Modified fusion proteins and uses thereof

Abstract

The present disclosure relates to modified fusion proteins that bind to vascular endothelial growth factor and placenta growth factor, and uses thereof. The modified fusion proteins according to one embodiment of the present disclosure can exhibit excellent productivity and stability. The modified fusion proteins according to one embodiment of the present disclosure can exhibit excellent stability in blood. The modified fusion proteins according to one embodiment of the present disclosure have acidic properties compared to previously developed substances, and can exhibit an increased ratio of charge variants in the acidic and neutral regions of the pI distribution.

Description

[Technical Field]

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

[0002] Vascular endothelial growth factor (VEGF) is a signaling protein that plays an important role in vasculogenesis and angiogenesis. Its general function is involved in embryonic development, muscle after injury or exercise, and the formation of new blood vessels to bypass infarcted blood vessels. However, abnormal increases in VEGF are closely related to tumor development and metastasis (Carmeliet, Py Jain, RK. 2000. Nature 407:249-257).

[0003] VEGFs are classified into five types: VEGF-A, -B, -C, -D, and PLGF (placenta growth factor), which are known to contribute to angiogenesis (Carmeliet, Py Jain, RK. 2000. Nature 407:249-257; Kuwano M, et al. 2001. Intern Med 40:565-572). There are three types of VEGF receptors: VEGF R1, R2, and R3. The types of ligands that bind to them and their binding affinities vary depending on the receptor. VEGFR-1 / -2, in particular, are known to be involved in angiogenesis (Veikkola et al. 2000. Cancer research 60:203-212).

[0004] Drug development has been ongoing to target angiogenesis by eliminating vascular endothelial growth factor (VEGF), a key factor in tumorigenesis. A representative example is bevacizumab, a monoclonal antibody targeting 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). Monoclonal antibody drugs have a mechanism of action that binds to a single VEGF. Because multiple growth factors are involved in cancer progression, monoclonal antibody drugs have not been effective in demonstrating the remarkable efficacy of VEGF. Instead, bevacizumab increases the expression of other non-targeted growth factors through intracellular compensatory mechanisms, resulting in resistance and other 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 these reasons, there is a growing need to develop drugs that target multiple vascular growth factors associated with carcinogenesis. In particular, PLGF, whose expression increases through compensatory pathways during late-stage carcinogenesis, is gaining importance. However, the majority of drugs currently developed or under development target VEGF, making the development of PLGF-targeting drugs even more necessary.

[0005] To address the shortcomings of monoclonal antibody drugs, drugs that target multiple vascular endothelial growth factors have been developed. Representative protein drugs include VEGF-Trap (Ciombor, et al., 2013, Clinical Cancer Research, 19(8):1920-1925, DrugBank Accession Number: DB08885) and VEGF-Grab (Lee, et al., 2015, Mol. Cancer. Ther., 14(2):470-9). Both of these drugs mimic a portion of a decoy receptor. VEGF-Trap is a heterologous protein of VGFR1 D2-VGFR2 D3, while VEGF-Grab is a recombinant fusion protein in the homologous protein form of VEGFR1 D2-D3 fused to immunoglobulin Fc. Efforts to improve the physical properties of these fusion proteins that target multiple vascular endothelial growth factors have been ongoing. In particular, there is a need for the development of fusion proteins that have high binding affinity to target substances, increased duration in the body, and physical and chemical stability sufficient to meet the high productivity requirements of protein therapeutics in commercial scale production.

[0006] technical challenges

[0007] Increasing the physical properties of proteins increases the physical and chemical stability of proteins during expression and purification, maximizing production efficiency, and reducing non-specific binding and degradation when proteins are introduced into the body, allowing them to achieve high in vivo durability.

[0008] The inventors of the present disclosure have made continuous efforts to efficiently increase the physical properties and stability of fusion proteins comprising the extracellular domain of VEGFR1.

[0009] As a result, the inventors of the present disclosure have developed a fusion protein comprising VEGFR1 domain D2 and domain D3, which is modified by amino acid mutation in a specific region of domain D3, adjusting or mutating the length of the linker, or introducing a disulfide bond into the fusion protein, and have confirmed that such a modified fusion protein exhibits excellent productivity and stability, thereby completing the present disclosure.

[0010] 1. One embodiment of the present disclosure may relate to 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, a linker is present between the Ig-like domain D3 and the multimerization domain, and the fusion protein has any one or more of the following 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 present in a region that is less than or equal to the net pI (net pi) of the protein; (b) the linker is about 13 to about 35 amino acids in length; and (c) a disulfide bond is present in the fusion protein, and one of the amino acid residues in the β1-β2 loop or 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 are substituted with cysteine.

