Stefin A protein variant that specifically binds to CD40L and uses thereof

Abstract

The present invention relates to a Stefin A protein variant that specifically binds to CD40L and uses thereof, and the Stefin A protein variant that specifically binds to CD40L of the present invention and / or a fusion protein or conjugate containing the same is a non-immunogenic polypeptide and can bind to CD40L with high affinity and specificity, and is therefore useful for targeting CD40L and various cells known to express CD40L. In addition, the Stefin A protein variant that specifically binds to CD40L of the present invention exhibits excellent CD40L activity suppression ability and can be usefully used in medical / pharmaceutical applications for the prevention and treatment of various diseases associated with CD40L.

Description

[Technical field]

[0001] The present invention relates to a Stefin A protein mutant that specifically binds to CD40L and uses thereof.

[0002] [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, as well as the signal transmission and interactions between each cell. The immune system maintains immune homeostasis by appropriately balancing the immune functions of immune tolerance, which suppresses and regulates immunity, and immune response, which promotes immunity. Immune tolerance and immune response can be imbalanced due to various causes, and such immune imbalance can lead to the development of various diseases. For example, when the immune tolerance mechanism becomes strong, cancer development or invasion of external infection sources is likely to occur, which can lead to the development of cancer, infectious diseases, etc., and conversely, when the immune response mechanism becomes strong, it can lead to inflammatory diseases such as allergies and 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 from 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 and 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. In recent years, 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. Cellular therapies based on specialized immune cells such as CAR-T or CAR-NK have limitations due to the high cost and limited targets associated with using autologous cells, while mesenchymal stem cells have limitations such as low therapeutic efficacy compared to the cost of treatment and difficulty in explaining a clear mechanism of action (Blood Cancer J.11,69(2021);World J Stem Cells.2019;11(4):212-221).

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

[0006]

[0007] In various immune homeostasis-related diseases, CD40L has been reported to play an important role in the interaction between antigen-presenting cells (APCs) and T cells. In particular, CD40L / CD40 interaction acts as a pathogenic factor in autoimmune or inflammatory diseases in which activation of T cells and B cells has a major impact on pathology. Specifically, it is a pathogenic factor in various diseases such as type 1 diabetes, thyroiditis, psoriasis, lupus (Systemic Lupus Erythematosus; SLE), rheumatoid arthritis (Rheumatoid Arthritis; RA), multiple sclerosis (Multiple Sclerosis; MS), etc. For the treatment of such diseases, various compounds or antibodies targeting CD40L have been developed (Semin Immunol. 2009; 21(5): 293-300; Advanced Drug Delivery Reviews Volume 141, 15February 2019, Pages 92-103).

[0008] 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, which allows it to bind to a target substance with high affinity and specificity in a manner similar to that of a monoclonal antibody. Affimer® exhibits significantly improved binding affinity and specificity compared to free peptide libraries, and is significantly smaller in size and more stable than antibodies, and is attracting great interest as a next-generation alternative pharmaceutical platform to replace antibodies (U.S. Patent No. 9447170, U.S. Patent No. 8853131, etc.).

[0009]

[0010] Under these circumstances, the present inventors have made intensive efforts to develop a polypeptide that specifically binds to CD40L by engineering based on the naturally occurring protein stefin protein, and as a result, have invented a stefin A protein mutant that can bind to CD40L with excellent affinity and specificity, and in particular, have confirmed that the stefin A protein mutant that specifically binds to CD40L exhibits excellent inhibitory activity against CD40L, thereby completing the present invention.

[0011]

[0012] Summary of the Invention

[0013] An object of the present invention is to provide Stefin A protein mutants that specifically bind to CD40L and uses thereof.

[0014] Another object of the present invention is to provide a fusion protein comprising the Stefin A protein mutant that specifically binds to CD40L.

[0015] It is yet another object of the present invention to provide a conjugate comprising the Stefin A protein mutant that specifically binds to CD40L.

[0016] It is still another object of the present invention to provide a nucleic acid encoding the Stefin A protein mutant that specifically binds to CD40L, a fusion protein or conjugate containing the same, and a vector containing the nucleic acid.

[0017] It is still another object of the present invention to provide a genetically engineered cell into which the nucleic acid and / or vector has been introduced, and uses thereof.

[0018]

[0019] In order to achieve the above-mentioned objects of the present invention, the present invention provides a Stefin A protein variant that specifically binds to CD40L.

[0020] The present invention also provides a fusion protein comprising the Stefin A protein variant that specifically binds to CD40L.

[0021] The present invention also provides a conjugate comprising the Stefin A protein variant that specifically binds to CD40L.

[0022] The present invention also provides a nucleic acid encoding the Stefin A protein variant that specifically binds to CD40L, or a fusion protein containing the same.

[0023] The present invention also provides a delivery vehicle comprising the nucleic acid.

[0024] The present invention also provides a genetically engineered cell into which the nucleic acid has been introduced.

[0025] The present invention also provides the use of said genetically engineered cell for the production of a Stefin A protein variant, a fusion protein or a conjugate.

[0026] The present invention also provides a method for producing a Stefin A protein variant, fusion protein or conjugate that specifically binds to CD40L, comprising the step of culturing the genetically engineered cells.

[0027] The present invention also provides pharmaceutical compositions comprising the Stefin A protein variant, fusion protein, conjugate, nucleic acid and / or delivery vehicle that specifically binds to CD40L.

[0028] The present invention also provides a use of the Stefin A protein variant, fusion protein, conjugate, nucleic acid and / or transfer vehicle that specifically binds to CD40L for the manufacture of a pharmaceutical composition.

[0029] The present invention also provides a method of treatment comprising the step of administering a Stefin A protein variant, a fusion protein, a conjugate, a nucleic acid and / or a delivery vehicle that specifically binds to CD40L.

[0030] [Brief description of the drawings]

[0031] [Figure 1] 1 shows the results of direct binding ELISA of monomeric clones to human CD40L.

[0032] [Diagram 2] Flow cytometry analysis shows that binding of monomeric clones confirmed human CD40L expression from HEK293 cells.

[0033] [Diagram 3] The results show that hCD40L expression is confirmed in hCD40L-HEK293 cells (top) compared with control HEK293 cells (bottom).

[0034] [Figure 4] Binding of clone 230 (SEQ ID NO: 249) to hCD40L-HEK293 cells at different concentrations.

[0035] [Diagram 5]1 shows the dose effect of the Stefin A protein mutants of the present invention that specifically bind to CD40L on blocking the binding of hCD40L to CD40 by CD40L HEK-BLUE reporter cell assay.

[0036] [Figure 6] Figure 1 shows various in line fusions (ILFs) used to increase avidity for CD40L. Concentrations of 1 μg (left) and 5 μg (right) of each format were tested. Each format is indicated on the gel (e.g., monomer, dimer, or trimer).

[0037] [Figure 7] Binding of different clones containing ILF dimeric and trimeric structures to hCD40L using a BIACORE™ assay.

[0038] [Figure 8] Binding of different clones containing ILF dimeric and trimeric structures to hCD40L is demonstrated by flow cytometry.

[0039] [Figure 9] FIG. 1 shows the dose effect of monomeric, dimeric and trimeric Stefin A protein variants or their ILFs on blocking the binding of hCD40L to CD40 by CD40L HEK-BLUE reporter cell assay.

[0040] [Figure 10] Competitive ELISA results for binding of clones of different formats (DT, trimer with rigid linker; XT75, trimer with rigid linker and HSA-binding Stefin A protein mutant; XT76, tetramer with rigid linker and HSA-binding Stefin A protein mutant).

[0041] [Figure 11] The dose effect of different formats (DT, trimer with rigid linker; XT75, trimer with rigid linker and HSA-binding Stefin A protein mutant; XT76, tetramer with rigid linker and HSA-binding Stefin A protein mutant) on blocking hCD40L binding to CD40 by CD40L HEK-BLUE reporter cell assay.

[0042] [Figure 12] The results of hCD40L / HSA bridging ELISAs were performed using clone-230 XT75 (ILF protein containing three clone 230 monomers and an HSA-binding stefin A protein variant (HSA AFFIMER®)) and clone-230 XT76 (ILF protein containing four clone-230 monomers and an HSA-binding stefin A protein variant). 3t0 Gly XT58 is an ILF protein containing a non-hCD40L targeting protein trimer and an HSA-binding stefin A protein variant (HSA AFFIMER®) linked to each other with a rigid linker. 3t0 Gly XT59 is an ILF protein containing two non-hCD40L targeting proteins and an HSA-binding stefin A protein variant (HSA AFFIMER®) linked to each other with a rigid linker. Figure 12A shows Stefin A protein variants binding to bound hCD40L (measured as anti-cystatin antibodies), Figure 12B shows Stefin A protein variants binding to bound hCD40L in the presence of HSA (measured as anti-cystatin antibodies), and Figure 12C shows Stefin A protein variants binding to bound hCD40L and HSA (measured as anti-HSA antibodies).

[0043] [Figure 13] FIG. 1 is a schematic diagram of a haploidentical GVHD mouse model.

[0044] [Figure 14] 1 shows the number of doses and intervals of doses of anti-CD40L Stefin A protein mutants (XT54, sequence number: 729; XT55, sequence number: 730) in a haploidentical GVHD mouse model.

[0045] [Figure 15] The efficacy of anti-CD40L Stefin A protein mutants (XT54, sequence number: 729; XT55, sequence number: 730) is shown in terms of weight change (top) and GVHD clinical score (bottom) using a haploidentical GVHD mouse model.

[0046]

[0047] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0048] Unless otherwise defined, 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.

[0049] As used herein, the term "protein" or "polypeptide" refers to a polymer of amino acids of any length. The polymer may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acids. In addition, the term protein or polypeptide includes amino acid polymers that have been modified naturally or by intervention. For example, it is intended to include polypeptides that contain at least one analog of an amino acid (including, for example, unnatural amino acids), as well as other modifications known in the art, such as disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling moiety.

[0050] For the most part, 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).

[0051] The specific amino acid residues described herein include not only natural 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, dienkolic acid, norleucine, 3-phosphoserine, homoserine, dihydroxy-phenylalanine, 5-hydroxytryptophan, 1-methylhistidine, 3-methylhistidine, diaminopimelic acid, ornithine, or diaminobutyric acid.

[0052] In the amino acid or nucleic acid sequences described herein, the term "identical" or "identity" expressed as a percentage 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 the present invention, two nucleic acid or amino acid sequences may be "substantially identical", which may mean that the two sequences match at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, the identity may be over a length of at least about 10, 20, 40-60, 60-80, 80-100 or more residues.

[0053] The proteins or polypeptides described herein, for example, Stefin A protein variants, fusion proteins, and fusion protein configurations, may include, in addition to the amino acid sequences described in connection therewith, proteins or polypeptides in which parts of the amino acid sequences have been substituted by conservative substitutions.

[0054] As used herein, "conservative substitution" refers to a modification of a polypeptide that involves replacing one or more amino acids with amino acids having similar biochemical properties that do not result in the loss of biological or biochemical function of the polypeptide. A "conservative amino acid substitution" is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Classes of amino acid residues having similar side chains are defined and well known in the art. These classes include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and 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 result in loss of their function. For example, Stefin A variants that specifically bind to CD40L do not abolish binding to CD40L due to the conservative substitutions. Methods for identifying such conservative substitutions are well known in the art.

[0055] 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. A material is considered to be substantially pure if it is at least 50% pure (e.g., free of contaminants), at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure. "CD40L (Cluster of Differentiation 40 ligand)" is a protein that, in humans, is encoded by the CD40L gene, and CD40L acts as a ligand for CD40 / TNFRSF5 and co-stimulates T cell proliferation and cytokine production. Cross-linking to T cells, together with TCR / CD3 ligation and CD28 costimulation, generates costimulatory signals that enhance the production of IL4 and IL10. CD40L induces activation of NK-kappa-B, and the activation of kinases MAPK8 and PAK2 in T cells, and induces tyrosine phosphorylation of CD28 isoform 3. CD40L also mediates B-cell proliferation in the presence of IL4 and in the absence of costimulation, as well as the production of IGE. B cells can present antigen to T cells, and if activated T cells recognize peptides presented by B cells, CD40L on T cells binds to CD40 on B cells, inducing B cell activation. T cells also produce IL-4, which directly affects B cells. As a result of this stimulation, B cells can undergo rapid cell division to form germinal centers where differentiation into plasma cells and memory B cells occurs, as well as antibody isotype switching and affinity maturation. The end result is a B cell that can mass-produce specific antibodies against antigen targets. Initial evidence for such an effect is that in CD40- or CD154-deficient mice, there is little class switching or germinal center formation and immune responses are severely suppressed.CD40L is also involved in immunoglobulin class switching.

[0056] Activation of endothelial cells by CD40L (e.g., in activated platelets) induces reactive oxygen species production, chemokine and cytokine production, and expression of adhesion molecules such as E-selectin, ICAM-1, and VCAM-1. Such inflammatory responses in endothelial cells can promote leukocyte recruitment to the lesion and potentially promote atherogenesis. CD40L has also been reported as a potential biomarker for atherosclerotic instability.

[0057] Although CD40L is generally considered to be a membrane-bound protein, naturally occurring proteolytically cleaved 15-18-kDa soluble forms of CD40L with full biological activity have been reported. CD40L can bind to the CD40 receptor on B cells and dendritic cells to provide important helper T cell signals required for germinal center formation, isotype class switching, and immunoglobulin antibody production. CD40L deficiency is the cause of immunoglobulin switch defects characterized by X-linked immune deficiency with high IgM (HIGM1), elevated IgM concentrations, and reduced levels of all other isotypes. CD40L knockout mice are unable to mount secondary antibody responses to T cell-dependent antigens and undergo isotype class switching. CD40L can also transmit signals back to T cells to induce short-term T cell proliferation. A role for CD40L in the proliferation of epithelial, fibroblastic, and smooth muscle cells has also been reported.

[0058]

[0059] In the present invention, we have invented a Stefin A protein mutant that can bind to CD40L with excellent affinity and specificity, and in particular, we have confirmed that the Stefin A protein mutant that specifically binds to CD40L exhibits excellent inhibitory activity against CD40L.

[0060] Thus, in one aspect, the present invention relates to a Stefin A protein variant that specifically binds to CD40L.

[0061]

[0062] Stefin A protein variant that specifically binds to CD40L

[0063] The term "Stefin A protein variant" according to the present invention refers to a scaffold of Stefin A protein, a sequence derived from Stefin A protein, for example Stefin A protein derived from a mammal, more preferably from a human. In the present invention, the term "Stefin A protein variant" may be used interchangeably with "Stefin A polypeptide variant" or "AFFIMER® protein" with substantially the same meaning.

[0064] In one embodiment of the present invention, a Stefin A protein variant that specifically binds to CD40L has been developed, and the term "Stefin A protein variant that specifically binds to CD40L" may be used interchangeably with "anti-CD40L Stefin A protein variant" and "anti-CD40L Affimer" to mean essentially the same thing.

[0065] "Stefin Protein" or "Stefin Polypeptides" includes a subgroup of proteins in the cystatin superfamily 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. They exhibit identical folding structures in various extracellular and intracellular environments due to the lack of post-translational modifications and the low number of disulfide bonds. Stefin A protein has been reported as a single entity, single chain, single domain protein composed of 98 amino acids. Avacta has developed the Affimer® platform using a mutational protein engineering approach 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.

[0066] The term "Stefin A protein variant" of the present invention refers to an engineered variant of a 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 sequence that allows 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 portions of the stefin A protein sequence may be made, which may improve properties such as increased stability against temperature and pH. In the present invention, the stefin A protein variant may include a sequence derived from a stefin A protein that shares substantial identity with a wild-type stefin A protein sequence, such as human stefin A protein. In particular, the Stefin A protein variant of the present invention may have at least 25%, 35%, 45%, 55% or 60% identity to the sequence corresponding to human Stefin A, e.g., 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., CD40L), e.g., do not restore or generate a biological function possessed by wild-type Stefin A but abolished in the mutational changes described herein. Such target protein-specific binding platforms utilizing Stefin A protein variants are disclosed in detail in U.S. Pat. No. 9,447,170, U.S. Pat. No. 8,853,131, etc.

[0067] In the present invention, the Stefin A protein mutant is characterized in that it is capable of binding to the target protein, CD40L, with high affinity and specificity by engineering the Stefin A protein.

[0068] In the present invention, a Stefin A protein mutant that specifically binds to CD40L may be characterized by specifically binding to CD40L and reducing or suppressing the activity of CD40L.

[0069] According to the present invention, the term "CD40L" or "CD40 ligand" refers to a protein that binds to its receptor, CD40, also named "CD154". CD40L may be used interchangeably with the terms TNF-Related Activation Protein, TRAP, Tumor Necrosis Factor (Ligand) Superfamily Member, TB Cell-Activating Molecule, CD40 Antigen Ligand, T- Cell Antigen Gp39, TNFSF5, HCD40L, CD154, Gp39, Tumor Necrosis Factor (Ligand) Superfamily Member 5 (Hyper-IgM Syndrome), Tumor Necrosis Factor (Ligand) Superfamily Hyper-IgM Syndrome, CD154, CD40LG, HIGM1, T-BAM, IMD3, IGM, and CD40-L, etc. Human CD40L amino acid and nucleic acid sequences can be found in public databases such as GenBank, UniProt, and Swiss-Prot. For example, the amino acid sequence of human CD40L can be found in UniProt / Swiss-Prot. Accession No. P29965, and the nucleic acid sequence encoding it can be found in Accession No. NM_000074.2.

[0070] The CD40L includes any native CD40L and mature CD40L produced in cells from CD40L precursor protein. The CD40L includes CD40L obtained from any organism, preferably an animal, unless otherwise indicated, including, but not limited to, CD40L obtainable from mammals such as primates (e.g., humans and cynomolgus monkeys) and rodents (e.g., mice and rats). The CD40L also includes, but is not limited to, any CD40L protein including mutations, such as point mutations, fragments, insertions, deletions, and splice variants of full-length wild-type CD40L.

[0071] In the present invention, the Stefin A protein mutant may be characterized in that it has at least one solvent accessible loop derived from the wild-type Stefin A protein which is capable of binding to CD40L.

[0072] In the present invention, the Stefin A protein mutant has a specific binding affinity to CD40L of 1×10 -6 It may be characterized by being capable of binding with a Kd value of M or less.

[0073] In the present invention, the Stefin A protein mutant may be characterized as being a mutant derived from Stefin A protein having a backbone sequence, in which any one or more of loop 2 and loop 4 are replaced with alternative loop sequences, (Xaa)n and (Xaa)m.

[0074] In the present invention, the Stefin A protein variant may be characterized in that it comprises an amino acid sequence as shown in formula I:

[0075] [Formula I]

[0076] FR1-(Xaa)n-FR2-(Xaa)m-FR3

[0077] wherein the FR1 may be characterized by comprising an amino acid sequence represented by MIPGGLSEAK PATPEIQEIV DKVKPQLEEK TGETYGKLEA VQYKTQVX (SEQ ID NO: 1), or 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 with the amino acid sequence of SEQ ID NO: 1, wherein the X in SEQ ID NO: 1 may be V;

[0078] The FR2 may be characterized by comprising an amino acid sequence set forth in 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;

[0079] The FR3 may be characterized as comprising 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

[0080] Xaa, in each occurrence, is individually any amino acid residue; n and m are each independently an integer from 3 to 20.

[0081] In the present invention, (Xaa)n and (Xaa)m may each independently be any amino acid sequence of 3 to 20 amino acids.

[0082] In the present invention, the FR1 may be characterized by having an amino acid sequence having at least 80%, 85%, 90%, 95%, or 98% homology with SEQ ID NO: 1. In the present invention, the FR1 may be characterized by having an amino acid sequence having at least 80%, 85%, 90%, 95%, or 98% identity with SEQ ID NO: 1.

[0083] In the present invention, the FR2 may be characterized by having an amino acid sequence having at least 80%, 85%, 90%, 95%, or 98% homology with SEQ ID NO: 2. In the present invention, the FR2 may be characterized by having an amino acid sequence having at least 80%, 85%, 90%, 95%, or 98% identity with SEQ ID NO: 2.

[0084] In the present invention, the FR3 may be characterized by having an amino acid sequence having at least 80%, 85%, 90%, 95%, or 98% homology with SEQ ID NO: 3. In the present invention, the FR3 may be characterized by having an amino acid sequence having at least 80%, 85%, 90%, 95%, or 98% identity with SEQ ID NO: 3.

[0085] In the present invention, said Stefin A protein variant may be characterized in that it comprises the amino acid sequence shown in formula II (SEQ ID NO: 4):

[0086] [Formula II]

[0087] MIP-Xaa1-GLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVV-(Xaa)n-Xaa2-TNYYIKVRAGDNKYMHLKVF-Xaa3-Xaa4-Xaa5-(Xaa)m-Xaa6-D-Xaa7-VLTGYQVDKNKDDELTGF (SEQ ID NO: 4)

[0088] wherein Xaa, in each occurrence, is individually any amino acid residue; n and m are each independently an integer from 3 to 20.

[0089] In the present invention, the Stefin A protein variant may be characterized in that it comprises 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 amino acid sequence set forth in SEQ ID NO:5 below:

[0090] MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVD-(Xaa)n-GTNYYIKVRAGDNKYMHLKVFKSL-(Xaa)m-EDLVLTGYQVDKNKDDELTGF (SEQ ID NO:5),

[0091] wherein Xaa, in each occurrence, is individually any amino acid residue; n and m are each independently an integer from 3 to 20.

[0092] In the present invention, said Xaa1 may be characterized as being Gly, Ala, Val, Arg, Lys, Asp or Glu, preferably Gly, Ala, Arg or Lys, more preferably Gly or Arg.

[0093] In the present invention, said Xaa2 may be characterized as being Gly, Ala, Val, Ser or Thr, preferably Gly or Ser.

[0094] In the present invention, Xaa3 may be characterized as being Arg, Lys, Asn, Gln, Ser or Thr, preferably Arg, Lys, Asn or Gln, more preferably Lys or Asn.

[0095] In the present invention, said Xaa4 may be characterized as being Gly, Ala, Val, Ser or Thr, preferably Gly or Ser.

[0096] In the present invention, said Xaa5 may be characterized as being Ala, Val, Ile, Leu, Gly or Pro, preferably Ile, Leu or Pro, more preferably Leu or Pro.

[0097] In the present invention, said Xaa6 may be characterized as being Gly, Ala, Val, Asp or Glu, preferably Ala, Val, Asp or Glu, more preferably Ala or Glu.

[0098] In the present invention, said Xaa7 may be characterized as being Ala, Val, Ile, Leu, Arg or Lys, preferably Ile, Leu or Arg, more preferably Leu or Arg.

[0099] In the present invention, n may be an integer of 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.

[0100] In the present invention, m may be an integer of 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.

[0101] In the present invention, said Xaa may each independently be any amino acid that can be added to a polypeptide upon expression in a prokaryotic or eukaryotic cell, preferably one of the 20 naturally occurring amino acids.

[0102] In the sequences and formulas of the present invention, the (Xaa)n may be characterized in that it comprises an amino acid sequence selected from SEQ ID NOs: 6 to 125, or a sequence having at least 80%, 85%, 90%, 95% or 98% homology to the amino acid sequence of SEQ ID NOs: 6 to 125. In the present invention, the (Xaa)n may be characterized in that it comprises an amino acid sequence selected from SEQ ID NOs: 6 to 125, or a sequence having at least 80%, 85%, 90%, 95% or 98% identity to the amino acid sequence selected from SEQ ID NOs: 6 to 125.

[0103] [Table 1]

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[0105] JPEG2025509078000004.jpg237128

[0106] JPEG2025509078000005.jpg107128

[0107] In the sequences and formulas of the present invention, the (Xaa)m may be characterized in that it comprises an amino acid sequence selected from SEQ ID NOs: 126 to 245, or a sequence having at least 80%, 85%, 90%, 95% or 98% homology to an amino acid sequence selected from SEQ ID NOs: 126 to 245. In the present invention, the (Xaa)m may be characterized in that it comprises an amino acid sequence selected from SEQ ID NOs: 126 to 245, or a sequence having at least 80%, 85%, 90%, 95% or 98% identity to an amino acid sequence selected from SEQ ID NOs: 126 to 245.

[0108] [Table 2]

[0109] JPEG2025509078000007.jpg237128

[0110] JPEG2025509078000008.jpg238128

[0111] JPEG2025509078000009.jpg91128

[0112] In the present invention, the Stefin A protein mutant may be characterized by comprising an amino acid sequence selected from SEQ ID NOs: 246-365.

[0113] In the present invention, the Stefin A protein mutant may be characterized by comprising a sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 98% identity to an amino acid sequence selected from SEQ ID NOs: 246 to 365.

[0114] [Table 3]

[0115] JPEG2025509078000011.jpg223153

[0116] JPEG2025509078000012.jpg224154

[0117] JPEG2025509078000013.jpg227154

[0118] JPEG2025509078000014.jpg223151

[0119] JPEG2025509078000015.jpg224153

[0120] JPEG2025509078000016.jpg203154

[0121] Nucleic acid encoding a Stefin A protein variant that specifically binds to CD40L

[0122] The term "nucleic acid" according to the present invention refers to a polynucleotide of any length and may include DNA, RNA, or a combination of DNA and RNA. In the present invention, the nucleic acid may be characterized as being deoxyribonucleotides, ribonucleotides, as well as modified nucleotides or bases, and / or their analogs, or any substrate that may be incorporated by DNA or RNA polymerase. In the present invention, the nucleic acid may 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 (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.

[0123] The term "encoding nucleic acid" as used herein refers to a nucleic acid sequence that codes for a specific protein or polypeptide. When the sequence of a specific protein or polypeptide is published, it is well known in the art how to design or derive a nucleic acid that codes for it.

[0124] Therefore, the nucleic acid encoding the Stefin A protein variant of the present invention that specifically binds to CD40L can be easily understood from the above description of "Stefin A protein variant that specifically binds to CD40L."

[0125] In the present invention, the Stefin A protein variant that specifically binds to CD40L may be characterized in that it is encoded by a nucleic acid comprising a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 98% identity to a nucleic acid sequence selected from SEQ ID NOs: 366 to 485.

[0126] In one embodiment of the present invention, a Stefin A protein variant that specifically binds to CD40L may be characterized in that it is encoded by a nucleic acid comprising a coding sequence that hybridizes to a nucleic acid sequence selected from SEQ ID NOs: 366 to 485. In the present invention, the hybridization may be characterized in that it is performed, for example, at 45° C. in the presence of 6× sodium chloride / sodium citrate (SSC), followed by washing at 65° C. with 0.2× SSC.

[0127] [Table 4]

[0128] JPEG2025509078000018.jpg229154

[0129] JPEG2025509078000019.jpg231154

[0130] JPEG2025509078000020.jpg231154

[0131] JPEG2025509078000021.jpg233154

[0132] JPEG2025509078000022.jpg232155

[0133] JPEG2025509078000023.jpg226152

[0134] JPEG2025509078000024.jpg234162

[0135] JPEG2025509078000025.jpg229152

[0136] JPEG2025509078000026.jpg223150

[0137] JPEG2025509078000027.jpg230153

[0138] JPEG2025509078000028.jpg230153

[0139] JPEG2025509078000029.jpg225153

[0140] JPEG2025509078000030.jpg234155

[0141] JPEG2025509078000031.jpg232154

[0142] JPEG2025509078000032.jpg228152

[0143] JPEG2025509078000033.jpg125146

[0144] In the present invention, in addition to the characteristics of loop 2 and loop 4 described above, the Stefin A protein mutant may also include a partial deletion or addition to the Stefin A protein or its mutant sequence described in the specification (e.g., addition or deletion of about 10 amino acids compared to the wild-type Stefin A protein).

[0145] In the present invention, the Stefin A protein mutant may be characterized by having a dissociation constant (KD) for human CD40L 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.

[0146] In the present invention, the Stefin A protein mutant is TM At least about 10 for human CD40L, as measured by such assays -3 s -1 , 10 -4 s -1 , or 10 -5 s -1 , 10 -6 s -1 Or, it may be characterized as having an off-rate constant Koff that is slower than any one of these.

[0147] In the present invention, the Stefin A protein mutant is TM At least about 10 for human CD40L, as measured by such assays 3 M -1 s -1 , 10 4 M -1 s -1 , 10 5 M -1 s -1 , 10 6 M -1 s -1 Or, it may be characterized by having a Kon (association constant) value faster than any one of these.

[0148] In the present invention, the Stefin A protein variant may be characterized by having an IC50 value of about 1 μM or less, about 100 nM or less, about 40 nM or less, about 20 nM or less, about 10 nM or less, about 1 nM or less, or about 0.1 nM or less against human CD40L in a competitive binding assay.

