Genetically modified cells comprising nucleic acid encoding TNFR2 binding agent and uses thereof

Abstract

To provide: a genetically modified cell in which a nucleic acid encoding a TNFR2 binding agent (e.g., a Stefin A protein variant that specifically binds to TNFR2) or a fusion protein including the same is introduced into a host cell; a conditioned cell culture medium thereof; pharmaceutical preparations of the genetically modified cell; and uses of the genetically modified cell for treatment of immune diseases, including inflammatory diseases, autoimmune diseases and cancers.SOLUTION: The invention provides population of genetically modified mesenchymal stem cells (MSCs), where the mesenchymal stem cells comprise an exogenous nucleic acid comprising a sequence that encodes a TNFR2 binding agent, where the TNFR2 binding agent comprises one or more binding domains from an antibody or antibody mimetic.SELECTED DRAWING: Figure 11

Description

[Technical field]

[0001] The present invention relates to a genetically modified cell in which a nucleic acid encoding an agent that specifically binds to TNFR2 (e.g., a Stefin A protein variant that specifically binds to TNFR2) and / or a fusion protein containing the same has been introduced into a host cell (e.g., a human mesenchymal stem cell or mesenchymal stromal cell), and uses thereof.

[0002] This research was conducted with funding from the government (Ministry of Science, ICT and Technology, Ministry of Health and Welfare) and support from the Regenerative Medicine Technology Development Foundation (24C0102L1). [Background technology]

[0003] The immune system is a collective term for a biological network composed of various cells and organs that is constructed to protect the organism from external invasion. The immune system operates based on the specific functions of the organs and cells that compose it, and the signal transmission and interaction between each cell. The immune system maintains immune homeostasis by maintaining an appropriate balance between immune tolerance, which suppresses and regulates immunity, and immune response, which promotes immunity. Immune tolerance and immune response can become unbalanced due to various causes, and such immune imbalance leads to the occurrence of various diseases. For example, when the immune tolerance mechanism becomes strong, it becomes easier for external infection sources to invade, causing cancer and infectious diseases, while when the immune response mechanism becomes strong, it can cause inflammatory diseases such as autoimmune diseases and allergic diseases.

[0004] In recent years, active research has been conducted on the immune synapse, which is a signal transmission system between immune cells that constitute the immune system or between target cells and immune cells. The immune synapse is composed of various cytokines and signal transmitters secreted by cells, as well as various costimulatory molecules and receptors expressed on the cell surface. As various factors involved in maintaining homeostasis of the immune system have been reported, interest in immunotherapeutic agents that target these factors to regulate immune responses is increasing. Most immunotherapeutic agents developed so far are antibody drugs against cytokines or cell surface molecules, but their use is greatly limited due to insufficient efficacy or side effects. Recently, cell therapy agents that utilize 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 methods for treating immune diseases. Differentiated immune cell-based cell therapy agents such as CAR-T or CAR-NK have limitations of high cost and limited targets due to the use of autologous cells, and mesenchymal stem cells have limitations of 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). Controlling cell-mediated and humoral immune responses is an important part of a healthy immune system. Abnormal regulation of immune responses centered on T cells and B cells is associated with a variety of human diseases, and inappropriate increased immune responses to various self- or foreign antigens play a causal role in the pathology of other immune diseases, including, for example, autoimmune diseases, asthma, allergic reactions, GvHD, graft rejection, and cancer.

[0005] These diseases are mediated by T and B lymphocytes that show reactivity against self antigens and antigens derived from non-threatening sources such as allergens or grafts. Regulatory T cells (Tregs) suppress the activity of immune cells that cross-react with self major histocomplex (MHC) proteins and other benign antigens, and the best-known Treg populations include CD4+, CD25+, FoxP3+ Tregs and CD17+ Tregs. TNFR2, which is involved in cell survival or growth, is an attractive target for treating diseases such as autoimmune diseases, GvHD, graft rejection, allergic reactions, asthma and cancer.

[0006] Under these circumstances, the present inventors have made extensive efforts to develop a new type of cell therapy agent that expresses a TNFR2-binding agent. Summary of the Invention [Problem to be solved by the invention]

[0007] An object of the present invention is to provide a recombinant cell expressing a TNFR2-binding agent and / or a fusion protein containing the same, and uses thereof.

[0008] Another object of the present invention is to provide recombinant cells containing nucleic acids encoding the TNFR2 binding agents and / or fusion proteins containing the same, and uses thereof.

[0009] It is still another object of the present invention to provide a recombinant cell in which a nucleic acid encoding a TNFR2-binding agent and / or a fusion protein containing the same is introduced into a host cell, and uses thereof.

[0010] A further object of the present invention is to provide a population or culture medium of said genetically modified cells, and uses thereof.

[0011] It is still another object of the present invention to provide a method for producing the genetically modified cell.

[0012] A further object of the present invention is to provide a population or culture medium of said genetically modified cells, and uses thereof. [Means for solving the problem]

[0013] In order to achieve the above-mentioned objects of the present invention, the present invention provides a recombinant cell expressing a TNFR2-binding agent and / or a fusion protein containing the same, and uses thereof.

[0014] The present invention also provides recombinant cells comprising nucleic acids encoding the TNFR2 binding agents and / or fusion proteins comprising the same, and uses thereof.

[0015] The present invention also provides recombinant cells in which a nucleic acid encoding a TNFR2 binding agent and / or a fusion protein comprising the same has been introduced into a host cell, and uses thereof.

[0016] The present invention also provides a population or culture medium of the genetically modified cells.

[0017] The present invention also provides a method for the preparation of a TNFR2-binding agent comprising the steps of: (a) introducing into a host cell a nucleic acid encoding a TNFR2-binding agent and / or a fusion protein comprising the same; and (b) selecting and harvesting host cells into which a nucleic acid encoding the TNFR2-binding agent and / or a fusion protein containing the same has been introduced.

[0018] The present invention also provides a cell therapy agent comprising the genetically modified cells and / or the conditioned cell culture medium thereof.

[0019] The present invention also provides a use of the genetically modified cells and / or the conditioned cell culture medium thereof for producing a cell therapy agent.

[0020] The present invention also provides a pharmaceutical composition for preventing and / or treating immune diseases or cancer, comprising the genetically modified cells of the present invention and / or a regulated cell culture medium thereof.

[0021] The present invention also provides the use of the genetically modified cells and / or the conditioned cell culture medium thereof for the prevention and / or treatment of immune diseases or cancer.

[0022] The present invention also provides the use of the genetically modified cells and / or the conditioned cell culture medium thereof for producing a pharmaceutical composition for the prevention and / or treatment of immune diseases or cancer.

[0023] The present invention also provides a method for preventing and / or treating an immune disease or cancer, comprising the step of administering genetically modified cells and / or a conditioned cell culture medium thereof to a subject.

[0024] The present invention also provides an immunoregulatory composition comprising the recombinant cell and / or a conditioned cell culture medium thereof.

[0025] The present invention also provides the use of said genetically modified cells and / or the conditioned cell culture medium thereof for immunomodulation.

[0026] The present invention also provides the use of the genetically modified cells and / or the conditioned cell culture medium thereof for the manufacture of a composition for immunomodulation.

[0027] The present invention also provides a method for immunomodulation comprising the step of administering to a subject genetically modified cells for immunomodulation and / or conditioned cell culture medium thereof.

[0028] The present invention also provides a composition or medium for culturing regulatory T cells, comprising the genetically modified cells and / or a conditioned cell culture medium thereof.

[0029] The present invention also provides a method for culturing regulatory T cells, comprising the step of culturing regulatory T cells in the presence of the genetically modified cells and / or a cell culture medium conditioned therewith.

[0030] The present invention also provides a use of the genetically modified cells and / or the conditioned cell culture medium thereof for culturing regulatory T cells.

[0031] The present invention also provides a use of the genetically modified cells and / or the conditioned cell culture medium thereof for producing a composition or medium for culturing regulatory T cells.

[0032] The present invention also provides a composition for drug delivery comprising the recombinant cell.

[0033] The present invention also provides a use of the genetically modified cell for drug delivery. Effect of the Invention

[0034] The present invention relates to a recombinant cell into which a nucleic acid encoding a TNFR2-binding agent and / or a fusion protein containing the same has been introduced, and the recombinant cell of the present invention can express the TNFR2-binding agent and bind to TNFR2 as a target, and exhibit improved immunomodulatory activity compared to host cells. Furthermore, when used as a cell therapy agent, the recombinant cell can exhibit excellent therapeutic effects against immune diseases such as inflammatory diseases and autoimmune diseases, and cancer, while reducing immune rejection reactions. [Brief description of the drawings]

[0035] The drawings included in this specification are used for illustrative purposes and are not used to limit the invention.

[0036] [Figure 1]FIG. 1 is a schematic diagram showing the selection strategy for Stefin A protein variants that specifically bind to human TNFR2. Both libraries were screened through solution and passive selection processes. The concentration of hTNFR2 was decreased as the rounds progressed, and the number of washing steps between rounds 1 and 2 was increased. In the passive selection, Avacta's proprietary Type 3 Affimer® phage library (US Pat. No. 9.932.575) was used, and deselection and competition methods were simultaneously utilized. In the deselection method, the Fc concentration in round n+1 was the hTNFR2 concentration in round n. In the competition method, the Fc concentration was set to a molar ratio of 10 times the hTNFR2 concentration specified in the round. For passive selection using Avacta's proprietary Type 1 Affimer® phage library (US Pat. No. 8,841,491), only the deselection method was performed, and round 4 was added because the initial screening of round 3 showed high diversity and hit rate. For solution selection, both libraries were subjected to a competitive method to decrease the enrichment of Fc-binding phage-displayed Affimer® polypeptides. Streptavidin- and neutravidin-coated beads were alternated in each round to decrease the enrichment of streptavidin- and / or neutravidin-binding phage-displayed peptides.

[0037] [Diagram 2] FIG. 1 shows Stefin A protein mutants binding to Expi293FTM cells overexpressing human TNFR2.

[0038] [Diagram 3] FIG. 1 shows titration data showing % inhibition and IC50 at 3 μM for the best blockers.

[0039] [Figure 4] FIG. 1 shows the results of screening 43 clones with agonistic activity through HEK-Blue TNFα SEAP reporter assay.

[0040] [Diagram 5] FIG. 1 shows the results of a HEK-Blue TNFα SEAP reporter assay with 20 clones ranked.

[0041] [Figure 6] FIG. 1 shows cross-reactivity of hTNFR2-binding stefin A protein mutant clones with Cynomolgus TNFR2.

[0042] [Figure 7-8] FIG. 7 shows the results of characterization of engineered Mesenchymal Stromal Cells (eMSCs). MSC-specific markers CD29, CD44, CD73, CD90, and CD105 (FIG. 7), and cell markers CD45, CD14, CD19, HLA-DR, SSEA-3, TRA-1-60, and TRA-1-81 (FIG. 8). (Data are shown for a representative experiment.)

[0043] [Figure 9] Stefin A mutants specifically bind to TNFR2 expressed on the cell surface. Surface expression was examined in cell lysates of eMSCs transfected with Stefin A mutants via Western blot and compared with the signal of Stefin A mutant clones on the same gel. β-actin Western blot was used as a loading control for protein lysates of various eMSCs.

[0044] [Figure 10A-10B]Figure 1 shows the results of quantification of Stefin A mutants on the cell surface. The number of Stefin A mutants on the cell surface was quantified by flow cytometry using the QuantiBRITE PE Fluorescence Quantification Kit. For this analysis, an antibody against Stefin A mutants conjugated with PE (phycoerythrin) was used.

[0045] [Figure 11] Figure 1. SEAP release of the HEK Blue™ TNFα SEAP reporter cell line induced by the seven best Stefin A protein variants on eMSC surfaces. HEK-Blue TNFα cells were plated at 20,000 cells per well and eMSCs were plated at 40,960 cells per well in 1 / 2 serial dilutions. Data are shown as mean ± SD from triplicates. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0046] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Generally, the nomenclature used herein is one that is well known and commonly used in the art.

[0047] As used herein, the terms "protein" or "polypeptide" refer to polymers of amino acids of any length. The polymers may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acids. The terms also include amino acid polymers that have been modified, either naturally or artificially, including, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or other manipulations, such as conjugation with a labeling moiety. The terms also include polypeptides that contain one or more analogs of an amino acid, including, for example, unnatural amino acids, as well as the various modifications known in the art.

[0048] 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, or isomers thereof (e.g., D- or L-isomers).

[0049] 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 variant of the present invention is produced by chemical synthesis, it may include amino acid analogs such as, but not limited to, cyanoalanine, canavanine, djenkolic acid, norleucine, 3-phosphoserine, homoserine, dihydroxy-phenylalanine, 5-hydroxytryptophan, 1-methylhistidine, 3-methylhistidine, diaminopimelic acid, ornithine, or diaminobutyric acid.

[0050] In the amino acid or nucleic acid sequences described herein, identity or percent identity refers to sequences that are identical or have a particular percentage match. When comparing and aligning two sequences for maximum match, conservative amino acid substitutions may not be considered as part of the sequence identity. In one embodiment of the invention, two nucleic acid or amino acid sequences may be "substantially identical", which may mean that the two sequences are at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical. In some embodiments, identity may exist over a length of at least about 10, 20, 40-60, 60-80, 80-100 or more residues. When calculating the percentage of identity, the residues defined as Xaa or X in the sequences of the invention may be included in the calculation of the percentage of identity. That is, all amino acids at this position in the comparison sequence may be calculated to match the sequences of the invention.

[0051] 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.

[0052] As used herein, "conservative substitution" refers to a modification of a polypeptide involving the replacement of one or more amino acids with amino acids having similar biochemical properties that does not result in the loss of biological or biochemical function of the polypeptide.

[0053] A "conservative amino acid substitution" is a substitution that replaces an amino acid residue with an amino acid residue having a similar side chain. Classes of amino acid residues having 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). In general, conservative substitutions in the sequences of the polypeptides and proteins described herein do not cause loss of their function. For example, Stefin A protein variants that specifically bind to TNFR2 have conservative substitutions that do not abolish binding to TNFR2. Methods for identifying conservative amino acid substitutions that do not abolish binding are well known in the art.

[0054] An "isolated" protein, polypeptide, antibody, polynucleotide, vector, cell or composition is a protein, polypeptide, antibody, polynucleotide, vector, cell or composition that is in a form not found in nature. An isolated protein, polypeptide, antibody, polynucleotide, vector, cell or composition includes one that has been purified to the extent that it is no longer in a form found in nature. For example, an isolated protein, polypeptide, antibody, polynucleotide, vector, cell or composition is substantially pure.

[0055] TNFR2 is a type 1 membrane-bound receptor that binds to TNFα together with TNFR1. TNFR2 is also known as p75, TNF receptor superfamily member 1B or CD120b, and is encoded by TNFRSF1B, which expresses a 48 kDa protein (UnipProt P20333) with a length of 461 amino acids in humans. In mice, the TNFR2 protein has a length of 474 amino acids and a molecular weight of 50 kDa.

[0056] In the present invention, embodiments described with the term "comprising" are understood to include similar embodiments described as "consisting of" and / or "essentially consisting of." Also, embodiments described as "essentially consisting of" are understood to include other embodiments described as "consisting of."

[0057] As used herein, a description of a value or parameter expressed as "about" includes an embodiment for that value or parameter. For example, "about X" includes a description of X.

[0058] As used herein, "and / or" such as "A and / or B" is used to mean A and B, A or B, A (single) and B (single). Similarly, "A, B and / or C" is used to mean A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A; B; and C.

[0059] As used herein, "at least one" may be used interchangeably with one or more.

[0060] Avacta has used its Affimer® platform technology to screen for stefin A protein variants that can bind to TNFR2 with excellent affinity and specificity, and has filed patent applications for stefin A protein variants that specifically bind to TNFR2 (e.g., PCT / GB2023 / 050850).

[0061] In some embodiments, the present invention describes a cell having excellent immunomodulatory activity through genetic recombination. In some embodiments, the present invention provides a genetically modified cell that stably expresses a Stefin A protein variant that specifically binds to TNFR2 by introducing a gene encoding the Stefin A protein variant that specifically binds to TNFR2.

[0062] In one embodiment of the present invention, a genetically engineered cell expressing a Stefin A protein mutant that specifically binds to TNFR2 can not only specifically bind to TNFR2, but can also have an agonistic or antagonistic effect.

[0063] In one embodiment of the present invention, it was confirmed that recombinant cells expressing a Stefin A protein mutant that specifically binds to TNFR2 exhibit excellent agonistic effects on TNFR2. In one embodiment of the present invention, the TNFR2-specific binding ability of the Stefin A protein mutant can be usefully used to culture or activate Tregs instead of TNFα, and to regulate (preferably suppress) the immune system.

[0064] Thus, one aspect of the present invention relates to a genetically modified cell in which a nucleic acid encoding an agent that specifically binds to TNFR2 (preferably a Stefin A protein mutant that specifically binds to TNFR2 or a fusion protein containing the same) has been introduced into a host cell.

[0065] TNFR2

[0066] In the present invention, the term "TNFR2" refers to a type 1 membrane-bound receptor that binds to TNFα together with TNFR1. TNFR2 is also known as p75, TNF receptor superfamily member 1B, or CD120b, and is encoded by TNFRSF1B, which expresses a 48 kDa protein (UnipProt P20333) with a length of 461 amino acids in humans. In mice, the TNFR2 protein has a length of 474 amino acids and a molecular weight of 50 kDa. In one embodiment of the present invention, TNFR2 sequences from various organisms are well known, and one of ordinary skill in the art can easily predict and modify the sequence therefrom. TNFR subtypes 1 and 2 have been reported to be specifically expressed on the surface of Tregs. Activation of TNFR2 by soluble TNFα induces signaling through the MAPK (mitogen activated protein kinase) signaling pathway, which regulates TRAF2 / 3 and NF-κB-mediated transcription, promoting cell proliferation and avoiding cell death.

[0067] Various studies have reported the importance of TNFR2 in Treg proliferation and function. TNFR2-deficient mice have reduced numbers of thymic and peripheral Tregs (2013_Chen X,et al.PMID:23277487), and TNFR2- / - Tregs fail to regulate inflammatory responses in vivo (2008_Van Mierlo GJ,et al.PMID:18292492). In humans, Tregs have been identified as expressing higher levels of TNFR2 than T effector cells (2013_Okubo Y,et al.PMID:24193319 & 2002_Annunziato F,et al.PMID:12163566). TNFR2+ Tregs were the most potent suppressors of proliferation and cytokine production in co-cultured T-responder cells (2010_Chen X, et al. PMID:20127680). TNFR2 has also been reported to play a major role in cancer development and growth by mediating TNF responses in immunosuppressive cells, allowing immune evasion and tumor development (Sheng Y., et al. doi:10.3389 / fimmu.2018.01170).

[0068] In autoimmune diseases affecting the central nervous system (CNS), especially multiple sclerosis (MS), pathogenic lymphocytes are activated in the periphery and then infiltrate the CNS, inducing local inflammation and demyelination. Demyelination means that the myelin sheath surrounding the nerve fibers in the brain is damaged. Preventing or reversing demyelination is an accessible strategy to treat MS. Interestingly, many studies have reported that TNFR2 is involved in neuroprotection. For example, in a mouse model of cuprizone-induced demyelination, it was reported that TNFR2 plays an important role in the regeneration of granule cells, which are cells responsible for maintaining and generating the myelin sheath surrounding the axon, while TNF signaling through TNFR1 promotes neuronal demyelination (2001_HA Arnett, et al. PMID:11600888).

[0069] TNFR2 binding agents

[0070] In one embodiment of the invention, a cell (eg, an MSC) is provided that contains an exogenous nucleic acid encoding a TNFR2 binding agent.

[0071] In one embodiment of the invention, the TNFR2 binding agent is a recombinant protein that contains one or more binding domains of an antibody or antibody mimetic that binds to TNFR2. In one embodiment of the present invention, the TNFR2 binding agent is a stefin A protein variant (Affimer®), Fab, 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), 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, or the like. In some embodiments, the antibody may be an affilin, an affitin, an adnectin, an atrimer, an evasin, a DARPin, an anticalin, an avimer, a fynomer, a versabody, a repebody, or a duocalin.

[0072] In one embodiment of the present invention, the TNFR2 binding domain may be selected from the group consisting of, but is not limited to, a Stefin A protein mutant, an antibody or 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 catalytic fragment thereof.

[0073] In one embodiment of the present invention, the TNFR2 binding agent may be an anti-TNFR2 antibody. In the present invention, the term "antibody" includes not only the complete antibody form that specifically binds to a target (antigen), but also an antigen-binding fragment of an antibody. A complete antibody includes a structure having two full-length light chains and two full-length heavy chains, each light chain being linked to a heavy chain by a disulfide bond. In the present invention, the term "heavy chain" refers to a full-length heavy chain and fragments thereof, including a variable region domain VH containing an amino acid sequence having three variable region sequences sufficient to confer specificity to an antigen, and three constant region domains CH1, CH2, and CH3. In the present invention, the term "light chain" refers to a full-length light chain and fragments thereof, including a variable region domain VL containing an amino acid sequence having three variable region sequences sufficient to confer specificity to an antigen, and a constant region domain CL. Whole antibodies include subtypes IgA, IgD, IgE, IgM, and IgG, and in particular, 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, and 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.

[0074] In one embodiment of the present invention, the TNFR2 binding agent is an anti-TNFR2 antigen-binding antibody fragment. An antigen-binding fragment of an antibody or antibody fragment refers to a fragment having an antigen-binding function, and includes Fab, F(ab'), F(ab')2, and Fv. Among the antibody fragments, Fab has a structure having a light chain and a heavy chain variable region, a light chain constant region, and the first constant region CH1 of the heavy chain, 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 the cysteine ​​residues in the hinge region of Fab'.

[0075] Fv is the smallest antibody fragment that contains only the heavy and light chain variable regions. Two-chain Fv (two-chain Fv) is a fragment in which the heavy and light chain variable regions are linked non-covalently, and scFv (single-chain Fv) is a fragment in which the heavy and light chain variable regions are linked covalently, generally via a peptide linker between them, or directly at the C-terminus, to form a dimeric structure like two-chain Fv. Such antibody fragments can be obtained using proteases (for example, Fab can be obtained by restricting and cleaving the whole antibody with papain, and F(ab')2 fragments can be obtained by restricting and cleaving the whole antibody with pepsin) or constructed through genetic recombination techniques.

[0076] 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.

[0077] 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 cysteines in the hinge region located at the C-terminus of the Fab' fragments.

[0078] "scFv (single-chain Fv)" antibody fragments are constructs made up of a single polypeptide chain containing the VH and VL domains of an antibody. scFvs can further contain a polypeptide linker between the VH and VL domains to form the desired structure for antigen binding.

[0079] Examples of antibodies suitable for expression in the cells of the invention (e.g., MSCs) may include, but are not limited to, monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, scFvs, Fab fragments, F(ab')2 fragments, disulfide-linked Fvs (sdFvs) and anti-idiotypic (anti-Id) antibodies, and antigen (epitope)-binding fragments thereof.

[0080] The heavy chain constant region can be selected from isotypes such as gamma (γ), mu (μ), alpha (α), delta (δ), epsilon (ε), etc. 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 of the kappa or lambda type.

[0081] A monoclonal antibody is an antibody obtained from a substantially homogeneous antibody population, in which the individual antibodies that make up the population are identical except for minor mutations that may occur naturally. Monoclonal antibodies are highly specific, being directed against a single epitope of the antigen. Unlike polyclonal antibodies, which generally contain different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen.

[0082] Examples of TNFR2 antibodies and antigen-binding antibody fragments are well known in the art. For example, antagonistic antibodies targeting TNFR2 have been reported to suppress regulatory T cell function and promote antitumor activity (Chen et al., (2022) Front.Immunol.13:835690.doi:10.3389 / fimmu.2022.835690; incorporated by reference). Exemplary anti-TNFR2 antibodies are described, for example, in WO2020061210A1, WO2014 / 124134 and WO2020 / 061210A1. Additional examples of antagonistic TNFR2 antibodies include BI-1808 (BioInvent International) and APX601 (Apexigen). Yet another example of a TNFR2 agonist antibody is HF200301 from HiFiBio Therapeutic.

