Single-chain variable fragment engineered proteins

Abstract

The present invention relates to a heavy chain variable domain (V H ) and the light chain variable domain (V L The present invention provides engineered proteins of single-chain variable (scFv) fragments having an albumin-binding portion fused to the single-chain variable fragment, wherein the albumin-binding portion is connected by a linker.

Description

[Technical Field]

[0001] The present disclosure relates to engineered single-chain variable fragment (scFv) proteins that can be used in the medical field. [Background technology]

[0002] Therapeutic antibodies were approved for use in 1986, and the market has grown rapidly since then. Existing monoclonal antibodies are expensive, require laborious mammalian expression systems, and have tissue penetration issues. These challenges have led to research into various antibody fragment types as alternatives.

[0003] Among all these antibody derivatives, scFvs have demonstrated clinical applicability, and several scFv-based therapeutics were approved by the FDA in 1988. scFvs are derived from the heavy chain variable domain (V) of immunoglobulin (Ig). H ) and the light chain variable domain (V L ) linked by a polypeptide linker. Such scFvs do not induce unwanted crystallizable fragment (Fc)-mediated immune responses and are more effective than full-length antibodies in tumor penetration and distribution. However, despite these advantages, scFvs typically suffer from the disadvantages of a very short half-life in serum due to their small size (approximately 25 kDa) and lack of an Fc region.

[0004] Therefore, despite their usefulness, the clinical application of scFvs is limited to a small number of cases, such as hematological malignancies and macular degeneration, etc. Therefore, there is a need to develop novel scFv-based therapeutic agents that can address this situation. Summary of the Invention [Problem to be solved by the invention]

[0005] An object of the present disclosure is to provide modified scFv proteins having novel structures.

[0006] An object of the present disclosure is to provide a pharmaceutical composition for the prevention or treatment of cancer, comprising a modified scFv protein. [Means for solving the problem]

[0007] 1. A single-chain variable fragment engineered protein in which an albumin-binding moiety is fused to a single-chain variable fragment (scFv) in which the heavy chain variable domain (VH) and light chain variable domain (VL) of an antibody are linked by a linker.

[0008] 2. A single chain variable fragment engineered protein according to 1 above, wherein the albumin binding moiety is fused within a linker or at the N-terminus of the scFv or at the C-terminus of the scFv.

[0009] 3. The single-chain variable fragment engineered protein according to 1 above, wherein the albumin binding moiety is fused within the linker and the biologically active agent is fused at the C-terminus of the scFv.

[0010] 4. The single chain variable fragment engineered protein according to paragraph 1 above, wherein the albumin binding moiety is fused at a position spaced at least 3 aa from the C-terminus of the heavy chain variable domain (VH) and the N-terminus of the light chain variable domain (VL), respectively.

[0011] 5. The single chain variable fragment engineered protein according to 1 above, wherein the albumin binding moiety is positioned in the opposite direction from the complementarity determining regions (CDRs) of the heavy chain variable domain (VH) and light chain variable domain (VL).

[0012] 6. The single-chain variable fragment modified protein according to 1 above, wherein the length of the albumin binding portion is 10 to 150 aa.

[0013] 7. The single-chain variable fragment engineered protein according to 1 above, wherein the linker has a length of 6 to 50 aa.

[0014] 8. The single-chain variable fragment engineered protein according to 1 above, wherein the length of the scFv is 150 to 350 aa.

[0015] 9. The single-chain variable fragment engineered protein according to 1 above, wherein the length of the heavy chain variable domain (VH) and the light chain variable domain (VL) are each 70 to 150 aa.

[0016] 10. The single-chain variable fragment engineered protein according to 1 above, wherein the albumin binding portion comprises an amino acid sequence selected from the sequences of SEQ ID NOs: 1 to 3.

[0017] 11. The single-chain variable fragment engineered protein according to 1 above, wherein the scFv comprises an amino acid sequence selected from the sequences of SEQ ID NOs: 4 to 6.

[0018] 12. The single chain variable fragment engineered protein according to 1 above, wherein the linker comprises the amino acid sequence (GS)n, (GGS)n, or (GGGGS)n.

[0019] 13. The single-chain variable fragment engineered protein of claim 3 above, wherein the biologically active agent is selected from the group consisting of cytokines, insulin, insulin-like growth factor (IGF)-1, IGF-2, epidermal growth factor (EGF), transforming growth factor (TGF), human growth hormone, and vascular endothelial growth factor (VEGF).

[0020] 14. A pharmaceutical composition for the treatment or prevention of cancer comprising any one of the single-chain variable fragment modified proteins described in 1 to 13 above. [Effects of the Invention]

[0021] The scFv engineered proteins of the present disclosure exhibit excellent antigen-binding ability because the albumin binding site does not interfere with the complementarity-determining regions (CDRs) of the heavy and light chain variable regions.

[0022] The scFv variant proteins of the present disclosure bind to albumin present in the body and therefore have an extended half-life.

[0023] The lengths of the linker and albumin binding moiety of the scFv modified proteins of the present disclosure can be adjusted appropriately, and therefore excellent binding ability to both antibody and albumin can be maintained.

[0024] The scFv variant proteins of the present disclosure can deliver bioactive agents, such as cytokines, to a target by conjugating them to the C-terminus.

[0025] The scFv modified proteins of the present disclosure have excellent cell-penetrating ability.

[0026] The scFv variant proteins of the present disclosure can be readily prepared using microbial production systems.

[0027] The scFv variant proteins of the present disclosure can be fused with albumin binding moieties while minimizing structural changes in known scFvs, and can therefore be suitably used to improve antibodies and fragments thereof. [Brief explanation of the drawings]

[0028] [Figure 1] 4D5scFv, the present examples and comparative examples, and their structures implemented by AlphaFold2 are illustrated. [Figure 2] 4D5scFv, the present examples and comparative examples, and their structures implemented by AlphaFold2 are illustrated. [Figure 3] 4D5scFv, the present examples and comparative examples, and their structures implemented by AlphaFold2 are illustrated. [Figure 4] 4D5scFv, the present examples and comparative examples, and their structures implemented by AlphaFold2 are illustrated. [Figure 5] 4D5scFv, the present examples and comparative examples, and their structures implemented by AlphaFold2 are illustrated. [Figure 6]4D5scFv, the present examples and comparative examples, and their structures implemented by AlphaFold2 are illustrated. [Figure 7] 4D5scFv, the present examples and comparative examples, and their structures implemented by AlphaFold2 are illustrated. [Figure 8] 4D5scFv, the present examples and comparative examples, and their structures implemented by AlphaFold2 are illustrated. [Figure 9] 4D5scFv, the present examples and comparative examples, and their structures implemented by AlphaFold2 are illustrated. [Figure 10] 4D5scFv, the present examples and comparative examples, and their structures implemented by AlphaFold2 are illustrated. [Figure 11] 4D5scFv, the present examples and comparative examples, and their structures implemented by AlphaFold2 are illustrated. [Figure 12] 4D5scFv, the present examples and comparative examples, and their structures implemented by AlphaFold2 are illustrated. [Figure 13] 4D5scFv, the present examples and comparative examples, and their structures implemented by AlphaFold2 are illustrated. [Figure 14] 4D5scFv, the present examples and comparative examples, and their structures implemented by AlphaFold2 are illustrated.

