Modified fusion proteins and uses thereof

Abstract

The present disclosure relates to a modified fusion protein in which a vascular endothelial growth factor is associated with a placental growth factor, and uses thereof. The modified fusion protein according to an embodiment of the present disclosure can exhibit excellent productivity and stability. The modified fusion proteins according to embodiments of the present disclosure may also exhibit excellent blood stability. The modified fusion proteins according to embodiments of the present disclosure have acidic properties compared to conventionally developed materials and can exhibit an increased proportion of charge isoforms in the acidic and neutral regions of the pI distribution.

Description

Technical Field

[0001] The present disclosure relates to modified fusion proteins that bind to vascular endothelial growth factor and placental growth factor and uses thereof. Background Art

[0002] Vascular endothelial growth factor (VEGF) is a signal transduction protein that plays an important role in angiogenesis and vasculogenesis. Its functions include the formation of new blood vessels during embryonic development, the formation of new blood vessels in muscles after injury or exercise, and the formation of new blood vessels that bypass blocked blood vessels, but the abnormal increase of VEGF is closely related to tumor development and metastasis (Carmeliet, P., Jain, RK. 2000. Nature 407: 249-257).

[0003] VEGF is classified into five types: VEGF-A, VEGF-B, VEGF-C, VEGF-D and PLGF (placental growth factor), which are known to contribute to angiogenesis (Carmeliet, P., Jain, RK. 2000. Nature 407: 249-257; Kuwano M et al. 2001. Intern Med 40: 565-572). On the other hand, there are three types of VEGF receptors: VEGFR1, R2 and R3. The type and binding affinity of the ligand vary depending on the type of receptor. Among these receptors, VEGFR-1 / -2 is known to be involved in angiogenesis (Veikkola et al., 2000. Cancer research 60: 203-212).

[0004] People have been working on developing drugs that inhibit angiogenesis by removing vascular endothelial growth factor, which is known to be a key factor in tumorigenesis. Bevacizumab (a drug in the form of a monoclonal antibody that targets VEGF-A) (Stacker et al., 2013. Chinese Journal of Cancer Research. 32(6): 297-302.; Kazazi-Hyseni et al., 2010. Oncologist. 15(8): 819-825; DrugBank accession number: DB00112) is a representative. The mechanism of action of monoclonal antibody drugs involves binding to a single vascular endothelial growth factor, but because multiple growth factors are involved in carcinogenesis, monoclonal antibodies are not effective drugs that exert significant therapeutic effects. Bevacizumab actually induces the increased expression of other types of non-targeted growth factors through intracellular compensatory mechanisms, thereby producing drug resistance side effects (Bagley et al., 2011. Clinical Cancer Research 17(5):976-988; Lieu et al., 2013. PLoS One 8:e77117; Cutsem et al., 2020 Clinical Cancer Research 26(3):717-725). For this reason, the demand for developing drugs targeting various vascular growth factors related to carcinogenesis is gradually increasing. In particular, PLGF is increasingly important because its expression rate increases due to compensatory pathways in the later stages of carcinogenesis, but most of the drugs that have been developed or are currently being developed target VEGF. Therefore, the demand for developing drugs targeting PLGF is growing.

[0005] In order to overcome the shortcomings of monoclonal antibody drugs, drugs targeting multiple vascular endothelial growth factors are being developed. Representative protein drugs are VEGF-Trap (Ciombor et al., 2013. Clinical Cancer Research 19 (8): 1920-1925. DrugBank Accession No.: DB08885) and VEGF-Grab (Lee et al., 2015. Molecular Cancer Therapy (Mol. Cancer. Ther.) 14 (2): 470-9). Both drugs simulate part of the decoy receptor and are recombinant fusion proteins, in which the Fc of the immunoglobulin is fused with the heterologous protein VGFR1 D2-VGFR2 D3 in VEGF-Trap, and with the homologous protein VEGFR1 D2-D3 in VEGF-Grab. People have been working hard to improve the physicochemical properties of such fusion proteins targeting multiple vascular endothelial growth factors. In particular, there is a need to develop fusion proteins that have high binding affinity for target substances and increased persistence in vivo, while also possessing physical and chemical stability to enable high productivity at the level of protein therapeutics in the context of commercial-scale production. Summary of the invention

[0006] Improved physicochemical properties of proteins can maximize production efficiency by enhancing the physical and chemical stability of proteins during expression and purification, and reduce nonspecific binding and degradation when the proteins are introduced into the body, thereby achieving high persistence in vivo.

[0007] The inventors of the present disclosure have been continuously working to effectively improve the physicochemical properties and stability of fusion proteins containing the VEGFR1 extracellular domain.

[0008] Therefore, the inventors of the present disclosure developed a modified fusion protein by introducing amino acid variation in a specific region of domain D3, length adjustment and variation of the linker, or disulfide bond into a fusion protein containing domain D2 and domain D3 of VEGFR1. The present disclosure was completed after determining that the modified fusion protein exhibited excellent productivity and stability.

[0009] 1. In one embodiment, the present disclosure relates to a modified fusion protein, the modified fusion protein comprising a VEGFR1 extracellular domain, a linker and a multimerization domain, wherein the VEGFR1 extracellular domain comprises an immunoglobulin (Ig)-like domain D2 and an Ig-like domain D3 of VEGFR1, and the linker is located between the Ig-like domain D3 and the multimerization domain, and wherein the modified fusion protein has one or more of the properties (a) to (c): (a) one or more amino acid residues on the β1-β2 loop of domain D3, one or more amino acid residues on the β2-β3 loop of domain D3, and / or one or more amino acid residues on the β5-β6 loop of domain D3 are substituted with amino acids that reduce the net pI of the protein, amino acids with negatively charged side chains, or amino acids with electronegative side chains, wherein the β1-β2 loop of domain D3 comprises SEQ ID NO:1, the β2-β3 loop includes amino acid residues T236 to T247 of the amino acid sequence shown in SEQ ID NO:1, the β2-β3 loop includes amino acid residues T256 to V262 of the amino acid sequence shown in SEQ ID NO:1, and the β5-β6 loop includes amino acid residues D299 to L308 of the amino acid sequence shown in SEQ ID NO:1; (b) the length of the linker is about 13 to about 35 amino acids; and (c) a disulfide bond is present in the fusion protein, and one of the amino acid residues on the β1-β2 loop of domain D3 or one of the amino acid residues on the β5-β6 loop of domain D3 and one of the amino acid residues at positions -2, -1, 0, +1, +2 and +3 relative to Y329 of domain D3 is substituted with cysteine.

[0010] 2. In one embodiment, the amino acid substitution in property (a) may be a substitution with: an amino acid residue of a VEGFR2 homologous sequence; an amino acid residue of serine, threonine, tyrosine, cysteine, asparagine, glutamine, aspartic acid or glutamic acid; or an amino acid residue of alanine or glycine having a short side chain.

[0011] 3. In one embodiment, in characteristic (a), one or more amino acid residues on the β1-β2 loop of domain D3 and one or more amino acid residues on the β5-β6 loop of domain D3 may be substituted with the following amino acids: amino acids that reduce the net pI of the protein, amino acids with negatively charged side chains, or amino acids with electronegative side chains.

[0012] 4. In one embodiment, in characteristic (a), one or more amino acid residues on the β2-β3 loop of the domain D3 may be replaced by an amino acid residue having a side chain smaller than the side chain of the existing amino acid residue or an amino acid residue having a polar side chain similar in size to the side chain of the existing amino acid residue.

[0013] 5. In one embodiment, characteristic (a) can be a substitution of: one or more of the amino acid residues K241, L243, R244 and H246 on the β1-β2 loop of the domain D3; the amino acid residue L258 on the β2-β3 loop of the domain D3; and / or one or more of the amino acid residues K300, Q302, K304 and K306 on the β5-β6 loop of the domain D3.

[0014] 6. In one embodiment, characteristic (a) may include: one or more amino acid substitutions selected from K241T, K241E, L243S, R244V and H246E on the β1-β2 loop of the domain D3; amino acid substitutions L258A, L258S or L258D on the β2-β3 loop of the domain D3; and / or one or more amino acid substitutions selected from K300G, Q302T, K304S and K306Q on the β5-β6 loop of the domain D3.

[0015] 7. In one embodiment, characteristic (a) may include: (i) amino acid substitutions L243S and R244V on the β1-β2 loop of the domain D3, or (ii) amino acid substitutions K241E, L243S, R244V and H246E on the β1-β2 loop of the domain D3; amino acid substitutions L258A, L258S or L258D on the β2-β3 loop of the domain D3; and / or (i) amino acid substitution K300G on the β5-β6 loop of the domain D3, (ii) amino acid substitutions K300G, Q302T and K304S on the β5-β6 loop of the domain D3, or (iii) amino acid substitutions K300G, Q302T, K304S and K306Q on the β5-β6 loop of the domain D3.

[0016] 8. In one embodiment, characteristic (a) may include: amino acid substitutions K241E, L243S, R244V and H246E on the β1-β2 loop of the domain D3; amino acid substitutions L258A, L258S or L258D on the β2-β3 loop of the domain D3; and / or amino acid substitutions K300G, Q302T and K304S on the β5-β6 loop of the domain D3.

[0017] 9. In one embodiment, the modified fusion protein may comprise amino acid substitutions R238S and R275N in domain D3.

[0018] 10. In one embodiment, in the modified fusion protein, the amino acid sequence of the C-terminus of domain D3 connected to the linker is KALE based on amino acid residue K331, provided that one or more of the amino acids K, A, L and E may be deleted.

[0019] 11. In one embodiment, in the modified fusion protein, the N-terminus of the domain D2 may start with the amino acid sequence SDT or the amino acid sequence EF.

[0020] 12. In one embodiment, in feature (b), the linker may be about 14 amino acids or more in length.

[0021] 13. In one embodiment, in characteristic (b), the linker comprises a GS repeat sequence, and the GS repeat sequence is an amino acid sequence of 2 to 60 amino acids long consisting only of G and S, and the N-terminal amino acid of the linker may be glutamic acid.

[0022] 14. In one embodiment, the GS repeat sequence is (GS) n , (GSSG) n 、(GGGGS) n or (GS) m (GGGGS) n , wherein n is an integer from 1 to 10, and m is an integer from 0 to 10.

[0023] 15. In one embodiment, the GS repeat sequence can be selected from the group consisting of: GS, GSSG, (GSSG)2, GGGGS, (GGGGS)4, GS(GGGGS) and GS(GGGGS)3.

[0024] 16. In one embodiment, in characteristic (b), the linker comprises an amino acid sequence derived from a hinge region of an immunoglobulin, and the amino acid sequence of the hinge region may be modified.

[0025] 17. In one embodiment, the hinge region derived from an immunoglobulin may include sequences derived from a CH1 region, an upper hinge and / or a core hinge.

[0026] 18. In one embodiment, the hinge region derived from immunoglobulin may be derived from human IgA, IgD, IgM, IgE or IgG.

[0027] 19. In one embodiment, in property (b), the papain recognition site or glycosylation site present in the hinge region derived from immunoglobulin may have an amino acid variation.

[0028] 20. In one embodiment, in characteristic (b), the amino acid variation in the papain recognition site may be substitution of one or more amino acid residues in the papain recognition site with alanine, serine, tyrosine, proline or threonine, or insertion of an amino acid sequence of 1 to 10 amino acids long comprising at least one amino acid having an aromatic or cyclic carbon in the side chain into the papain recognition site, and the amino acid variation in the glycosylation site may be (i) deletion of a serine or threonine present in the hinge region derived from the immunoglobulin, or (ii) substitution of a serine or threonine present in the hinge region derived from the immunoglobulin with an amino acid other than serine, asparagine or threonine.

[0029] 21. In one embodiment, the amino acid other than serine, asparagine or threonine may be glycine or alanine.

[0030] 22. In one embodiment, the cysteine ​​present in the core hinge may be substituted by serine or glycine.

[0031] 23. In one embodiment, in characteristic (b), the linker may include: (i) an amino acid sequence selected from the group consisting of GS, GSSG, (GSSG)2, GGGGS, (GGGGS)4, GS(GGGGS) and GS(GGGGS)3; and (ii) a hinge region derived from an immunoglobulin selected from the group consisting of human IgG1, IgG4 or IgD / G1, wherein the amino acid sequence of the hinge region may be modified.

[0032] 24. In one embodiment, in characteristic (b), the linker can be an amino acid sequence selected from the group consisting of GSSGDKTHTSPPSP, GSSGEPKSSDKTYTSPPSP, GSKVDKKVEPKSSDKTHTCPPCP, GSKVDKKVEPKSSDKTYTCPPCP, GSKVDKKVEPKSSDTPPTCPPCP, GSGGGGSGGGGSGGGGSAESKYGPPCPPCP, GSNTGSGGEEKKKEKEKEEQEERSSDKTYTCPPCP, GSNTGSGGEEKKKEKEKEEQEERSSDTPPTCPPCP, EGSSGGSSGEPKSDATPTCPPCP, EGSSGGSSGEPKSDSTYTCPPCP, GCKVD KKVEPKSSDKTYTCPPCP, CSKVDKKVEPKSSDKTYTCPPCP, GGGGSAEPKAGDKAPPGPPGP, GGGGSAEPKSSDKTYTCPPCP, GGGGSGGGGSGGGGSGGGGSAEPKSSDKTYTCPPCP, CSSGDATPTSPPSP, CSKVDKKVEPKSSD TPPTCPPCP, CSGGGGSAEPKAGDATPPTCPPCP, CSGGGGSGGGGSGGGGSAESKYGPPCPPCP, CSNTGSGGEEKKKEKEEQEERSSDTPPTCPPCP, CGSSGGSSGEPKSDATPTCPPCP, and CSGGGGSAEPKAGDATPPTCPPCPPCP.

[0033] 25. In one embodiment, in characteristic (c), the one of the amino acid residues on the β1-β2 loop may be one of the amino acid residues on the amino acid sequence from V240 to T247.

[0034] 26. In one embodiment, in characteristic (c), one of the amino acid residues T241, L243 and R244 on the β1-β2 loop of the domain D3 or the amino acid residue N303 on the β5-β6 loop can be substituted by cysteine, and one of the amino acid residues at positions 0, +2 and +3 relative to Y329 of the domain D3 can be substituted by cysteine.

[0035] 27. In one embodiment, in feature (c), T241 and the amino acid residue at position +3 relative to domain D3 of Y329 are substituted by cysteine; the amino acid variants L243C and Y329C are introduced; L243 and the amino acid residue at position +2 relative to domain D3 of Y329 are substituted by cysteine; the amino acid variants R244C and Y329C are introduced; or the amino acid variants N303C and Y329C are introduced.

[0036] 28. In one embodiment, the modified fusion protein may have all of the following properties (a) to (c): (a) introducing: one or more amino acid substitutions selected from K241T, K241E, L243S, R244V and H246E on the β1-β2 loop of the domain D3, amino acid substitutions L258A, L258S or L258D on the β2-β3 loop of the domain D3 and / or one or more amino acid substitutions selected from K300G, Q302T, K304S and K306Q on the β5-β6 loop of the domain D3; (b) the linker is an amino acid sequence selected from the group consisting of: GSSGD KTHTSPPSP,GSSGEPKSSDKTYTSPPSP,GSKVDKKVEPKSSDKTHTCPPCP,GSKVDKKVEPKSSDKTYTCPPCP,GSKVDKKVEPKSSDTPPTCPPCP,GSGGGGSGGGGSG E GSSGGSSGEPKSDSTYTCPPCP, GCKVDKKVEPKSSDKTYTCPPCP, CSKVDKKVEPKSSDKTYTCPPCP, GGGGSAEPKAGDKAPPGPPGP, GGGGSAEPKSSDKTYTCPPCP, GGGGSGGGGSGGGGSGGGGSAEPKSSDKTYTCPPCP, CSSGDATPTSPPSP, CSKVDKKVEPKSSDTPPTCPPCP, CSGGGGSAEPKAGDATPPTCPPCP, CSGGGGSGGGGSGG GGSAESKYGPPCPPCP, CSNTGSGGEEKKKEKEKEEQEERSSDTPPTCPPCP, CGSSGGSSGEPKSDATPTCPPCP and CSGGGGSAEPKAGDATPPTCPPCPPCP; and (c) one of the amino acid residues T241, L243 and R244 on the β1-β2 loop of said domain D3 or the amino acid residue N303 on the β5-β6 loop is substituted with cysteine, and one of the amino acid residues at positions 0, +2 and +3 relative to Y329 of said domain D3 is substituted with cysteine.

[0037] 29. In one embodiment, the modified fusion protein may have all of the following properties (a) to (c): (a) introducing: (i) amino acid substitutions L243S and R244V on the β1-β2 loop of the domain D3, or (ii) amino acid substitutions K241E, L243S, R244V and H246E on the β1-β2 loop of the domain D3, amino acid substitutions L258A, L258S or L258D on the β2-β3 loop of the domain D3, and / or (i) amino acid substitution K300G on the β5-β6 loop of the domain D3, (ii) amino acid substitutions K300G, Q302T and K304S on the β5-β6 loop of the domain D3, or (iii) amino acid substitutions K300G, Q302T, K304S and K306Q; (b) the linker is an amino acid sequence selected from the group consisting of CSSGDATPTSPPSP, CSKVDKKVEPKSSDTPPTCPPCP, CSGGGGSAEPKAGDATPPTCPPCP, CSGGGGSGGGGSGGGGSAESKYGPPCPPCP, CSNTGSGGEEKKKEKEKEEQEERSSDTPPTCPPCP, CGSSGGSSGEPKSDATPTCPPCP, CSKVDKKVEPKSSDKTYTCPPCP and CSGGGGSAEPKAGDATPPTCPPCPPCP; and (c) L243 and the amino acid residue at position +2 relative to Y329 of domain D3 are substituted by cysteine.