[0083] In one embodiment of the invention, a TNFR2 binding agent (e.g., a TNFR2 antibody or antigen-binding fragment thereof) can bind to human TNFR2 monomer with a dissociation constant Kd of about 2 μM or less, 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.

[0084] In one embodiment of the present invention, the TNFR2 binding agent (e.g., a TNFR2 antibody or antigen-binding fragment thereof) can bind to human TNFR2 monomer with an off-rate constant Koff of about 2 x 10-1s-1, about 10-1s-1, about 10-3s-1 (e.g., 1 / second units); about 10-4s-1, about 10-5s-1 or slower, as measured by a method such as a BIACORE™ assay.

[0085] In one embodiment of the invention, the TNFR2 binding agent (e.g., a TNFR2 antibody or antigen-binding fragment thereof) can bind to human TNFR2 monomer with a binding constant K on of at least about 10 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 or faster, as measured by a method such as a BIACORE™ assay.

[0086] In one embodiment of the invention, the TNFR2 binding agent (e.g., a TNFR2 antibody or antigen-binding fragment thereof) can be characterized as binding to human TNFR2 monomer with an IC50 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, when measured by methods such as competitive binding assays. For example, in one embodiment of the invention, the TNFR2 binding agent (e.g., a TNFR2 antibody or antigen-binding fragment thereof) can be characterized as binding to human TNFR2 monomer with an IC50 of 1 nM to 1 μM, preferably 10 nM to 500 nM, when measured by methods such as competitive binding assays.

[0087] In one embodiment of the invention, a TNFR2 binding agent (e.g., a TNFR2 antibody or an antigen-binding fragment thereof) can be expressed in a genetically engineered cell (e.g., MSC) and can be secreted and / or anchored to the cell membrane and displayed on the cell surface.

[0088] In one embodiment of the present invention, a TNFR2 binding agent (e.g., a TNFR2 antibody or an antigen-binding fragment thereof) can be immobilized on the membrane of a genetically modified MSC or presented on the cell surface of the MSC. In one embodiment of the present invention, when the TNFR2 binding agent is presented on the membrane or surface of a genetically modified cell (e.g., MSC), the genetically modified cell (e.g., MSC) can have an agonistic effect on TNFR2.

[0089] In one embodiment of the invention, a TNFR2 binding agent (eg, a TNFR2 antibody or antigen-binding fragment thereof) can be expressed in genetically engineered cells (eg, MSCs) and localized to a specific organelle or location.

[0090] In one embodiment of the present invention, when a TNFR2-binding agent (e.g., a TNFR2 antibody or an antigen-binding fragment thereof) is anchored to a cell membrane or expressed on a cell surface, the TNFR2-binding agent may be directly / indirectly linked (or fused) to a transmembrane domain, or may be expressed in the form of a fusion protein that includes a transmembrane domain. Membrane-anchored or cell-surface expressed TNFR2-binding fusion proteins are described in detail in the fusion protein section.

[0091] In one embodiment of the invention, the TNFR2 binding agent can be monomeric.

[0092] In one embodiment of the invention, the TNFR2 binding agent is capable of forming multimers.

[0093] In one embodiment of the invention, the TNFR2 binding agent can form dimers, trimers, tetramers, pentamers or higher multimers, which can be formed covalently or non-covalently by interactions between amino acid residues of the TNFR2 binding agent.

[0094] In one embodiment of the invention, the multimer may be a fusion protein formed through a fusion domain fused to a TNFR2 antibody or antigen-binding antibody fragment.

[0095] In one embodiment of the invention, the multimers may be formed through inline fusion of TNFR2 antibodies or antigen-binding antibody fragments.

[0096] Stefin A protein variant that specifically binds to TNFR2

[0097] In the present invention, the term "Stefin A protein variant" refers to a scaffold based on Stefin A protein, and the sequence is derived from Stefin A protein, for example, Stefin A protein derived from a mammal, more preferably from a human. In one embodiment of the present invention, the "Stefin A protein variant" may be used interchangeably with "Stefin A protein variants" or "Affimer® protein" in a substantially identical sense. Unless otherwise specified, the Stefin A protein variant of the present invention refers to a Stefin A protein variant that specifically binds to TNFR2 as described herein.

[0098] Affimer®, developed by Avacta Life Sciences Limited, is a small and stable protein molecule engineered based on the Stefin A protein, a biological protein. Affimer® contains two short peptide sequences with random sequences and an N-terminal sequence, through which it can bind to a target substance with high affinity and specificity in a manner similar to that of a monoclonal antibody. Affimer® has shown significantly improved binding affinity and specificity compared to free peptide libraries, and is significantly smaller in size and more stable than antibodies, and is therefore attracting great interest as a next-generation alternative pharmaceutical platform to replace antibodies (U.S. Patent Nos. 9,447,170 and 8,853,131, etc.).

[0099] Avacta Life Sciences Limited has engineered the native Stefin A protein to develop a polypeptide that specifically binds to TNFR2, resulting in the invention of a Stefin A protein variant that can bind to TNFR2 with excellent affinity and specificity.

[0100] In one embodiment of the present invention, a stefin A protein variant that specifically binds to TNFR2 has been invented. The term "stefin A protein variant that specifically binds to TNFR2" may be used interchangeably with "TNFR2 stefin A protein variant," "stefin A protein variant that binds to TNFR2," and "TNFR2 Affimer (registered trademark)" in substantially the same sense.

[0101] "Stefin Protein" or "Stefin Polypeptides" includes a subgroup of proteins in the cystatin superfamily, a family that includes proteins containing multiple cystatin-like sequences. The stefin subgroup of the cystatin family includes relatively small (approximately 100 amino acids) single domain proteins. These lack post-translational modifications and have few disulfide bonds, allowing them to exhibit identical folded structures in diverse extracellular and intracellular environments. Stefin A protein has been reported to be a single, single chain, single domain protein composed of 98 amino acids. Avacta has developed the Affimer® platform using protein engineering techniques through mutations based on the structure of Stefin A. The only known biological activity of cystatins is the inhibition of cathepsin activity, which allows extensive testing of engineered proteins for residual biological activity.

[0102] In the present invention, the term "Stefin A protein variant" refers to an engineered variant of the stefin protein, which is a small and highly stable protein. For example, the stefin A protein variant of the present invention can present two peptide loops and an N-terminal sequence of random sequences that allow it to bind to a target protein with high affinity and specificity in a manner similar to an antibody. Stabilization of the two peptides by the stefin A protein scaffold limits the possible conformations that the peptides can take, increasing the binding affinity and specificity compared to a free peptide library. Such engineered non-antibody binding proteins are designed to mimic the molecular recognition properties of monoclonal antibodies in various applications. Additional modifications to other parts of the stefin A protein sequence can be made, which can improve properties such as increased stability against temperature and pH. In one embodiment of the present invention, the stefin A protein variant can include a sequence derived from a stefin A protein that shares substantial identity with the wild-type sequence of the stefin A protein, such as human stefin A protein. Those skilled in the art can easily modify the scaffold sequence based on the contents of the present invention. In particular, the Stefin A protein variant of the present invention can have at least 25%, 35%, 45%, 55% or 60% identity to the sequence corresponding to human Stefin A, for example, at least 70%, at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95% identity. Such modifications do not negatively affect the ability of the scaffold to bind to a desired target (e.g., TNFR2), and may not restore or generate, for example, a biological function possessed by wild-type Stefin A but abolished by the mutational changes described herein. Such target protein-specific binding platforms utilizing Stefin A protein variants are disclosed in detail in U.S. Pat. Nos. 9,447,170 and 8,853,131, among others.In one embodiment of the present invention, the Stefin A protein mutant may be a Stefin A protein mutant fragment comprising a portion of the Stefin A protein mutant that binds to human TNFR2.

[0103] In one embodiment of the present invention, the Stefin A protein variant can specifically bind to a target protein, TNFR2, with high affinity and specificity through engineering of the Stefin A protein.

[0104] In one embodiment of the present invention, the Stefin A protein mutant may not bind to TNFR1.

[0105] In one embodiment of the present invention, the Stefin A protein mutant may exhibit an agonistic effect on TNFR2.

[0106] In one embodiment of the present invention, "agonist" and "agonistic" refer to an agent that can directly or indirectly and substantially induce, activate, promote, increase or enhance the biological activity of a target or target pathway. In the present invention, the term "agonist" is used to include all agonists that can partially or fully induce, activate, promote, increase or enhance the activity of a protein or other target.

[0107] In one embodiment of the present invention, the Stefin A protein mutant that specifically binds to TNFR2 can bind to TNFR2 and activate the downstream pathway of TNFR2.

[0108] In one embodiment of the present invention, the Stefin A protein mutant that specifically binds to TNFR2 may contain at least one solvent accessible loop from a wild-type Stefin A protein that is capable of binding to TNFR2.

[0109] In one embodiment of the present invention, the Stefin A protein mutant that specifically binds to TNFR2 may bind to TNFR2 with a Kd value of about 2×10 −6 M or less, about 10 −6 M or less, about 10 −7 M or less, or about 10 −8 M or less.

[0110] In one embodiment of the present invention, the Stefin A protein mutant that specifically binds to TNFR2 may be a mutant derived from Stefin A protein having a backbone sequence and in which any one or more of loop 2 and loop 4 are replaced with alternative loop sequences, (Xaa)n and (Xaa)m.

[0111] In one embodiment of the present invention, the Stefin A protein variant that specifically binds to TNFR2 may comprise an amino acid sequence represented by Formula I:

[0112] [Number I] FR1-(Xaa)n-FR2-(Xaa)m-FR3

[0113] wherein FR1 comprises or represents an amino acid sequence comprising: MIPGGLSEAK PATPEIQEIV DKVKPQLEEK TGETYGKLEA VQYKTQVX (SEQ ID NO: 1); MIPGGLSEAK PATPEIQEIV DKVKPQLEEK TNETYGKLEA VQYKTQVLA (SEQ ID NO: 470); a 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 is any number of independently selected amino acids, more preferably 3 or less independently selected amino acids, and more preferably X is V or D; and / or The FR2 may include or represent 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; The FR3 may include or represent 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 individually any amino acid residue; n and m are each independently an integer from 3 to 20.

[0114] In one embodiment of the present invention, the FR1 may be an amino acid sequence having at least 80%, 85%, 90%, 95%, or 98% homology with SEQ ID NO: 1. In one embodiment of the present invention, the FR1 may be an amino acid sequence having at least 80%, 85%, 90%, 95%, or 98% identity with SEQ ID NO: 1.

[0115] In one embodiment of the present invention, the FR2 may be an amino acid sequence having at least 80%, 85%, 90%, 95%, or 98% homology to SEQ ID NO: 2. In one embodiment of the present invention, the FR2 may be an amino acid sequence having at least 80%, 85%, 90%, 95%, or 98% identity to SEQ ID NO: 2.

[0116] In one embodiment of the present invention, the FR3 may be an amino acid sequence having at least 80%, 85%, 90%, 95%, or 98% homology to SEQ ID NO: 3. In one embodiment of the present invention, the FR3 may be an amino acid sequence having at least 80%, 85%, 90%, 95%, or 98% identity to SEQ ID NO: 3.

[0117] In one embodiment of the present invention, the Stefin A protein variant that specifically binds to TNFR2 may include an amino acid sequence represented by Formula II:

[0118] [Mathematics II] MIP-Xaa1-GLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQV-Xaa2-(Xaa)n-Xaa3-TNYYI KVRAGDNKYMHLKVF-Xaa4-Xaa5-Xaa6-(Xaa)m-Xaa7-D-Xaa8-VLTGYQVDKNKDDELTGF (SEQ ID NO: 4)

[0119] Here, Xaa is, in each case, independently any number of any amino acid residues, more preferably 3 or less (preferably 1 or 2); n and m are each independently an integer of 3 to 20.

[0120] In one embodiment of the present invention, the Stefin A protein variant that specifically binds to TNFR2 may include 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:

[0121] MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVV-(Xaa)n-GTNYYIKVRAGDNKYMHLKVFKSL-(Xaa)m-EDLVLTGYQVDKNKDDELTGF (SEQ ID NO: 487); MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVD-(Xaa)n-GTNYYIKVRAGDNKYMHLKVFKSL-(Xaa)m-EDLVLTGYQVDKNKDDELTGF (SEQ ID NO: 5); or MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVLA-(Xaa)n-GTNYYIKVRAGDNKYMHLKVFKSL-(Xaa)m-EDLVLTGYQVDKNKDDELTGF (SEQ ID NO: 488), Xaa is, in each case, independently any number of any amino acid residues, more preferably 3 or less (preferably 1); n and m are each independently an integer of 3-20.

[0122] In one embodiment of the invention, Xaa1 can be Gly, Ala, Val, Arg, Lys, Asp, or Glu, preferably Gly, Ala, Arg, or Lys, more preferably Gly or Arg; Xaa2 can be Val, Asp, or "Leu-Ala"; Xaa3 can be Gly, Ala, Val, Ser, or Thr, preferably Gly or Ser; Xaa4 can be Arg, Lys, Asn, Gln, Ser, or Thr, preferably Arg, Lys, Asn, or Gln, more preferably Lys or Asn; Xaa5 can be Xaa6 can be Ala, Val, Ser or Thr, preferably Gly or Ser; Xaa7 can be Gly, Ala, Val, Asp or Glu, preferably Ala, Val, Asp or Glu, more preferably Ala or Glu; Xaa8 can be Ala, Val, Ile, Leu, Arg or Lys, preferably Ile, Leu or Arg, more preferably Leu or Arg.

[0123] In one embodiment of the present invention, n may be an integer from 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.

[0124] In one embodiment of the present invention, m may be an integer from 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.

[0125] In one embodiment of the present invention, the Xaa may be an amino acid that can be added independently to a polypeptide by recombinant expression in a prokaryotic or eukaryotic cell, preferably one of the 20 naturally occurring amino acids.

[0126] In the sequences and formulas of the present invention, the (Xaa)n may be or may include an amino acid sequence selected from SEQ ID NOs: 6 to 102. In one embodiment of the present invention, the (Xaa)n may be a sequence having at least 80%, 85%, 90%, 95%, or 98% homology or identity to an amino acid sequence selected from SEQ ID NOs: 6 to 102.

[0127] In one embodiment of the present invention, the (Xaa)n may be characterized by specifically binding to human TNFR2, and for example, the (Xaa)n may be an amino acid sequence selected from SEQ ID NOs: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 25, 28, 29, 30, 32, 33, 34, 38, 39, 40, 41, 42, 43, 44, 45, 47, 48, 49, 52, 54, 55, 56, 59, 60, 65, 66, 67, 68, 69, 70, 72, 73, 74, 75, 77, 79, and 80, or a sequence including the same, or having at least 80%, 85%, 90%, 95%, or 98% homology or identity thereto.

[0128] In one embodiment of the present invention, it may be characterized by having an agonistic effect on TNFR2, for example, (Xaa)n may be an amino acid sequence selected from SEQ ID NOs: 6, 11, 13, 16, 17, 24, 25, 29, 33, 41, 47, 54, 65, 67, 73 and 75, or a sequence including the same, or having at least 80%, 85%, 90%, 95% or 98% homology or identity thereto.

[0129] In one embodiment of the present invention, it may be characterized by having a TNFR signal competition effect, for example, the (Xaa)n may be an amino acid sequence selected from SEQ ID NOs: 6, 7, 11, 13, 14, 15, 16, 17, 21, 24, 25, 28, 29, 30, 33, 41, 42, 47, 49, 55, 65, 69, and 75, or a sequence including the same, or having at least 80%, 85%, 90%, 95%, or 98% homology or identity thereto.

[0130] In the sequences and formulas of the present invention, the (Xaa)m may be or may include an amino acid sequence selected from SEQ ID NOs: 103 to 199. In one embodiment of the present invention, the (Xaa)m may be a sequence having at least 80%, 85%, 90%, 95% or 98% homology or identity to an amino acid sequence selected from SEQ ID NOs: 103 to 199.

[0131] In one embodiment of the present invention, the (Xaa)m may be characterized as specifically binding to human TNFR2, and for example, the (Xaa)m may be selected from the group consisting of SEQ ID NOs: 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 121, 122, 125, 126, 127, 129, 130, 131, 135, 136, 137, 138, 139, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 35 38, 139, 140, 141, 142, 144, 145, 146, 149, 151, 152, 153, 156, 157, 162, 163, 164, 165, 166, 167, 169, 170, 171, 172, 174, 176 and 177 or a sequence which comprises the same or has at least 80%, 85%, 90%, 95% or 98% homology or identity thereto.

[0132] In one embodiment of the present invention, it may be characterized by having an agonistic effect on TNFR2, for example, the (Xaa)m may be an amino acid sequence selected from SEQ ID NOs: 103, 108, 110, 113, 114, 121, 122, 126, 130, 138, 144, 151, 162, 164, 170 and 172, or a sequence including the same, or having at least 80%, 85%, 90%, 95% or 98% homology or identity thereto.

[0133] In one embodiment of the present invention, it may be characterized by having a TNFR signal competitive effect, for example, the (Xaa)m may be an amino acid sequence selected from SEQ ID NOs: 103, 104, 108, 110, 111, 112, 113, 114, 118, 121, 122, 125, 126, 127, 130, 138, 139, 144, 146, 152, 162, 166 and 172, or a sequence including the same, or having at least 80%, 85%, 90%, 95% or 98% homology or identity thereto.

[0134] In one embodiment of the present invention, the Stefin A protein variant that specifically binds to TNFR2 may have or include an amino acid sequence selected from SEQ ID NOs: 200 to 296, wherein the sequence may optionally exclude one or more of the 21 residues at the C'-terminus. In one embodiment of the present invention, the Stefin A protein variant that specifically binds to TNFR2 may have at least 80%, 85%, 90%, 95%, or 98% homology or identity to an amino acid sequence selected from SEQ ID NOs: 200 to 296.

[0135] In one embodiment of the present invention, the Stefin A protein variant that specifically binds to TNFR2 may be characterized by specifically binding to human TNFR2, and may be, for example, any of SEQ ID NOs: 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 218, 219, 222, 223, 224, 226, 227, 228, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, , 233, 234, 235, 236, 237, 238, 239, 241, 242, 243, 246, 248, 249, 250, 253, 254, 259, 260, 261, 262, 263, 264, 266, 267, 268, 269, 271, 273, and 274 or a sequence comprising the same or having at least 80%, 85%, 90%, 95% or 98% homology or identity thereto.

[0136] In one embodiment of the present invention, the Stefin A protein variant that specifically binds to TNFR2 may preferably be characterized by having an agonistic effect on TNFR2, and may be, for example, an amino acid sequence selected from SEQ ID NOs: 200, 205, 207, 210, 211, 218, 219, 223, 227, 235, 241, 248, 259, 261, 267 and 269, or a sequence comprising the same, or having at least 80%, 85%, 90%, 95% or 98% homology or identity thereto.

[0137] In one embodiment of the present invention, the Stefin A protein variant that specifically binds to TNFR2 may be characterized by having a TNFR signal competitive effect, and may be, for example, an amino acid sequence selected from SEQ ID NOs: 200, 201, 205, 207, 208, 209, 210, 211, 215, 218, 219, 222, 223, 224, 227, 235, 236, 241, 243, 249, 259, 263 and 269, or a sequence including the same, or having at least 80%, 85%, 90%, 95% or 98% homology or identity thereto.

[0138] The 21 amino acids at the C'-terminus of the Stefin A protein variant sequence that specifically binds to TNFR2 were added for cloning and analysis. The 3x Ala linker, the 10 amino acid Myc tag, the 2x Ala linker and the 6x His tag are all options and are not essential. [Table 1] TIFF2025069065000003.tif248170TIFF2025069065000004.tif248170TIFF2025069065000005.tif248170 TIFF2025069065000006.tif248170TIFF2025069065000007.tif248170TIFF2025069065000008.tif133170

[0139] The C'-terminal sequence of SEQ ID NOs: 200 to 296, AAAEQKLISEEDL AAHHHHHH (SEQ ID NO: 489), is a sequence for purifying the Stefin A protein mutant and is not essential.

[0140] In one embodiment of the present invention, the amino acid sequence "AAAEQKLISEEDLAAHHHHHH" (SEQ ID NO: 489) may be excluded from SEQ ID NOs: 200-296.

[0141] In addition to the characteristics of loop 2 and loop 4 described above, the Stefin A protein or mutant sequence described in the specification may further include minor modifications such as partial deletions or additions (e.g., additions or deletions of about 10 amino acids compared to the wild-type Stefin A protein or mutant).

[0142] In one embodiment of the present invention, the Stefin A protein variant that specifically binds to TNFR2 can bind to human TNFR2 monomer with a dissociation constant Kd of about 2 μM or less, 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.

[0143] In one embodiment of the present invention, the Stefin A protein mutant that specifically binds to TNFR2 can bind to human TNFR2 monomer with an off-rate constant Koff of about 2 x 10-1s-1, about 10-1s-1, about 10-3s-1 (e.g., 1 / second unit); about 10-4s-1, about 10-5s-1 or slower, when measured by a method such as a BIACORETM assay.

[0144] In one embodiment of the present invention, the Stefin A protein variant that specifically binds to TNFR2 can bind to human TNFR2 monomer with a binding constant K on of at least about 10 3 M-1s-1; at least about 10 4 M-1s-1; at least about 10 5 M-1s-1; at least about 10 6 M-1s-1 or faster, when measured by a method such as a BIACORE™ assay.

[0145] In one embodiment of the present invention, the Stefin A protein variant that specifically binds to TNFR2 may be characterized as binding to human TNFR2 monomer with an IC50 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 when measured by a method such as a competitive binding assay. For example, in one embodiment of the present invention, the Stefin A protein variant that specifically binds to TNFR2 may be characterized as binding to human TNFR2 monomer with an IC50 of 1 nM to 1 μM, preferably 10 nM to 500 nM, when measured by a method such as a competitive binding assay.

[0146] In one embodiment of the present invention, the Stefin A protein variant that specifically binds to TNFR2 may be characterized as having a melting temperature (Tm, i.e., the temperature at which both the folded and unfolded states are equally packed) of 65° C. or more, preferably at least 70° C., 75° C., 80° C., or 85° C. or more. The melting temperature is a useful indicator of protein stability, and the relative proportion of folded and unfolded protein can be determined using known techniques such as differential scanning calorimetry, UV difference spectroscopy, fluorescence, CD (circular dichroism), and NMR (Pace et al. (1997) “Measuring the conformational stability of a protein” in Protein structure: A practical approach 2:299-321).

[0147] In one embodiment of the present invention, the Stefin A protein variant that specifically binds to TNFR2 can be expressed in a genetically engineered cell and can be secreted and / or anchored to the cell membrane and displayed on the cell surface.

[0148] In one embodiment of the present invention, the Stefin A protein variant that specifically binds to TNFR2 can be immobilized on the membrane or presented on the cell surface of a genetically modified cell. In one embodiment of the present invention, when the Stefin A protein variant that specifically binds to TNFR2 is presented on the membrane or surface of a genetically modified cell, the genetically modified cell can have an agonistic effect on TNFR2.

[0149] In one embodiment of the present invention, the Stefin A protein variant that specifically binds to TNFR2 can be expressed in genetically engineered cells and localized to a specific organelle or location.

[0150] In one embodiment of the present invention, when the Stefin A protein variant that specifically binds to TNFR2 is fixed to a cell membrane or expressed on a cell surface, the TNFR2 binding agent may be directly / indirectly linked (or fused) to a transmembrane domain, or may be expressed in the form of a fusion protein containing a transmembrane domain. In the case of Stefin A protein variant that is fixed to a membrane or expressed on a cell surface, the fusion protein containing a membrane-spanning portion is described in detail in the fusion protein section.

[0151] In one embodiment of the present invention, the Stefin A protein mutant that specifically binds to TNFR2 may be a monomer.

[0152] In one embodiment of the present invention, the Stefin A protein mutant that specifically binds to TNFR2 is capable of forming multimers.

[0153] In one embodiment of the present invention, the Stefin A protein variant that specifically binds to TNFR2 can form a dimer, trimer, tetramer, pentamer or higher multimer. In one embodiment of the present invention, the multimer can be formed by covalent or non-covalent interactions between amino acid residues of the Stefin A protein variant.

[0154] In one embodiment of the present invention, the multimer may be a fusion protein formed through a fusion domain fused to a Stefin A protein mutant.

[0155] In one embodiment of the present invention, the multimers may be formed through in-line fusion of Stefin A protein, and such multimeric forms of in-line fusion proteins are described in detail in the section on fusion proteins.