[0029] [Figure 15] Computational evaluation of structure predictions for 4D5scFv and D5-ABD variants. (a) Model structures predicted by AlphaFold2 and visualized by PyMOL. (b) Position-wise pLDDT scores for structure predictions reported by AlphaFold2.

[0030] [Figure 16]4D5scFv and 4D5-ABD variants aligned and visualized by PyMOL are shown.

[0031] [Figure 17] Figure 1 shows the complex conformational structures of two 4D5-ABD variants and HER2 (PDB ID: 1N8Z) visualized by PyMOL.

[0032] [Figure 18a] Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the expression and purification of 4D5scFv and 4D5-ABD variants. Coomassie blue-stained SDS-PAGE gels of cell lysate samples obtained during expression (MW: molecular weight standards; BI: cell lysate samples before induction; AI: cell lysate samples after induction). [Figure 18b] 1 shows sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis results for the expression and purification of 4D5scFv and 4D5-ABD variants. Coomassie blue stained SDS-PAGE gel of purified samples.

[0033] [Figure 19] The results of SDS-PAGE analysis of 4D5scFv and two 4D5-ABD variants under reducing (left) and non-reducing (right) conditions are shown (MW: molecular weight standard).

[0034] [Figure 20] 1 shows the results of MALDI-TOF analysis of purified 4D5scFv and 4D5-ABD variants.

[0035] [Figure 21]Size-exclusion chromatograms of 4D5scFv, 4D5-ABD variants, and reference proteins (4D5scFv: 27 kDa; 4D5scFv dimer: 54 kDa; 4D5-S-ABD: 32 kDa; 4D5-L-ABD: 33 kDa; ovalbumin (OA): 43 kDa; conalbumin (CA): 75 kDa; aldolase (Ald): 158 kDa) are shown.

[0036] [Figure 22] 1 shows anti-HER2 enzyme-linked immunosorbent assay (ELISA) results for 4D5scFv and 4D5-ABD variants in the presence and absence of HSA.

[0037] [Figure 23] 1 shows the anti-HSA ELISA results for 4D5scFv and 4D5-ABD variants.

[0038] [Figure 24] Pharmacokinetic profiles of 4D5scFv and 4D5-ABD variants are shown. DETAILED DESCRIPTION OF THE INVENTION

[0039] The antibody may be selected appropriately depending on the use of the modified protein of the present disclosure, and is not limited to a particular type.

[0040] For example, the antibody may be an antibody against a disease antigen, and the disease may be an autoimmune disease or cancer.

[0041] The antibody may be an anti-cancer agent or an immunosuppressant. Specifically, the antibody may be selected from the group consisting of, but not limited to, trastuzumab, naxitamab, cetuximab, telisotuzumab, tisotumab, pinatuzumab, rifastuzumab, indusatumab, bundletuzumab, sofituzumab, borcetuzumab, trastuzumab, mirvetuximab, cortuximab, naratuximab, indatuximab, anetuzumab, lorvotuzumab, cantuzumab, laprituximab, bivatuzumab, vadastuximab, rovalpituzumab, inotuzumab, sacituzumab, labetuzumab, milatuzumab, rupartuzumab, apultuzumab, pembrolizumab, atezolizumab, and OKT3 (an anti-CD3 monoclonal antibody).

[0042] The scFv is not limited to having a particular amino acid length, so long as it can be fused with an albumin binding moiety for the purposes of this disclosure.

[0043] The amino acid length of the scFv can be, for example, at least 100 aa, at least 110 aa, at least 120 aa, at least 130 aa, at least 140 aa, at least 150 aa, at least 160 aa, at least 170 aa, at least 180 aa, at least 190 aa, at least 200 aa, at least 210 aa, at least 220 aa, at least 230 aa, at least 240 aa, at least 250 aa, at least 260 aa, at least 270 aa, at least 280 aa, at least 290 aa, or at least It may be at least 300 aa, at least 310 aa, at least 320 aa, at least 330 aa, at least 340 aa, at least 350 aa, at least 360 aa, at least 370 aa, at least 380 aa, at least 390 aa, at least 400 aa, at least 410 aa, at least 420 aa, at least 430 aa, at least 440 aa, at least 450 aa, at least 460 aa, at least 470 aa, at least 480 aa, at least 490 aa, or at least 500 aa.

[0044] The amino acid length of the scFv may be, for example, 500 aa or less, 490 aa or less, 480 aa or less, 470 aa or less, 460 aa or less, 450 aa or less, 440 aa or less, 430 aa or less, 420 aa or less, 410 aa or less, 400 aa or less, 390 aa or less, 380 aa or less, 370 aa or less, 360 aa or less, 350 aa or less, 340 aa or less, 330 aa or less, 320 aa or less, 310 aa or less, It may be 300aa or less, 290aa or less, 280aa or less, 270aa or less, 260aa or less, 250aa or less, 240aa or less, 230aa or less, 220aa or less, 210aa or less, 200aa or less, 190aa or less, 180aa or less, 170aa or less, 160aa or less, 150aa or less, 140aa or less, 130aa or less, 120aa or less, 110aa or less, or 100aa or less.

[0045] The scFv may have a length of, for example, 150 to 350 aa, 160 to 340 aa, 170 to 330 aa, 180 to 320 aa, 190 to 310 aa, or 200 to 300 aa.

[0046] Heavy chain variable domain (V H ) and the light chain variable domain (V L ) is not limited to having a particular amino acid length.

[0047] Heavy chain variable domain (V H ) and the light chain variable domain (V L) each have an amino acid length of, for example, at least 50 aa, at least 55 aa, at least 60 aa, at least 65 aa, at least 70 aa, at least 75 aa, at least 80 aa, at least 85 aa, at least 90 aa, at least 95 aa, at least 100 aa, at least 105 aa, at least 110 aa, at least 115 aa, at least 120 aa, at least 125 aa, at least 130 aa, at least 135 aa, at least 140 aa, at least 145 aa, at least 1 It may be 50 aa, at least 155 aa, at least 160 aa, at least 165 aa, at least 170 aa, at least 175 aa, at least 180 aa, at least 185 aa, at least 190 aa, at least 195 aa, at least 200 aa, at least 205 aa, at least 210 aa, at least 215 aa, at least 220 aa, at least 225 aa, at least 230 aa, at least 235 aa, at least 240 aa, at least 245 aa, or at least 250 aa.