[0011] 2. In one embodiment, the amino acid substitution in characteristic (a) may be with an amino acid residue of a VEGFR2 homologous sequence, an amino acid residue that is serine, threonine, tyrosine, cysteine, asparagine, glutamine, aspartic acid, or glutamic acid, or an amino acid residue with a short side chain that is alanine or glycine.

[0012] 3. In one embodiment, property (a) may be that one or more amino acid residues in the β1-β2 loop of domain D3 and one or more amino acid residues in the β5-β6 loop of domain D3 have been substituted with an amino acid that reduces the net pI of the protein, an amino acid whose side chain carries a negative charge, or an amino acid whose side chain carries an electrostatic negative charge.

[0013] 4. In one embodiment, characteristic (a) may be that one or more amino acid residues on the β2-β3 loop of domain D3 have been replaced with an amino acid residue having a side chain smaller than the existing amino acid residue or an amino acid residue having a polar side chain of similar size.

[0014] 5. In one embodiment, characteristic (a) may be a substitution of any one or more of the amino acid residues K241, L243, R244, and H246 on the β1-β2 loop of domain D3, the amino acid residue L258 on the β2-β3 loop of domain D3, and / or the amino acid residues K300, Q302, K304, and K306 on the β5-β6 loop of domain D3.

[0015] 6. In one embodiment, characteristic (a) may include any one or more amino acid substitutions of K241T, K241E, L243S, R244V, and H246E in the β1-β2 loop of domain D3, any one or more amino acid substitutions of L258A, L258S, or L258D in the β2-β3 loop of domain D3, and / or any one or more amino acid substitutions of K300G, Q302T, K304S, and K306Q in the β5-β6 loop of domain D3.

[0016] 7. In one embodiment, characteristic (a) may include (i) L243S and R244V amino acid substitutions, or (ii) K241E, L243S, R244V, and H246E amino acid substitutions in the β1-β2 loop of domain D3, L258A, L258S, or L258D amino acid substitutions in the β2-β3 loop of domain D3, and / or (i) K300G amino acid substitution, (ii) K300G, Q302T, and K304S amino acid substitutions, or (iii) K300G, Q302T, K304S, and K306Q amino acid substitutions in the β5-β6 loop of domain D3.

[0017] 8. In one embodiment, characteristic (a) may include K241E, L243S, R244V, and H246E amino acid substitutions on the β1-β2 loop of domain D3; L258A, L258S, or L258D amino acid substitutions on the β2-β3 loop of domain D3; and K300G, Q302T, and K304S amino acid substitutions on the β5-β6 loop of domain D3.

[0018] 9. In one embodiment, the modified fusion protein may contain R238S and R275N amino acid substitutions in domain D3.

[0019] 10. In one embodiment, the modified fusion protein has an amino acid sequence at the C-terminus of domain D3 linked to the linker that is KALE based on the K331 amino acid residue, although in this case, any one or more of the amino acids K, A, L, and E may be deleted.

[0020] 11. In one embodiment, the N-terminus of the D2 domain in the modified fusion protein may begin with the amino acid sequence SDT or the amino acid sequence EF.

[0021] 12. In one embodiment, in property (b), the linker may have a length of about 14 or more amino acids.

[0022] 13. In one embodiment, in the characteristic (b), the linker may contain a GS repeat sequence, which is an amino acid sequence of 2 to 60 amino acids consisting only of G and S, and the N-terminal amino acid of the linker may be glutamic acid.

[0023] 14. In one embodiment, the GS repeat sequence is (GS) n , (GSSG) n , (GGGGS) n , or (GS) m (GGGGS) n where n may be an integer from 1 to 10, and m may be an integer from 0 to 10.

[0024] 15. In one embodiment, the GS repeat sequence may be selected from the group consisting of GS, GSSG, (GSSG)2, GGGGS, (GGGGS)4, GS(GGGGS), and GS(GGGGS)3.

[0025] 16. In one embodiment, in property (b), the linker comprises an amino acid sequence of a hinge region derived from an immunoglobulin, and the amino acid sequence of the hinge region may be modified.

[0026] 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.

[0027] 18. In one embodiment, the hinge region derived from an immunoglobulin may be derived from human IgA, IgD, IgM, IgE, or IgG.

[0028] 19. In one embodiment, the antibody having property (b) may have an amino acid mutation at a papain recognition site or a glycosylation site present in a hinge region derived from an immunoglobulin.