[0038] 30. In one embodiment, the modified fusion protein can have all of the following properties (a) to (c): (a) introducing: amino acid substitutions K241E, L243S, R244V and H246E on the β1-β2 loop of the domain D3, amino acid substitutions L258A, L258S or L258D on the β2-β3 loop of the domain D3, and amino acid substitutions K300G, Q302T and K304S on the β5-β6 loop of the domain D3; (b) the linker is an amino acid sequence selected from the group consisting of: CSSGDATPTSPPSP, CSKVDKKVEPKS SDTPPTCPPCP, CSGGGGSAEPKAGDATPPTCPPCP, CSGGGGSGGGGSGGGGSAESKYGPPCPPCP, CSNTGSGGEEKKKEKEKEEQEERSSDTPPTCPPCP, CGSSGGSSGEPKSDATPTCPPCP, CSKVDKKVEPKSSDKTYTCPPCP and CSGGGGSAEPKAGDATPPTCPPCPPCP; and (c) L243 and the amino acid residue at position +2 relative to Y329 of domain D3 are substituted by cysteine.

[0039] 31. In one embodiment, the modified fusion protein may consist of an amino acid sequence selected from the group consisting of: SEQ ID NO: 10 to SEQ ID NO: 70, SEQ ID NO: 77 to SEQ ID NO: 93 and SEQ ID NO: 98.

[0040] 32. In one embodiment, the multimerization domain can be: (a) the Fc region of an immunoglobulin; (b) the CH3 region of IgG1, IgG2, IgG3 or IgG4; (c) the CH2 region and CH3 region of IgG1, IgG2, IgG3 or IgG4; (d) the Fc region of an immunoglobulin comprising an amino acid sequence that is at least 85% identical to SEQ ID NO:99; or (e) the Fc region of an immunoglobulin comprising the amino acid sequence shown in SEQID NO:99.

[0041] 33. In one embodiment, the multimerization domain may be the Fc region of an immunoglobulin and may start from the lower hinge region of the Fc region.

[0042] 34. In one embodiment, the multimerization domain can be the Fc region of IgG1 consisting of SEQ ID NO:99.

[0043] 35. In one embodiment, the multimerization domain may have an amino acid substitution D126E, an amino acid substitution L128M and / or a deletion of amino acid K217 based on SEQ ID NO:99.

[0044] 36. In one embodiment, the modified fusion protein may be in the form of a dimer or a multimer.

[0045] 37. In one embodiment, the present disclosure relates to a pharmaceutical composition for preventing or treating autoimmune diseases, inflammatory diseases, tumor diseases, cancer, angiogenesis-related diseases or eye diseases, wherein the pharmaceutical composition comprises a modified fusion protein according to one embodiment of the present disclosure as an active ingredient.

[0046] 38. In one embodiment, the present disclosure relates to a nucleic acid molecule encoding a modified fusion protein according to an embodiment of the present disclosure.

[0047] 39. In one embodiment, the present disclosure relates to a host cell comprising a nucleotide sequence encoding a modified fusion protein according to an embodiment of the present disclosure.

[0048] 40. In one embodiment, the present disclosure relates to a vector comprising a nucleotide sequence encoding a modified fusion protein according to an embodiment of the present disclosure.

[0049] 41. In one embodiment, the vector may be a recombinant viral vector.

[0050] 42. In one embodiment, the present disclosure relates to a pharmaceutical composition for administering a viral vector to a subject, the pharmaceutical composition comprising a recombinant viral vector according to an embodiment of the present disclosure, wherein the fusion protein encoded by the recombinant viral vector is expressed in the subject and is intended for preventing or treating autoimmune diseases, inflammatory diseases, neoplastic diseases, cancer, angiogenesis-related diseases or eye diseases.

[0051] 43. In one embodiment, the recombinant viral vector may be a recombinant adeno-associated viral vector.

[0052] The modified fusion protein according to one embodiment of the present disclosure may exhibit excellent productivity, physical stability, and chemical stability.

[0053] The modified fusion protein according to one embodiment of the present disclosure may exhibit excellent serum stability.

[0054] Compared with previously developed substances, the modified fusion proteins according to the embodiments of the present disclosure have acidic properties and can show an increased charge variant ratio in the acidic and neutral regions of the pI distribution. Therefore, the modified fusion proteins can have an improved effect of non-specific interactions with the matrix and cell surface in vivo. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1a is a 3D structural diagram showing the surface charge distribution of the D2-D3 domains of VEGFR1 and VEGFR2. The diagram shows the binding surface of the ligand VEGF (indicated by the green circle) and the region where the loop of the D3 domain is located (indicated by the red circle) where variations can be introduced.

[0056] FIG. 1 b is a 3D structural diagram showing the positions of exemplary amino acid residues that may be mutated in domain D3 of VEGFR1.

[0057] Fig. 1c is a 3D structural diagram showing exemplary amino acid residues and their structures that can be varied in domain D3 of VEGFR1. In Fig. 1c, (A) is a diagram showing the positions of amino acid residues and their side chains in the variant sites using stick representation, and (B) shows a view obtained by rotating (A) 45 degrees.

[0058] Fig. 2 is a 3D structural diagram showing the extracellular domain of VEGFR1. Fig. 2a shows the structure of VEGFR1 when it is bound to a VEGF ligand. Figs. 2b and 2c respectively show, by 3D modeling, the fusion protein (e.g., VEGF-Grab3) formed by the binding of domain D2 and domain D3 of VEGFR1 to the Fc region when it is in an open structure before binding to the ligand (Fig. 2b) and when it is in a closed structure after binding to the ligand (Fig. 2c). Fig. 2d shows the distance from the end of domain D3 to the dimer formation site of domain D4 when VEGFR1 in the native state forms a dimer.

[0059] Figure 3 is an exemplary schematic diagram used to design linkers introduced in the present disclosure.

[0060] FIG. 4a and FIG. 4b are graphs showing serum stability and VEGF-A binding ability of proteins.

[0061] 5a to 5c are graphs showing serum stability of proteins.

[0062] 6a to 6f are graphs showing the isoelectric points of proteins.

[0063] 7a to 7d are graphs showing the pI distribution of analyzed proteins.

[0064] 8a to 8d are graphs analyzing the thermal stability of proteins.

[0065] 9a to 9c are graphs showing the VEGF-A binding ability of proteins. DETAILED DESCRIPTION

[0066] The various embodiments or examples described herein are merely exemplary and explanatory and do not limit the technical concept of the present disclosure. The technical concept of the present disclosure encompasses various modifications, equivalents and alternatives of each embodiment or example described herein, as well as all or part of the selective combination of a single embodiment or example.

[0067] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0068] Unless the context requires otherwise, the use of the singular herein may include the plural, and this also applies to singular expressions recited in the claims.

[0069] I. Definitions

[0070] In the present disclosure, the term "about" may represent a typical error range for a particular value recited, as is well known to those of ordinary skill in the art. When used in the context of a numerical value or range recited in the present disclosure, the term may mean ±20%, ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% of a numerical value or range recited in a certain embodiment or claimed. With respect to the length of a nucleotide or amino acid sequence, "about" may indicate that a nucleic acid or protein is not limited to the number of nucleotides or amino acids recited, and may include the addition or removal of several nucleotides or amino acids from either end, as long as the functional activity is not hindered.

[0071] As used herein, expressions such as "comprising," "including," "having," and the like should be interpreted as open-ended terms meaning the possibility of including other embodiments in a manner similar to "comprising," unless otherwise stated in the phrase or sentence containing these expressions.

[0072] As used herein, with respect to items associated with terms, the term "and / or" may mean any one or more of the items, any combination of the items, or all of the items.

[0073] As used herein, the term "amino acid" may refer to all naturally occurring L-α-amino acids. This definition is intended to include norleucine, ornithine, and homocysteine.

[0074] As used herein, the term "variation" or "amino acid variation" may refer to a substitution, insertion, deletion, or a combination thereof in an amino acid sequence compared to a reference (e.g., native sequence) polypeptide or protein, and the term "variant" may refer to a molecule having certain differences in its amino acid sequence compared to a reference polypeptide or protein due to such variation.

[0075] The scope of variants herein also includes proteins or fragments or derivatives thereof that exhibit the same or similar biological activity, as well as derivatives that have been modified differently during or after translation, such as by glycosylation, proteolytic cleavage, linkage to antibody molecules or other cellular ligands, etc.

[0076] As used herein, the term "carrier" may include pharmaceutically acceptable carriers, excipients or stabilizers that are non-toxic to cells or mammals at the dosages and concentrations employed. Typically, a pharmaceutically acceptable carrier is a pH buffered aqueous solution. Examples of pharmaceutically acceptable carriers include buffers such as phosphates, citrates and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium ions; and / or nonionic surfactants such as Polyethylene glycol (PEG) and But it is not limited to this.

[0077] As used herein, the term "effective amount" may refer to an amount sufficient to achieve a beneficial or desired clinical or biochemical outcome. An effective amount may be administered in one or more administrations. An effective amount may refer to an amount sufficient to alleviate, improve, stabilize, slow down or delay the progression of a disease state.

[0078] As used herein, the term "ligand" may refer to a molecule that binds to a modified fusion protein with high affinity (e.g., VEGF-A, VEGF-B, VEGF-C, VEGF-D, and PLGF). In other cases, a "ligand" may refer to any molecule or agent, or compound that can specifically bind to a molecule such as a polypeptide or protein, either covalently or transiently.

[0079] As used herein, the term "individual" or "subject" may refer to a mammal. The mammal includes, but is not limited to, livestock (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In one embodiment, the individual or subject may be a human.

[0080] As used herein, the term "pharmaceutically acceptable carrier and / or diluent" may include, but is not limited to, any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and adsorption delaying agents, and the like.

[0081] The embodiments of the present disclosure described herein should be understood to encompass “comprising,” “consisting of,” and / or “consisting essentially of.

[0082] II. Modified Fusion Proteins and Components of Modified Fusion Proteins

[0083] 1. Vascular endothelial growth factor receptor (VEGFR)

[0084] In one embodiment, the modified fusion protein of the present invention may include a VEGFR1 extracellular domain as its component. VEGFR1 is also known as fms-related tyrosine kinase (FLT-1) and is encoded by the FLT1 gene. The amino acid sequence of the VEGFR1 is shown in SEQ ID NO:1, which can be found in UniProtKB, #P17948. In one embodiment, the VEGFR1 is from a mammal, such as a human. VEGFR1 has seven immunoglobulin (Ig)-like domains, which are numbered 1, 2, 3, 4, 5, 6 and 7 from the N-terminus to the C-terminus of the extracellular region, as are VEGFR2 and VEGFR3.

[0085] In one embodiment, the VEGFR1 extracellular domain may include Ig-like domain D2 and Ig-like domain D3. As used herein, the term "Ig-like domain D2" of VEGFR1 refers to the second Ig-like domain found at the N-terminus of the VEGFR1 extracellular region, and "Ig-like domain D3" of VEGFR1 refers to the third Ig-like domain found at the N-terminus of the VEGFR1 extracellular region, but as long as the functions of these domains (e.g., binding to VEGF ligands and / or inhibiting the activation of VEGFR pathways) are maintained, these terms cover the corresponding variants. Since the amino acid sequences of active proteins of different species may differ, the Ig-like domains D2 and D3 of VEGFR1 are not limited by their source or sequence, and may include wild type or its active variants.

[0086] In one embodiment, the Ig-like domain D2 of VEGFR1 may comprise an amino acid sequence of G132 to H214 of SEQ ID NO: 1 or an amino acid sequence variant having a binding affinity similar to or equivalent to that exhibited by the amino acid sequence for VEGF ligand. Specifically, in one embodiment, the Ig-like domain D2 of VEGFR1 may comprise an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with an amino acid sequence of G132 to H214 of SEQ ID NO: 1.

[0087] In one embodiment, the Ig-like domain D3 of VEGFR1 may include the amino acid sequence L215 to A332 of SEQ ID NO: 1, or an amino acid sequence variant having a binding affinity similar to or equivalent to the binding affinity exhibited by the amino acid sequence for VEGF ligand. Specifically, in one embodiment, the Ig-like domain D3 of VEGFR1 may include an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with the amino acid sequence of L215 to A332.

[0088] In one embodiment, the modified fusion protein of the present disclosure may further comprise other VEGFR extracellular domains in addition to the Ig-like domain D2 and the Ig-like domain D3 of VEGFR1.

[0089] Although it is not desired to be bound by theory, it is generally believed that the extracellular domain of VEGFR inhibits the activation of VEGF path by combining with VEGF ligand, thereby blocking the interaction between VEGF ligand and VEGFR. In addition, although it is not desired to be bound by theory, it is generally believed that the extracellular domain of VEGFR can be combined with VEGFR to achieve the dominant negative inhibitory effect on VEGF signal transduction pathway. In one embodiment, the extracellular domain of the VEGFR can be combined with one or more VEGF ligands selected from the group consisting of: VEGF-A, VEGF-B, VEGF-C, VEGF-D and PLGF. In one embodiment, the extracellular domain of the VEGFR can be combined with VEGFR (for example, VEGFR1, VEGFR2 and / or VEGFR3).

[0090] In one embodiment, the VEGFR extracellular domain may or may not include a signal peptide, which may be used as a signal sequence for secretion from a host cell of a VEGFR extracellular domain or a modified fusion protein comprising the same domain. The signal peptide may be operably connected to a nucleic acid encoding a protein of interest (e.g., the extracellular domain of VEGFR1).

[0091] 2. Linker

[0092] Each component of the modified fusion protein (e.g., the extracellular domain or multimerization domain of VEGFR1) can be connected by a connecting portion (e.g., a peptide linker). The linker increases the flexibility of each component of the fusion protein and does not significantly interfere with the structure of each functional component in the fusion protein.

[0093] In one embodiment, the linker can be used to connect the C-terminus of the VEGFR1 extracellular domain (eg, the C-terminus of the Ig-like domain D3) to the N-terminus of the multimerization domain (eg, the IgG1 Fc region).

[0094] In one embodiment, the linker may include an amino acid sequence derived from a hinge region of an immunoglobulin. The hinge region derived from an immunoglobulin may include a hinge region present in the N-terminal region of the Fc region of an immunoglobulin. In one embodiment, the hinge region derived from an immunoglobulin may be derived from human IgA, IgD, IgM, IgE or IgG. In one embodiment, the hinge region derived from an immunoglobulin may include an amino acid sequence extending to a core hinge region of the Fc region of an immunoglobulin, the sequence excluding the lower hinge region of the Fc region. For example, if the hinge region is from IgG1, the lower hinge region may be an amino acid sequence corresponding to APELLGGP. In one embodiment, the linker may have the amino acid sequence EDKTHTCPPCP or LEDKTHTCPPCP.

[0095] 3. Multimerization domain

[0096] The disclosure provides a multimerization domain (e.g., the Fc region of an immunoglobulin), which can be a component of any modified fusion protein. The multimerization domain is a part of a multimeric protein that promotes subunit association to form, for example, dimers, trimers, tetramers, etc. As used herein, the term "multimerization domain" can be used to refer to a dimerization domain, a trimerization domain, a tetramerization domain, etc. The fusion protein comprising the multimerization domain can interact with another fusion protein comprising the multimerization domain to produce a fusion protein polymer (e.g., a fusion protein dimer). For example, the IgG Fc region is a dimerization domain that can be fused with the extracellular domain of VEGFR1 as described herein. The fusion protein comprising the extracellular domain of VEGFR1 and the IgG Fc region can dimerize with another fusion protein comprising the IgG Fc region, thereby forming a fusion protein dimer that can be combined with a VEGF ligand and a PLGF ligand simultaneously.

[0097] In one embodiment, the multimerization domain can be the Fc region of the immunoglobulin. In an additional embodiment, the Fc region of the immunoglobulin can be selected from the group consisting of: IgG Fc region, IgA Fc region, IgM Fc region, IgD Fc region and IgE Fc region. In another additional embodiment, the Fc region of the immunoglobulin can be selected from the group consisting of: IgG1 Fc region, IgG2 Fc region, IgG3 Fc region and IgG4 Fc region. In one embodiment, the Fc region can include the CH3 region of IgG1, IgG2, IgG3 or IgG4. In one embodiment, the Fc region can include the CH2 region and CH3 region of IgG1, IgG2, IgG3 or IgG4. The amino acid sequence encoding the immunoglobulin including the Fc region is well known in the art. In one embodiment, the multimerization domain can be the Fc region of the immunoglobulin comprising the amino acid sequence of SEQ ID NO:99. In one embodiment, the multimerization domain may be the Fc region of IgG1 consisting of the amino acid sequence of SEQ ID NO:99.

[0098] In one embodiment, the Fc region may comprise an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with the amino acid sequence of the CH3 region or the CH2 region and the CH3 region of the IgG1, IgG2, IgG3 or IgG4. In one embodiment, the multimerization domain may comprise an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 99.

[0099] In one embodiment, the multimerization domain can start from the lower hinge region of the immunoglobulin Fc region. The lower hinge region of the Fc region can start from Ala244 and can correspond to the amino acid sequence of Ala244 to Pro251. In this article, the position of the amino acid residue follows the Kabat numbering scheme (Kabat et al., "Sequences of Proteins of Immunological Interest" 5th edition, US Department of Health and Human Services, NIH Publication No. 91-3242, 1991). For example, the lower hinge region of the human IgG1 Fc region can correspond to the 1st to 8th amino acid residues based on the amino acid sequence of SEQ ID NO:99 or the amino acid sequence corresponding to APELLGGP. The amino acid sequence of the human IgG1 Fc region starting from the lower hinge region is shown in SEQ ID NO:99.

[0100] In one embodiment, the Fc region may be partially glycosylated, or completely aglycosylated.