[0149] In the present invention, the Stefin A protein variant may be characterized by having a melting temperature (Tm, the temperature at which the folded and unfolded states are the same) of about 65° C. or higher, about 70° C. or higher, about 75° C. or higher, about 80° C. or higher, or about 85° C. or higher, about 65° C. or higher. The relative proportion of folded and unfolded protein may be measured by various methods known in the art ("Measuring the conformational stability of a protein" in Protein structure: A practical approach 2:299-321).

[0150]

[0151] In the present invention, the Stefin A protein mutant may be characterized in that it forms a multimer.

[0152] In the present invention, the Stefin A protein mutant may be characterized in that it forms a dimer, a triplet, a tetramer, a quintuplet or higher multimer.

[0153] In the present invention, the multimer may be formed by covalent or non-covalent binding through interactions between amino acid residues of the Stefin A protein mutant.

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

[0155] In the present invention, the multimer may be characterized as being formed by in-line fusion of Stefin A protein mutants, and such a multimer form of an in-line fusion protein is described in detail in the fusion protein section below.

[0156]

[0157] Fusion proteins

[0158] In another aspect, the present invention relates to a fusion protein comprising a Stefin A protein variant that specifically binds to CD40L.

[0159] In still another aspect, the present invention relates to use of the Stefin A protein variant that specifically binds to CD40L for producing a fusion protein.

[0160] In the present invention, the fusion protein may be characterized in that a peptide containing one or more amino acids is added, substituted, and / or deleted to adjust the biological activity of the Stefin A protein variant or to add further biological activity. For example, the Stefin A protein variant may be fused to another protein for adjusting the binding affinity to CD40L, adjusting the half-life, adjusting the stability, adjusting the cleavage by proteases, adjusting the dosage, adjusting the release or bioavailability, facilitating purification, reducing deamidation, improving storage stability, or changing the administration route. For example, the fusion protein may further include, in addition to the Stefin A protein variant, a protease cleavage sequence, a reactive functional group, an antibody binding domain, a fusion domain for protein expression and purification (e.g., FLAG, poly-His, GST, c-myc, etc.), a binding molecule (e.g., biotin, etc.), or another therapeutic peptide or protein, but is not limited thereto.

[0161] In the present invention, the fusion protein may be characterized in that an additional peptide sequence (fusion domain) is fused to one and / or the other end of the Stefin A protein mutant.

[0162] The term "fusion domain" according to the present invention refers to an additional domain or moiety that may be included, directly or indirectly fused to the Stefin A protein variant of the present invention that specifically binds to CD40L.

[0163] In the present invention, for example, the fusion domain may be fused to a protein or peptide having a function, such as, but not limited to, imparting expression characteristics such as secretion from a cell, or anchoring to the cell surface, or intracellular localization; adding a substrate or other recognition sequence for post-translational modification; generating multimeric structures that are aggregated by protein-protein interactions; extending or decreasing half-life; or altering tissue localization, tissue exclusion, or other ADME properties.

[0164] In the present invention, a signal sequence may be included in the fusion protein for transport and secretion of the Stefin A protein variant. In the present invention, when the Stefin A protein variant is to be expressed in a membrane-anchored or cell surface form, the fusion protein may include a transmembrane domain or a cell surface-retaining signal sequence.

[0165] In the present invention, the signal sequence (signal peptide or leader sequence) may be located at the N-terminus of the Stefin A protein variant. The signal sequence functions to target the Stefin A protein variant to the endoplasmic reticulum for secretion. In general, the signal sequence may be cleaved after transport to the endoplasmic reticulum, and may be characterized by being cleaved at a residue within the signal sequence, but is not limited thereto.

[0166] In the present invention, the signal sequence may be characterized by being 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, or about 25 to about 30, about 30 to about 35, or about 35 to about 40 amino acids in length), but is not limited thereto.

[0167] In the present invention, the signal sequence may be characterized, for example, as a native signal peptide of human origin. In the present invention, the signal sequence may be characterized, for example, as a non-natural signal peptide. The non-natural signal peptide may be characterized, for example, as a variant comprising one or more substitutions, insertions and / or deletions in a signal peptide derived from a corresponding native secreted human protein.

[0168] In the present invention, the signal sequence may be characterized as being, but is not limited to being, a signal peptide or a variant thereof from the same non-igSF protein family as a signal sequence from an immunoglobulin (e.g., IgG heavy chain or IgG-kappa light chain), a cytokine (e.g., IL-2 or CD33), plasma albumin, a human azurocidin preprotein signal sequence, luciferase, trypsinogen, chymotrypsinogen or other secreted protein.

[0169] In the present invention, the following Table 5 lists examples of signal peptides that can be used for secretion of the Stefin A protein mutant of the present invention or its fusion protein, but the present invention is not limited thereto.

[0170] [Table 5]

[0171] In yet another example, the fusion domain may be fused for isolation and / or purification of the fusion protein. Fusion domains for such uses may be affinity tags such as, but are not limited to, 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.

[0172]

[0173] In the present invention, the fusion protein may be characterized by comprising at least one linker linking the Stefin A protein variant and the fusion domain. In the present invention, the "linker" is inserted between the first polypeptide (e.g., the Stefin A protein variant that specifically binds to CD40L) and the second polypeptide (e.g., further Stefin A protein variant or the fusion domain) of the fusion protein of the present invention.

[0174] 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.

[0175] In the present invention, the linker may have additional functions such as improved biological activity, increased expression yield, and improved pharmacokinetic profile in addition to fusing the Stefin A protein mutant of the present invention with a functional domain.

[0176] In the present invention, it is preferred that the linker does not negatively affect the expression, secretion or functional activity of the respective domains of the fusion protein. In the present invention, it is preferred that the linker does not exhibit immunogenicity.

[0177] In the present invention, the linker may be characterized as being preferably a GS linker containing glycine and serine residues, but is not limited thereto, and the linker may contain threonine-alanine residues in addition to the glycine-serine linker, but is not limited thereto.

[0178] In the present invention, the linker may be characterized by being, for example, about 1 to 50 amino acids in length, 1 to 22 amino acids in length, 1 to 10 amino acids in length, 1 to 5 amino acids in length, or 1 to 3 amino acids in length, but is not limited thereto.

[0179] In the present invention, the linker may comprise a cleavage site.

[0180] In the present invention, the linker may be characterized as being a flexible linker. The flexible linker is preferably used when the fusion domain bound to the Stefin A protein variant of the present invention requires a certain degree of movement or interaction, but is not limited thereto. In the present invention, examples of the flexible linker are described in Argos P. (1990) "An investigation of oligopeptides linking domains in protein tertiary structures and possible candidates for general gene fusion" J Mol Biol. 211: 943-958, but are not limited thereto. In the present invention, the flexible linker confers flexibility to the fusion protein and mobility to the linked fusion domain. The inclusion of serine or threonine may be characterized as maintaining the stability of the linker in an aqueous solution. The most commonly used flexible linker is mainly a GS linker, which may be characterized as being represented by (Gly-Gly-Gly-Gly-Ser)n (SEQ ID NO: 508), but is not limited thereto. In SEQ ID NO: 508, n is an integer of 1 or more, and may be optimized for appropriate distance and interaction of the fusion domain by adjusting the number of repeats of Gly-Gly-Gly-Gly-Ser. The flexible linker may further include amino acids such as Thr, Ala, Lys, and Glu in addition to Gly and Ser.

[0181] In the present invention, the linker may be characterized as being a rigid linker. A flexible linker has the advantage of linking functional fusion domains and allowing a certain degree of movement, but the lack of rigidity of the linker may act as a limiting factor in certain fusion protein embodiments, such as expression yield or biological activity. In this case, a rigid linker may be used to maintain a fixed distance between each domain of the fusion protein and maintain independent functions.

[0182] Most 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. Therefore, a stiff α-helical linker may be used as a rigid spacer 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, thereby 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 may be used when spatial separation of domains is important to preserve the stability or biological activity of the fusion protein. In this regard, an alpha helix-forming linker having the sequence (EAAAK)n (SEQ ID NO:509) is commonly used in the construction of many fusion proteins, with other examples of rigid linkers including, but not limited to, Pro-rich sequences (XP)n, where X is any amino acid, preferably Ala, Lys, or Glu.

[0183] In the present invention, Table 6 below lists examples of linkers that can be used for secretion of the Stefin A protein mutant of the present invention or its fusion protein, but is not limited thereto.

[0184] [Table 6]

[0185] Further examples of linkers that can be used in the fusion proteins of the present invention include, but are not limited to, the following:

[0186] SerGly, GGSG (SEQ ID NO: 519), GSGS (SEQ ID NO: 520), GGGS (SEQ ID NO: 521), S(GGS)n (SEQ ID NO: 522) (n is 1 to 7), GRA, poly(Gly), poly(Ala), GGGSGGG (SEQ ID NO: 523), ESGGGGVT (SEQ ID NO: 524), LESGGGGVT (SEQ ID NO: 525), GRAQVT (SEQ ID NO: 526), ​​WRAQVT (SEQ ID NO: 527), and ARGRAQVT (SEQ ID NO: 528).

[0187] In the present invention, when the Stefin A protein mutant is fused to an Fc domain, the hinge regions may be regarded as a linker.

[0188] In the present invention, the fusion protein may be characterized in that it comprises one or more linker domains selected from the following:

[0189] SGTTSGTTRLLSGHTCFTLTGLLGTLVTMGLLT(SEQ ID NO:529)

[0190] SGTSPGLSAGATVGIMIGVLVGVALI(SEQ ID NO:530)

[0191] SAPVLSAVATVGITIGVLARVALI(SEQ ID NO:531)

[0192] SSPDLSAGTAVSIMIGVLAGMALI(SEQ ID NO:532)

[0193] TLGGNSASYTFVSLLFSAVTLLLLC(SEQ ID NO:533)

[0194] SGTSPGLSAGATVGIMIGVLVGVALI(SEQ ID NO:534)

[0195] Yet another variation applicable to the flanking polypeptide moiety provided as part of the fusion protein in the present invention is that it contains one or more sequences as sites for enzymatic post-translational modification, which may be characterized as, but are not limited to, domains for modifications such as glycosylation, acetylation, acylation, lipid modification, palmitoylation, addition of palmitate, phosphorylation, glycolipid conjugation, etc.

[0196]

[0197] In the present invention, the fusion protein may be characterized in that it is localized to a specific organ or location within a cell.

[0198] In the present invention, the fusion protein may be characterized as being a secreted fusion protein and / or a membrane-anchored fusion protein.

[0199]

[0200] In the present invention, the fusion protein may be characterized in that it further comprises one or more fusion domains.

[0201] In 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.

[0202]

[0203] In the present invention, the fusion protein may further comprise a therapeutic peptide or protein.

[0204] In the present invention, the fusion domain may be characterized in that it further comprises a therapeutic peptide or protein.

[0205] In 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 the present invention, the therapeutic peptide or protein may be a Stefin A protein mutant (which may be the same as or different from the Stefin A protein mutant that specifically binds to CD40L of the present invention), and may also include, without limitation, peptides and proteins reported in the art as having a preventive or therapeutic effect against a specific disease.

[0206]

[0207] In the present invention, the fusion protein may be characterized in that it comprises a binding domain (or binding moiety).

[0208] In the present invention, when the fusion protein contains a binding domain, it may be characterized as having multispecificity capable of binding to one or more target molecules in addition to CD40L.

[0209] In the present invention, the binding domain may be characterized as being selected from the group consisting of, for example, a Stefin A protein mutant (which may be the same as or different from the Stefin A protein mutant of the present invention that specifically binds to CD40L), an antibody or a fragment thereof, an antibody analog, an antigen-binding peptide, a ligand-binding site of a receptor (e.g., a receptor trap polypeptide), a receptor-binding ligand (e.g., a cytokine, a growth factor), an engineered T-cell receptor, and an enzyme or a catalytic fragment thereof, but is not limited thereto.

[0210] Further examples of the binding domain in the present invention include, but are not limited to, adnectins / monobodies, affilins, affibodies, affitins, anticalins, atrimers, avimers, bicyclic peptides, C7 peptide, centyrin, carbohydrate-binding modules (CBM), Cys-knots, darpin, El-tandem, fynomers, knottin, Kunitz domains, O-bodies, Pronectin, ScFv, Sac7d, Sso7d, and Tn3.

[0211] In the present invention, the fusion domain fused to the Stefin A protein variant may be the same or a different Stefin A protein variant that specifically binds to CD40L and / or may be a Stefin A protein variant that specifically binds to another target.

[0212] In the present invention, when the fusion domain is a Stefin A protein mutant that specifically binds to CD40L, the two Stefin A protein mutants contained in the fusion protein of the present invention may be characterized in that they bind to the same or different sites on CD40L. In the present invention, the fusion protein may be characterized in that it binds to two sites (biparatopic) or two or more sites (multiparatopic) on CD40L.

[0213] The term "antibody" as used herein includes complete antibody forms that specifically bind to a target (antigen) as well as antigen-binding fragments of the antibody molecule. The complete antibody has a structure with two full-length light chains and two full-length heavy chains, with each light chain linked to a heavy chain by a disulfide bond. The term "heavy chain" as used herein means either a full-length heavy chain or a fragment thereof, including a variable region domain VH containing an amino acid sequence with sufficient variable region sequence to confer specificity to an antigen, and three constant region domains CH1, CH2, and CH3. The term "light chain" as used herein means either a full-length light chain or a fragment thereof, including a variable region domain VL containing an amino acid sequence with sufficient variable region sequence to confer specificity to an antigen, and a constant region domain CL. The whole antibody includes IgA, IgD, IgE, IgM, and IgG subtypes, and in particular, IgG includes IgG1, IgG2, IgG3, and IgG4. Heavy chain constant regions are of gamma (γ), mu (μ), alpha (α), delta (δ) and epsilon (ε) types, with subclasses gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3), gamma 4 (γ4), alpha 1 (α1) and alpha 2 (α2), whereas light chain constant regions are of kappa (κ) and lambda (λ) types.

[0214] Antigen-binding fragment of an antibody or antibody fragment means a fragment that has an antigen-binding function, and includes Fab, F(ab'), F(ab')2, and Fv. Among antibody fragments, Fab has a structure that has 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 one or more cysteine ​​residues at the C-terminus of the heavy chain CH1 domain. F(ab')2 is generated when the cysteine ​​residues in the hinge region of Fab' form disulfide bonds.

[0215] Fv corresponds to the minimum antibody fragment having only the heavy chain variable region and the light chain variable region. In a two-chain Fv, the heavy chain variable region and the light chain variable region are linked by a non-covalent bond, and in a single-chain Fv (single-chain Fv, scFv), the heavy chain variable region and the light chain variable region are generally linked by a covalent bond via a peptide linker or directly at the C-terminus, so that they can form a dimer-like structure like the two-chain Fv. Such antibody fragments can be produced by using proteolytic enzymes (for example, Fab can be obtained by restrictively cleaving an intact antibody with papain, or F(ab')2 can be obtained by cleaving with pepsin) or by using gene recombination technology.

[0216] An "Fv" fragment is an antibody fragment that contains a complete antigen recognition and binding site. Such a region is a dimer of one heavy-chain variable domain and one light-chain variable domain.

[0217] 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.

[0218] "Single-chain Fv (scFv)" antibody fragments are structures consisting of a single polypeptide chain comprising the VH and VL domains of an antibody and may further comprise a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding.

[0219] In one embodiment, the antibodies of the present invention include, but are not limited to, monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, scFv, Fab fragments, F(ab')2 fragments, disulfide-linked Fvs (sdFv) and anti-idiotypic (anti-Id) antibodies, or epitope-binding fragments of these antibodies.

[0220] The heavy chain constant region may be selected from any one of the gamma (γ), mu (μ), alpha (α), delta (δ) or epsilon (ε) isotypes. For example, the constant region is gamma 1 (IgG1), gamma 2 (IgG2), gamma 3 (IgG3) or gamma 4 (IgG4). The light chain constant region may be of the kappa or lambda type.

[0221] The term monoclonal antibody refers to an antibody obtained from a substantially homogeneous antibody population, i.e., the individual antibodies populating the population are identical except for possible naturally occurring mutations that may be present in minute amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Typically, in contrast to conventional (polyclonal) antibodies, which contain different antibodies against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen.

[0222] In the present invention, the fusion protein may include the Fc portion of an immunoglobulin. For example, when the fusion protein includes the Fc portion, it can activate Fc receptor-positive cells by binding to the Fc receptor, thereby initiating or increasing the expression of cytokines and / or costimulatory antigens, and inducing antibody-dependent cellular cytotoxicity (ADCC).

[0223] In 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 the present invention, the fusion protein can bind to the Fc receptor to activate the Fc receptor-positive cell and initiate or increase the expression of cytokines and / or costimulatory antigens. The cytokines and / or costimulatory antigens can transmit signals that are more than the secondary activation signal required for T cell activation. In the present invention, the fusion protein can possess antibody-dependent cellular cytotoxicity (ADCC) function by binding the Fc region to other cells that express Fc receptors present on the surface of immune cells, hepatocytes, and effector cells of the immune system, such as endothelial cells.

[0224] In the present invention, the Fc region of the fusion protein can positively affect the retention of serum levels of the fusion protein, independent of Fc-mediated cytotoxicity, which can mean improved stability and persistence in vivo. For example, when the Fc portion binds to Fc receptors on endothelial cells and phagocytes, the AFFIMER® formulation can be internalized and recycled back into the bloodstream, improving its half-life in vivo.

[0225] In the present invention, other exemplary targets of the multispecific fusion protein may be, but are not limited to, immune checkpoint proteins and immune co-stimulatory receptors, receptors, cytokines, growth factors, or tumor-associated antigens. In the present invention, the binding domain may be a monoclonal antibody or fragment thereof against at least one autoimmune target (e.g., TNFR2 or IL6-R). In the present invention, the fusion protein may comprise one or more binding domains that bind to a protein upregulated in an autoimmune state (e.g., TNFR2 or IL6-R). Various forms of the fusion proteins of the present invention are described in detail in William BA et al. J. Clin. Med. 2019;8(8):1261.

[0226]

[0227] In the present invention, the fusion domain may be, for example, but is not limited to, an immune checkpoint protein, an immune co-stimulatory receptor, a receptor (or a receptor agonist), a cytokine, a growth factor, or a tumor-associated antigen.

[0228] In the present invention, the cytokine is used as a general 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.

[0229] In 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, such as, but not limited to, GH, EGF, VEGF, FGF, bFGF, HGF, BMPs, M-CSF, G-CSF, GM-CSF, EPO, GDNF, IGF, KGF, BDNF, NGF, PDGF, TPO, and TGF.

[0230] In the present invention, the term "enzyme" is used as a general term for proteins that catalyze biological reactions. For example, the enzyme may be, but is not limited to, α-chymotrypsin, lysozyme, urate oxidase, acetylcholinesterase, Thermomyces lanuginosus lipase, glucose oxidase, superoxide dismutase, caspase, β-glucosidase, Trametes versicolor laccase, alcohol oxidase, Cas9, Cas12, Cas13, Cas14, zinc finger nuclease, TALLEN, dimethylsulfoxide, uricase, agalsidase beta, agalsidase alpha, imiglucerase, thaliglycerase alpha, velaglucerase alpha, alglucerase, sebelipase alpha, laronidase, idursulfase, elosulfase alpha, galsulfase, and alglucosidase alpha.

[0231] In 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 Stefin A protein mutant may be characterized as being absorbed into cells. For example, the cell-penetrating peptide may be, but is not limited to, Tat, Penetrantin, Transparent, Pept1, Pept2, pVEC, DPV3, DPV6, R8, R9, MPG, MAP, Bip4, C105Y, Melittin, etc.

[0232]

[0233] In the present invention, the fusion protein may be characterized as being anchored to the cell membrane of the genetically engineered cell or being expressed on the cell surface.

[0234] In the present invention, when the fusion protein is fixed to a cell membrane or expressed on the cell surface, it may be characterized by further comprising a transmembrane domain. The term "transmembrane domain" in the present invention means a protein domain that spans the cell membrane. In the present invention, the transmembrane domain preferably has an alpha helix structure, but is not limited thereto.

[0235] In the present invention, the transmembrane domain may be characterized as being a transmembrane domain derived from, for example, CD3, CD4, CD5, CD8, CD28, CD99, immunoglobulins (e.g., IgG1, IgG4, IgD, etc.), PDGFR, PTGFRN, etc., or a mutant thereof, but is not limited thereto.

[0236] In the present invention, the fusion protein may further comprise a hinge domain in addition to the transmembrane domain. The term "hinge domain" in the present invention means a series of amino acid sequences present between the transmembrane domain and the extracellular domain of a membrane-anchored protein. In the present invention, the hinge domain may be characterized as being located between the Stefin A protein mutant that specifically binds to CD40L and the transmembrane domain.

[0237] In the present invention, the hinge domain may be characterized as being a hinge domain derived from, for example, CD3, CD4, CD5, CD8, CD28, CD99, immunoglobulins (e.g., IgG1, IgG4, IgD, etc.), PDGFR, PTGFRN, etc., or a mutant thereof, but is not limited thereto.

[0238] In the present invention, the fusion protein may further comprise a coiled-coil domain. The term "coiled-coil domain" in the present invention means a structural motif of a protein in which 2 to 7 alpha helices are wound like a rope. Preferably, the coiled-coil domain may be characterized by having 2 or 3 alpha helices wound around it.

[0239] In 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.

[0240] In the present invention, the coiled-coil domain may be characterized as being located between the Stefin A protein mutant that specifically binds to CD40L and the transmembrane domain.

[0241] In the present invention, the fusion protein may further include a virus-derived peptide or protein. In 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 G 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 G proteins, or F and HN ... The virus F and HN proteins may be characterized as, but are not limited to, the F and HN proteins of a rubulavirus, or the F and HN proteins of an avulavirus, or variants or combinations thereof.

[0242] In the present invention, the fusion protein may further comprise an immunomodulatory domain or an intracellular signal transduction domain.

[0243] In the present invention, the immune modulatory domain or intracellular signal transduction domain refers to a domain located in the cytoplasmic direction of a membrane-anchored protein, and means a site that activates or suppresses an immune response when a target antigen binds to the extracellular domain.

[0244] In the present invention, the immunoregulatory domain or intracellular signaling domain may be characterized as being an immunoregulatory domain derived from CD3, CD28, CD40L, ICOS, OX40, 4-1BB, TNFR2, etc., but is not limited thereto.

[0245] In the present invention, the fusion protein may be a chimeric antigen receptor (CAR). In the present invention, when the fusion protein is a chimeric antigen receptor, the Stefin A protein mutant that specifically binds to CD40L may function as an extracellular binding domain.

[0246] In the present invention, when the fusion protein is a chimeric antigen receptor, it may further comprise the above-mentioned transmembrane domain, hinge domain, and intracellular signal transduction domain, but is not limited thereto. The extracellular antigen-binding domain of various chimeric antigen receptors or analogs thereof known in the art can be modified to the Stefin A protein mutant of the present invention that specifically binds to CD40L, and can be easily designed and produced.

[0247] In the present invention, the fusion protein may be characterized in that it further comprises a localization domain. In the present invention, when the fusion protein is expressed intracellularly, 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 the present invention, the localization domain may be characterized in that it is an organ-specific localization domain or an intracellular protein localization domain.

[0248] In the present invention, the localization domain may be characterized as being, for example, a nucleus-specific localization domain derived from VACM-1 / CUL5, CXCR4, VP1, 53BP1, ING4, IER5, ERK5, Hrp1, UL79, EWS, PTHrP, Pho4, 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.

[0249]

[0250] In the present invention, the fusion protein may further comprise a half-life extension domain. In the present invention, the half-life extension domain refers to a domain or moiety fused to the Stefin A protein variant of the present invention for the purpose of extending the half-life of the Stefin A protein variant of the present invention, and for example, the half-life extension domain may be, 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.

[0251]

[0252] Pharmacokinetic and ADME (Absorption, Distribution, Metabolism, Excretion) Properties Engineered Fusion Proteins

[0253] The term "half-life" as used herein means the time it takes for an active ingredient, such as the Stefin A protein variant or fusion protein of the present invention, to lose half of its pharmacological or physiological activity or concentration. Biological half-life 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 the present invention, biological half-life may be assessed by determining the time it takes for the plasma concentration of the substance to reach half of its normal state level.

[0254] In the present invention, the Stefin A protein mutant or a fusion protein containing the same may not have an appropriate half-life and / or pharmacokinetic profile (PK profile), and may further comprise a half-life extending domain (or half-life extending moiety) to further improve the half-life or PK profile.

[0255] The term "half-life prolonging domain" or "half-life extending moiety" of the present invention refers to a pharma- ceutically acceptable moiety, domain, or molecule that is directly or indirectly conjugated or fused to the Stefin A protein variant of the present invention via a linker or the like for extending the half-life of the Stefin A protein variant of the present invention. In the present invention, the half-life prolonging domain can prevent in vivo proteolysis or activity reduction or modification of the Stefin A protein variant, or improve or modify pharmacokinetic or biophysical properties such as mitigation, half-life extension, increased absorption rate, reduced toxicity, improved solubility, reduced protein aggregation, increased biological activity and / or target selectivity, increased productivity, and reduced immunogenicity, but is not limited thereto.

[0256] In the present invention, the "half-life prolonging domain" includes not only proteins or peptides but also non-proteinaceous half-life prolonging domains, 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, and branched or unbranched C8-C30 alkyl groups; and proteinaceous half-life prolonging domains, such as serum albumin, transferrin, adnectins (e.g., albumin-binding adnectins or pharmacokinetically extended adnectins (PKE adnectins)), Fc domains, unstructured polypeptides, such as XTEN and PAS polypeptides (e.g., amino acids Pro, Ala and / or Ser) and fragments thereof.

[0257] In the present invention, the half-life prolonging domain may be characterized by extending the half-life of the Stefin A protein variant of the present invention circulating in the serum of a subject, compared to the half-life of a fusion protein not including the half-life prolonging domain. 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, 6.0 times, or more, but is not limited thereto. In the present invention, the half-life may be extended by 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, but is not limited thereto.

[0258] In the present invention, examples of the preparation of the fusion protein containing the half-life prolonging domain are as follows, but are not limited thereto:

[0259] - Genetic fusions of the Stefin A protein variant sequence with naturally long half-life proteins or protein domains (eg, Fc domain fusions, transferrin (Tf) fusions, or albumin fusions). The fusion technology with the proteinaceous half-life extending domain is well known in the art (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. etc. See).

[0260] - A genetic fusion of a Stefin A protein variant sequence with an inert polypeptide (e.g., XTEN (also known as recombinant PEG or rPEG)), homoamino acid polymer (HAP), proline-alanine-serine polymer (PAS; PASylation), or elastin-like peptide (ELP; ELPylation), the fusion techniques of said inactive polypeptides are well known in the art (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 or PEGylation for extending the plasma half-life of soluble active proteins. Protein Eng Des Sel. 2007;20:273-84). Sel.26:489-501.Floss et al.(2012)“Elastin-like polypeptides revolutionize recombinant protein expression and their biomedical application.Trends Biotechnol.28:37-45.Floss et al.“ELP-fusion technology for biopharmaceuticals.In:Schmidt S, editor.Fusion protein technologies for biopharmaceuticals:application and challenges.Hoboken:Wiley;2013.p.372-98. etc.).

[0261] - increasing the hydrodynamic radius by chemical conjugation of pharmacologically active peptides or proteins to repeat chemical moieties such as PEG (PEGylation) or hyaluronic acid. The techniques of increasing the hydrodynamic radius by chemical conjugation are well known in the art (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 or 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. (see, for example, et al. (2013) “Conjugation of hyaluronan or proteins” Carb Polymers. 92: 2163-70).

[0262]

[0263] - Significantly increasing the negative charge of a pharmacologically active peptide or protein fusion by polysialylation; or alternatively, (b) 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 by polysialylation is well known in the art. (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 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; through (See, e.g., O-glycosylation.)

[0264] - non-covalent binding via peptide or protein binding domains to proteins that generally have a long half-life, such as HSA, IgG, transferrin or fibronectin, such methods being well known in the art (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.)

[0265] 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. Besides increasing the size of peptide drugs and extending their half-life, Fc and albumin fusion proteins also take advantage of 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 3-16 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 the same protein that is utilized by fatty acylated peptides, and is yet another fusion domain used for half-life extension. An example based on the HSA binding platform is the GLP-1 receptor agonist Albiglutide. The main difference between Fc domain and albumin is that Fc is generally used in dimerization, while HSA is used in monomeric structure. The fusion protein of the Stefin A protein variant and Fc domain of the present invention may be used in dimerization, but is not limited thereto.