[0048] Heavy chain variable domain (V H ) and the light chain variable domain (V L ) may have an amino acid length of, for example, 250 aa or less, 245 aa or less, 240 aa or less, 235 aa or less, 230 aa or less, 225 aa or less, 220 aa or less, 215 aa or less, 210 aa or less, 205 aa or less, 200 aa or less, 195 aa or less, 190 aa or less, 185 aa or less, 180 aa or less, 175 aa or less, 170 aa or less, 165 aa or less, 160 aa or less, 155 It may be aa or less, 150aa or less, 145aa or less, 140aa or less, 135aa or less, 130aa or less, 125aa or less, 120aa or less, 115aa or less, 110aa or less, 105aa or less, 100aa or less, 95aa or less, 90aa or less, 85aa or less, 80aa or less, 75aa or less, 70aa or less, 65aa or less, 60aa or less, 55aa or less, or 50aa or less.

[0049] The heavy chain variable domain and the light chain variable domain may each have a length of, for example, 70 to 150 aa, 80 to 140 aa, 90 to 130 aa, or 100 to 120 aa.

[0050] The linker is a peptide linker that maintains structural stability and links the heavy chain variable domain and the light chain variable domain, allowing for proper orientation for antigen binding by controlling the distance and orientation.

[0051] For example, the amino acid length of the linker may be at least 3 aa, at least 4 aa, at least 5 aa, at least 6 aa, at least 7 aa, at least 8 aa, at least 9 aa, at least 10 aa, at least 11 aa, at least 12 aa, at least 13 aa, at least 14 aa, at least 15 aa, at least 16 aa, at least 17 aa, at least 18 aa, at least 19 aa, at least 20 aa, at least 21 aa, at least 22 aa, at least 23 aa, at least 24 aa, at least 25 aa, at least 26 aa, at least 27 aa, at least 28 aa, at least 29 aa, or at least 30 aa.

[0052] In addition, the amino acid length of the linker may be 100 aa or less, 95 aa or less, 90 aa or less, 85 aa or less, 80 aa or less, 75 aa or less, 70 aa or less, 65 aa or less, 60 aa or less, 55 aa or less, 50 aa or less, 55 aa or less, 50 aa or less, 45 aa or less, 40 aa or less, 35 aa or less, 30 aa or less, 25 aa or less, 20 aa or less, 15 aa or less, or 10 aa or less.

[0053] For example, the linker may have a length of 6 to 50 aa, 6 to 40 aa, 6 to 30 aa, or 6 to 20 aa.

[0054] The linker is not limited to a particular sequence, as long as it can maintain the flexibility and structural stability of the scFv.

[0055] For example, the linker may be (GS(SEQ ID NO:7)) n , (GGS (SEQ ID NO: 8)) n , (GGGGS (SEQ ID NO: 9)) n In this case, the above-mentioned n is a natural number from 1 to 10, and may be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. The order and repetition of these combinations are not limited, and at least two types may be combined.

[0056] The linker may have the sequence of SEQ ID NO:10-12.

[0057] The scFv may further comprise an initiation restriction sequence (eg, MG) at the N-terminus.

[0058] The C-terminus of the scFv may further comprise a His tag sequence (eg, HHHHHH) for isolating and purifying the modified scFv protein.

[0059] For example, the scFv may have the sequence of SEQ ID NO: 4 (4D5scFv), SEQ ID NO: 5 (OKTscFv), or SEQ ID NO: 6 (3F8scFv).

[0060] As used herein, the term albumin binding moiety (ABM) refers to a moiety that interacts with the albumin protein and may be, for example, an antibody, an antibody analog, a protein domain, a protein motif, a peptide, a chemical compound, an aptamer, an oligonucleotide, or a saccharide.

[0061] The amino acid length of the ABM can be, for example, at least 5 aa, at least 6 aa, at least 7 aa, at least 8 aa, at least 9 aa, at least 10 aa, at least 11 aa, at least 12 aa, at least 13 aa, at least 14 aa, at least 15 aa, at least 16 aa, at least 17 aa, at least 18 aa, at least 19 aa, at least 20 aa, at least 21 aa, at least 22 aa, at least 23 aa, at least 24 aa, at least 25 aa, at least 26 aa, at least 27 aa, It may be at least 28 aa, at least 29 aa, or at least 30 aa, at least 31 aa, at least 32 aa, at least 33 aa, at least 34 aa, at least 35 aa, at least 36 aa, at least 37 aa, at least 38 aa, at least 39 aa, at least 40 aa, at least 41 aa, at least 42 aa, at least 43 aa, at least 44 aa, at least 45 aa, at least 46 aa, at least 47 aa, at least 48 aa, at least 49 aa, or at least 50 aa.

[0062] The amino acid length of the ABM may be, for example, 200 aa or less, 195 aa or less, 190 aa or less, 185 aa or less, 180 aa or less, 175 aa or less, 170 aa or less, 165 aa or less, 160 aa or less, 155 aa or less, 150 aa or less, 145 aa or less, 140 aa or less, 135 aa or less, 130 aa or less, 125 aa or less, 120 aa or less, 115 aa or less, 110 aa or less, 105 aa or less, 100 aa or less, 95 aa or less, 90 aa or less, 85 aa or less, 80 aa or less, 75 aa or less, 70 aa or less, 65 aa or less, 60 aa or less, 55 aa or less, or 50 aa or less.

[0063] The ABM may have a length of, for example, 10 to 150 aa, 30 to 100 aa, or 30 to 50 aa.

[0064] For example, the ABM may have the sequence of SEQ ID NO:1 (ABD), SEQ ID NO:2 (VNAR (variable domain of novel antigen receptor)), or SEQ ID NO:3 (ABP (albumin binding protein)).

[0065] The ABM is introduced at a selected position that does not significantly impair the structure or antigen-binding function of the scFv. The ABM may be fused within a linker or at the N- or C-terminus of the scFv.

[0066] Depending on the binding site of the ABM, the scFv can be: (i) ABM-V H -Linker-V L , (ii) V H -Linker-V L -ABM, (iii)V H -ABM-Linker-V L , (iv) V H - linker1-ABM-linker2-VL, and (v) V H -Linker-ABM-V L It may be diagrammed as:

[0067] When the ABM is fused at the N-terminus or C-terminus as in (i) and (ii), a separate linker may be added to link the ABMs. An example of such a form is (vi) ABM-Linker 1-V H -Linker 2-V L and (vii) V. H -Linker 1-V L - Linker 2-ABM may be included.

