exhibited lower scores than the control group. Statistical significance was confirmed by two-way ANOVA, with P values ​​<0.0001 for interaction, column factor, and row factor. The group treated with XT75-expressing eMSCs exhibited similar scores compared to the positive control group treated with 5c8 (Figure 28).

[0503] Example 12: Therapeutic effects of anti-CD40L Stefin A protein variants on a haploidentical genetic GVHD animal model Example 12-1: Experimental Method One hundred and ninety-two healthy female C57BL / 6 mice (6 weeks old) were purchased from Janvier (France) and used for splenocyte extraction for GVHD induction. Twelve healthy female B6D2F1 mice (6 weeks old) were purchased from Charles River and used for GVHD induction (syngeneic control). Seventy healthy female B6D2F1 mice (6 weeks old) were purchased from Charles River and used as donor mice. Stefin A protein variants that specifically bind to mouse CD40L were expressed in the form of trimeric in-line fusion proteins (Table 17).

[0504] [Table 17]

[0505] Spleens were removed from C57BL / 6 and B6D2F1 mice, and spleen cells were extracted and prepared in HBSS buffer. Erythrocytes contained in the extracted spleen cells were lysed using an erythrocyte lysis solution (BD Pharma) and then washed to prepare the spleen. Sixty-four B6D2F1 mice (G4-G11) were treated with 6 × 10 spleen cells isolated from C57BL / 6 mice. 7 Three B6D2F1(G3) mice were intravenously injected with 6 × 10 spleen cells isolated from the spleens of C57BL / 6 mice. 7 Three B6D2F1(G2) mice were intravenously injected with 6 × 10 spleen cells isolated from the spleen of the same B6D2F1 mouse. 7 The mice were intravenously injected at 100 cells / head (FIG. 29).

[0506] Each animal in this study was randomly assigned to the study based on individual body weight, and analysis of variance (ANOVA) was performed to determine homogeneity between groups to confirm the absence of statistical significance.

[0507] The test substances were administered intraperitoneally. The pH of the formulation was adjusted to 7.3–7.4 for intraperitoneal administration, and the volume was 20 mL / kg. The types and amounts of the substances administered to the test animals are shown in the table. The first administration of the test substance was performed 1 hour after GVHD induction, and the administration intervals for each substance were as follows: The Stefin A protein mutant administration group, excluding the antibody administration group, received six doses at two-day intervals after GVHD induction, and the MR-1 antibody was administered three doses at two-day intervals after GVHD induction (Figure 30, Table 18). The survival and behavior of the animals were monitored daily, and clinical observations were recorded daily. Body weights and clinical scores were recorded daily. The clinical score of GVHD was evaluated by visually observing the GVHD status using the following evaluation method and recording the sum of the observed scores. ANOVA was performed using GraphPad Prism to determine statistical significance between groups. Statistical significance between groups was determined to be significant when p-value was <0.05.

[0508] [Table 18]

[0509] Example 12-2: Results After randomization, the overall mean weight of the animal groups was 21.5g, with a weight range of 18.8g to 23.8g. Statistical analysis revealed no significant differences between groups. The weight changes of the experimental animals were monitored throughout the study period. To confirm weight changes, the mean weight change (MBWC%) was compared based on the change rate on day 18. The mean weight change of the G1 group increased to 9.7%, and the mean weight change of the G2 group increased to 15.6%. The mean weight change of the G3 group, the GVHD control group, tended to decrease to 24.4%. The mean weight changes of the drug-treated groups G4 to G11 were as follows:

[0510] G4 group: 1.6% increase, G5 group: 4.1% decrease, G6 group: 14.6% decrease, G7 group: 2.8% increase, G8 group: 6.4% decrease, G9 group: 9.0% decrease, G10 group: 4.4% decrease, G11 group: 21.7% decrease. The groups administered the stefin A protein mutant showed better average weight changes than the G3 group.