[0266]

[0267] Fc domain fusion

[0268] In the present invention, the fusion protein may comprise an immunoglobulin Fc domain (Fc domain) or a fragment or variant thereof, such as a functional Fc region. In the present invention, the Fc region may be characterized as being an FcγR null-binding Fc region. In the present invention, the fusion protein may comprise at least one Stefin A protein variant that binds to CD40L, linked directly or indirectly by a peptide backbone to an immunoglobulin Fc region. In the present invention, the fusion protein may comprise an antibody Fc region (which enhances effector functions and pharmacokinetic properties) and a Stefin A protein variant that is part of the same peptide and binds to CD40L. The immunoglobulin Fc region may also be indirectly linked to at least one Stefin A protein variant that binds to CD40L via a linker. Various linkers that can be used in the fusion protein of the present invention are known in the art. In the present invention, the fusion protein comprising an Fc domain may be used in a dimerized form, and may be used in a homodimerized or heterodimerized form.

[0269] In the present invention, when the fusion protein contains an Fc domain, the stability can be improved and the antibody-like properties provided by the Fc region can be utilized. In the present invention, when the fusion protein contains an Fc domain, the salvage neonatal FcRn receptor pathway, which involves FcRn-mediated recycling of the fusion protein to the cell surface after intracellular introduction, can proceed, thereby avoiding lysosomal degradation and being released into the bloodstream, thereby extending the half-life. Fusion of the Fc domain can be useful not only for extending the half-life but also for separating and purifying the fusion protein of the present invention.

[0270] In the present invention, the Fc domain may comprise the constant region of an antibody excluding the first constant region immunoglobulin domain.

[0271] In the present invention, the Fc domain may include the last two constant region immunoglobulin domains of IgA, IgD and IgG, and the flexible hinge N-terminal to those domains. In the case of IgA and IgM, the Fc domain may include the J chain. In the case of IgG, the Fc domain may include the immunoglobulin domains Cγ2, Cγ3, and the hinge between Cγ1 and Cγ2. Although the boundaries of the Fc domain may 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 uses Kabat numbering. In the present invention, the terms "Fc", "Fc domain" and "Fc region" may be used interchangeably, and the Fc domain may refer to the above regions separately, or may refer to a part of an antibody, a fragment thereof, or a fusion protein. Polymorphisms of the Fc domain have been reported at various positions and may be used as the fusion domain of the fusion protein of the present invention without limitation.

[0272] In the present invention, the Fc domain may be characterized as 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 standard binding assays known in the art. In the present invention, the functional Fc region may be characterized as binding to FcRn or having no 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 by various validation methods known in the art for evaluating it.

[0273] In the present invention, the Fc domain may be characterized as being derived from IgG1, IgG2, IgG3, IgG4, or the like.

[0274] In the present invention, said Fc domain may be characterized in that it is derived from an IgG1 immunoglobulin Fc domain, for example, the Fc domain may be characterized in that it comprises the following sequence:

[0275] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:535).

[0276] In the present invention, the Fc region contained in the fusion protein may be characterized by comprising a hinge region. For example, the hinge region may be characterized by comprising core hinge residues at sequence positions 1-16 of a human IgG1 Fc domain (e.g., DKTHTCPPCPAPELLG (SEQ ID NO: 536)). In the present invention, the fusion protein may be characterized by forming a multimeric structure (e.g., a dimer) in part by cysteine ​​residues at positions 6 and 9 in the hinge region of a human IgG1 immunoglobulin Fc domain sequence. In the present invention, the hinge region may further comprise residues from the CH1 and CH2 regions flanking the core hinge sequence of an IgG immunoglobulin Fc domain sequence. In the present invention, the hinge sequence may comprise or consist of GSTHTCPPCPAPELLG (SEQ ID NO: 537) or EPKSCDKTHTCPPCPAPELLG (SEQ ID NO: 538).

[0277] In the present invention, the hinge region may be characterized in that it preferably comprises one or more substitutions that confer pharmacokinetic, biophysical and / or biological properties. For example, the hinge region may comprise or consist of the following sequence:

[0278] EPKSCDKTHTCPPCPAPELLGGPS (SEQ ID NO:539);

[0279] EPKSSDKTHTCPPCPAPELLGGPS (SEQ ID NO:540);

[0280] EPKSSDKTHTCPPCPAPELLGGSS(SEQ ID NO:541);

[0281] EPKSSGSTHTCPPCPAPELLGGSS (SEQ ID NO:542);

[0282] DKTHTCPPCPAPELLGGPS (SEQ ID NO:543); and

[0283] DKTHTCPPCPAPELLGGSS (sequence number 544).

[0284] In the present invention, the 18th residue P of the human IgG1 immunoglobulin Fc domain sequence may be substituted with S to remove Fc effector function; an example of such a substitution is as follows:

[0285] EPKSSDKTHTCPPCPAPELLGGSS(SEQ ID NO:541);

[0286] EPKSSGSTHTCPPCPAPELLGGSS (SEQ ID NO:542); and

[0287] DKTHTCPPCPAPELLGGSS (sequence number 544).

[0288] In the present invention, the DK at the 1st to 2nd positions 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:

[0289] EPKSSGSTHTCPPCPAPELLGGSS (sequence number 542).

[0290] In the present invention, the 103rd residue C of the heavy chain constant region (e.g., CH1-CH3) of human IgG may be substituted with S to prevent inappropriate cysteine ​​bond formation in the absence of a light chain, and an example of such a substitution is as follows:

[0291] EPKSSDKTHTCPPCPAPELLGGPS (SEQ ID NO:540),

[0292] EPKSSDKTHTCPPCPAPELLGGSS (SEQ ID NO:541), and

[0293] EPKSSGSTHTCPPCPAPELLGGSS (sequence number 542).

[0294] In the present invention, the Fc domain may be characterized as being an Fc domain derived from a mammal, preferably a human Fc domain. In the present invention, the Fc domain may be characterized as being an Fc domain derived from IgG1, IgG2, IgG3 or IgG4. In the present invention, the Fc domain may be characterized as having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity with a native Fc domain and / or an Fc region of a parent polypeptide. In the present invention, the Fc domain may have 90% or more sequence identity with a native Fc domain and / or an Fc domain of a certain polypeptide.

[0295] In the present invention, the Fc domain may comprise an amino acid sequence selected from SEQ ID NOs: 545 to 558, or an Fc domain of SEQ ID NOs: 545 to 558 described in the Examples. In the present invention, lysine at the C-terminus of an Fc domain should be understood as a selective configuration in a fusion protein comprising an Fc domain. In the present invention, the Fc domain may be characterized by comprising an amino acid sequence selected from SEQ ID NOs: 545 to 558. In the present invention, the Fc domain may be characterized by comprising an amino acid sequence selected from SEQ ID NOs: 545 to 558 from which the lysine present at the C-terminus is omitted.

[0296] [Table 7]

[0297] JPEG2025509078000037.jpg170154

[0298] As used herein, the term "antibody-dependent cellular cytotoxicity" or "ADCC" refers to the specific binding of cytotoxic effector cells of secretory immunoglobulins bound to Fc receptors (FcRs) present on specific cytotoxic cells to antigen-bearing targets, thereby exerting cytotoxicity and killing the cells.

[0299] In the present invention, the fusion protein may be characterized by comprising an Fc domain with no or reduced ADCC and / or complement activation or effector function. For example, the Fc domain may be characterized by comprising a naturally inactivated constant region of an IgG2 or IgG4 isotype or a mutated IgG1 constant region. Such modified Fc domains are described, for example, in EP0307434. For example, the Fc domain may comprise, but is not limited to, substitutions of alanine residues at positions 235 and 237 (EU index numbering).

[0300] In the present invention, the fusion protein may be characterized in that it comprises an Fc domain that retains some or all of the functions of Fc (e.g., ADCC and / or CDC). For example, the fusion protein may comprise, but is not limited to, 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, it 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 mutations may be any one or more selected from S239D, I332E and A330L. In the present invention, the Fc domain may be characterized in that the glycosylation profile is altered to reduce fucosylation of the Fc region.

[0301]

[0302] Albumin fusion

[0303] In an embodiment of the present invention, the fusion protein may be characterized in that it comprises an albumin sequence or a fragment thereof. In the present invention, the albumin sequence or a fragment thereof may be characterized in that it is fused or conjugated by incorporation or chemical linkage to a polypeptide sequence comprising a Stefin A protein variant. In the present invention, the albumin, albumin variant or albumin fragment may be characterized in that it is human serum albumin (HSA), a variant or a fragment thereof. In the present invention, the albumin serum protein other than HSA may be characterized in that it is derived from, for example, cynomolgus monkeys, cows, dogs, rabbits and mice, for example, BSA is structurally most similar to HSA (Kosa et al., (2007) J Pharm Sci. 96(11):3117-24). In the present invention, the albumin may be a non-human serum albumin such as, but not limited to, cyno serum albumin or bovine serum albumin.

[0304] Mature HSA, a 585 amino acid polypeptide (about 67 kDa) with a serum half-life of about 20 days, is mainly 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), mostly exhibits an alpha-helical conformation, and is highly stabilized by 17 disulfide bonds. In the present invention, the fusion protein may be characterized by comprising at least one Stefin A protein variant; and mature human serum albumin (e.g., SEQ ID NO: 559) or a variant or fragment thereof. In the present invention, the albumin of the fusion protein may be characterized by retaining a desired level of PK and / or biodistribution properties.

[0305] (SEQ ID NO:559)

[0306] In the present invention, the albumin sequence may be separated from other flanking sequences of the Stefin A protein variant or fusion protein by a linker sequence.

[0307] Unless otherwise specified, in the present invention, albumin or mature albumin means human serum albumin. However, full-length HSA may be characterized by including a signal peptide (MKWVTFISLLFLFSSAYS (SEQ ID NO: 486)) consisting of 18 amino acids and a prodomain sequence (RGVFRR (SEQ ID NO: 560)) consisting of the following 6 amino acids, and the total of 24 amino acids may be named a preprodomain. In the present invention, the fusion protein may be characterized by being expressed and secreted by the HSA preprodomain. In the present invention, the fusion protein comprising the Stefin A protein variant and HSA may be characterized by including the signal sequence described in the Stefin A protein variant, and being expressed and secreted by this.

[0308] In the present 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, for example, chemical conjugation via an amino acid side chain of the Stefin A protein variant or a fusion protein comprising the same.

[0309] Serum Binding Domain

[0310] In the present invention, the fusion protein may be characterized by comprising a serum binding domain (or moiety), which may be included as part of the fusion protein sequence or may be chemically conjugated by other moieties.

[0311] In the present invention, the serum-binding domain may be characterized as being an albumin-binding domain. Albumin contains multiple hydrophobic binding pockets and can naturally act as a carrier of various ligands such as fatty acids and steroids, and the surface of albumin is negatively charged, making it highly water-soluble.

[0312] The term "albumin binding domain" of the present invention means any group capable of binding to albumin, for example, a compound having albumin binding affinity. Albumin can bind to endogenous ligands such as fatty acids, and can also interact with exogenous ligands such as warfarin, penicillin, and diazepam. The binding of such drugs to albumin is reversible, and albumin-drug complexes can act as drug depots or drug carriers that can improve the biodistribution and bioavailability of drugs.

[0313] In the present invention, the chemical modification applicable to the fusion protein of the present invention to increase the protein half-life may be characterized as lipidation, which covalently binds a fatty acid to a peptide side chain. Such a method is well known in the art as a method for extending the half-life of insulin, and may be characterized as increasing the hydrodynamic radius and reducing renal filtration, such as PEGylation. The lipid moiety itself is relatively small and can indirectly extend the half-life by non-covalent binding with circulating albumin. Lipidation may reduce the water solubility of the peptide, but this can be adjusted by engineering the linker between the peptide and the fatty acid. Linker engineering and lipid modification can contribute to an increase in the in vivo half-life independent of albumin (Jonassen et al. (2012) Pharm Res. 29 (8): 2104-14).

[0314] In the present invention, the albumin-binding domain may be characterized as being an albumin-binding adnectin (albumin-binding (PKE2) adnectin; see WO2011140086 "Serum Albumin Binding Molecules", WO2015143199 "Serum albumin-binding Fibronectin Type III Domains", and WO2017053617 "Fast-off rate serum albumin binding fibronectin type iii domains"), albumin-binding domain 3 (ABD3) of Protein G of Streptococcus G148, an albumin-binding domain antibody (e.g., GSK2374697, AlbudAb), or an albumin-binding nanobody (e.g., ATN103, Ozolralizumab).

[0315] AFFIMER® XT

[0316] In the present invention, the fusion protein may be characterized in that it comprises a Stefin A protein mutant capable of binding to a serum protein.In the present invention, the fusion protein may be characterized in that it comprises a Stefin A protein mutant capable of binding to HSA.

[0317] In the present invention, at least one of the solvent accessible loops of the Stefin A protein variant capable of binding to HSA (HSA AFFIMER®) may be characterized as being derived from the wild-type Stefin A protein, which allows it to bind to HSA. In the present invention, the Stefin A protein variant capable of binding to HSA may be characterized as being 10 -6 It may be characterized as binding to HSA with a Kd of less than or equal to M

[0318] In the present invention, the Stefin A protein mutant capable of binding to HSA has a concentration of 1×10 -9 M~1×10 -6In the present invention, the Stefin A protein mutant capable of binding to HSA may be characterized by having a Kd of 1×10 M at pH 7.4 to 7.6. -6 In the present invention, the Stefin A protein mutant capable of binding to HSA may be characterized by having a Kd of 1×10 M or less at pH 7.4 to 7.6. -7 In the present invention, the Stefin A protein mutant capable of binding to HSA may be characterized by having a Kd of 1×10 M or less at pH 7.4 to 7.6. -8 In the present invention, the Stefin A protein mutant capable of binding to HSA may be characterized by having a Kd of 1×10 M or less at pH 7.4 to 7.6. -9 In the present invention, the Stefin A protein mutant capable of binding to HSA may be characterized by having a Kd of 1×10 M or less at pH 7.4 to 7.6. -10 In the present invention, the Stefin A protein mutant capable of binding to HSA may be characterized by having a Kd of 1×10 M or less at pH 7.4 to 7.6. -11 In the present invention, the Stefin A protein mutant capable of binding to HSA may be characterized as having a Kd of 1×10 at pH 7.4 or less. -9 M~1×10 -6 In the present invention, the Stefin A protein mutant capable of binding to HSA may be characterized as having a Kd of 1×10 M at pH 7.4. -6 In the present invention, the Stefin A protein mutant capable of binding to HSA may be characterized as having a Kd of 1×10 at pH 7.4 or less. -7 In the present invention, the Stefin A protein mutant capable of binding to HSA may be characterized as having a Kd of 1×10 at pH 7.4 or less. -8 In the present invention, the Stefin A protein mutant capable of binding to HSA may be characterized as having a Kd of 1×10 at pH 7.4 or less. -9In the present invention, the Stefin A protein mutant capable of binding to HSA may be characterized as having a Kd of 1×10 at pH 7.4 or less. -10 In the present invention, the Stefin A protein mutant capable of binding to HSA may be characterized as having a Kd of 1×10 at pH 7.4 or less. -11 It may be characterized as binding to HSA with a Kd of less than or equal to M

[0319] In the present invention, the Stefin A protein mutant capable of binding to HSA may be characterized as being a mutant derived from a wild-type human Stefin A protein having a backbone sequence, in which any one or more of loops 2 and 4 are replaced with alternative loop sequences, (Xaa)n and (Xaa)m.

[0320] In the present invention, the Stefin A protein variant capable of binding to HSA may be characterized in that it comprises an amino acid sequence as shown in formula I:

[0321] [Formula I]

[0322] FR1-(Xaa)n-FR2-(Xaa)m-FR3 (I)

[0323] Here, the FR1 may be characterized as being represented by 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 with the amino acid sequence represented by MIPGGLSEAK PATPEIQEIV DKVKPQLEEK TNETYGKLEA VQYKTQVLA (SEQ ID NO: 1A); and the FR2 may be characterized as being represented by 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 with the amino acid sequence of SEQ ID NO: 2;

[0324] The FR3 may be characterized by being represented by 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

[0325] Xaa, in each occurrence, is individually any amino acid residue; n and m are each independently an integer from 3 to 20.

[0326] In the present invention, the FR1 may be a polypeptide having at least 80% to 98%, 82% to 98%, 84% to 98%, 86% to 98%, 88% to 98%, 90% to 98%, 92% to 98%, 94% to 98%, or 96% to 98% homology with SEQ ID NO: 1A. In the present invention, the FR1 may be a polypeptide having at least 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, or 98% homology with SEQ ID NO: 1A. In the present invention, the FR1 may be a polypeptide of SEQ ID NO: 1A. In the present invention, the FR2 may be a polypeptide having at least 80% to 96%, 84% to 96%, 88% to 96%, or 92% to 96% homology with SEQ ID NO: 2. In the present invention, the FR2 may be a polypeptide having at least 80%, 84%, 88%, 92%, or 96% homology with SEQ ID NO: 2. In the present invention, the FR2 may be a polypeptide having at least 80%, 85%, 90%, 95%, or 98% homology with SEQ ID NO: 2. In the present invention, the FR3 may be a polypeptide having at least 80% to 95%, 85% to 95%, or 90% to 95% homology with SEQ ID NO: 3. In the present invention, the FR3 may be a polypeptide having at least 80%, 85%, 90%, or 95% homology with SEQ ID NO: 3. In the present invention, the FR3 may be a polypeptide of SEQ ID NO: 3.

[0327] In the present invention, the Stefin A protein variant capable of binding to HSA may be characterized in that it comprises the amino acid sequence shown in formula II (SEQ ID NO: 4):

[0328] [Formula II]

[0329] MIP-Xaa1-GLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVV-(Xaa)n-Xaa2-TNYYIKVRAGDNKYMHLKVF-Xaa3-Xaa4-Xaa5-(Xaa)m-Xaa6-D-Xaa7-VLTGYQVDKNKDDELTGF (SEQ ID NO: 4)

[0330] wherein Xaa, in each occurrence, is individually any amino acid residue; n and m are each independently an integer from 3 to 20.

[0331] In the present invention, the Stefin A protein variant capable of binding to HSA may be characterized in that it comprises an amino acid sequence represented by formula III.

[0332] [Formula III]

[0333] MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVD-(Xaa)n-GTNYYIKVRAGDNKYMHLKVFKSL-(Xaa)m-EDLVLTGYQVDKNKDDELTGF (SEQ ID NO: 5)

[0334] wherein Xaa, in each occurrence, is individually any amino acid residue; n and m are each independently an integer from 3 to 20.

[0335] In the present invention, Xaa1 may be characterized as being Gly, Ala, Val, Arg, Lys, Asp, or Glu; Xaa2 is Gly, Ala, Val, Ser, or Thr; Xaa3 is Arg, Lys, Asn, Gln, Ser, or Thr; Xaa4 is Gly, Ala, Val, Ser, or Thr; Xaa5 is Ala, Val, Ile, Leu, Gly, or Pro; Xaa6 is Gly, Ala, Val, Asp, or Glu; and Xaa7 is Ala, Val, Ile, Leu, Arg, or Lys.

[0336] In the present invention, Xaa1 may be characterized as being Gly, Ala, Arg or Lys. In the present invention, Xaa1 may be characterized as being Gly or Arg. In the present invention, Xaa2 may be characterized as being Gly, Ala, Val, Ser or Thr. In the present invention, Xaa2 may be characterized as being Gly or Ser. In the present invention, Xaa3 may be characterized as being Arg, Lys, Asn, Gln, Ser or Thr. In the present invention, Xaa3 may be characterized as being Arg, Lys, Asn or Gln. In the present invention, Xaa3 may be characterized as being Lys or Asn. In the present invention, Xaa4 may be characterized as being Gly, Ala, Val, Ser or Thr. In the present invention, Xaa4 may be characterized as being Gly or Ser. In the present invention, Xaa5 may be characterized as Ala, Val, Ile, Leu, Gly or Pro. In the present invention, Xaa5 may be characterized as Ile, Leu or Pro. In the present invention, Xaa5 may be characterized as Leu or Pro. In the present invention, Xaa6 may be characterized as Gly, Ala, Val, Asp or Glu. In the present invention, Xaa6 may be characterized as Ala, Val, Asp or Glu. In the present invention, Xaa6 may be characterized as Ala or Glu. In the present invention, Xaa7 may be characterized as Ala, Val, Ile, Leu, Arg or Lys. In the present invention, Xaa7 may be characterized as Ile, Leu or Arg. In the present invention, Xaa7 may be characterized as Leu or Arg.

[0337] In the present invention, n may be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In the present invention, n may be 8 to 10, 7 to 11, 6 to 12, 5 to 13, 4 to 14, or 3 to 15. In the present invention, m may be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In the present invention, m may be 8 to 10, 7 to 11, 6 to 12, 5 to 13, 4 to 14, or 3 to 15.

[0338] In the present invention, (Xaa)n may be characterized as being represented by formula IV:

[0339] [Formula IV]

[0340] aa1-aa2-aa3-aa4-aa5-aa6-aa7-aa8-aa9

[0341] aa3 is an amino acid having a neutral non-polar hydrophobic side chain; aa4 is an amino acid having a neutral polar hydrophilic side chain; aa5 is an amino acid having a positively charged polar hydrophilic side chain; aa6 is an amino acid having a positively charged polar hydrophilic side chain; aa7 is an amino acid having a neutral non-polar hydrophobic side chain; aa8 is an amino acid having a neutral non-polar hydrophobic side chain; and aa9 is an amino acid having a neutral non-polar hydrophobic side chain.

[0342] In the present invention, (Xaa)m may be characterized as being represented by formula V:

[0343] [Formula V]

[0344] aa1-aa2-aa3-aa4-aa5-aa6-aa7-aa8-aa9 (V)

[0345] aa2 is an amino acid with a positively charged polar hydrophilic side chain; aa3 is an amino acid with a neutral nonpolar hydrophobic side chain; aa4 is an amino acid with a positively charged polar hydrophilic side chain; aa5 is an amino acid with a neutral polar hydrophilic side chain; aa6 is an amino acid with a neutral polar hydrophilic side chain; aa7 is an amino acid with a negatively charged polar hydrophilic side chain; aa8 is an amino acid with a positively charged polar hydrophilic side chain; and aa9 is an amino acid with a neutral nonpolar hydrophilic side chain.

[0346] Examples of amino acids having a neutral non-polar hydrophilic side chain may include cysteine ​​(Cys) and glycine (Gly). In the present invention, an amino acid having a neutral non-polar hydrophilic side chain may be Cys. In the present invention, an amino acid having a neutral non-polar hydrophilic side chain may be Gly.

[0347] Examples of amino acids having a neutral non-polar hydrophobic side chain may include alanine (Ala), isoleucine (Ile), leucine (Leu), methionine (Met), phenylalanine (Phe), proline (Pro), tryptophan (Trp), and valine (Val). In the present invention, an amino acid having a neutral non-polar hydrophobic side chain may be Ala. In the present invention, an amino acid having a neutral non-polar hydrophobic side chain may be Ile. In the present invention, an amino acid having a neutral non-polar hydrophobic side chain may be Leu. In the present invention, an amino acid having a neutral non-polar hydrophobic side chain may be Met. In the present invention, an amino acid having a neutral non-polar hydrophobic side chain may be Phe. In the present invention, an amino acid having a neutral non-polar hydrophobic side chain may be Pro. In the present invention, an amino acid having a neutral non-polar hydrophobic side chain may be Trp. In the present invention, an amino acid having a neutral non-polar hydrophobic side chain may be Val.

[0348] Examples of amino acids having a neutral polar hydrophilic side chain may include asparagine (Asn), glutamine (Gln), serine (Ser), threonine (Thr), and tyrosine (Tyr). In the present invention, an amino acid having a neutral polar hydrophilic side chain may be Asn. In the present invention, an amino acid having a neutral polar hydrophilic side chain may be Gln. In the present invention, an amino acid having a neutral polar hydrophilic side chain may be Ser. In the present invention, an amino acid having a neutral polar hydrophilic side chain may be Thr. In the present invention, an amino acid having a neutral polar hydrophilic side chain may be Tyr.

[0349] Examples of amino acids having a positively charged polar hydrophilic side chain may include arginine (Arg), histidine (His), and lysine (Lys). In the present invention, an amino acid having a positively charged polar hydrophilic side chain may be Arg. In the present invention, an amino acid having a positively charged polar hydrophilic side chain may be His. In the present invention, an amino acid having a positively charged polar hydrophilic side chain may be Lys.

[0350] Examples of amino acids having a negatively charged polar hydrophilic side chain may include aspartate (Asp) and glutamate (Glu). In the present invention, an amino acid having a negatively charged polar hydrophilic side chain may be Asp. In the present invention, an amino acid having a negatively charged polar hydrophilic side chain may be Glu.

[0351] In the present invention, (Xaa)n may be characterized as being represented by formula IV:

[0352] [Formula IV]

[0353] aa1-aa2-aa3-aa4-aa5-aa6-aa7-aa8-aa9 (IV)

[0354] Here, aa1 is an amino acid selected from Asp, Gly, Asn, and Val; aa2 is an amino acid selected from Trp, Tyr, His, and Phe; aa3 is an amino acid selected from Trp, Tyr, Gly, Trp, and Phe; aa4 is an amino acid selected from Gln, Ala, and Pro; aa5 is an amino acid selected from Ala, Gln, Glu, Arg, and Ser; aa6 is an amino acid selected from Lys, Arg, and Tyr; aa7 is an amino acid selected from Trp and Gln; aa8 is an amino acid selected from Pro and His; and / or aa9 is an amino acid selected from His, Gly, and Gln. In the present invention, aa1 may be Asp. In the present invention, aa1 may be Gly. In the present invention, aa1 may be Asn. In the present invention, aa2 may be Trp. In the present invention, aa2 may be Tyr. In the present invention, aa2 may be His. In the present invention, aa2 may be Phe. In the present invention, aa3 may be Trp. In the present invention, aa3 may be Tyr. In the present invention, aa3 may be Gly. In the present invention, aa3 may be Trp. In the present invention, aa3 may be Phe. In the present invention, aa4 may be Gln. In the present invention, aa4 may be Ala. In the present invention, aa4 may be Pro. In the present invention, aa5 may be Ala. In the present invention, aa5 may be Gln. In the present invention, aa5 may be Glu. In the present invention, aa5 may be Arg. In the present invention, aa5 may be Ser. In the present invention, aa6 may be Lys. In the present invention, aa6 may be Arg. In the present invention, aa6 may be Tyr. In the present invention, aa7 may be Trp. In the present invention, aa7 may be Gln. In the present invention, aa8 may be Pro. In the present invention, aa8 may be His. In the present invention, aa9 may be His. In the present invention, aa9 may be Gly. In the present invention, aa9 may be Gln.

[0355] In the present invention, (Xaa)m may be characterized as being represented by formula IV:

[0356] [Formula IV]

[0357] aa1-aa2-aa3-aa4-aa5-aa6-aa7-aa8-aa9 (IV)

[0358] aa2 is an amino acid selected from Lys, Pro, His, Ala, and Thr; aa3 is an amino acid selected from Val, Asn, Gly, Gln, Ala, and Phe; aa4 is an amino acid selected from His, Thr, Lys, Trp, Lys, Val, and Arg; aa5 is an amino acid selected from Gln, Ser, Gly, Pro, and Asn; aa6 is an amino acid selected from Ser, Tyr, Glu, Leu, Lys, and Thr; aa7 is an amino acid selected from Ser, Asp, Val, and Lys; aa8 is an amino acid selected from Gly, Leu, Ser, Pro, His, Asp, and Arg; and / or aa9 is an amino acid selected from Gly, Gln, Glu, and Ala.