[0068] Linker 1 and linker 2 may differ from each other in amino acid sequence and length.

[0069] The amino acid length of each of Linker 1 and Linker 2 may be at least 3 aa, at least 5 aa, at least 7 aa, 9 aa, or at least 10 aa, and the upper limit may be, but is not limited to, 15 aa or less, 20 aa or less, 25 aa or less, 30 aa, or 50 aa or less.

[0070] When the ABM is fused within a linker, it is preferably fused at a position spaced at least 2 aa, at least 3 aa, at least 4 aa, at least 5 aa, at least 6 aa, at least 7 aa, at least 8 aa, at least 9 aa, or at least 10 aa from the C-terminus of the heavy chain variable domain and the N-terminus of the light chain variable domain, respectively.

[0071] For example, the ABM may be fused between one of the fourth to eighth amino acids and the adjacent amino acid of a linker having a length of 10 to 15 aa. Alternatively, the ABM may be fused between one of the seventh to 14th amino acids and the adjacent amino acid of a linker having a length of 16 to 20 aa.

[0072] Specifically, the ABM may be fused between one of the 5th to 8th amino acids of the linker of SEQ ID NO: 10 or 11 and the adjacent amino acid, or between one of the 11th to 16th amino acids of the linker of SEQ ID NO: 12 and the adjacent amino acid.

[0073] Alternatively, the ABM may be fused between the 6th and 7th amino acids of the linker of SEQ ID NO: 10 or 11, or between the 13th and 14th amino acids of the linker of SEQ ID NO: 12, without limitation.

[0074] If the ABM is not fused at a position at least 3 aa away from the C-terminus of the heavy chain variable domain and the N-terminus of the light chain variable domain, interference with adjacent CDRs may occur, resulting in poor binding ability of the antibody.

[0075] When the ABM is fused within a linker or at the C-terminus of the scFv, the ABM is positioned in the opposite direction to the CDRs of the heavy and light chain variable domains. In this case, opposite direction means, for example, that when the CDRs are positioned at the bottom of the modified scFv protein, the ABM is positioned at the top. Additionally, opposite direction means, for example, that when the CDRs are positioned at the left side of the modified scFv protein, the ABM is positioned at the right side.

[0076] The albumin may be human serum albumin (HSA).

[0077] When administered in vivo, the modified proteins of the present disclosure bind to serum albumin via the ABM, thus slowing the metabolism and degradation of the modified protein and increasing cancer cell permeability.

[0078] When the ABM is fused within the linker of the scFv, the biologically active substance may additionally be attached to the N-terminus or C-terminus of the scFv. Additionally, when the ABM is fused at a position other than the C-terminus of the scFv, the biologically active substance may be attached to the C-terminus of the scFv. In this case, the modified scFv protein of the present disclosure functions as a carrier to deliver the biologically active substance to the target.

[0079] The bioactive substance refers to any material capable of exhibiting a predetermined activity when administered in vivo. The bioactive substance may be, for example, any one selected from the group consisting of cytokines, insulin, insulin-like growth factor-1 (IGF-1), insulin-like growth factor-2 (IGF-2), epidermal growth factor (EGF), transforming growth factor (TGF), human growth hormone, and vascular endothelial growth factor (VEGF).

[0080] Examples of such cytokines may include interleukins (IL-1 to IL-17), tumor necrosis factors (TNF), interferons (IFN), erythropoietin (EPO), and thrombopoietin (TPO).

[0081] The ABM may be fused to the center of the amino acid sequence of the linker in the scFv. Alternatively, the ABM may be fused between an amino acid separated by 1 to 5 aa or 1 to 10 aa from the center or between adjacent amino acids.

[0082] The ABM may be, for example, an albumin protein domain, an affibody, or a peptide. The albumin protein domain, affibody, or peptide is small in size compared to an antibody (IgG) and is fused to an scFv to form a single polypeptide chain. The albumin protein domain, affibody, or peptide may have a structure that does not contain disulfide bonds.

[0083] An ABM may be a portion of a particular protein, or may be an artificially designed domain.

[0084] For example, the ABM may be a small triple-helical protein domain found in various surface proteins expressed by Gram-positive bacteria. Specifically, the ABM may be derived from streptococcal protein G or Peptostreptococcal albumin-binding protein (PAB) of Finegoldia magna, or may be modified to further increase albumin-binding affinity.

[0085] For example, the ABM may be a VNAR (V domain of cartilage oligomeric matrix protein derived from chondrocytes) derived from sea sharks, or may be modified to further increase albumin binding affinity.

[0086] Specifically, the ABM may have the amino acid sequence of SEQ ID NO:1-3.

[0087] The variant proteins of the disclosure may be prepared in a microorganism in which a sequence encoding the protein is expressed.

[0088] The microorganism may be any microorganism known in the art, without limitation, for example, but not limited to, Escherichia coli.

[0089] When preparing engineered proteins using a microbial system, expression is facilitated only if the protein does not involve post-translational modifications (PTMs) and contains no or few disulfide bonds. Antibody forms such as IgG contain many disulfide bonds and involve PTMs, requiring additional coupling and purification processes when compounds are used. The engineered proteins of the present disclosure are characterized by being in the form of fused polypeptides, not involving PTMs, and containing few disulfide bonds, and therefore can facilitate microbial system-based expression.

[0090] In the preparation process, the linker sequence in the scFv described above may be partially replaced with or further contain a site recognizable by a restriction enzyme, so that the ABM is inserted during gene cloning. For example, the linker sequences located on both sides of the ABM may be partially replaced with or further contain a TS sequence, which is a site recognizable by a restriction enzyme (SpeI). The residues in the TS sequence have the same benefits as those in the GS sequence of the linker.

[0091] The present disclosure provides a pharmaceutical composition for the prevention or treatment of cancer, comprising the above-mentioned protein. All details explained with respect to the above-mentioned protein strictly apply to the protein acting as an active ingredient in the pharmaceutical composition of the present application.