[0511] Changes in clinical scores for individual animals were recorded using a score sheet that included body, skin, hair, and mobility criteria. The mean GVHD scores for the G1 and G2 groups did not increase throughout the study period. The G3 group began to increase on day 15, reaching a mean score of 8.0 on day 18. Similar to the G1 and G2 groups, the G4 group did not show an increase in GVHD scores throughout the study period. The G5-G7 groups began to increase between days 9 and 14 after GVHD induction, with GVHD scores ranging from 0.3 to 1.1 by day 18, the final day of the experiment. The clinical scores for the G8-G10 groups began to increase between days 7 and 10 after GVHD induction, with GVHD scores ranging from 0.3 to 1.4 by day 18, the final day of the experiment (Figure 31).

[0512] Considering the above, it was confirmed that the therapeutic effect of the stefin A protein mutant was statistically significantly suppressed compared to the GVHD control group.

[0513] As described above, the recombinant cells of the present invention can express a CD40L-binding agent, e.g., a Stefin A protein variant that specifically binds to CD40L, and / or a fusion protein containing the same, thereby enabling secretion, expression on the cell membrane, and / or intracellular expression of CD40L. It has been confirmed that the CD40L-binding agent (e.g., a Stefin A protein variant) and / or a fusion protein containing the same expressed by the recombinant cells can specifically bind to CD40L and reduce or inhibit CD40L activity. Therefore, the recombinant cells of the present invention can exhibit excellent immunomodulatory effects, such as suppression of T cell activity and B cell activity, and are therefore useful for the prevention or treatment of immune diseases such as autoimmune diseases and inflammatory diseases.

[0514] The above description of the present invention is for illustrative purposes only, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical concept or essential features of the present invention. Therefore, it should be understood that the above-described embodiments are illustrative in all respects and are not limiting.

Claims

1. A population of genetically modified mesenchymal stromal cells (MSCs) comprising an exogenous nucleic acid containing a coding sequence encoding a CD40L-binding agent, wherein the CD40L-binding agent comprises one or more binding domains of an antibody or antibody mimetic.

2. The CD40L-binding agent may be a Stefin A protein variant, Fab, Fab', F(ab')2, Fv, Fd, scFv, sdFv), VL, VH, Camel Ig, V-NAR, VHH, trispecific (Fab3), bispecific (Fab2), diabody ((VL-VH)2 or (VH-VL)2), triabody (trivalent), tetrabody (tetravalent), minibody ((scFv-CH3) ２ ), bispecific single chain Fv (Bis-scFv), shark heavy chain only antibody (VNAR), microprotein (cysteine ​​knot protein, knottin), affibody, aptamer, avimer, nanobody, unibody, single domain antibody, affilin, affitin, adnectin, atrimer, evasin, DARPin, anticalin, fynomer, versabody, lipibody or duocalin.

3. 3. The population of genetically modified mesenchymal stem cells (MSCs) of claim 1 or 2, wherein the CD40L-binding agent is or comprises a Stefin A protein variant.

4. The population of genetically modified mesenchymal stem cells (MSCs) of any one of claims 1 to 3, wherein the exogenous nucleic acid comprises a transcriptional regulatory sequence operably linked to the coding sequence.

5. 5. The population of genetically modified mesenchymal stem cells (MSCs) of claim 4, wherein the transcriptional regulatory sequence is a promoter selected from the group consisting of a CMV promoter, an EFS promoter, a CBh promoter, an MSCV promoter, an SFFV promoter, and an E1FA promoter.

6. The population of genetically modified mesenchymal stem cells (MSCs) of any one of claims 1 to 5, wherein the exogenous nucleic acid comprises (i) an IRES or 2A sequence and / or (ii) a selection gene.

7. 7. The population of genetically modified mesenchymal stem cells (MSCs) of any one of claims 1 to 6, wherein the exogenous nucleic acid sequentially comprises a promoter, a coding sequence encoding a CD40L-binding agent, an IRES or 2A sequence, and an antibiotic selection gene.