[0156] Nucleic Acids Encoding TNFR2 Binding Agents

[0157] In the present invention, the term "nucleic acid" refers to a polynucleotide of any length, including DNA, RNA, or a combination of DNA and RNA. In one embodiment of the present invention, the nucleic acid can 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. In one embodiment of the present invention, the nucleic acid can be, but is not limited to, RNA, DNA, threese nucleic acids (TNA), glycol nucleic acids (GNA), peptide nucleic acids (PNA), locked nucleic acids (LNA, e.g., LNA with β-D-ribo sequence, a-LNA (a partial stereoisomer of LNA) with aL-ribo sequence, 2'-amino-LNA or 2'-amino-a-LNA with 2'-amino functionalization), ethylene nucleic acid (ENA), cyclohexenyl nucleic acids (CeNA), or hybrids or combinations thereof.

[0158] In the present invention, the term "encoding nucleic acid" refers to a nucleic acid sequence that encodes a specific protein or polypeptide. If the sequence of a specific protein or polypeptide is published in the art, it is well known how to design or derive a nucleic acid that encodes it.

[0159] Thus, a nucleic acid encoding a TNFR2 binding agent of the present invention (e.g., a Stefin A protein mutant that specifically binds to TNFR2) can be readily understood from the above description of "Stefin A protein mutant that specifically binds to TNFR2."

[0160] In one embodiment of the present invention, the Stefin A protein variant that specifically binds to TNFR2 may be encoded by a nucleic acid comprising a nucleic acid sequence selected from SEQ ID NOs: 297 to 393 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 98% identity thereto, and may optionally exclude the nucleic acid encoding the C'-terminal 21 amino acids.

[0161] In one embodiment of the present invention, the nucleic acid encoding the Stefin A protein mutant that specifically binds to TNFR2 may include a sequence that hybridizes to a nucleic acid selected from SEQ ID NOs: 297 to 393, and the hybridization may be characterized by being performed, for example, in the presence of 6X sodium chloride / sodium citrate (SSC) at 45°C, followed by washing with 0.2X SSC at 65°C.

[0162] Nucleic acid sequence encoding a Stefin A protein variant that specifically binds to TNFR2. The 5'-terminal 63 nucleic acids encoding the C'-terminal 21 amino acids are added sequences for cloning and analysis: 3x Ala linker, 10 amino acid Myc tag, 2x Ala linker and 6x His tag are all optional and not essential. [Table 2] TIFF2025069065000010.tif255167TIFF2025069065000011.tif255167TIFF20250690650 00012.tif255167TIFF2025069065000013.tif255167TIFF2025069065000014.tif255167 TIFF2025069065000015.tif255167TIFF2025069065000016.tif255167TIFF20250690650 00017.tif255167TIFF2025069065000018.tif255167TIFF2025069065000019.tif231170

[0163] Fusion Proteins - General

[0164] In one embodiment of the present invention, the genetically modified cell may be characterized in that a nucleic acid encoding a fusion protein containing a TNFR2 binding agent is introduced into the genetically modified cell. In one embodiment of the present invention, the fusion protein may be a TNFR2 binding agent (e.g., a Stefin A protein mutant) fused to one or more of both ends of the TNFR2 binding agent (e.g., a Stefin A protein mutant) with an additional peptide sequence (fusion domain).

[0165] In the present invention, the term "fusion domain" refers to an additional domain or moiety that may be included in, directly / indirectly fused to, a TNFR2 binding agent (eg, a Stefin A protein variant) of the present invention.

[0166] In one embodiment of the present invention, the fusion protein may comprise at least one fusion domain.

[0167] In one embodiment of the present invention, the fusion protein may include additions, substitutions, and / or deletions in a peptide containing one or more amino acids to modulate the biological activity of the TNFR2 binding agent (e.g., a Stefin A protein variant) or to add additional biological activity. For example, the additions, substitutions, and / or deletions may modulate at least one property or activity of the altered TNFR2 binding agent (e.g., a Stefin A protein variant). For example, the additions, substitutions, and / or deletions may adjust affinity, half-life, stability, protease cleavage, dosage, release or bioavailability, facilitate purification, reduce deamidation, improve storage stability, or change the administration route for binding to TNFR2 and pathway activation of the TNFR2 binding agent. For example, the fusion protein may further comprise, but is not limited to, a protease cleavage sequence, a reactive functional group, an antibody binding domain, a fusion domain for protein expression and purification (e.g., but not limited to, FLAG, poly-His, GST, c-myc, etc.), or a linking molecule (e.g., but not limited to, biotin, etc.).

[0168] In one embodiment of the invention, the fusion domains can be fused to proteins or peptides that confer expression characteristics such as secretion from a cell, or anchoring to the cell surface (e.g., cell membrane), or subcellular localization; add substrate or other recognition sequences for post-translational modification; generate multimeric structures that aggregate through protein-protein interactions; modulate (extend or decrease) serum half-life; or alter tissue localization, tissue clearance, or other ADME properties, or to add other functions.

[0169] For example, some fusion domains are particularly useful for isolation and / or purification of the fusion protein, such as by affinity chromatography. Well-known examples of fusion domains that facilitate expression or purification include, but are not limited to, affinity tags such as polyhistidine tags, Strep II tags, streptavidin-binding peptide (SBP) tags, calmodulin-binding peptide (CBP) tags, S-tags, HA tags, c-Myc tags, thioredoxin, protein A, and protein G.

[0170] In one embodiment of the present invention, if the TNFR2 binding agent (e.g., Stefin A protein variant or fusion protein) is to be secreted, it may contain a signal sequence that directs the protein to be transported to the lumen of the endoplasmic reticulum and ultimately secreted (or to be retained on the cell surface if it has a transmembrane domain or cell surface retention signal). The signal sequence (signal peptide or leader sequence) may be located at the N-terminus of the nascent polypeptide. The signal sequence targets the nascent polypeptide to the endoplasmic reticulum, where the protein is sorted to a destination, such as the internal space of an organelle, an internal membrane, the extracellular membrane, or the outside of the cell via secretion. In general, the signal sequence may be cleaved after transport to the endoplasmic reticulum, and may be characterized as being cleaved at a residue within the signal sequence, but is not limited thereto.

[0171] A signal sequence (also called a signal peptide or leader sequence) may be located at the N-terminus of the nascent peptide. The signal sequence functions to target the polypeptide to the endoplasmic reticulum so that the protein can be sorted to a destination, such as the internal space of an organelle, the internal membrane, the extracellular membrane, or the outside of the cell via secretion. Generally, the signal sequence may be cleaved by a signal peptidase after transport to the endoplasmic reticulum, and cleavage of the signal sequence generally occurs at a specific site in the amino acid sequence and varies depending on the amino acid residue in the signal sequence.

[0172] In one embodiment of the invention, the signal sequence can be 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).

[0173] In one embodiment of the present invention, the signal sequence may be a native signal peptide derived from, for example, a human. In one embodiment of the present invention, the signal sequence may be a non-native signal peptide. For example, in one embodiment of the present invention, the non-native signal peptide may be a signal peptide derived from, for example, a corresponding native secreted human protein, and may include one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) substitutions, insertions, and / or deletions.

[0174] In one embodiment of the invention, the signal sequence may be other signal peptides such as, but not limited to, signal sequences derived from immunoglobulins (e.g., IgG heavy chain or IgG-kappa light chain), cytokines (e.g., IL-2 or CD33), plasma albumin proteins (e.g., HSA or albumin), a human azurocidin preprotein signal sequence, luciferase, trypsinogen, chymotrypsinogen, or other secreted proteins. Exemplary signal sequences include, but are not limited to, the following:

[0175] Table 3 below lists non-limiting examples of signal peptide sequences that can be used to secrete a TNFR2 binding agent of the invention (eg, a Stefin A protein variant) or a fusion protein thereof.

[0176] [Table 3]

[0177] In one embodiment of the present invention, the fusion protein may further comprise at least one linker separating the TNFR2 binding agent (e.g., a Stefin A protein variant) and / or the fusion domain. In one embodiment of the present invention, the "linker" may be inserted between the first polypeptide (e.g., a Stefin A protein variant that specifically binds to TNFR2) and the second polypeptide (e.g., another Stefin A protein variant or a fusion domain) of the fusion protein of the present invention.

[0178] In the present invention, the linker may be classified into a flexible linker, a rigid linker, and an in vivo cleavable linker depending on the structure.

[0179] Many natural linkers have an α-helical structure. The α-helical structure forms a rigid and stable structure with intrasegment hydrogen bonds and a closely packed backbone. Thus, 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). In general, rigid linkers adopt an α-helical structure or contain a large number of Pro residues, exhibiting a relatively rigid structure. 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 the optimal distance between the domains. As a result, rigid linkers can be used when spatial separation of domains is important to preserve the stability or bioactivity of the fusion protein. In this regard, an α-helix forming linker having the sequence (EAAAK)n (SEQ ID NO:421) is commonly used in the construction of many fusion proteins, and other examples of rigid linkers include, but are not limited to, Pro-rich sequences such as (XP)n, where X is any amino acid, preferably Ala, Lys, or Glu.

[0180] Representative linkers for the purpose of illustration include, but are not limited to, GRA, poly(Gly), poly(Ala) and the linkers shown in Table 4 below.

[0181] [Table 4]

[0182] In one embodiment of the present invention, in addition to its basic role of linking the fusion domains, the linker can provide various advantages to the fusion protein, such as improving biological activity, increasing expression yield, and achieving a favorable pharmacokinetic profile.

[0183] In one embodiment of the present invention, the linker preferably does not negatively affect the expression, secretion or activity of each domain. In one embodiment of the present invention, the linker preferably is not antigenic and does not induce an immune response.

[0184] In one embodiment of the present invention, the fusion protein may be characterized by comprising a fusion domain selected from the group consisting of an antigen binding protein (domain), a cytokine, a half-life extension domain, a growth factor, an enzyme, and a cell penetrating domain, but is not limited thereto. In one embodiment of the present invention, the fusion protein may further comprise a therapeutic peptide or protein.

[0185] In one embodiment of the present invention, the "therapeutic peptide or protein" refers to any peptide or protein having a preventive or therapeutic effect against a specific disease. In one embodiment of the present invention, 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 TNFR2 of the present invention), and may also include, without limitation, peptides and proteins reported in the art to have a preventive or therapeutic effect against a specific disease.

[0186] In one embodiment of the present invention, the fusion protein may comprise a binding domain.

[0187] In one embodiment of the present invention, when the fusion protein contains a binding domain, it may have multispecificity, capable of binding to at least one target molecule other than TNFR2.

[0188] In one embodiment of the present invention, the binding domain may be characterized as being selected from the group consisting of, for example, a Stefin A protein variant (which may be the same or different from the Stefin A protein variant of the present invention that specifically binds to TNFR2), 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, a growth factor), an engineered T-cell receptor, and an enzyme or a catalytic fragment thereof, but is not limited thereto.

[0189] In an embodiment of the present invention, 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 module (CBM), Cys-knots, darpin, El-tandem, fynomers, knottin, Kunitz domains, O-bodies, Pronectin, ScFv, Sac7d, Sso7d, and Tn3.

[0190] In one embodiment of the invention, the fusion domain fused to the TFNR2 binding agent (e.g., a Stefin A protein variant) may be the same or a different binding protein (e.g., a Stefin A protein variant) and / or may be a Stefin A protein variant that specifically binds to another target.

[0191] In one embodiment of the present invention, when the fusion domain is a Stefin A protein variant that specifically binds to TNFR2, two or more Stefin A protein variants contained in the fusion protein of the present invention can bind to the same or different sites of TNFR2. In one embodiment of the present invention, the fusion protein can be characterized by binding to two sites (biparatopic) or two or more sites (multiparatopic) of TNFR2.

[0192] In an embodiment of the present invention, exemplary targets of the fusion protein other than TNFR2 may be, but are not limited to, immune checkpoint proteins and immune co-stimulatory receptors, receptors, cytokines, growth factors, or tumor-associated antigens.

[0193] In one embodiment of the present invention, the cytokine is used as a collective term for secretory proteins that play an important role in intercellular signal transmission, including, but not limited to, chemokines, interferons, lymphokines, interleukins, tumor necrosis factors, etc.

[0194] In one embodiment of the present invention, the growth factor refers to a naturally occurring substance or a 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, TGF, etc.

[0195] In an embodiment of the present invention, the enzyme is used as a general term for proteins that catalyze biological reactions. For example, the enzyme may be α-chymotrypsin, lysozyme, urate oxidase, acetylcholinesterase, Thermomyces lanuginose 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 Examples of such enzyme inhibitors include, but are not limited to, Agalsidase alfa, Imiglucerase, Taliglucerase alfa, Velaglucerase alfa, Alglucerase, Sebelipase alpha, Laronidase, Idursulfase, Elosulfase alpha, Galsulfase, Alglucosidase alpha and the like.

[0196] In one embodiment of the present invention, the cell-penetrating peptide refers to a short peptide that promotes cellular uptake and absorption of various molecules. In the present invention, when the fusion protein includes a cell-penetrating peptide, the TNFR2 binding agent (e.g., a Stefin A protein mutant) can be absorbed into cells. For example, the cell-penetrating peptide includes, but is not limited to, Tat, Penetratin, Transporant, Pept1, Pept2, pVEC, DPV3, DPV6, R8, R9, MPG, MAP, Bip4, C105Y, Melittin, etc.

[0197] In one embodiment of the present invention, a nucleic acid encoding the fusion protein is introduced into a genetically engineered cell, which can express the fusion protein and secrete it extracellularly or, preferably, anchor it to the cell membrane and display it on the cell surface.

[0198] In one embodiment of the present invention, the fusion protein can be expressed in genetically engineered cells and localized to a specific organ or location within the cell.

[0199] In one embodiment of the present invention, the fusion protein may be a secreted fusion protein and / or a membrane-anchored fusion protein.

[0200] In one embodiment of the present invention, when the fusion protein is anchored to a cell membrane or expressed on a cell surface, it may further comprise a transmembrane domain. In the present invention, the term "transmembrane domain" refers to a protein domain that spans the membrane of a cell. In one embodiment of the present invention, the transmembrane domain preferably has an α-helical structure, but is not limited thereto.

[0201] In one embodiment of the present invention, the transmembrane domain may be, for example, a transmembrane domain derived from CD3, CD4, CD5, CD8, CD28, CD99, immunoglobulins (e.g., IgG1, IgG4, IgD, etc.), PDGFR, PTGFRN, etc., or a variant thereof, but is not limited thereto.

[0202] In one embodiment of the present invention, the fusion protein may further include a hinge domain in addition to the transmembrane domain. In the present invention, the term "hinge domain" refers to a series of amino acid sequences present between the transmembrane domain and the extracellular domain of a membrane-anchored protein. In one embodiment of the present invention, the hinge domain may be located between a TNFR2 binding agent (e.g., a Stefin A protein mutant that specifically binds to TNFR2) and the transmembrane domain.

[0203] In one embodiment of the present invention, the hinge domain may be, but is not limited to, a hinge domain derived from CD3, CD4, CD5, CD8, CD28, CD99, immunoglobulin (e.g., IgG1, IgG4, IgD, etc.), PDGFR, PTGFRN, etc., or a variant thereof. In one embodiment of the present invention, the TNFR2-binding fusion protein comprises a hinge domain derived from an immunoglobulin (e.g., IgG1, IgG4, IgD, etc.).

[0204] In one embodiment of the present invention, the fusion protein may further include a coiled-coil domain. In the present invention, the term "coiled-coil domain" refers to a structural motif of a protein in which two to seven α-helices are wound like a rope. Preferably, the coiled-coil domain may be characterized by having two or three α-helices wound around it.

[0205] In one embodiment of the present invention, the coiled-coil domain may be characterized as being 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, vasodilator-stimulated phosphoprotein (VASP), or a variant thereof, but is not limited thereto.

[0206] In one embodiment of the present invention, the coiled-coil domain can be located between a TNFR2 binding agent (eg, a Stefin A protein mutant that specifically binds to TNFR2) and a transmembrane domain.

[0207] In an embodiment of the present invention, the fusion protein may further include a virus-derived peptide or protein. In an embodiment of the present invention, the virus-derived peptide or protein may be, for example, 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 H proteins, or Sendai virus F and H proteins. The F and HN proteins of the present invention may be characterized as, 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 or combinations thereof.

[0208] In an embodiment of the present invention, the fusion protein may further comprise an immunomodulatory domain or an intracellular signaling domain.

[0209] In one embodiment of the present invention, the immune regulatory domain or intracellular signal transduction 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.

[0210] In one embodiment of the present invention, the immunomodulatory domain or intracellular signaling domain may be characterized as being an immunomodulatory domain derived from CD3, CD28, CD40L, ICOS, OX40, 4-1BB, TNFR2, etc., but is not limited thereto.

[0211] In one embodiment of the present invention, the fusion protein may be a chimeric antigen receptor (CAR). In one embodiment of the present invention, when the fusion protein is a chimeric antigen receptor, the TNFR2 binding agent (e.g., a Stefin A protein mutant that specifically binds to TNFR2) may function as an extracellular binding domain.

[0212] In one embodiment of the present invention, when the fusion protein is a chimeric antigen receptor, it may further include the above-mentioned transmembrane domain, hinge domain, and intracellular signaling domain, but is not limited thereto, and the extracellular antigen binding domain of various chimeric antigen receptors or analogs thereof known in the art can be modified to easily design and prepare a TNFR2 binding agent of the present invention (e.g., a Stefin A protein mutant that specifically binds to TNFR2).

[0213] In one embodiment of the present invention, the fusion protein may further comprise a localization domain. In one embodiment of the present invention, when the fusion protein is expressed in a cell, it is preferable that the fusion protein further comprises a localization domain. In the present invention, the term "localization domain" refers to a peptide or protein sequence that functions to localize a protein to a specific organ or a specific location within a cell. In one embodiment of the present invention, the localization domain may be an organ-specific localization domain or an intracellular protein localization domain.

[0214] In one embodiment of the present invention, the localization domain may be characterized as, 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, etc.; a mitochondrial-specific localization domain derived from ATP synthase F1b, cytochrome c oxidase polypeptide VIII, SOD2, citrate synthase, Tu translation elongation factor, etc.; or a peroxisome localization domain derived from PTS1, PTS2, etc., but is not limited thereto.

[0215] In one embodiment of the present invention, the fusion protein may further comprise a half-life extension domain. In one embodiment of the present invention, the half-life extension domain refers to a domain or moiety fused to the TNFR2 binding agent (e.g., Stefin A protein variant) of the present invention for the purpose of extending the half-life of the TNFR2 binding agent (e.g., Stefin A protein variant), and 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, a transferrin binding protein or peptide, etc. Yet another modification that may be made to the TNFR2 binding agent (e.g., Stefin A protein variant), the fusion protein sequence, or a flanking polypeptide moiety provided as part of the fusion protein is to include one or more sequences as sites for post-translational modification by enzymes. This may be characterized by, but is not limited to, a domain for modifications such as glycosylation, acetylation, acylation, lipid-modification, palmitoylation, addition of palmitate, phosphorylation, glycolipid-binding, etc.

[0216] Multispecific fusion proteins

[0217] In one embodiment of the invention, the fusion protein can be, for example, a multispecific polypeptide comprising a first TNFR2 binding agent (e.g., a Stefin A protein variant that specifically binds to TNFR2) and at least one additional binding domain. The additional binding domain can be, for example, a polypeptide sequence selected from the group consisting of a second binding domain (e.g., a second Stefin A protein variant, which can be the same or different from the first Stefin A protein variant), an antibody or antigen-binding polypeptide thereof, a ligand binding site of a receptor (e.g., a receptor trap polypeptide), a receptor-binding ligand (e.g., a cytokine, growth factor or analog thereof), an engineered T-cell receptor, and an enzyme or catalytic fragment thereof.

[0218] In one embodiment of the invention, the fusion protein can include at least two TNFR2 binding agents. The fusion proteins can bind to identical or overlapping sites, or can bind to two sites (biparatopic) or more than two sites (multiparatopic). In one embodiment of the invention, the fusion protein can include at least one additional Stefin A protein variant sequence directed to TNFR2 or another target. The additional Stefin A protein variant that specifically binds to TNFR2 can be the same or different from the first Stefin A protein variant that specifically binds to TNFR2 to generate a multispecific fusion protein.

[0219] In one embodiment of the present invention, the fusion protein may contain one or more antigen-binding sites of an antibody. The fusion protein may be a single chain containing all of the Stefin A protein variants that specifically bind to TNFR2 and the antigen-binding sites (e.g., scFv), or may be a multimeric protein complex, such as an antibody assembled with antibody heavy and / or light chains.

[0220] In one embodiment of the present invention, when the fusion protein comprises a full-length immunoglobulin, the fusion protein can preserve the Fc function of the Fc region of the immunoglobulin. For example, the fusion protein can bind to the Fc receptor of an Fc receptor-positive cell via the Fc region. In one embodiment of the present invention, the fusion protein can bind to the Fc receptor and activate the Fc receptor-positive cell, and can initiate or increase the expression of cytokines and / or costimulatory antigens. The cytokines and / or costimulatory antigens can transmit signals beyond the secondary activation signal required for T cell activation. In one embodiment of the present invention, the fusion protein can possess antibody-dependent cellular cytotoxicity (ADCC) function by binding of the Fc region to other cells expressing Fc receptors present on the surface of effector cells of the immune system, such as immune cells, hepatocytes, and endothelial cells. This is a cell-mediated immune defense mechanism in which effector cells of the immune system actively lyse target cells whose membrane surface antigens are bound by antibodies, thereby inducing cell death (e.g., tumor cell death) through ADCC.

[0221] In one embodiment of the present invention, the ADCC function of the fusion protein was demonstrated.

[0222] As explained above, the Fc region of the fusion protein, apart from Fc-mediated cytotoxicity, can positively affect the maintenance of serum levels of the fusion protein, which may mean improved stability and persistence in vivo. For example, when the Fc portion binds to Fc receptors on endothelial cells and phagocytes, the fusion protein can be internalized and then recycled back into the bloodstream, improving its half-life in vivo.

[0223] In one embodiment of the present invention, other exemplary targets of the additional binding domain (e.g., Stefin A protein variant) include, but are not limited to, immune checkpoint proteins and immune co-stimulatory receptors, receptors, cytokines, growth factors, or tumor-associated antigens. In one embodiment of the present invention, the binding domain may be a monoclonal antibody or fragment thereof against at least one autoimmune target. In one embodiment of the present invention, the TNFR2 binding agent (e.g., Stefin A protein variant that specifically binds to TNFR2) may be part of a fusion protein that binds to a protein upregulated in an autoimmune state. Various forms of Stefin A protein variants and fusion proteins containing the same are described in detail in William BA et al. J. Clin. Med. 2019;8(8):1261.

[0224] In one embodiment of the present invention, the multispecific fusion protein may further comprise a half-life extending moiety as described herein. For example, at least one TNFR2 binding agent (e.g., a Stefin A protein variant that specifically binds to TNFR2) of the fusion protein may be linked to at least one immune cell (e.g., T cell and / or NK cell) binding domain (e.g., CD3ε chain or CD16) specific binding domain via a peptide linker, and may additionally be linked to a half-life extending moiety, such as a fragment crystallizable (Fc) domain (e.g., FcγR null-binding Fc), human serum albumin (HSA), or a Stefin A protein variant that specifically binds to HSA. In one embodiment of the present invention, the half-life extending moiety may be a fragment crystallizable (Fc) domain. In one embodiment of the present invention, the half-life extending moiety may be human serum albumin (HSA). In one embodiment of the present invention, the half-life extending moiety may be a Stefin A protein variant that specifically binds to human serum albumin.

[0225] Engineering PK and ADME properties

[0226] In one embodiment of the present invention, the fusion protein may not have a half-life and / or pharmacokinetic profile (PK profile) suitable for administration routes such as parenteral therapeutic administration. In the present invention, the term "half-life" refers to the time it takes for an active ingredient, such as a TNFR2 binding agent (e.g., a Stefin A protein variant) or a fusion protein of the present invention, to lose half of its pharmacological or physiological activity or concentration. Biological half-life may 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. In one embodiment of the present invention, biological half-life may be evaluated by determining the time it takes for the plasma concentration of the substance to reach half of its normal state level (plasma half-life). To address this shortcoming, a common strategy used to extend the half-life of other protein therapeutics involves integrating a half-life extending moiety as part of the fusion protein.