[0092] Examples of such cancers include brain cancer, head and neck cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, colorectal cancer, endometrial cancer, esophageal cancer, leukemia, lung cancer, liver cancer, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, kidney cancer, stomach cancer, testicular cancer, uterine cancer, vascular tumors, squamous cell carcinoma, adenocarcinoma, small cell carcinoma, melanoma, glioma, neuroblastoma, sarcoma, laryngeal cancer, parotid gland cancer, biliary tract cancer, thyroid cancer, actinic keratosis, acute lymphoblastic leukemia, acute myeloid leukemia, adenoid cystic carcinoma, adenoma, adenosquamous carcinoma, anal canal cancer, anal cancer, Anorectal cancer, astrocytoma, Bartholin's adenocarcinoma, basal cell carcinoma, biliary tract cancer, bone cancer, bone marrow cancer, bronchial cancer, bronchial adenocarcinoma, carcinoid, cholangiocarcinoma, chronic lymphoblastic leukemia, chronic myeloid leukemia, clear cell carcinoma, connective tissue carcinoma, cystadenoma, digestive system cancer, Duodenal cancer, endocrine system cancer, endodermal sinus tumor, endometrial hyperplasia, endometrioid adenocarcinoma, endothelial cell carcinoma, ependymal cell, epithelial cell carcinoma, orbital cancer, focal nodular hyperplasia, gallbladder cancer, pyloric cancer, gastric fundus cancer, gastrinoma, glioblastoma, glucagonoma, heart cancer, Hemangioblastoma, hemangioendothelioma, hemangioma, hepatic adenoma, hepatic adenomatosis, hepatobiliary cancer, hepatocellular carcinoma, Hodgkin's disease, ileal cancer, insulinoma, intraepithelial neoplasm, intraepithelial squamous neoplasm, intrahepatic biliary tract cancer, invasive squamous cell carcinoma, jejunal cancer, joint cancer, pelvic cancer, giant cell carcinoma, Colorectal cancer, lymphoma, malignant mesothelioma, medulloblastoma, medulloepithelioma, meningeal cancer, mesothelial cancer, metastatic cancer, oral cancer, mucoepidermoid cancer, multiple myeloma, muscle cancer, nasal cavity cancer, nervous system cancer, non-epithelial skin cancer, non-Hodgkin's lymphoma, oat cell carcinoma, oligodendroglioma cancer, oral cancer , osteosarcoma, papillary serous adenocarcinoma, penile cancer, pharyngeal cancer, pituitary tumor, plasmacytoma, pseudosarcoma, pulmonary blastoma, rectal cancer, renal cell carcinoma, respiratory system cancer, retinoblastoma, serous carcinoma, sinus cancer, skin cancer, small cell carcinoma, small intestine cancer, leiomyosarcoma, soft tissue cancer, somatostatin-secreting tumor, spinal cord cancer, squamous cell carcinoma, rhabdomyocarcinoma, submesothelial cell carcinoma, T-cell leukemia, tongue cancer, ureter cancer, urethral cancer, cervical cancer, corpus cancer, vaginal cancer, VIP-secreting tumor, vulvar cancer, well-differentiated carcinoma, and Wilms' tumor.

[0093] The pharmaceutical compositions of the present disclosure may include a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier or diluent that does not impair the biological activity and properties of the administered component and does not cause significant irritation in living organisms. The pharmaceutically acceptable carrier of the present disclosure may be saline, sterile water, Ringer's solution, buffered saline, glucose solution, maltodextrin solution, glycerol, ethanol, or a mixture of one or more of the above components. Additionally, the pharmaceutically acceptable carrier may be formulated as an injectable form suitable for injection into tissues or organs by adding other common additives, such as antioxidants, buffers, and bacteriostatic agents, as needed. Furthermore, the pharmaceutically acceptable carrier may be formulated as an isotonic sterile solution, or in some cases, as a dried preparation (particularly a lyophilized preparation) that can be made into an injectable solution by adding sterile water or normal saline. To specifically function in the target organ, an antibody or other ligand specific to the target organ may also be bound to the carrier used.

[0094] Additionally, the compositions of the present disclosure may further comprise fillers, excipients, disintegrants, binders, or lubricants. Furthermore, the compositions of the present disclosure may be formulated using methods known in the art to provide quick, sustained, or delayed release of the active ingredient after administration to a mammal.

[0095] In one embodiment, the pharmaceutical composition is an injectable formulation and may be administered intravenously, but without limitation.

[0096] As used herein, the term "effective amount" refers to the amount required to delay or completely prevent the onset or progression of the particular disease that is the target of treatment.

[0097] In the present disclosure, a pharmaceutically effective amount of the composition may be administered. It will be apparent to those skilled in the art that an appropriate total daily dosage of the pharmaceutical composition may be determined by a prescribing physician within the scope of sound medical judgment.

[0098] For purposes of this disclosure, a particular pharmaceutically effective amount for a particular patient will preferably vary depending on a variety of factors such as the type and degree of response to be achieved, the particular composition, including in some cases whether other preparations are used, the patient's age, weight, general health, sex, and diet, the time of administration, the route of administration, the excretion rate of the composition, the duration of treatment, and drugs used in conjunction with or concurrently with the particular composition, and similar factors well known in the medical arts.

[0099] In the present disclosure, pharmaceutical compositions may optionally be accompanied by instructions for use associated with the packaging in a form mandated by a government agency regulating the preparation, use, and sale of pharmaceuticals. In addition, the instructions may represent approval by a civil agency for the composition or form of administration to humans or animals, such as labeling approved by the U.S. FDA for prescribing drugs.

[0100] The present disclosure provides a method for treating cancer, comprising administering the above-mentioned protein. All details explained with respect to the above-mentioned protein apply strictly to the protein serving as the active ingredient in the method for treating cancer of the present application.

[0101] The methods of the disclosure include administering a protein to a subject with cancer.

[0102] The subject with cancer may be an animal with cancer, particularly a mammal with cancer, more particularly a human with cancer.

[0103] A therapeutically effective amount of the protein may be administered.

[0104] As used herein, the term "administration" refers to introducing a composition of the present disclosure into a patient by any suitable method. With regard to the route of administration, the composition of the present disclosure may be administered via various oral or parenteral routes, as long as the composition can reach the target tissue. The composition of the present disclosure may be administered intraperitoneally, intravenously, intramuscularly, subcutaneously, intradermally, orally, topically, intranasally, intrapulmonary, or rectally, without limitation.

[0105] In the following, the present disclosure will be explained in more detail with reference to examples. [Example]

[0106] Examples 1 to 9 and Comparative Examples 1 to 4 were designed by varying the scFvs, the type of ABD, and the location of the ABD, and then their structural models were identified. The amino acid sequence of the scFv protein consists of an N-terminal V linked by a linker. H Domain and C-terminal V L The nucleotide sequence "MG" was inserted at the N-terminus as a restriction site for the NcoI enzyme, and a hexahistidine tag (His tag) sequence was inserted at the C-terminus as a protein purification tag. The SpeI restriction site encoding the amino acid residues "TS" was used for ABD insertion.