8. The population of genetically modified mesenchymal stem cells (MSCs) of any one of claims 1 to 7, wherein the mesenchymal stem cells are derived from panpotent stem cells.

9. 9. The population of genetically modified mesenchymal stem cells (MSCs) according to any one of claims 1 to 8, wherein the mesenchymal stem cells are derived from induced pluripotent stem cells (iPS cells) or embryonic stem cells (ES cells).

10. 10. The population of genetically modified mesenchymal stem cells (MSCs) of any one of claims 1 to 9, wherein the mesenchymal stem cells express at least one cell surface marker selected from CD29, CD44, CD73, CD90, and CD105.

11. A population of genetically modified mesenchymal stem cells (MSCs) according to any one of claims 1 to 10, wherein the expression of cell surface markers is maintained at least 90% or more in the mesenchymal stem cells for 15 or more passages.

12. 12. The population of genetically modified mesenchymal stem cells (MSCs) of any one of claims 1 to 11, wherein the mesenchymal stem cells do not express at least one cell surface marker selected from CD11b, CD14, CD34, CD45, CD79, HLA-DR, TRA-1-60, and TRA-1-81.

13. 12. The population of genetically modified mesenchymal stem cells (MSCs) of any one of claims 1 to 11, wherein 95% or more of the mesenchymal stem cells are CD73+ and CD105+, and less than 1% express CD45, SSEA-3, TRA-1-60, TRA-1-81, and HLA-DR.

14. The CD40L-binding agent was administered at 1×10 －６ A population of genetically modified mesenchymal stem cells (MSCs) according to any one of claims 1 to 13, which exhibits a Kd value of M or less.

15. 15. The population of genetically modified mesenchymal stem cells (MSCs) of any one of claims 3 to 14, wherein the Stefin A protein variant comprises the following amino acid sequence: (i) MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVV-(Xaa)n-GTNYYIKVRAGDNKYMHLKVFKSL-(Xaa)m-EDLVLTGYQVDKNKDDELTGF; (ii) MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVD-(Xaa)n-GTNYYIKVRAGDNKYMHLKVFKSL-(Xaa)m-EDLVLTGYQVDKNKDDELTGF; or (iii) MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVLA-(Xaa)n-GTNYYIKVRAGDNKYMHLKVFKSL-(Xaa)m-EDLVLTGYQVDKNKDDELTGF; Here, Xaa is an amino acid residue, and n and m are each independently an integer of 3 to 20.

16. 16. The population of genetically modified mesenchymal stem cells (MSCs) of any one of claims 3 to 15, wherein the Stefin A protein variant comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 246 to 365.

17. (i) the (Xaa)n comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 6-125; and / or (ii) the (Xaa)m comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 126-245.

18. 18. The population of genetically modified mesenchymal stem cells (MSCs) of any one of claims 3 to 17, wherein the Stefin A protein mutant further comprises a signal peptide.

19. 19. The population of genetically modified mesenchymal stem cells (MSCs) of any one of claims 3 to 18, wherein the CD40L-binding agent comprises a triplet or quadruplet of a Stefin A protein variant.

20. 20. A population of genetically modified mesenchymal stem cells (MSCs) according to any one of claims 1 to 19, wherein 90% or more of the mesenchymal stem cells in said population contain said exogenous nucleic acid.

21. 21. The population of genetically modified mesenchymal stem cells (MSCs) of any one of claims 1 to 20, wherein the CD40L-binding agent is expressed on the surface of the MSCs.

22. The CD40L-binding agent is secreted extracellularly and selectively: (i) the population of genetically modified mesenchymal stem cells secretes an average level of 200 fg / cell / day or greater; (ii) The population of genetically modified mesenchymal stem cells (MSCs) of any one of claims 1 to 21, wherein the population of genetically modified mesenchymal stem cells secretes an average level of 200 to 1500 fg / cell / day or more.