[0359] In the present invention, aa1 may be Tyr. In the present invention, aa1 may be Phe. In the present invention, aa1 may be Trp. In the present invention, aa1 may be Asn. In the present invention, aa2 may be Lys. In the present invention, aa2 may be Pro. In the present invention, aa2 may be His. In the present invention, aa2 may be Ala. In the present invention, aa2 may be Thr. In the present invention, aa3 may be Val. In the present invention, aa3 may be Asn. In the present invention, aa3 may be Gly. In the present invention, aa3 may be Gln. In the present invention, aa3 may be Ala. In the present invention, aa3 may be Phe. In the present invention, aa4 may be His. In the present invention, aa4 may be Thr. In the present invention, aa4 may be Lys. In the present invention, aa4 may be Trp. In the present invention, aa4 may be Lys. In the present invention, aa4 may be Val. In the present invention, aa4 may be Arg. In the present invention, aa5 may be Gln. In the present invention, aa5 may be Ser. In the present invention, aa5 may be Gly. In the present invention, aa5 may be Pro. In the present invention, aa5 may be Asn. In the present invention, aa6 may be Ser. In the present invention, aa6 may be Tyr. In the present invention, aa6 may be Glu. In the present invention, aa6 may be Leu. In the present invention, aa6 may be Lys. In the present invention, aa6 may be Thr. In the present invention, aa7 may be Ser. In the present invention, aa7 may be Asp. In the present invention, aa7 may be Val. In the present invention, aa7 may be Lys. In the present invention, aa8 may be Gly. In the present invention, aa8 may be Leu. In the present invention, aa8 may be Ser. In the present invention, aa8 may be Pro. In the present invention, aa8 may be His. In the present invention, aa8 may be Asp. In the present invention, aa8 may be Arg. In the present invention, aa9 may be Gly.In the present invention, aa9 may be Gln. In the present invention, aa9 may be Glu. In the present invention, aa9 may be Ala.

[0360] In the present invention, (Xaa)n may be characterized as being represented by formula V:

[0361] [Formula V]

[0362] Asn-aa1-aa2-Gln-Gln-Arg-Arg-Trp-Pro-Gly (V)

[0363] wherein aa1 is an amino acid selected from Trp and Phe; and aa2 is an amino acid selected from Tyr and Phe. In the present invention, aa1 may be Trp. In the present invention, aa1 may be Phe. In the present invention, aa2 may be Tyr. In the present invention, aa2 may be Phe.

[0364] In the present invention, (Xaa)n may be characterized as being represented by formula VI:

[0365] [Formula VI]

[0366] aa1-aa2-Trp-aa3-aa4-Lys-Trp-Pro-aa5 (VI)

[0367] Here, aa1 is an amino acid selected from Asp and Gly; aa2 is an amino acid selected from Trp, Tyr, and Phe; aa3 is an amino acid selected from Gln and Ala; aa4 is an amino acid selected from Ala and Ser; and aa5 is an amino acid selected from His and Gly. In the present invention, aa1 may be Asp. In the present invention, aa1 may be Gly. In the present invention, aa2 may be Trp. In the present invention, aa2 may be Tyr. In the present invention, aa2 may be Phe. In the present invention, aa3 may be Gln. In the present invention, aa3 may be Ala. In the present invention, aa4 may be Ala. In the present invention, aa4 may be Ser. In the present invention, aa5 may be His. In the present invention, aa5 may be Gly.

[0368] In the present invention, (Xaa)n may be characterized as having the formula VII:

[0369] [Formula VII]

[0370] aa1-aa2-aa3-aa4-aa5-aa6-Trp-Pro-Gly (VII)

[0371] Here, aa1 is an amino acid selected from Gly and Asn; aa2 is an amino acid selected from Tyr, Phe, Trp, and His; aa3 is an amino acid selected from Trp, Tyr, and Phe; aa4 is an amino acid selected from Ala and Gln; aa5 is an amino acid selected from Ala, Ser, Gln, and Arg; and aa6 is an amino acid selected from Lys, Arg, and Tyr. In the present invention, aa1 may be Gly. In the present invention, aa1 may be Asn. In the present invention, aa2 may be Tyr. In the present invention, aa2 may be Phe. In the present invention, aa2 may be Trp. In the present invention, aa2 may be His. In the present invention, aa3 may be Trp. In the present invention, aa3 may be Tyr. In the present invention, aa3 may be Phe. In the present invention, aa4 may be Ala. In the present invention, aa4 may be Gln. In the present invention, aa5 may be Ala. In the present invention, aa5 may be Ser. In the present invention, aa5 may be Gln. In the present invention, aa5 may be Arg. In the present invention, aa6 may be Lys. In the present invention, aa6 may be Arg. In the present invention, aa6 may be Tyr.

[0372] In the present invention, (Xaa)n may be characterized as being represented by formula VIII:

[0373] [Formula VIII]

[0374] Gly-aa1-aa2-Ala-aa3-aa4-Trp-Pro-Gly (VIII) (SEQ ID NO: 561)

[0375] Here, aa1 is an amino acid selected from Tyr, Phe, and His; aa2 is an amino acid selected from Trp and Tyr; aa3 is an amino acid selected from Ala, Ser, and Arg; and aa4 is an amino acid selected from Lys and Tyr. In the present invention, aa1 may be Tyr. In the present invention, aa1 may be Phe His. In the present invention, aa1 may be His. In the present invention, aa2 may be Trp. In the present invention, aa2 may be Tyr. In the present invention, aa3 may be Ala. In the present invention, aa3 may be Ser. In the present invention, aa3 may be Arg. In the present invention, aa4 may be Lys. In the present invention, aa4 may be Tyr.

[0376] In the present invention, (Xaa)n may be characterized as being represented by formula IX:

[0377] [Formula IX]

[0378] aa1-aa2-aa3-Gln-aa4-aa5-Trp-Pro-aa6 (IX)

[0379] Here, aa1 is an amino acid selected from Asp and Asn; aa2 is an amino acid selected from Trp and Phe; aa3 is an amino acid selected from Trp, Tyr, and Phe; aa4 is an amino acid selected from Ala, Gln, and Arg; aa5 is an amino acid selected from Lys and Arg; and aa6 is an amino acid selected from His and Gly. In the present invention, aa1 may be Asp. In the present invention, aa1 may be Asn. In the present invention, aa2 may be Trp. In the present invention, aa2 may be Phe. In the present invention, aa3 may be Trp. In the present invention, aa3 may be Tyr. In the present invention, aa3 may be Phe. In the present invention, aa4 may be Ala. In the present invention, aa4 may be Gln. In the present invention, aa4 may be Arg. In the present invention, aa5 may be Lys. In the present invention, aa5 may be Arg. In the present invention, aa6 may be His. In the present invention, aa6 may be Gly.

[0380] In the present invention, the Stefin A protein mutant capable of binding to HSA may be characterized by comprising any one of loop 2 amino acid sequences selected from the amino acid sequences of SEQ ID NOs: 562 to 614 (Table 8).In the present invention, the Stefin A protein mutant capable of binding to HSA may be characterized by comprising any one of loop 4 amino acid sequences selected from the amino acid sequences of SEQ ID NOs: 615 to 667 (Table 8).

[0381] [Table 8]

[0382] JPEG2025509078000039.jpg197146

[0383] In the present invention, the (Xaa)n may be characterized in that it comprises an amino acid sequence having at least 80% or at least 90% identity with the amino acid sequence of any one of SEQ ID NOs: 562 to 614. In the present invention, the (Xaa)n may be characterized in that it comprises an amino acid sequence having 80% to 90% identity with the amino acid sequence of any one of SEQ ID NOs: 562 to 614. In the present invention, the (Xaa)n may be characterized in that it comprises an amino acid sequence of any one of SEQ ID NOs: 562 to 614.

[0384] In the present invention, the (Xaa)m may be characterized in that it comprises an amino acid sequence having at least 80% or at least 90% identity with the amino acid sequence of any one of SEQ ID NOs: 615 to 667. In the present invention, the (Xaa)m may be characterized in that it comprises an amino acid sequence having 80% to 90% identity with the amino acid sequence of any one of SEQ ID NOs: 615 to 667. In the present invention, the (Xaa)m may be characterized in that it comprises an amino acid sequence of any one of 615 to 667.

[0385] In the present invention, the Stefin A protein mutant capable of binding to HSA may be characterized by comprising any one of the amino acid sequences of SEQ ID NOs: 668 to 674 (Table 9).

[0386] [Table 9]

[0387] In the present invention, the Stefin A protein mutant capable of binding to HSA may be characterized by comprising an amino acid sequence having at least 80% or at least 90% identity with any one of the amino acid sequences of SEQ ID NOs: 668 to 674.

[0388] In the present invention, the Stefin A protein mutant capable of binding to HSA may be characterized by comprising an amino acid sequence having 80% to 90% identity with any one of the amino acid sequences of SEQ ID NOs: 668 to 674.

[0389] In the present invention, the Stefin A protein variant capable of binding to HSA can be linked to another molecule (e.g., a therapeutic polypeptide or a Stefin A protein variant of the present invention that specifically binds to CD40L) to extend the half-life of the molecule. In the present invention, the Stefin A protein variant capable of binding to HSA can be characterized by having various binding affinities that cross-react with other species, such as mouse and cyno monkey. In the present specification, the Stefin A protein variant capable of binding to HSA can be characterized by having various binding affinities that cross-react with other species, such as mouse and cyno monkey. TM In the present invention, the HSA-binding stefin A protein variant can extend the half-life of other stefin A protein variants or their fusion proteins conjugated by a single gene fusion method in a controlled manner based on in vivo pharmacokinetic (PK) studies. In the present invention, the HSA-binding stefin A protein variant (AFFIMER XT TM ) may be used to extend the half-life of other peptide or protein therapeutics.

[0390] In the present invention, the Stefin A protein variant capable of binding to HSA may be characterized by increasing the serum half-life in vivo of a molecule, for example, a therapeutic protein such as a Stefin A protein variant of the present invention that specifically binds to CD40L or a fusion protein comprising the same. For example, the Stefin A protein variant capable of binding to HSA may be characterized by increasing the serum half-life of a molecule, for example, a therapeutic protein such as a Stefin A protein variant of the present invention that specifically binds to CD40L or a fusion protein comprising the same, by at least 2-fold compared to a molecule to which the Stefin A protein variant capable of binding to HSA is not linked.

[0391] In the present invention, when the fusion protein comprises the Stefin A protein variant capable of binding to HSA, the fusion protein may be characterized by increasing the serum half-life by at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 20-fold, or at least 30-fold, as compared to a molecule to which the Stefin A protein variant capable of binding to HSA is not linked. In the present invention, when the fusion protein comprises the Stefin A protein variant capable of binding to HSA, the fusion protein may be characterized by increasing the serum half-life by 2-fold to 5-fold, 2-fold to 10-fold, 3-fold to 5-fold, 3-fold to 10-fold, 15-fold to 5-fold, 4-fold to 10-fold, or 5-fold to 10-fold, as compared to a molecule to which the Stefin A protein variant capable of binding to HSA is not linked. In the present invention, when the fusion protein contains the Stefin A protein variant capable of binding to HSA, it may be characterized in that the serum half-life is increased by at least 6 hours, at least 12 hours, at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, for example, at least one week after in vivo administration, compared to a molecule to which the Stefin A protein variant capable of binding to HSA is not linked.

[0392] In the present invention, the fusion protein may be characterized by having an increased serum half-life compared to a Stefin A protein mutant that specifically binds to CD40L, and comprising any one of the amino acid sequences that are at least 70%, 75%, 80%, 85%, 90%, 95% or 98% identical to the sequences of SEQ ID NOs: 667 to 702 (Table 10).In the present invention, the fusion protein may be characterized by having an increased serum half-life compared to a Stefin A protein mutant that specifically binds to CD40L, and comprising any one of the amino acid sequences that are at least 70%, 75%, 80%, 85%, 90%, 95% or 98% identical to the sequences of SEQ ID NOs: 667 to 702 (Table 10) and SEQ ID NOs: 703 to 728 (Table 11).

[0393]

[0394] PEGylation, XTEN, PAS and other polymers

[0395] A wide variety of macromolecular polymers and other molecules can be linked to the CD40L-specific binding Stefin A protein variants of the present invention to modulate and / or provide new biological properties.

[0396] In the present invention, the polymer may be linked to a Stefin A protein variant of the present invention that specifically binds to CD40L by a naturally encoded amino acid, a non-naturally encoded amino acid, or any functional substituent of a natural or non-natural amino acid, or any substituent or functional group added to a natural or non-natural amino acid.

[0397] In the present invention, the molecular weight of the polymer is about 100 Da to about 100,000 Da, but is not limited thereto. In the present invention, the molecular weight of the polymer is 100,000 Da, 95,000 Da, 90,000 Da, 85,000 Da, 80,000 Da, 75,000 Da, 70,000 Da, 65,000 Da, 60,000 Da, 55,000 Da, 50,000 Da, 45,000 Da, 40,000 Da, 35,000 Da, 30,000 Da, 25,000 Da, 20,000 Da, 15,000 Da, and the like. The molecular weight may be about 100 Da to about 100,000 Da, including, but not limited to, Da, 10,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000 Da, 5,000 Da, 4,000 Da, 3,000 Da, 2,000 Da, 1,000 Da, 900 Da, 800 Da, 700 Da, 600 Da, 500 Da, 400 Da, 300 Da, 200 Da, and 100 Da. In the present invention, the molecular weight of the polymer may be characterized as being about 100 Da to about 50,000 Da, about 100 Da to about 40,000 Da, about 1,000 Da to about 40,000 Da, about 5,000 Da to about 40,000 Da, or about 10,000 Da to about 40,000 Da.

[0398] To this end, methods have been developed that involve PEGylation, polysialylation, HESylation, glycosylation, or recombinant PEG analogs fused to flexible, hydrophilic amino acid chains (500-600 amino acids) (see, e.g., Chapman, (2002) Adv Drug Deliv Rev. 54. 531-545; Schlapschy et al., (2007) Prot Eng Des Sel. 20, 273-283; Contermann (2011) Curr Op Biotechnol. 22, 868-876; Jevsevar et al., (2012) Methods Mol Biol. 901, 233-246).

[0399] In the present invention, the polymer may be, for example, polyalkyl ethers and their alkoxy-capped analogs (e.g., polyoxyethylene glycol, polyoxyethylene / polypropylene glycol and its methoxy- or ethoxy-capped analogs, especially polyoxyethylene glycol, the latter also named polyethylene glycol); dPEG (discrete PEG); polyvinylpyrrolidones; polyvinylalkyl ethers; polyoxazolines, polyalkyl oxazolines, and polyhydroxyalkyl oxazolines; polyacrylamides, polyalkyl acrylamides, and polyhydroxyalkyl acrylamides (e.g., polyhydroxypropylmethacrylamide and its derivatives); polyhydroxyalkyl acrylates; polysialic acids. acids and their analogues; hydrophilic peptide sequences; dextran and dextran derivatives (e.g., carboxymethyl dextran polysaccharides and their derivatives), polysaccharides and their derivatives, including amide dextrans; cellulose and its derivatives, e.g., carboxymethyl cellulose, hydroxyalkyl celluloses;chitin and its derivatives (e.g., chitosan, succinyl chitosan, carboxymethylchitin, carboxymethylchitosan); hyaluronic acid and its derivatives; starches; alginates; chondroitin sulfate; albumin; pullulan and carboxymethyl pullulan; polyaminoacids and derivatives thereof (e.g., polyglutamic acids, polylysines, polyaspartic acids, polyaspartamides); maleic anhydride copolymers (e.g., styrene maleic anhydride copolymers); Examples of the copolymer include, but are not limited to, divinylethyl ether maleic anhydride copolymer; polyvinyl alcohols; copolymers thereof; terpolymers thereof; mixtures thereof; and derivatives thereof.

[0400] In the present invention, the polymer may be characterized as being a water-soluble polymer such that the fusion protein does not precipitate in an aqueous environment, such as a physiological environment. In the present invention, the water-soluble polymer may have any structure, including but not limited to linear, forked, or branched. Typically, the water-soluble polymer is a polyalkylene glycol, such as polyethylene glycol (PEG), but may be other water-soluble polymers, including but not limited to these. For example, PEG is used in some embodiments of the present invention. In the present invention, the polymer may be characterized as being pharma- ceutically acceptable.

[0401] The term "polyethylene glycol (PEG)" as used herein is used broadly to encompass any polyethylene glycol molecule, regardless of size or the presence or absence of modification at the PEG terminus, and may be characterized as being linked to the Stefin A protein variant of the present invention as shown in the following formula:

[0402] XO-(CH 2 CH 2 O) n-CH2CH2-, or

[0403] XO-(CH 2 CH 2 O)n-

[0404] where n is 2 to 10,000, and X is H or, as a non-limiting example, C 1-4 These are terminal modifications such as alkyl, blocking groups, or terminal functional groups.

[0405] In the present invention, the PEG may be terminated at one end with hydroxy or methoxy, for example, X is H or CH 3 The present invention may be characterized in that:

[0406] The other end of the PEG, designated in the formula by the terminal "-", may be attached to the Stefin A protein variant of the invention through a naturally occurring or non-naturally encoded amino acid. For example, the attachment may be to an amine group (e.g., including but not limited to, the epsilon amine of lysine, or the N-terminus) through an amide, carbamate, or urea linkage. Alternatively, the polymer may be linked to a thiol group (e.g., including but not limited to, the thiol group of cysteine) through a maleimide bond, allowing the residue of the Stefin A protein variant of the invention to be replaced with a cysteine ​​when the polymer is conjugated to a fusion protein.

[0407] The number of water-soluble polymers (e.g., the degree of PEGylation or glycosylation) linked to the Stefin A protein variant or fusion protein thereof of the present invention may be adjusted to provide or modify pharmacological, pharmacokinetic, or pharmacodynamic properties, such as half-life. In the present invention, the half-life of the Stefin A protein variant or fusion protein thereof comprising said polymer may be characterized as being increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 2-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 50-fold, or at least 100-fold.

[0408] A polymer useful for modifying the PK or biological properties of the Stefin A protein variant or fusion protein of the present invention is one that is part of the fusion protein of the present invention and uses a hydrophilic amino acid polymer that is functionally similar to PEG. In the present invention, the fusion protein may be characterized as comprising a hydrophilic amino acid polymer. The inherent biodegradability of polypeptides has highlighted them as potential alternatives to PEG. In addition, in contrast to the supposed acidity of PEG, the hydrophilic amino acid polymer has a precise molecular structure.

[0409] Unlike fusions of HSA and Fc domains, which must retain the tertiary fold of the fusion domain, fusion proteins containing unstructured domains have the advantage that most of their structure can be exposed to harsh conditions such as higher temperatures or HPLC purification.

[0410] As a non-limiting example of such a polypeptide, XTEN (Amunix) is composed of 864 amino acids consisting of A, E, G, P, S and T (see Schellenberger et al. "A recombinant polypeptide extends the in vivo half-life of peptides and proteins in a tunable manner" 2009 Nat Biotechnol. 27(12):1186-90). The biodegradable properties of XTEN are far greater than those of the commonly used 40KDa PEG, and are accompanied by an extended half-life. Fusion proteins containing XTEN can extend their half-life by 60- to 130-fold compared to those not containing XTEN.

[0411] Yet another example of such a polypeptide is PAS (XL-Protein GmbH) (see Schlapschy et al. "PASYlation: a biological alternative to PEGylation for extending the plasma half-life of soluble active proteins" 2013 Protein Eng Des Sel. 26(8):489-501). The PAS is a random coil polymer composed of a limited set of uncharged amino acids, proline, alanine and serine. As with Fc domains, HSA and XTEN, PAS may be genetically encoded together with a nucleic acid encoding a Stefin A protein variant or fusion protein to be produced as an in-line fusion protein when expressing a Stefin A protein variant or fusion protein that specifically binds CD40L in the genetically engineered cells of the invention.

[0412]

[0413] In line fusion protein

[0414] In the present invention, the fusion protein may be characterized as being any one of the polypeptides in Table 10 below.

[0415] [Table 10]

[0416] JPEG2025509078000042.jpg229154

[0417] JPEG2025509078000043.jpg229152

[0418] JPEG2025509078000044.jpg230154

[0419] JPEG2025509078000045.jpg225149

[0420] JPEG2025509078000046.jpg104152

[0421] In the present invention, the nucleic acid encoding the fusion protein may be characterized by being selected from the nucleic acids provided in Table 11, but is not limited thereto. It can be obviously understood that the nucleic acid sequence encoding the fusion protein can be designed and predicted based on the amino acid sequence of the Stefin A protein of the present invention that specifically binds to CD40L and the nucleic acid sequence encoding the same, as well as the polypeptide sequence of the fusion domain contained in the fusion protein of the present invention and the nucleic acid sequence encoding the same.

[0422] [Table 11]

[0423] JPEG2025509078000048.jpg228152

[0424] JPEG2025509078000049.jpg228153

[0425] JPEG2025509078000050.jpg227152

[0426] JPEG2025509078000051.jpg227152

[0427] JPEG2025509078000052.jpg228151

[0428] JPEG2025509078000053.jpg228152

[0429] JPEG2025509078000054.jpg228151

[0430] JPEG2025509078000055.jpg228152

[0431] JPEG2025509078000056.jpg228151

[0432] JPEG2025509078000057.jpg228152

[0433] JPEG2025509078000058.jpg229152

[0434] JPEG2025509078000059.jpg227152

[0435] JPEG2025509078000060.jpg184152

[0436] Stefin A protein variant-Conjugate

[0437] In another aspect, the present invention relates to a conjugate comprising a Stefin A protein variant that specifically binds to CD40L.

[0438] In still another aspect, the present invention relates to use of the Stefin A protein variant capable of specifically binding to CD40L for producing a conjugate.

[0439] The term "conjugate" or "cojugation" as used herein means linking or joining two or more compounds together to form another compound by any linking or joining method known in the art, or a compound produced by binding or joining two or more compounds. In the present invention, the Stefin A protein variant-conjugate may be understood to include a fusion protein in a broad sense. More specifically, the Stefin A protein variant-conjugate may mean a conjugate comprising at least one moiety conjugated by chemical conjugation in addition to the formation of a continuous peptide bond via the C-terminus and / or N-terminus of the Stefin A protein variant or fusion protein of the present invention that specifically binds to CD40L. The conjugate may be, for example, a Stefin A protein variant-drug conjugate, and the Stefin A protein variant-drug conjugate may comprise at least one pharmacologically active moiety.

[0440] In the present invention, the conjugate may include at least one functional moiety, such as a variety of functional groups, substituents or moieties. In the present invention, the functional moiety may be, for example, a label; a dye; an immunoadhesion molecule; a radionuclide; a cytotoxic compound; a drug; an affinity label; a photoaffinity label; a reactive compound; a resin; a second protein or a polypeptide or a polypeptide analog; an antibody or an antibody fragment; a metal chelator; a cofactor; a fatty acid; a carbohydrate; a polynucleotide; DNA; RNA; an antisense polynucleotide; a saccharide; a water-soluble dendrimer; a cyclodextrin; an inhibitory ribonucleic acid; a biomaterial; a nanoparticle; a spin label; a fluorophore, a metal-containing moiety; a radioactive moiety; a novel functional group; a functional group that interacts covalently or non-covalently with other molecules; a photocaged moiety. moiety;actinic radiation excitable moiety;photoisomerizable moiety;biotin;derivative of biotin;biotin analogue;moiety incorporating a heavy atom;chemically cleavable group;photocleavable group;elongated side chain;carbon-linked sugar;redox-active agent;The moieties may include, but are not limited to, an amino thioacid; a toxic moiety; an isotopically labeled moiety; a biophysical probe; a phosphorescent group; a chemiluminescent group; an electron dense group; a magnetic group; an intercalating group; a chromophore; an energy transfer agent; a biologically active agent; a detectable label; a small molecule; a quantum dot; a nanotransmitter; a radionucleotide; a radiotransmitter; a neutron-capture agent; or a combination thereof, or any other suitable compound or substance;

[0441] - Labels and Detectable Moieties

[0442] In the present invention, the conjugate may comprise a functional moiety. In the present invention, the functional moiety may be a detectable label. For example, the detectable label may be a fluorescent label, a radioactive label, an enzyme label, or any other label known in the art. In the present invention, the functional moiety may be a detectable label that may be included as part of a conjugate for use in medical imaging. The term medical imaging in the present invention refers to any technique that can be used to visualize internal regions of the human or animal body for diagnostic, research, or therapeutic purposes. For example, it may be radioscintigraphy, magnetic resonance imaging (MRI), computed tomography (CT scan), nuclear imaging, positron emission comprising a metal tomography (PET) contrast agents, optical imaging (e.g., fluorescence imaging, including near-infrared fluorescence (NIRF) imaging), bioluminescence imaging, or a combination thereof. In the present invention, the functional moiety may optionally be a contrast agent for X-ray imaging. Substances useful for enhancing such imaging techniques allow visualization of specific locations, organs, or diseased areas in the body and / or improve the quality of the images produced by the imaging techniques, allowing for improved or easier analysis. Such agents may be referred to as contrast agents, and the use of contrast agents increases the contrast between the imaged area and other areas, making it easier to distinguish specific areas. Thus, the term contrast agent is used to include agents for improving image quality (e.g., MRI, etc.) as well as agents that are a prerequisite for image production (e.g., nuclear imaging, etc.).

[0443] In the present invention, the detectable label comprises a chelating moiety for chelating a metal, e.g., a chelator for a radiometal or a paramagnetic ion. In the present invention, the detectable label may be a chelator for a radionuclide useful in radiotherapy or imaging. In the present invention, the useful radionuclides include gamma-emitters, positron-emitters, Auger electron-emitters, X-ray emitters and fluorescence-emitters, including therapeutic beta- or alpha-emitters. Examples of radionuclides useful as toxins in radiotherapy are: 43 K, 47 Sc, 51 Cr, 57 Co, 58 Co, 59 Fe, 64 Cu, 67 Ga, 67 Cu, 68 Ga, 71 Ge, 75 Br, 76 Br, 77 Br, 77 As, 81 Rb, 90 Y, 97 Ru, 99m Tc, 100 Pd, 101 Rh, 103 Pb, 105 Rh, 109 Pd, 111 Ag, 111 In, 113 In, 119 Sb 121 Sn, 123 I, 125 I, 127 Cs, 128 Ba, 129 Cs, 131 I, 131 Cs, 143 Pr, 153 Sm, 161 Tb,166 Ho, 169 EU, 177 Lu, 186 Re, 188 Re, 189 Re, 191 Os, 193 Pt, 194 Ir, 197 Hg, 199 Au, 203 Pb, 211 At, 212 Pb, 212 Bi and 213 Bi. Conditions for the coordination of the chelator with the metal are as described, for example, in U.S. Pat. Nos. 4,831,175, 4,454,106, and 4,472,509 to Gansow et al. Examples of the chelator in the present invention include, but are not limited to, NOTA (1,4,7-triazacyclononane-N,N',N"-triacetic acid), DOTA (1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid), and TETA (1,4,8,11-tetraazacyclotetradecane-N,N',N",N"'-tetraacetic acid).

[0444] Still other detectable isotopes that may be incorporated directly into amino acid residues of the polypeptide in the present invention or that do not require a chelator are: 3 H, 14 C. 32 P, 35 S and 36 Examples include, but are not limited to, Cl.

[0445] In the present invention, the paramagnetic ion may be usefully used for diagnosis. In the present invention, examples of the paramagnetic ion may be, but are not limited to, chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III), erbium (III), or combinations thereof.

[0446] In the present invention, examples of the fluorescent label include, but are not limited to, organic dyes (e.g., cyanine, fluorescein, rhodamine, Alexa Fluors, Dylight fluors, ATTO dyes, BODIPY dyes, etc.), biological fluorophores (e.g., GFP (green fluorescent protein), R-Phycoerythrin, etc.), and quantum dots.

[0447] Non-limiting fluorescent compounds that may be used in the present invention are well known in the art. For example, Cy5, Cy5.5 (also known as Cy5++), Cy2, FITC (fluorescein isothiocyanate), TRITC (tetramethylrhodamine isothiocyanate), phycoerythrin, Cy7, FAM (fluorescein), Cy3, Cy3.5 (also known as Cy3++), Texas Red, LightCycler-Red 640, LightCycler Red 705, TMR (tetramethylrhodamine), rhodamine, rhodamine derivative (ROX), HEX (hexachlorofluorescein), R6G (rhodamine 6G), rhodamine derivative JA133, Alexa fluorescent dyes (e.g., Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 633, Alexa Fluor 555, and Alexa Fluor 647, DAPI (4',6-diamidino-2-phenylindole), propidium iodide, AMCA, Spectrum Green, Spectrum Orange, Spectrum Aqua, Lissamine, and fluorescent transition metal complexes such as europium, but are not limited thereto. Other examples of fluorescent compounds in the present invention include, but are not limited to, fluorescent proteins, such as green fluorescent protein (GFP), enhanced GFP (EGFP), blue fluorescent protein and its derivatives (BFP, EBFP, EBFP2, Azurite, mKalama1), cyan fluorescent protein and its derivatives (CFP, ECFP, Cerulean, CyPet), and yellow fluorescent protein and its derivatives (YFP, Citrine, Venus, YPet).Further examples of fluorescent compounds are also described in detail in the following patent documents: WO2008142571, WO2009056282, and WO9922026.