[0107] [Table 1]

[0108] Figures 1-14 illustrate the structures of 4D5 scFv (SEQ ID NO: 4, Figure 1), Examples 1-9, and Comparative Examples 1-4, calculated using AlphaFold2. In the figures, the three bright regions within each heavy and light chain variable domain represent the CDRs. All examples confirmed that the protein structure folds normally without compromising the structure formed by the heavy and light chain variable domains fused within the linker. Figure 1 illustrates the structure of the original 4D5 scFv without the ABD, which was performed by AlphaFold2 using the ColabFold web server. In comparison, Examples 1-5 confirmed that the original structure of 4D5 Ab was substantially preserved, and that anti-HER2 binding affinity would be maintained.

[0109] In addition, 4D5-S-ABD and 4D5-L-ABD were analyzed by pTM score and pLDDT. These structures were computationally predicted using AlphaFold2 and analyzed by pTM score and pLDDT to identify the structural integrity of these 4D5-ABD variants in their unfamiliar forms (Figure 15, Table 3). The model structures of the 4D5-ABD variants were then aligned with those of the 4D5scFv and ABD (Figure 16, Table 4). The TM scores for all alignments were 0.94 or higher, which was significantly higher. In other words, the 4D5scFv and ABD structures were substantially retained in the constructed 4D5-ABD variants, meaning their functions were also maintained. Additionally, as expected, the predicted model structures indicated that the ABD insertion site was separated from the CDRs of the 4D5scFv. This fact is also observable in the visualization of the 4D5-ABD and HER2 complex (Figure 17), where the ABD is clearly separated from the HER2-binding site. Therefore, it can be predicted that the antigen-binding affinity of 4D5scFv will not be significantly hindered by the presence of the ABD or by albumin binding to the ABD. In conclusion, the high structural consistency and location independence of each component allow 4D5scFv and ABD to substantially retain their functions.

[0110] [Table 2]

[0111] [Table 3]

[0112] Experimental example

[0113] 1. Protein Expression and Purification

[0114] 1.1. Plasmids

[0115] Among the engineered proteins constructed according to the above design, those in Examples 1 to 5 were subjected to cytoplasmic expression in a bacterial host to produce the proteins. Specifically, genes encoding the above amino acid sequences were optimized using ExpOptimizer (NovoPro Bioscience, Shanghai, China), synthesized by Macrogen (Seoul, Korea), subcloned, and inserted into the NcoI / KpnI restriction sites of the pBAD vector to produce the pBAD_4D5scFv plasmid.

[0116] Plasmids from Examples 1-5 and 4D5scFv were transformed into E. coli TOP10 host cells. Transformed cells were incubated at 37°C for 16 hours in standard 2xYT medium containing 100 μg / mL ampicillin with shaking at 200 rpm. These cells were inoculated into the same fresh medium and incubated until their optical density at 600 nm (OD600) reached 0.5-0.6. Protein expression was induced with L-(+)-arabinose at a final concentration of 0.2% (w / v). After cooling to 23°C, the culture was incubated for 24 hours, and the grown cells were collected by centrifugation at 8000 rpm at 4°C. The cell pellet was then stored at -80°C. Samples for each variant before induction (BI) and after induction (AI) were collected, centrifuged at 13,000 rpm for 1 minute, and resuspended in PBS (pH 7.4) containing 2 M urea, whereby SDS-PAGE analysis was performed.

[0117] The His-tagged examples described above were purified by metal affinity chromatography using Ni-NTA. Cell pellets were lysed with 1 mg / mL lysozyme and 5 mg / mL DNase in lysis buffer (10 mM imidazole, 50 mM NaH2PO4, 300 mM NaCl, pH 8.0) and incubated on ice for at least 5 minutes. The resulting solution was sonicated at 28% amplitude (500 W, 20 kHz) with a 1-second pulse and a 2-second pause for a total of 15 minutes. After a 5-minute pause, this process was repeated. The resulting mixture was then centrifuged at 10,000 x g for 20 minutes at 4°C, and the supernatant was incubated with Ni-NTA agarose resin for 30 minutes at 4°C. The incubated resin was packed into a polypropylene column equipped with a filter, washed with wash buffer (20 mM imidazole, 50 mM NaH2PO4, 300 mM NaCl, pH 8.0), and eluted with elution buffer (250 mM imidazole, 50 mM NaH2PO4, 300 mM NaCl, pH 8.0). The buffer was exchanged with PBS (pH 7.4) using a PD-10 desalting column according to the manufacturer's protocol. The purified protein was stored at 4 °C and analyzed by SDS-PAGE.

[0118] 1.2.SDS-PAGE analysis

[0119] The prepared proteins, including the resuspended BI and AI samples and purified proteins, were analyzed using SDS-PAGE. For reduction, all samples were mixed with 2x sample loading dye (0.2% bromophenol blue, 4% SDS, 20% glycerol, 100 mM Tris-HCl, pH 6.8) containing 100 mM dithiothreitol (DTT). Non-reduced samples were prepared by omitting DTT. The samples were boiled at 100°C for 10 min and then loaded onto a 12% SDS-PAGE gel. After electrophoresis, the gel was stained with Coomassie Brilliant Blue (0.25% Coomassie Blue, 50% ethanol, 10% acetic acid) and destained (50% ethanol, 10% acetic acid). The resulting gel was imaged and visualized using a ChemiDoc XRS+ system (Bio-Rad, Hercules, CA, USA).

[0120] Figure 18a shows the results of SDS-PAGE analysis of cell lysates collected during expression. Bands were detected in the lanes loaded with AI samples having molecular weights of 27, 32, and 33 kDa, corresponding to the molecular weights of 4D5scFv, 4D5-S-ABD, and 4D5-L-ABD, respectively. Purification was performed using the interaction between Ni-NTA in the agarose resin and the His-tag affinity tag at the C-terminus of each protein. SDS-PAGE analysis of the purified proteins revealed bands at the same positions as in the expression gel (Figure 18b).

[0121] In addition, as confirmed by Examples 1 and 2, the modified protein showed only a monomeric protein band under both reducing and non-reducing conditions (Figure 19), indicating that no substantial intermolecular disulfide bonds were formed.

[0122] 1.3.MALDI-TOF analysis

[0123] MALDI-TOF analysis was performed. Each purified sample was desalted using a Zeba spin desalting column (7K MWCO) in PBS (pH 7.4) and prepared with 0.1% TFA solution. The sample was mixed with SA matrix saturated in TA30 (30% acetonitrile, 0.1% TFA) solution at a 1:1 (v / v) ratio. SA matrix saturated in ethanol solution was pre-deposited onto a target steel plate. The mixed sample solution was then deposited onto a layer, and molecular weight analysis was performed using an Autoflex Speed ​​(Bruker, Billerica, MA, USA). Protein Standard II was used as a calibration standard. These three proteins were additionally identified by MALDI-TOF analysis (Figure 20).