23. Use of the population of genetically modified mesenchymal stem cells (MSCs) according to any one of claims 1 to 22 for the prevention or treatment of immune diseases.

24. A pharmaceutical composition for preventing or treating immune diseases, comprising a population of genetically modified mesenchymal stem cells (MSCs) according to any one of claims 1 to 22.

25. The immune disease may be lupus (SLE), lupus nephritis (e.g., drug-induced lupus nephritis), immune thrombocytopenia (ITP), rheumatoid arthritis (RA), multiple sclerosis (MS), inflammatory bowel disease (IBD) (e.g., Crohn's disease and ulcerative colitis), graft-versus-host disease (GvHD) or allograft rejection, transplantation / solid organ transplantation (T&C).

25. The use or pharmaceutical composition according to claim 23 or 24, wherein the disease is selected from the group consisting of primary biliary cholangitis (SOT), primary biliary cholangitis (PBC), psoriasis, psoriatic arthritis, collagen-induced arthritis, oophoritis, allergic rhinitis, asthma, Sjogren's syndrome, atopic eczema, myasthenia gravis, Graves' disease, and glomerulosclerosis.

26. 26. A method for producing a population of genetically modified mesenchymal stem cells (MSCs) according to any one of claims 1 to 25, comprising the steps of: contacting the population of mesenchymal stem cells with a lentiviral vector comprising an exogenous nucleic acid containing a coding sequence encoding a CD40L-binding agent; and Culturing said population of mesenchymal stem cells.

27. 27. The method of claim 26, wherein a population of mesenchymal stem cells is unattached when the contacting step is initiated.

28. 28. The method of claim 26 or 27, wherein the exogenous nucleic acid comprises an antibiotic selection gene, and further comprising the step of selecting cells that express the selection gene.

29. The method according to any one of claims 26 to 28, wherein a population of mesenchymal stem cells is produced in which 90% or more of the mesenchymal stem cells contain the exogenous nucleic acid.

30. The method of any one of claims 26 to 29, wherein 90% or more of the expression of cell surface markers is maintained in the population of mesenchymal stem cells even after 15 or more passages in culture.

31. The method of any one of claims 26 to 30, wherein the CD40L-binding agent is expressed on the surface of the mesenchymal stem cells.

32. The method of any one of claims 26 to 30, wherein the CD40L-binding agent is secreted extracellularly.

33. A lentiviral vector comprising a nucleic acid sequence comprising a transcriptional regulatory sequence operably linked to a sequence encoding a CD40L-binding agent, an IRES or 2A sequence, and a selection gene.

34. The lentiviral vector of claim 33, wherein the transcriptional regulatory sequence is a promoter selected from a CMV promoter, an EFS promoter, a CBh promoter, an MSCV promoter, an SFFV promoter, and an E1FA promoter.

35. 35. The lentiviral vector of claim 33 or 34, wherein the nucleic acid sequentially comprises a promoter, a sequence encoding a CD40L-binding agent, an IRES or 2A sequence, and an antibiotic selection gene.

36. A method for treating an immune disease, comprising administering the population of genetically modified mesenchymal stem cells according to any one of claims 1 to 22 to a subject in need thereof.

37. The immune disease may be lupus (SLE), lupus nephritis (e.g., drug-induced lupus nephritis), immune thrombocytopenia (ITP), rheumatoid arthritis (RA), multiple sclerosis (MS), inflammatory bowel disease (IBD) (e.g., Crohn's disease and ulcerative colitis), graft-versus-host disease (GvHD) or allograft rejection, transplantation / solid organ transplantation (T&C).

37. The method of claim 36, wherein the disease is selected from the group consisting of primary biliary cholangitis (SOT), primary biliary cholangitis (PBC), psoriasis, psoriatic arthritis, collagen-induced arthritis, oophoritis, allergic rhinitis, asthma, Sjogren's syndrome, atopic eczema, myasthenia gravis, Graves' disease, and glomerulosclerosis.