[0029] 20. In one embodiment, the amino acid mutation in the papain recognition site in property (b) may be a substitution of any one or more amino acid residues present in the papain recognition site with alanine, serine, tyrosine, proline, or threonine, or an insertion into the papain recognition site of a sequence of 1 to 10 amino acids containing at least one amino acid having an aromatic carbon or cyclic carbon in its side chain; and the amino acid mutation in the glycosylation site may be (i) a deletion of serine or threonine present in the hinge region derived from an immunoglobulin, or (ii) a substitution with an amino acid other than serine, asparagine, or threonine.

[0030] 21. In one embodiment, the amino acid other than serine, asparagine, or threonine may be glycine or alanine.

[0031] 22. In one embodiment, the cysteine ​​present in the core hinge may be substituted with serine or glycine.

[0032] 23. In one embodiment, in property (b), the linker may comprise, from the N-terminus, (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, in that order, and the amino acid sequence of the hinge region may be modified.

[0033] 24. In one embodiment, in characteristic (b), the linker is selected from the group consisting of GSSGDKTHTSPPSP, GSSGEPKSSDKTYTSPPSP, GSKVDKKVEPKSSDKTHTCPPCP, GSKVDKKVEPKSSDKTYTCPPCP, GSKVDKKVEPKSSDTPPTCPPCP, GSGGGGSGGGGSGGGGSAESKYGPPCPPCP, GSNTGSGGEEKKKEKEKEEQEERSSDKTYTCPPCP, GSNTGSGGEEKKKEKEKEEQEERSSDTPPTCPPCP, EGSSGGSSGEPKSDATPTCPPCP, EGSSGGSSGEPKSDSTYTCPPCP, GCKVDKKVEPKSSDKTYTCPPCP, It may be an amino acid sequence selected from the group consisting of CSKVDKKVEPKSSDKTYTCPPCP, GGGGSAEPKAGDKAPPGPPGP, GGGGSAEPKSSDKTYTCPPCP, GGGGSGGGGSGGGGSGGGGSAEPKSSDKTYTCPPCP, CSSGDATPTSPPSP, CSKVDKKVEPKSSDTPPTCPPCP, CSGGGGSAEPKAGDATPPTCPPCP, CSGGGGSGGGGSGGGGSAESKYGPPCPPCP, CSNTGSGGEEKKKEKEKEEQEERSSDTPPTCPPCP, CGSSGGSSGEPKSDATPTCPPCP, and CSGGGGSAEPKAGDATPPTCPPCPPCP.

[0034] 25. In one embodiment, in property (c), any one of the amino acid residues in the β1-β2 loop may be any one of the amino acid residues in the amino acid sequence of V240 to T247.

[0035] 26. In one embodiment, in characteristic (c), any 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 may be substituted with cysteine, and any one of the amino acid residues at positions 0, +2, and +3 relative to Y329 of domain D3 may be substituted with cysteine.

[0036] 27. In one embodiment, the fusion protein modified in characteristic (c) may have the amino acid residues T241 and +3 relative to Y329 in domain D3 substituted with cysteine; L243C and Y329C amino acid mutations; L243 and +2 relative to Y329 in domain D3 substituted with cysteine; R244C and Y329C amino acid mutations; or N303C and Y329C amino acid mutations.

[0037] 28. In one embodiment, the modified fusion protein comprises: (a) one or more amino acid substitutions of K241T, K241E, L243S, R244V, and H246E in the β1-β2 loop of domain D3; L258A, L258S, or L258D in the β2-β3 loop of domain D3; and / or one or more amino acid substitutions of K300G, Q302T, K304S, and K306Q in the β5-β6 loop of domain D3; and (b) a linker selected from the group consisting of GSSGDKTHTSPPSP, GSSGEPKSSDKTYT SPPSP, GSKVDKKVEPKSSDKTHTCPPCP, GSKVDKKVEPKSSDKTYTCPPCP, GSKVDKKVEPKSSDTPPTCPPCP, GSGGGGSGGGGSGGGGSAESKYGPPCPPCP, GSNTGSGGEEKKK EKEKEEQEERSSDKTYTCPPCP, GSNTGSGGEEKKKEKEKEEQEERSSDTPPTCPCP, EGSSGGSSGEPKSDATPTCPPCP, EGSSGGSSGEPKSDSTYTCPPCP, GCKVDKKVEPKSSDKT YTCPPCP, CSKVDKKVEPKSSDKTYTCPPCP, GGGGSAEPKAGDKAPPGPPGP, GGGGSAEPKSSDKTYTCPPCP, GGGGSGGGGSGGGGGSGGGGSAEPKSSDKTYTCPPCP, CSSGDATPT SPPSP, CSKVDKKVEPKSSDTPPTCPPCP, CSGGGGSAEPKAGDATPPTCPPCP, CSGGGGSGGGGSGGGGSAESKYGPPCPPCP, CSNTGSGGEEKKKEKEKEEQEERSSDTPPTCPCP, C (c) an amino acid sequence selected from the group consisting of GSSGGSSGEPKSDATPTCPPCP and CSGGGGSAEPKAGDATPPTCPPCPPCP, in which any 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 any one of the amino acid residues at positions 0, +2, and +3 relative to Y329 of domain D3 is substituted with cysteine, and the domain may have all of the above properties (a) to (c).