[0101] In one embodiment, the Fc region may have an amino acid substitution D126E, an amino acid substitution L128M and / or a deletion of amino acid K217 based on SEQ ID NO: 99. These amino acid variations can enhance the half-life of the fusion protein by interacting with FcRn. The amino acid variations can occur in the same manner in other immunoglobulin Fc regions having a sequence corresponding to the amino acid sequence of SEQ ID NO: 99, and the modified fusion proteins of the present disclosure can include another immunoglobulin Fc region having amino acid variations as a multimerization domain component.

[0102] The following describes the properties of the modified fusion proteins according to the present disclosure. In these characteristic amino acid variations, the positions of the amino acid residues correspond to the positions of the amino acid residues in the complete amino acid sequence of VEGFR1 in SEQ ID NO:1.

[0103] 4. Characteristics of surface charge transition variation

[0104] In one embodiment, the modified fusion protein of the present disclosure may have the property of surface charge shift variation. In the present disclosure, the modified fusion protein having the property of surface charge shift variation can be used interchangeably with "surface charge shift variant" or "surface charge variant".

[0105] In the characteristics of the surface charge conversion variation according to one embodiment, the modified fusion protein may have one or more amino acid residues on the β1-β2 loop of the Ig-like domain D3, one or more amino acid residues on the β2-β3 loop of the domain D3, and / or one or more amino acid residues on the β5-β6 loop of the domain D3 replaced by amino acids that reduce the net pI of the protein, amino acids with negatively charged side chains, or amino acids with electronegative side chains. In one embodiment, amino acids with electronegative side chains may mean that the amino acid becomes electronegative after substitution according to the properties of adjacent amino acid residues or molecules, and may refer to a relative concept generated by the distribution of electrons within the molecule. For example, an amino acid residue containing a hydroxyl group (-OH) may become electronegative due to the lone pair of electrons present on the oxygen atom, and this phenomenon may reduce the net pI of the protein.

[0106] In one embodiment, the β1-β2 loop of the Ig-like domain D3 may include amino acid residues T236 to T247 of the amino acid sequence shown in SEQ ID NO: 1. In one embodiment, the β2-β3 loop of the Ig-like domain D3 may include amino acid residues T256 to V262 of the amino acid sequence shown in SEQ ID NO: 1. In one embodiment, the β5-β6 loop of the Ig-like domain D3 may include amino acid residues D299 to L308 of the amino acid sequence shown in SEQ ID NO: 1. As described in the present disclosure, the β1-β2 loop may be referred to as "site 1", the β2-β3 loop may be referred to as "site 2", and the β5-β6 loop may be referred to as "site 3". The inventors of the present disclosure have determined that the amino acid residues present in the loop are exposed on the surface of the domain D3, and surface charge stabilization and structural stabilization of the modified fusion protein have been achieved by variation of the amino acid residues present in the loop.

[0107] In one embodiment, the amino acid residues present in site 1 and site 3 affect each other through interactions between the amino acid residues due to their structural positions, and therefore, double, triple or quadruple amino acid variations (e.g., amino acid substitutions) can be introduced at each of the sites.

[0108] In one embodiment, the amino acid residue at position 2 is exposed on the domain surface on the side of domain D3 that binds to the VEGF ligand or PLGF ligand, and the surface exposure of the residue may affect the binding affinity or solubility to the ligand. In one embodiment, the amino acid residue at position 2 can be substituted with an amino acid residue having a smaller side chain than the side chain of the existing amino acid residue (e.g., Ala), or an amino acid residue having a polar side chain similar in size to the side chain of the existing amino acid residue (e.g., Ser or Asp), as long as the protein structure or the binding affinity and solubility to the ligand are not significantly affected.

[0109] In one embodiment, the three loops should be understood to encompass amino acid sequence variants that maintain their function. For example, in one embodiment, the β1-β2 loop, the β2-β3 loop, and the β5-β6 loop can include amino acid sequences having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with the amino acid sequences TPRPVKLLRGHT, TPLNTRV, and DKMQNKDKGL, respectively.

[0110] In the characteristics of the surface charge transition variation according to one embodiment, the amino acid substitution can be a substitution with: (i) an amino acid residue of a VEGFR2 homologous sequence, (ii) an amino acid residue of serine, threonine, tyrosine, cysteine, asparagine, glutamine, aspartic acid or glutamic acid, or (iii) an amino acid residue of alanine or glycine with a short side chain, but is not limited thereto.

[0111] In the linker variation characteristics according to one embodiment, one or more amino acid residues on the β1-β2 loop of the Ig-like domain D3 and one or more amino acid residues on the β5-β6 loop of domain D3 of the modified fusion protein may have the following substitutions: amino acids that reduce the net pI of the protein, amino acids with negatively charged side chains, or amino acids with electronegative side chains.

[0112] In the characteristics of the surface charge shift variation according to one embodiment, the modified fusion protein may include (i) amino acid substitutions of one or more of amino acid residues K241, L243, R244 and H246 on the β1-β2 loop of the Ig-like domain D3, but is not limited thereto. In one embodiment, the amino acid residues on the β1-β2 loop may be substituted by any one of threonine, glutamic acid, serine and valine.

[0113] In the characteristics of the surface charge conversion variation according to one embodiment, the modified fusion protein may include (ii) an amino acid substitution of amino acid residue L258 on the β2-β3 loop of the Ig-like domain D3, but is not limited thereto. In one embodiment, the amino acid residue on the β2-β3 loop may be substituted by any one of alanine, serine and aspartic acid.

[0114] In the characteristics of the surface charge conversion variation according to one embodiment, the modified fusion protein may include (iii) amino acid substitution of one or more of amino acid residues K300, Q302, K304 and K306 on the β5-β6 loop of the domain D3, but is not limited thereto. In one embodiment, the amino acid residues on the β5-β6 loop may be substituted by any one of glycine, threonine, serine and glutamine.

[0115] In the characteristics of the surface charge transition variation according to one embodiment, the modified fusion protein may comprise (i) one or more amino acid substitutions selected from K241T, K241E, L243S, R244V and H246E on the β1-β2 loop of the domain D3, (ii) amino acid substitutions L258A, L258S or L258D on the β2-β3 loop of the domain D3, and / or (iii) one or more amino acid substitutions selected from K300G, Q302T, K304S and K306Q on the β5-β6 loop of the domain D3.

[0116] In the characteristics of the surface charge transition variation according to one embodiment, the modified fusion protein may comprise (i) amino acid substitutions L243S and R244V on the β1-β2 loop of the domain D3, or (ii) amino acid substitutions K241E, L243S, R244V and H246E on the β1-β2 loop of the domain D3; amino acid substitutions L258A, L258S or L258D on the β2-β3 loop of the domain D3; and / or (i) amino acid substitution K300G on the β5-β6 loop of the domain D3, (ii) amino acid substitutions K300G, Q302T and K304S on the β5-β6 loop of the domain D3, or (iii) amino acid substitutions K300G, Q302T, K304S and K306Q on the β5-β6 loop of the domain D3.

[0117] In the characteristics of the surface charge transition variation according to one embodiment, the modified fusion protein may include amino acid substitutions K241E, L243S, R244V and H246E on the β1-β2 loop of the domain D3; amino acid substitutions L258A, L258S or L258D on the β2-β3 loop of the domain D3; and / or amino acid substitutions K300G, Q302T and K304S on the β5-β6 loop of the domain D3.

[0118] In one embodiment, the modified fusion protein may comprise the amino acid substitutions R238S and / or R275N in the Ig-like domain D3.

[0119] In one embodiment, the modified fusion protein may be such that the amino acid sequence of the C-terminus of the Ig-like domain D3 connected to the linker is a KALE based on the amino acid residue K331, provided that one or more of the amino acids K, A, L, and E may be deleted. The deletion is possible. In one embodiment, the C-terminus of the Ig-like domain D3 of the modified fusion protein may have a deleted K and A; a deleted K, A, and L; a deleted A, L, and E; or a deleted K, A, L, and E. In one embodiment, the C-terminal amino acid of the Ig-like domain D3 of the modified fusion protein of the present disclosure may be D330.

[0120] In one embodiment, the N-terminus of the Ig-like domain D2 of the modified fusion protein can be further modified. The modified fusion protein of the present invention can be produced by inserting the nucleic acid sequence or nucleic acid molecule encoding the fusion protein into a vector of various sources. In this case, depending on the type or source of the vector into which the nucleic acid sequence or nucleic acid molecule is inserted, the amino acid sequence at the N-terminus of the Ig-like domain D2 may vary, wherein the N-terminus starts from an amino acid sequence (e.g., amino acid sequence EF) derived from the vector, rather than starting from G132. In addition, in one embodiment, the modified fusion protein of the present invention may further include a sequence from the C-terminus of the Ig-like domain D1 of VEGFR1 at the N-terminus of the Ig-like domain D2. In one embodiment, the N-terminus of the Ig-like domain D2 of the modified fusion protein may start with an amino acid sequence SDT or an amino acid sequence EF. However, this variation of the amino acid sequence derived from the vector to the N-terminal sequence may not affect the productivity or physicochemical properties of the modified fusion protein of the present invention.

[0121] 5. Linker variation characteristics

[0122] In one embodiment, the modified fusion protein of the present invention may have a linker variation as a characteristic. In the present disclosure, the modified fusion protein having a linker variation characteristic may be used interchangeably with a "linker variant". Due to the linker variation characteristic, (i) the modified fusion protein of the present invention may closely mimic the structure of domain D2 and domain D3 in the natural VEGFR1 extracellular domain binding to a ligand, (ii) domain D3 in the natural VEGFR1 extracellular domain may have a length extending to the dimer formation site of domain D4, as a structural characteristic required to ensure smooth ligand binding, and (iii) the linker itself may have excellent structural stability.

[0123] In the variant characteristics of the linker according to one embodiment, the length of the linker can be about 14 amino acids or longer. In one embodiment, the length of the linker can be about 14 to about 70 amino acids, about 14 to about 65 amino acids, about 14 to about 60 amino acids, about 14 to about 55 amino acids, about 14 to about 50 amino acids, about 14 to about 45 amino acids, about 14 to about 40 amino acids, about 14 to about 38 amino acids, or about 14 to about 36 amino acids. In one embodiment, the linker can comprise or consist of the following number of amino acids: 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65 or 70 (inclusive of the end values, and including any values ​​between these numbers).

[0124] In the variant characteristics of the linker according to one embodiment, the linker may include a GS repeat sequence. In one embodiment, the GS repeat sequence may include an amino acid sequence consisting only of G and S of 2 to 60 amino acids in length. The N-terminal amino acid of the GS repeat sequence may be glutamic acid. In one embodiment, the GS repeat sequence may include or consist of the following number of amino acids: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55 or 60 (including the end values, and including any value between these numbers). In one embodiment, the GS repeat sequence may include or consist of 2 to 20 amino acids.

[0125] In the linker variation characteristics according to one embodiment, the GS repeat sequence can be (GS) n , (GSSG) n 、(GGGGS) n or (GS) m (GGGGS) n , where n can be an integer from 1 to 10, and m can be an integer from 0 to 10.

[0126] In the linker variation characteristics according to one embodiment, the GS repeat sequence can be selected from the group consisting of: GS, GSSG, (GSSG)2, GGGGS, (GGGGS)4, GS (GGGGS) and GS (GGGGS)3, but is not limited thereto.

[0127] In the linker variation characteristics according to one embodiment, the linker may include an amino acid sequence derived from a hinge region of an immunoglobulin. In one embodiment, the amino acid sequence of the hinge region may be modified.

[0128] In the present disclosure, the linker is located between the C-terminus of the VEGFR1 domain D3 and the Fc region of the human immunoglobulin produced by papain digestion, and can refer to the entire region extending between P243 at the N-terminus of the CH2 region (A244-K360) and the C-terminus of the domain D3. The positions of the amino acid residues in the Fc region and the CH2 region included in the Fc region follow the Kabat numbering scheme (Kabat et al., "Immunology Protein Sequences" 5th Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, 1991). In one embodiment, the linker may include certain regions derived from human immunoglobulin, and the region may be the amino acid sequence of CH1 (K218-V223) or hinge region (E226-P243), or a portion of such an amino acid sequence.

[0129] In the present disclosure, when the linker includes an amino acid sequence of a hinge region derived from an immunoglobulin, the characteristics of the hinge region included are retained in the linker. For example, the hinge region of the Fc region has cleavage sites for various proteases, and can induce oligomerization by O-glycosylation on Ser / Thr amino acids after being introduced into the fusion protein (Song et al., 2020. "Comput Struct Biotechnol J. " 18: 3925-3935). This glycosylation in the hinge region can affect its physicochemical properties during protein expression or purification, and can produce structural interference to disulfide bond pairing in the hinge region, which occurs reversibly during purification, thereby hindering normal disulfide bond formation. In the modified fusion protein with the linker variation characteristics disclosed herein, the amino acid sequence of the hinge region derived from immunoglobulin is modified to achieve the excellent structural stability of the linker itself and improve its physicochemical properties.

[0130] In the linker variation characteristics according to one embodiment, the hinge region derived from an immunoglobulin may include a sequence derived from a CH1 region, an upper hinge and / or a core hinge. In one embodiment, certain amino acid sequences derived from the CH1 region of an immunoglobulin may form a β-sheet or a β-fold. In one embodiment, the hinge region derived from an immunoglobulin may be derived from human IgA, IgD, IgM, IgE, IgG or a combination thereof.

[0131] In the linker variation feature according to one embodiment, the papain recognition site or glycosylation site derived from the hinge region of an immunoglobulin contained in the linker of the modified fusion protein may have an amino acid variation.

[0132] In the linker variation characteristics according to one embodiment, the amino acid variation in the papain recognition site can be replacing one or more amino acid residues present in the papain recognition site with alanine, serine, tyrosine, proline or threonine, or inserting an amino acid sequence of 1 to 10 amino acids long containing at least one amino acid having an aromatic or cyclic carbon in the side chain into the papain recognition site.

[0133] In one embodiment, the hinge region included in the linker may be derived from IgG1. In this case, the amino acid sequence near the cleaved amino acid in the papain recognition site may be KTHT. In one embodiment, when the hinge region is derived from IgG1, the histidine corresponding to the cleaved amino acid in the papain recognition site may be substituted by proline, proline-proline or tyrosine.

[0134] In one embodiment, when the hinge region is derived from IgG1, an amino acid sequence of 1 to 10 amino acids long containing at least one amino acid having aromatic or cyclic carbon in the side chain can be inserted before the histidine corresponding to the cleaved amino acid. In one embodiment, the amino acid having aromatic or cyclic carbon in the side chain can be proline or tyrosine. In one embodiment, the sequence inserted before the cleaved amino acid can include cysteine. If the sequence inserted into the papain recognition site contains cysteine, when a dimer is formed, the sequence can form a disulfide bond with the cysteine ​​at the same site in the opposite monomer, thereby giving rigidity to the linker, thereby stabilizing the ring structure of the linker. In one embodiment, the amino acid sequence inserted before the cleaved amino acid can be the sequence PPTC, but is not limited thereto. In the sequence inserted into the papain recognition site, due to the amino acid having aromatic or cyclic carbon in the side chain, hydrophobic interactions may occur between the aromatic carbon or cyclic carbon, and the interaction can stabilize the ring structure of the linker.

[0135] In one embodiment, when the hinge region is derived from IgG1, the amino acid sequence KTHT having the variant papain recognition site may be modified to an amino acid sequence selected from the group consisting of KTYT, TPP, ATPT, STYT, KAPP and ATPPTCP.

[0136] In the linker variation characteristics according to one embodiment, the amino acid variation in the glycosylation site may be a deletion of a serine or threonine present in the hinge region derived from the immunoglobulin, or a substitution of a serine or threonine present in the hinge region derived from the immunoglobulin with an amino acid other than serine or threonine. In one embodiment, the amino acid other than serine or threonine may be glycine or alanine. O-glycosylation that may occur in the hinge region may be prevented by amino acid substitution of serine or threonine.

[0137] In the linker variation characteristics according to one embodiment, the cysteine ​​present in the core hinge can be replaced by serine or glycine. In this case, the disulfide bond present in the core hinge can be removed to increase the flexibility of the linker, and it can be expected to achieve the same effect as increasing the length of the linker.

[0138] In the linker variation characteristics according to one embodiment, the linker may include, starting from the N-terminus, in the following order: (i) an amino acid sequence selected from the group consisting of GS, GSSG, (GSSG)2, GGGGS, (GGGGS)4, GS(GGGGS) and GS(GGGGS)3; and (ii) a hinge region derived from an immunoglobulin selected from the group consisting of human IgG1, IgG4 or IgD / G1, and the amino acid sequence of the hinge region may be modified.

[0139] In the present disclosure, the hinge region derived from IgD / G1 may refer to a combination of the hinge sequences of IgD and IgG1. Specifically, the hinge region derived from IgD / G1 may be NTGSGGEEKKKEKEKEEQEERSSDKTHTCPPCP. In one embodiment, the hinge sequence of the IgD may be NTGSGGEEKKKEKEKEEQEERSS, NTGRGGEEKKKEKEKEQEER or a variant thereof. In one embodiment, the hinge sequence of the IgG1 may be DKTHTCPPCP or a variant thereof.