[0448] In the present invention, the enzyme label may be, for example, horseradish peroxidase (HRP), alkaline phosphatase (AP), glucose oxidase, and β-galactosidase, but is not limited thereto.

[0449] In the present invention, the label may be a biotin label. The biotin label generally consists of a biotinyl group, a spacer, and a reactive group responsible for attachment to a target moiety of a cancer or protein. The biotin may be usefully used to attach the labeled protein to other moieties, including an avidin moiety.

[0450] - Stefin A protein mutant-drug conjugate

[0451] In the present invention, the conjugate may include one or more therapeutic agents. In the present invention, the conjugate may be a Stefin A protein variant-drug conjugate. In the present invention, the "therapeutic agent" refers to a substance that can be used to cure, mitigate, treat, or prevent a disease in humans or other animals. Such therapeutic agents may include therapeutic substances approved by official national agencies, including Korea, and may include, but are not limited to, small molecules, nucleotides, oligopeptides, polypeptides, proteins, etc. In the present invention, examples of therapeutic agents that can be conjugated to the conjugate may include, but are not limited to, cytotoxic agents, anti-metabolites, alkylating agents, antibiotics, growth factors, cytokines, anti-angiogenic agents, anti-mitotic agents, toxins, apoptotic agents, etc.More specific examples include, but are not limited to, DNA alkylating agents, topoisomerase inhibitors, microtubule inhibitors (e.g., DM1, DM4, MMAF, and MMAE), endoplasmic reticulum stress inducing agents, platinum compounds, antimetabolites, vincalkaloids, taxanes, epothilones, enzyme inhibitors, receptor antagonists, therapeutic antibodies, tyrosine kinase inhibitors, radiosensitizers, and chemotherapy combination therapies such as illustrators.

[0452] To prepare the conjugates of the invention, any method known in the art for conjugation of antibodies and proteins such as the Stefin A protein variants of the invention may be used. For example, such methods are described, but are not limited to, in Hunter, et al., (1962) Nature 144:945; David, et al., (1974) Biochemistry 13:1014; Pain, et al., (1981) J. Immunol. Meth. 40:219; and Nygren, J., (1982) Histochem. and Cytochem. 30:407. Methods for conjugating peptides, polypeptides, and organic / inorganic moieties to antibodies and proteins are well known in the art and can be readily used to adapt the conjugates of the invention. For example, when the conjugated moiety is a peptide or polypeptide, it may be chemically crosslinked to the Stefin A protein variant of the invention or may be included as part of a fusion protein. As an example of such a compound, diptheria toxin-AFFIMER® manufactured by Avacta has been known. In the case of non-peptide moieties, they may generally be conjugated to the stefin A protein mutant by chemical conjugation (e.g., functional groups on amino acid side chains, carboxyl groups at the C-terminus, or amino groups at the N-terminus, etc.). In the present invention, the conjugated moieties, regardless of their type, may include at least one moiety that is sensitive to environmental conditions (e.g., pH, etc.). The moiety that is sensitive to environmental conditions may be characterized by releasing the conjugated moiety in target tissues such as diseased tissues or protective tissues.

[0453] Spacer

[0454] In the present invention, the conjugate comprises a spacer or linkage (L) between the substrate recognition sequence (SRS) and the half-life extension moiety that is cleavable by an enzyme present in the inflammatory microenvironment. 1 ) may be included.

[0455] In the present invention, the spacer may be any molecule, for example, one or more nucleotides, amino acids, chemical functional groups. In the present invention, the spacer is a peptide linker (for example, two or more amino acids). In the present invention, the spacer may not adversely affect the expression, secretion or biological activity of the polypeptide. In the present invention, the spacer may not be antigenic and may not induce an immune response. The immune response includes a response from the innate immune system and / or the adaptive immune system. Thus, the immune response may be a cell-mediated response and / or a humoral immune response. The immune response may be, for example, a T cell response, a B cell response, a natural killer (NK) cell response, a monocyte response and / or a macrophage response. In the present invention, other cellular responses may be considered. In the present invention, the linker may be non-protein-coding.

[0456] In the present invention, the L 1 is a hydrocarbon (linear or cyclic) such as 6-maleimidocaproyl, maleimidopropanoyl, and maleimidomethyl cyclohexane-l-carboxylate, or said L1 can be N-succinimidyl 4-(2-pyridylthio)pentanoate, N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, N-succinimidyl(4-iodo-acetyl)aminobenzoate.

[0457] In the present invention, the L 1L may be a polyether such as poly(ethylene glycol) or other hydrophilic linker. For example, if the site or residue to which L is attached contains a thiol (e.g., a cysteine ​​residue), L may be a polyethylene glycol linked to the thiol group by a maleimide moiety. Linkers that can be used in the present invention are described, for example, but not limited to, in WO2019 / 236567, which is incorporated by reference.

[0458] In the present invention, the L 1 may be characterized, for example, by the following chemical formula:

[0459] JPEG2025509078000061.jpg25128

[0460] Here, p represents an integer of 1 to 100, preferably 6 to 50, and more preferably 6 to 12.

[0461] In the present invention, the L 1 The site or residue to which L is attached contains a thiol, 1 When L is a hydrocarbon moiety linked to a thiol group by a maleimide moiety, 2 may be characterized by being represented by the chemical formula:

[0462] JPEG2025509078000062.jpg62128

[0463] Here, p is an integer of 1 to 20, preferably 1 to 4.

[0464]

[0465] Self-Immolative Linkers

[0466] In the present invention, the conjugate has a self-immolative linker (L) between the substrate recognition sequence (SRS) for the enzyme and the drug moiety. 2 ).

[0467] In the present invention, the self-immolative moiety may be defined as a bifunctional chemical group capable of covalently bonding two separate chemical moieties in a generally stable molecule, which may be characterized in that it releases one of the separate chemical moieties from the molecule upon enzymatic cleavage and then cleaves itself from the remainder of the bifunctional chemical group after enzymatic cleavage to release the other one of the separate residues. Thus, in the present invention, the self-immolative moiety may be covalently bonded at one end to a ligand via an amide bond, either directly or indirectly via a spacer unit, and at the other end to a chemical reactive site (functional group) of a drug moiety. In the present invention, derivatization of a drug moiety with a self-immolative moiety may reduce or mask the pharmacological activity of the drug until the drug is cleaved.

[0468] In the present invention, the conjugate may generally be stable in the circulatory system, or at least stable in the absence of an enzyme capable of cleaving the amide bond between the substrate recognition sequence (enzyme-cleavable linker) and the self-immolative moiety. In the present invention, when the Stefin A protein variant-drug conjugate is exposed to a suitable enzyme, the amide bond can be cleaved, initiating a spontaneous self-immolation reaction, leading to cleavage of the covalent bond between the self-immolative moiety and the drug moiety and release of the free drug moiety in an underivatized or pharmacologically active form. In the present invention, the self-immolative moiety of the conjugate may incorporate one or more heteroatoms, which may exhibit enhanced solubility, improved cleavage rate, and reduced tendency of the conjugate to aggregate.

[0469] In the present invention, the L 2 In the present invention, the self-immolative linker L 2 is -NH-(CH 2 ) 4 -C(=O)- or -NH-(CH 2 ) 3In the present invention, the L 2 In the present invention, the L 2 may be 2,4-bis(hydroxymethyl)aniline.

[0470] In the present invention, the Stefin A protein mutant-drug conjugate can employ a heterocyclic self-immolative moiety covalently linked to a therapeutic moiety and a cleavable substrate recognition sequence.

[0471] In the present invention, the L 2 may be a benzyloxycarbonyl group.

[0472] In the present invention, the L 2 may be of the formula:

[0473] JPEG2025509078000063.jpg35128

[0474] Here, the R 1 is hydrogen, unsubstituted or substituted C 1-3 In the present invention, R 1 may be hydrogen. 1 may be methyl.

[0475] In the present invention, the L 2 may be characterized by being selected from the following:

[0476] JPEG2025509078000064.jpg55170

[0477] In the present invention, the L 2 may be characterized by being selected from the following:

[0478] JPEG2025509078000065.jpg24154

[0479] where U is O, S or NR 6 and;

[0480] Q is CR 4 or N;

[0481] V 1 , V 2 and V 3 are CR 4 or N, provided that Q, V 1 , V 2 at least one of is N;

[0482] T is an NH, NR 6 , O or S;

[0483] R 1 , R 2 , R 3 and R 4 are selected from the following: H, F, Cl, Br, I, OH, -N(R 5 ) 2 , -N(R 5 ) 3 + , C 1 -C 8 Alkyl halide (C 1 -C 8 alkylhalide, carboxylate, sulfate, sulfamate, sulfonate, -SO 2 R 5 , -S(=O)R 5 , -SR 5 , -SO 2 N(R 5 ) 2 , -C(=O)R 5 , -CO 2 R 5 , -C(=O)N(R 5 ) 2 , -CN, -N3 , -NO 2 , C 1 -C 8 Alkoxy, C 1 -C 8 Halo-substituted alkyl (C 1 -C 8 halosubstituted alkyl, polyethyleneoxy, phosphonate, phosphate, C 1 -C 8 Alkyl, C 1 -C 8 Substituted Alkyl, C 2 -C 8 Alkenyl, C 2 -C 8 Substituted alkenyl, C 2 -C 8 Alkynyl, C 2 -C 8 Substituted alkynyl, C 6 -C 20 Aryl, C 6 -C 20 Substituted Aryl, C 1 -C 20 Heterocycles, and C 1 -C 20 a substituted heterocycle; or R 2 and R 3 are bonded together to form a carbonyl (=O) or a spiro carbocyclic ring having 3 to 7 carbon atoms; and

[0484] R 5 and R 6 are selected from the following, H, C 1 -C 8 Alkyl, C 1 -C 8 Substituted Alkyl, C 2 -C 8 Alkenyl, C 2 -C 8 Substituted alkenyl, C 2 -C 8 Alkynyl, C 2 -C 8Substituted alkynyl, C 6 -C 20 Aryl, C 6 -C 20 Substituted Aryl, C 1 -C 20 Heterocycles, and C 1 -C 20 Substituted heterocycles;

[0485] Here, C 1 -C 8 Substituted Alkyl, C 2 -C 8 Substituted alkenyl, C 2 -C 8 Substituted alkynyl, C 6 -C 20 Substituted aryl, and C 2 -C 20 Each substituted heterocycle is substituted with one or more substituents selected from the following: F, Cl, Br, I, OH, -N(R 5 ) 2 , -N(R 5 ) 3 + , C 1 -C 8 Alkyl halide (C 1 -C 8 alkylhalide), carboxylate, sulfate, sulfamate, sulfonate, C 1 -C 8 Alkyl sulfonates, C 1 -C 8 Alkylamino, 4-dialkylaminopyridinium, C 1 -C 8 Alkyl hydroxyl, C 1 -C 8 Alkylthiol, -SO 2 R 5 , -S(=O)R 5 , -SR 5 , -SO 2 N(R 5 ) 2 , -C(=O)R 5 , -CO 2 R 5, -C(=O)N(R 5 ) 2 , -CN, -N 3 , -NO 2 , C 1 -C 8 Alkoxy, C 1 -C 8 Trifluoroalkyl, C 1 -C 8 Alkyl, C 3 -C 12 Carbocycle (C 3 -C 12 carbocycle), C 6 -C 20 Aryl, C 2 -C 20 Heterocycles, polyethyleneoxy, phosphonates, and phosphates.

[0486] In the present invention, when T is NH, it is a primary amine (-NH) that is tethered to the therapeutic moiety (prior to being coupled to the self-immolative moiety). 2 ), and when the T is N, it may be understood to be derived from a secondary amine (-NH-). Similarly, when the T is O or S, it is derived from a hydroxyl (-OH) or sulfhydryl (-SH) group, respectively, that is tethered from the therapeutic moiety prior to coupling to the self-immolative moiety.

[0487] In the present invention, the self-immolative linker L 2 is -NH-(CH 2 ) 4 -C(=O)- or -NH-(CH 2 ) 3 It may be -C(=O)-.

[0488] In the present invention, the self-immolative linker L 2 may be PABC (p-aminobenzyloxycarbonyl).

[0489] In the present invention, the self-immolative linker L 2may be 2,4-bis(hydroxymethyl)aniline.

[0490]

[0491] Other examples of self-immolative linkers readily adaptable for use in the Stefin A protein mutant-drug conjugates according to the present invention are taught, for example but not limited to, U.S. Pat. No. 7,754,681, WO2012 / 074693; US9,089,614; WO2019236567A; EP1,732,607; WO2015 / 038426A1, Walther et al. "Prodrugs in medicinal chemistry and enzyme prodrug therapies" Adv Drug Deliv Rev. 2017 Sep 1;118:65-77; and Tranoy-Opalinski et al. "Design of self- immolative linkers for tumor-activated prodrug therapy", Anticancer Agents Med Chem. 2008 Aug;8(6):618-37;

[0492] Further examples of self-immolative linkers readily adaptable for use in the Stefin A protein mutant-drug conjugates described in the present invention are described in, but not limited to, International Publication WO2019 / 236567.

[0493]

[0494] In still another aspect, the present invention relates to a nucleic acid encoding the Stefin A protein mutant that specifically binds to CD40L, or a fusion protein or conjugate containing the same.

[0495] In yet another aspect, the present invention relates to a vector comprising the nucleic acid.

[0496] In yet another aspect, the present invention relates to a delivery vehicle comprising the nucleic acid.

[0497] In the present invention, the delivery vehicle may be characterized as being used for in vivo delivery.

[0498] In yet another aspect, the present invention relates to a genetically engineered cell into which the nucleic acid or vector has been introduced.

[0499] In the present invention, the nucleic acid may be characterized in that it is delivered in vivo.

[0500]

[0501] In vivo delivery of nucleic acids encoding the Stefin A protein variants of the invention or fusion proteins or conjugates containing same

[0502] In the present invention, an alternative approach to the delivery of the Stefin A protein variant that specifically binds to CD40L or a fusion protein or conjugate containing the same is to rely on the body itself to produce the therapeutic polypeptide. Numerous clinical studies have reported the usefulness of in vivo gene delivery to cells using various delivery systems. In vivo gene delivery is the administration of a nucleic acid encoding the Stefin A protein variant, rather than the Stefin A protein variant itself, to a patient. This allows the patient's body to produce the Stefin A protein variant that specifically binds to CD40L of the present invention, or a fusion protein or conjugate containing the same, over a long period of time and secrete it systemically or locally depending on the production site. Genetically encoded nucleic acid can provide a labor- and cost-efficient alternative to the existing production, purification, and administration of proteins. A variety of in vivo antibody expression platforms can be used to deliver the Stefin A protein variant or a nucleic acid encoding a fusion protein or conjugate containing the same, including, for example, viral vectors, naked DNA, or RNA. Gene delivery of nucleic acid can reduce the cost of goods and production, thereby enabling lower costs and less frequent drug administration. Overall, the in vivo production of the Stefin A protein variants that specifically bind to CD40L of the present invention, or fusion proteins or conjugates containing the same, by expression of nucleic acids has the advantages of (i) serving a wide range of therapeutic or prophylactic applications at low cost, (ii) accessibility in developed and developing countries, and (iii) being a more effective and inexpensive method of treatment. In addition to in vivo gene transfer, cell therapies genetically engineered with the nucleic acids of the present invention can be produced based on cells harvested from a host (or donor) and administered to a patient.

[0503] Intramuscular antibody gene administration has been most widely evaluated (Deal et al. (2015) "Engineering humoral immunity as prophylaxis or therapy" Curr Opin Immunol. 35: 113-22.), and may be applied as an administration method for in vivo delivery of the nucleic acid of the present invention. In fact, the inherent anatomical, cellular and physiological properties of skeletal muscle provide a stable environment for expression and systemic circulation of long-term encoded nucleic acid, and allow easy repeated administration. The abundant blood vessels provide a system in which the Stefin A protein variant of the present invention that specifically binds to CD40L, or a fusion protein or conjugate containing the same, expressed and secreted in vivo, can be efficiently transported to the circulatory system. In the present invention, the in vivo delivered nucleic acid may be integrated into the host genome, but integration in the host genome is not a prerequisite for achieving long-term protein expression. The liver is yet another site that is frequently used for preclinical antibody gene delivery, and may generally be transfected by intravenous (iv) injection. The liver may be a gene delivery site for vascular secretion and systemic circulation of the Stefin A protein variant of the present invention and fusion protein containing the same. The liver synthesizes plasma proteins and has various physiological functions, and therefore may be suitable for expressing the Stefin A protein mutant and a fusion protein containing the same by transferring the nucleic acid of the present invention.

[0504] The success of gene therapy has been mainly driven by the improvement of non-viral and viral gene transfer vectors. A series of physical and chemical non-viral methods have been used to transfer DNA and mRNA into mammalian cells, a number of which have been developed as clinical stage techniques for gene therapy in vitro and in vivo and are readily applicable to gene therapy using the nucleic acids of the present invention. Cationic liposome technology can be used, in which amphipathic lipids with positively charged head groups and hydrophobic lipid tails have the ability to bind negatively charged DNA or RNA, forming particles that generally enter cells by intracellular transfer. Some cationic liposomes also contain neutral co-lipids, which are understood to improve uptake by mammalian cells. (Felgner et al. (1987) Lipofection: a highly, lipid-mediated DNA-transfection procedure. MNAS84:7413-7417; San et al. (1983) “Safety and short-term efficient toxicity of a novel cationic lipid formulation for human gene therapy” Hum. Gene Ther.4:781-788; Xu et al. (1996) “Mechanism of DNA release from cationic liposome / DNA complexes used in cell transfection” Biochemistry 35:5616-5623; and Legendre et al. (1992) “Delivery of plasmid DNA into mammalian cell lines using pH -sensitive liposomes: comparison with cationic liposomes” Pharm.Res. 9, 1235-1242.).

[0505] Similarly, other polycations such as poly-l-lysine and polyethylene-imine may be used to deliver the nucleic acids of the invention. Such polycations complex with the nucleic acid through charge interactions and help condense the DNA or RNA into nanoparticles, which then become substrates for endosome-mediated uptake. Several such cationic nucleic acid complex technologies have been developed as potential clinical products, including complexes with plasmid DNA, oligodeoxynucleotides, and various forms of synthetic RNA. Modified and unmodified (or "naked") DNA and RNA have also been shown to mediate successful gene transfer in a variety of situations and may also be used as systems for delivery of encoded nucleic acids. This includes the use of plasmid DNA by direct intramuscular injection.For example, reference is made to Rodrigo et al. (2012) “De novo automated design of small RNA circuits for engineering synthetic riboregulation in living cells” PNAS 109:15271-15276; Oishi et al. (2005) “Smart polyion complex micelles for targeted intracellular delivery of PEGylated antisense oligonucleotides containing acid-labile linkages” Chembiochem. 6:718-725; Bhatt et al. (2015) “Microbeads mediated oral plasmid DNA delivery using polymethacrylate vectors: an effectual groundwork for colorectal cancer” Drug Deliv. 22:849-861; Ulmer et al. (1994) “Protective immunity by intramuscular injection of low doses of influenza virus DNA vaccines” Vaccine 12:1541-1544; and Heinzerling et al. (2005), etc.

[0506] Viral vectors are currently used as a delivery vehicle in most preclinical and clinical gene therapy trials and are the first approved gene therapies (Gene Therapy Clinical Trials Worldwide 2017 (abedia.com / wiley / )). The main driver for this is the excellent gene transfer efficiency that reflects natural evolutionary development. Viral vector systems are attractive for gene transfer because viruses have evolved the ability to cross cell membranes upon infection and deliver the nucleic acid of the present invention to target cells. The field of viral vector-mediated antibody gene transfer pioneered by the adenovirus system has made considerable development in recent decades. Numerous administration routes, preclinical models, and disease indications that have been successfully evaluated have fully demonstrated the antibody gene transfer capabilities, allowing skilled technicians to easily select and apply the antibody gene transfer system as a technique for in vivo delivery of the nucleic acid of the present invention. Muscle has emerged as the administration site of choice for extended mAb expression and may be a suitable target tissue for expression of the nucleic acid of the present invention.

[0507] In vivo gene transfer of the nucleic acids of the invention can also be achieved using non-viral vectors, such as expression plasmids. Non-viral vectors are reported to be easily produced and do not induce specific immune responses. Muscle tissue is the most frequently used target tissue for transfection because it is well vascularized and easily accessible, and muscle cells are long-lived cells. Intramuscular injection of naked plasmid DNA transfects a proportion of muscle cells. Using this approach, plasmid DNA encoding cytokines and cytokine / IgG1 chimeric proteins has been reported to have a positive impact on disease outcome (autoimmunity) in vivo.

[0508] In some cases, to increase the transfection efficiency using intravascular delivery, which has shown improved gene transfer and expression levels, short-term transient hypertension may be induced in the vein. Such a transient increase in vascular pressure can enhance local absorption, and a specialized blood pressure cuff for this purpose has been reported (Zhang et al. (2001) "Efficient expression of naked DNA delivered intraarterially to limb muscles of nonhuman primates" Hum. Gene Ther., 12: 427-438).

[0509] The efficiency of in vivo gene delivery can also be increased by other techniques such as improving nucleic acid delivery using chemical carriers (cationic polymers or lipids) or physical access methods (gene gun delivery or electroporation) (Tranchant et al. (2004) "Physicochemical optimization of plasmid delivery by cationic lipids" J. Gene Med., 6(Suppl. 1):S24-S35; and Niidome et al. (2002) "Gene therapy progress and prospects: nonviral vectors" Gene Ther., 9:1647-1652. Electroporation is especially regarded as an interesting technique for nonviral gene delivery. Somiari, et al. (2000) "Theory and in vivo application of electroporative gene delivery" Mol. Ther. 2:178-187; and Jaroszeski et al. (1999) "In vivo gene delivery by electroporation" Adv. Drug Delivery Rev., 35:131-137). With electroporation, a pulsed electrical current is applied to a local tissue area to increase cell permeability, allowing gene transfer across the membrane.Studies have shown that in vivo gene transfer using electroporation can be at least 10-100 times more efficient than without electroporation (e.g., Aihara et al. (1998) "Gene transfer into muscle by electroporation in vivo" Nat. Biotechnol. 16:867-870; Mir, et al. (1999) "High-efficiency gene transfer into skeletal muscle mediated by electric pulses" PNAS 96:4262-4267; Rizzuto, et al. (1999) "Efficient and regulated erythropoietin production by naked DNA injection and muscle electroporation" PNAS 96:6417-6422; and Mathiesen (1999) "Electropermeabilization of skeletal muscle enhances gene transfer in vivo" Gene Ther., 6:508-514.).

[0510] In the present invention, the encoding nucleic acid may be delivered by a wide variety of gene delivery systems commonly used in viral, non-viral or physical gene therapy, e.g., Rosenberg et al., Science, 242:1575-1578, 1988, and Wolff et al., Proc. Natl. Acad. Sci. USA 86:9011-9014 (1989). Discussion of methods and compositions for use in gene therapy include Eck et al., in Goodman & Gilman's The Pharmacological Basis of Therapeutics, Ninth Edition, Hardman et al., eds., McGraw-Hill, New York,(1996),Chapter5,pp.77-101;Wilson,Clin.Exp.Immunol.107(Suppl.1):31-32,1997;Wivel et al.,Hematology / Oncology Clinics of North America,Gene Therapy,SLEck,ed.,12(3):483-501,1998;Romano et al.,Stem Cells, 18:19-39, 2000, and U.S. Pat. No. 6,080,728.

[0511] Delivery routes include, for example, systemic administration and in situ administration. For efficient gene delivery, it should be targeted to the specific tissue / cell required, and the resulting transgene expression should be at a level appropriate for the specific application. The promoter is the key cis-acting element within the vector genome design, which can dictate the overall strength of expression as well as cell specificity.

[0512] [Table 12]

[0513] JPEG2025509078000067.jpg189128

[0514] JPEG2025509078000068.jpg96134

[0515] In the present invention, ubiquitous expression of the encoded nucleic acid in all cell types would be preferred. Constitutive promoters such as EF1α (Human elongation factor 1α-subunit), CMV (Immediate-early cytomegalovirus), CBA (chicken β-actin) and its derivatives CAG, GUSB (β glucuronidase) or ubiquitin C (UBC) are used to drive expression of the encoded nucleic acid in most tissues. In general, CBA and CAG drive stronger expression among constitutive promoters. However, their size of ∼1.7 kbs compared to CMV (∼0.8 kbs) or EF1α (∼1.2 kbs) may limit their use in vectors with packaging constraints such as AAV, especially when the size of the nucleic acid of the present invention is large. The GUSB or UBC promoters can provide ubiquitous gene expression at small sizes of 378 bps and 403 bps, respectively, but are weaker than the CMV or CBA promoters. Therefore, modifications to constitutive promoters have been pursued to reduce their size while not affecting expression, and examples such as CBh (~800 bps) and miniCBA (~800 bps) can drive similar and much higher expression in selected tissues (Gray et al., Hum Gene Ther. 2011 22:1143-1153).

[0516] In the present invention, when expression of the encoded nucleic acid needs to be restricted to a particular cell type within an organ, a promoter may be used to mediate this specificity. For example, within the nervous system, a promoter may be used to restrict expression to neurons, astrocytes, or oligodendrocytes. In neurons, the neuron-specific enolase (NSE) promoter induces stronger expression than ubiquitous promoters. Also, the platelet-derived growth factor B-chain (PDGF-β), synapsin (Syn), and methyl-CpG binding protein 2 (MeCP2) promoters can induce neuron-specific expression at lower levels than NSE. In astrocytes, a 680 bps long truncated version of the glial fibrillary acidic protein (GFAP, 2.2 kbs) promoter [gfaABC(1)D] can confer higher levels of expression with the same astrocyte specificity as the GFAP promoter. Selection of the myelin basic protein (MBP) promoter, which restricts expression to glial cells, can confer expression targeted to oligodendrocytes, but its use is limited by its 1.9 kbs size and low expression levels.

[0517] When expressing the nucleic acid of the present invention in skeletal muscle cells, an exemplary promoter based on muscle creatine kinase (MCK) and desmin (1.7 kbs) shows a high ratio of specificity (with minimal expression in liver, if desired). The promoter of α-myosin heavy chain (α-MHC; 1.2 kbs) showed considerable cardiac specificity compared to other muscle promoters (Lee et al., 2011 J Cardiol. 57(1):115-22). In hematopoietic stem cells, the synthetic MND promoter (Li et al., 2010 J Neurosci Methods. 189(1): 56-64) and the promoter containing the 2AUCOE (ubiquitous chromatin opening element) were found to induce higher transgene expression in all cell lineages when compared to the EF1α and CMV promoters, respectively (Zhang et al., 2007 Blood. 110(5): 1448-57; Koldej 2013 Hum Gene Ther Clin Dev. 24(2): 77-85; Dighe et al., 2007 Blood. 110(5): 1448-57; al., 2014 PLoS One. 9(8): e104805.). Conversely, the use of promoters that restrict expression to hepatocytes after vector-mediated gene transfer has been shown to reduce transgene-specific immune responses in at-risk systems and even induce immune tolerance to the expressed protein (Zhang et al., 2012 Hum Gene Ther. 23(5):460-72). The α1-antitrypsin (hAAT; 347 bps) and thyroxine-binding globulin (TBG; ~400 bps) promoters drive gene expression restricted to the liver while minimizing penetration into other tissues (Yan et al., 2012 Gene. 506(2):289-94; Cunningham et al., 2008 Mol Ther. 16(6):1081-8).

[0518] In the present invention, a mechanism is required to control the duration and amount of expression of the encoded nucleic acid delivered in vivo, and the art is aware of a variety of inducible promoters that can be used in viral vectorization and plasmid DNA-based gene delivery: Fang et al. (2007) Mol Ther. 5(6): 1153-9; and Perez et al. (2004) Genet Vaccines Ther. 2(1): 2. An example of a regulatable mechanism currently in clinical trials is an ecdysone-based gene switch that is activated by a small molecule ligand: Cai et al. (2016) Clin Pharmacol Drug Dev. 2016.