[0227] In the present invention, the term "half-life extending moiety" refers to a pharma- ceutically acceptable moiety, domain, or molecule that is covalently linked (chemically conjugated or fused) to a TNFR2 binding agent (e.g., a Stefin A protein variant) of the invention to form a fusion protein, optionally conjugated or fused directly via a non-naturally encoded amino acid or indirectly via a linker or the like, to improve or alter the pharmacokinetic or biophysical properties of the TNFR2 binding agent (e.g., a Stefin A protein variant) of the invention, including, but not limited to, preventing or mitigating reduced activity or alteration of the TNFR2 binding agent (e.g., a Stefin A protein variant), extending half-life, increasing absorption rate, reducing toxicity, improving solubility, reducing protein aggregation, increasing biological activity and / or target selectivity of the altered TNFR2 binding agent (e.g., a Stefin A protein variant), increasing productivity, reducing immunogenicity, etc. For example, the increased productivity and / or reduced immunogenicity of the fusion protein is compared to a fusion protein that does not contain the fusion protein.

[0228] In one embodiment of the present invention, the "half-life extending moiety" includes not only proteins or peptides but also non-proteinaceous half-life extending moieties, such as, but not limited to, water-soluble polymers, such as polyethylene glycol (PEG) or discrete PEG, HES (hydroxyethyl starch), lipids, branched or unbranched acyl groups, branched or unbranched C8-C30 acyl groups, branched or unbranched alkyl groups, branched or unbranched C8-C30 alkyl groups, and unstructured polypeptides, such as serum albumin, transferrin, adnectins (e.g., albumin-binding adnectins or pharmacokinetically extended adnectins (PKE adnectins)), Fc domains, XTEN and PAS polypeptides, and fragments thereof.

[0229] In one embodiment of the present invention, the half-life extending moiety may extend the half-life of the fusion protein circulating in the serum of a subject, compared to the half-life of the fusion protein not containing the half-life extending moiety. For example, the half-life may be extended by about 1.2 times, 1.5 times, 2.0 times, 3.0 times, 4.0 times, 5.0 times, or 6.0 times or more, but is not limited thereto. In one embodiment of the present invention, the half-life may be extended by, but is not limited to, 6 hours or more, 12 hours or more, 24 hours or more, 72 hours or more, 96 hours or more, or 1 week or more after administration.

[0230] By way of additional example, half-life extending moieties that may be included in the fusion proteins of the invention include, but are not limited to, the following:

[0231] - A genetic fusion of a TNFR2 binding agent sequence and a naturally long half-life protein or protein domain (e.g., an Fc domain fusion, a transferrin (Tf) fusion, or an albumin fusion). Fusion technologies with said proteinaceous half-life extending moieties are well known in the art: see, 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. (2009) "Receptor-Fc fusion therapeutics, traps, and Mimetibody 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. (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.p.325-43。.

[0232] - A genetic fusion of a TNFR2 binding agent sequence and an inert polypeptide (e.g., XTEN (also known as recombinant PEG or rPEG), homoamino acid polymer (HAP, HAPylation), proline-alanine-serine polymer (PAS, PASylation), or elastin-like peptide (ELP, ELPylation)).The fusion technique with the inactive polypeptide is well known in the art: see, for example, 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 soluble 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.

[0233] - Increasing the hydrodynamic radius by chemical conjugation of pharmacologically active peptides or proteins to repeating chemical moieties such as PEG (PEGylation) or hyaluronic acid. Techniques for increasing hydrodynamic radius by chemical conjugation are well known in the art: see, 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 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.

[0234] - significantly increasing the negative charge of the fusion of a pharmacologically active peptide or protein by polysialylation; or alternatively, (b) the fusion of a negatively charged highly sialylated peptide (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). The technology of half-life extension through polysialylation is well known in the art: see, 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 dose pharmacokinetics and effects on follicular growth and serum hormones of a long-acting recombinant FSH preparation (FSHCTP) in healthy pituitary-suppressed females" (2002) Hum Reprod. 17:1987-93; and Fares et al. "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; and Fares “Half-life extension through O-glycosylation.

[0235] - Non-covalent binding through peptide or protein-binding domains to proteins that generally have a long half-life, such as HSA, IgG, transferrin or fibronectin. Such methods are well known in the art: see, 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 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.

[0236] Classical genetic fusion to long-lived serum proteins offers an alternative method of half-life extension that is different from chemical conjugation to PEG or lipids. Antibody Fc domain and human serum albumin have been used as traditional fusion domains for half-life extension. Fc fusions include fusion of peptides, proteins, or receptor exodomains to the Fc portion of an antibody. Fc and albumin fusion proteins not only increase the size and extend the half-life of peptide drugs, but also use the body's natural recycling mechanism, FcRn (neonatal Fc receptor). The pH-dependent binding of these proteins to FcRn prevents degradation of the fusion protein in endosomes. Fusion proteins fused with such proteins can have a half-life of about 3 to 16 days, which is much longer than the half-life 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 an Fc domain fusion is dulaglutide, a GLP receptor agonist. Human serum albumin is yet another fusion domain used for half-life extension, the same protein used by fatty acylated peptides. An example based on the HSA binding platform is the GLP-1 receptor agonist Albiglutide. The main difference between Fc domains and albumin is that Fc is generally used dimerized, whereas HSA is a monomeric structure, and the fusion protein can be used as a dimer or monomer depending on the choice of fusion partner. The dimeric nature of Fc fusions may or may not be preferred depending on the target, although it may show better efficacy when the antibody and / or target are closely spaced but are themselves dimeric.

[0237] Fc fusion

[0238] In one embodiment of the present invention, the fusion protein may comprise an immunoglobulin Fc domain (Fc domain) or a fragment or variant thereof, e.g., a functional Fc region. In one embodiment of the present invention, the Fc region may be an FcγR null-binding Fc region. In one embodiment of the present invention, the fusion protein may comprise at least one Stefin A protein variant that specifically binds to at least one TNFR2 linked directly or indirectly via a peptide backbone to an immunoglobulin Fc region. In one embodiment of the present invention, the fusion protein may comprise an antibody Fc region (to enhance effector functions and pharmacokinetic properties) and a TNFR2 binding agent (e.g., a Stefin A protein variant that specifically binds to TNFR2) as part of the same peptide. Alternatively, the immunoglobulin Fc region may be indirectly linked to at least one Stefin A protein variant that specifically binds to TNFR2 via a linker. Various linkers for use in the fusion proteins of the present invention are known in the art. In one embodiment of the present invention, a fusion protein containing an Fc domain may be used in a dimerized form, and may be used as a homodimer or a heterodimer.

[0239] In one embodiment of the present invention, various Fc domain sequences and their functions that can be used for Fc domain fusion are well known in the art, and a person of ordinary skill in the art can select an appropriate Fc domain according to the purpose and fuse it to the TNFR2 binding agent (e.g., Stefin A protein mutant) of the present invention.

[0240] In one embodiment of the invention, the TNFR2 binding agent (eg, a Stefin A protein variant) can be part of an immunoglobulin Fc domain (Fc domain) or a fragment or variant thereof, such as a fusion protein having a functional Fc region.

[0241] In one embodiment of the present invention, the Fc fusion homodimer comprises a dimer of a fusion protein comprising an Fc domain linked between two Stefin A protein mutants that specifically bind to TNFR2 (e.g., TNFR2 Affimer®-Fc domain-TNFR2 Affimer®).

[0242] There are several reasons for choosing the Fc region of a human antibody for use in a fusion protein. The main reason is to produce a stable protein large enough to exhibit a pharmacokinetic profile similar to that of an antibody, and to take advantage of the properties that the Fc region confers. This may include the salvage neonatal FcRn receptor pathway, which involves FcRn-mediated recycling of the fusion protein to the cell surface after intracellular entry, through which it may avoid lysosomal degradation and have an extended half-life while being released into the bloodstream. Yet another advantage is that the Fc domain binds to Protein A, simplifying downstream steps in isolation and purification of the fusion protein, allowing for the production of highly pure fusion protein.

[0243] Generally, the Fc domain can include the constant region of an antibody excluding the first constant region immunoglobulin domain. Thus, the Fc domain can include the last two constant region immunoglobulin domains of IgA, IgD, and IgG, the last three constant region immunoglobulin domains of IgE, and IgM, and a flexible hinge N-terminal to these domains. For IgA and IgM, the Fc domain can include the J chain. For IgG, the Fc domain can include immunoglobulin domains Cγ2, Cγ3, and the hinge between Cγ1 and Cγ2. Although the boundaries of the Fc domains can vary, the human IgG heavy chain Fc region is generally defined to include the carboxyl-terminal residues C226 or P230, where the amino acid numbering is according to the EU index as set forth in Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, NIH, Bethesda, Md. (1991)). In the present invention, the terms "Fc", "Fc domain" and "Fc region" may be used interchangeably, and the Fc domain may refer to the region in isolation or in the context of an antibody, fragment thereof, or fusion protein. Polymorphisms in the Fc domain have been reported at various locations and may be used as fusion domains in the fusion proteins of the present invention, without limitation.

[0244] In one embodiment of the present invention, the Fc domain may comprise a "functional Fc region". The functional Fc region refers to an Fc domain or a fragment thereof capable of binding to FcRn. The ability of the Fc region or a fragment thereof to bind to FcRn may be determined by a standard binding assay known in the art. In one embodiment of the present invention, the functional Fc region may be characterized as binding to cRn but not having an effector function. The "effector function" may 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 may be evaluated using various validation methods known in the art for evaluating the effector function.

[0245] In one embodiment of the invention, the Fc domain is derived from the IgG1 subtype, although other subclasses (e.g., IgG2, IgG3, or IgG4) may also be used. Examples of IgG1 immunoglobulin Fc domains that may be used are as follows:

[0246] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (sequence number 442).

[0247] In one embodiment of the invention, the Fc region in the fusion protein can include a hinge region. For example, the hinge region can include core hinge residues at positions 1-16 of the human IgG1 Fc domain sequence (e.g., DKTHTCPPCPAPELLG (SEQ ID NO: 536)). In one embodiment of the invention, the fusion protein can form a multimeric structure (e.g., a dimer) in part through cysteine ​​residues at positions 6 and 9 in the hinge region of the human IgG1 immunoglobulin Fc domain sequence. In one embodiment of the invention, 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 of the invention, the hinge sequence can include or consist of GSTHTCPPCPAPELLG (SEQ ID NO: 443) or EPKSCDKTHTCPPCPAPELLG (SEQ ID NO: 444).

[0248] In one embodiment of the invention, the hinge region may preferably include one or more substitutions that confer pharmacokinetic, biophysical and / or biological properties. For example, the hinge region may include or consist of the following sequence:

[0249] EPKSCDKTHTCPPCPAPELLGGPS (SEQ ID NO:445); EPKSSDKTHTCPPCPAPELLGGPS (SEQ ID NO:446); EPKSSDKTHTCPPCPAPELLGGSS(SEQ ID NO:447); EPKSSGSTHTCPPCPAPELLGGSS (SEQ ID NO: 448); DKTHTCPPCPAPELLGGPS (SEQ ID NO: 449); and DKTHTCPPCPAPELLGGSS (sequence number 450).

[0250] In one embodiment of the present invention, 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:

[0251] EPKSSDKTHTCPPCPAPELLGGSS (sequence number 451), EPKSSGSTHTCPPCPAPELLGGSS (sequence number 452), and DKTHTCPPCPAPELLGGSS (sequence number 453).

[0252] In one embodiment of the present invention, 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:

[0253] EPKSSGSTHTCPPCPAPELLGGSS (sequence number 448).

[0254] In one embodiment of the present invention, the 103rd residue 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:

[0255] EPKSSDKTHTCPPCPAPELLGGPS (SEQ ID NO: 446), EPKSSDKTHTCPPCPAPELLGGSS (SEQ ID NO: 451), and EPKSSGSTHTCPPCPAPELLGGSS (sequence number 452).

[0256] In one embodiment of the present invention, the Fc domain may be an Fc domain derived from a mammal, preferably a human Fc domain. In one embodiment of the present invention, the Fc domain may be an Fc domain derived from IgG1, IgG2, IgG3 or IgG4. In one embodiment of the present invention, 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 of the present invention, the Fc domain may have 90% or more sequence identity with a native Fc domain and / or the Fc domain of a parent polypeptide.

[0257] In one embodiment of the present invention, the Fc domain may comprise an amino acid sequence selected from SEQ ID NOs: 454 to 467, or an Fc domain of SEQ ID NOs: 454 to 467 described in the Examples. In one embodiment of the present invention, the C-terminal lysine of the Fc domain should be understood as an optional configuration for a fusion protein containing an Fc domain. In one embodiment of the present invention, the Fc domain may comprise an amino acid sequence selected from SEQ ID NOs: 454 to 467. In one embodiment of the present invention, the Fc domain may comprise an amino acid sequence selected from SEQ ID NOs: 454 to 467, with the lysine present at the C-terminus omitted.

[0258] [Table 5] TIFF2025069065000023.tif126165

[0259] As used herein, the term "antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which cytotoxic effector cells specifically bind to antigen-bearing targets via secreted immunoglobulins bound to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., NK cells, neutrophils, and macrophages), thereby exerting cytotoxicity and killing the cells.

[0260] In one embodiment of the present invention, the fusion protein may comprise an Fc domain that has no or reduced ADCC and / or complement activation or effector function. For example, the Fc domain may be characterized as comprising a naturally inactivated constant region of an IgG2 or IgG4 isotype, or a mutated IgG1 constant region. Examples of suitable modifications are described, for example, in EP0307434. By way of example, and not by way of limitation, the Fc domain may comprise substitutions of alanine residues at positions 235 and 237 (EU index numbering).

[0261] In one embodiment of the present invention, the fusion protein may include an Fc domain that partially or entirely retains Fc function. For example, the fusion protein may perform ADCC and / or CDC activity, such as when the fusion protein includes an Fc domain of human IgG1 or IgG3. The level of effector function of the Fc domain of the present invention may be adjusted by known methods. For example, the fusion protein may have a mutation in the CH2 domain, for example, one or more mutations selected from positions 239, 332, and 330 of CH2 of IgG1, for example, the mutation may be any one or more selected from S239D, I332E, and A330L. In one embodiment of the present invention, the glycosylation profile of the Fc domain may be altered to reduce fucosylation of the Fc region.

[0262] Albumin fusion

[0263] In one embodiment of the present invention, the fusion protein can include an albumin sequence or a fragment thereof in addition to at least one TNFR2 binding agent (e.g., a Stefin A protein variant). In one embodiment of the present invention, the albumin sequence or a fragment thereof can be conjugated through incorporation or chemical linkage to a polypeptide sequence that includes a TNFR2 binding agent (e.g., a Stefin A protein variant). In one embodiment of the present invention, the albumin, albumin variant, or albumin fragment can be human serum albumin (HSA), a variant thereof, or a fragment thereof. In one embodiment of the present invention, the albumin serum protein other than HSA can be characterized as being derived from, for example, cynomolgus monkey, cow, dog, rabbit, and mouse, and for example, BSA is structurally most similar to HSA (Kosa et al., (2007) J Pharm Sci. 96(11):3117-24). In one embodiment of the present invention, the albumin may be a non-human serum albumin such as cyno serum albumin or bovine serum albumin, but is not limited thereto.

[0264] Mature HAS, 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 protein has three structurally homologous domains (domains I, II, and III), exhibits mostly α-helical conformation, and is highly stabilized by 17 disulfide bonds. In one embodiment of the present invention, the fusion protein may be characterized as comprising at least one Stefin A protein variant; and mature human serum albumin (e.g., SEQ ID NO: 468) or a variant or fragment thereof. In one embodiment of the present invention, the albumin of the fusion protein may be characterized as maintaining a desired level of PK and / or biodistribution properties.

[0265] (SEQ ID NO:468)

[0266] In one embodiment of the invention, the albumin sequence may be separated from other flanking sequences of the TNFR2 binding agent (eg, a Stefin A protein variant) or fusion protein by a linker sequence.

[0267] Unless otherwise specified, in the present invention, albumin or mature albumin refers to human serum albumin (HSA). However, full-length HSA is known to include a signal peptide (MKWVTFISLLFLFSSAYS (SEQ ID NO: 483)) consisting of 18 amino acids, followed by a prodomain sequence (RGVFRR (SEQ ID NO: 469)) consisting of 6 amino acids. The total of 24 amino acids may be named a preprodomain. In one embodiment of the present invention, a fusion protein containing HSA may be characterized as being expressed and secreted by the HSA preprodomain. In one embodiment of the present invention, a fusion protein containing HSA may include the above-mentioned signal sequence, and may be characterized as being expressed and secreted through the signal sequence.

[0268] In one embodiment of the invention, the serum albumin may be covalently coupled to the Stefin A protein variant or a fusion protein comprising the same by a bond other than an amide bond, such as by chemical conjugation through an amino acid side chain, rather than being provided as part of the Stefin A protein variant or a fusion protein comprising the same, for example by cross-linking through a chemical bond between an albumin polypeptide and an amino acid side chain of a polypeptide comprising the Stefin A protein variant.

[0269] In one embodiment of the present invention, a chemical modification applicable to generate TNFR2 binding agents (e.g., stefin A protein variants) or fusion proteins to increase protein half-life is lipidation, which involves covalently attaching fatty acids to peptide side chains. It was developed as a method to extend the half-life of insulin and shares the same half-life extension mechanism as PEGylation. Specifically, it increases the hydrodynamic radius to reduce renal filtration. However, the lipid moiety itself is relatively small, and its effect is indirectly mediated through the non-covalent binding of the lipid moiety to circulating albumin. One consequence of lipidation is a decrease in the water solubility of the peptide, but this can be regulated by engineering the linker between the peptide and the fatty acid. For example, glutamic acid or mini-PEG in the linker can be used. Linker engineering and modification using lipid moieties can affect self-aggregation, delay biodistribution independent of albumin, and increase half-life (Jonassen et al. (2012) Pharm Res. 29(8):2104-14.).

[0270] In one embodiment of the present invention, the albumin-binding domain may be albumin-binding (PKE2) adnectins (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 of Streptococcus G148, albumin-binding domain antibody (e.g., GSK2374697, AlbudAb), or albumin-binding nanobody (e.g., ATN103, Ozolralizumab).

[0271] Affimer® XT

[0272] In one embodiment of the present invention, the serum protein binding molecule such as HSA may include a stefin A protein variant that specifically binds to HSA. Examples of stefin A protein variants that specifically bind to HSA are described in WO2022 / 023540, etc. In some embodiments, the stefin A protein variant that specifically binds to HSA may be included in a fusion protein to extend the half-life of the fusion protein. It has been confirmed in an in vivo pharmacokinetic (PK) study that the stefin A protein variant that specifically binds to HSA can extend the serum half-life of other stefin A protein variants that are monogenically fused to the stefin A protein variant (e.g., produced in E. coli) in a controlled manner. In addition, Affimer® XT polypeptide may be used to extend the half-life of a protein therapeutic agent such as a TNFR2 binding agent (e.g., a stefin A protein variant that specifically binds to TNFR2) or a fusion protein of the present invention.

[0273] In one embodiment of the present invention, the Stefin A protein variant that binds to HSA can increase the serum half-life of a protein molecule, for example, a therapeutic protein, such as a TNFR2 binding agent of the present invention (e.g., a Stefin A protein variant that specifically binds to TNFR2) or a fusion protein containing the same, in vivo. For example, the fusion protein containing the Stefin A protein variant that binds to HSA can be characterized as having at least a two-fold increase in serum half-life compared to a molecule to which the Stefin A protein variant that binds to HSA is not linked. In one embodiment of the present invention, the Stefin A protein variant that binds to HSA can be characterized as having at least a three-fold, at least a four-fold, at least a five-fold, at least a six-fold, at least a seven-fold, at least a eight-fold, at least a nine-fold, at least a ten-fold, at least a twenty-fold, or at least a thirty-fold increase in serum half-life compared to a molecule to which the Stefin A protein variant that binds to HSA is not linked. In one embodiment of the present invention, the HSA-binding Stefin A protein variant may be characterized by increasing serum half-life by 2-5 times, 2-10 times, 3-5 times, 3-10 times, 15-5 times, 4-10 times, or 5-10 times, compared to a molecule to which the HSA-binding Stefin A protein variant is not linked. In one embodiment of the present invention, the HSA-binding Stefin A protein variant may increase serum half-life 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 one week, after in vivo administration, compared to a molecule to which the HSA-binding Stefin A protein variant is not linked.

[0274] In one embodiment of the present invention, at least one of the solvent accessible loops of the HSA-binding Stefin A protein variant (HSAAffimer®) can be derived from a wild-type Stefin A protein, allowing it to bind to HSA. In one embodiment of the present invention, the HSA-binding Stefin A protein variant can bind to HSA with a Kd of 10-6 M or less.

[0275] In one embodiment of the present invention, the HSA-binding Stefin A protein mutant may be characterized as being a mutant derived from a wild-type human Stefin A protein having a backbone sequence, and in which any one or more of loop 2 and loop 4 are replaced with alternative loop sequences, (Xaa)n and (Xaa)m.

[0276] In one embodiment of the present invention, the Stefin A protein variant may be characterized by having an amino acid sequence represented by Formula I:

[0277] FR1-(Xaa)n-FR2-(Xaa)m-FR3(I)

[0278] wherein the FR1 is an amino acid sequence having at least 70%, 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: 470); The 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; The FR3 is an amino acid sequence represented by EDLVLTGYQV DKNKDDELTG F (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, at each occurrence, is individually any amino acid residue; and n and m are each independently an integer from 3 to 20. Additional variations of such structures are described in WO2022 / 023540.

[0279] In all embodiments, each amino acid of (Xaa)n may be the same or selected from any amino acid, and this also applies equally to (Xaa)m.

[0280] In one embodiment of the present invention, the fusion protein can comprise a Stefin A protein variant that specifically binds to HSA, comprising an amino acid sequence selected from SEQ ID NOs: 471-477 (Table 6). In one embodiment of the present invention, the Stefin A protein variant that specifically binds to TNFR2 has an extended serum half-life and can comprise an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95% or 98% identical to an amino acid sequence selected from SEQ ID NOs: 471-477 (Table 6). Stefin A protein variants that specifically bind to HSA for use in the present invention are described in WO2022 / 023540.

[0281] [Table 6]

[0282] Host Cells and Genetically Modified Cells

[0283] In one embodiment of the invention, a nucleic acid encoding the TNFR2 binding agent (eg, a Stefin A protein variant that specifically binds to TNFR2) and / or a fusion protein comprising the same is introduced into a host cell.

[0284] In the present invention, the term "host cell" refers to a cell prior to introduction of a nucleic acid encoding a TNFR2 binding agent of the present invention (e.g., a Stefin A protein mutant that specifically binds to TNFR2) and / or a fusion protein containing the same.

[0285] As used herein, the term "genetically modified cell" refers to a cell that has been engineered to contain an exogenous nucleic acid. A genetically modified cell includes a cell that contains an exogenous nucleic acid, regardless of whether the exogenous nucleic acid has been integrated into the genome of the cell. In one embodiment of the invention, the genetically modified cell is modified to express a TNFR2 binding agent (e.g., a Stefin A protein variant) and / or a fusion protein. In one embodiment of the invention, the genetically modified cell can be produced by introducing a nucleic acid encoding a TNFR2 binding agent (e.g., a Stefin A protein variant) and / or a fusion protein of the invention.

[0286] In one embodiment of the present invention, the genetically modified cells are capable of expressing a TNFR2 binding agent, preferably, the TNFR2 binding agent is membrane anchored or displayed on the cell surface.

[0287] In one embodiment of the present invention, the genetically modified cells can express TNFR2 binding agent molecules at an average level of about 2,500 or more, about 5,000 or more, about 10,000 or more, about 20,000 or more, about 30,000 or more, or about 40,000 or more molecules per cell.

[0288] In one embodiment of the present invention, the genetically modified cells may contain additional genetic modifications in addition to the introduction of a nucleic acid encoding a TNFR2 binding agent of the present invention (e.g., a Stefin A protein variant that specifically binds to TNFR2) and / or a fusion protein containing the same.