[0038] 29. In one embodiment, the modified fusion protein comprises: (a) (i) L243S and R244V amino acid substitutions, or (ii) K241E, L243S, R244V, and H246E amino acid substitutions on the β1-β2 loop of domain D3; L258A, L258S, or L258D amino acid substitutions on the β2-β3 loop of domain D3; and / or (i) K300G amino acid substitutions, (ii) K300G, Q302T, and K304S amino acid substitutions, or (iii) K300G, Q302T, K304S, and K306Q amino acid substitutions on the β5-β6 loop of domain D3; and (b) the linker is CSSGDATPTSPPSP, CSKVDKK. (c) an amino acid sequence selected from the group consisting of VEPKSSDTPPTCPPCP, CSGGGGSAEPKAGDATPPTCPPCP, CSGGGGSGGGGSGGGGSAESKYGPPCPPCP, CSNTGSGGEEKKKEKEKEEQEERSSDTPPTCPPCP, CGSSGGSSGEPKSDATPTCPPCP, CSKVDKKVEPKSSDKTYTCPPCP, and CSGGGGSAEPKAGDATPPTCPPCPPCP; and (c) an amino acid sequence in which the amino acid residues at positions +2 relative to L243 and Y329 in domain D3 are substituted with cysteine, and which has all of the above properties (a) to (c).

[0039] 30. In one embodiment, the modified fusion protein comprises: (a) K241E, L243S, R244V, and H246E amino acid substitutions on the β1-β2 loop of domain D3, L258A, L258S, or L258D amino acid substitutions on the β2-β3 loop of domain D3, and K300G, Q302T, and K304S amino acid substitutions on the β5-β6 loop of domain D3; and (b) a linker comprising: CSSGDATPTSPPSP, CSKVDKKVEPKSSDTPPTCPPCP, CSGGGGSAEPKAGDATPPTCPPCP, CSG (c) an amino acid sequence selected from the group consisting of GGGSGGGGSGGGGSAESKYGPPCPPCP, CSNTGSGGEEKKKEKEKEEQEERSSDTPPTCPPCP, CGSSGGSSGEPKSDATPTCPPCP, CSKVDKKVEPKSSDKTYTCPPCP, and CSGGGGSAEPKAGDATPPTCPPCPPCP; and (c) an amino acid sequence in which the amino acid residues at positions +2 relative to L243 and Y329 in domain D3 are substituted with cysteine, and which has all of the above properties (a) to (c).

[0040] 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.

[0041] 32. In one embodiment, the multimerization domain may be (a) an Fc portion of an immunoglobulin; (b) a CH3 portion of an IgG1, IgG2, IgG3, or IgG4; (c) a CH2 and CH3 portion of an IgG1, IgG2, IgG3, or IgG4; (d) an Fc portion of an immunoglobulin comprising an amino acid sequence having at least 85% identity to SEQ ID NO: 99; or (e) an Fc portion of an immunoglobulin comprising the amino acid sequence of SEQ ID NO: 99.

[0042] 33. In one embodiment, the multimerization domain is the Fc portion of an immunoglobulin and may begin in the lower hinge region of the Fc portion.

[0043] 34. In one embodiment, the multimerization domain may be the Fc portion of IgG1 consisting of SEQ ID NO:99.

[0044] 35. In one embodiment, the multimerization domain may have a D126E amino acid substitution, an L128M amino acid substitution, and / or a K217 amino acid deletion relative to SEQ ID NO:99.

[0045] 36. In one embodiment, the modified fusion protein may be in a dimeric or multimeric form.