[0140] In the linker variation characteristics according to one embodiment, the linker can be an amino acid sequence selected from the group consisting of GSSGDKTHTSPPSP, GSSGEPKSSDKTYTSPPSP, GSKVDKKVEPKSSDKTHTCPPCP, GSKVDKKVEPKSSDKTYTCPPCP, GSKVDKKVEPKSSDTPPTCPPCP, GSGGGGSGGGGSGGGGSAESKYGPPCPPCP, GSNTGSGGEEKKKEKEKEEQEERSSDKTYTCPPCP, GSNTGSGGEEKKKEKEKEEQEERSSDTPPTCPPCP, EGSSGGSSGEPKSDATPTCPPCP, EGSSGGSSGEPKSDSTYTCPPCP, GCKVDK KKVEPKSSDKTYTCPPCP, CSKVDKKVEPKSSDKTYTCPPCP, GGGGSAEPKAGDKAPPGPPGP, GGGGSAEPKSSDKTYTCPPCP, GGGGSGGGGSGGGGSGGGGSAEPKSSDKTYTCPPCP, CSSGDATPTSPPSP, CSKVDKKVEPKSSD TPPTCPPCP, CSGGGGSAEPKAGDATPPTCPPCP, CSGGGGSGGGGSGGGGSAESKYGPPCPPCP, CSNTGSGGEEKKKEKEEQEERSSDTPPTCPPCP, CGSSGGSSGEPKSDATPTCPPCP, and CSGGGGSAEPKAGDATPPTCPPCPPCP.

[0141] 6. Disulfide bond variation characteristics

[0142] In one embodiment, the modified fusion protein of the present invention may have the property of disulfide bond variation. In the present disclosure, the modified fusion protein having the disulfide bond variation property may be used interchangeably with "disulfide bond variant". Since the modified fusion protein of the present invention is a fusion of heterologous proteins, it is expected that its stability will be reduced at the site where different types of proteins are connected. Therefore, the disulfide bond variation property is introduced to prevent physical cleavage of the modified fusion protein and improve its stability.

[0143] In order to introduce disulfide bond variation characteristics, the positions of amino acid residues that can introduce disulfide bond variation were searched according to the following criteria, centered on the C-terminal region of the Ig-like domain D3 in the fusion protein where the heterologous protein connection starts and may lack secondary structure: (1) the candidate amino acid residue should be exposed on the surface of the fusion protein so that it can form a disulfide bond, (2) the candidate amino acid residue should be present on the flexible loop-linker that connects the heterologous protein, and (3) the amino acid residues located at two different sites must be within the distance that they can form a disulfide bond. Based on these criteria, the candidate amino acid residues were selected using a tertiary structure energy prediction program (FoldX in YASARA, Schymkowitz et al. 2005 "Nucleic Acids Research". 33: W382-388), and these amino acid residues were replaced by cysteine ​​to determine that the formation of disulfide bonds can improve the properties of the modified fusion protein (e.g., productivity, physicochemical properties, ligand binding) (see Example 2.3). In one embodiment, the disulfide bond variant property can be introduced by substituting a cysteine ​​residue at one position in the VEGFR1 domain and one position in the linker, thereby achieving excellent stability of the modified fusion protein through the stable bonding form of covalent bond.

[0144] In one embodiment, for the disulfide bond variation property, (i) one of the amino acid residues on the β1-β2 loop of the Ig-like domain D3 or one of the amino acid residues on the β5-β6 loop of the Ig-like domain D3 and (ii) one of the amino acid residues at positions -2, -1, 0, +1, +2 and +3 relative to Y329 of the Ig-like domain D3 can be substituted with cysteine. Through this substitution, the modified fusion protein of the present disclosure can have a disulfide bond inside the protein. In one embodiment, the amino acid residues present at the positions described in (i) and (ii) for introducing the disulfide bond variation property can be selected according to the above criteria (1) to (3).

[0145] In one embodiment, the one amino acid residue among the amino acid residues on the β1-β2 loop in (i) may be one amino acid residue among the amino acid residues on the amino acid sequence from V240 to T247.

[0146] In one embodiment, for the disulfide bond variation feature, one of the amino acid residues T241, L243 and R244 on the β1-β2 loop of the Ig-like domain D3 or the amino acid residue N303 on the β5-β6 loop of the Ig-like domain D3 can be substituted with cysteine, and one of the amino acid residues at positions 0, +2 and +3 relative to Y329 of the Ig-like domain D3 can be substituted with cysteine.

[0147] In one embodiment, for the disulfide bond variation characteristics, the modified fusion protein of the present invention may have: T241 and the amino acid residue at position +3 relative to domain D3 of Y329 are replaced by cysteine; amino acid variations L243C and Y329C; L243 and the amino acid residue at position +2 relative to domain D3 of Y329 are replaced by cysteine; amino acid variations R244C and Y329C; or amino acid variations N303C and Y329C.

[0148] 7. Fusion Protein

[0149] In one embodiment of the present disclosure, a modified fusion protein capable of binding to a VEGF ligand and a PLGF ligand simultaneously may be provided. In one embodiment, the modified fusion protein may have a first binding specificity to a VEGF ligand and a second binding specificity to a PLGF ligand. Here, the VEGF ligand may be one or more of VEGF-A, VEGF-B, VEGF-C, and VEGF-D, and may specifically be VEGF-A.

[0150] In one embodiment, the modified fusion protein may include a VEGFR1 extracellular domain, a linker, and a multimerization domain. In one embodiment, the modified fusion protein may include a VEGFR1 extracellular domain, a linker, and a multimerization domain as components arranged in order from the N-terminus to the C-terminus. In one embodiment, the VEGFR1 extracellular domain may include an Ig-like domain D2 and an Ig-like domain D3 of VEGFR1.

[0151] In one embodiment, the modified fusion protein may have one or more of the following: (a) surface charge conversion properties, (b) linker variation properties, and (c) disulfide bond variation properties.

[0152] In one embodiment, the modified fusion protein may have all of the following properties (a), (b) and (c):

[0153] (a) Introduction:

[0154] one or more amino acid substitutions selected from the group consisting of K241T, K241E, L243S, R244V and H246E on the β1-β2 loop of domain D3,

[0155] The amino acid substitutions in the β2-β3 loop of domain D3 are L258A, L258S or L258D, and / or

[0156] one or more amino acid substitutions selected from the group consisting of K300G, Q302T, K304S and K306Q on the β5-β6 loop of domain D3;

[0157] (b) the linker is an amino acid sequence selected from the group consisting of GSSGDKTHTSPPSP, GSSGEPKSSDKTYTSPPSP, GSKVDKKVEPKSSDKTHTCPPCP, GSKVDKKVEPKSSDKTYTCPPCP, GSKVDKKVEPKSSDTPPTCPPCP, GSGGGGSGGGGSGGGGSAESKYGPPCPPCP, GSNTGSGGEEKKKEKEKEEQEERSSDKTYTCPPCP, GSNTGSGGEEKKKEKEKEEQEERSSDTPPTCPPCP, EGSSGGSSGEPKSDATPTCPPCP, EGSSGGSSGEPKSDSTYTCPPCP, GCKVDKKVEPKSSD KTYTCPPCP, CSKVDKKVEPKSSDKTYTCPPCP, GGGGSAEPKAGDKAPPGPPGP, GGGGSAEPKSSDKTYTCPPCP, GGGGSGGGGSGGGGSGGGGSAEPKSSDKTYTCPPCP, CSSGDATPTSPPSP, CSKVDKKVEPKSSDTPPTC PPCP, CSGGGGSAEPKAGDATPPTCPPCP, CSGGGGSGGGGSGGGGSAESKYGPPCPPCP, CSNTGSGGEEKKKEKEEQEERSSDTPPTCPPCP, CGSSGGSSGEPKSDATPTCPPCP, and CSGGGGSAEPKAGDATPPTCPPCPPCP;

[0158] (c) one of the amino acid residues T241, L243 and R244 on the β1-β2 loop of domain D3 or the amino acid residue N303 on the β5-β6 loop is substituted with cysteine, and

[0159] One of the amino acid residues at positions 0, +2 and +3 relative to Y329 of domain D3 is substituted by cysteine.

[0160] In one embodiment, the modified fusion protein may have all of the following properties (a), (b) and (c):

[0161] (a) Introduce the following:

[0162] (i) amino acid substitutions L243S and R244V on the β1-β2 loop of domain D3, or (ii) amino acid substitutions K241E, L243S, R244V and H246E on the β1-β2 loop of domain D3,

[0163] The amino acid substitutions in the β2-β3 loop of domain D3 are L258A, L258S or L258D, and / or

[0164] (i) amino acid substitution K300G on the β5-β6 loop of domain D3, (ii) amino acid substitution K300G, Q302T and K304S on the β5-β6 loop of domain D3, or (iii) amino acid substitution K300G, Q302T, K304S and K306Q on the β5-β6 loop of domain D3;

[0165] (b) the linker is an amino acid sequence selected from the group consisting of CSSGDATPTSPPSP, CSKVDKKVEPKSSDTPPTCPPCP, CSGGGGSAEPKAGDATPPTCPPCP, CSGGGGSGGGGSGGGGSAESKYGPPCPPCP, CSNTGSGGEEKKKEKEKEEQEERSSDTPPTCPPCP, CGSSGGSSGEPKSDATPTCPPCP, CSKVDKKVEPKSSDKTYTCPPCP and CSGGGGSAEPKAGDATPPTCPPCPPCP;

[0166] (c) L243 and the amino acid residue at position +2 relative to Y329 of domain D3 are substituted with cysteine.

[0167] In one embodiment, the modified fusion protein may have all of the following properties (a), (b) and (c):

[0168] (a) Introduction:

[0169] The amino acid substitutions on the β1-β2 loop of domain D3 are K241E, L243S, R244V and H246E,

[0170] The amino acid substitutions in the β2-β3 loop of domain D3 are L258A, L258S or L258D, and / or

[0171] Amino acid substitutions K300G, Q302T and K304S in the β5-β6 loop of domain D3;

[0172] (b) the linker is an amino acid sequence selected from the group consisting of CSSGDATPTSPPSP, CSKVDKKVEPKSSDTPPTCPPCP, CSGGGGSAEPKAGDATPPTCPPCP, CSGGGGSGGGGSGGGGSAESKYGPPCPPCP, CSNTGSGGEEKKKEKEKEEQEERSSDTPPTCPPCP, CGSSGGSSGEPKSDATPTCPPCP, CSKVDKKVEPKSSDKTYTCPPCP and CSGGGGSAEPKAGDATPPTCPPCPPCP;

[0173] (c) L243 and the amino acid residue at position +2 relative to Y329 of domain D3 are substituted with cysteine.

[0174] In one embodiment, the modified fusion protein may comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 10 to SEQ ID NO: 70, SEQ ID NO: 77 to SEQ ID NO: 93, and SEQ ID NO: 98. In one embodiment, the modified fusion protein may consist of an amino acid sequence selected from the group consisting of SEQ ID NO: 10 to SEQ ID NO: 70, SEQ ID NO: 77 to SEQ ID NO: 93, and SEQ ID NO: 98.

[0175] In one embodiment, the modified fusion protein can have at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 10 to SEQ ID NO: 70, SEQ ID NO: 77 to SEQ ID NO: 93, and SEQ ID NO: 98. Such variants may also include amino acid variations (e.g., conservative amino acid substitutions) in additional amino acid sequences while retaining or maintaining the amino acid variations defined in the properties of the modified fusion proteins of the present disclosure.

[0176] In one embodiment, the components of the modified fusion protein of the present disclosure (VEGFR1 extracellular domain, linker and multimerization domain) may carry post-translational modifications, including, for example, glycosylation, sialylation, acetylation and phosphorylation.

[0177] In one embodiment, the modified fusion protein of the present invention can include conservative amino acid substitutions at positions other than the positions of amino acid variations defined in the characteristics described in the present invention. These conservative amino acid substitutions can be introduced into any individual component of the modified fusion protein of the present invention (e.g., VEGFR1 extracellular domain, linker, and multimerization domain).

[0178] In one embodiment, the modified fusion protein of the present disclosure may include a signal peptide or signal sequence for secreting the protein from the cell. For example, the modified fusion protein of the present disclosure may further include a peptide of heterologous origin, specifically, a signal sequence or other peptide with a specific cleavage site at the N-terminus of the mature fusion protein. In one embodiment, the heterologous signal sequence may be a sequence that can be recognized and processed (i.e., cut by a signal peptide hydrolase) by a eukaryotic host cell. Information about signal peptides is well known in the art, such as in Korean Patent No. 10-2228921, there is a relevant elaboration, but is not limited thereto. In one embodiment, the signal peptide may be a human interleukin signal sequence. In one embodiment, the signal peptide can be composed of the following amino acid sequence: MVSYWDTGVLLCALLSCLLLTGSSSG (tPA), MEFGLSWVFLVALFRGVQC (H7), MKWVTFISLLFLFSSAYS (human serum albumin), MGWSCIILFLVATATGVHS (mouse Ig heavy chain), MDWTWRVFCLLAVAPGAHS (human Ig heavy chain) or MYRMQLLSCIALSLALVTNS (human interleukin-2), but any signal peptide that can be used by a person of ordinary skill in the art to produce a fusion protein can be used without limitation.

[0179] In one embodiment of the present disclosure, a multimeric fusion protein comprising two or more modified fusion proteins of the present disclosure may be provided. Multimers (e.g., dimers, trimers, tetramers, etc.) may be formed by the same fusion protein (e.g., homopolymers), or may form fusion proteins of heterologous origin (e.g., heteropolymers). In one embodiment, the multimeric fusion protein may include at least one modified fusion protein, the at least one modified fusion protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 10 to SEQ ID NO: 70, SEQ ID NO: 77 to SEQ ID NO: 93 and SEQ ID NO: 98, or an amino acid sequence having at least 90% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 10 to SEQ ID NO: 70, SEQ ID NO: 77 to SEQ ID NO: 93 and SEQ ID NO: 98. In one embodiment, such fusion proteins are recovered as protein fusion multimers from host cells containing nucleic acids encoding modified fusion proteins of the present disclosure. In one embodiment, such multimeric fusion proteins may be glycosylated. For example, after such multimeric fusion proteins are released from host cells, glycosylation may occur at the extracellular domain and / or at the multimerization domain of VEGFR.

[0180] In one embodiment, the IC of the modified fusion protein of the present disclosure for inhibition of ligand activity (eg, inhibition of VEGF activity or PLGF activity) is 50 It can be less than or equal to about 1 mg / ml, 500 ng / ml, 300 ng / ml, 100 ng / ml, 70 ng / ml, 50 ng / ml, 45 ng / ml, 40 ng / ml, 35 ng / ml, 30 ng / ml, 25 ng / ml, 20 ng / ml, 10 ng / ml, 5 ng / ml or 1 ng / ml (including the end values, including any values ​​between these values).

[0181] In one embodiment, the Kd of the modified fusion protein of the present disclosure may be less than any of the following: 1.0 mM, 500 μM, 100 μM, 50 μM, 25 μM, 10 μM, 5 μM, 1 μM, 900 nM, 800 nM, 700 nM, 600 nM, 500 nM, 400 nM, 350 nM, 300 nM, 250 nM, 200 nM, 150 nM, 100 nM, 95 nM, 90 nM, 85 nM, 80 nM, 75 nM, 70 nM, 65 nM, 60 nM, 55 nM. pM, 1pM, 0.5pM, 0.1pM, 0.05pM or 0.01pM (inclusive of any values ​​between these values).

[0182] III. Nucleic Acids, Vectors and Host Cells

[0183] Nucleic Acids

[0184] In one embodiment of the present disclosure, an isolated nucleic acid encoding any modified fusion protein component of the present disclosure (such as VEGFR1 extracellular domain and / or multimerization domain) can be provided. For all receptor types, nucleic acids encoding mammalian VEGFRs have been described. Exemplary nucleic acid sequences can be found in U.S. Patent No. 7,928,072 and WO2006 / 113277, but are not limited thereto. The mRNA encoding human VEGFR1 and VEGFR2 can be found under GenBank accession numbers NM_002019.4 and NM_002253.2, respectively. In one embodiment, an isolated nucleic acid can encode Ig-like domains D2 and D3 of VEGFR1 comprising an amino acid sequence consisting of SEQID NO:100. In one embodiment, the isolated nucleic acid can encode the Ig-like domains D2 and D3 of VEGFR1, which comprise an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the amino acid sequence consisting of SEQ ID NO: 100.

[0185] In the present disclosure, isolated nucleic acids encoding multimerization domains (e.g., Fc regions) may be provided. In one embodiment, the isolated nucleic acid may encode a multimerization domain comprising an amino acid sequence of SEQ ID NO: 99. In some embodiments, the isolated nucleic acid may encode a multimerization domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 99 having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity.

[0186] The present disclosure can provide an isolated nucleic acid encoding a modified fusion protein of the present disclosure. In one embodiment, the isolated nucleic acid can encode a modified fusion protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 10 to SEQ ID NO: 70, SEQ ID NO: 77 to SEQ ID NO: 93 and SEQ ID NO: 98. In one embodiment, the isolated nucleic acid can encode a modified fusion protein comprising an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 10 to SEQ ID NO: 70, SEQ ID NO: 77 to SEQ ID NO: 93 and SEQ ID NO: 98.

[0187] The isolated nucleic acid sequence encoding the modified fusion protein or component of the fusion protein of the present disclosure (eg, the extracellular domain or multimerization domain of VEGFR1) can further comprise a nucleic acid sequence encoding a linker.

[0188] In one embodiment, the isolated nucleic acid may further comprise a sequence encoding a signal peptide, which serves as a signal sequence for secreting the modified fusion protein from a host cell. In one embodiment, the isolated nucleic acid may not comprise a sequence encoding a signal peptide.

[0189] The nucleic acid molecules of the separation of the modified fusion protein of the present disclosure or the component of the fusion protein (for example, the extracellular domain of VEGFR1, linker or polymerizing domain) can be in the form of RNA, such as mRNA, hnRNA, tRNA or any other form, or in the form of DNA, including but not limited to cDNA and genomic DNA obtained by cloning or synthetically produced, or any combination thereof. Such isolated nucleic acid molecules can be prepared by various methods known in the art (see "Molecular Cloning: Laboratory Manual (Molecular Cloning:A Laboratory Manual)" (Sambrook et al., 4th edition, Cold Spring Harbor Laboratory Press (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY), 2012) and "Current Protocols in Molecular Biology Laboratory Guide (Current Protocols in Molecular Biology)" (FM Ausubel et al., 2003)).