[0289] In one embodiment of the invention, the host cell is genetically engineered to contain a nucleic acid encoding a TNFR2 binding agent (e.g., a Stefin A protein variant that specifically binds to TNFR2) and / or a fusion protein comprising the same, and is capable of expressing the TNFR2 binding agent and / or a fusion protein comprising the same. In one embodiment of the invention, the host cell for use in producing the genetically engineered cell of the invention may be characterized as not expressing TNFR2.

[0290] In general, the recombinant cell may be any type of cell, including eukaryotic cells and prokaryotic cells. Examples of host cells that may be used include, but are not limited to, bacterial cells such as Escherichia coli, Streptomyces, Salmonella typhimurium, etc.; yeast cells; fungal cells such as Pichia pastoris, etc.; insect cells such as Drosophila melanogaster and Fallopsis amplexicaule Sf9 cells; animal cells such as CHO, COS, NSO, 293, Bowes melanoma cells, etc.; or plant cells.

[0291] In one embodiment of the present invention, the host cells may be selected from the group consisting of stem cells, immune cells, and somatic cells.

[0292] In one embodiment of the present invention, the host cell may be a naturally derived cell, for example, a cell derived from an animal, preferably a mammal, more preferably a human; or a cell engineered through cell engineering or genetic engineering methods.

[0293] In the present invention, the term "stem cell" refers to a cell that can differentiate into various cells that constitute biological tissues, and collectively refers to each undifferentiated cell in the stage before differentiation obtained from each tissue of an embryo, fetus, and living organism. Stem cells are characterized by having the property of proliferation (expansion) since they can produce identical cells by cell division (self-renewal) unlike cells that undergo differentiation into specific cells in response to differentiation stimuli (environment) and stop cell division after differentiation is completed, and by having plasticity in differentiation since they can differentiate into other cells in response to different environments or different differentiation stimuli.

[0294] In one embodiment of the present invention, the stem cells include, but are not limited to, pluripotent stem cells, multipotent stem cells, and unipotent stem cells according to their differentiation potential.

[0295] In one embodiment of the present invention, the pluripotent stem 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) or induced pluripotent stem cells (iPS).

[0296] In some embodiments, the present invention provides transgenic pan-potent stem cells expressing a TNFR2 binding agent and / or a fusion protein. In some embodiments, the transgenic PS cells may be iPS cells. In some embodiments, the transgenic PS cells are embryonic stem cells.

[0297] In one embodiment of the invention, a genetically modified PS cell expressing a TNFR2 binding agent (e.g., a Stefin A protein mutant) and / or a fusion protein is produced by introducing a nucleic acid encoding the TNFR2 binding agent and / or a fusion protein containing the same into a PS cell (e.g., an iPS cell or an Es cell). In one embodiment of the invention, the genetically modified PS cell may not express TNFR2.

[0298] In one embodiment of the present invention, the multipotent stem cell refers to a cell that has the potential to differentiate from a progenitor cell into a cell belonging to a specific type family. For example, the multipotent stem cell may be, but is not limited to, hematopoietic stem cells, mesenchymal stem cells, and neural stem cells.

[0299] In one embodiment of the present invention, the stem cells may be mesenchymal stem cells. In the present invention, the term "mesenchymal stem cells (MSCs)" refers to cells that can differentiate into osteoblasts, adipocytes, chondrocytes, etc., which can be differentiated from the mesoderm, one of the three germ layers of embryonic tissue. The mesenchymal stem cells can 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 capable of allotransplantation and xenotransplantation because they have the immunoregulatory ability to 1) inhibit the activity and proliferation of T lymphocytes and B lymphocytes, 2) inhibit the activity of natural killer cells (NK cells), and 3) regulate the functions of dendritic cells and macrophages. In the present invention, the mesenchymal stromal cells may be used interchangeably with the term "mesenchymal stromal cells" in substantially the same sense.

[0300] In one embodiment of the present invention, genetically modified mesenchymal stem cells (MSCs) are provided that contain a nucleic acid encoding a TNFR2 binding agent. In one embodiment of the present invention, genetically modified MSCs expressing a TNFR2 binding agent are provided, where the MSCs can be derived from induced pluripotent stem cells. In one embodiment of the present invention, genetically modified MSCs expressing a TNFR2 binding agent are provided, where the MSCs can have pluripotency to differentiate into cells selected from the group consisting of adipocytes, osteocytes, chondrocytes, muscle cells, nerve cells, and cardiomyocytes. In one embodiment of the present invention, genetically modified MSCs expressing a TNFR2 binding agent are provided, where the MSCs can be stored for a long period of time and / or subcultured. In one embodiment of the present invention, genetically modified MSCs expressing a TNFR2 binding agent are provided, where the cell surface marker expression of the MSCs can be maintained at 90% or more in mesenchymal stem cells for 20 or more passages. In one embodiment of the invention, genetically modified mesenchymal stem cells expressing a TNFR2 binding agent (e.g., a Stefin A protein variant) or a fusion protein comprising the same can be generated by introducing a nucleic acid encoding the TNFR2 binding agent and / or the fusion protein into a mesenchymal stem cell. In one embodiment of the invention, a population of MSCs is provided, at least 90% of which can be genetically modified to express the TNFR2 binding agent and / or the fusion protein comprising the same. In one embodiment of the invention, at least 95% of the population of MSCs expresses the TNFR2 binding agent and / or the fusion protein comprising the same.

[0301] In one embodiment of the present invention, the genetically modified mesenchymal stem cells may be characterized in that they do not express TNFR2, preferably endogenous TNFR2.

[0302] In one embodiment of the present invention, a TNFR2 binding agent (e.g., a Stefin A protein variant that specifically binds to TNFR2) exhibits an agonistic effect on TNFR2, and thus exhibits an immune suppressing effect such as suppressing T cell and / or B cell activity. In one embodiment of the present invention, a nucleic acid encoding a TNFR2 binding agent (e.g., a Stefin A protein variant that specifically binds to TNFR2) is introduced into a host cell, a mesenchymal stem cell, and it has been confirmed that a novel immune suppressing activity is obtained through the TNFR2 agonistic effect of the TNFR2 binding agent while maintaining the immune suppressing effect (e.g., T cell activity suppressing effect) of the mesenchymal stem cell, and thus immune responses can be suppressed significantly and effectively. It can be obviously understood that such an immune suppressing effect also exhibits an excellent therapeutic effect on inflammatory diseases or autoimmune diseases.

[0303] In one embodiment of the present invention, the mesenchymal stem cells may be characterized as being derived from omnipotent (pluripotent) stem cells. In one embodiment of the present invention, the mesenchymal stem cells may be characterized as being capable of long-term subculture. The method for producing mesenchymal stem cells from pluripotent stem cells and the method for long-term subculture are well known in the art. For example, Korean Patent Publication No. 10-2021-0072734 and Korean Patent Registration No. 10-1135636 disclose in detail a method for maintaining undifferentiated potential and marker expression characteristics even after several dozen or more subcultures.

[0304] In one embodiment of the present invention, the mesenchymal stem cells may be characterized by expressing one or more of CD29, CD44, CD73, CD90, and CD105. In one embodiment of the present invention, the mesenchymal stem cells may be characterized by not expressing one or more of cell surface markers of CD14, CD19, CD34, CD45, HLA-DR, SSEA-3, TRA-1-60, TRA-1-81, Nanog, and Oct3 / 4.

[0305] In one embodiment of the present invention, the mesenchymal stem cells may be characterized by expressing one or more cell surface markers among CD29, CD44, CD73, CD90, and CD 105. In one embodiment of the present invention, 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 express CD90.

[0306] In one embodiment of the present invention, the mesenchymal stem cells may be characterized by not expressing any one or more cell surface markers including CD11b, CD14, CD34, CD45, CD79, HLA-DR, TRA-1-60, and TRA-1-81.

[0307] In one embodiment of the invention, less than 1% of the mesenchymal stem cells in the population may not express at least one, at least two, at least three, or at least four cell surface markers selected from the group consisting of CD14, CD19, CD34, CD45, HLA-DR, SSEA-3, TRA-1-60, TRA-1-81, Nanog, and Oct3 / 4.

[0308] In one embodiment of the present invention, the mesenchymal stem cells may not express at least one cell surface marker selected from CD34, CD45, HLA-DR, TRA-1-60, and TRA-1-81.

[0309] In one embodiment of the present invention, the host cell may be an immune cell.

[0310] In the present invention, the term "immune cells" refers to all types of cells that constitute the immune system. Cell therapy agents using the immunoregulatory ability of immune cells are used to treat various diseases such as cancer and autoimmune diseases, and engineered immune cell therapy agents that enable target-specific immunoregulation such as chimeric antigen receptors through non-specific effects have attracted much attention.

[0311] The present invention provides the insight that expression of a TNFR2 binding agent (e.g., a Stefin A protein variant that specifically binds to TNFR2), in particular expression of a TNFR2 binding agent (e.g., a Stefin A protein variant) that has an agonistic effect on TNFR2, can improve the immunomodulatory effect. In one embodiment of the present invention, a TNFR2 binding agent (e.g., a Stefin A protein variant that specifically binds to TNFR2) can be introduced into a host cell (e.g., MSC) to improve the immunomodulatory effect.

[0312] In one embodiment of the present invention, the immune cells may be characterized as being selected from the group consisting of T cells, B cells, natural killer (NK) cells, NKT cells, monocytes, macrophages, regulatory T cells, and antigen-presenting cells (e.g., dendritic cells), but are not limited thereto.

[0313] In one embodiment of the present invention, the immune cells may be cells isolated from the human body, blood or peripheral blood mononuclear cells (PBMCs), or may be differentiated from stem cells (preferably, totipotent stem cells), but are not limited thereto.

[0314] In one embodiment of the present invention, when a nucleic acid encoding a fusion protein (e.g., a chimeric antigen receptor) containing a TNFR2-binding agent (e.g., a Stefin A protein mutant that specifically binds to TNFR2) as an extracellular binding domain is introduced into the immune cells, it can be used as a cell therapy agent such as CAR-T, CAR-NK, etc.

[0315] In one embodiment of the present invention, the host cell may be a somatic cell.

[0316] In the present invention, the term "somatic cells" refers to general cells, excluding reproductive 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 membranes can be used for the purpose of skin burns, scars, and cosmetics, and chondrocytes, adipocytes, pancreatic islet cells, and skeletal hair cells can be used to treat diseases such as degenerative arthritis and subcutaneous fat deficiency, but are not limited thereto.

[0317] When the host cell of the present invention is used as a somatic cell and a gene encoding the TNFR2 binding agent (e.g., a Stefin A protein mutant that specifically binds to TNFR2) and / or a fusion protein containing the same is introduced, an immunosuppressive effect can be exhibited through regulating the activity of TNFR2.

[0318] In one embodiment of the present invention, the genetically modified cells can secrete, express in the cell membrane, and / or localize to a specific site within the cell the TNFR2 binding agent (e.g., a Stefin A protein mutant that specifically binds to TNFR2) and / or a fusion protein containing the same.

[0319] In one embodiment of the present invention, most preferably, the genetically modified cell is capable of expressing the TNFR2 binding agent (e.g., a Stefin A protein mutant that specifically binds to TNFR2) and / or a fusion protein containing the same and displaying it on the cell surface (or cell membrane).

[0320] Method for introducing nucleic acid into host cells

[0321] In the present invention, the term "introduction" means enabling a host cell to accommodate a foreign gene (nucleic acid) that the host cell does not have.

[0322] In one embodiment of the present invention, a nucleic acid encoding the TNFR2 binding agent (e.g., a Stefin A protein mutant that specifically binds to TNFR2) or a fusion protein containing the same can be introduced into a host cell via various methods known in the art.

[0323] In one embodiment of the present invention, for example, a nucleic acid encoding the TNFR2 binding agent or a fusion protein comprising the same may be introduced into a host cell using a gene editing system. In one embodiment of the present invention, a nucleic acid encoding the TNFR2 binding agent or a fusion protein comprising the same may be introduced into a host cell through gene editing and integrated into an endogenous gene of the host cell.

[0324] The gene editing may use any gene editing system currently known in the art or that may be developed later, and preferably, the gene editing system may introduce the nucleic acid of the present invention in a site-specific or sequence-specific manner. For example, the gene editing system may include, but is not limited to, a CRISPR / Cas system meganuclease editing system, a zinc finger nuclease (ZFN) editing system, a transcription activator-like effector nuclease (TALEN) editing system, a base editing system, and a prime editing system.

[0325] In one embodiment of the invention, for example, a nucleic acid encoding the TNFR2 binding agent or a fusion protein containing the same can be introduced into a host cell via a vehicle (or construct) such as a vector. In the present invention, the term "vector" 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)), poxvirus (GCE, NJL, Krupa M, Esteban M., Curr Gene Ther 8(2):97-120(2008)), reovirus, measles virus, Semliki Forest virus and poliovirus derived vectors; non-viral vectors such as plasmid vectors (Sambrook et al., 1989) and minicircles (Yew et al. 2000 Mol Ther 1(3),255-62).

[0326] The vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more antibiotic resistance marker genes, an enhancer element, a promoter, and a transcription termination sequence. A nucleic acid encoding a TNFR2 binding agent (Stephen A protein variant) of the present invention or a fusion protein containing the same can be operably linked to such a promoter and transcription termination sequence.

[0327] "Operably linked" refers to a functional connection between a nucleic acid expression control sequence (e.g., a promoter, a 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.

[0328] When a prokaryotic cell is used as the host, it generally contains a strong promoter capable of 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 the initiation of translation, and a transcription / translation termination sequence. Furthermore, for example, when a eukaryotic cell is used as the host, a promoter derived from the genome of a mammalian cell (e.g., metallothionine promoter, β-actin promoter, human hemoglobin promoter, and human muscle creatine promoter) or a promoter derived from a mammalian virus (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) may be used, which generally has a polyadenylation sequence as a transcription termination sequence.

[0329] In one embodiment of the present invention, the promoter may be a eukaryotic promoter, preferably selected from the group consisting of CMV A promoter, CMV B promoter, PGK promoter, EF1α promoter, EFS promoter, CBh promoter, MSCV promoter, SFFV promoter, and UbC promoter, and most preferably selected from the group consisting of the cytomegalovirus (CMV) promoter, EF1α promoter, and CBh promoter, but is not limited thereto.

[0330] In one embodiment of the present invention, the promoter may further include an enhancer sequence, but is not limited thereto.

[0331] In some cases, the vector may be fused to other sequences to facilitate purification of the antibodies expressed therefrom, such as glutathione S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA), FLAG (IBI, USA) and 6x His (hexahistidine; Quiagen, USA).

[0332] The vector may contain an antibiotic resistance gene commonly used in the art as a selective marker, such as, but not limited to, resistance genes to ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin, and tetracycline.

[0333] In one embodiment of the present invention, the nucleic acid encoding the TNFR2 binding agent (e.g., a Stefin A protein mutant that specifically binds to TNFR2) or a fusion protein containing the same may be introduced by mixing into the genes of a host cell.

[0334] In one embodiment of the present invention, the nucleic acid encoding the TNFR2 binding agent (e.g., a Stefin A protein variant that specifically binds to TNFR2) or a fusion protein containing the same can be prepared by chemical synthesis using an oligonucleotide synthesizer. The oligonucleotide can be designed by selecting the amino acid sequence of the desired polypeptide and the codons that are preferred in the host cell in which the recombinant polypeptide of interest is to be produced. Standard methods can be applied to synthesize polynucleotide sequences encoding isolated polypeptides of interest. For example, the complete amino acid sequence can be used to construct a reverse-translated gene. Also, DNA oligomers can be synthesized that contain nucleoside sequences that encode a particular isolated polypeptide. For example, multiple small oligonucleotides encoding portions of the desired polypeptide can be synthesized and then ligated. Individual oligonucleotides generally include 5' or 3' overhangs for complementary assembly.

[0335] In one embodiment of the present invention, once a nucleic acid sequence encoding the TNFR2 binding agent (e.g., a Stefin A protein variant that specifically binds to TNFR2) or a fusion protein containing the same is obtained, a vector containing the same can be produced by recombinant DNA technology using techniques well known in the art. Using methods well known to those skilled in the art, an expression vector containing a sequence encoding the TNFR2 binding agent of the present invention (a Stefin A protein variant or a fusion protein containing the same) and appropriate transcriptional and translational control signals can be constructed. Such methods include, for example, in vitro recombinant DNA technology, synthetic technology, and in vivo genetic recombination (e.g., Sambrook et al, 1990, MOLECULAR CLONING, A LABORATORY MANUAL, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, and Ausubel et al. eds., 1998, CURRENT PROTOCOLS IN Molecular Biology, John Wiley & Sons, NY).

[0336] In one embodiment of the present invention, a nucleic acid encoding the TNFR2 binding agent (e.g., a Stefin A protein mutant that specifically binds to TNFR2) or a fusion protein containing the same, or a non-viral expression vector containing the same, can be delivered to a host cell by conventional techniques (e.g., electroporation, liposome transfection, and calcium phosphate precipitation).

[0337] The vector can be introduced into a host cell by a method such as transformation or transfection. As used herein, the term "transformation" refers to introducing DNA into a host cell so that the DNA is replicable as an extrachromosomal element or by chromosomal integration. As used herein, the term "transfection" refers to the host cell receiving an expression vector, regardless of whether any coding sequence is 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, or lipofection.

[0338] Of course, it should be understood that not all vectors and expression control sequences function equally well to express the DNA sequence of the present invention. Similarly, not all hosts function equally well with the same expression system. However, one skilled in the art can make an appropriate selection from a number of vectors, expression control sequences, and hosts without undue experimental burden and without departing from the scope of the present invention. For example, the host must be considered when selecting a vector, since the vector must replicate therein. The number of copies of the vector, the ability to regulate the number of copies, and the expression of other proteins encoded by the vector, such as antibiotic markers, must also be considered. Various factors must also be considered when selecting an expression control sequence, such as the relative strength of the sequence, its regulatability, and its compatibility with the DNA sequence of the present invention, particularly in relation to possible secondary structures. A unicellular host must be selected taking into consideration factors such as the toxicity of the product encoded by the DNA sequence of the present invention, secretion characteristics, ability to correctly fold the protein, culture and fermentation requirements, and ease of purifying the product encoded by the DNA sequence of the present invention from the host. Within these ranges, one skilled in the art can select various combinations of vectors / expression regulatory sequences / hosts that can ferment or express the DNA sequences of the present invention in large-scale animal culture. When attempting to clone cDNA by expression cloning, the binding method, panning method, film emulsion method, etc. can be applied as a screening method.

[0339] In one embodiment of the invention, a lentivirus is used to introduce into a host cell a nucleic acid encoding a TNFR2 binding agent (eg, a Stefin A protein mutant that specifically binds to TNFR2) or a fusion protein comprising the same.

[0340] In particular, when gene is introduced using lentivirus as in one embodiment of the present invention, a transduction enhancer can be used.

[0341] In one embodiment of the present invention, the transformation enhancer may be selected from, for example, polyberene, protamine sulfate, and Sirion's Lentiboost, and is most preferably, but is not limited to, polyberene.

[0342] In one embodiment of the present invention, the method may be characterized in that the transformation enhancing agent and the lentivirus are simultaneously mixed and infected into attached or non-attached host cells, and preferably, the infection is performed before the attachment of the cells, but is not limited thereto.

[0343] In yet another aspect, the invention relates to a recombinant cell expressing a TNFR2 binding agent (eg, a Stefin A protein mutant that specifically binds to TNFR2) or a fusion protein comprising the same.

[0344] Method for producing recombinant cells and culture medium for recombinant cells

[0345] In another aspect, the present invention provides a method for producing a method for manufacturing a semiconductor device comprising the steps of: (a) introducing into a host cell a nucleic acid encoding a TNFR2 binding agent (e.g., a Stefin A protein variant that specifically binds to TNFR2) and / or a fusion protein comprising the same; and (b) selecting and harvesting a host cell into which a nucleic acid encoding the TNFR2-binding agent (e.g., a Stefin A protein variant that specifically binds to TNFR2) and / or a fusion protein containing the same has been introduced; The present invention relates to methods for producing recombinant cells into which nucleic acids encoding TNFR2 binding agents and / or fusion proteins containing the same have been introduced.

[0346] In one embodiment of the present invention, step (a) may be carried out through various means known in the art.

[0347] In one embodiment of the present invention, step (a) may be carried out using a lentivirus containing nucleic acid encoding a TNFR2 binding agent (e.g., a Stefin A protein mutant that specifically binds to TNFR2) and / or a fusion protein containing the same.

[0348] In one embodiment of the present invention, the lentivirus may be characterized in that a vector containing a nucleic acid encoding a TNFR2 binding agent (e.g., a Stefin A protein mutant that specifically binds to TNFR2) and / or a fusion protein containing the same is introduced.

[0349] In one embodiment of the present invention, 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.

[0350] In one embodiment of the present invention, the promoter may be a eukaryotic promoter, preferably selected from the group consisting of CMV A promoter, CMV B promoter, PGK promoter, EF1α promoter, EFS promoter, CBh promoter, MSCV promoter, SFFV promoter, and UbC promoter, and most preferably selected from the group consisting of the cytomegalovirus (CMV) promoter, EF1α promoter, and CBh promoter, but is not limited thereto.

[0351] In one embodiment of the present invention, the promoter may further include an enhancer sequence, but is not limited thereto.

[0352] In one embodiment of the present invention, the enhancer is a short DNA region of about 50 bp to 1500 bp in length that can bind to a transcriptional regulatory protein. The enhancer may be located upstream or downstream of the transcription initiation site or promoter. Enhancers for various promoters are well known in the art, and those skilled in the art can select and apply them without restrictions.

[0353] In one embodiment of the present invention, step (a) may include infecting a host cell with the lentivirus to transform the host cell.

[0354] In one embodiment of the present invention, the step of infecting a host cell with the lentivirus and transforming the host cell may be performed with the addition of a transformation enhancer.

[0355] In one embodiment of the present invention, the "transformation enhancer" is preferably a cationic polymer, which facilitates the incorporation of negatively charged nucleic acids or genes into host cells.

[0356] In one embodiment of the present invention, the transformation enhancer may be characterized as a cationic polymer, for example, selected from polyberene, protamine sulfate, and Sirion's Lentiboost, and most preferably, polyberene, but is not limited thereto.

[0357] In one embodiment of the present invention, the step of infecting a host cell with the lentivirus and transforming the host cell may be characterized in that the lentivirus is treated after attachment of the host cell, or the lentivirus is treated and infected into the host cell before attachment of the host cell.

[0358] In one embodiment of the present invention, the method of treating the host cells with a lentivirus prior to attachment and infecting the host cells during the attachment process is also known as reverse transduction.

[0359] In one embodiment of the present invention, the step of infecting a host cell with the lentivirus and transforming the host cell is preferably performed by treating and infecting the host cell with the lentivirus prior to attachment of the host cell, but is not limited thereto.

[0360] In one embodiment of the present invention, step (b) may be characterized by using an antibiotic and its resistance gene to select a host cell into which a nucleic acid encoding the TNFR2 binding agent (e.g., a Stefin A protein mutant that specifically binds to TNFR2) and / or a fusion protein containing the same has been introduced.

[0361] Methods for selecting transformed cells into which an antibiotic and its resistance gene have been introduced using a vector are well known in the art.

[0362] In one embodiment of the present invention, step (b) may be characterized in that the recombinant cell into which the nucleic acid has been introduced is treated with an aminoglycoside-based antibiotic and then selected.

[0363] In one embodiment of the present invention, examples of the antibiotic include, but are not limited to, ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, puromycin, blasticidin, hygromycin, geneticin, neomycin, and tetracycline.

[0364] In one embodiment of the present invention, the genetically modified cell may further contain a neomycin resistance gene in addition to a nucleic acid encoding a TNFR2 binding agent (e.g., a Stefin A protein mutant that specifically binds to TNFR2) and / or a fusion protein containing the same.

[0365] In one embodiment of the present invention, the neomycin 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.

[0366] In another aspect, the present invention relates to a population or culture medium of the genetically modified cells. In one embodiment of the present invention, the culture medium may be prepared by culturing the genetically modified cells using appropriate conditions and medium depending on the type of host cell.

[0367] In one embodiment of the present invention, the genetically modified cells may express and secrete a TNFR2 binding agent (e.g., a Stefin A protein variant that specifically binds to TNFR2) and / or a fusion protein containing the same. In this case, the culture medium of the genetically modified cells may contain the TNFR2 binding agent (e.g., a Stefin A protein variant that specifically binds to TNFR2) and / or a fusion protein containing the same secreted together with the genetically modified cells.