[0046] 37. An embodiment of the present disclosure may relate to a pharmaceutical composition for the prevention or treatment of an autoimmune disease, an inflammatory disease, a neoplastic disease, a cancer, an angiogenesis-related disease, or an eye disease, comprising a modified fusion protein according to an embodiment of the present disclosure as an active ingredient.

[0047] 38. One embodiment of the present disclosure may relate to a nucleic acid molecule encoding a modified fusion protein according to one embodiment of the present disclosure.

[0048] 39. One embodiment of the present disclosure may relate to a host cell comprising a nucleotide sequence encoding a modified fusion protein according to one embodiment of the present disclosure.

[0049] 40. One embodiment of the present disclosure may relate to a vector comprising a nucleotide sequence encoding a modified fusion protein according to one embodiment of the present disclosure.

[0050] 41. In one embodiment, the vector may be a recombinant viral vector.

[0051] 42. One embodiment of the present disclosure may relate to a pharmaceutical composition for delivering a viral vector to a subject, comprising a recombinant viral vector according to one embodiment of the present disclosure, wherein a fusion protein encoded by the recombinant viral vector is expressed in the subject, and the pharmaceutical composition is for the prevention or treatment of an autoimmune disease, an inflammatory disease, a neoplastic disease, a cancer, an angiogenesis-related disease, or an eye disease.

[0052] 43. In one embodiment, the recombinant viral vector may be a recombinant adeno-associated viral vector. Summary of the Invention [Effects of the Invention]

[0053] The modified fusion protein according to one embodiment of the present disclosure can exhibit excellent productivity, physical stability, and chemical stability.

[0054] The modified fusion protein according to one embodiment of the present disclosure can exhibit excellent stability in blood.

[0055] The modified fusion protein according to one embodiment of the present disclosure has acidic properties compared to previously developed substances, and may exhibit an increased ratio of charge variants in the acidic and neutral regions of the pI distribution, thereby improving non-specific interactions with matrices and cell surfaces in vivo. [Brief explanation of the drawings]

[0056] [Figure 1A] Figure 1A shows the 3D structure of the surface charge distribution of the D2-D3 domains of VEGFR1 and VEGFR2, including the binding surface of the ligand VEGF (shown as a green circle) and the location of loops in the D3 domain where mutations can be introduced (shown as a red circle). [Figure 1B]FIG. 1B is a 3D structure diagram showing exemplary amino acid residue locations that can be mutated in domain D3 of VEGFR1. [Figure 1C] Figure 1C shows exemplary amino acid residues that can be mutated in domain D3 of VEGFR1 and their structures. (A) of Figure 1C shows the positions of the amino acid residues and side chains at the mutation site in stick figures, and (B) is a 45° rotated version of (A). [Figure 2] Figure 2 shows the 3D structure of the extracellular domain of VEGFR1. Figure 2A shows the structure of VEGFR1 when bound to a VEGF ligand. Figures 2B and 2C show 3D modeling of a fusion protein (e.g., VEGF-Grab3) in which domains D2 and D3 of VEGFR1 are linked to an Fc region, in an open conformation before ligand binding (Figure 2B) and a closed conformation after ligand binding (Figure 2C). Figure 2D shows the distance from the end of D3 to the dimerization site of domain D4 when dimerization occurs in native VEGFR1. [Figure 3] FIG. 3 is an exemplary schematic diagram for designing the linkers introduced in this disclosure. [Figure 4] 4A and 4B are graphs showing the serum stability and VEGF-A binding ability of the protein. [Figure 5] 5A to 5C are graphs showing the measurement of the serum stability of proteins. [Figure 6] 6A to 6F are graphs showing the results of measuring the isoelectric points of proteins. [Figure 7] 7A to 7D are graphs analyzing the pI distribution of proteins. [Figure 8] 8A to 8D are graphs analyzing the thermal stability of proteins. [Figure 9] 9A to 9C are graphs showing the measurement of the VEGF-A binding ability of proteins. MODE FOR CARRYING OUT THE INVENTION

[0057] The various embodiments or examples described in this document are exemplified for the purpose of clearly explaining the technical idea of ​​the present disclosure, and are not intended to limit the technical idea to specific embodiments. The technical idea of ​​the present disclosure includes various modifications, equivalents, alternatives, and embodiments or examples that are selectively combined from all or part of the embodiments or examples described in this document.

[0058] Unless otherwise defined, all technical and scientific terms used in this disclosure have the meaning commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0059] As used in this document, the singular terms "a," "an," and "the" may also include the plural meaning unless the context clearly dictates otherwise, and this also applies to the singular terms used in the claims.