[0124] In Figure 20, peaks at 26,645, 32,013, and 32,816 m / z were detected in the mass spectra of intact 4D5scFv, 4D5-S-ABD, and 4D5-L-ABD. These results aligned well with the expected values ​​for the 4D5scFv variants at 26,801, 32,155, and 32,759 m / z, respectively, with a deviation of less than 1%. The production yields of purified 4D5scFv, 4D5-L-ABD, and 4D5-S-ABD were 22.0 ± 0.5, 18.8 ± 1.3, and 21.5 ± 0.8 mg / L, respectively. Overall, these results indicate that the 4D5scFv variant samples were successfully expressed and purified.

[0125] 1.4. Size Exclusion Chromatography (SEC) Analysis

[0126] Additionally, the purified samples of Examples 1 and 2 were characterized by fast protein liquid chromatography (SEC) (size exclusion chromatography). Equimolar (5 μM) samples prepared in PBS (pH 7.4) were injected onto a Superdex75 Increase 10 / 300GL column (Cytiva, Uppsala, Sweden) and eluted at a flow rate of 0.2 mL / min. Ovalbumin, conalbumin, and aldolase from the Gel Filtration Calibration Kit HMW (Cytiva, Uppsala, Sweden) were used as reference proteins. Analysis was performed using a chromatography system, NGC Quest 10 (Bio-Rad, Hercules, CA, USA).

[0127] The SEC analysis results in Figure 21 showed that the 4D5scFv was stable in a mixture of monomers and dimers, and no disulfide bonds were formed by non-reducing SDS-PAGE. In contrast, the 4D5-ABD variant did not form a dimer but existed only as a monomer, which may be due to steric hindrance created by the ABD fusion.

[0128] 2. Binding affinity analysis

[0129] (1) Binding affinity analysis for HER2

[0130] The anti-HER2 targeting efficiency of the purified 4D5scFv variants was analyzed by anti-HER2 ELISA in the presence or absence of HSA. Specifically, immunoplates were coated with recombinant HER2 antigen (500 pg / μL, 100 μL / well) incubated overnight at 4°C in coating buffer (PBS, pH 7.4). The plates were shaken and washed three times with 200 μL / well of PBS containing 0.05% Tween® 20 (PBST). The plates were then blocked by adding 200 μL / well of blocking buffer (5% skim milk in PBST) for room temperature incubation, followed by four washes. The purified proteins were incubated at room temperature in PBS (pH 7.4) with or without HSA at a 1:2 molar ratio. The incubated proteins were then prepared in blocking buffer to create a 50 nM stock, which was then subjected to three-fold serial dilutions. The prepared samples (100 μL / well, n=2) were loaded onto the plate and incubated at room temperature. Unbound proteins were then washed four times. The plate was incubated with 100 μL / well of rabbit anti-His tagged antibody diluted 1:1500 in blocking buffer. After incubation, each well was washed four times. Finally, 100 μL / well of HRP-conjugated anti-rabbit IgG antibody diluted 1:3000 in blocking buffer was incubated together. Each well was washed four times. Detection was performed by adding 100 μL / well of TMB, and the reaction was quenched with 2 M HCl. The absorbance of the bound protein was measured at 450 nm for quantification.

[0131] The experimental results are shown in Figure 22 and Table 4 below. All proteins showed similar concentration-dependence curves, indicating that the ABD insertion did not significantly distort the 4D5scFv structure. In conclusion, this suggests that the anti-HER2 binding affinity of the 4D5-ABD variants is maintained.

[0132] Specifically, to compare the binding ability between albumin and ABD, we compared an experimental group in which the antibody and HSA were preincubated (HSA+) and an experimental group in which the antibody and HSA were not preincubated (HSA-). The HER2 binding of 4D5scFv without ABD did not change significantly, indicating that the anti-HER2 binding affinity of 4D5scFv is not hindered or blocked by the presence of HSA. When HSA was bound to the 4D5-ABD variant, the anti-HER2 binding affinity was slightly reduced. This may be due to weaker steric hindrance to HER2 when bulky HSA binds to 4D5scFv. Both in the presence and absence of HSA, 4D5-L-ABD and 4D5-S-ABD, which have an ABD located in the center of the linker, showed relatively strong binding ability, and among them, 4D5-L-ABD, which has the longest linker length, showed the strongest binding ability. LinkC and TermN, which are expected to have low binding affinity due to the ABD being positioned close to the CDR based on the structure predicted by AlphaFold2, surprisingly showed higher binding affinity than the LinkN variant, in which the ABD is located away from the CDR. This is presumably because the linker residues are minimally inserted at the C-terminus of the ABD in LinkC and TermC, whereas LinkN does not contain additional linker residues at the N-terminus. Such differences arise during the cloning process to create each variant. This means that fewer or no linkers are required between the ABD and the antibody when steric hindrance occurs, and a linker is minimally required between the antibody and the ABD for binding capacity. Therefore, 4D5-S-ABD and 4D5-L-ABD, with the ABD inserted in the center of the linker region, have the highest binding capacity because the antibody and ABD are least affected by steric hindrance.

[0133] [Table 4]

[0134] (2) HSA binding affinity analysis

[0135] The anti-HSA efficacy of the purified 4D5-ABD variants was also investigated by ELISA. Specifically, plates were coated with HSA (500 pg / μL) in coating buffer. The purified proteins were prepared at 20 nM in blocking buffer, and then subjected to 3-fold serial dilutions. All other conditions and procedures were carried out as described above.

[0136] To investigate the binding affinity of 4D5scFv variants in the presence of HSA, all variants were pre-incubated with HSA at a molar ratio of 1:2, followed by ELISA. The results of anti-HSA binding affinity are shown in Figure 23 and Table 5 below.

[0137] 4D5-L-ABD and 4D5-S-ABD were shown to have the strongest binding affinity. The binding affinity of ABD tended to decrease slightly, and therefore appeared to be less affected by linker length than that of antibody, but the tendency was the same as that of antibody.

[0138] [Table 5]

[0139] 2.9. Pharmacokinetic Analysis

[0140] For pharmacokinetic analysis, the serum half-life of the 4D5scFv variants was investigated using mice. Experiments were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee of the Gwangju Institute of Science and Technology (GIST). Specifically, purified 4D5scFv and 4D5-ABD variants (100 μg / mL) in 200 μL of PBS (pH 7.4) were injected into the tail vein of 9-week-old female BALB / c mice (n=4). Blood samples (<100 μL) were collected by retroorbital bleeding at 3 minutes, 1, 2, 4, 8, 24, and 48 hours post-injection. Serum was separated from blood by centrifugation at 10,000 × g for 10 minutes at 4°C and then stored at -20°C until further use. Serum protein concentrations at each time point were measured by ELISA as described above. Concentrations were calculated by interpolating a standard calibration curve.