[0368] In one embodiment of the present invention, when the host cells are mesenchymal stem cells, methods for long-term subculture thereof are well known in the art. For example, Korean Patent Publication No. 10-2021-0072734 and Korean Patent Registration No. 10-1135636 disclose in detail a method for maintaining undifferentiated potential and marker expression characteristics even after several dozens of subcultures.

[0369] In one embodiment of the invention, the population of genetically modified cells can express TNFR2 binding proteins on the cell surface at an average level of about 2,500 to 250,000, about 10,000 to 200,000, about 20,000 to 150,000, or about 30,000 to 100,000 molecules per cell.

[0370] Use - Cellular therapy agent or pharmaceutical composition

[0371] In one embodiment of the present invention, a nucleic acid encoding a TNFR2-binding agent (e.g., a Stefin A protein mutant that specifically binds to TNFR2) and / or a fusion protein containing the same was introduced into a PSC-derived mesenchymal stem cell to produce a genetically modified cell. The TNFR2-binding agent (e.g., a Stefin A protein mutant) expressed by the genetically modified cell is fused to the transmembrane domain of human PDGFR and presented on the cell surface. In the above embodiment, it was confirmed that the genetically modified cell (e.g., MSC) expressed and presented a high level of the TNFR2-binding agent (e.g., a Stefin A protein mutant) on its surface while maintaining the existing properties maintained by the host cell (e.g., expression of MSC surface markers).

[0372] In one embodiment of the invention, a composition is provided that includes genetically modified cells (eg, MSCs) that contain or express a TNFR2 binding agent.

[0373] In one embodiment of the invention, a pharmaceutical composition is provided that includes genetically modified cells (eg, MSCs) that contain or express a TNFR2 binding agent.

[0374] In one embodiment of the invention, genetically modified cells containing or expressing the TNFR2 binding agent can retain the properties (eg, surface marker expression) of the host cell.

[0375] In addition, the present invention evaluated TNFR2 agonist activity through co-culture of genetically modified cells expressing a TNFR2 binding agent with HEK-Blue-TNF-alpha cells, and confirmed that a TNFR2 binding agent (e.g., a stefin A protein mutant) expressed and surface-displayed in genetically modified cells (e.g., MSCs) binds to TNFR2 in HEK-Blue-TNF-alpha cells and exhibits excellent agonist effects.

[0376] Various studies have reported the importance of TNFR2 activation in Treg proliferation and function. TNFR2-deficient mice have reduced numbers of thymic and peripheral Tregs (2013_Chen X,et al.PMID:23277487), and TNFR2- / - Tregs fail to regulate inflammatory responses in vivo (2008_Van Mierlo GJ,et al.PMID:18292492). In humans, Tregs were found to express higher levels of TNFR2 than T effector cells (2013_Okubo Y,et al.PMID:24193319 & 2002_Annunziato F,et al.PMID:12163566). TNFR2+ Tregs were the most potent suppressors of proliferation and cytokine production of co-cultured T-responder cells (2010_Chen X,et al.PMID:20127680).

[0377] Therefore, the genetically modified cells of the present invention can induce the proliferation and activation of Tregs, and the agonistic activity of the genetically modified cells of the present invention against TNFR2 can reduce immune responses or suppress the immune system.

[0378] In conclusion, the recombinant cells of the present invention can be used to prevent and / or treat various immune diseases caused by abnormal immune homeostasis, preferably excessive or abnormal reactions of the immune system. More specifically, the recombinant cells of the present invention can be used to prevent and / or treat various diseases that have been reported to be treatable using the proliferation and activation of Tregs as a mechanism.

[0379] In another aspect, the present invention relates to a cell therapeutic agent comprising the genetically modified cells and / or the conditioned cell culture medium thereof.

[0380] In yet another aspect, the present invention relates to the use of genetically modified cells and / or regulated cell culture medium thereof for producing a cell therapeutic agent.

[0381] In yet another aspect, the present invention relates to a pharmaceutical composition for preventing and / or treating immune diseases or cancer, comprising the genetically modified cell of the present invention and / or a regulated cell culture medium thereof.

[0382] In yet another aspect, the present invention relates to the use of the genetically modified cells and / or the conditioned cell culture medium thereof for the prevention and / or treatment of immune diseases or cancer.

[0383] In yet another aspect, the present invention relates to the use of genetically modified cells and / or regulated cell culture medium thereof for producing a pharmaceutical composition for the prevention and / or treatment of immune diseases or cancer.

[0384] In still another aspect, the present invention relates to a method for preventing and / or treating an immune disease or cancer, comprising the step of administering genetically modified cells and / or a regulated cell culture medium thereof to a subject.

[0385] Immune diseases for which the recombinant cells of the present invention can be used to provide preventive and / or therapeutic effects include, but are not limited to, inflammatory diseases, autoimmune diseases, and cancers.

[0386] In the present invention, the term "immune disease" refers to a disease that can occur directly or indirectly due to an abnormality in the immune system, and examples thereof include, but are not limited to, dermatitis, allergies, rhinitis, gout, ankylosing spondylitis, rheumatic fever, lupus, fibromyalgia, tendonitis, type 1 diabetes, scleroderma, neurodegenerative disease, type 2 diabetes, silicosis, atherosclerosis, vitiligo, conjunctivitis, and autoimmune disease.

[0387] In the present invention, the term "autoimmune disease" refers to a disease that occurs when an organism's immune cells recognize and attack the organism's own tissues or cells as antigens rather than foreign invaders. Examples of the autoimmune disease include rheumatoid arthritis, systemic scleroderma, atopic dermatitis, psoriasis, asthma, Guillain-Barre syndrome, myasthenia gravis, dermatomyositis, polymyositis, multiple sclerosis, autoimmune encephalomyelitis, polyarteritis nodosa, temporal arteritis, childhood diabetes, alopecia areata, blisters, aphthous stomatitis, Crohn's disease, and Behcet's disease. Diseases) but are not limited to these.

[0388] In the present invention, the term "inflammatory disease" means a disease in which inflammation is the main lesion, and examples of the inflammatory disease include edema, allergy, asthma, conjunctivitis, periodontitis, rhinitis, otitis media, sore throat, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn's disease, colitis, hemorrhoids, gout, ankylosing spondylitis, rheumatic fever, lupus, fibromyalgia, psoriatic arthritis, osteoarthritis, rheumatoid arthritis, periarthritis of the shoulder, and the like. Examples of conditions that may be treated include, but are not limited to, shoulder pain, tendinitis, tenosynovitis, myositis, hepatitis, cystitis, nephritis, Sjogren's syndrome, severe myasthenia gravis, and multiple sclerosis.

[0389] Further examples of diseases for which the present invention can be used include, but are 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) (stem cell transplantation associated) or allograft rejection, transplantation / solid organ transplantation (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, glomerulosclerosis, and cancer.

[0390] In the present invention, the term "prevention" refers to any action of administering the pharmaceutical composition provided by the present invention to an individual who is suspected of developing an immune disease or cancer, thereby suppressing or delaying the onset of the immune disease or cancer.

[0391] In the present invention, the term "treatment" refers to any action of clinical intervention to change the natural process of the subject or cells to be treated, which may be performed during the progression of a clinical pathological condition or to prevent it. The intended therapeutic effects include prevention of the onset or recurrence of the disease, alleviation of symptoms, inhibition of all direct or indirect pathological consequences of the disease, prevention of metastasis, reduction in the rate of disease progression, relief or temporary alleviation of the disease state, and improvement of prognosis. For the purposes of the present invention, the treatment may be interpreted as including any action of administering the pharmaceutical composition of the present invention to a patient suffering from an autoimmune disease, including psoriasis, to improve the course of the autoimmune disease, but is not particularly limited thereto.

[0392] Lupus

[0393] Systemic lupus erythematosus: 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:

[0394] Cutaneous lupus: This type of lupus affects the skin. "Cutaneous" is the term that means skin. People with cutaneous lupus can experience skin problems such as sensitivity to the sun and rashes. Hair loss can also be a symptom of the disease.

[0395] 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.

[0396] Neonatal lupus: A rare type of lupus, neonatal lupus is a disease that is found in infants at birth. Babies born with neonatal lupus inherit antibodies from their mothers, who either had lupus during pregnancy or are at risk for developing the disease later in life. Not all babies born to mothers with lupus will develop lupus.

[0397] 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 (corticosteroids, including prednisone); hydroxychloroquine (Plaquenil®); azathioprine (Imuran®); methotrexate (Rheumatrex®); cyclophosphamide (Cytoxan®) and mycophenolate mofetil (CellCept®); belimumab (Benlysta®); and / or rituximab (Rituxan®).

[0398] Lupus nephritis

[0399] Lupus nephritis develops as a complication of lupus. It occurs when lupus autoantibodies affect the kidney structures that filter waste products. This causes kidney inflammation and can lead to blood in the urine, protein in the urine, high blood pressure, kidney dysfunction or kidney failure. About half of adults with systemic lupus develop lupus nephritis. Systemic lupus causes immune system proteins to damage the kidneys and impair their ability to filter waste products.

[0400] Rheumatoid arthritis

[0401] 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 disease progression, especially joint damage.

[0402] Arthritis is a general term that describes inflammation of the joints. Rheumatoid arthritis is a type of chronic (ongoing) arthritis (inducing pain and swelling) that generally occurs symmetrically in joints (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.

[0403] Besides affecting the joints, rheumatoid arthritis can sometimes affect the skin, eyes, lungs, heart, blood, nerves, or kidneys.

[0404] Therapies that may be used in combination with the pharmaceutical compositions of the present invention to treat rheumatoid arthritis include, for example:

[0405] Pain relievers 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. Yet another class of drugs, COX-2 inhibitors, also fall 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 were designed to reduce gastrointestinal bleeding side effects.

[0406] 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).

[0407] Biologics: Besides these "traditional" DMARDs, new drugs have been approved. Currently, there are seven classes of drugs, with different types in each category depending on the case (some of which have been used since 2000 as the anti-TNF series). 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 induces the joint damage seen in rheumatoid arthritis. By attacking cells at a more specific level than the inflammation itself, biologics are considered to be more effective and more specifically targeted. Biologics include etanercept (ENBREL®), infliximab (REMICADE®), adalimumab (HMIRA®), 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.

[0408] Multiple sclerosis

[0409] 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, a protective membrane that surrounds the nerves in the brain and spinal cord. When myelin is damaged, nerve signals that go from the brain to the rest of the body are blocked. The damage can trigger symptoms that affect the brain, spinal cord, and eyes.

[0410] There are four types of multiple sclerosis.

[0411] Clinically isolated syndrome (CIS): When the first symptoms of MS are present, health care providers classify this as primarily CIS. Not everyone with CIS will progress to multiple sclerosis.

[0412] Relapsing-remitting MS (RRMS): This is the most frequent form of multiple sclerosis. People with RRMS have flares of new or worsening symptoms (also called relapses or exacerbations). There are continued periods of remission (when symptoms stabilize or go away).

[0413] Primary progressive MS (PPMS): People diagnosed with PPMS have relapses, or symptoms that gradually worsen without remission.

[0414] 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 get worse. You may experience some relapses or flares (when symptoms increase), but then there are no more periods of remission (when symptoms stabilize or go away).

[0415] Therapies that may be used in combination with the pharmaceutical compositions of the present invention include, for example: Disease-modifying therapies (DMT): A number of drugs have received FDA approval for long-term MS treatment. Such drugs help reduce relapses (also called flares or attacks). They can slow the progression of the disease and prevent new lesions from forming in the brain and spinal cord.

[0416] Relapse Management Drug Treatment: If you have severe attacks, your neurologist may recommend high doses of corticosteroids. The drugs can quickly reduce inflammation and slow damage to the myelin sheath that surrounds nerve cells.

[0417] Physical Rehabilitation Therapy: Multiple sclerosis can affect your physical function. Staying physically healthy and strong helps you maintain mobility.

[0418] Mental health consultations: 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.

[0419] inflammatory bowel disease

[0420] Inflammatory bowel diseases (IBD) are a group of disorders that cause chronic inflammation (pain and edema) of the intestine.

[0421] Crohn's disease and ulcerative colitis are the main types of IBD. The types are:

[0422] Crohn's disease induces 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 the upper part of the large intestine.

[0423] Ulcerative colitis causes swelling and sores (ulcers) in the large intestine (colon and rectum).

[0424] Microcolitis induces intestinal inflammation that is only detectable under a microscope.

[0425] Therapies that may be used in combination with the pharmaceutical compositions of the present invention include, for example: Aminosalicylates (anti-inflammatories such as sulfasalazine, mesalamine, or balsalazide) to minimize irritation to internal organs; antibiotics to treat infections and abscesses; biologics to block immune system signals that trigger inflammation; corticosteroids such as prednisone to suppress the immune system and manage flare-ups; immunomodulators to calm an overactive immune system; anti-diarrheals; nonsteroidal anti-inflammatory drugs (NSAIDs); and supplements such as vitamins and probiotics.

[0426] Graft-versus-host disease (GvHD)

[0427] Graft-versus-host disease (GvHD) is a condition that can occur after allogeneic transplantation, in which the donated bone marrow and / or peripheral blood stem cells view the recipient's body as foreign and the donated cells / bone marrow attack the body.

[0428] GvHD can include acute graft-versus-host disease (aGvHD); and chronic graft-versus-host disease (cGvHD).

[0429] psoriasis

[0430] Psoriasis is a chronic skin disorder, meaning a skin disease 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 usually starts early in adulthood, but can also start later. Besides the red and scaly patches, symptoms of psoriasis include itchy, cracked, dry skin, a scaly scalp, skin pain, pitted nails, nails that crack or crumble easily, and joint pain.

[0431] Treatments that may be used in conjunction 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 cleaners, 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.

[0432] Sjögren's syndrome

[0433] Sjögren's syndrome is a lifelong autoimmune disease that reduces the amount of moisture produced by the sweat glands in the eyes and mouth, and 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.

[0434] There are two forms of Sjogren's syndrome: primary Sjogren's syndrome, which occurs on its own and not due to other health conditions, and secondary Sjogren's syndrome, which occurs in addition to other autoimmune diseases such as rheumatoid arthritis, lupus and psoriatic arthritis.

[0435] 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), treatments for joint or organ problems (e.g., pain relievers, anti-rheumatic agents, immunosuppressants, steroids, anti-fungal agents, and agents to treat vaginal dryness).

[0436] myasthenia gravis

[0437] Myasthenia gravis (MG) is an autoimmune disease in which the body's immune system mistakenly attacks its own parts. MG affects the transmission of signals between nerves and muscles (neuromuscular junctions).

[0438] 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 the muscles of the eyes, face, neck, and limbs. MG is a lifelong neuromuscular disorder.

[0439] Myasthenia gravis affects about 20 out of every 100,000 people. Experts estimate that 36,000 to 60,000 Americans have this neuromuscular disease. The actual number of people affected may be higher, as some people with mild symptoms 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. About 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.

[0440] Autoimmune MG is the most frequent form of this neuromuscular disease. Autoimmune MG is as follows:

[0441] Ocular: The muscles that move the eye and eyelids become weak. The eyelids may droop or the eye may not be able to open. Some people have double vision. Weakened vision is often the first sign of MG. Nearly half of people with ocular MG develop the generalized form within 2 years after the initial symptoms.

[0442] Generalized: Muscle weakness affects the eyes, face, neck, arms, legs, and other parts of the body. It can become difficult to speak or drink, lift your arms above your head, stand from a sitting position, walk long distances, or climb stairs.

[0443] 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 (plasma exchange), and / or surgery.

[0444] cancer

[0445] The binding of TNF to TNFR2 has been reported to promote the activation of tumor cells and tumor-associated cell types, affecting immune responses such as tumor-infiltrating lymphocytes, creating an environment in which tumors can progress. In fact, TNFR2 expression in tumors is associated with disease progression and poor clinical outcomes (Sheng Y, et al.). Thus, the recombinant cells of the present invention can be used in cancer therapy as a monotherapy or in combination with other cancer therapies, such as immunotherapy, e.g., CAR-T therapy, checkpoint inhibitors, and conventional cancer therapies.

[0446] The cancer may include, for example, but is not limited to, positive, precancerous and malignant tumors or proliferative disorders, a precancerous condition.

[0447] In one embodiment of the present invention, the cancer is selected from the group consisting of chronic lymphocytic leukemia (CLL), acute leukemia, acute lymphocytic leukemia (ALL), B-cell acute lymphocytic leukemia (B-ALL), T-cell acute lymphocytic leukemia (T-ALL), chronic myeloid leukemia (CML), B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell- or a large cell-follicular lymphoma, malignant lymphoproliferative disorders, and the like. The cancer may be, but is not limited to, hematological cancer such as, but not limited to, myeloma, myelodysplasia, osteomyelitis, myelodysplasia syndrome, myelodysplastic syndrome ...

[0448] In one embodiment of the present invention, the cancer is selected from the group consisting of colon cancer, rectal cancer, renal cell carcinoma, liver cancer, non-small cell lung cancer, small intestine cancer, esophageal cancer, melanoma, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, anal region cancer, gastric cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, and penile cancer. The cancer may be, but is not limited to, pediatric solid tumors, bladder cancer, kidney or ureter cancer, renal pelvis cancer, central nervous system (CNS) neoplasms, primary central nervous system lymphoma, tumor angiogenesis, spinal tumors, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers, combinations of said cancers, and metastatic lesions of said cancers.

[0449] The pharmaceutical formulations and dosage forms of the present invention may be prepared for storage or use of the genetically modified cells using a pharma- ceutically acceptable vehicle (e.g., carrier or excipient). One of ordinary skill in the art would generally consider pharma- ceutically acceptable carriers, excipients and / or stabilizers to be inactive ingredients of the dosage form or pharmaceutical composition.

[0450] In one embodiment of the present invention, the pharmaceutical composition may be stored in a lyophilized form. In one embodiment of the present invention, the dosage form comprising the recombinant cells of the present invention may be lyophilized.

[0451] Suitable pharma- ceutically acceptable vehicles include non-toxic buffers such as phosphates, citrates, and other organic acids; salts such as sodium chloride; antioxidants including ascorbic acid and methionine; preservatives such as octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol; low molecular weight polypeptides (e.g., less than about 10 amino acid residues); serum albumin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; carbohydrates such as monosaccharides, disaccharides, glucose, mannose or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt forming counterions such as sodium; metal complexes such as Zn-protein complexes; and non-ionic surfactants such as WEEN or polyethylene glycol (PEG) (Remington: The Science and Practice of Pharmacy, 22.sup.nd Edition, 2012, Pharmaceutical Press, London).

[0452] The disclosed pharmaceutical compositions can be administered in a variety of ways for local or systemic treatment: topical administration via epidermal or transdermal patches, ointments, lotions, creams, gels, suppositories, sprays, liquids and powders; pulmonary administration via inhalation or injection of powders or aerosols, including nebulizers, intranasal and intranasal; oral administration; parenteral administration, including but not limited to intravenous, intraarterial, intratumoral, subcutaneous, intraperitoneal, intramuscular (e.g., injection or infusion) or intracranial (e.g., intrathecal or intraventricular).

[0453] The genetically modified cells described herein may also be entrapped in microcapsules, which may be prepared, for example, by coacervation techniques or interfacial polymerization, such as, for example, hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively, and may be prepared in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or macroemulsions, but are not limited thereto (Remington: The Science and Practice of Pharmacy, 22.sup.nd Edition, 2012, Pharmaceutical Press, London).

[0454] In one embodiment of the present invention, the pharmaceutical composition may be in the form of a formulation containing recombinant cells complexed with liposomes. Methods for producing liposomes are well known in the art. For example, some liposomes can be produced by reverse phase evaporation of a lipid composition containing phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes can be filtered through a filter with a defined pore size to obtain liposomes of a desired diameter.

[0455] In one embodiment of the present invention, a sustained release dosage form can be prepared comprising the genetically modified cells of the present invention. Suitable examples of sustained release dosage forms include semipermeable matrices of solid hydrophobic polymers comprising the genetically modified cells of the present invention, which matrices can be in the form of shaped articles (e.g., films or microcapsules). Examples of sustained release matrices include, but are not limited to, hydrogels such as polyester, poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol), polylactide, copolymers of L-glutamic acid and 7-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT.TM. (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprorelin acetate), sucrose acetate isobutyrate, and poly-D-(-)-3-hydroxybutyric acid.

[0456] In one embodiment of the present invention, the method may further include administering at least one additional therapeutic agent in addition to administering the genetically modified cells of the present invention. An additional treatment may be provided before, simultaneously with, and / or after administration of the genetically modified cells of the present invention. In one embodiment of the present invention, the pharmaceutical composition may include the genetically modified cells of the present invention and an additional therapeutic agent.

[0457] In general, combination therapy using two or more therapeutic agents preferably, but not necessarily, uses preparations that act through different mechanisms of action. Combination therapy using preparations with different mechanisms of action may show positive or synergistic effects. Combination therapy may be used at lower doses of each preparation than in monotherapy, reducing toxicity and side effects and / or increasing the therapeutic index of the genetically modified cells.

[0458] In some embodiments of the invention, combination therapy of the genetically modified cells of the invention with at least one additional therapeutic agent may exhibit positive or synergistic effects. In some embodiments of the invention, combination therapy may increase the therapeutic index of the additional therapeutic agent and / or the genetically modified cells. In some embodiments of the invention, combination therapy may reduce the toxicity and / or side effects of the additional therapeutic agent and / or the genetically modified cells.

[0459] Concomitant administration may include co-administration using a single pharmaceutical dosage form or separate dosage forms, or sequential administration in either order, but is generally preferably administered within a period that allows all active agents to simultaneously exert their biological activity.

[0460] The combination of the genetically modified cells of the present invention and at least one additional therapeutic agent may be administered in any order or simultaneously. In one embodiment of the present invention, the genetically modified cells may be administered to a patient who has previously been treated with a second therapeutic agent. In one embodiment of the present invention, the genetically modified cells and the second therapeutic agent may be administered substantially simultaneously or simultaneously. For example, a subject may receive the genetically modified cells during a course of treatment with the second therapeutic agent (e.g., chemotherapy). In one embodiment of the present invention, the genetically modified cells may be administered within one year after treatment with the second therapeutic agent. In one embodiment of the present invention, the genetically modified cells may be administered within 10 months, 8 months, 6 months, 4 months, or 2 months after treatment with the second therapeutic agent. In one embodiment of the present invention, the genetically modified cells may be administered within 4 weeks, 3 weeks, 2 weeks, or 1 week after treatment with the second therapeutic agent. In one embodiment of the present invention, the genetically modified cells may be administered within 5 days, 4 days, 3 days, 2 days, or 1 day after treatment with the second therapeutic agent. The two (or more) agonists or therapies may be administered to a subject within a matter of hours or minutes (eg, substantially simultaneously).

[0461] Uses - Compositions or methods for immunomodulation

[0462] As described above, it is well known in the art that activation of the TNFR2 pathway can lead to suppression of the immune system. The TNFR2 binding agent (e.g., Stefin A protein mutant) and / or fusion protein expressed by the recombinant cell of the present invention can exhibit the effect of proliferation and activation of Treg cells by binding to and activating the TNFR2 pathway, and as a result, can exhibit the effect of immunomodulation, i.e., suppression of the immune system or immune response.

[0463] Therefore, in another aspect, the present invention relates to an immunoregulatory composition comprising the genetically modified cells and / or the conditioned cell culture medium thereof.

[0464] In another aspect, the present invention relates to the use of said genetically modified cells and / or the conditioned cell culture medium thereof for immunomodulation.

[0465] In another aspect, the invention relates to the use of the genetically modified cells and / or the conditioned cell culture medium thereof for the manufacture of a composition for immunomodulation.

[0466] In another aspect, the present invention relates to a method for immunomodulation comprising the step of administering to a subject genetically modified cells for immunomodulation and / or conditioned cell culture medium thereof.

[0467] In the present invention, the term "immunomodulation" means alleviating immune imbalance in blood and maintaining immune homeostasis. Maintaining immune homeostasis refers to a state in which immune tolerance, which is a mechanism that suppresses immunity, and immunity that promotes immune response are in balance, and maintaining such a state is an essential factor in the treatment of immune diseases, particularly autoimmune diseases.