[0060] I. Definition

[0061] In the present disclosure, the term "about" can represent a normal error range for each value, as is widely known to those skilled in the art. In the context of numerical values ​​or ranges described in this disclosure, this can mean ±20%, ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% of the numerical value or range stated or claimed in one embodiment. In the length of a nucleotide or amino acid sequence, "about" can also indicate that the nucleic acid or protein is not limited to the number of nucleotides or amino acids listed, and that adding or removing some nucleotides or amino acids to or from either end may be included within a range that does not inhibit functional activity.

[0062] As used in this disclosure, expressions such as "including," "comprising," "having," and the like should be understood as open-ended terms that include the possibility of including other embodiments in a manner similar to "including," unless otherwise stated in the phrase or sentence in which the expression is included.

[0063] As used in this disclosure, the term "and / or" may mean any one or more of the items, any combination of the items, or all of the items with which the term is associated.

[0064] The term "amino acid" as used in this disclosure can refer to any naturally occurring L-α-amino acid. This definition can include norleucine, ornithine, and homocysteine.

[0065] As used in this disclosure, the term "mutation" or "amino acid mutation" can refer to an amino acid sequence substitution, insertion, deletion, or a combination thereof, compared to a reference (e.g., native sequence) polypeptide or protein, and the term "variant" can refer to a molecule that has a partial difference in amino acid sequence compared to the reference polypeptide or protein due to such a mutation.

[0066] The scope of variants of the present disclosure may also include proteins or fragments or derivatives thereof that exhibit the same or similar biological activity, and derivatives that are otherwise modified during or after translation, for example, by glycosylation, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc.

[0067] The term "carriers" as used in this disclosure may include pharmaceutically acceptable carriers, excipients, or stabilizers that are nontoxic to cells or mammals at the dosages and concentrations applied. Often, the pharmaceutically acceptable carrier is an aqueous pH buffer solution. Examples of pharmaceutically acceptable carriers include, but are not limited to, buffers such as phosphate, citrate, 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; and / or nonionic surfactants such as TWEEN®, polyethylene glycol (PEG), and PLURONICS®.

[0068] As used in this disclosure, the term "effective amount" can mean an amount sufficient to bring about beneficial or desired clinical or biochemical results. An effective amount can be administered in one or more doses. An effective amount can mean an amount sufficient to palliate, ameliorate, stabilize, slow, or delay the progression of a disease state.

[0069] As used in this disclosure, the term "ligand" can refer to a molecule (e.g., VEGF-A, -B, -C, -D, and PLGF) that binds with high affinity to a modified fusion protein. In other contexts, "ligand" can refer to any molecule, agent, or compound that specifically binds, covalently or transiently, to a molecule such as a polypeptide or protein.

[0070] As used in this disclosure, the term "individual" or "subject" can refer to a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, 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 can be a human.

[0071] As used in this disclosure, 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 absorption delaying agents, and the like.

[0072] References to an embodiment of the present disclosure described herein are understood to include "comprising," "consisting of," and / or "consisting essentially of" an embodiment.

[0073] II. MODIFIED FUSION PROTEINS AND MODIFIED FUSION PROTEIN COMPONENTS

[0074] 1.Vascular endothelial growth factor receptor (VEGFR)

[0075] In one embodiment, the modified fusion protein of the present disclosure may comprise a VEGFR1 extracellular domain as a component. VEGFR1, also known as fms-related tyrosine kinase (FLT-1), is encoded by the FLT1 gene. The amino acid sequence of VEGFR1 is set forth as SEQ ID NO: 1 and can be found at UniProtKB.#P17948. In one embodiment, VEGFR1 is derived from a mammal, such as a human. VEGFR1 has seven immunoglobulin (Ig)-like domains, numbered 1, 2, 3, 4, 5, 6, and 7, from the N-terminus of the extracellular region to the C-terminus, as are VEGFR2 and VEGFR3.

[0076] In one embodiment, the VEGFR1 extracellular domain may comprise 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 extracellular region of VEGFR1, and the term "Ig-like domain D3" of VEGFR refers to the third Ig-like domain found at the N-terminus of the extracellular region of VEGFR1. However, this concept encompasses variants that retain the functionality of these domains (e.g., binding to VEGF ligands and / or inhibiting VEGFR pathway activation). Because the amino acid sequences of active proteins may differ depending on the species, the VEGFR1 Ig-like domains D2 and D3 are not limited by their origin or sequence, and may include wild-type or active variants.