[0190] Carrier

[0191] In one embodiment, the present disclosure relates to a nucleic acid delivery vehicle or its use for introducing one or more nucleic acid sequences encoding modified fusion proteins or fusion protein components into cells for expressing the protein. The nucleic acid sequence may be the sequence of the above-mentioned isolated nucleic acid.

[0192] In one embodiment, examples of nucleic acid delivery vehicles include liposomes, biocompatible polymers (including natural polymers and synthetic polymers); lipoproteins; polypeptides; polysaccharides; lipopolysaccharides; artificial viral envelopes; metal particles; and bacteria, viruses (such as baculovirus, adenovirus and retrovirus), phages, clays, plasmids, fungal vectors, and other recombinant vehicles commonly used in the art expressed in various eukaryotic and prokaryotic hosts. In one embodiment, the nucleic acid delivery vehicle can be an expression vector, such as a plasmid. In one embodiment, the nucleic acid sequence contained in the expression vector can be operably connected to an expression control sequence.

[0193] In one embodiment, the vector may include any element for realizing the conventional functions of an expression vector, such as a promoter, a ribosome binding element, a terminator, an enhancer, a selection marker, and a replication origin. The promoter may be a constitutive promoter, an inducible promoter, or a repressible promoter. Exemplary promoters are provided in Korean Patent No. 10-0659477, but are not limited thereto.

[0194] A variety of expression vectors capable of delivering nucleic acids to cells (e.g., derived from bacterial cells, yeast cells, plant cells, or mammalian cells) are known in the art and can be used herein to produce modified fusion proteins or fusion protein components in cells. For example, if E. coli is transferred into an engineered plasmid (e.g., pBR322) containing a nucleic acid encoding a fusion protein (Mandel et al., J. Mol. Biol., 1970, 53: 154), the E. coli can be used to produce a modified fusion protein. The expressed modified fusion protein or fusion protein component can be harvested from the cell and purified according to conventional techniques known in the art and the methods described in the present disclosure.

[0195] In one embodiment, an expression vector comprising a nucleic acid encoding a modified fusion protein and capable of replicating in a bacterial or eukaryotic host can be used to transfect a host, thereby directing the expression of the nucleic acid to produce a modified fusion protein, and then recovering the modified fusion protein in a biologically active form. As used herein, a biologically active form includes a form that can bind to a VEGF ligand or a PLGF ligand.

[0196] In one embodiment, the expression vector containing the nucleic acid molecule encoding the modified fusion protein or fusion protein component can be identified by at least three general methods, but not limited to: (a) DNA-DNA hybridization, (b) the presence or absence of "marker" gene function, and (c) expression of the inserted sequence. Detailed information on the above three methods is provided in Korean Patent No. 10-0659477 or No. 10-2228921, but not limited thereto.

[0197] Host cells

[0198] In one embodiment of the present disclosure, a host cell containing an expression vector encoding the modified fusion protein of the present disclosure can be provided. Such host cells are suitable for expressing the modified fusion protein of the present disclosure, and can constitute a host-vector system for producing the modified fusion protein. In one embodiment, the host cell can be used to produce viral particles (e.g., recombinant viral vectors).

[0199] As used herein, the term "host cell" may include cells that are or may be receptors for the disclosed vectors and their daughter cells. Due to natural, accidental or artificial mutations, daughter cells are not necessarily identical to the original parent cells in morphology or in the total DNA complementary genome (). In one embodiment, the host cell may be a bacterial cell (such as Escherichia coli), a yeast cell (such as Pichia pastoris), an insect cell (such as Spodoptera frugiperda), or a mammalian cell (such as COS, HEK or CHO cells), but is not limited thereto.

[0200] In one embodiment, the modified fusion proteins of the present disclosure may be expressed transiently, constitutively, or permanently in a host cell.

[0201] In one embodiment of the present disclosure, a method for producing a modified fusion protein of the present disclosure may be provided, the method comprising growing a host cell or a cell of the above-mentioned host-vector system under conditions that allow the production of the modified fusion protein, and then recovering the produced fusion protein. The modified fusion protein for implementing the present disclosure may be produced by expressing in a prokaryotic or eukaryotic expression system. Methods for culturing host cells or producing fusion proteins from host cells are well known in the art, such as the method disclosed in Korean Patent No. 10-2228921, but are not limited thereto.

[0202] In one embodiment, the modified fusion protein of the present disclosure produced from the above-mentioned host cells can be purified and identified by a variety of methods. The method for purifying and identifying the fusion protein produced from the host cells is well known in the art, such as the method provided in Korean Patent No. 10-2228921, but is not limited thereto. For example, the fusion protein produced from the host cells can be recovered from the cells as a soluble protein, or recovered as an inclusion body, and the fusion protein can be quantitatively extracted from the inclusion body by 8M guanidine hydrochloride and dialysis. In order to further purify the fusion protein, any number of purification methods can be used, including but not limited to conventional ion exchange chromatography, affinity chromatography, different sugar chromatography, hydrophobic interaction chromatography, reverse phase chromatography or gel filtration.

[0203] IV. Viral Particles and Methods for Producing Viral Particles

[0204] In one embodiment of the present disclosure, a viral particle (or virion) comprising a nucleic acid encoding a modified fusion protein of the present disclosure may be provided.

[0205] Viral vectors can be used to deliver nucleic acids encoding fusion proteins or fusion protein components to express the protein in target cells within a specific target tissue (e.g., diseased tissue). Many types of viruses are known, and many viruses have been studied for the purpose of delivering nucleic acids to target cells. Exogenous nucleic acids can be inserted into vectors (such as adenoviruses, partially deleted adenoviruses, completely deleted adenoviruses, adeno-associated viruses (adeno-associated virus, AAV), retroviruses, slow viruses, etc.) for delivery to cells.

[0206] In one embodiment, the cell is located in the individual body, and the virus can be delivered intravenously, intramuscularly, intraportally or by other administration routes. In one embodiment, the viral vector may include a viral vector derived from adenovirus, adeno-associated virus (AAV) and retrovirus (including slow virus, such as human immunodeficiency virus (human immunodeficiencyvirus, HIV)). For exemplary viral vectors, see but are not limited to U.S. Patent No. 7,928,072 and WO 2006 / 113277, which are incorporated herein by reference in their entirety. Viral particles containing nucleic acids encoding fusion proteins and methods for producing the viral particles are well known in the art, such as methods disclosed in Korean Patent No. 10-2228921, but are not limited thereto.

[0207] V. Therapeutic Methods Using Fusion Proteins and Viral Particles

[0208] In one embodiment, the present disclosure relates to a pharmaceutical composition for preventing, ameliorating or treating a VEGF ligand or VEGF receptor-related disease or at least one of the symptoms of the disease, the pharmaceutical composition comprising the modified fusion protein of the present disclosure as an active ingredient.

[0209] In one embodiment, the present disclosure relates to a method for preventing, ameliorating or treating a VEGF ligand or VEGF receptor-related disease or at least one of the symptoms of the disease, the method comprising administering the modified fusion protein of the present disclosure.

[0210] In one embodiment, the present disclosure relates to the use of the modified fusion protein of the present disclosure for preventing, ameliorating or treating a VEGF ligand or VEGF receptor-related disease or at least one of the symptoms of the disease.

[0211] In one embodiment, the present disclosure relates to the modified fusion protein of the present disclosure or a pharmaceutical composition comprising the fusion protein, which is used in a method for preventing, ameliorating or treating a VEGF ligand or VEGF receptor-related disease or at least one of the symptoms of the disease.

[0212] In one embodiment, the modified fusion protein in the form of a nucleic acid encoding the modified fusion protein of the present invention can be used in a nucleic acid delivery vehicle or a viral particle (e.g., a recombinant viral vector) for preventing, improving or treating at least one symptom of a VEGF ligand or VEGF receptor-related disease or the symptoms of the disease. In one embodiment, the nucleic acid delivery vehicle or viral particle can be applied to gene therapy for preventing, improving or treating at least one symptom of a VEGF ligand or VEGF receptor-related disease or the symptoms of the disease. In one embodiment, the nucleic acid can produce an encoded protein that mediates a therapeutic effect, i.e., a modified fusion protein of the present invention. According to the present disclosure, any gene therapy method available in the art can be used. In one embodiment, the nucleic acid is introduced into the cell before the resulting recombinant cell is administered in vivo. Cells into which nucleic acids are introduced for gene therapy purposes encompass all available cell types, and include, for example, epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes, blood cells (e.g., T lymphocytes, B lymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes or granulocytes), various stem cells or progenitor cells, such as hematopoietic stem cells or progenitor cells obtained from bone marrow, umbilical cord blood, peripheral blood or fetal liver, etc., but are not limited thereto. In one embodiment, the cells used for gene therapy can be autologous cells of the patient.

[0213] In one embodiment, the modified fusion protein can bind to VEGF protein and / or PLGF protein. In one embodiment, the modified fusion protein can have one or more of the following properties: (a) binding to one or more proteins of the VEGF family, such as VEGF-A, VEGF-B, VEGF-C, VEGF-D or PLGF; (b) blocking the binding of VEGF family proteins to VEGF receptors; (c) inhibiting the activation of VEGF signaling pathways; (d) preventing, improving and / or treating diseases such as eye diseases, autoimmune diseases, inflammatory diseases, neoplastic diseases or cancer; (e) weakening, curbing or preventing the growth of tumors or cancers; (f) inhibiting the metastasis of tumors or cancers. The activity of the modified fusion protein can be measured in vivo and / or in vitro.

[0214] In one embodiment, the pharmaceutical composition may include a pharmaceutically acceptable carrier and / or diluent. In one embodiment, the pharmaceutically acceptable carrier may be a sterile liquid, such as water and oil, including oils from petroleum sources, animal sources, plant sources or synthetic sources, such as peanut oil, soybean oil, mineral oil, etc. Physiological saline and aqueous glucose solutions, polyethylene glycol (PEG) and glycerol solutions may also be used as liquid carriers, particularly for injection solutions. Such pharmaceutical compositions may further include additional ingredients, such as preservatives, buffers, isotonic agents, antioxidants and stabilizers, nonionic wetting agents or clarifying agents, thickening agents, etc. Pharmaceutical compositions of the present disclosure may be packaged in single unit doses or in multiple doses. These compositions are typically formulated into sterile and substantially isotonic solutions. In addition, compositions may be formulated into osmotic pressure values ​​compatible with aqueous humor and ophthalmic tissues. Such osmotic pressure values ​​are generally in the range of about 200 millimoles per kilogram of water ("mOsm / kg") to 400 mOsm / kg, or about 300 mOsm / kg. The osmotic pressure value of the retina is believed to be approximately 283 mOsm / kg.

[0215] In one embodiment, the pharmaceutical composition may be a parenteral composition. It may be advantageous to formulate parenteral compositions in dosage unit form to ensure uniformity of dosage and ease of administration.

[0216] Disease or condition

[0217] In one embodiment, the VEGF ligand or VEGF receptor-related disease may include an autoimmune disease (e.g., rheumatoid arthritis, multiple sclerosis, or systemic lupus erythematosus), an inflammatory disease (inflammatory arthritis, osteoarthritis, or psoriasis), a neoplastic disease, a cancer (e.g., breast cancer, lung cancer, gastric cancer, pancreatic cancer, or leukemia), an angiogenesis-related disease (e.g., atherosclerosis), or an eye disease (e.g., age-related macular degeneration, choroidal neovascularization, or uveitis), but is not limited thereto.

[0218] In one embodiment, the cancer may include prostate cancer, urethral cancer, penile cancer, breast cancer, lung cancer, esophageal cancer, small intestine cancer, colorectal cancer, rectal cancer, colon cancer, liver cancer, urinary system cancer (e.g., bladder cancer), kidney cancer, lung cancer (e.g., non-small cell lung cancer), ovarian cancer, cervical cancer, endometrial cancer, vaginal cancer, vulvar cancer, pancreatic cancer, gastric cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, skin cancer (e.g., melanoma), Hodgkin's disease (Hodgkin's disease), bone cancer, lymphatic or myeloid hematopoietic cancer, chronic or acute leukemia, head and neck cancer, nasopharyngeal carcinoma (NPC), glioblastoma, teratocarcinoma, neuroblastoma, adenocarcinoma, cancer of mesenchymal origin (e.g., fibrosarcoma or rhabdomyosarcoma), soft tissue sarcoma and carcinoma, choriocarcinoma, hepatoblastoma, central nervous system (CNS) tumor, primary central nervous system lymphoma, spinal cord tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma or Wilms' tumor, but not limited thereto. The modified fusion protein disclosed herein can be used as an anti-angiogenic agent or an anti-VEGF agent to improve, prevent and / or treat cancer or cancer symptoms.

[0219] Method of administration and dosage

[0220] In one embodiment of the present disclosure, a method for delivering an effective amount of a modified fusion protein to a subject can be provided. The modified fusion protein can be delivered to a subject in the form of a composition. The modified fusion protein can also be delivered to a subject by a nucleic acid delivery vehicle or a viral particle (e.g., a recombinant viral vector) containing a nucleic acid encoding the modified fusion protein. In one embodiment of the present disclosure, a composition can be provided, the composition comprising a modified fusion protein or a nucleic acid delivery vehicle or a viral particle (e.g., a recombinant viral vector), the nucleic acid delivery vehicle or the viral particle comprising a nucleic acid encoding a modified fusion protein.

[0221] In one embodiment, the compositions of the present disclosure can be applied to an individual by any approach, including but not limited to: intravenous (e.g., by an infusion pump), intraperitoneal, intraocular, intraarterial, intrapulmonary, oral, inhalation, intravesical, intramuscular, intratracheal, subcutaneous, intraocular, intrathecal, transdermal, transpleural, intraarterial, local, inhalation (e.g., as a spray mist), mucosa (e.g., by nasal mucosa), subcutaneous, transdermal, gastrointestinal, intraarticular, intracisternal, intraventricular, intracranial, intraurethral, ​​intrahepatic and intratumoral. In one embodiment, the compositions of the present disclosure can be applied by any conventional route, for example, by infusion or bolus injection, by absorption via epithelial or mucocutaneous lining (e.g., oral mucosa, rectal mucosa, intestinal mucosa, etc.), and can also be applied together with other bioactive ingredients. Application can be systemic or local. In one embodiment, the compositions of the present disclosure may be introduced into the central nervous system by any suitable route, including intraventricular injection and intrathecal injection, wherein intraventricular injection may be administered via an intraventricular catheter, for example, connected to a reservoir such as an Ommaya reservoir.

[0222] In one embodiment, the optimal effective amount of the modified fusion protein, the nucleic acid delivery vehicle encoding the modified fusion protein, or the composition comprising the modified fusion protein can be determined empirically, and can depend on the type and severity of the disease, route of administration, disease progression and health status, individual body weight and body surface area. Such determination is within the technical scope of ordinary technicians in the field.

[0223] In one embodiment, the administration of the modified fusion protein can be, for example, about 0.05 ng to about 20 mg per kilogram of body weight per day. In one embodiment, the dosage of the modified fusion protein can be at least about 0.1 mg / kg, at least about 0.5 mg / kg, at least about 1.0 mg / kg, at least about 1.5 mg / kg, at least about 2.0 mg / kg, at least about 2.5 mg / kg, at least about 3.0 mg / kg, at least about 3.5 mg / kg, at least about 4.0 mg / kg, at least about 4.5 mg / kg, at least about 5.0 mg / kg, at least about 6.0 mg / kg, at least about 8.0 mg / kg, at least about 10.0 mg / kg, at least about 15.0 mg / kg, or about 2 In one embodiment, the dosage of the modified fusion protein can be about 1.0 mg / kg to about 10.0 mg / kg or about 1.0 mg / kg to about 5.0 mg / kg.

[0224] In one embodiment, the amount of nucleic acid delivery vehicle (eg, recombinant viral vector) containing a nucleic acid encoding a modified fusion protein of the present disclosure can be administered at about 10 μg / mL per dose. 4 to about 10 14 In one embodiment, the amount of nucleic acid delivery vehicle comprising a nucleic acid encoding a modified fusion protein may be administered to an individual at a dose of about 10 5 to about 10 13 , about 10 6 to about 10 12 , about 10 7 to about 10 11 , about 10 8 to about 10 10 , about 10 9 to about 10 10 , about 10 10 to about 10 11 or about 10 11 to about 10 12 The drp is administered to an individual.

[0225] In one embodiment, the composition comprising the modified fusion protein of the present disclosure or the nucleic acid delivery medium encoding the modified fusion protein can be applied daily with a single dose, or the daily total dose can be applied in divided doses of 2 times, 3 times or 4 times a day. In one embodiment, the composition comprising the modified fusion protein of the present disclosure can be applied 6 times a week, 5 times a week, 4 times a week, 3 times a week, 2 times a week, once a week, once every 2 weeks, once every 3 weeks, once a month, once every 2 months, once every 3 months, once every 6 months, once every 9 months, or once a year. In one embodiment, the composition comprising the nucleic acid delivery medium comprising the nucleic acid encoding the modified fusion protein of the present disclosure can be applied less frequently, for example, once every 3 months, once every 4 months, once every 5 months, once every 6 months, once every 7 months, once every 8 months, once every 9 months, once every 10 months, once every 11 months, or once a year.

[0226] Diagnosis or testing

[0227] The modified fusion proteins disclosed herein can be labeled with a detectable marker, such as a radioisotope, a fluorescent marker, a toxin marker, an enzyme marker, a chemiluminescent marker, or a nuclear magnetic resonance contrast agent to confirm ligand-receptor binding interactions. Detection systems applied to chimeric molecules can also be considered. Such detectable markers are well known in the art.