[0468] As used herein, the term "immunosuppression" or "suppression of the immune system" refers to a variety of substances used to reduce or block the host's ability to produce antibodies (humoral immune response) or to elicit a cellular immune response to the action of an antigen.

[0469] In one embodiment of the present invention, the genetically modified cells and / or the cell culture medium conditioned therewith may exhibit an excessive immunosuppressive effect, which is an effect of suppressing excessive expression of lymphocytes induced by non-specific stimulatory factors of splenocytes, thereby suppressing excessive immunity.

[0470] The immunosuppressive composition of the present invention may be administered in combination with immune-related proteins, particularly autoimmune or allergy-related proteins. Specific examples of such proteins include autoantigens associated with autoimmune diseases. For example, proteins may include autoantigens associated with rheumatoid arthritis, such as heat shock proteins (HSPs), citrullinated filaggrin, glucose-6-phosphate isomerase, p205, and collagen; autoantigens associated with type 1 diabetes, such as insulin, zinc transporter 8 protein (ZnT8), pancreatic and duodenal homeobox 1 (PDX1), chromogranin A (CHGA), and isletamyloid polypeptide (IAPP); and autoantigens associated with myasthenia gravis, such as acetylcholine receptors. In addition to all types of autoantigens known to cause autoimmune diseases, proteins may include a variety of allergens known to cause food allergies, such as peanuts, milk, eggs, nuts, beans, shellfish such as shrimp, and fish-derived substances.

[0471] Uses - Compositions and methods for culturing regulatory T cells

[0472] As explained above, various studies have reported the importance of TNFR2 in Treg proliferation and function. TNFR2-deficient mice have reduced numbers of thymic and peripheral Tregs (2013_Chen X,et al.PMID:23277487), and TNFR2- / - Tregs fail to regulate inflammatory responses in vivo (2008_Van Mierlo GJ,et al.PMID:18292492). In humans, Tregs have been confirmed to express higher levels of TNFR2 than T effector cells (2013_Okubo Y,et al.PMID:24193319 & 2002_Annunziato F,et al.PMID:12163566). TNFR2+ Tregs most potently suppressed proliferation and cytokine production of co-cultured T-responder cells (2010_Chen X, et al. PMID:20127680).

[0473] Therefore, in another aspect, the present invention relates to a composition or medium for culturing regulatory T cells, which comprises the genetically modified cells and / or a regulated cell culture medium thereof.

[0474] In yet another aspect, the present invention relates to a method for culturing regulatory T cells, comprising the step of culturing regulatory T cells in the presence of the genetically modified cells and / or the regulated cell culture medium thereof.

[0475] In yet another aspect, the present invention relates to use of the genetically modified cells and / or the conditioned cell culture medium thereof for culturing regulatory T cells.

[0476] In yet another aspect, the present invention relates to use of the genetically modified cells and / or the conditioned cell culture medium thereof for producing a composition or medium for culturing regulatory T cells.

[0477] In one embodiment of the present invention, the composition or medium for culturing regulatory T cells may further include any one or more selected from the group consisting of an energy source, an amino acid, a sugar, an inorganic salt, a vitamin, FBS (fetal bovine serum), HEPES, a protein, a carbohydrate, mercaptoethanol, and a growth factor.

[0478] In one embodiment of the present invention, the composition or medium may further include components necessary for cell growth and survival in vitro or components promoting cell growth and survival, such as, but not limited to, vitamins, essential or non-essential amino acids, trace elements, etc.

[0479] In the present invention, the term "regulatory T cells (Treg or Treg cells)" includes natural regulatory T cells (nTreg) or induced regulatory T cells (iTreg). Regulatory T cells maintain immune homeostasis and block autoimmune reactions by suppressing immune responses. In one embodiment of the present invention, the regulatory T cells may be genetically engineered regulatory T cells. Examples of genetically engineered regulatory T cells may include, but are not limited to, CAR-Treg and TCR-Treg.

[0480] In one embodiment of the present invention, the regulatory T cells may be preferably, but are not limited to, CD4+Foxp3+ regulatory T cells.

[0481] In one embodiment of the present invention, the regulatory T cells may express TNFR2, and preferably, the TNFR2 may be present on the surface (or membrane) of the regulatory T cells. In one embodiment of the present invention, the regulatory T cells may be isolated regulatory T cells.

[0482] In one embodiment of the present invention, the regulatory T cells are cultured in the presence of genetically modified cells and / or their conditioned cell culture medium.

[0483] In one embodiment of the present invention, culturing the regulatory T cells in the presence of the genetically modified cells and / or the cell culture medium conditioned therewith may be performed in vitro or ex vivo. In one embodiment of the present invention, culturing the regulatory T cells may be performed by co-culturing the regulatory T cells and the genetically modified cells. In the present invention, the genetically modified cells and / or the cell culture medium conditioned therewith may bind to TNFR2 presented on the surface of Treg cells and activate the TNFR2 pathway.

[0484] In one embodiment of the present invention, induced regulatory T cells (iTreg) are obtained by co-culturing CD4+ T cells with tolerant antigen-presenting cells, but is not limited thereto.

[0485] In one embodiment of the present invention, the use, composition, medium or method for culturing Treg cells of the present invention may induce the proliferation and / or activation of regulatory T cells.

[0486] Uses - Drug delivery compositions

[0487] In still another aspect, the present invention relates to a composition for drug delivery comprising the genetically modified cell.

[0488] In one embodiment of the present invention, the genetically modified cells may be characterized in that they carry or have one or more drugs attached to their surface.

[0489] In one embodiment of the present invention, the drug may further include at least one drug selected from the group consisting of genes, viruses, and small molecule compounds.

[0490] In one embodiment of the present invention, the drug may be characterized as having an immunomodulatory ability, and more preferably, as a drug having an immunosuppressant effect.

[0491] The recombinant cell of the present invention can express Stefin A protein mutant that specifically binds to TNFR2, and can thereby target and bind to TNFR2 of immune cells.Preferably, the recombinant cell of the present invention can bind to TNFR2 and exhibit agonistic activity.

[0492] 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 to biologically search for damaged or infected sites in the body and have excellent targeting ability, so that drugs can be effectively and accurately delivered to the desired sites in the body.

[0493] In one embodiment of the present invention, the drug may be carried inside the mesenchymal stem cells, attached to the surface, or carried inside and attached to the surface, but is not limited thereto. The drug may be directly carried / carried inside the mesenchymal stem cells, or attached to the surface, but is not limited thereto. The drug may be loaded into a nanostructure, or may be carried inside the mesenchymal stem cells in a state of being attached to a specific molecule, or may be attached to the surface, but is not limited thereto. In one embodiment of the present invention, the nanostructure may include inorganic nanoparticles, polymer 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 in a form of being carried 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 mainly made of polymers and lipids.

[0494] In one embodiment of the present invention, the genetically modified cells may be characterized by releasing a drug from a target site. In one embodiment of the present invention, a drug loaded in the genetically modified cells may be released. In one embodiment of the present invention, a drug attached to the surface of the genetically modified cells may be detached from the surface and released by the target environment. In one embodiment of the present invention, when a drug is loaded on the nanostructure, the drug may be released due to temperature-specific and pH-specific structural changes of the nanostructure.

[0495] EXAMPLES

[0496] The present invention will be described in more detail with reference to the following 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 are not intended to limit the scope of the present invention.

[0497] Example 1: Selection of Stefin A protein variants that specifically bind to TNFR2 in a phage display library

[0498] Candidate clone identification

[0499] The Stefin A protein variants of the present invention that specifically bind to TNFR2 are identified by screening a Stefin A protein variant library with two random loop sequences, each loop being displayed on a fixed Stefin A protein variant framework scaffold based on the amino acid sequence of Stefin A and having a length of about 9 amino acids. Such screening 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 phages expressing Stefin A protein variants (Affimer®) is cultured with human or mouse TNFR2 as needed, the sequences of which are as shown in Table 7 below.

[0500] [Table 7]

[0501] Commercial antigens used during selection. The TNFR2 antigens having the sequences set forth in Table 7 and other proteins set forth below were tested in a variety of formats for use in biotinylation, selection of Stefin A protein variants that specifically bind to TNFR2, and other analyses. [Table 8]

[0502] In some cases, TNFR2 was biotinylated and alternately captured on streptavidin and neutravidin beads, and in other cases, TNFR2 was passively absorbed to the surface. Unbound phage particles were then washed, and bound phage were eluted after washing. Elution of bound phage was achieved by exposure to trypsin. The eluted phage particles were then used to infect E. coli, and the infected bacteria were cultured under conditions appropriate for bacteriophage replication. After release of bacteriophage particles from these infected bacteria, the phage particles were allowed to bind to target antigens and the bound phage particles were eluted. Cycles of propagating the eluted phage particles on bacteria and isolating the released phage particles from the infected bacteria were repeated to enrich the bacteriophage population for phage particles displaying proteins that bind to the target antigen. In this cycle, certain conditions were modified, such as increasing the number of washing steps, decreasing the amount of available antigen, or adding a blocking agent, to select for phage particle-displayed proteins that bind more strongly or specifically to the target antigen.

[0503] After multiple rounds of phage display library selection and amplification, the proteins expressed by the phages were expressed and screened through 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 the ELISA, human, murine, and cynomolgus TNFR2 were immobilized on plates, lysates were added, and the amount of Stefin A protein variants that specifically bound to TNFR2 on each plate was measured using a detection antibody specific to the 6xMyc tag expressed on the candidate Stefin A protein variants. The phagemid vectors encoding the Stefin A protein variants with the best human TNFR2 binding activity were sequenced to confirm the DNA sequence of the candidate clones for further development. TNFR1 polypeptide was also used to confirm specificity. The amino acid sequences of loop 2 and loop 4 of each of these candidate clones are presented in Table 1.

[0504] Example 2: Production and screening of Stefin A protein variants as water-soluble proteins in mammalian cells

[0505] Cloning of the sequence of interest into a vector and characterization

[0506] To evaluate the ability of the selected Stefin A protein variants to bind to TNFR2 when expressed on the surface of mammalian cells, a cell pool was prepared by transforming 30 fast-tracked clones in order of the most repeated passive selection. These clones were also used for initial characterization and assay optimization. TNFR2 and Stefin A protein variants that specifically bind to TNFR2 were cloned into the mammalian expression vector pD609kanR (ATM custom vector) in the phagemid of Example 1 for expression in Expi293FTM cells (ThermoFisher). The average concentration obtained through expression was 2.8 mg / mL, and 20 clones were selected based on purity. Of these, 19 clones that existed as monomers were selected and evaluated by ELISA (Figure 2).

[0507] The 19 clones were screened by flow cytometry on Expi293FTM hTNFR2 cells expressing hTNFR2 at 1 μM and 0.1 μM, compared to the negative control groups SQT-GLY and 3T0-GLY, which contain glycines only in loops 2 and 4 and are not specific binders. The signal detected from transfected cells excluded the signal obtained from untransfected Expi293FTM cells. Of these, 16 clones were confirmed to bind hTNFR2-Expi293FTM cells at 1 μM > 10% after binding to parental cells, while clones 15, 22, and 23 showed no such binding (Figure 2). Stefin A protein variants that bind to hTNFR2 purified in mammalian cells were screened in the HEK BlueTM TNFα SEAP reporter assay (Invivogen) according to the manufacturer's protocol and evaluated for their ability to bind to TNFR2 and induce the NFκB pathway and SEAP production when clustered by anti-his antibody coated on the plate. In this evaluation, five of the rapidly expanding clones (clones 01, 09, 12, 21, and 26) induced SEAP release, with clone 12 in particular showing the most consistent agonist activity.

[0508] An additional 90 Stefin A protein variants were cloned into phagemids and characterized in the same manner as described above. Fifty-five clones were selected and confirmed to bind to TNFR2 via the Mirrorball assay.

[0509] Additional characterization was performed to test the ability of 75 Stefin A protein variants from the rapid selection (20) and the additional selection (55) to block the interaction between hTNFR2-hFc-his-Avi and TNF-alpha. A series of experiments was performed to determine the EC80 of hTNFR2-hFc-his-Avi when binding to coated hTNF-alpha (593 pM). Stefin A protein variants were tested at three concentrations (10 μM, 1 μM, and 0.1 μM) in the presence of hTNFR2 antigen at the determined EC80. Detection of bound hTNFR2-hFc-his-Avi was performed with anti-hFc-HRP antibody. Thirty-two of the 75 clones tested showed more than 65% inhibition of the interaction between hTNFR2 and TNF-alpha at 10 μM, and these 32 clones were further analyzed to determine the IC50 (Figure 3). Clones 06, 21, 26, 27, 44, 108 and 115 were confirmed to completely block the interaction between TNFα and hTNFR2 at the concentrations tested.

[0510] Co-culture of Expi293F and HEK-Blue TNFα cells to assess the agonist activity of plasma membrane-expressed TNFR2 binding agents

[0511] Based on the above experiments, among the Stefin A protein mutant clones that specifically bind to TNFR2, cell membrane-expressing TNFR2 binding agents were selected for agonist activity evaluation, and then cloned into pDisplay vector and transiently transduced into Expi293F cells to express TNFR2-specific Stefin A protein mutants on the cell surface. After about 29 hours of transduction, cell viability was measured using a cell counter, and expression of Stefin A protein mutants was confirmed using a polyclonal anti-cystatin A antibody.

[0512] After 48 hours of transduction, Expi293F cells expressing the regulated Stefin A protein variants at four different cell concentrations were co-cultured with HEK Blue™ TNFα SEAP reporter cells, and after 24 hours, SEAP release in the supernatant was quantified using QUANTI-Blue™ and absorbance was measured at 640 nm. Clones with an absorbance of 0.4 or higher were selected as clones with agonistic activity (43 clones, Figure 4).

[0513] Example 3: Characterization of 43 selected clones

[0514] ELISA cross-reactivity binding validation

[0515] The specificity of the 43 clones selected in Example 2 to hTNFR2 was evaluated by ELISA by comparing the Stefin A protein mutant clones that bind to TNFR2 with TNFR1. None of the clones bound to the recombinant antigen TNFR1. No clones were found to bind to hTNFR1 expressed in Expi293FTM cells, confirming that the Stefin A protein mutants of the present invention do not show cross-reactivity between hTNFR2 and hTNFR1.

[0516] In addition to human TNFR2, cross-reactivity with mouse TNFR2 was also confirmed by the same method. Since the homology between the two proteins is about 68% in the extracellular domain, cross-reactivity was expected to be low. As expected, it was confirmed that none of the clones bound to mTNFR2 expressed in Expi293FTM cells.

[0517] HEK293-HEK Blue™ TNFα SEAP Reporter Assay

[0518] The 43 clones selected in Example 2 were transfected into HEK293 cells together with four control groups, SQT-Gly (SEQ ID NO: 484), 3t0-Gly (SEQ ID NO: 485), empty vector and negative clone 06. Approximately 29 hours after transfection, cell viability was determined using a cell counter, and expression of Stefin A protein variants was determined using an anti-HA antibody with a flow cytometer. 48 hours after transfection, HEK293 cells expressing Stefin A protein variants were co-cultured with HEK Blue™ TNFα SEAP reporter cells at four cell densities (40000, 20000, 10000 and 5000), and the number of HEK Blue cells was fixed at 50000 cells / well. In addition, the positive control group MR2-1 was also cultured with HEK Blue cells at various concentrations for 24 hours. After 24 hours, the release of SEAP in the supernatant was quantified using Quanti-Blue and the absorbance at 640 nm was measured. Stefin A protein mutants were stained with a single clone anti-HA antibody, and the expression level was found to be over 90% in both experiments. Stefin A protein, which specifically binds to TNFR2, was expressed on the surface of HEK293 cells and co-cultured with HEK Blue™ TNFα SEAP reporter cells to confirm the agonist effect. Finally, 16 clones that were consistently included in the top 20 rankings in both experiments were selected, and as shown in Figure 5, the selected clones were as follows: clones 001, 007, 009, 012, 013, 026, 027, 037, 044, 059, 069, 079, 100, 103, 112, and 115.

[0519] Analysis of binding to cells expressing human and cynomolgus monkey TNFR2

[0520] The 16 selected agonist clones were analyzed in soluble form to confirm binding to human TNFR2-overexpressing Expi293FTM and cynomolgus TNFR2-overexpressing Expi293FTM. The experiment was repeated three times and two other batches of transfected cells were used. All clones consistently showed specific binding to hTNFR2-Expi293FTM cells, and in particular, clones 26 and 69 showed the best binding to both human and cynomolgus TNFR2-expressing cells. Thirteen of the 16 clones tested showed cross-reactivity with cynomolgus TNFR2, while clones 001, 007, and 009 showed no cross-reactivity (Figure 6).

[0521] Example 4. In-line fusion homodimers

[0522] Clones 001, 009, and 012 were selected for production in homodimeric form as in-line fusions (ILFs) and evaluated in comparison to the monomers after expression as soluble proteins or on the cell surface. Two constructs were used to express the ILF homodimers using flexible linkers, one codon-optimized for mammalian expression and the other not. Both constructs were cloned into a mammalian secretion vector (pD609_KanR) and the recombinant proteins were purified and evaluated for expression levels, protein quality, binding kinetics with hTNFR2-hFc-his-avi by Biacore and agonist activity in a reporter assay using HEK Blue™ TNFα SEAP reporter cells. Both constructs were also expressed on the surface of Expi293FTM cells and cloned into the pDisplay vector to assess expression levels, binding to hTNFR2-hFc-his-avi, and agonist activity in a co-culture reporter assay with HEK BlueTM TNFα SEAP reporter cells. Codon optimization failed to improve the yield or purity of the soluble ILF homodimer, as confirmed by HPLC.

[0523] Biacore analysis showed that clones 001 and 009 had increased affinity in the soluble ILF form compared to the soluble monomeric form, but clone 12 showed no increased affinity. When activity against agonists was examined through SEAP assays in HEK Blue™ TNFα SEAP reporter cells, only clone 12 showed increased activity in the soluble ILF form, but when expressed on the cell surface, SEAP analysis and flow cytometry showed no difference between the ILF and monomeric forms.

[0524] Summary of selection and characterization of Stefin A protein variants that specifically bind to hTNFR2

[0525] More than 2500 single clone phages were screened by phage ELISA with a hit-rate of more than 60% and good diversity (20%-40%). Using crude cell extracts, more than 400 clones were confirmed to bind to the hTNFR2 antigen as a result of ELISA screening. At this stage, an additional step was introduced in the selection screening campaign to reduce the number of clones to be cloned into mammalian expression vectors. In parallel, 30 rapidly evolving clones that directly expressed in soluble Stefin A protein variants in passive selection were selected to facilitate assay development and screening step construction for the remaining clones. 20 of these clones passed the protein production quality standard of more than 80% purity by SEC-HPLC and were used to analyze the properties of the material through various assays.

[0526] After experiments with the rapidly developed clones, Stefin A protein variants that specifically bind to TNFR2 were further screened. A total of 364 Stefin A protein variants that specifically bind to human TNFR2 were purified with phagemid and tested for binding to TNFR2-overexpressing cell lines. Following this assay, 90 Stefin A protein variant clones that specifically bind to human TNFR2 were reformatted in mammalian expression vectors. 55 of these clones passed the protein production quality standard of ≥80% purity by SEC-HPLC and were subsequently used for analysis.

[0527] From the total clones (75 test clones that were rapidly developed later), 32 clones were selected that competitively bound to TNFR2 with TNF-alpha through the binding ability test of soluble Stefin A protein mutants to hTNFR2 overexpressing cells, agonist activity assay using HEK Blue™ TNFα SEAP reporter cells, and competitive binding analysis, and 43 clones with agonist activity to TNFR2 were further selected through agonist activity assay using HEK Blue™ TNFα SEAP reporter cells co-cultured with Stefin A protein mutants expressed on the surface of Expi293F™ cells. Of the 43 clones, 16 clones that were selected as those that showed the best TNFR2 agonist activity in repeated tests successfully calculated Kd using a 1:1 binding model. None of the clones bound to human TNFR1 by ELISA or to mouse TNFR2 by cell binding. Forty-three Stefin A mutants were expressed on the surface of HEK293 cells and co-cultured with HEK Blue™ TNFα SEAP reporter cells to assess TNFR2 agonist activity. Most of the clones tested showed dose-dependent agonist effects.

[0528] Of the 43 clones with agonistic activity against TNFR2, 16 Stefin A protein variants were selected as those that showed the best TNFR2 agonistic activity in repeated tests, and further characterization was performed to evaluate their binding to cynomolgus monkey TNFR2 and human TNFR1 expressed in cells.

[0529] In the characteristic analysis, The 32 TNFR2 Stefin A protein variants that competitively bind to hTNFR2 with TNF-alpha in a water-soluble form are clones 001, 003, 004, 006, 007, 008, 009, 010, 011, 012, 013, 014, 015, 021, 026, 027, 036, 037, 038, 044, 045, 058, 059, 060, 069, 072, 080, 100, 108, 115, 117 and 120.

[0530] The 43 Stefin A protein variants analyzed as specifically binding to hTNFR2 polypeptide and exhibiting agonist activity upon expression in the cell membrane are clones 001, 003, 007, 009, 010, 011, 012, 013, 021, 026, 027, 036, 037, 038, 043, 044, 056, 057, 059, 060, 063, 064, 065, 069, 071, 072, 077, 079, 080, 081, 088, 089, 100, 102, 103, 105, 108, 109, 111, 112, 113, 115 and 119.

[0531] Among the 43 Stefin A protein variants that specifically bind to the hTNFR2 polypeptide, 16 Stefin A protein variants were selected in repeated experiments as having particularly excellent agonistic activity, namely clones 001, 007, 009, 012, 013, 026, 027, 037, 044, 059, 069, 079, 100, 103, 112 and 115.

[0532] The use of in-line fusion homodimers in the form of water-soluble proteins demonstrated an avidity advantage over monomers in recombinant TNFR2 binding by Biacore and a potential advantage in increased activity in anti-His clustering assays using HEK Blue™ TNFα SEAP reporter cells, but no advantage was observed over monomers in activating HEK Blue™ TNFα SEAP reporter cells when in-line fusion Stefin A protein mutants were displayed on the cell surface.

[0533] Example 5. Stefin A protein mutants that specifically bind to mouse TNFR2

[0534] Phage selection and screening

[0535] The mTNFR2 antigen, which has the sequence shown in Table 7 but exists in three forms (mFc, hFc and His tag), was analyzed and mTNFR2-mFc was selected as the form suitable for selection of Stefin A protein mutants.

[0536] choice

[0537] Passive and solution selections were performed with two Stefin A protein mutant phage libraries, similar to the method used in screening for Stefin A protein mutants that specifically bind to hTNFR2. Enrichment was observed when the mTNFR2 concentration was maintained at 10 nM in round 3. More than 2000 phage-displayed Stefin A protein mutants in rounds 2 and 3 were screened by single-clone phage ELISA. More than 900 of these clones showed mTNFR2 binding, with no detectable nonspecific binding to mouse Fc, plastic, streptavidin, or neutravidin. Each of the clones was defined as a "hit" by the criteria of an absorbance at 450 nm to 630 nm of 0.5 or greater on mTNFR2-coated plates and less than 0.05 on control antigen (e.g., mouse Fc, plastic, neutravidin)-coated plates. Sequencing of the phage ELISA hits identified 281 unique clone sequences, of which 109 showed mTNFR2 binding after ELISA with crude cell extracts. 58 clones were selected for further binding analysis using mTNFR2-Expi293FTM cells, and the selected clones are shown in Table 2 as clones 122 to 179.

[0538] Water-soluble protein characterization

[0539] As was done with the Stefin A protein variants that specifically bind to hTNFR2, the Stefin A protein variants that specifically bind to mTNFR2 were cloned into a mammalian vector by phagemid and expressed in Expi293FTM cells. The expressed proteins were assessed for purity by SDS-PAGE and HPLC, for affinity by Biacore, and for cell-based binding of mTNFR2 in mTNFR2-Expi293FTM cells. Specificity for mTNFR2 was tested using ELISA and cell-based assays using mTNFR1 and mTNFR2 as targets to confirm the specificity of the identified clones. For characterization of the soluble proteins, the Stefin A protein variants were tested for their ability to block mTNFR2-mFC binding to mTNFα in ELISA, and finally, four clones (157, 160, 171, and 172) showed a clear competitive response in the range tested.