[0077] In one embodiment, the Ig-like domain D2 of VEGFR1 may comprise the amino acid sequence of G132 to H214 of SEQ ID NO: 1, or may comprise an amino acid sequence variant having similar or equivalent binding affinity to a VEGF ligand expressed by such an amino acid sequence. Specifically, in one embodiment, the Ig-like domain D2 of VEGFR1 may comprise an amino acid sequence exhibiting 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 of G132 to H214 of SEQ ID NO: 1.

[0078] In one embodiment, the Ig-like domain D3 of VEGFR1 may comprise the amino acid sequence of L215 to A332 of SEQ ID NO: 1, or may comprise an amino acid sequence variant having similar or equivalent binding affinity to a VEGF ligand expressed by such an amino acid sequence. Specifically, in one embodiment, the Ig-like domain D3 of VEGFR1 may comprise an amino acid sequence showing 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 of L215 to A332.

[0079] 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 Ig-like domain D3 of VEGFR1.

[0080] Without being bound by theory, the present disclosure contemplates that the extracellular domain of a VEGFR inhibits activation of the VEGF pathway by binding to a VEGF ligand and blocks the interaction between the VEGF ligand and the VEGFR. Also, without being bound by theory, the present disclosure contemplates that the extracellular domain of a VEGFR may bind to a VEGFR to effect dominant-negative inhibition of the VEGF signaling pathway. In one embodiment, the extracellular domain of a VEGFR may bind to 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 a VEGFR may bind to a VEGFR (e.g., VEGFR1, VEGFR2, and / or VEGFR3).

[0081] In one embodiment, the VEGFR extracellular domain may or may not include a signal peptide that serves as a signal sequence for secretion of the VEGFR extracellular domain or a modified fusion protein comprising the same from a host cell. Such a signal peptide may be operably linked to a nucleic acid encoding a protein of interest (e.g., the extracellular domain of VEGFR1).

[0082] 2. Linker

[0083] The components of the modified fusion protein (e.g., the extracellular domain or multimerization domain of VEGFR1) may be linked by a linking moiety, such as a peptide linker, which increases the flexibility of the fusion protein components and does not significantly interfere with the structure of each functional component within the fusion protein.

[0084] In one embodiment, a linker may be used to link the C-terminal end of the VEGFR1 extracellular domain (e.g., the C-terminus of the Ig-like domain D3) to the N-terminal end of the multimerization domain (e.g., the IgG1 Fc portion).

[0085] In one embodiment, the linker may comprise the amino acid sequence of a hinge region derived from an immunoglobulin. The hinge region derived from an immunoglobulin may comprise the hinge region present in the N-terminal region of the Fc portion of the 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 comprise the amino acid sequence up to the core hinge region of the Fc portion of the immunoglobulin, excluding the lower hinge region of the Fc portion. For example, if the hinge region is derived from IgG1, the lower hinge region may be the amino acid sequence corresponding to APELLGGP. In one embodiment, the linker may be the amino acid sequence EDKTHTCPPCP or LEDKTHTCPPCP.

[0086] 3. Multimerization domain

[0087] The present disclosure provides multimerization domains (e.g., immunoglobulin Fc moieties) that can be components of any modified fusion protein. A multimerization domain is a portion of a multimeric protein that promotes subunit assembly, forming, for example, dimers, trimers, tetramers, etc. As used in the present disclosure, the term "multimerization domain" may refer to a dimerization domain, trimerization domain, tetramerization domain, etc. Fusion proteins containing a multimerization domain can interact with other fusion proteins containing a multimerization domain to generate fusion protein multimers (e.g., fusion protein dimers). For example, an IgG Fc moiety is a dimerization domain that can be fused to the extracellular domain of VEGFR1 described in the present disclosure. A fusion protein containing the extracellular domain of VEGFR1 and an IgG Fc moiety can dimerize with yet another fusion protein containing an IgG Fc moiety to generate a fusion protein dimer that can simultaneously bind to a VEGF ligand and a PLGF ligand.

[0088] In one embodiment, the multimerization domain may be an Fc portion of an immunoglobulin. In another embodiment, the Fc portion of an immunoglobulin may be selected from the group consisting of an IgG Fc portion, an IgA Fc portion, an IgM Fc portion, an IgD Fc portion, and an IgE Fc portion. In yet another embodiment, the Fc portion of an immunoglobulin may be selected from the group consisting of an IgG1 Fc portion, an IgG2 Fc portion, an IgG3 Fc portion, and an IgG4 Fc portion. In one embodiment, the Fc portion may comprise a CH3 portion of an IgG1, IgG2, IgG3, or IgG4. In one embodiment, the Fc portion may comprise a CH2 and CH3 portion of an IgG1, IgG2, IgG3, or IgG4. Amino acid sequences encoding immunoglobulins containing Fc portions are well known in the art. In one embodiment, the multimerization domain may be an Fc portion of an immunoglobulin comprising the amino acid sequence of SEQ ID NO:99. In one embodiment, the multimerization domain may be the Fc portion of IgG1 consisting of the amino acid sequence of SEQ ID NO:99.