[0228] VI. Products and Kits

[0229] In one embodiment, the present disclosure relates to a product or kit, which contains a modified fusion protein of the present disclosure, a recombinant viral vector expressing the modified fusion protein, or a composition containing the above components, and is packaged appropriately. In one embodiment, the suitable packaging is well known in the art, for example, including vials (e.g., sealed vials), containers, ampoules, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), etc., but is not limited thereto. These products can be further sterilized and / or sealed.

[0230] In one embodiment, the kit may further include instructions for how to use the composition for purposes described herein. In one embodiment, the kit may further include other components required from a commercial and user perspective, such as other buffers, diluents, filters, needles, syringes, and package inserts (PIs) with instructions for performing any method described herein. For example, in one embodiment, the kit may include (i) fusion proteins as described herein and / or recombinant viral vectors encoding fusion proteins as described herein, (ii) a pharmaceutically acceptable carrier, and (iii) any one or more of the following: buffers, diluents, filters, needles, syringes, and package inserts with instructions for administration.

[0231] Hereinafter, the technical features and effects of the present disclosure will be described in more detail in conjunction with working examples. However, these examples are provided only for illustrative purposes to help understand the present disclosure, and the scope of the present disclosure is not limited by the following examples.

[0232] [Example 1] Protein production and evaluation of physicochemical properties and binding capacity

[0233] Example 1.1-Preparation of expression vector

[0234] The coding sequence region (CDS) of the modified fusion proteins designed in the following examples were codon optimized to a form suitable for CHO production cells and prepared by gene synthesis (Geneuniversal) in the final pcDNA3.1(+) vector (Invitrogen). The existing fusion proteins VEGF-Grab1 and VEGF-Grab3 were also produced using the same method.

[0235] Example 1.2 - Production of modified fusion proteins and evaluation of physicochemical properties

[0236] ExpiCHO for protein production TM Cells (Gibco) were used for transient expression at passages 10 to 20. At each step, VEGF-Grab1 or VEGF-Grab3 was cultured and purified under the same conditions and used as a control to determine the improvement of the physicochemical properties of each modified fusion protein. Transient expression of VEGF-Grab1, VEGF-Grab3 and the modified fusion proteins was performed according to the maximum titer protocol using Expifectamine ELISA provided by the manufacturer (Gibco).TM (Gibco). One day before transfection, TM The cells were subcultured to a cell density of 3.0 x 10 6 The next day, cell density and viability were determined again and ExpiCHO TM Dilute the cells to a cell density of 6.0 x 10 6 Cells / ml. Aliquot 25 ml of cell culture into 125 ml conical flasks and store the cells in an incubator at 37°C until the transfection mixture is prepared. Mix 25 μg of plasmid gene with 1 ml of OptiPro TM SFM medium was thoroughly mixed. 80 μl of Expifectamine TM CHO cells and 920 μl OptiPro TM SFM medium was mixed thoroughly and incubated at room temperature for 2 minutes. TM Add the SFM mixture to the previously mixed plasmid-OptiPro TM SFM, mix thoroughly, and leave at room temperature for 2 minutes. After 2 minutes, slowly add the mixture to the previously aliquoted cell culture and incubate in an incubator set at 37°C, 8% CO2, and 130 rpm. 18-22 hours after transfection, add 150 μl of Expifectamine TM CHO Enhancer and 4ml ExpiCHO TM The feed medium was mixed and added to the culture flasks, and the cell culture was continued in an incubator set at 32°C, 8% CO2 and 130 rpm. On day 5 post-transfection, an additional 4 ml of ExpiCHO TM Feed. Starting from the 6th day after transfection, the cell density and viability were determined by trypan blue staining using Countess II (Invitrogen). When the cell viability dropped to less than 70% to 80%, the final titer was determined using Cedex (Roche) equipment, and then centrifuged at a centrifugal force of 3000xg for 30 minutes to recover the cell culture containing protein. The recovered supernatant was filtered through a 0.22 μm syringe filter, and the culture solution was stored at -80°C.

[0237] Proteins were purified from the culture solution by affinity chromatography using an AKTA avant25 instrument equipped with a HiTrap Fibro PrismA column. The culture solution filtered through a 0.22 μm syringe filter was passed through the column at a rate of 16 ml / min, and then the column was washed with a buffer containing 50 mM Na-Pi, 50 mM NaCl (pH 7.0) for 15 column volumes. In order to remove non-specific binding, the column was first washed with a buffer containing 50 mM Na-Pi, 1 M NaCl (pH 5.5), followed by a buffer containing 50 mM Na-OAc (pH 4.6) for 15 column volumes. 15 column volumes of elution buffer containing 100 mM glycine (pH 3.0) were passed through the column to elute the bound protein, and the protein was collected in a deep well plate or a 15 ml test tube. For the collected protein, 1 M 1% of the collected protein volume was added. Alkaline (pH 11.0) solution was used to neutralize the protein solution, and the precipitate was removed using a 0.22 μm syringe filter. The samples before and after neutralization were quantified by UV measurement, analyzed by SE-HPLC, and the purity was determined. In order to determine the binding capacity, for samples with a purity of less than 90%, further purification was performed by size exclusion chromatography using a Superdex200 increase10 / 300 column. The protein was concentrated to a volume of less than 500 μ l in a 30 MWCO concentrator and then injected into a chromatographic column balanced with 250 mM K-Pi, 200 mM KCl (pH 6.2) or 250 mM K-Pi, 200 mM KCl (pH 5.5). The eluted protein was collected in a pipe and its purity was checked using SE-HPLC, which was then concentrated. The final concentrated protein was analyzed by UV quantitative analysis, and its purity was confirmed by SEC-HPLC and SDS-PAGE detection.

[0238] By the above method, one or more of the titer, elution amount, step yield, high molecular weight species (HMWS) content, low molecular weight species (LMWS) content and functional monomer content of each protein are determined as its physicochemical properties. Here, the titer refers to the concentration of the Fc fusion protein present in the culture medium measured when the cell culture is recovered; the elution amount refers to the total amount of protein recovered after the affinity chromatography as the first purification step. The yield refers to the value obtained by dividing the total amount of protein measured in the culture medium by the total amount of protein recovered after the first purification, and the yield represents the recovery rate of the protein recovered by the chromatographic column during the first purification. High molecular weight species (HMWS) are protein species with a mass greater than the predicted functional unit of the protein, and low molecular weight species (LMWS) are protein species with a mass less than the predicted functional unit of the protein. Both represent parts with abnormal protein folding. Functional monomers refer to parts with the structure and function of normal proteins.

[0239] Example 1.3 - Evaluation of Binding Affinity of VEGF-A and PLGF

[0240] According to previous literature (Kamat et al., 2017), The binding affinity of each protein to VEGF-A and PLGF ligands was determined using a RED96e (ForteBIO) instrument.

[0241] First, before determining the binding affinity, the AHC sensor was immersed in 1x KB buffer (Forte Bio) to stabilize the AHC sensor. The substances to be analyzed, i.e., VEGF-Grab1 or VEGF-Grab-3 and the modified fusion protein, flowed through at a concentration of 20 nM to bind to the sensor, and then washed with 1x KB buffer to remove the residual protein after binding. Then, VEGF-A and PLGF (VEGF-A: 40 nM, 20 nM, 10 nM; PLGF: 5 nM, 2.5 nM, 1.25 nM) were provided in 3 concentration ranges, respectively, to determine the binding affinity. In the binding affinity measurement results, the average value of the binding affinity measured at two concentrations was calculated, where the indicators of reliability Full X^2 ​​(3 or less) and Full R^2 (0.95 or more) were required to be within the acceptable range, and the obtained average value was used for relative comparison.

[0242] [Example 2] Design of modified fusion protein

[0243] Example 2.1 - Preparation and characterization of surface charge switching variants

[0244] In order to ensure that a fusion protein containing VEGFR receptor components with enhanced physicochemical properties is obtained, the inventors of the present disclosure used the structure of the entire extracellular domain of VEGFR1 (PDB entry .5t89) as a template, analyzed the surface charge distribution of the protein, and deduced the locations of positively charged dense areas and hydrophobic surface charges ( Figure 1a ).

[0245] The results showed that in the D3 domain of VEGFR1, the side chains of the amino acid residues in the β1-β2 loop (named as site 1) corresponding to amino acid residues T236 to T247, the β2-β3 loop (named as site 2) corresponding to amino acid residues T256 to V262, and the β5-β6 loop (named as site 3) corresponding to amino acid residues D299 to L308 were exposed on the surface of the fusion protein. Focusing on the amino acid residues exposed on the surface, the positions of the amino acid residues that can introduce mutations were screened and deduced by replacing the positions deduced from the surface charge distribution of the protein (Figures 1b and 1c). The researchers tried to stabilize the surface charge and structure of the fusion protein by changing the amino acid residues located in the flexible loop exposed on the surface. For example, an attempt was made to replace the amino acid residues exposed on the surface with amino acid residues present at the homologous position of VEGFR2. In addition, the amino acid residues at site 2 are exposed on the protein surface on the side that binds to the ligand (VEGF or PLGF), and mutations at this site affect the binding to the ligand or the solubility of the protein due to the surface exposure of the residues. Therefore, for the amino acid residue at position 2, attempts are made to replace the amino acid residue with, for example, an amino acid residue having a smaller side chain than the side chain of the existing amino acid residue or a polar side chain of similar size to the side chain of the existing amino acid residue, to an extent that does not significantly affect the protein structure or ligand binding.

[0246] As shown in Tables 32 and 33, modified fusion proteins in which mutations were introduced at different positions were prepared, and the mutation positions of each variant are shown in Table 1. Variants were prepared according to Example 1, and the physicochemical properties and binding affinity of the variants were evaluated. The physicochemical property evaluation results of the variants prepared based on the VEGFR wild-type protein (C88) are shown in Table 2, and the binding affinity evaluation results are shown in Tables 3 to 4. The physicochemical property evaluation results of the variants prepared based on VEGF-Grab3 are shown in Table 5, and the binding affinity evaluation results are shown in Table 6. The binding affinity evaluation results show the relative binding affinity evaluation results compared to VEGF-Grab3 (manufactured by Samsung Biologics).

[0247] Regarding the surface charge variations or positions of disulfide bonds listed in the following examples or tables, for variants C10 to C87 (SEQ ID NO: 10 to SEQ ID NO: 70), the positions of the variations and the amino acid residues are described based on the amino acid sequence of SEQ ID NO: 3, and for variants C97 to C114 and C119 (SEQ ID NO: 77 to SEQ ID NO: 93 and SEQ ID NO: 98), the positions of the variations and the amino acid residues are described based on the amino acid sequence of SEQ ID NO: 71.

[0248] Table 1

[0249]

[0250]

[0251] Table 2:

[0252]

[0253] Table 3

[0254]

[0255]

[0256] Table 4

[0257]

[0258]

[0259] Table 5

[0260]

[0261] Table 6

[0262]

[0263] With reference to the results in Tables 2 to 6, the results of titer and protein elution amount indicate that the modified fusion protein according to the embodiment of the present disclosure exhibits excellent productivity, and the results of functional monomers indicate that the modified fusion protein has excellent physicochemical properties. In addition, for example, the results of Kon et al. indicate that the modified fusion protein also exhibits excellent properties in terms of binding affinity to the ligand.

[0264] Example 2.2 - Preparation and characterization of linker variants

[0265] The human VEGF receptor binds to the ligand VEGF or PLGF through the interaction with the immunoglobulin-like domains D2 and D3 (see Figure 2a). For the fusion protein using the domain D2 and the domain D3 (e.g., VEGF-Grab3), the domain D2 and the domain D3 are in an open structure before binding to the ligand, and then form a closed structure when binding to the ligand (see Figures 2b and 2c). If the construct of the domain D2 and the domain D3 cannot ensure sufficient space to bind to the ligand according to the binding length of the domain D3 and the multimerization domain, or mutual interference occurs between the constructs, the binding affinity of the fusion protein to the ligand may be reduced. Therefore, the binding length and binding mode of the domain D3 and the multimerization domain in the fusion protein can affect the structure of the fusion protein when bound to the ligand and its binding affinity to the ligand.

[0266] On the other hand, for the human VEGF receptor in the native state, in the structure in which domain D3 and domain D4 are connected, the length from the end of domain D3 to the dimerization site of domain D4 is about (See Figure 2d), and for the human VEGF receptor, the domains from domain D4 and the downstream exist in a state where the same domains are bound to each other. Domain D4 and the downstream part can be regarded as a structure similar to the multimerization domain in the fusion protein. Therefore, the inventors changed the length and type of the linker in the modified fusion protein of the present invention to simulate and ensure the structural characteristics of the natural human VEGF receptor. Specifically, a linker composed of a polypeptide of a Gly-Ser repeating sequence, an amino acid sequence derived from the hinge region of IgG1, IgG4 or IgD, and a variant sequence of the hinge was introduced. Such a linker is intended to increase physical stability by reducing the tension generated in the loop (linker) formed between domain D3 and the multimerization domain (Fc region) when the ligand binds.

[0267] In addition, the amino acid sequence derived from the hinge region of the immunoglobulin has a cleavage site for multiple proteases due to its own characteristics (Vlasak and Ionescu. 2011. "Monoclonal Antibodies (Mabs)" 3: 253-263) and can be introduced into the fusion protein. After glycosylation, it can cause oligomerization through glycosylation such as O-glycosylation (Song et al. 2020. "Computational and Structural Biotechnology Journal" 18: 3925-3935). In order to improve the in vivo stability of the modified fusion protein disclosed in the present invention, the inventors further modified the amino acid sequence at the proteolytic enzyme cleavage site (e.g., the cleavage site of papain), and attempted to modify the glycosylated amino acid residues Ser or Thr. In addition, in order to increase the structural flexibility of the linker, it is also attempted to replace the cysteine ​​present in the sequence of the core hinge (e.g., CPPCP) derived from the Fc hinge region. Figure 3 An exemplary schematic diagram of the design of the linker introduced in the present disclosure is shown in FIG.

[0268] As shown in Tables 32 and 33, modified fusion proteins introducing linkers were prepared, and the linker properties of each variant are shown in Table 7. In Table 7, bold and underlined amino acids refer to amino acid residues at amino acid variation positions.

[0269] Variants were prepared according to Example 1, and the physicochemical properties and binding affinity of the variants were evaluated. The physicochemical property evaluation results of the variants prepared based on the VEGFR wild-type protein are shown in Table 8, and the binding affinity evaluation results are shown in Tables 9 to 10. The physicochemical property evaluation results of the variants prepared based on VEGF-Grab3 are shown in Table 11, and the binding affinity evaluation results are shown in Tables 12 and 13. Table 12 shows the results of the relative binding affinity evaluation compared with VEGF-Grab3 (manufactured by Panolos Bioscience), and Table 13 shows the results of the relative binding affinity evaluation compared with VEGF-Grab3 (manufactured by Samsung Bioscience). When the relative binding affinity evaluation is performed with C88 or VEGF-Grab3 (manufactured by Panolos Bioscience), the protein produced in the same batch as the variant is used for comparison under the same conditions.

[0270] Table 7

[0271]

[0272]

[0273] Table 8

[0274] Protein code HMWS(%) Functional monomer content (%) LMWS(%) C88 70.1 22.5 7.4 C108 61.9 25.1 13.1 C109 65.2 26.9 7.9 C111 65.9 27.7 6.5 C115 65.4 18.5 16.1 C116 70.1 24.0 5.9 C117 78.0 15.2 6.7 C118 68.7 25.0 6.3

[0275] Table 9

[0276]

[0277] Table 10

[0278]

[0279] Table 11

[0280]

[0281] Table 12

[0282]

[0283]

[0284] Table 13

[0285]

[0286] With reference to the results in Tables 8 to 13, the results of titer and protein elution amount show that the modified fusion protein according to the embodiment of the present disclosure exhibits excellent productivity, and the functional monomer results show that the modified fusion protein has excellent physicochemical properties. In addition, for example, the results of Kon et al. show that the modified fusion protein also exhibits excellent properties in terms of binding affinity with the ligand.

[0287] On the other hand, for variants (C115 to C118) having linker sequences that deviate from the linker lengths of the linker variants according to the embodiments of the present disclosure, the results show that the variants exhibit significantly reduced titers or functional monomer ratios compared to the modified fusion proteins of the present disclosure (e.g., variant C108).

[0288] Example 2.3 - Preparation and characterization of disulfide bond variants

[0289] Since the fusion protein is a combination of heterologous proteins, it is expected that stability will be reduced due to nonspecific cleavage at the attachment site. The end of domain D3 is a looped portion that may lack secondary structure and is used as the site where the multimerization domain (a heterologous protein) begins to attach, and is expected to have poor stability. By introducing a covalent disulfide bond, the inventors aim to induce conjugation between domains near the attachment site of the heterologous protein, thereby preventing physical breakage of the protein and improving its stability.

[0290] The positions where disulfide bonds could be introduced were identified by tertiary structure-based analysis. Among the residues located in the variable loop adjacent to the terminal region of domain D3, those whose side chains extended toward the linker and whose terminal atoms were within 1.5 m from the Cα atom of Y329 were selected. The residues within 100 μg / mL were used as the variant residues for forming disulfide bonds. After ensuring the three-dimensional structure of the fusion protein by a structural modeling program (Phyre2, Swiss Model), the structural model of the disulfide bond was obtained by computer simulation of the introduced site. Thereafter, the change in the energy level of the fusion protein caused by the introduction of the disulfide bond was predicted by a tertiary structure energy prediction program (FoldX in YASARA, Schymkowitz et al. 2005 "Nucleic Acids Research". 33: W382-388).

[0291] As positions that are physically close to the end of domain D3 in the fusion protein and capable of forming a disulfide bond, amino acid residues on the β1-β2 loop or the β5-β6 loop of domain D3 were selected, while for the end of domain D3, amino acid residues present at positions -2, -1, 0, +1, +2 and +3 relative to Y329 were selected.