[0519] In the in vivo delivery and expression of the nucleic acids of the invention, viral post-transcriptional regulatory elements (PREs) may be used; such cis-acting elements are required for the nuclear export of intronless viral RNA (Huang and Yen, 1994 J Virol. 68(5):3193-9; and 1995 Mol Cell Biol. 15(7):3864-9). Examples include HPRE (Hepatitis B Virus PRE, 533 bps) and WPRE (Woodchuck Hepatitis Virus PRE, 600 bps), which can increase the expression level of a delivered gene by approximately 10-fold in certain cases (Donello et al., 1998 J Virol. 72(6):5085-92). Furthermore, when using lentivirus and AAV vectors, WPRE has been shown to increase the expression of PPE, PDGF, and NSE promoter-driven transgenes in addition to increasing the expression of CMV promoter-driven transgenes. Yet another effect of the WPRE may be to protect the encoded nucleic acid gene from silencing (Paterna et al., 2000 Gene Ther. 7(15):1304-11; Xia et al., 2007 Stem Cells Dev. 2007Feb;16(1):167-76).

[0520] Polyadenylation of the encoded nucleic acid transcript may also be important for nuclear exit, translation and mRNA stability. Thus, in the present invention, the nucleic acid may comprise a polyadenylation signal sequence. Various studies are known that have determined the effect of various polyA signals on gene expression and mRNA stability. Exemplary polyadenylation signal sequences include the SV40 late or bovine growth hormone polyA (bGHpA) signal sequences as well as the minimal synthetic polyA (SPA) signal (Levitt et al., 1989 Genes Dev. 3(7): 1019-25; Yew et al., 1997 Hum Gene Ther. 19978(5): 575-84). The efficiency of polyadenylation is increased by the SV40 late polyA signal upstream enhancer (USE) located upstream of other polyA signals (Schek et al., 1992 Mol Cell Biol. 12(12): 5386-93). In the present invention, for example, the nucleic acid may include, but is not limited to, the SV40late+2xUSE polyA signal.

[0521] [Table 13]

[0522] In the present invention, the nucleic acid may further include at least one regulatory enhancer in addition to any promoter sequence. The CMV enhancer is upstream of the CMV promoter at -598 to -68 (Boshart et al., 1985 Cell. 41(2):521-30) and contains a (-600 bps) transcription binding site. In the present invention, the CMV enhancer may be included in the construct to increase tissue-specific promoter-driven transgene expression, such as the atrial natriuretic factor (ANF) promoter, the club cell 10 (CC10) promoter, the surfactant protein C (SP-C) promoter, or the platelet-derived growth factor-β (PDGF-β) promoter. Overall, the CMV enhancer is a broadly applicable tool to increase transgene expression under different cell-specific promoters and different cell types, and to increase transgene expression levels. In muscle, transgene expression using, for example, a muscle-specific promoter together with a CMV enhancer in an AAV expression system can increase the expression level of the protein encoded by the transgene, and thus can be particularly useful for expressing the nucleic acid of the present invention in muscle cells. In the present invention, the nucleic acid may further comprise at least one intron sequence. The presence of introns or intervening sequences in mRNA has been reported to be important for mRNA processing and increased transgene expression in vitro (Huang and Gorman, 1990 Mol Cell Biol. 10(4):1805-10; Niwa et al., 1990 Genes Dev. 4(9):1552-9). The intron may be located within the nucleic acid sequence encoding the Stefin A protein variant of the present invention, or a fusion protein or conjugate containing the same, and / or may be located between the promoter and the transgene.Table 14 compares various introns placed between the promoter and the transgene in mice using AAV2 for hepatic transgene expression (Wu et al., 2008). It was reported that the MVM (Microvirus of Mice) intron increased transgene expression more than any other intron tested, increasing it by more than 80-fold compared to the absence of an intron (Wu et al., 2008). However, in cultured neurons using AAV expression cassettes, transgene expression was less observed under the CaMPKII promoter with a chimeric intron (human β-globin donor and immunoglobulin heavy chain acceptor) between the transgene and the polyA signal compared to WPRE (Choi et al., 2014). In the present invention, the intron can be an important element included in the expression cassette to increase transgene expression.

[0523] [Table 14]

[0524] In the present invention, the vector may be an episomal vector. In the present invention, when the vector is an episomal vector, it may contain at least one origin of replication, a minichromosome retention element (MME) and / or a nuclear localization element. In the present invention, the episomal vector may contain a portion of a viral genomic DNA encoding an origin of replication (ori) necessary for the vector to replicate autonomously and persist in a host cell for multiple generations. In the present invention, the episomal vector may contain at least one gene encoding at least one viral protein required for replication, such as a replicator protein. Optionally, the replicator protein that helps initiate replication may be expressed in trans on yet another DNA molecule, such as yet another vector or host genomic DNA, in a host cell containing the self-replicating episomal expression vector of the present invention. In the present invention, preferably, the self-replicating episomal LCR-containing expression vector may not contain viral sequences that are not necessary for long-term stable maintenance in eukaryotic host cells, such as viral genomic DNA encoding core or capsid proteins that generate infectious viral particles or viral oncogenic sequences that may be present in full-length viral genomic DNA molecules. In the present invention, "stable maintenance" refers to the ability of a self-replicating episomal expression vector to persist or be maintained without significant loss (e.g., >50%) in the progeny of a dividing cell in the absence of non-dividing cells or continuous selection in the copy number of the vector for 2, 3, 4, or 5 or more generations. In the present invention, the vector may be maintained for 10-15 or more generations. In contrast, "transient" or "short term" persistence of a plasmid in a host cell means that the vector cannot replicate and segregate in a stable manner in the host cell. In this case, the vector is lost after 1-2 generations or shows a >51% loss in copy number between successive generations.

[0525] Some representative self-replicating, LCR-containing, episomal vectors useful in the present invention are further described below. The self-replicating function may alternatively be provided by at least one mammalian sequence, and may optionally be combined with at least one sequence required for nuclear retention, as described in Wohlgeuth et al., 1996, Gene Therapy 3:503; Vos et al., 1995, Jour. Cell. Biol., Supp. 21A, 433; and Sun et al., 1994, Nature Genetics 8:33. The advantage of using mammalian, and particularly human, sequences to provide the self-replicating function is that no exogenous activators are required, which may have toxic or carcinogenic properties. One of ordinary skill in the art will appreciate that the present invention is not limited to one origin of replication or any one episomal vector, but may include combinations of tissue-restricted regulation of LCRs in episomal vectors. The present invention also makes reference to WO1998007876 and U.S. Patent No. 7,790,446.

[0526] In the present invention, the episomal expression vector may be, for example, an Epstein-Barr virus-based self-replicating episomal expression vector (Yates et al., Proc. Natl. Acad. Sci. USA 81:3806-3810 (1984); Yates et al., Nature 313:812-815 (1985); Krysan et al., Mol. Cell. Biol. 9:1026-1033 (1989); James et al. Gene 86:233-239 (1990); Peterson and Legerski, Gene 107:279-284 (1991); and Pan et al., Som. Cell 107:279-284 (1992)). Molec. Genet. 18:163-177 (1992); papillomavirus-based self-replicating episomal expression vectors (e.g., bovine papillomavirus and human papillomavirus (Ustav et al., EMBO J. 10:449-457 (1991); Ustav et al., EMBO J. 10:4231-4329, (1991); Ustav et al., Proc. Natl. Acad. Sci. USA 90:898-902 (1993)); or parvovirus-based self-replicating episomal expression vectors (Papovavirus-Based, Self-Replicating, Episomal Expression Vectors, De Benedetti and Rhoads, Nucleic Acids Res. 19:1925 (1991), as can a 3.2 kb fragment of the BK virus (Cooper and Miron, Human Gene Therapy 4:557(1993)), but is not limited thereto.

[0527] The nucleic acid encoding the Stefin A protein variant specifically binding to CD40L of the present invention or a fusion protein or conjugate comprising the same may be provided as a circular or linear nucleic acid, said circular or linear nucleic acid being capable of directing the expression of the coding sequence in an appropriate target cell. In the present invention, at least one of the nucleic acid systems for expression may be chimeric, meaning that at least one of such components is heterologous relative to at least one of the other components.

[0528]

[0529] Method for introducing nucleic acid (or vector)

[0530] As used herein, the term "introduction" refers to making a host cell receive an exogenous gene (nucleic acid or vector) that the host cell does not possess.

[0531] In the present invention, the nucleic acid encoding the Stefin A protein variant that specifically binds to CD40L or a fusion protein or conjugate containing the same may be characterized in that it is introduced into a host cell using a vector containing the same.

[0532] The term "vector" according to the present invention refers to a means for expressing a gene of interest in a host cell, and may be a viral vector or a non-viral vector. In the present invention, the viral vector may be, for example, an adenovirus vector, a retrovirus vector, an adeno-associated viral vector, a retro-associated viral vector (e.g., a gamma retrovirus vector), a 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)), a lentivirus (Wang G. et al., J. Clin. Invest., 104 (11): R55-62 (1999)), a 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, but are not limited to these.

[0533] In the present invention, the non-viral vector includes, but is not limited to, naked plasmid DNA (pDNA), linear nucleic acid or linear expression cassette (LEC), a plasmid vector (Sambrook et al., 1989) and a minicircle (Yew et al., 2000 Mol Ther 1(3), 255-62).

[0534] 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 Stefin A protein variant of the invention, or a fusion protein or conjugate containing the same, may be operably linked to such a promoter and transcription termination sequence.

[0535] "Operably linked" refers to the functional association of a nucleic acid expression control sequence (e.g., a promoter, a signal sequence, or an array of transcription factor binding sites) with another nucleic acid sequence, whereby the control sequence controls the transcription and / or translation of the other nucleic acid sequence.

[0536] When a prokaryotic cell is used as the host, it generally contains a strong promoter capable of driving transcription (e.g., tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pLλ promoter, pRλ promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter, T7 promoter, etc.), a ribosome binding site for 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.

[0537] In the present invention, the promoter may be characterized as being a eukaryotic promoter, preferably selected from the CMV promoter, PGK promoter, EF1α promoter, EFS promoter, CBh promoter, MSCV promoter, SFFV promoter, and UbC promoter, and most preferably selected from the cytomegalovirus (CMV) promoter, EF1α promoter, and CBh promoter, but is not limited thereto.

[0538] In the present invention, the promoter may further be characterized in that it contains an enhancer sequence, but is not limited thereto.

[0539] 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).

[0540] The vector may contain an antibiotic resistance gene commonly used in the art as a selection marker, such as, but not limited to, resistance genes for ampicillin, gentamycin, carbenicillin, chloramphenicol, streptomycin, kanamycin, puromycin, blasticidin, hygromycin, geneticin, neomycin, and tetracycline.

[0541] In the present invention, the nucleic acid encoding the Stefin A protein variant that specifically binds to CD40L or a fusion protein containing the same may be introduced by being mixed into a gene of a host cell.

[0542]

[0543] In the present invention, the nucleic acid encoding the Stefin A protein variant that specifically binds to CD40L or a fusion protein containing the same may be prepared by chemical synthesis using an oligonucleotide synthesizer. The oligonucleotide may be designed by selecting the amino acid sequence of the desired polypeptide and the codons preferred in the host cell in which the recombinant polypeptide of interest is to be produced. Standard methods may be applied to synthesize a polynucleotide sequence encoding an isolated polypeptide of interest. For example, a complete amino acid sequence may be used to construct a reverse-translated gene. Alternatively, a DNA oligomer containing a nucleotide sequence encoding a specific isolated polypeptide may be synthesized. For example, several small oligonucleotides encoding portions of the desired polypeptide may be synthesized and then ligated. The individual oligonucleotides generally contain 5' or 3' overhangs for complementary assembly.

[0544] In the present invention, once a nucleic acid sequence encoding the Stefin A protein variant that specifically binds to CD40L or a fusion protein containing the same is obtained, a vector containing the same may be produced by recombinant DNA technology using techniques widely known in the art. Expression vectors containing a sequence encoding the Stefin A protein variant of the present invention or a fusion protein containing the same and appropriate transcriptional and translational control signals can be constructed using methods well known to those skilled in the art. Such methods include, for example, in vitro recombinant DNA technology, synthetic technology, and in vivo genetic recombination. (For example, 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).

[0545] In the present invention, introduction of a nucleic acid or vector into a host cell may be carried out using various methods known to those skilled in the art.

[0546] In the present invention, a nucleic acid encoding the Stefin A protein variant that specifically binds to CD40L or a fusion protein containing the same, or a non-viral expression vector containing the same, may be delivered and introduced into a host cell by conventional techniques (e.g., RNA-mediated gene delivery, electroporation, liposome transfection, and calcium phosphate precipitation).

[0547] When the gene delivery system of the present invention is constructed based on a viral vector construct, delivery may be performed by a conventional infection method known in the art, such as, but not limited to, electroporation (Neumann, E. et al., EMBO J., 1:841 (1982); and Tur-Kaspa et al., Mol. Cell Biol., 6:716-718 (1986)), gene bombardment (Yang et al., Proc. Natl. Acad. Sci., 87:9568-9572 (1990) in which DNA particles (e.g., gold) are loaded and allowed to enter cells), sonoporation, magnetofection, and hydrodynamic delivery.

[0548] In the present invention, the nucleic acid or vector may be delivered to the host cell by RNA-mediated gene transfer (see, e.g., "mRNA-based therapeutics: developing a new class of drugs" Nat Rev Drug Discov. 13(10):759-80.; Pardi et al. 2015 J Control Release 217:345-51; WO2017 / 162266; Stadler et al. (2017) Nature Medicine 23:815-817; WO / 2017 / 036889; WO2015 / 034928; WO2015 / 034925; WO2013 / 151666). For exemplary mRNA and other nucleic acids in the present invention, reference is made to the specifications and drawings described in WO2017 / 049275, WO2016 / 118724, WO2016 / 118725, WO2016 / 011226, WO2015 / 196128, WO / 2015 / 196130, WO / 2015 / 196118, WO / 2015 / 089511, and WO2015 / 105926, among others.

[0549] In the present invention, the nucleic acid or vector may be introduced into the host cell by electroporation (Kim et al. Cancer Gene Ther, (2016) 23(10):341-347; in the present invention, electroporation can be carried out by electroporation, for example, as described in U.S. Pat. Nos. 7,245,963; 6,302,874; 5,676,646; 6,241,701; 6,233,482; 6,216,034; 6,208,893; 6,192,270; 6,181,964; 6,150,148; 6,1 and 5,702,359; International Publication Nos. WO / 2017 / 106795, WO / 2016 / 161201, WO / 2016 / 154473, WO / 2016 / 112359, and WO / 2014 / 066655, without limitation.

[0550] In the present invention, Transfection Enhancing Formulations may be used for the delivery of the nucleic acid. In the present invention, the nucleic acid may be encapsulated in liposomes, preferably cationic liposomes ((Wong, TK et al., Gene, 10:87 (1980); Nicolau and Sene, Biochim. Biophys. Acta, 21:185-190 (1982); and Nicolau et al., Methods Enzymol., 149:157-176 (1987)), which can interact with cell membranes and deliver the nucleic acid to cells via fusion or intracellular entry, or polymersomes (synthetic liposomes). In the present invention, DNA may also be formed as a complex with polymers (polyplexes) or dendrimers, which can directly release the load into the cytoplasm of cells. Exemplary carriers include, but are not limited to, microparticles of poly(lactide-co-glycolide), polyacrylate, latex, starch, cellulose, dextran, and the like. Still other exemplary carriers include, but are not limited to, supramolecular biovectors that include a non-liquid hydrophilic core (e.g., cross-linked polysaccharides or oligosaccharides) and an outer layer that includes an amphiphilic compound, such as an optional phospholipid (see, e.g., U.S. Pat. No. 5,151,254, WO94 / 20078, WO / 94 / 23701, and WO96 / 06638). Biodegradable microspheres (e.g., polylactate, polyglycolate) may be used as carriers for the compositions. In the present invention, examples of suitable biodegradable microspheres are as described in U.S. Patent Nos. 4,897,268, 5,075,109, 5,928,647, 5,811,128, 5,820,883, 5,853,763, 5,814,344, 5,407,609, and 5,942,252.The modified hepatitis B core protein carrier system described in WO / 9940934 and references cited therein may also be usefully applied. Yet another exemplary carrier / delivery system uses a carrier comprising a microparticle-protein complex as described in U.S. Pat. No. 5,928,647, which provides additional advantages when used to deliver the nucleic acids of the invention. Biodegradable polymeric nanoparticles can facilitate non-viral nucleic acid delivery to cells. Small (approximately 200 nm), positively charged (approximately 10 mV) particles are formed by self-assembly of cationic, hydrolyzable poly(beta-amino esters) and plasmid DNA.

[0551] The nucleic acids of the invention may also be introduced into cells by direct microinjection, transient cell permeabilization (eg, co-administration of inhibitors and / or activators with a cell permeabilizing agent), fusion to a membrane translocating peptide, and the like.

[0552] Lipid-mediated nucleic acid delivery and expression of foreign nucleic acids, including mRNA, in vitro and in vivo has been very successful. Lipid-based non-viral formulations offer an alternative to viral gene therapy. Currently, in vivo lipid delivery methods use subcutaneous, intravascular, intratumoral, or intracranial injections. Developments in lipid formulations have improved the efficiency of in vivo gene delivery (see WO98 / 07408). For example, lipid formulations composed of an equimolar ratio of 1,2-bis(oleoyloxy)-3-(trimethylammonio)propane (DOTAP) and cholesterol can significantly improve systemic in vivo gene delivery. DOTAP:cholesterol lipid formulations form unique structures known as "sandwich liposomes." These formulations have been reported to "sandwich" DNA between an invaginated bilayer or "vase" structure. Such lipid structures have advantages including positive p, colloidal stabilization by cholesterol, two-dimensional nucleic acid packing, and increased serum stability.

[0553] Cationic liposome technology is based on the ability of amphipathic lipids with positively charged head groups and hydrophobic lipid tails to bind negatively charged DNA or RNA, forming particles that typically enter cells by endocytosis. Some cationic liposomes also contain neutral co-lipids, which are understood to improve liposome uptake by mammalian cells. Similarly, other polycations such as poly-L-lysine and polyethylene-imine form complexes with nucleic acids through charge interactions and help DNA or RNA condense into nanoparticles that serve as substrates for endosome-mediated uptake. Several such cationic-nucleic acid complex technologies have been developed as potential clinical products, including complexes with plasmid DNA (pDNA), oligodeoxynucleotides, and various forms of synthetic RNA, and may be used as part of the encoded nucleic acid delivery system of the present invention.

[0554] The encoded nucleic acid of the invention may be associated with a polycationic molecule that serves to improve uptake into cells. Complexing the nucleic acid construct with a polycationic molecule also serves to package the construct such that its size is reduced, which is believed to aid in cellular uptake. Once inside the endosome, the complex dissociates due to low pH and the polycationic molecule can disrupt the endosomal membrane, facilitating the escape of the DNA into the cytoplasm before it is degraded. Existing data shows that when embodiments of the nucleic acid construct are complexed with the polycationic molecules polylysine or polyethyleneimine, there is improved uptake into SC over DC. An example of a polycationic molecule useful for complexing with a nucleic acid construct includes a cell-penetrating peptide (CPP), examples of which include polylysine (discussed above), polyarginine, and Tat peptide. Cell-penetrating peptides (CPPs) are small peptides that can bind to DNA and, once released, cross the cell membrane to facilitate the escape of DNA from the endosome into the cytoplasm. Yet another example of a CPP is related to a 27-residue chimeric peptide called MPG, which has previously been shown to bind to ss- and ds-oligonucleotides in a stable manner, resulting in non-covalent complexes that protect nucleic acids from degradation by DNase and effectively deliver oligonucleotides to cells in vitro (Mahapatro A, et al., J Nanobiotechnol, 2011, 9:55). The non-covalent complexes formed small particles of about 150nm to 1um when different peptide:DNA ratios, 10:1 and 5:1 ratios (150nm and 1um, respectively), were examined. Yet another CPP is related to the modified tetrapeptide TL-GCP (tetralysine containing guanidinocarbonylpyrrole (GCP) groups), which was reported to bind with high affinity to 6.2 kb plasmid DNA and generate positively charged aggregates of 700-900 nm (Li et al., Agnew Chem Int Ed Enl 2015;54(10):2941-4). RNA may also be complexed with such multi-cationic molecules for in vivo delivery.Other examples of polycationic molecules that can be complexed with the nucleic acid constructs described in the present invention include commercially available polycationic polymers such as JETPRIME® and In vivo JET (Polypus-transfection, SA, Illkirch, France).

[0555] The present invention contemplates a method for delivering mRNA (or other polynucleotide) encoding a Stefin A protein variant or a fusion protein or conjugate comprising same to cells of a patient by administering nanoparticles comprising: (i) a lipid component comprising a compound of formula I, a phospholipid, a structural lipid, and a PEG-lipid; and (ii) mRNA (or other polynucleotide), said administration comprising contacting said mammalian cells with said nanoparticle composition, whereby said mRNA (or other polynucleotide) is delivered to said cells.

[0556] For example, in the present invention, the PEG lipid is selected from the group consisting of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol.For example, in the present invention, the structural lipid is selected from the group consisting of cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, and alpha-tocopherol.In some embodiments, the structural lipid is cholesterol.

[0557] For example, in the present invention, the phospholipid may comprise a moiety selected from the group consisting of phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl glycerol, phosphatidyl serine, phosphatidic acid, 2-lysophosphatidyl choline, and sphingomyelin. In the present invention, the phospholipid may comprise at least one fatty acid moiety selected from the group consisting of lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, arachidic acid, arachidonic acid, phytanoic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid. For example, in the present invention, the phospholipid may be selected from the group consisting of 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-0-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), and sphingomyelin. In the present invention, the phospholipid may be DOPE or DSPC.,

[0558] As an additional example, the phospholipid may be DOPE and the lipid component may include about 35 mol% to about 45 mol% of the compound described above, about 10 mol% to about 20 mol% of DOPE, about 38.5 mol% to about 48.5 mol% of a structured lipid, and about 1.5 mol% of a PEG lipid. The lipid component may include about 40 mol% of the compound described above, about 15 mol% of a phospholipid, about 43.5 mol% of a structured lipid, and about 1.5 mol% of a PEG lipid.

[0559] In the present invention, the weight / weight ratio of lipid component to Stefin A protein variant or fusion protein or conjugate-encoding mRNA (or other polynucleotide) that specifically binds to CD40L may be from about 5:1 to about 50:1, or from about 10:1 to about 40:1.

[0560] In the present invention, the average size of the nanoparticle composition may be from about 50 nm to about 150 nm, or from about 80 nm to about 120 nm.

[0561] In the present invention, the polydispersity index of the nanoparticle composition may be from about 0 to about 0.18, or from about 0.13 to about 0.17.

[0562] In the present invention, the nanoparticle composition may have a zeta potential of about -10 to about +20 mV.

[0563] In the present invention, the nanoparticle composition may further comprise a cationic and / or ionizable lipid selected from the group consisting of 3-(didodecylamino)-N1,N1,4-tridodecyl-1-piperazineethanamine (KL10), 14,25-ditridecyl-15,18,21,24-tetraaza-octatriacontane (KL25), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin- K-DMA), heptatriaconta-6,9,28,31-tetraen-19-yl4-(dimethylamino)butanoate(DLin-MC3-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane(DLin-KC2- DMA), 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA), and (2R)-2-({8-[(3P)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yl oxy]propan-1-amine(Octyl-CLinDMA(2R)).

[0564]

[0565] The nucleic acid or vector may 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, either as an extrachromosomal element or by chromosomal integration. As used herein, the term "transfection" refers to the acceptance of an expression vector by a host cell, without any coding sequence actually being expressed. To introduce the vector, various techniques commonly used to introduce exogenous nucleic acid (DNA or RNA) into prokaryotic or eukaryotic host cells may be used, including, but not limited to, electrophoresis, calcium phosphate precipitation, DEAE-dextran transfection, or lipofection.

[0566] Of course, it should be understood that not all vectors and expression control sequences are equally functional in expressing the DNA sequences of the present invention. Similarly, not all hosts will function equally well with the same expression system. However, one skilled in the art can make an appropriate selection among various vectors, expression control sequences and hosts without undue experimental burden and within the scope of the present invention. For example, in selecting a vector, the host must be considered since the vector must replicate therein. The number of copies of the vector, the ability to regulate that number, and the expression of other proteins encoded by the vector, such as antibiotic markers, must also be considered. In selecting an expression control sequence, various factors must also be considered, such as the relative strength, regulatability and compatibility of the sequence with the DNA sequences of the present invention, particularly in relation to potential secondary structures. A unicellular host must be selected taking into account factors such as the selected vector, the toxicity of the product encoded by the DNA sequences of the present invention, secretion characteristics, ability to fold the protein correctly, culture and fermentation requirements, and ease of purification of the product encoded by the DNA sequences of the present invention from the host. Within these ranges, one skilled in the art can select various vector / expression regulatory sequence / host combinations that can express the DNA sequence of the present invention in fermentation or large-scale animal culture. Screening methods for cloning cDNA by expression cloning include the binding method, panning method, film emulsion method, etc.

[0567] In the present invention, a "transformation enhancing agent" may be further used to increase the efficiency of transformation. The "transformation enhancing agent" is generally preferably a cationic polymer, which facilitates the introduction of negatively charged nucleic acids or genes into host cells.

[0568] In the present invention, the transformation enhancer may be characterized as a cationic polymer, for example, selected from polybrene, protamine sulfate, and Sirion's Lentiboost, and most preferably, polybrene, but is not limited thereto.

[0569] Expression methods and systems

[0570] The Stefin A protein variant of the present invention that specifically binds to CD40L and / or a fusion protein or conjugate containing the same may be produced by any suitable method known in the art. Such methods include various methods, such as direct protein synthesis methods, in which the nucleic acid of the present invention is introduced into a suitable host and expressed. When the conjugate containing the Stefin A protein variant of the present invention that specifically binds to CD40L contains various modifications such as chemical modification or conjugation, it may be produced by isolating it from a host cell or chemically synthesizing it, and then further chemically or enzymatically manipulating it.

[0571] Therefore, in another aspect, the present invention relates to a method for producing a Stefin A protein variant that specifically binds to CD40L and / or a fusion protein or conjugate containing the same, comprising the steps of culturing the genetically engineered cells and producing a Stefin A protein variant that specifically binds to CD40L and / or a fusion protein or conjugate containing the same.

[0572] In the present invention, when the conjugate contains various modifications such as chemical modifications or conjugations, it may be prepared by isolating it from a host cell or by chemical synthesis followed by further chemical or enzymatic manipulation.

[0573] Methods for introducing and expressing sequences encoding proteins or polypeptides into host cells and producing them can be found in, for example, WO04 / 041862, WO2006 / 122786, WO2008 / 020079, WO2008 / 142164, and WO2009 / 068627, but are not limited thereto.

[0574] In the present invention, the nucleic acid encoding the Stefin A protein variant specifically binding to CD40L and / or the fusion protein or conjugate containing the same may be constructed by chemical synthesis using an oligonucleotide synthesis machine. The oligonucleotide may be designed based on the sequence of the desired protein or polypeptide, and may be codon optimized to select codons preferred in the host cell. Standard methods may be applied to synthesize the nucleic acid of the present invention. For example, a complete amino acid sequence may be used to construct a reverse-translated gene. Alternatively, a DNA oligomer containing a nucleotide sequence encoding an isolated specific polypeptide may be synthesized. For example, multiple small oligonucleotides encoding portions of the desired polypeptide may be synthesized and then ligated. Individual oligonucleotides may generally include 5' or 3' overhangs for complementary assembly.

[0575] The nucleic acid encoding the Stefin A protein mutant of the present invention that specifically binds to CD40L may be, for example, any one of those listed in Table 4, but is not limited thereto.

[0576] Based on the nucleic acid encoding the Stefin A protein variant that specifically binds to CD40L of the present invention, an expression vector containing the same can be constructed using recombinant DNA technology known in the art. A vector for producing a protein may be produced by recombinant DNA technology using techniques well known in the art. A person skilled in the art can use known methods to construct a vector containing a sequence encoding the Stefin A protein variant that specifically binds to CD40L of the present invention and / or a fusion protein or conjugate containing the same and appropriate transcriptional and translational control signals. Such methods may include, but are not limited to, in vitro recombinant DNA technology, synthetic technology, and in vivo gene recombination technology (see 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).

[0577] In the present invention, the expression vector may be delivered to a host cell by a conventional technique (e.g., electroporation, liposome transfection, calcium phosphate precipitation, etc.), and the transfected cell may be cultured by a conventional method known in the art to express the Stefin A protein variant that specifically binds to CD40L and / or a fusion protein or conjugate containing the same. In the present invention, the expression of the protein may be regulated by a constitutive, inducible or tissue-specific promoter.