[0141] The serum half-lives of 4D5scFv and 4D5-ABD variants were measured after intravenous injection into BALB / c mice (n=4). ELISA was performed to quantify the protein remaining in the serum (Figure 23). While 4D5scFv was rapidly cleared from the blood with a serum half-life of 18 minutes, 4D5-S-ABD and 4D5-L-ABD exhibited a significantly extended half-life of 34 hours, which is 114-fold greater than that of 4D5scFv. In addition, the AUC values ​​were increased approximately 70-fold for both ABD fusion variants, calculated to be 42, 2887, and 3008 for 4D5scFv, 4D5-S-ABD, and 4D5-L-ABD, respectively. Considering that the 4D5-ABD variant did not reach the detection limit, the actual AUC increase is expected to be much higher.

[0142] [Table 6]

[0143] In the above, 1 0-2h; 2 0~48h.

[0144] The significant increase in half-life of the 4D5-ABD variant can be attributed to the FcRn-mediated recycling of the 4D5-ABD molecule bound to albumin. This result also indicates that the internally inserted ABD allows the binding affinity to serum albumin to be maintained, which is consistent with the structure calculations and ELISA results. In addition, no differences were observed between the 4D5-ABD variants, which also corresponds to the ELISA results. This also suggests that the linker length of 15 amino acid residues in the 4D5-S-ABD variant is sufficient to allow the ABD moiety to move freely to bind to albumin.

[0145] [Item 1] A single-chain variable fragment engineered protein in which an albumin-binding moiety is fused to a single-chain variable fragment (scFv) in which the heavy chain variable domain (VH) and light chain variable domain (VL) of an antibody are linked by a linker. [Item 2] 2. The single chain variable fragment engineered protein of item 1, wherein the albumin binding moiety is fused within the linker or at the N-terminus of the scFv or at the C-terminus of the scFv. [Item 3] the albumin binding moiety is fused within the linker; and 2. The single-chain variable fragment engineered protein of item 1, wherein a biologically active agent is fused at the C-terminus of the scFv. [Item 4] 2. The single-chain variable fragment engineered protein of claim 1, wherein the albumin binding moiety is fused at least 3 aa from the C-terminus of the heavy chain variable domain (VH) and the N-terminus of the light chain variable domain (VL). [Item 5] 2. The single chain variable fragment engineered protein of claim 1, wherein the albumin binding moiety is positioned in the opposite direction from the complementarity determining regions (CDRs) of the heavy chain variable domain (VH) and the light chain variable domain (VL). [Item 6] 2. The single-chain variable fragment engineered protein according to item 1, wherein the albumin binding portion is 10 to 150 aa in length. [Item 7] 2. The single-chain variable fragment engineered protein according to Item 1, wherein the linker has a length of 6 to 50 aa. [Item 8] 2. The single-chain variable fragment engineered protein according to Item 1, wherein the length of the scFv is 150 to 350 aa. [Item 9] 2. The single-chain variable fragment engineered protein according to Item 1, wherein the heavy chain variable domain (VH) and the light chain variable domain (VL) each have a length of 70 to 150 aa. [Item 10] 2. The single-chain variable fragment engineered protein according to Item 1, wherein the albumin binding portion comprises an amino acid sequence selected from the sequences of SEQ ID NOs: 1 to 3. [Item 11] 2. The single-chain variable fragment engineered protein according to Item 1, wherein the scFv comprises an amino acid sequence selected from the sequences of SEQ ID NOs: 4 to 6. [Item 12] 2. The single chain variable fragment engineered protein of item 1, wherein the linker comprises the amino acid sequence (GS)n, (GGS)n, or (GGGGS)n. [Item 13] 4. The single-chain variable fragment engineered protein of item 3, wherein the bioactive agent is selected from the group consisting of cytokines, insulin, insulin-like growth factor (IGF)-1, IGF-2, epidermal growth factor (EGF), transforming growth factor (TGF), human growth hormone, and vascular endothelial growth factor (VEGF). [Item 14] 14. A pharmaceutical composition for treating or preventing cancer, comprising the single-chain variable fragment engineered protein according to any one of items 1 to 13.

Claims

1. A single-chain variable fragment engineered protein in the form of a single-chain variable fragment (scFv) in which an antibody heavy chain variable domain (VH) and light chain variable domain (VL) are linked by a linker, and an albumin-binding moiety is fused to the scFv.

2. 2. The single chain variable fragment engineered protein of claim 1, wherein the albumin binding moiety is fused within the linker or at the N-terminus of the scFv or at the C-terminus of the scFv.

3. the albumin binding moiety is fused within the linker; and 2. The single-chain variable fragment engineered protein of claim 1, wherein a biologically active agent is fused at the C-terminus of the scFv.

4. 2. The single chain variable fragment engineered protein of claim 1, wherein the albumin binding moiety is fused at least 3 aa from the C-terminus of the heavy chain variable domain (VH) and the N-terminus of the light chain variable domain (VL).

5. 2. The single chain variable fragment engineered protein of claim 1, wherein said albumin binding moiety is positioned in the opposite direction from the complementarity determining regions (CDRs) of said heavy chain variable domain (VH) and said light chain variable domain (VL).

6. 2. The single-chain variable fragment engineered protein of claim 1, wherein the albumin binding portion is 10-150 aa in length.

7. 2. The single-chain variable fragment engineered protein of claim 1, wherein the linker is 6 to 50 aa in length.

8. 2. The single-chain variable fragment engineered protein of claim 1, wherein the length of the scFv is 150-350 aa.

9. 2. The single-chain variable fragment engineered protein of claim 1, wherein the length of said heavy chain variable domain (VH) and said light chain variable domain (VL) is 70 to 150 aa each.

10. 2. The single chain variable fragment engineered protein of claim 1, wherein said albumin binding moiety comprises an amino acid sequence selected from the sequences of SEQ ID NOs: 1-3.

11. 2. The single chain variable fragment engineered protein of claim 1, wherein the scFv comprises an amino acid sequence selected from the sequences of SEQ ID NOs: 4-6.

12. 2. The single chain variable fragment engineered protein of claim 1, wherein the linker comprises the amino acid sequence (GS), (GGS), or (GGGGS).

13. 4. The single-chain variable fragment engineered protein of claim 3, wherein the bioactive agent is selected from the group consisting of cytokines, insulin, insulin-like growth factor (IGF)-1, IGF-2, epidermal growth factor (EGF), transforming growth factor (TGF), human growth hormone, and vascular endothelial growth factor (VEGF).

14. A pharmaceutical composition for the treatment or prevention of cancer, comprising the single-chain variable fragment engineered protein of any one of claims 1 to 13.

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