[0292] As shown in Tables 32 and 33, modified fusion proteins introducing disulfide bonds were prepared, and the disulfide bond characteristics of each variant are shown in Table 14. The energy values ​​of each variant calculated by the tertiary structure energy prediction program are shown in Table 15. Variants were prepared according to Example 1, and the physicochemical properties and binding affinity of the variants were evaluated. The physicochemical property evaluation results of the variants prepared based on the VEGFR wild-type protein are shown in Table 16, and the binding affinity evaluation results are shown in Tables 17 to 18. The physicochemical property evaluation results of the variants prepared based on VEGF-Grab3 are shown in Table 19, and the binding affinity evaluation results are shown in Tables 20 and 21. Table 20 shows the results of the relative binding affinity evaluation compared to VEGF-Grab3 (manufactured by PanolosBioscience), and Table 21 shows the results of the relative binding affinity evaluation compared to VEGF-Grab3 (manufactured by Samsung Bio). When relative binding affinity was assessed with VEGF-Grab3 (manufactured by Panolos Bioscience), comparison was made with proteins produced in the same batch as the variants under the same conditions.

[0293] Table 14

[0294]

[0295]

[0296] Table 15

[0297] Protein code Protein structure energy value (kcal / mol) C37 322.64 C47 320.01 C48 323.96 C49 326.73 C50 315.08 C51 320.02

[0298] Table 16

[0299]

[0300] Table 17

[0301]

[0302] Table 18

[0303]

[0304]

[0305] Table 19

[0306]

[0307] Table 20

[0308]

[0309] Table 21

[0310]

[0311]

[0312] Referring to the results in Tables 16 to 21, the results of the titer and protein elution amount indicate that the modified fusion protein according to the embodiment of the present disclosure exhibits excellent productivity, and the functional monomer results indicate that the modified fusion protein has excellent physicochemical properties. This is also demonstrated by the calculation results of the energy level of the structural model. According to Table 15, the energy value of the fusion protein structure with the introduction of disulfide bonds is equal to or lower than the energy value of the structure without the introduction of disulfide bonds, which indicates that the stability of the structure can be ensured by introducing disulfide bonds. In addition, for example, the results of kon et al. show that the modified fusion protein also exhibits excellent properties in terms of binding affinity with the ligand.

[0313] Example 2.4 - Preparation and characterization of combinatorial variants

[0314] Composition variants were prepared, and the composition variants had a combination of the properties of the variants that showed relatively good properties among the variants obtained from Examples 2.1 to 2.3. The properties of each variant are shown in Table 22. Variants were prepared according to Example 1, and the physicochemical properties and binding affinity of the variants were evaluated. The physicochemical property evaluation results of the variants prepared based on the VEGFR wild-type protein are shown in Table 23, and the binding affinity evaluation results are shown in Tables 24 to 25 (as a comparison, the VEGFR wild-type protein (C88) and variants C65 and C73 were evaluated together). The physicochemical property evaluation results of the variants prepared based on VEGF-Grab3 are shown in Table 26, and the binding affinity evaluation results are shown in Tables 27 and 28. Table 27 shows the results of the relative binding affinity evaluation compared to VEGF-Grab3 (manufactured by Panolos Bioscience), and Table 28 shows the results of the relative binding affinity evaluation compared to VEGF-Grab3 (manufactured by Samsung Bio). When relative binding affinity was assessed with VEGF-Grab3 (manufactured by Panolos Bioscience), comparison was made with protein produced in the same batch as the variant under the same conditions.

[0315] Table 22

[0316]

[0317]

[0318] Table 23

[0319]

[0320] Table 24

[0321]

[0322] Table 25

[0323]

[0324] Table 26

[0325]

[0326]

[0327] Table 27

[0328]

[0329]

[0330] Table 28

[0331]

[0332]

[0333] With reference to the results in Tables 23 to 28, the results of titer and protein elution amount indicate that the modified fusion protein according to the embodiments of the present disclosure exhibits excellent productivity, and the functional monomer results indicate that the modified fusion protein has excellent physicochemical properties. In addition, for example, the results of Kon et al. show that the modified fusion protein also exhibits excellent properties in terms of binding affinity to the ligand. These results indicate that the characteristics introduced during the structural design of the modified fusion protein in Examples 2.1 to 2.3 are effective, and the combination of these characteristics produces a synergistic effect.

[0334] Example 2.5 - Preparation and characterization of other variants

[0335] In the fusion protein, the amino acid sequence DKA located at the end of D3 is structurally the site where the connection with the multimerization domain begins, and because the amino acid sequence is connected to the linker to form a loop, the amino acid sequence is a physically weak site and is easily broken during protein expression and purification. During protein expression and purification, the presence of physically weak areas may lead to the generation of fragments. Therefore, in the above-mentioned Examples 2.1 to 2.3, modified fusion proteins in which KA is deleted from the end of domain D3 in most sequences were prepared. In order to determine that the deletion of the amino acid sequence at the end of domain D3 does not affect the improvement of the physicochemical properties of the modified fusion protein of the present disclosure, deletion variants involving the deletion of the amino acid sequence KALE (the site where the C-terminus of domain D3 is connected to the linker) were prepared, and the physicochemical properties of the deletion variants were determined. Specifically, a variant (C89) in which the amino acid KA is deleted from the VEGFR wild-type protein (C88) and a variant (C90 and C91) in which KA and KAL are deleted from VEGF-Grab3 were prepared.

[0336] When a fusion protein containing the VEGFR1 extracellular domain is produced, the N-terminal sequence of domain D2 may vary depending on the type or characteristics of the expression vector. In order to determine that such variation does not affect the physicochemical properties of the fusion protein, variants with amino acid variations at the N-terminus of domain D2 were prepared. Specifically, considering that the variants in Examples 2.1 to 2.3 all contain the amino acid sequence EF derived from the vector at the N-terminus of domain D2, variants (C4 and C5) containing the amino acid sequence EF, variants (C8 and C9) lacking the amino acid sequence EF, and variants (C6 and C7) starting with SDT containing the original VEGFR1 sequence at the N-terminus were prepared.

[0337] In order to determine that the amino acid substitution D126E, amino acid substitution L128M and / or amino acid deletion K217 based on SEQ ID NO: 99 in the multimerization domain used to generate the fusion protein does not affect the physicochemical properties or therapeutic efficacy of the fusion protein, variants were made. Specifically, a variant (C95) having an amino acid substitution D126E and an amino acid substitution L128M and a variant (C96) having an amino acid deletion K217 were prepared based on VEGF-Grab3.

[0338] As shown in Table 33, modified fusion proteins introducing the above-mentioned additional mutations were prepared. Variants were prepared according to Example 1, and the physicochemical properties, serum stability or VEGF-A inhibitory ability of the variants were evaluated. Physicochemical properties were evaluated according to the method described in Example 1.2, serum stability was evaluated according to the method described in Example 3, and VEGF-A inhibitory ability was evaluated according to the method described in Example 6. The evaluation results are shown in Tables 29 to 31 and Figure 4.

[0339] Table 29

[0340]

[0341] Table 30

[0342]

[0343] Table 31

[0344]

[0345] The results in Tables 29 to 31 indicate that variations at the end of domain D3, variations at the N-terminus of domain D2, and variations in the multimerization domain do not affect the improvement of the physicochemical properties, serum stability, or VEGF-A inhibitory ability of the modified fusion proteins.

[0346] The properties of the variants described in Examples 2.1 to 2.4 above are summarized in Table 32 below.

[0347] Table 32

[0348]

[0349]

[0350]

[0351]

[0352] Table 33 lists the identification codes, amino acid sequences and SEQ ID NOs of the proteins described in the present disclosure.

[0353] Table 33

[0354]

[0355]

[0356]

[0357]

[0358]

[0359]

[0360]

[0361]

[0362]

[0363]

[0364]

[0365]

[0366]

[0367]

[0368]

[0369]

[0370]

[0371] [Example 3] Stability evaluation in a blood simulation environment

[0372] In an indirect analysis of the in vivo stability of the modified fusion protein disclosed herein, the concentration of the protein variant remaining at a specific time point after the modified fusion protein variant reacted with blank serum collected from rats was measured by ELISA. VEGF-Grab3 (a modified fusion protein) and Aflibercept (SANOFI; SEQ ID NO: 101) were mixed with rat blank serum to a final concentration of 10 μg / ml, respectively, and stored in an incubator at 37°C 168 hours, 96 hours, 48 ​​hours, 24 hours, 4 hours, 2 hours, 1 hour and 0 hours before the start of the test. One day before the test, human VEGF-A (R&D systems) was coated on an ELISA plate and incubated overnight at 4°C, and then washed with 0.1% PBS-T washing buffer to remove insufficiently bound human VEGF-A. Afterwards, a blocking treatment was performed with 0.1% PBS-T containing 5% skim milk for 1 hour at room temperature. VEGF-Grab3 and aflibercept were serially diluted in the concentration range of 50 μg / ml to 0.39 μg / ml to prepare standards for concentration comparison. The serial dilutions of the standards and the protein-serum mixtures prepared at different time points were diluted 200 times with blocking buffer, and 100 μl of each was added to the wells of the ELISA plate and reacted at room temperature for 2 hours. Subsequently, all the reaction solutions in the wells were removed and the ELISA plate was washed with washing buffer. The detection antibody HRP-labeled goat anti-human IgG Fc cross-adsorbed secondary antibody (Invitrogen) was reacted at room temperature for 1 hour and then washed to remove the unreacted detection antibody. Then, 100 μl of 3,3',5,5'-tetramethylbenzidine (TMB) solution (Sigma) was added to each well and reacted at room temperature for 10 minutes, and then the stop solution (Sigma) was added to stop the reaction. The absorbance was measured at 450 nm using an ELISA reader. A standard curve was drawn based on the absorbance of the measured standards (VEGF-Grab3 and Aflibercept), and the remaining amount of the modified fusion protein was calculated for relative comparison. From the calculated results, the ratio of the concentration at 168 hours (after 7 days) to the concentration at 0 hours was calculated, and the relative absorbance % is shown in Table 34, Table 35 and FIG. 5 .

[0373] Table 34

[0374] Protein code Relative absorbance of variants (after 7 days) C88 6.6 VEGF-Grab3 (from Panolos Bioscience) 12.2 VEGF-Grab3 (from Samsung Biopharma) 35.5 VEGF-Grab1 (from Panolos Bioscience) 34.4 C98 62.7 C119 16.1 C100 49.9 C101 5.5 C102 13.4 C104 83.3 C105 74.0 C107 61.6 C108 8.5 C109 6.7 C111 8.6 C112 84.0 C113 97.7 C114 99.2 C115 8.8 C116 5.8 C118 6.9 C65 103.1 C73 105.1

[0375] Table 35

[0376]

[0377]

[0378] According to Table 34 and Figure 5, compared with the VEGFR wild-type protein, the modified fusion protein of the present invention shows superior serum stability. The serum stability of the variant C112 introduced with a disulfide bond is significantly better than that of the variant C109 (6.7%) having the same linker sequence but without the introduction of a disulfide bond. Compared with the VEGFR wild-type protein, variants C115, C116 and C118 (their linker sequences deviate from the linker length of the linker variant in the embodiment of the present disclosure) do not show superior serum stability.

[0379] According to Table 35, the modified fusion protein of the present disclosure showed a serum stability of more than about 97%, which indicates that its serum stability is equivalent to or better than that of commercially available drugs. In all these experiments, no protein degradation was observed during the experimental period (7 days). From these results, it can be seen that the modified fusion protein disclosed in the present invention has excellent stability in vivo and is comparable to In comparison, its stability is equivalent to that of the drug product. It can be indirectly predicted that when the modified fusion protein of the present disclosure is introduced into the body, it can improve the therapeutic effect by prolonging the retention time of the protein.

[0380] [Example 4] Isoelectric point analysis

[0381] icIEF (imaged capillary isoelectric focusing) was used to analyze the isoelectric points of VEGF-Grab3 and the modified fusion proteins disclosed herein. 40 μl of the modified fusion protein diluted to a concentration of 2 mg / ml and 160 μl of a pre-prepared master mix solution (SERVALYT TM+ low pI marker (5.85) (ProteinSimple) + high pI marker (9.99) (ProteinSimple) + 1% methylcellulose (ProteinSimple) + deionized water (DW) + 500mM arginine (Sigma) + 200mM iminodiacetic acid (Sigma) + 10M urea (Sigma)) mixed, centrifuged to remove precipitates and bubbles, and then injected into each well of the instrument (Maurice, ProteinSimple). Take 2ml of the cathode electrolyte solution and the anode electrolyte solution respectively and place them at the OH- and H+ positions of the cartridge, and install the cartridge on the instrument. Afterwards, measure the isoelectric point according to the operating manual of the instrument. Since the measured pI does not appear in a single peak form, but in a multi-peak form within a certain range, the pI distribution of the protein is divided into three intervals for definition: acidic (pI 6-7), neutral (pI7-8) and alkaline (pI>8). The highest percentage distributions were compared in each interval. The results are shown in Table 36 and Figures 6 and 7.

[0382] Table 36

[0383] total% Acidic neutral Alkaline sum VEGF-Grab3 (from Panolos Bioscience) 18.6 81.3 99.9 VEGF-Grab3 (from Samsung Biopharma) 48.6 51.3 99.9 C61 74.5 25.5 100 C62 51 29.8 19.3 100.1 C64 48.8 29.2 21.8 99.8 C65 96.8 3.4 100.2 C69 66.5 28.1 5.5 100.1 C71 57.6 42.5 100.1 C72 51.3 48.7 100 C73 71.1 28.8 99.9 Aflibercept 61.8 38.2 100

[0384] As shown in Table 36 and Figures 6 and 7, it is shown that the existing material VEGF-Grab3 has a wider and higher pI than aflibercept. The highest proportion segment of the charge variant is the basic segment (pI>8.0), which is in sharp contrast to aflibercept, which has the highest distribution in the acidic segment (pI 6.0-7.0). For the modified fusion protein of the present disclosure, its distribution in the basic segment is reduced (C62) or completely disappeared (C61, C65, C71, C72, C73), and the segment with the largest distribution moves to the acidic segment. Therefore, it is expected that the modified fusion protein of the present disclosure will reduce nonspecific interactions with the matrix and cell surface. In addition, due to the reduction of such nonspecific interactions, the intracellular recycling of the modified fusion protein of the present disclosure may be improved, and the in vivo half-life and therapeutic efficacy of the modified fusion protein of the present disclosure may also be improved.

[0385] [Example 5] Thermal stability analysis

[0386] VEGF-Grab3 and the modified fusion proteins of the present disclosure (C62, C72, C73) were diluted to a final concentration of 2 mg / ml using 1x PBS buffer. 2.5 μl of 8X Protein Thermal Shift TMDye (Life Technologies) and 5.0 μl Protein Thermal Shift TM Buffer (Life Technologies) was added to 12.5 μl of the protein solution to prepare the final reaction solution. 20 μl of the prepared reaction solution was placed in a PCR tube and placed in the QuantStudio TM The Tm value was measured on the tray of the real-time PCR device. All analyses were repeated four times. The temperature curve diagram and Tm1 value, Tm2 value and Tm3 value of each protein are shown in Figure 8 and Table 37.

[0387] Table 37

[0388] Protein code Tm1 Tm2 Tm3 VEGF-Grab3 41.83 48.50 67.55 C62 61.51 - - C72 55.12 - - C73 52.90 - -

[0389] As shown in FIG8 and Table 37, the existing material VEGF-Grab3 was observed to have melting points in three intervals: Tm1 = 41.83° C., Tm2 = 48.50° C., and Tm3 = 67.55° C. For each of the modified fusion proteins (C62, C72, and C73) in the modified fusion proteins of the present disclosure, only a single Tm point was observed, and the Tm was increased by at least 11° C. and at most 20° C. compared to the first Tm of VEGF-Grab3. This indicates that the modified fusion proteins of the present disclosure have excellent thermal stability.

[0390] [Example 6] Analysis of VEGF-A inhibitory ability

[0391] To quantify the inhibitory capacity of VEGF-A, a genetically engineered cell line (KDR / NFAT-RE HEK293, Promega) was used, into which a luciferase reporter system based on VEGFR-2 (KDR) expression and VEGF-A / VEGFR-2 interaction had been introduced. 6 Cell suspensions of 10 cells / ml were added to a 96-well plate, and then 25 μl of VEGF-Grab3 or the modified fusion protein of the present invention diluted to different concentrations was added to react in an incubator at 37°C, 5% CO2 for 6 hours. The plate was then cooled to room temperature for 15 minutes, and then 75 μl of Bio-Glo reagent (Promega, luciferase assay buffer + substrate mixture) was added. After reacting for 10 minutes in a light-shielded state, the luminescence intensity was measured using an ELISA reader. The IC values ​​of each substance were calculated by GraphPad Prism analysis. 50The values ​​were obtained and the results are shown in Tables 38 to 41 and Figure 9. In Figure 9c, the graphs of VEGFA stimulation and VEGF stimulation show the results when no variants were added and only VEGFA or VEGF was used for treatment.

[0392] The data in Tables 38 to 41 respectively describe the relevant values ​​of proteins produced under the same conditions and batches, and therefore, the relevant values ​​of VEGF-Grab3 (manufactured by Panolos Bioscience) used as a control are listed respectively.

[0393] Table 38

[0394]

[0395]

[0396] Table 39

[0397] Protein code <![CDATA[IC 50 (μg / ml)]]> VEGF-Grab3 (from Panolos Bioscience) 0.04353 C61 0.03218 C62 0.02953 C63 0.03275 C64 0.03324 C65 0.04045 C66 0.03915 C67 0.02917 C68 0.03036 C69 Undetermined C70 0.0349 C71 0.03073 C72 0.03669 C73 0.02925

[0398] Table 40

[0399]

[0400]

[0401] Table 41

[0402] Protein code <![CDATA[IC 50 (μg / ml)]]> VEGF-Grab3 (from Panolos Bioscience) 0.003805 C81 0.00268 C84 0.006157

[0403] According to FIG. 9 and Tables 38 to 41, it can be seen that both VEGF-Grab3 and the modified fusion protein of the present disclosure exhibit low IC 50 For the variants C115 and C116 whose linker sequences deviate from the linker length described in the embodiments of the present disclosure, they do not show VEGF-A inhibitory ability superior to that of the VEGFR wild-type protein.