[0578] In the present invention, the expression vector may include a replication origin that may be selected based on the type of host cell to be used. For example, pBR322 (Product No. 303-3s, New England Biolabs, Beverly, Mass.) is mostly useful for Gram-negative bacteria, whereas SV40, polyoma, adenovirus, VSV (vesicular stomatitus virus) or papillomavirus (HPV, BPV) are useful for cloning vectors in mammalian cells. In general, a replication origin may not be necessary for mammalian expression vectors (for example, the SV40 replication origin can be used because it contains an early promoter).

[0579] In the present invention, the expression vector may contain at least one selectable marker gene, e.g., a genetic element encoding a protein necessary for the survival and growth of a host cell grown in a selective culture medium. Exemplary selectable marker genes may encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, tetracycline, or kanamycin, to prokaryotic host cells; (b) complement auxotrophic deficiencies of cells; or (c) supply critical nutrients not available from complex media. In the present invention, preferred selectable markers may be, but are not limited to, kanamycin resistance genes, ampicillin resistance genes, or tetracycline resistance genes. Neomycin resistance genes may be used for selection in prokaryotic and eukaryotic host cells. Other selectable genes may be used to amplify the gene to be expressed. Amplification refers to the process in which genes in high demand for growth-critical protein production are repeated side-by-side in the chromosomes of successive recombinant cell generations. Examples of selectable markers for mammalian cells include dihydrofolate reductase (DHFR) and thymidine kinase. The mammalian cell transformants are placed under selection pressure to which only the transformants are uniquely adapted to survive, by virtue of the marker present in the vector. Selection pressure is exerted by culturing the transformed cells under conditions in which the concentration of the selection agent in the medium is successively changed, thereby allowing both the selection gene and the DNA encoding the Stefin A protein variant of the present invention or a fusion protein or conjugate containing the same to be amplified, and thus allowing the synthesis of increased amounts of protein from the amplified DNA.

[0580] In the present invention, the expression vector may contain at least one ribosome binding site. The ribosome binding site may be transcribed into an mRNA containing a coding sequence for a protein. For example, the ribosome binding site may be a Shine-Dalgarno sequence (prokaryotes) or a Kozak sequence (eukaryotes). In the present invention, the binding site may typically be located 3' to the promoter of the polypeptide to be expressed and 5' to the coding sequence. There are various Shine-Dalgarno sequences, but they are generally polypurines (high AG content). Many Shine-Dalgarno sequences have been identified and can be readily synthesized using the methods described above and used in prokaryotic vectors.

[0581] In the present invention, the expression vector may typically contain a promoter that is recognized by the host organism and is operably linked to the nucleic acid encoding the Stefin A protein variant of the present invention or a fusion protein or conjugate containing the same. Depending on the host cell used for expression and the yield desired, native or heterologous promoters may be used.

[0582] Promoters for use with prokaryotic hosts in the present invention include the beta-lactamase and lactose promoter systems; the alkaline phosphatase, tryptophan (trp) promoter system; and hybrid promoters such as the tac promoter. Other known bacterial promoters may also be suitable. The sequences of such promoters are publicly available and may be ligated to the desired nucleic acid sequence using linkers or adapters as desired to provide restriction sites.

[0583] Promoters for use with yeast hosts are also known in the art. Yeast enhancers may be advantageously used with yeast promoters. Suitable promoters for use with mammalian host cells are well known and include, but are not limited to, promoters derived from polyoma virus, fowlpox virus, adenovirus (e.g., adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis-B virus, and most preferably, Simian virus 40 (SV40). Other suitable mammalian promoters include heterologous mammalian promoters, such as heat-shock promoters and actin promoters.

[0584] Additional promoters that can be used in the present invention include, for example, the SV40 early promoter region (Bernoist and Chambon, Nature, 290:304-310, 1981); the CMV promoter; the promoter containing the 3' long terminal repeat of Rous sarcoma virus (Yamamoto et al. (1980), Cell 22:787-97); the herpes thymidine kinase promoter (Wagner et al. (1981), Proc. Natl. Acad. Sci. USA 78:1444-5); the regulatory sequence of the metallothionine gene (Brinster et al., Nature, 296;39-42, 1982); and prokaryotic expression vectors such as the beta-lactamase promoter (Villa-Kamaroff, et al. al., Proc. Natl. Acad. Sci. USA, 75;3727-3731, 1978); or the tac promoter (DeBoer, et al. (1983), Proc. Natl. Acad. Sci. USA, 80:21-5). Also, animal transcriptional control regions that exhibit tissue specificity and have been exploited in transgenic animals may be used: the elastase I gene control region, which is activated in pancreatic acinar cells (Swift et al. (1984), Cell 38:639-46; Ornitz et al. (1986), Cold Spring Harbor Symp. Quant. Biol. 50:399-409; MacDonald (1987), Hepatology 7:425-515); the insulin gene control region, which is activated in pancreatic beta cells (Hanahan (1985), Nature 315:115-22); the immunoglobulin gene control region, which is activated in lymphoid cells (Grosschedl et al. (1984), Cell 38;647-58; Adames et al. (1985), Nature 318;533-8; Alexander et al. (1987), Hepatology 7:425-515); al. (1987), Mol. Cell. Biol.7:1436-44); mouse mammary tumor virus control region, which is activated in testis, breast, lymphocytes, and mast cells (Leder et al. (1986), Cell 45:485-95); albumin gene control region, which is activated in liver (Pinkert et al. (1987), Genes and Devel. 1:268-76); alpha fetal protein gene control region, which is activated in liver (Krumlauf et al. (1985), MoI. Cell. Biol. 5:1639-48; Hammer et al. (1987), Science, 235:53-8); alpha 1-antitrypsin gene control region, which is activated in liver (Kelsey et al. (1987), Genes and Devel. 1:161-71); beta-globin gene control region, which is activated in bone marrow cells (Mogram et al., Nature, 315 338-340, 1985; Kollias et al. (1986), Cell 46:89-94); the myelin basic protein gene control region, which is activated in brain oligodendrocytes (Readhead et al. (1987), Cell, 48:703-12); the myosin light chain-2 gene control region, which is activated in skeletal muscle (Sani (1985), Nature, 314:283-6); and the gonadotropin releasing hormone gene control region, which is activated in the hypothalamus (Mason et al. (1986), Science 234:1372-8).

[0585] Enhancer sequences may be inserted into vectors to increase transcription in eukaryotic host cells. A variety of enhancer sequences are known that can be used in mammalian genes (e.g., globin, elastase, albumin, alpha-feto-protein, and insulin). However, viral enhancers are commonly used. SV40 enhancer, cytomegalovirus early promoter enhancer, polyoma enhancer, and adenovirus enhancer are exemplary enhancers for the activation of eukaryotic promoters.

[0586] The enhancer may be spliced ​​into the vector at a position 5' or 3' to the polypeptide coding region, but is generally located 5' from the promoter.

[0587] In the present invention, vectors for expressing nucleic acids may include those compatible with bacterial, insect and mammalian host cells, including, but not limited to, inter alia, pCRII, pCR3, and pcDNA3.1 (Invitrogen Company, San Diego, Calif.), pBSII (Stratagene Company, La Jolla, Calif.), pET15 (Novagen, Madison, Wis.), pGEX (Pharmacia Biotech, Piscataway, NJ), pEGFP-N2 (Clontech, Palo Alto, Calif.), pETL (BlueBacII; Invitrogen), pDSR-alpha (PCT Publication No. WO90 / 14363), and pFastBacDual (Gibco / BRL, Grand Island, NY).

[0588] In the present invention, possible additional vectors include, but are not limited to, cosmids, plasmids, or modified viruses, provided that the vector is compatible with the selected host cell. Such vectors include Bluescript® plasmid derivatives (high copy number ColE1-based phagemid, Stratagene Cloning Systems Inc., La Jolla Calif.), PCR cloning plasmids designed for cloning Taq amplified PCR products (e.g., TOPO TM TA Cloning(R) Kit, PCR 2Expression vectors include, but are not limited to, plasmids such as pBacPAK plasmid derivatives, Invitrogen, Carlsbad, CA), and mammalian, yeast, or viral vectors such as baculovirus expression systems (pBacPAK plasmid derivatives, Clontech, Palo Alto, CA). In the present invention, recombinant molecules such as vectors may be introduced into host cells by transformation, transfection, infection, electroporation, or other known techniques.

[0589] Eukaryotic and prokaryotic host cells, including mammalian cells, as hosts for the expression of the Stefin A protein of the present invention or fusion proteins or conjugates containing the same are well known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC). They may include, among others, Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells, HEK-293 cells, and many other cell lines. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, cow, horse, and hamster cells. A cell line of particular preference may be selected by determining which cell line has a high expression level. Other cell lines that may be used include insect cell lines such as Sf9 cells, amphibian cells, bacterial cells, plant cells, and fungal cells.The fungal cells include yeast and filamentous fungus cells, such as Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia minuta (Ogataea minuta, Pichia lindneri), Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia sp., Saccharomyces cerevisiae, Saccharomyces sp., Hansenula polymorpha, Kluyveromyces sp., Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium sp., Fusarium gramineum, Fusarium venenatum, Physcomitrella patens and Neurospora crassa. Pichia sp., any Saccharomyces sp., Hansenula polymorpha, any Kluyveromyces sp., Candida albicans, any Aspergillus sp., Trichoderma reesei, Chrysosporium lucknowense, any Fusarium sp., Yarrowia lipolytica, and Neurospora crassa may be used, but are not limited to these.

[0590] In the present invention, various host expression vector systems can be used to express the Stefin A protein of the present invention or a fusion protein or conjugate comprising the same. In the present invention, the host expression system refers to a vehicle capable of producing and purifying the sequence encoding the Stefin A protein of the present invention or a fusion protein or conjugate comprising the same, but it can also refer to a cell capable of expressing the Stefin A protein of the present invention or a fusion protein or conjugate comprising the same in situ when transformed or transfected with a suitable nucleic acid coding sequence. This includes microorganisms such as bacteria (e.g., E. coli and B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors comprising the nucleic acid sequences of the invention; yeast (e.g., Saccharomyces pichia) transformed with recombinant yeast expression vectors containing the nucleic acid sequences of the invention; insect cell systems infected with recombinant viral expression vectors (e.g., baculovirus) comprising the nucleic acid sequences of the invention; plant cell systems infected with recombinant viral expression vectors (e.g., Coli Flower Mosaic Virus (CMV) and Tobacco Mosaic Virus (TMV)) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) comprising the nucleic acid sequences of the invention; or recombinant expression constructs comprising promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., adenovirus late promoter; vaccinia virus 7.5K promoter), mammalian cell systems (e.g., COS, CHO, BHK, 293, 293T, 3T3 cells, lymphoid cells (see U.S. Pat. No. 5,807,715), Perl ... C.6 cells (rat retinal cells developed at Crucell))).

[0591] Depending on the use of the protein expressed in the bacterial system, a number of expression vectors may be advantageously selected. For example, if such a protein must be produced in large quantities for the production of a pharmaceutical composition, a vector directing the expression of a high level fusion protein product that is easily purified would be preferred. Such vectors include, but are not limited to, the expression vector pUR278 (Ruther et al. (1983) EMBO J. 2:1791-1794), in which the Stefin A protein variant coding region of the invention is individually ligated in frame with the lac Z coding region to produce a fusion protein; pIN vectors (Inouye et al. (1985) Nucleic Acids Res. 13:3101-3110; Van Heeke et al. (1989) J. Biol. Chem. 24:5503-5509); and the like. pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and may be easily purified from lysed cells by adsorption and binding to matrix glutathione-agarose beads followed by elution in the presence of free glutathione. The pGEX vectors are designed to contain thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.

[0592] In an insect system, AcNPV (Autographa californica nuclear polyhedrosis virus) is used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. Sequences encoding the Stefin A protein of the invention or fusion proteins or conjugates containing same may be cloned individually into non-essential regions (e.g. the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (e.g. the polyhedrin promoter).

[0593] In mammalian host cells, a number of virus-based expression systems may be used. When adenovirus is used as an expression vector, the protein coding sequence may be ligated to an adenovirus transcription / translation control complex, e.g., the late promoter and triple leader sequence. Such a chimeric gene may then be inserted into the adenovirus genome by in vitro or in vivo recombination. Insertion into non-essential regions of the viral genome (e.g., regions E1 or E3) produces recombinant viruses that are viable in infected hosts and capable of expressing immunoglobulin molecules (see, e.g., Logan et al. (1984) Proc. Natl. Acad. Sci. (USA) 81:3655-3659). Specific initiation signals may be required for efficient translation of inserted nucleic acid sequences. Such signals include the ATG initiation codon and adjacent sequences. The initiation codon must also be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. Such exogenous translational control signals and initiation codons may be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see Bitter et al. (1987 Methods in Enzymol. 153:516-544).

[0594] In addition, a host cell strain can be selected which modulates the expression of the inserted sequences and modifies and processes the gene product in the specific manner desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of proteins can be important for the function of the protein. Different host cells have characteristic and specific mechanisms for post-translational processing and modification of proteins and gene products. An appropriate cell line or host system can be selected to ensure the correct modification and processing of the expressed foreign protein. To this end, eukaryotic host cells that possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used. Such eukaryotic host cells include, but are not limited to, CHO, VERY, BHK, Hela, COS, MDCK, 293, 293T, 3T3, WI38, BT483, Hs578T, HTB2, BT20 and T47D, CRL7030, and Hs578Bst.

[0595] Stable expression is contemplated for long-term, high-yield production of recombinant proteins. For example, cell lines that stably express antibodies may be engineered. Instead of using expression vectors containing viral origins of replication, host cells may be transformed with DNA controlled by appropriate expression control elements (e.g., promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc.) and selectable markers. After introduction of the foreign DNA, engineered cells may be allowed to grow in enriched medium for 1-2 days and then switched to selective medium. The selectable marker on the recombinant plasmid confers resistance to selection, allowing the cells to stably integrate the plasmid into their chromosomes and grow to form foci that can be cloned and expanded as cell lines. This method may be advantageously used to engineer cell lines that express the Stefin A protein of the invention or fusion proteins or conjugates containing the same. Such engineered cell lines may be particularly useful for screening and evaluating compounds that interact directly or indirectly with recombinant Stefin A protein or fusion proteins or conjugates containing the same.

[0596] In the present invention, various selection systems may be used, for example, but not limited to, herpes simplex virus thymidine kinase (Wigler et al. (1977) Cell 11:223-232), hypoxanthine-guanine phosphoribosyltransferase (Szybalska et al. (1962), Proc. Natl. Acad. Sci. (USA) 48:2026-2034), and adenine phosphoribosyltransferase (Lowy et al. (1980), Cell 22:817-823) genes may be used for tk-, hgprt-, or aprt- cells, respectively. Antimetabolite resistance may also be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al. (1980) Proc. Natl. Acad. Sci. (USA) 77:3567-3570; O'Hare et al. (1981) Proc. Natl. Acad. Sci. (USA) 78:1527-1531); gpt, which confers resistance to mycophenolic acid (Mulligan et al. (1981) Proc. Natl. Acad. Sci. (USA) 78:2072-2076); neo, which confers resistance to the aminoglycoside G-418 (Tachibana et al. al.(1991)Cytotechnology6(3):219-226;Tolstoshev(1993)Ann.Rev.Pharmacol.Toxicol.32:573-596;Mulligan(1993)Science 260:926-932;and Morgan et al.(1993)Ann.Rev.Biochem.62:191-217).Methods generally known in the art of recombinant DNA technology that can be used are described in Ausubel et al. (eds.), 1993, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, NY; Kriegler, 1990, GENE TRANSFER AND EXPRESSION, A LABORATORY MANUAL, Stockton Press, NY; and in Chapters 12 and 13, Dracopoli et al. (eds), 1994, CURRENT PROTOCOLS IN HUMAN GENETICS, John Wiley & Sons, NY.; Colbere-Garapin et al. (1981) J. Mol. Biol. 150:1-14; and hygro, which confers resistance to hygromycin (Santerre et al. (1984) Gene 30:147-156).

[0597] The expression level of the Stefin A protein of the present invention or a fusion protein or conjugate containing the same can be increased by vector amplification (see Bebbington and Hentschel, in DNA CLONING, Vol. 3 (Academic Press, New York, 1987)). When the marker of the vector system expressing the Stefin A protein or a fusion protein or conjugate containing the same is amplified, an increase in the inhibitor level present in the culture of the host cells can increase the number of copies of the marker gene. Since the amplified region is associated with the nucleic acid sequence encoding the Stefin A protein or a fusion protein or conjugate containing the same, the production of the Stefin A protein or a fusion protein or conjugate containing the same can also be increased (Crouse et al. (1983) Mol. Cell. Biol. 3:257-266).

[0598] When the fusion protein of the invention is an antibody fusion or other multi-protein complex, the host cell may be co-transfected with two expression vectors, for example a first vector encoding the heavy chain and a second vector encoding the light chain derived polypeptide. Either or both of the two expression vectors may contain the Stefin A protein variant coding sequence of the invention. The two vectors may contain identical selection markers allowing for identical expression of heavy and light chain polypeptides. Alternatively, a single vector may be used that encodes both heavy and light chain polypeptides. In such a situation, the light chain must precede the heavy chain to avoid excessive toxicity of free heavy chains (Proudfoot (1986) Nature 322:562-565; Kohler (1980) Proc. Natl. Acad. Sci. (USA) 77:2197-2199). The coding sequences for the heavy and light chains may comprise cDNA or genetic DNA.

[0599] In general, glycoproteins produced in a particular cell line or transgenic animal may have a glycosylation pattern characteristic of the glycoprotein produced in that cell line or transgenic animal. Thus, the particular glycosylation pattern of a Stefin A protein or a fusion protein or conjugate comprising the same may vary depending on the particular cell line or transgenic animal used to produce the protein. In some embodiments of the Stefin A protein variant-antibody fusion protein of the invention, a glycosylation pattern that includes only non-fucosylated N-glycans may be advantageous, since antibodies have been found to typically exhibit stronger potency both in vitro and in vivo compared to their fucosylated counterparts. (See, e.g., Shinkawa et al., J. Biol. Chem. 278:3466-3473 (2003); U.S. Patent Nos. 6,946,292 and 7,214,775).

[0600] Also, expression of the Stefin A protein or fusion proteins or conjugates comprising the same from the production cell line may be enhanced using a number of known techniques. For example, the Glutamine Synthetase Gene Expression System (GS System) is a common approach for enhancing expression in certain conditions. The GS System is described in whole or in part in connection with European Patent Nos. 0216846, 0256055, and 0323997 and European Patent Application No. 89303964.4. Thus, in the present invention, a mammalian host cell (e.g., CHO) lacks a glutamine synthetase gene and grows in the absence of glutamine in the medium, but a polynucleotide encoding an immunoglobulin chain comprises a glutamine synthetase gene, which can complement the genetic deficiency of the host cell. Such host cells containing the Stefin A protein or fusion proteins or conjugates comprising the same of the present invention, nucleic acids or vectors encoding the same, as well as expression methods for producing the Stefin A protein or fusion proteins or conjugates comprising the same using such host cells are provided as part of the scope of the present invention.

[0601] Expression of recombinant proteins in insect cell culture systems (e.g., baculovirus) also provides a powerful method for producing correctly folded, biologically functional proteins. The baculovirus system for producing heterologous proteins in insect cells is well known to those skilled in the art.

[0602] The Stefin A protein produced by the transformed host cell or a fusion protein or conjugate containing same may be purified by any suitable method. Standard methods include chromatography (e.g., ion exchange, affinity and sizing column chromatography), centrifugation, differential solubility or other standard techniques for protein purification. Affinity tags such as hexahistidine, maltose binding domain, influenza-coat sequence, and glutathione-S-trancafelase can be attached to the protein and readily purified by passage through an appropriate affinity column. The isolated protein may also be physically characterized using techniques such as proteolysis, mass spectrometry (MS), nuclear magnetic resonance (NMR), high performance liquid chromatography (HPLC), and x-ray crystallography.

[0603] In the present invention, Stefin A protein produced in bacterial culture or a fusion protein or conjugate containing same may be isolated, for example, by initial extraction from cell pellets followed by at least one concentration, salting-out, aqueous ion exchange, or size-exclusion chromatography step. HPLC can be used for final purification steps. Microbial cells used for expression of recombinant proteins may be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents.

[0604]

[0605] Purpose

[0606] Uses - Pharmaceutical uses (methods, pharmaceutical compositions, etc.)

[0607] In particular, it is well known in the art that activation of T cells and B cells by CD40L / CD40 interaction acts as a pathogenic factor of autoimmune or inflammatory diseases that has a major impact on pathology. Specifically, it is a pathogenic factor of various diseases such as type 1 diabetes, thyroiditis, psoriasis, lupus (Systemic Lupus Erythematosus; SLE), rheumatoid arthritis (RA), multiple sclerosis (Multiple Sclerosis; MS), etc. For the treatment of such diseases, various compounds or antibodies targeting CD40L have been developed (Semin Immunol. 2009; 21(5): 293-300; Advanced Drug Delivery Reviews Volume 141, 15 February 2019, Pages 92-103).

[0608]

[0609] In the examples of the present invention, it was confirmed that administration of the Stefin A protein mutant that specifically binds to CD40L exhibited a remarkable therapeutic effect in an animal model with graft-versus-host disease (GVHD).

[0610] Therefore, in another aspect, the present invention relates to a pharmaceutical composition containing a fusion protein containing the Stefin A protein mutant that specifically binds to CD40L, the fusion protein, the conjugate, the nucleic acid, and / or the delivery vehicle as an active ingredient, and / or a conjugate containing the same.

[0611] In the present invention, the pharmaceutical composition may be characterized in that it is used for the prevention or treatment of various diseases associated with CD40L, particularly diseases known in the art in which a preventive or therapeutic effect can be achieved by targeting or inhibiting CD40L.

[0612] Therefore, in yet another aspect, the present invention relates to a pharmaceutical composition for preventing or treating immune diseases, comprising as an active ingredient a Stefin A protein mutant that specifically binds to CD40L, the fusion protein, the conjugate, the nucleic acid, and / or the delivery vehicle.

[0613] In yet another aspect, the present invention relates to a method for preventing or treating an immune disease, comprising the step of administering to a subject the Stefin A protein mutant that specifically binds to CD40L, the fusion protein, the conjugate, the nucleic acid, and / or the delivery vehicle.

[0614] In yet another aspect, the present invention relates to use of the Stefin A protein mutant that specifically binds to CD40L, the fusion protein, the conjugate, the nucleic acid, and / or the transfer vehicle for the prevention or treatment of immune diseases.

[0615] In yet another aspect, the present invention relates to a use of the Stefin A protein variant that specifically binds to CD40L, the fusion protein, the conjugate, the nucleic acid, and / or the delivery vehicle for the manufacture of a pharmaceutical composition. In the present invention, the pharmaceutical composition may be used for the prevention or treatment of an immune disease.

[0616]

[0617] The term "prevention" as used herein means any action of administering the pharmaceutical composition provided by the present invention to an individual who is expected to develop an immune disease, thereby suppressing or delaying the onset of the disease.

[0618] The term "treatment" as used herein means any action of clinical intervention to alter the natural process of the individual or cell to be treated, which may be performed during or to prevent the progression of a clinical pathological condition. The intended therapeutic effects include prevention of disease onset or recurrence, 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, but is not limited to, 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.

[0619] In the present invention, the pharmaceutical composition may further comprise one or more pharma- ceutically acceptable carriers.

[0620] The pharma- ceutically acceptable carriers contained in the composition of the present invention are those commonly used in formulation, and include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil, etc. In addition to the above-mentioned components, the composition of the present invention may further include lubricants, wetting agents, sweeteners, flavorings, emulsifiers, suspending agents, preservatives, etc.

[0621] The pharmaceutical composition of the present invention can be administered orally or parenterally. In the case of parenteral administration, it can be administered by intravenous injection, subcutaneous injection, intramuscular injection, peritoneal injection, intradermal administration, topical administration, intranasal administration, intrapulmonary administration, intrarectal administration, etc.

[0622] Upon oral administration, proteins or peptides are digested, so oral compositions must be formulated to coat or protect the active agent from degradation in the stomach, and the pharmaceutical composition may be administered by any device that allows the active agent to be delivered to the target cells.

[0623] The appropriate dosage of the composition of the present invention varies depending on factors such as the formulation method, administration method, age, weight, sex, pathological condition, diet, administration time, administration route, excretion rate and reaction sensitivity of the patient, and a skilled general physician can easily determine and prescribe an effective dosage for the desired treatment or prevention. As used herein, the term "pharmaceutical effective amount" means an amount sufficient to prevent or treat an immune disease.

[0624] The pharmaceutical composition of the present invention may be prepared in unit dose form or in multi-dose containers by formulating it with pharma- ceutically acceptable carriers and / or excipients in a manner easily practicable by a person of ordinary skill in the art to which the invention pertains, and may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or in the form of an extract, powder, suppository, powder, granule, tablet or capsule, and may further include a dispersant or stabilizer.

[0625] The pharmaceutical composition of the present invention may be administered in parallel or in combination with known pharmaceuticals or pharmaceutical compositions having the effect of preventing, ameliorating or treating symptoms of immune diseases, for example, in combination with one or more other immunotherapeutic agents, chemotherapeutic agents, antibody therapeutic agents, etc.

[0626] As a specific example, the pharmaceutical composition of the present invention may be characterized by being used in combination with a drug selected from the group consisting of disease modifying antirheumatic drugs (DMARDs); nonsteroidal anti-inflammatory medications (NSAIDs); corticosteroids; Janus kinase inhibitors; calcineurin inhibitors; mTOR inhibitors; IMDH inhibitors; and biological agents, but is not limited thereto.

[0627] In the present invention, examples of the disease modifying antirheumatic drugs (DMARDs) include, but are not limited to, Actarit, Auranofin, Azathioprine, Bucillamine, Cyclophophamide, D-penicillamine, Leflunomide, Lobenzarit disodium, Methotrexate, Minocycline hydrochloride, Mizoribine, and Salazosulfapyridine.

[0628] In the present invention, the nonsteroidal anti-inflammatory medication (NSAID) includes, but is not limited to, Celecoxib, Diclofenac sodium, Ibuprofen, Ketoprofen, Meloxicam, Naproxen, Piroxicam, etc.

[0629] In the present invention, the corticosteroids include, but are not limited to, prednisone (Deltasone, Orasone), budesonide (Entocort EC), prednisolone (Millipred), methylprednisolone, etc.

[0630] In the present invention, the Janus kinase inhibitors include approved drugs such as tofacitinib, abrocitinib, baricitinib, delgocitinib, fedratinib, filgotinib, oclacitinib, peficitinib, ruxolitinib, and upadacitinib, and drugs in clinical trials such as cerdulatinib, gandotinib, lestaurtinib, momelotinib, pacritinib, and deucravacitinib, but are not limited thereto.

[0631] In the present invention, the calcineurin inhibitors include, but are not limited to, cyclosporine, tacrolimus, etc.

[0632] In the present invention, the mTOR inhibitor includes, but is not limited to, sirolimus (Rapamune), everolimus (Afinitor, Zortress), etc.

[0633] In the present invention, the IMDH inhibitor includes, but is not limited to, azathioprine (Azasan, Imuran), mycophenolate (CellCept, Myfortic), etc.

[0634] In the present invention, the biological agents include, but are not limited to, abatacept, adalimumab, anakinra, certolizumab, etanercept, golimumab, infliximab, ixekizumab, natalizumab, rituximab, secukinumab, tocilizumab, ustekinumab, vedolizumab, basiliximab, daclizumab, etc.

[0635]

[0636] In the present invention, the immune disease may be characterized as being an autoimmune disease or an inflammatory disease.

[0637] In the present invention, said immune disease may be characterized as being selected from the group consisting of, but is not limited to, lupus (SLE), lupus nephritis (e.g. drug-induced lupus nephritis), immune thrombocytopenia (ITP), rheumatoid arthritis (RA), multiple sclerosis (MS), inflammatory bowel disease (IBD) (e.g. Crohn's disease and colitis / ulcerative colitis), graft versus host disease (GvHD) or allograft rejection, 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 / or glomerulosclerosis.

[0638]

[0639] Lupus

[0640] Lupus, also known as systemic lupus erythematosus (SLE), is a chronic autoimmune disease that can cause swelling (inflammation) and pain throughout the body. There are different types of lupus, with systemic lupus erythematosus being the most common. Other types of lupus include:

[0641] Cutaneous lupus: This type of lupus affects the skin. Cutaneous is the word for skin. People with cutaneous lupus may experience skin problems such as sensitivity to the sun and rashes. Hair loss can also be a symptom of the disease.

[0642] 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 they are usually temporary.

[0643] Neonatal lupus: A rare form of lupus, neonatal lupus, is a disease that is detected in infants at birth. Babies born with neonatal lupus receive antibodies from their mothers, who may have had lupus during pregnancy or may develop the disease later in life. Not all infants born to mothers with lupus will develop lupus.