38. A population of genetically engineered cells comprising an exogenous nucleic acid comprising a coding sequence encoding a CD40L binding agent, said CD40L binding agent comprising one or more binding domains derived from an antibody or antibody mimetic.

39. 39. The population of genetically modified cells of claim 38, wherein the cells are animal cells.

40. 39. The population of genetically modified cells of claim 38, wherein the cells are mammalian cells.

41. 39. The population of genetically modified cells of claim 38, wherein the cells are human cells.

42. 42. The population of genetically modified cells of any one of claims 38 to 41, wherein the cells are selected from the group consisting of stem cells, immune cells, and somatic cells.

43. 43. The population of genetically modified cells of claim 42, wherein the cells are panpotent stem cells.

44. 43. The population of genetically modified cells of claim 42, wherein the cells are pluripotent stem cells.

45. 43. The population of genetically modified cells of claim 42, wherein the cells are immune cells selected from T cells, B cells, NK cells, monocytes, macrophages, Treg cells, and antigen-presenting cells (e.g., dendritic cells).

46. The CD40L-binding agent may be a Stefin A protein variant, Fab, Fab', F(ab')2, Fv, Fd, scFv, sdFv), VL, VH, Camel Ig, V-NAR, VHH, trispecific (Fab3), bispecific (Fab2), diabody ((VL-VH)2 or (VH-VL)2), triabody (trivalent), tetrabody (tetravalent), minibody ((scFv-CH3) ２ 46. ​​The population of genetically modified cells of any one of claims 38 to 45, which is or comprises a bispecific single chain Fv (Bis-scFv), shark heavy chain only antibody (VNAR), microprotein (cysteine ​​knot protein, knottin), affibody, aptamer, avimer, nanobody, unibody, single domain antibody, affilin, affitin, adnectin, atrimer, evasin, DARPin, anticalin, fynomer, versabody, lipibody or duocalin.

47. 47. The population of genetically modified cells of any one of claims 38 to 46, wherein the CD40L-binding agent is or comprises a Stefin A protein variant.

48. 48. The population of genetically modified cells of any one of claims 38 to 47, wherein the exogenous nucleic acid comprises a transcriptional regulatory sequence operably linked to the coding sequence.

49. 49. The population of genetically modified cells of claim 48, wherein the transcriptional regulatory sequence is a promoter selected from the group consisting of a CMV promoter, an EFS promoter, a CBh promoter, an MSCV promoter, an SFFV promoter, and an E1FA promoter.

50. 50. The population of genetically modified cells of any one of claims 38 to 49, wherein the exogenous nucleic acid comprises (i) an IRES or 2A sequence and / or (ii) a selection gene.

51. 51. The population of genetically modified cells of any one of claims 38 to 50, wherein the exogenous nucleic acid sequentially comprises a promoter, a coding sequence encoding a CD40L-binding agent, an IRES or 2A sequence, and an antibiotic selection gene.

52. The CD40L-binding agent was administered at 1×10 －６ 52. The population of genetically modified cells according to any one of claims 47 to 51, which exhibits a Kd value of M or less.

53. 53. The population of genetically modified cells of any one of claims 47 to 52, wherein the mutant Stefin A protein comprises the amino acid sequence: (i) MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVV-(Xaa)n-GTNYYIKVRAGDNKYMHLKVFKSL-(Xaa)m-EDLVLTGYQVDKNKDDELTGF; (ii) MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVD-(Xaa)n-GTNYYIKVRAGDNKYMHLKVFKSL-(Xaa)m-EDLVLTGYQVDKNKDDELTGF; or (iii) MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVLA-(Xaa)n-GTNYYIKVRAGDNKYMHLKVFKSL-(Xaa)m-EDLVLTGYQVDKNKDDELTGF; Here, Xaa is an amino acid residue, and n and m are each independently an integer of 3 to 20.