[0540] T cell stimulation analysis

[0541] A T cell stimulation assay was used as a screening tool because activated T cells express high levels of TNFR2 and stimulation with HM102 (a TNFR2 agonist) increases CD25 expression.

[0542] Stefin A protein mutants that specifically bind to mTNFR2 were expressed on the surface of HEK293 cells. The HEK293 cells were then co-cultured with BALB / c splenocytes for 48 hours, and the increased expression of CD25 was evaluated on CD4 and CD8 positive T cells. To evaluate the effect of Stefin A protein mutant-expressing HEK cells on CD4+ and CD8+ cells, after 48 hours of culture, the cells were stained with the following marker panel: Live / Dead staining, CD90.2, CD4, CD8, and CD25.

[0543] CD25 expression was observed in both CD4+ and CD8+ T cells in a dose-dependent manner in response to HEK293 expressing a mutant Stefin A protein that specifically binds to mTNFR2, which was similar to the results with the HM102tNFR2 agonist. The response observed in CD8+ T cells was stronger than that observed in CD4+ T cells. The top six clones consistently ranked in two replicates were clones 174, 160, 175, 125, 157, and 179.

[0544] Summary of selection and characterization of Stefin A protein variants that specifically bind mTNFR2

[0545] More than 2000 single clone phages were screened by phage ELISA, showing a reasonable hit rate of 35%-60% and good diversity (25%-35%). 109 clones were confirmed to bind to the mTNFR2 antigen by ELISA screening as crude cell extracts. Using the same screening procedure as the human program, phagemid-identified binders were purified and tested for binding to TNFR2-overexpressing cell lines. From this analysis, 60 Stefin A protein variants that specifically bind to mTNFR2 were reconstituted in mammalian expression vectors and tested for their biological and physical properties as soluble proteins. 22 Stefin A protein variants passed the protein purity standard of 80% or higher. The 22 clones were characterized as soluble proteins by various assays and then expressed on the HEK293 surface for co-culture assays with splenocytes.

[0546] Only 10 of the 22 selected Stefin A protein variants showed triple-digit nanomolar binding affinities (KD) when tested by Biacore, but Stefin A protein variants with higher KD values ​​also showed good agonist activity when expressed on the cell surface. Also, the performance of the clones as soluble proteins appeared differently depending on whether they were native dimers or trimers. For this reason, the Biacore data was considered in the context of mTNFR2 agonist activity but was not used to select the clones. When tested on cells expressing mTNFR2, 20 of the 22 clones showed specific binding with a wide range of EC50 values. None of the clones bound to recombinant protein or cell surface expressed TNFR1. Five of the 22 clones were confirmed to completely inhibit the interaction between TNF-alpha and TNFR2 in a competitive ELISA.

[0547] Twenty-two mouse clones were expressed on the surface of HEK293 cells for co-culture assay with splenocytes. Most of the clones tested were effective in increasing CD25 expression in a dose-dependent manner (>70%) in both CD4+ and CD8+ T cells. Clones were ranked by CD25 expression in both CD4+ and CD8+ T cells, and based on the results, clones 174, 160, 175, 125, 157, and 179 were selected as Stefin A protein variants that specifically bind to mTNFR2.

[0548] Examples 1 to 5 were carried out by Avacta Life Sciences Limited.

[0549] Example 6: Characterization of Stefin A protein mutants that specifically bind to cell surface expressed TNFR2

[0550] Stable genetic modification of PSC-derived MSCs (totipotent stem cell-derived mesenchymal stem cells) using lentiviral vectors

[0551] The top seven clones (clones 001, 009, 100, 026, 012, 044, and 007) with high agonistic activity among the Stein A protein mutant clones that specifically bind to human TNFR2 were cloned into a plasmid vector so that they could be expressed by the CBh promoter, and lentiviral vectors were constructed for each. The lentiviral vectors were constructed using a vector containing a nucleic acid sequence encoding an exemplary TNFR2 binding agent, i.e., a Stein A protein mutant that specifically binds to TNFR2 fused to a transmembrane domain (derived from PDGFR, SEQ ID NO: 486, Biotechnology and Bioengineering, 73(4), 313-323), and a nucleic acid sequence encoding a neomycin resistance gene (Gene Volume 85, Issue 2, 28 December 1989, Pages 421-426). Frozen naive PSC-derived MSCs were thawed, mixed with 1 MOI lentiviral vector and 2 μg / mL-8 μg / mL polybrene, and inoculated into a cell culture flask. After culturing for 16-20 hours at 37°C and 5% CO2, the lentivirus-containing culture medium was completely removed and replaced with new culture medium, and then cultured for 48 hours at 37°C and 5% CO2. The cells were then harvested, re-inoculated at a cell density of 0.4-1.0×104cells / cm2, and cultured for 18-24 hours at 37°C and 5% CO2. The culture medium was replaced with a culture medium containing 100 μg / mL-250 μg / mL G418, and then cultured for 5 days, with the culture medium being replaced with a culture medium containing G418 every 2 days. When the cell density reached 90% or more, the cells were harvested and frozen.

[0552] Confirmation of the maintenance of stemness of PSC-derived MSCs after lentiviral nucleic acid transfer

[0553] Flow cytometry was performed on eMSCs that had been cultured for five passages after transduction with lentivirus encoding a stefin A protein mutant that specifically binds to TNFR2 to analyze whether they maintained the characteristics of naive PSC-derived MSCs. Cells frozen at each passage were thawed and analyzed for purity and immune markers through flow cytometry. The expression of mesenchymal stem cell surface markers CD29, CD44, CD73, and CD105, as well as the 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 compared with naive PSC-derived MSCs. The results are shown in Figures 7 and 8. It was confirmed that the expression of mesenchymal stem cell surface markers CD29, CD44, CD73, and CD105 was maintained at 95% regardless of gene transduction. In addition, the expression of CD45, SSEA-3, TRA-1-60, TRA-1-81, and HLA-DR was maintained at less than 1%, confirming that the characteristics of mesenchymal stem cells were well maintained.

[0554] Analysis of eMSCs expressing Stein A protein variants that specifically bind to TNFR2

[0555] The extracellular expression of the selected TNFR2-binding fusion protein from eMSCs was confirmed. The human PDGFR (platelet-derived growth factor receptor) transmembrane domain was fused to the C-terminus of a stefin A protein variant that specifically binds to TNFR2, a cell membrane protein that faces the extracellular direction. The functional expression of the stefin A protein variant that specifically binds to TNFR2 was confirmed by Western blot and flow cytometry.

[0556] The lentiviral vector-transduced eMSCs were harvested and protein lysates were prepared after 5 passages. Western analysis showed that all cells transduced with the PDGFR transmembrane domain-fused Stefin A protein mutants showed high expression of Stefin A protein mutants that specifically bind to TNFR2 (Figure 9).

[0557] The percentage of cells expressing stefin A protein variants was determined through flow cytometry using picoerythrin (PE)-labeled antibodies against Affimer® (stefin A protein variants) and the average absolute number of membrane-anchored stefin A protein variants per cell using QuantiBRITE PE calibration beads (BD Biosciences, San Jose, CA, USA). FACS analysis results show that stefin A protein variants were introduced and expressed in more than 95% of MSCs (Figure 9B). The number of stefin A protein variant molecules expressed on the cell surface was analyzed using QuantiBRITE PE beads. Compared with naive PSC-derived MSCs (non-genetically modified MSCs), it was confirmed that both stefin A protein variants that specifically bind to TNFR2 and eMSCs transduced with hPDGFR-fused genes showed high expression (Table 9, Figures 10A and 10B).

[0558] [Table 9]

[0559] Co-culture of eMSCs and HEK-Blue TNFα cells for evaluation of TNFR2 agonist activity

[0560] Seven stefin A mutant clones selected as shown in Table 9 were stably transduced into PSC-derived MSCs to generate cell lines. SQT-Gly and 3t0-Gly were also generated as negative controls. eMSCs expressing stefin A mutants were co-cultured with HEK Blue™ TNFα SEAP reporter cells at 10 different cell densities (1 / 2 dilution) ranging from 80 to 40,960 cells / well, and the number of HEK Blue cells was fixed at 20,000 cells / well. After 24 hours, the release of SEAP from the supernatant was quantified using Quanti-Blue and the absorbance at 640 nm was measured. AFX002-C010 and AFX002-C011 cells used as negative controls did not show agonist activity. In the case of the four cell lines, AFX002-C012, AFX002-C014, AFX002-C015, and AFX002-C018, excellent TNFR2 agonist activity was shown in a dose-dependent manner (Figure 11). A water-soluble form of the Stefin A protein mutant (AFFIMER®), which specifically binds to human TNFR2, was unable to induce activation of HEK-Blue TNFα cells. However, it was confirmed that PSC-derived MSCs transfected with the Stefin A protein mutant gene induced TNFR2 activation. According to previous reports, secondary clustering of the initially formed trimeric TNF-TNFR2 complex is required for triggering of the TNFR2-related signaling pathway (Front.Immunol.10:2040,2019), so the above results imply that cell membrane expression of the Stefin A protein mutant can more effectively induce clustering between the Stefin A protein mutant and TNFR2 complex and transmit signals.

[0561] 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 stem cells (MSCs), the mesenchymal stem cells comprising an exogenous nucleic acid comprising a sequence encoding a TNFR2 binding agent, wherein the TNFR2 binding agent comprises one or more binding domains of an antibody or antibody mimetic.

2. The TNFR2 binding agents include Stefin A protein variants, Fab, Fab', F(ab')2, Fv, Fd, scFv, sdFv, VL, VH, Camel Ig, V-NAR, VHH, trispecific (Fab3), bispecific (Fab2), diabody, triabody, tetrabody, minibody, bispecific scFv (Bis-scFv), VNAR (shark heavy-chain-only 2. The population of mesenchymal stem cells of claim 1, wherein the antibody is a mAb, a mAb, a mAb antibody, a microprotein, an affibody, an aptamer, an avimer, a nanobody, a unibody, a single domain antibody, an affilin, an affitin, an adnectin, an atrimer, an evasin, a DARPin, an anticalin, a finomer, a versabody, a lipibody, or a duocalin.

3. The population of mesenchymal stem cells of claim 1 or 2, wherein the TNFR2 binding agent is or comprises a Stefin A protein mutant.

4. The population of mesenchymal stem cells according to any one of claims 1 to 3, wherein the TNFR2 binding agent has a Kd value of 2 x 10-6 M or less for TNFR2.

5. The TNFR2 binding agent may be membrane anchored or cell surface expressed, and optionally (i) the genetically modified MSCs express an average of 2500 or more TNFR2-binding agents per cell; and / or (ii) The population of mesenchymal stem cells according to any one of claims 1 to 4, wherein the genetically modified MSCs express the TNFR2-binding agent at an average level of between 2500 and 250,000 per cell.

6. The population of mesenchymal stem cells according to any one of claims 1 to 5, wherein the TNFR2 binding agent is a fusion protein that binds to TNFR2.

7. The population of mesenchymal stem cells described in claim 6, wherein the fusion protein that binds to TNFR2 includes any one or more 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.

8. The population of mesenchymal stem cells described in claim 6, wherein the fusion protein that binds to TNFR2 includes any one or more selected from the group consisting of a signal peptide, an Fc domain, a binding domain, a cytokine, a half-life extension domain, a growth factor, an enzyme, a cell penetrating domain, a therapeutic peptide and a therapeutic protein.

9. The population of mesenchymal stem cells of any one of claims 1 to 8, wherein the genetically altered MSCs do not express TNFR2 (eg, endogenous TNFR2).

10. 8. The population of mesenchymal stem cells of claim 6 or 7, wherein the TNFR2-binding fusion protein comprises a transmembrane domain derived from the group consisting of CD3, CD4, CD5, CD8, CD28, CD99, PDGFR and PTGFRN, and optionally, the TNFR-binding fusion protein comprises a hinge domain derived from an immunoglobulin (e.g., IgG1, IgG4, IgD, etc.).

11. The population of mesenchymal stem cells according to any one of claims 1 to 10, wherein the MSCs are derived from totipotent stem cells.

12. The population of mesenchymal stem cells according to any one of claims 1 to 11, wherein the MSCs are derived from iPSCs or embryonic stem cells.

13. The population of mesenchymal stem cells according to any one of claims 1 to 12, wherein the MSCs express at least one cell surface marker selected from the group consisting of CD29, CD44, CD73, CD90 and CD105.

14. The population of mesenchymal stem cells according to any one of claims 1 to 13, wherein the population of genetically modified MSCs maintains at least 90% cell surface marker expression after at least 5 passages or at least 15 passages.

15. The population of mesenchymal stem cells according to any one of claims 1 to 14, wherein the MSCs do not express one or more surface markers selected from the group consisting of CD11b, CD14, CD34, CD45, CD79, HLA-DR, TRA-1-60 and TRA-1-81.

16. 15. The population of mesenchymal stem cells of any one of claims 1 to 14, wherein at least 95% of the MSCs in said population are CD73+ and CD105+, and less than 1% express CD45, SSEA-3, TRA-1-60, TRA-1-81 and HLA-DR.

17. The population of mesenchymal stem cells according to any one of claims 3 to 14, wherein the Stefin A protein variant comprises an amino acid sequence having at least 95% identity with the following amino acid: MIP-Xaa1-GLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQV-Xaa2-(Xaa)n-Xaa3-TNYYIKV RAGDNKYMHLKVF-Xaa4-Xaa5-Xaa6-(Xaa)m-Xaa7-D-Xaa8-VLTGYQVDKNKDDELTGF (SEQ ID NO: 4), wherein each Xaa is independently any number of any amino acid residues, n and m each independently represent an integer of 3 to 20.

18. The population of mesenchymal stem cells according to claims 3 to 16, wherein said Stefin A protein variant comprises an amino acid sequence having at least 95% identity with the following amino acid: MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVV-(Xaa)n-GTNYYIKVRAGDNKYMHLKVFKSL-(Xaa)m-EDLVLTGYQVDKNKDDELTGF (SEQ ID NO:487); MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVD-(Xaa)n-GTNYYIKVRAGDNKYMHLKVFKSL-(Xaa)m-EDLVLTGYQVDKNKDDELTGF (SEQ ID NO:5); or MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVLA-(Xaa)n-GTNYYIKVRAGDNKYMHLKVFKSL-(Xaa)m-EDLVLTGYQVDKNKDDELTGF (SEQ ID NO: 488); wherein each Xaa is independently any number of any amino acid residues, n and m each independently represent an integer of 3 to 20.

19. (i) the (Xaa)n comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 6-102; and / or (ii) The mesenchymal stem cell population according to claim 17 or 18, wherein (Xaa)m comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 103 to 199.

20. The population of mesenchymal stem cells according to any one of claims 3 to 19, wherein the Stefin A protein mutant comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 200 to 296.

21. The population of mesenchymal stem cells according to any one of claims 3 to 19, wherein the Stefin A protein variant comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 200 to 296, excluding one or more of the 21 carboxy-terminal residues.

22. The population of mesenchymal stem cells according to any one of claims 3 to 21, wherein the TNFR2 binding agent comprises a trimer or tetramer of a Stefin A protein mutant.

23. The population of mesenchymal stem cells according to any one of claims 1 to 22, wherein the exogenous nucleic acid comprises a transcriptional regulatory sequence operably linked to a coding sequence.

24. 24. The population of mesenchymal stem cells of claim 23, wherein the transcriptional regulatory sequence is a promoter selected from the group consisting of a cytomegalovirus (CMV) promoter, a PGK promoter, an EF1α promoter, an EFS promoter, a CBh promoter, an MSCV promoter, an SFFV promoter and an UbC promoter.

25. The population of mesenchymal stem cells according to any one of claims 1 to 24, wherein the exogenous nucleic acid comprises: (i) an IRES or 2A sequence; and / or (ii) a selection gene.

26. Use of the population of genetically modified mesenchymal stem cells according to any one of claims 1 to 25 for the prevention or treatment of immune diseases.

27. A pharmaceutical composition for preventing or treating an immune disease, comprising a population of genetically modified mesenchymal stem cells according to any one of claims 1 to 25.

28. 28. The use or pharmaceutical composition according to claim 26 or 27, wherein 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) (stem cell transplantation associated) or allograft rejection, transplantation / solid organ transplantation (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 glomerulosclerosis.

29. contacting the MSC population with a lentiviral vector comprising an exogenous nucleic acid comprising a sequence encoding a TNFR2-binding agent; and A method for producing a population of genetically modified mesenchymal stem cells according to any one of claims 1 to 28, comprising the step of culturing said population of MSCs.

30. 30. The method of claim 29, wherein the exogenous nucleic acid comprises an antibiotic selection gene, further comprising the step of selecting cells that express the selection gene.

31. 31. The method of claim 29 or 30, wherein a minimum of 95% of the MSCs in the population produced by said method comprise the exogenous nucleic acid.

32. The method of any one of claims 29 to 31, wherein the population of MSCs and / or the population of genetically modified MSCs do not express TNFR2.

33. The method according to any one of claims 29 to 32, characterized in that MSCs are cultured for at least five passages.

34. The method of claim 33, wherein the expression of cell surface markers is maintained by at least 90% in the population of MSCs after at least 5 passages or at least 15 passages.

35. The method of any one of claims 29 to 33, wherein the TNFR2 binding agent is expressed on the surface of MSCs.

36. A method for treating an immune disease, comprising administering the genetically modified MSC of any one of claims 1 to 28 to a subject in need thereof.

37. 37. The method of claim 36, wherein 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) (stem cell transplantation-associated) or allograft rejection, transplantation / solid organ transplantation (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 glomerulosclerosis.

38. A population of genetically modified cells comprising an exogenous nucleic acid comprising a sequence encoding a TNFR2 binding agent, said TNFR2 binding agent comprising one or more binding domains of an antibody or antibody mimetic.

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

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

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

42. 42. The population of genetically modified cells according to 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 totipotent 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 the group consisting of T cells, B cells, NK cells, monocytes, macrophages, Treg cells and antigen presenting cells (e.g., dendritic cells).

46. The TNFR2 binding agents include Stefin A protein variants, Fab, Fab', F(ab')2, Fv, Fd, scFv, sdFv, VL, VH, Camel Ig, V-NAR, VHH, trispecific (Fab3), bispecific (Fab2), diabody, triabody, tetrabody, minibody, bispecific scFv (Bis-scFv), VNAR (shark heavy-chain-only 46. ​​The population of genetically modified cells of any one of claims 38 to 45, wherein the genetically modified cell is a polypeptide that is a polypeptide of the invention, the polypeptide of the invention being ...

47. 47. The population of genetically modified cells of any one of claims 38 to 46, wherein the TNFR2 binding agent is or comprises a Stefin A protein mutant.

48. 48. The population of genetically modified cells of any one of claims 38 to 47, wherein the TNFR2 binding agent has a Kd value of 1 x 10-6 M or less for TNFR2.

49. 49. The population of genetically modified cells of any one of claims 38 to 48, wherein the TNFR2 binding agent is membrane anchored or expressed on the cell surface.

50. 50. The population of genetically modified cells of any one of claims 38-49, wherein the TNFR2 binding agent is a fusion protein that binds to TNFR2.

51. The population of genetically modified cells described in claim 50, wherein the fusion protein that binds to TNFR2 includes any one or more selected from the group consisting of a transmembrane domain, a hinge domain, a coiled-coil domain, a viral-derived domain, an intracellular signaling domain, and a localization domain.

52. The population of genetically modified cells described in claim 50, wherein the fusion protein that binds to TNFR2 includes any one or more selected from the group consisting of a signal peptide, an Fc domain, a binding domain, a cytokine, a half-life extension domain, a growth factor, an enzyme, a cell-penetrating domain, a therapeutic peptide and a therapeutic protein.

53. 53. The population of genetically modified cells of any one of claims 38-52, wherein the genetically altered cells do not express TNFR2 (e.g., endogenous TNFR2).

54. 53. The population of genetically modified cells of any one of claims 50-52, wherein the TNFR2-binding fusion protein comprises a transmembrane domain derived from the group consisting of CD3, CD4, CD5, CD8, CD28, CD99, PDGFR and PTGFRN, and optionally, the TNFR-binding fusion protein comprises a hinge domain derived from an immunoglobulin (e.g., IgG1, IgG4, IgD, etc.).

55. 55. The population of genetically modified cells of any one of claims 47 to 54, wherein the Stefin A protein variant comprises an amino acid sequence having at least 95% identity with the following amino acid: MIP-Xaa1-GLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQV-Xaa2-(Xaa)n-Xaa3-TNYYIKV RAGDNKYMHLKVF-Xaa4-Xaa5-Xaa6-(Xaa)m-Xaa7-D-Xaa8-VLTGYQVDKNKDDELTGF (SEQ ID NO: 4), wherein each Xaa is independently any number of any amino acid residues, n and m each independently represent an integer of 3 to 20.

56. 55. The population of genetically modified cells of any one of claims 47 to 54, wherein the Stefin A protein variant comprises an amino acid sequence having at least 95% identity with the following amino acid: MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVV-(Xaa)n-GTNYYIKVRAGDNKYMHLKVFKSL-(Xaa)m-EDLVLTGYQVDKNKDDELTGF (SEQ ID NO:487); MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVD-(Xaa)n-GTNYYIKVRAGDNKYMHLKVFKSL-(Xaa)m-EDLVLTGYQVDKNKDDELTGF (SEQ ID NO:5); or MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVLA-(Xaa)n-GTNYYIKVRAGDNKYMHLKVFKSL-(Xaa)m-EDLVLTGYQVDKNKDDELTGF (SEQ ID NO: 488); wherein each Xaa is independently any number of any amino acid residues, n and m each independently represent an integer of 3 to 20.

57. (i) the (Xaa)n comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 6-102; and / or (ii) the (Xaa)m comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 103-199.

58. 55. The population of genetically modified cells of any one of claims 47-54, wherein the Stefin A protein variant comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 200-296.

59. 55. The population of genetically modified cells of any one of claims 47 to 54, wherein the Stefin A protein variant comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 200 to 296, excluding one or more of the 21 carboxy-terminal residues.

60. 60. The population of genetically modified cells of any one of claims 47-59, wherein the TNFR2 binding agent comprises a trimer or tetramer of a Stefin A protein mutant.

61. 61. The population of genetically modified cells of any one of claims 38 to 60, wherein the exogenous nucleic acid comprises a transcriptional regulatory sequence operably linked to a coding sequence.

62. 62. The population of genetically modified cells of claim 61, wherein the transcriptional regulatory sequence is a promoter selected from the group consisting of a cytomegalovirus (CMV) promoter, a PGK promoter, an EF1α promoter, an EFS promoter, a CBh promoter, an MSCV promoter, an SFFV promoter, and an UbC promoter.

63. 63. The population of genetically modified cells of any one of claims 38 to 62, wherein the exogenous nucleic acid comprises: (i) an IRES or 2A sequence; and / or (ii) a selection gene.

64. Use of the population of genetically modified cells according to any one of claims 38 to 36 for the prevention or treatment of an immune disease.

65. A pharmaceutical composition for preventing or treating an immune disease, comprising a population of genetically modified cells according to any one of claims 38 to 36.

66. 66. The use or pharmaceutical composition according to claim 64 or 65, wherein 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) (stem cell transplantation associated) or allograft rejection, transplantation / solid organ transplantation (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 glomerulosclerosis.

67. contacting the cell population with a lentiviral vector comprising an exogenous nucleic acid comprising a sequence encoding a TNFR2 binding agent; and A method for producing a population of genetically modified cells according to any one of claims 38 to 63, comprising the step of culturing said population of cells.

68. 68. The method of claim 67, wherein the exogenous nucleic acid comprises an antibiotic selection gene, further comprising the step of selecting cells that express the selection gene.

69. 69. The method of claim 67 or 68, wherein a minimum of 95% of the MSCs in the population produced by said method comprise the exogenous nucleic acid.

70. The method of any one of claims 67 to 69, wherein the population of MSCs and / or the population of genetically modified MSCs do not express TNFR2.

71. The method of any one of claims 67 to 70, wherein the TNFR2 binding agent is expressed on the surface of MSCs.

72. A method for treating an immune disorder comprising administering to a subject in need thereof a population of genetically modified cells according to any one of claims 38 to 63.

73. 73. The method of claim 72, wherein 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) (stem cell transplantation-associated) or allograft rejection, transplantation / solid organ transplantation (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, Grave's disease, and glomerulosclerosis.

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