[0644] Therapies that can 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®).

[0645]

[0646] Lupus nephritis

[0647] Lupus nephritis develops as a complication of lupus. It occurs when lupus autoantibodies affect the kidney structures that filter waste products. This can lead to kidney inflammation, which can lead to blood in the urine, protein in the urine, high blood pressure, kidney dysfunction, or kidney failure. Lupus nephritis develops in about half of adults with systemic lupus. Systemic lupus causes immune system proteins to damage the kidneys, impairing their ability to filter waste products.

[0648]

[0649] Rheumatoid arthritis

[0650] Rheumatoid arthritis is a type of chronic (ongoing) arthritis that occurs in both joints of the body, such as the hands, wrists, and knees. The short-term goal of rheumatoid arthritis medication is to reduce joint pain and swelling and improve joint function. The long-term goal is to slow or stop the progression of the disease, especially joint damage.

[0651] Arthritis is a general term that describes inflammation of the joints. Rheumatoid arthritis is a type of chronic (ongoing) arthritis (causing pain and swelling) that generally occurs symmetrically in the joints (both in the body, such as the hands, wrists, and knees). This involvement of various joints helps distinguish rheumatoid arthritis from other types of arthritis.

[0652] In addition to affecting the joints, rheumatoid arthritis can sometimes affect the skin, eyes, lungs, heart, blood, nerves, or kidneys.

[0653] Therapies that can be used in combination with the pharmaceutical compositions of the present invention to treat rheumatoid arthritis may include, for example:

[0654] 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 are designed to have fewer gastrointestinal bleeding side effects.

[0655] Disease modifying antirheumatic drugs (DMARDs): Unlike other NSAIDs, DMARDs can actually modify the immune system and slow the progression of the disease. Earlier DMARDs included methotrexate (TREXALL®), gold salts, penicillamine (CUPRIMINE®), hydroxychloroquine (PLAQUENIL®), sulfasalazine (AZULFIDINE®), cyclophosphorine (SANDIMMUNE®), cyclophosphamide (CYTOXAN®), and leflunomide (ARAVA®). Currently, methotrexate, leflunomide, hydroxychloroquine, and sulfasalazine are the most commonly used (cyclophosphorine, cyclophosphamide, gold salts, and penicillamine are no longer in common use).

[0656] Biological agents. Besides these "traditional" DMARDs, new drugs have been approved. Currently, there are seven drugs, with different types in each category (some of them have been used since 2000 as anti-TNF series). Collectively, such DMARDs are known by another name: biological agents (or biological reactants). 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. Biological agents reduce the inflammatory process that ultimately induces the joint damage seen in rheumatoid arthritis. By attacking cells at a specific level rather than the inflammation itself, biological agents are considered to be more effective and more specifically targeted. Biologics include etanercept (Enbrel®), infliximab (Remicade®), adalimumab (Humira®), anakinra (Kinaret®), abatacept (Orencia®), rituximab (Rituxan®), certolizumab pegol (Cimzia®), golimumab (Symponi®), tocilizumab (Actemra®), and tofacitinib (Xeljanj®). Some biologics are used in combination with traditional DMARDs, particularly methotrexate.

[0657] Multiple sclerosis

[0658] Multiple sclerosis (MS) is an autoimmune disease. In this condition, the immune system mistakenly attacks healthy cells. In people with multiple sclerosis, the immune system attacks myelin cells, the protective membrane that surrounds 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.

[0659] There are four types of multiple sclerosis.

[0660] Clinically isolated syndrome (CIS): When the first symptoms of MS appear, health care providers typically classify this as CIS. Not everyone with CIS will progress to multiple sclerosis.

[0661] Relapsing-remitting MS (RRMS): This is the most common form of multiple sclerosis. People with RRMS have flare-ups (also called relapses or exacerbations) of new or worsening symptoms, followed by periods of remission (when symptoms stabilize or go away).

[0662] Primary progressive MS (PPMS): People diagnosed with PPMS have relapsing or slowly worsening symptoms that are not relieved.

[0663] 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 get progressively worse. Some may experience relapses or exacerbations (when symptoms increase) that are followed by no further periods of relief (when symptoms stabilize or go away).

[0664] Treatments that can be used in combination with the pharmaceutical compositions of the present invention include, for example:

[0665] Disease-modifying therapies (DMT): A variety of drugs have been approved by the FDA for long-term treatment of MS. These drugs help reduce relapses (also called flare-ups or attacks). They slow the progression of the disease and can prevent new lesions from forming in the brain and spinal cord.

[0666] Relapse Management Drug Treatment: If you have severe attacks, your neurologist may recommend high doses of corticosteroids. The drugs can quickly reduce inflammation. They slow the damage to the myelin sheath that surrounds the nerve cells.

[0667] Physical rehabilitation: Multiple sclerosis can affect your physical function. Staying physically healthy and strong can help you maintain your mobility.

[0668] Mental health consultations: Emotionally, chronic illness can be difficult to cope with. MS can sometimes affect mood and memory. Working with a neuropsychologist or other emotional support is essential to managing the disease.

[0669]

[0670] inflammatory bowel disease

[0671] Inflammatory Bowel Disease (IBD) is a group of disorders that cause chronic inflammation (pain and swelling) of the intestine.

[0672] Crohn's disease and ulcerative colitis are the main types of IBD. The types are:

[0673] Crohn's disease causes pain and swelling in the digestive tract. It can affect any part of the digestive tract from the mouth to the anus. It most commonly affects the small intestine and upper part of the large intestine.

[0674] Ulcerative colitis causes boils and sores (ulcers) in the large intestine (colon and rectum).

[0675] Microcolitis induces intestinal inflammation that can only be detected under a microscope.

[0676] Treatments that can be used in combination with the pharmaceutical compositions of the present invention include, for example:

[0677] Aminosalicylates (anti-inflammatories such as sulfasalazine, mesalamine, or balsalazide) minimize irritation to internal organs; antibiotics treat infections and abscesses; biologics block immune system signals that trigger inflammation; corticosteroids such as prednisone suppress the immune system and manage flare-ups; immunomodulators calm an overactive immune system; antidiarrheals; nonsteroidal anti-inflammatory drugs (NSAIDs); and supplements such as vitamins and probiotics.

[0678]

[0679] Graft-versus-host disease (GvHD)

[0680] 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.

[0681] GvHD includes acute graft-versus-host disease (aGvHD) and chronic graft-versus-host disease (cGvHD).

[0682]

[0683] psoriasis

[0684] Psoriasis is a chronic skin disorder, meaning it is 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 develops in early adulthood, but it can develop later in life. Besides the red scaly patches, symptoms of psoriasis include itchy, cracked, dry skin, a scaly scalp, sore skin, pitted nails, nails that crack or break easily, and joint pain.

[0685] Treatments that can be used in combination with the pharmaceutical compositions of the present invention may include, but are not limited to, the following: steroid creams, moisturizers for dry skin, anthralin (a drug that slows skin cell production), medicated lotions, shampoos and bath solutions to improve scalp psoriasis, 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, cyclophosphorine and / or immunotherapy.

[0686]

[0687] Sjögren's syndrome

[0688] Sjögren's syndrome is a lifelong autoimmune disease that reduces the amount of moisture produced by the exocrine glands in the eyes and mouth. It is named after 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.

[0689] There are two forms of Sjogren's syndrome: primary Sjogren's syndrome, which occurs on its own and not due to another health condition, and secondary Sjogren's syndrome, which occurs in addition to other autoimmune diseases such as rheumatoid arthritis, lupus, and psoriatic arthritis.

[0690] Treatments that can 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).

[0691]

[0692] myasthenia gravis

[0693] Myasthenia gravis (MG) is an autoimmune disease in which the body's immune system mistakenly attacks the body's own immune system. MG affects the transmission of signals between nerves and muscles (neuromuscular junctions).

[0694] Patients with myasthenia gravis lose the ability to 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 legs. MG is a lifelong neuromuscular disease.

[0695] Myasthenia gravis affects approximately 20 out of every 100,000 people. Experts estimate that 36,000 to 60,000 Americans suffer from this neuromuscular disease. Some people with mild cases may not know they have the disease, so the actual number affected may be much higher. 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 (teenage MG). The disease can affect people of all ages, but is rare in children.

[0696] Autoimmune MG is the most common form of this neuromuscular disease. Autoimmune MG is as follows:

[0697] Ocular: The muscles that move the eye and eyelids become weak. The eyelids may droop or become unable to open the eye. Some people may have double vision. Weakened vision is often the first sign of MG. About half of people with ocular MG develop generalized MG within 2 years of the initial symptoms.

[0698] Generalized: Muscle weakness affects the eyes and other parts of the body such as the face, neck, arms, and legs. Speech, swallowing, lifting the arms, standing, walking long distances, and climbing stairs can become difficult.

[0699] Therapies that can 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.

[0700]

[0701] Working Example

[0702] The present invention will be described in more detail below with reference to examples. It will be apparent 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.

[0703]

[0704] Example 1: Selection of anti-CD40L Stefin A protein variants from a phage display library

[0705] Candidate clone identification

[0706] The CD40L-binding stefin A protein variants of the present disclosure (hereinafter, anti-CD40L stefin A protein variants) were identified by selection from a stefin A protein variant library having 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 selection procedures are well known in the art (see Tiede et al. Protein Eng Des Sel. 2014.27(5):145-155 and Hughes et al. Sci Signal. 2018.10(505):eaaj 2005). According to such procedures, a suspension of phage expressing a stefin A protein variant (AFFIMER®) was cultured with human CD40L (ACROBiosystems, CDL-H82Q8) or mouse CD40L (ACROBiosystems, CDL-M5248) as required. In some cases, CD40L was biotinylated and alternately captured on streptavidin and neutravidin beads, and in other cases, CD40L 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 incubation of the antigen in a low pH solution followed by exposure to a high pH solution and trypsin. The eluted phage particles were then used to infect Escherichia coli (E. coli), and the infected bacteria were incubated under conditions suitable for bacteriophage replication. After release of the bacteriophage particles from the infected bacteria, the phage particles were allowed to bind to the target antigen, and the bound phage particles were eluted. The cycle of propagating the eluted phage particles to bacteria and isolating the released phage particles from the infected bacteria was repeated, enriching 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, reducing the amount of antigen available, or adding a blocking agent, to select for phage particle-displayed proteins that bind more strongly or specifically to the target antigen.

[0707] After repeated rounds of phage display library selection and amplification, the proteins expressed by the phages were expressed and screened by 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 CD40L was immobilized on a plate, lysates were added, and the amount of CD40L-binding Stefin A protein variants was measured on each plate using a detection antibody specific for the 6xMyc tag expressed on the candidate Stefin A protein variants. The phagemid vectors encoding the Stefin A protein variants with the best human CD40L binding activity were sequenced to confirm the DNA sequences of the candidate clones for further development. The loop 2 and loop 4 amino acid sequences of each of the candidate clones are presented in Tables 1 and 2, respectively.

[0708]

[0709] Example 2: Screening of anti-CD40L Stefin A protein mutants by direct ELISA

[0710] A binding ELISA was performed to measure the affinity of individual monomeric DAW01 clones for hCD40L. Specifically, plates were coated with 5 μg / ml hCD40L antigen and incubated overnight at 4°C. Plates were washed twice with 150 μl of wash buffer (PBS, Tween 20 0.1%) in a plate washer and saturated with 5% casein (Sigma) in PBS for 90 min at room temperature (25 ± 1°C). For binding, each DAW01 clone and rhCD40Fc was added to the plate at 1 μM, 1 / 3 to 300 nM, 1 / 3, respectively. Plates were washed 3 times as previously described. Plates were then loaded with anti-human CD40 biotinylated polyclonal antibody and incubated for 90 min. Cystatin A biotinylated polyclonal antibody (BAF1407) was then diluted in dilution buffer (PBS, 1% casein, 0.01% Tween20) to a concentration of 0.05 μg / ml and the plate was incubated at room temperature (25 ± 1 °C) for 90 min. Poly-HRP-streptavidin was then diluted in dilution buffer and the plate was incubated at room temperature (25 ± 1 °C) for 90 min. The plate was then washed three times as described above and substrate (TMB, Pierce Thermo-Scientific) was added to the plate for 10 min. The reaction was stopped using an acidic solution and the plate was read at 450-630 nm. The results are shown in Figure 1, with EC50 values ​​ranging from 0.67 to 60 nM.

[0711]

[0712] Example 3: Anti-CD40L Stefin A protein mutant screening in hCD40L-HEK293 cells by flow cytometry

[0713] To investigate the binding capacity of DAW01 monomeric stefin A protein variants to cell surface expressed hCD40L, a flow cytometric cell binding assay was performed. Briefly, hCD40L-HEK-293 cells (Crown Biosciences, C2041) were harvested by centrifugation at 300 rpm for 5 min. Cells were resuspended in PBS and aliquoted at 200,000 cells per well in a round-bottom 96-well plate. Cells were washed with PBS. Stefin A protein variants and controls were incubated in duplicate with 1% BSA, 0.01% sodium azide (NaN 3), diluted in staining buffer containing 2 mM EDTA, added to cells and stained for approximately 60 min at 4 ± 1 °C. Cells were washed and anti-cystatin A (R&D, AF1407) secondary antibody was diluted to 0.2 mg / ml in staining buffer and added to cells and stained for approximately 45 min at 4 ± 1 °C. Cells were washed again and A488 anti-goat (ThermoFisher, A21467) detection antibody was diluted 1:500 in staining buffer and added to cells and stained (approximately 30 min at 4 ± 1 °C). Finally, cells were washed and L / D stain Zombie Yellow (Biolegend, 423103) diluted in staining buffer was used to stain live and dead cells for 10 min at 4 ± 1 °C. The cells were washed again and each well was added with fixation buffer (R&D) for 10 min at 4±1°C, followed by addition of PBS containing EDTA (Lonza) and reading the plate on a flow cytometer (Guava 12 HT, Millipore). Dead cells were excluded and the green fluorescence channel (488 nm / 525 / 30) was acquired. Results were analyzed using Incyte and data were displayed as a dot plot using GraphPad. An example of the results at 1 μM is shown in Figure 2. Stefin A protein mutants were found to bind specifically to hCD40L-HEK293 cells (dark grey). HEK-293 negative cells were also used in the experiment to assess non-specific binding (light grey). HuCD40L HEK293 cells and control HEK293 cells were assessed for hCD40L expression using BV711 mAb (clone 24-31). The results are shown in Figure 3. The binding of clone 230 (SEQ ID NO: 249) to hCD40L-HEK293 cells was evaluated at different doses ranging from 0.7 to 500 nM. As can be seen in Figure 4, the binding of clone 230 was dose-dependent between 0.7 and 55 nM, and reached saturation above 55 nM.

[0714]

[0715] Example 4: Anti-CD40L Stefin A protein mutant screening in the CD40-HEK Blue reporter assay

[0716] HEK-Blue CD40 expressing cells (Invivogen) are capable of detecting bioactive CD40L by activating NF-κB after CD40 stimulation. Activation of the NF-κB pathway can be determined by measuring the levels of secreted embryonic alkaline phosphatase (SEAP) in the cell culture medium. The assay was performed according to the manufacturer's instructions.

[0717] Briefly, cells were plated in test medium (DMEM high glucose containing Blasticin and Zeocin) and aliquoted at 20,000 cells per well of a 96-well flat-bottom tissue culture plate and incubated at 37°C and 5% CO. 2 Afterwards, 50 μl of test medium was removed from the cells and cells were treated by adding 50 μl of 4X dilutions of test Stefin A protein variants or controls containing hCD40L (final concentration 0.8 nM). Plates were then incubated at 37°C, 5% CO 2 The plates were incubated at 37°C for 22 hours. The next day, the supernatant was collected and 30 μl of each well was mixed with 200 μl of HEK-Blue detection reagent (Invivogen) to detect SEAP activity. The mixture was incubated at 37°C and monitored periodically for any color changes. The absorbance (640 nm) was measured for 3 hours using a Pherastar plate reader. The data was displayed in a dot plot. IC50 was then calculated using an interpolated non-linear four-parameter curve as OD=f(log concentration). 5C8, a clinical grade monoclonal anti-hCD40L antibody, was used as a positive control for this assay. The calculated IC50 ranged from 11.6 to 100 nM (e.g., clone 230 (SEQ ID NO: 249) and clone 248 (SEQ ID NO: 267)). An example of the results is shown in FIG. 5.

[0718]

[0719] Example 5: Formatting of Stefin A protein mutants in dimeric or trimeric configurations for increased binding to hCD40L

[0720] The Stefin A protein variants can be designed to assemble into stable multimeric oligomers for increased binding avidity. The Stefin A protein variants generated are In Line Fusion (ILF) proteins and may be dimers or trimers with various linkers (rigid linkers, see e.g., SEQ ID NOs: 508, 510-514; or flexible linkers, see e.g., SEQ ID NOs: 509, 515-518) as shown in FIG. 6.

[0721] The binding of different Stefin A protein variants (monomer, dimer, trimer) to hCD40L was examined as described above. The results, shown in Figure 7, indicate that the ILF proteins have the same or greater binding affinity to their target ligands compared to the monomeric forms. This binding was further analyzed by flow cytometry (Figure 8), and it was found that the Stefin A protein variants (monomer, dimer, trimer forms) bound to hCD40L at similar levels as the anti-CD40L antibody.

[0722] Other formats were also screened using the HEK-Blue cell-based assay as described above, and the results are shown in Figure 9, showing that the clone 230DJ format (trimer) was the best with an EC50 of 5.99 nM.

[0723]

[0724] Example 6: Characterization of Stefin A protein variants in trimer or tetramer configurations using HSA-binding Stefin A protein variants.

[0725] In additional experiments, Stefin A protein variants were designed to assemble into stable multimeric oligomers. The Stefin A protein variants generated were ILF proteins and could be trimers or tetramers with various linkers (rigid linkers, see e.g., SEQ ID NOs: 508, 510-514; or flexible linkers, see e.g., SEQ ID NOs: 509, 515-518). Four Stefin A protein variants were tested: Clone-230DT (trimer with rigid linker), Clone-230 XT75 (trimer with rigid linker and HSA Stefin A protein variant), Clone-230DS (tetramer with rigid linker), and Clone-230 XT76 (tetramer with rigid linker and HSA Stefin A protein variant).

[0726] To evaluate the binding potential of the different formats, a CD40L competitive ELISA was performed. Briefly, plates were coated with 1 μg / ml rhCD40Fc and incubated overnight at 4°C. Plates were washed twice with 150 μl of wash buffer (PBS, Tween 20 0.1%) in a plate washer and saturated with PBS with 5% casein (Sigma) for 90 min at room temperature (25±1°C). Human CD40L at 2x EC80 (1 nM) was combined with anti-hCD40L monoclonal antibodies or each of the tested ILF clones starting at 10 nM. The resulting solutions were then mixed and added to the plates. Plates were washed three times as described above. Biotinylated anti-hCD40L polyclonal antibodies were then diluted in dilution buffer and added to the plates. Plates were incubated for 90 min at room temperature (25±1°C). Plates were then washed. Poly-HRP-streptavidin was then diluted in dilution buffer and added to the plate. The plate was incubated for an additional 90 min at room temperature (25±1°C). The plate was washed three times as described above and substrate (TMB, Pierce Thermo-Scientific) was added to the plate for 10 min. The reaction was stopped using an acidic solution, the plate was read at 450-630 nm and the final percentage of inhibition was calculated. The results are shown in Figure 10.

[0727] The CD40L inhibitory activity of the ILF fusion proteins containing the Stefin A protein mutants was evaluated by HEK-Blue assay in the same manner as described in Example 4. The results are shown in FIG.

[0728] To demonstrate that different ILF proteins can simultaneously bind two targets (human CD40L and HSA), a bridging ELISA was performed. In this assay, hCD40L is used to capture the bispecific stefin A protein variants and anti-HSA antibodies are used to detect the stefin A protein variants, i.e., HSA-bound stefin A protein variants. Briefly, 96-well plates were coated with 0.5 mg / ml human CD40L in carbonate buffer. After saturation with 5% casein / PBS buffer, the plates were washed and dilutions of stefin A protein variants or controls were incubated with HSA at a final concentration of 10 μM for 90 min. The plates were then washed and biotinylated polyclonal antibodies to HSA (HRP-conjugated) (Abcam) were added for 90 min. After the final washing step, TMB was added for experimental development and the plates were read at 450 nm. EC50 was then calculated using an interpolated non-linear four-parameters standard curve (Figure 12). In control experiments, an anti-cystatin antibody bound to the Stefin A framework of Stefin A protein variants was added instead of anti-HSA antibody in the absence (Figure 12A) or presence (Figure 12B) of HSA, demonstrating that all Stefin A protein variants bound to hCD40L in both conditions. Bridge ELISA data showed that participation of HSA did not affect CD40L binding in either of the two Stefin A protein variant formats tested.

[0729]

[0730] Example 7: Therapeutic effect of anti-CD40L Stefin A protein mutant on haploid GVHD animal model

[0731] Example 7-1: Experimental Method

[0732] 192 healthy female C57BL / 6 mice (6 weeks old) were purchased from Janvier (France) and used for splenocyte extraction for GVHD induction. 12 healthy female B6D2F1 mice (6 weeks old) were purchased from Charles River and used for GVHD induction (syngeneic control). 70 healthy female B6D2F1 mice (6 weeks old) were purchased from Charles River and used as donor mice.

[0733] Stefin A protein mutants that specifically bind to mouse CD40L were expressed in the form of trimeric in-line fusion proteins (Table 15).

[0734] [Table 15]

[0735] The spleens of C57BL / 6 and B6D2F1 mice were removed, and spleen cells were extracted and prepared in HBSS buffer. Erythrocytes contained in the extracted spleen cells were lysed using an erythrocyte lysis solution (BD Pharma) and then washed to prepare the spleen cells. Sixty-four B6D2F1 mice (G4-G11) were cultured with 6 × 10 spleen cells isolated from C57BL / 6 spleens. 7 Three B6D2F1(G3) mice were injected intravenously with 6 × 10 spleen cells isolated from the spleens of C57BL / 6 mice. 7 Three B6D2F1(G2) mice were injected intravenously with 6 × 10 spleen cells isolated from the spleen of the same B6D2F1 mouse. 7 The mice were intravenously injected at 100 cells / head (Figure 13).

[0736] The animals in this study were randomly allocated to the experiment based on their individual body weights, and the homogeneity between groups was confirmed by analysis of variance (ANOVA) to show no statistical significance.

[0737] The administration route of the test substance was ip, and the pH of the dosage form was adjusted to 7.3-7.4 for ip administration, and the volume was 20 mL / kg. The types and amounts of the administered substances administered to the test animals were as shown in the table. The administration of the test substance was performed 1 hour after GVHD induction, and the administration intervals for the administered substances were as follows. The Stefin A protein mutant administration group, except for the antibody administration group, was administered 6 times at 2-day intervals after GVHD induction, and the MR-1 antibody was administered 3 times at 2-day intervals after GVHD induction (Figure 14, Table 16). The survival and behavior of the animals were observed every day, and the clinical measures were recorded every day. Body weight and clinical score were recorded every day. The clinical score of GVHD was evaluated by macroscopic observation according to the following evaluation format, and the total of the observed points was recorded. ANOVA was performed using GraphPad Prism to determine the statistical significance between the groups. The statistical significance between the groups was determined to be significant when p value<0.05 or less.

[0738] [Table 16]

[0739] Example 12-2: Results

[0740] After random allocation, the overall mean weight of the animal groups was 21.5g, with a weight range of 18.8g-23.8g. Statistical analysis showed no significant differences between groups. The weight changes of the experimental animals were monitored throughout the study period. To confirm the weight changes, the mean weight change (MBWC%) was compared based on the rate of change on D18. The mean weight change of the G1 group increased by 9.7%, and the mean weight change of the G2 group increased by 15.6%. The GVHD control group, G3, showed a tendency for the mean weight change to decrease by 24.4%. The mean weight changes of the drug-administered groups, G4-G11, were as follows:

[0741] G4 group: increase of 1.6%, G5 group: decrease of 4.1%, G6 group: decrease of 14.6%, G7 group: increase of 2.8%, G8 group: decrease of 6.4%, G9 group: decrease of 9.0%, G10 group: decrease of 4.4%, and G11 group: decrease of 21.7%, indicating that the groups administered the Stefin A protein mutant had better average body weight changes than the G3 group.

[0742] Changes in clinical scores for individual animals were recorded using a score sheet that included body, skin, hair, and mobility criteria. It was confirmed that the mean GVHD scores of the G1 and G2 groups did not increase throughout the study period. The G3 group began to increase on D15 and recorded a mean score of 8.0 on D18. The G4 group, similar to the G1 and G2 groups, did not increase in GVHD scores throughout the study period, and the G5-G7 groups began to increase on days 9-14 after GVHD induction and showed GVHD scores ranging from 0.3 to 1.1 on day 18, the day the experiment was terminated. The clinical scores of the G8-G10 groups began to increase on days 7-10 after GVHD induction and showed GVHD scores ranging from 0.3 to 1.4 on day 18, the day the experiment was terminated (Figure 15).

[0743] From this, it was confirmed that the inhibitory effect of the stefin A protein mutant on CD40L activity was statistically significant compared to the GVHD control.

[0744] [Industrial Applicability]

[0745] The Stefin A protein variants of the present invention that specifically bind to CD40L and / or fusion proteins or conjugates containing the same are non-immunogenic polypeptides that can bind to CD40L with high affinity and specificity, and are therefore useful for targeting CD40L and various cells known to express CD40L. Furthermore, the Stefin A protein variants of the present invention that specifically bind to CD40L exhibit excellent CD40L activity suppression ability and can be usefully used in medical / pharmaceutical applications for the prevention and treatment of various diseases associated with CD40L.

[0746]

[0747] 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, the above-described embodiments should be understood to be illustrative and not restrictive in all respects.

[0748] Sequence Catalog Free Text

[0749] Electronic file attachments

Claims

1. A Stefin A protein variant that specifically binds to CD40L.

2. The Stefin A protein mutant binds to CD40L at 1×10 －６ The Stefin A protein variant of claim 1, which exhibits a Kd value of M or less.

3. 2. The Stefin A protein variant of claim 1, characterized in that it comprises the following amino acid sequence: MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVD-(Xaa)n-GTNYYIKVRAGDNKYMHLKVFKSL-(Xaa)m-EDLVLTGYQVDKNKDDELTGF (SEQ ID NO:5), or MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVV-(Xaa)n-GTNYYIKVRAGDNKYMHLKVFKSL-(Xaa)m-EDLVLTGYQVDKNKDDELTGF (SEQ ID NO: 733) Here, each Xaa is independently an amino acid residue, and each n and m is independently an integer of 3 to 20.

4. The Stefin A protein variant according to claim 1, characterized in that the Stefin A protein variant comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 246 to 365.

5. The Stefin A protein variant according to claim 3, wherein (Xaa)n comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 6 to 125.

6. The Stefin A protein mutant according to claim 3, wherein (Xaa)m comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 126 to 245.

7. The Stefin A protein variant of claim 1, further comprising a signal peptide.

8. A fusion protein comprising the Stefin A protein variant of claim 1.

9. The fusion protein according to claim 8, characterized in that the fusion protein comprises an amino acid sequence having an amino acid sequence of SEQ ID NOs: 677 to 702.

10. The fusion protein of claim 8, wherein the fusion protein is a triplet or quadruplet of Stefin A protein variants, and the triplet or quadruplet is characterized in that the Stefin A protein variants are linked by a linker.

11. The fusion protein of claim 8, further comprising at least one selected from the group consisting of a binding domain, a cytokine, a half-life extension domain, a growth factor, an enzyme, and a cell penetration domain.

12. The fusion protein of claim 8, further comprising at least one 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.

13. A conjugate comprising the Stefin A protein mutant of claim 1.

14. A nucleic acid encoding the Stefin A protein variant of claim 1 or the fusion protein of claim 8.

15. A delivery vehicle comprising the nucleic acid of claim 14.

16. An expression vector comprising the nucleic acid of claim 14.

17. A genetically engineered cell into which the nucleic acid of claim 14 has been introduced.

18. Culturing the genetically engineered cell of claim 17 to produce a Stefin A protein variant or fusion protein; and obtaining the produced Stefin A protein variant or fusion protein.

19. A pharmaceutical composition containing as an active ingredient any one or more selected from the group consisting of the Stefin A protein mutant described in claim 1, the fusion protein described in claim 8, the conjugate described in claim 13, and the nucleic acid described in claim 14.