54. 53. The population of genetically modified cells of any one of claims 47-52, wherein the Stefin A protein variant comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 246-365.

55. (i) the (Xaa)n comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 6-125; and / or (ii) the (Xaa)m comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 126-245.

56. 56. The population of genetically modified cells of any one of claims 47 to 55, wherein the mutant Stefin A protein further comprises a signal peptide.

57. 57. The population of genetically modified cells of any one of claims 47 to 56, wherein the CD40L-binding agent comprises a triplet or quadruplet of a Stefin A protein variant.

58. 58. The population of genetically modified cells of any one of claims 38 to 57, wherein 90% or more of the cells in the population contain the exogenous nucleic acid.

59. 59. The population of genetically modified cells of any one of claims 38 to 58, wherein the CD40L-binding agent is expressed on the cell surface.

60. 60. The population of genetically modified cells of any one of claims 38 to 59, wherein the CD40L-binding agent is secreted extracellularly.

61. Use of the population of genetically modified cells according to any one of claims 38 to 60 for the prevention or treatment of immune diseases.

62. A pharmaceutical composition for preventing or treating an immune disease, comprising the population of genetically modified cells according to any one of claims 38 to 60.

63. The immune disease may be lupus (SLE), lupus nephritis (e.g., drug-induced lupus nephritis), immune thrombocytopenia (ITP), rheumatoid arthritis (RA), multiple sclerosis (MS), inflammatory bowel disease (IBD) (e.g., Crohn's disease and ulcerative colitis), graft-versus-host disease (GvHD) or allograft rejection, transplantation / solid organ transplantation (T&C).

63. The use or pharmaceutical composition according to claim 61 or 62, wherein the disease is selected from the group consisting of primary hepatic ulcerative colitis (SOT), primary biliary cholangitis (PBC), psoriasis, psoriatic arthritis, collagen-induced arthritis, oophoritis, allergic rhinitis, asthma, Sjogren's syndrome, atopic eczema, myasthenia gravis, Graves' disease, and glomerulosclerosis.

64. A method for producing a population of genetically modified cells according to any one of claims 38 to 60, comprising the steps of: contacting the population of cells with a lentiviral vector comprising an exogenous nucleic acid containing a coding sequence encoding a CD40L-binding agent; and Culturing said population of cells.

65. 65. The method of claim 64, wherein the exogenous nucleic acid comprises an antibiotic selection gene, further comprising selecting for cells that express the selection gene.

66. 66. The method of claim 64 or 65, which produces a population in which 90% or more of the cells contain the exogenous nucleic acid.

67. 67. The method of any one of claims 64 to 66, wherein the CD40L-binding agent is expressed on the surface of the cell.

68. The method of any one of claims 64 to 66, wherein the CD40L-binding agent is secreted extracellularly.

69. 61. A method for treating an immune disease, comprising administering to a subject in need thereof the population of genetically modified cells of any one of claims 38 to 60.

70. The immune disease may be lupus (SLE), lupus nephritis (e.g., drug-induced lupus nephritis), immune thrombocytopenia (ITP), rheumatoid arthritis (RA), multiple sclerosis (MS), inflammatory bowel disease (IBD) (e.g., Crohn's disease and ulcerative colitis), graft-versus-host disease (GvHD) or allograft rejection, transplantation / solid organ transplantation (T&C).

70. The method of claim 69, wherein the disease is selected from the group consisting of primary biliary cholangitis (PBC), primary hepatic cholangitis (SOT), primary biliary cholangitis (PBC), psoriasis, psoriatic arthritis, collagen-induced arthritis, oophoritis, allergic rhinitis, asthma, Sjogren's syndrome, atopic eczema, myasthenia gravis, Graves' disease, and glomerulosclerosis.

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