[0404] According to the above description, it will be understood by those skilled in the art that the present disclosure may be implemented in other specific forms without changing the technical concept or basic features of the present disclosure. In this regard, the above embodiments should be understood as exemplary and non-restrictive in all aspects. The scope of the present disclosure should be interpreted according to the meaning and scope of the claims, rather than the detailed description above, and includes all variations or modifications of equivalent concepts.

Claims

1. A modified fusion protein comprising a VEGFR1 extracellular domain, a linker and a multimerization domain, wherein the VEGFR1 extracellular domain comprises an immunoglobulin (Ig)-like domain D2 and an Ig-like domain D3 of VEGFR1, and the linker is located between the Ig-like domain D3 and the multimerization domain, and Wherein the modified fusion protein has one or more of properties (a) to (c): (a) one or more amino acid residues in the β1-β2 loop of domain D3, one or more amino acid residues in the β2-β3 loop of domain D3, and / or one or more amino acid residues in the β5-β6 loop of domain D3 are substituted with an amino acid that reduces the net pI of the protein, an amino acid with a negatively charged side chain, or an amino acid with an electronegative side chain, in, The β1-β2 loop of domain D3 includes amino acid residues T236 to T247 of the amino acid sequence set forth in SEQ ID NO:1, the β2-β3 loop includes amino acid residues T256 to V262 of the amino acid sequence set forth in SEQ ID NO:1, and the β5-β6 loop includes amino acid residues D299 to L308 of the amino acid sequence set forth in SEQ ID NO:1; (b) the linker has a length of about 13 to about 35 amino acids; and (c) a disulfide bond is present in the fusion protein, and one of the amino acid residues on the β1-β2 loop of domain D3 or one of the amino acid residues on the β5-β6 loop of domain D3 and one of the amino acid residues at positions -2, -1, 0, +1, +2 and +3 relative to Y329 of domain D3 is substituted with cysteine.

2. The modified fusion protein according to claim 1, wherein the amino acid substitution in property (a) is a substitution with: The amino acid residues of the VEGFR2 homologous sequence, an amino acid residue of serine, threonine, tyrosine, cysteine, asparagine, glutamine, aspartic acid or glutamic acid, or An amino acid residue of alanine or glycine with a short side chain.

3. The modified fusion protein according to claim 1, wherein in characteristic (a), one or more amino acid residues on the β1-β2 loop of domain D3 and one or more amino acid residues on the β5-β6 loop of domain D3 are substituted by the following amino acids: amino acids that reduce the net pI of the protein, amino acids with negatively charged side chains, or amino acids with electronegative side chains.

4. The modified fusion protein according to claim 3, wherein in characteristic (a), one or more amino acid residues on the β2-β3 loop of domain D3 are substituted by amino acid residues having side chains smaller than the side chains of existing amino acid residues or by amino acid residues having polar side chains similar in size to the side chains of existing amino acid residues.

5. The modified fusion protein of claim 1, wherein property (a) is a substitution of: one or more of amino acid residues K241, L243, R244 and H246 on the β1-β2 loop of domain D3; Amino acid residue L258 on the β2-β3 loop of domain D3; and / or One or more of amino acid residues K300, Q302, K304 and K306 on the β5-β6 loop of domain D3.

6. The modified fusion protein of claim 5, wherein property (a) comprises: one or more amino acid substitutions selected from the group consisting of K241T, K241E, L243S, R244V and H246E on the β1-β2 loop of domain D3; Amino acid substitution L258A, L258S or L258D on the β2-β3 loop of domain D3; and / or One or more amino acid substitutions selected from K300G, Q302T, K304S and K306Q on the β5-β6 loop of domain D3.

7. The modified fusion protein of claim 6, wherein property (a) comprises: (i) amino acid substitutions L243S and R244V on the β1-β2 loop of domain D3, or (ii) amino acid substitutions K241E, L243S, R244V and H246E on the β1-β2 loop of domain D3; Amino acid substitution L258A, L258S or L258D on the β2-β3 loop of domain D3; and / or (i) amino acid substitution K300G on the β5-β6 loop of domain D3, (ii) amino acid substitution K300G, Q302T and K304S on the β5-β6 loop of domain D3, or (iii) amino acid substitution K300G, Q302T, K304S and K306Q on the β5-β6 loop of domain D3.

8. The modified fusion protein of claim 7, wherein property (a) comprises: Amino acid substitutions K241E, L243S, R244V and H246E on the β1-β2 loop of domain D3; Amino acid substitution L258A, L258S or L258D on the β2-β3 loop of domain D3; and The amino acid substitutions on the β5-β6 loop of domain D3 are K300G, Q302T and K304S.

9. The modified fusion protein of claim 1, wherein the modified fusion protein comprises amino acid substitutions R238S and R275N in domain D3.

10. The modified fusion protein according to claim 1, wherein the amino acid sequence of the C-terminus of domain D3 connected to the linker is KALE based on amino acid residue K331, with the proviso that one or more of the amino acids K, A, L and E may be deleted. The modified fusion protein according to claim 1 , wherein the N-terminus of the domain D2 starts with the amino acid sequence SDT or the amino acid sequence EF.

12. The modified fusion protein of claim 1, wherein in property (b), the linker has a length of about 14 or more amino acids.

13. The modified fusion protein according to claim 1, wherein in characteristic (b), The linker comprises a GS repeat sequence, and The GS repeat sequence is an amino acid sequence consisting of only G and S and is 2 to 60 amino acids long, and the N-terminal amino acid of the linker may be glutamic acid.

14. The modified fusion protein according to claim 13, wherein the GS repeat sequence is (GS) n , (GSSG) n 、(GGGGS) n or (GS) m (GGGGS) n , wherein n is an integer from 1 to 10, and m is an integer from 0 to 10.

15. The modified fusion protein of claim 14, wherein the GS repeat sequence is selected from the group consisting of GS, GSSG, (GSSG)2, GGGGS, (GGGGS)4, GS(GGGGS) and GS(GGGGS)3. 16 . The modified fusion protein according to claim 1 , wherein in characteristic (b), the linker comprises an amino acid sequence derived from a hinge region of an immunoglobulin, and the amino acid sequence of the hinge region can be modified. 17 . The modified fusion protein according to claim 16 , wherein the hinge region derived from an immunoglobulin comprises a sequence derived from a CH1 region, an upper hinge and / or a core hinge.

18. The modified fusion protein according to claim 17, wherein the hinge region derived from immunoglobulin is derived from human IgA, IgD, IgM, IgE or IgG.

19. The modified fusion protein according to claim 16, wherein in characteristic (b), the papain recognition site or the glycosylation site present in the hinge region derived from immunoglobulin has an amino acid variation.

20. The modified fusion protein according to claim 19, wherein in characteristic (b), The amino acid variation in the papain recognition site is to replace one or more amino acid residues in the papain recognition site with alanine, serine, tyrosine, proline or threonine, or to insert an amino acid sequence of 1 to 10 amino acids long containing at least one amino acid having an aromatic or cyclic carbon in the side chain into the papain recognition site, and The amino acid variation in the glycosylation site is (i) deleting the serine or threonine present in the hinge region derived from the immunoglobulin, or (ii) substituting the serine or threonine present in the hinge region derived from the immunoglobulin with an amino acid other than serine, asparagine or threonine.

21. The modified fusion protein of claim 20, wherein the amino acid other than serine, asparagine or threonine is glycine or alanine.

22. The modified fusion protein of claim 17, wherein the cysteine ​​present in the core hinge is substituted with serine or glycine.

23. The modified fusion protein of claim 1, wherein in characteristic (b), the linker comprises, starting from the N-terminus, in the following order: (i) an amino acid sequence selected from the group consisting of GS, GSSG, (GSSG)2, GGGGS, (GGGGS)4, GS(GGGGS) and GS(GGGGS)3; and (ii) a hinge region derived from an immunoglobulin selected from the group consisting of human IgG1, IgG4 or IgD / G1, The amino acid sequence of the hinge region may be modified.

24. The modified fusion protein according to claim 1, wherein in characteristic (b), The linker is an amino acid sequence selected from the group consisting of: GSSGDKTHTSPPSP, GSSGEPKSSDKTYTSPPSP, GSKVDKKVEPKSSDKTHTCPPCP, GSKVDKKVEPKSSDKTYTCPPCP, GSKVDKKVEPKSSDTPPTCPPCP, GSGGGGSGGGGSGGGGSAESKYGPPCPPCP, GSNTGSGGEEKKKEKEKEEQEERSSDKTYTCPPCP, GSNTGSGGEEKKKEKEKEEQEERSSDTPPTTCPPCP, EGSSGGSSGEPKSDATPTCPPCP, EGSSGGSSGEPKSDSTYTCPPCP, GCKVDKKVEPKSSDKTYTCPPCP, CSKVDKKVEPKSSDKTYTCPPCP, GGGGSAEPKAGDKAPPGPPGP,GGGGSAEPKSSDKTYTCPPCP, GGGGSGGGGSGGGGSGGGGSAEPKSSDKTYTCPPCP, CSSGDATPTSPPSP, CSKVDKKVEPKSSDTPPTCPPCP, CSGGGGSAEPKAGDATPPTCPPCP, CSGGGGSGGGGSGGGGSAESKYGPPCPPCP, CSNTGSGGEEKKKEKEKEEQEERSSDTPPTTCPPCP, CGSSGGSSGEPKSDATPTCPPCP and CSGGGGSAEPKAGDATPPTCPPCPPCP.

25. The modified fusion protein according to claim 1, wherein in property (c), the one of the amino acid residues on the β1-β2 loop is one of the amino acid residues on the amino acid sequence of V240 to T247.

26. The modified fusion protein according to claim 1, wherein in characteristic (c), One of the amino acid residues T241, L243 and R244 on the β1-β2 loop of the domain D3 or the amino acid residue N303 on the β5-β6 loop is substituted with cysteine, and One of the amino acid residues at positions 0, +2 and +3 relative to Y329 of domain D3 is substituted by cysteine.

27. The modified fusion protein of claim 26, wherein in characteristic (c), T241 and the amino acid residue at position +3 relative to Y329 of domain D3 were substituted with cysteine; The amino acid mutations L243C and Y329C were introduced; L243 and the amino acid residue at position +2 relative to Y329 of domain D3 were substituted with cysteine; introducing the amino acid variants R244C and Y329C; or Amino acid mutations N303C and Y329C were introduced.

28. The modified fusion protein of claim 1, wherein the modified fusion protein has all of the following properties (a) to (c): (a) Introduction: one or more amino acid substitutions selected from the group consisting of K241T, K241E, L243S, R244V and H246E on the β1-β2 loop of domain D3, Amino acid substitutions L258A, L258S or L258D on the β2-β3 loop of domain D3, and / or one or more amino acid substitutions selected from the group consisting of K300G, Q302T, K304S and K306Q on the β5-β6 loop of domain D3; (b) the linker is an amino acid sequence selected from the group consisting of: GSSGDKTHTSPPSP, GSSGEPKSSDKTYTSPPSP, GSKVDKKVEPKSSDKTHTCPPCP, GSKVDKKVEPKSSDKTYTCPPCP, GSKVDKKVEPKSSDTPPTCPPCP, GSGGGGSGGGGSGGGGSAESKYGPPCPPCP, GSNTGSGGEEKKKEKEKEEQEERSSDKTYTCPPCP, GSNTGSGGEEKKKEKEKEEQEERSSDTPPTTCPPCP, EGSSGGSSGEPKSDATPTCPPCP, EGSSGGSSGEPKSDSTYTCPPCP, GCKVDKKVEPKSSDKTYTCPPCP, CSKVDKKVEPKSSDKTYTCPPCP, GGGGSAEPKAGDKAPPGPPGP,GGGGSAEPKSSDKTYTCPPCP, GGGGSGGGGSGGGGSGGGGSAEPKSSDKTYTCPPCP, CSSGDATPTSPPSP, CSKVDKKVEPKSSDTPPTCPPCP, CSGGGGSAEPKAGDATPPTCPPCP, CSGGGGSGGGGSGGGGSAESKYGPPCPPCP, CSNTGSGGEEKKKEKEKEEQEERSSDTPPTTCPPCP, CGSSGGSSGEPKSDATPTCPPCP and CSGGGGSAEPKAGDATPPTCPPCPPCP; and (c) one of the amino acid residues T241, L243 and R244 on the β1-β2 loop of domain D3 or the amino acid residue N303 on the β5-β6 loop is substituted with cysteine, and One of the amino acid residues at positions 0, +2 and +3 relative to Y329 of domain D3 is substituted by cysteine.

29. The modified fusion protein of claim 1, wherein the modified fusion protein has all of the following properties (a) to (c): (a) Introduction: (i) amino acid substitutions L243S and R244V on the β1-β2 loop of domain D3, or (ii) amino acid substitutions K241E, L243S, R244V and H246E on the β1-β2 loop of domain D3, amino acid substitutions L258A, L258S or L258D on the β2-β3 loop of domain D3, and / or (i) amino acid substitution K300G on the β5-β6 loop of domain D3, (ii) amino acid substitutions K300G, Q302T and K304S on the β5-β6 loop of domain D3, or (iii) amino acid substitutions K300G, Q302T, K304S and K306Q on the β5-β6 loop of domain D3; (b) the linker is an amino acid sequence selected from the group consisting of: CSSGDATPTSPPSP, CSKVDKKVEPKSSDTPPTCPPCP, CSGGGGSAEPKAGDATPPTCPPCP, CSGGGGSGGGGSGGGGSAESKYGPPCPPCP, CSNTGSGGEEKKKEKEKEEQEERSSDTPPTTCPPCP, CGSSGGSSGEPKSDATPTCPPCP, CSKVDKKVEPKSSDKTYTCPPCP and CSGGGGSAEPKAGDATPPTCPPCPPCP; and (c) L243 and the amino acid residue at position +2 relative to Y329 of domain D3 are substituted with cysteine.

30. The modified fusion protein of claim 1, wherein the modified fusion protein has all of the following properties (a) to (c): (a) Introduction: Amino acid substitutions K241E, L243S, R244V and H246E on the β1-β2 loop of domain D3, L258A, L258S or L258D on the β2-β3 loop of domain D3, and / or K300G, Q302T and K304S on the β5-β6 loop of domain D3; (b) the linker is an amino acid sequence selected from the group consisting of: CSSGDATPTSPPSP, CSKVDKKVEPKSSDTPPTCPPCP, CSGGGGSAEPKAGDATPPTCPPCP, CSGGGGSGGGGSGGGGSAESKYGPPCPPCP, CSNTGSGGEEKKKEKEKEEQEERSSDTPPTTCPPCP, CGSSGGSSGEPKSDATPTCPPCP, CSKVDKKVEPKSSDKTYTCPPCP and CSGGGGSAEPKAGDATPPTCPPCPPCP; and (c) L243 and the amino acid residue at position +2 relative to Y329 of domain D3 are substituted with cysteine.

31. The modified fusion protein of claim 1, wherein the modified fusion protein consists of an amino acid sequence selected from the group consisting of: SEQ ID NO:10 to SEQ ID NO:70, SEQ ID NO:77 to SEQ ID NO:93 and SEQ ID NO:

98.

32. The modified fusion protein of claim 1, wherein the multimerization domain is: (a) Fc region of an immunoglobulin; (b) the CH3 region of IgG1, IgG2, IgG3 or IgG4; (c) the CH2 region and CH3 region of IgG1, IgG2, IgG3 or IgG4; (d) an Fc region of an immunoglobulin comprising an amino acid sequence that is at least 85% identical to SEQ ID NO: 99; or (e) an Fc region of an immunoglobulin comprising the amino acid sequence shown in SEQ ID NO:

99.

33. The modified fusion protein of claim 1, wherein the multimerization domain is an Fc region of an immunoglobulin and starts from the lower hinge region of the Fc region.

34. The modified fusion protein of claim 1, wherein the multimerization domain is the Fc region of IgG1 consisting of SEQ ID NO:

99.

35. The modified fusion protein of claim 34, wherein the multimerization domain has an amino acid substitution D126E, an amino acid substitution L128M and / or a deletion of amino acid K217 based on SEQ ID NO:

99.

36. The modified fusion protein according to any one of claims 1 to 35, which is in the form of a dimer or a multimer.

37. A pharmaceutical composition for preventing or treating an autoimmune disease, an inflammatory disease, a tumor disease, cancer, an angiogenesis-related disease or an eye disease, the pharmaceutical composition comprising the modified fusion protein according to any one of claims 1 to 35 as an active ingredient.

38. A nucleic acid molecule encoding the modified fusion protein according to any one of claims 1 to 35.

39. A host cell comprising a nucleotide sequence encoding the modified fusion protein according to any one of claims 1 to 35.

40. A vector comprising a nucleotide sequence encoding the modified fusion protein according to any one of claims 1 to 35.

41. The vector according to claim 40, which is a recombinant viral vector.

42. A pharmaceutical composition for delivering a viral vector to a subject, the pharmaceutical composition comprising the recombinant viral vector according to claim 41, wherein the fusion protein encoded by the recombinant viral vector is expressed in the subject and is intended for preventing or treating autoimmune diseases, inflammatory diseases, tumor diseases, cancer, angiogenesis-related diseases or eye diseases.

43. The pharmaceutical composition of claim 42, wherein the recombinant viral vector is a recombinant adeno-associated viral vector.

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