[0089] In one embodiment, the Fc portion may comprise an amino acid sequence that exhibits 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 of the CH3 portion or the CH2 and CH3 portions of IgG1, IgG2, IgG3, or IgG4. In one embodiment, the multimerization domain may comprise an amino acid sequence that exhibits 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 of SEQ ID NO:99.

[0090] In one embodiment, the multimerization domain may begin at the lower hinge region of the immunoglobulin Fc region. The lower hinge region of the Fc region may begin at Ala244 or correspond to the amino acid sequence from Ala244 to Pro251. Here, the positions of the amino acid residues are according to the Kabat numbering system (Kabat et al., "Proteins of Immunological Interest," 5th Ed., US Department of Health and Human Services, NIH Publication No. 91-3242, 1991). For example, the lower hinge region of a human IgG1 Fc region may correspond to amino acid residues 1 to 8 of the amino acid sequence of SEQ ID NO: 99, or may correspond to the amino acid sequence of APELLGGP. The human IgG1 Fc region beginning at the lower hinge region is represented by the amino acid sequence of SEQ ID NO: 99.

[0091] In one embodiment, the Fc portion of the protein may be in a partially glycosylated or entirely non-glycosylated form.

[0092] In one embodiment, the Fc portion may have a D126E amino acid substitution, an L128M amino acid substitution, and / or a K217 amino acid deletion based on SEQ ID NO: 99. Such amino acid mutations can increase the half-life of the fusion protein through interaction with FcRn. The amino acid mutations may be identically present in other immunoglobulin Fc portions having a sequence corresponding to the amino acid sequence of SEQ ID NO: 99, and the modified fusion proteins of the present disclosure may include other immunoglobulin Fc portions having the amino acid mutations as a multimerization domain component.

[0093] The following describes the properties of the modified fusion protein of the present disclosure. In the amino acid mutations for such properties, the positions of the amino acid residues are described corresponding to the positions of the amino acid residues in the entire amino acid sequence of VEGFR1 of SEQ ID NO: 1.

[0094] 4. Surface Charge Transfer Mutation Characteristics

[0095] In one embodiment, the modified fusion proteins of the present disclosure may have a surface charge transfer mutation property. In the present disclosure, the modified fusion proteins having a surface charge transfer mutation property may be used interchangeably as "surface charge transfer mutants" or "surface charge mutants."

[0096] In one embodiment, the surface charge transfer mutation in the modified fusion protein may involve substitution of one or more amino acid residues in the β1-β2 loop of the Ig-like domain D3, one or more amino acid residues in the β2-β3 loop of the domain D3, and / or one or more amino acid residues in the β5-β6 loop of the domain D3 with amino acids that decrease the net pI of the protein, amino acids that impart a negatively charged side chain, or amino acids that result in an electrostatically negative side chain. In one embodiment, an amino acid that results in an electrostatically negative side chain may refer to an amino acid that, after substitution, will have an electrostatically negative charge due to the properties of the adjacent amino acid residue or molecule, or may refer to a relative concept resulting from intramolecular electron distribution. For example, an amino acid residue containing a hydroxyl (-OH) may become electrostatically negative due to the lone electron pair present on the oxygen atom, which may result in a decrease in the net pI of the protein.

[0097] In one embodiment, the β1-β2 loop of Ig-like domain D3 may comprise amino acid residues T236 to T247 of the amino acid sequence of SEQ ID NO: 1. In one embodiment, the β2-β3 loop of Ig-like domain D3 may comprise amino acid residues T256 to V262 of the amino acid sequence of SEQ ID NO: 1. In one embodiment, the β5-β6 loop of Ig-like domain D3 may comprise amino acid residues D299 to L308 of the amino acid sequence of SEQ ID NO: 1. In the present disclosure, the β1-β2 loop may be referred to as "Site 1," the β2-β3 loop as "Site 2," and the β5-β6 loop as "Site 3." The inventors of the present disclosure confirmed that the amino acid residues in these loops are exposed on the surface of domain D3, and achieved stabilization of the surface charge and structure of the modified fusion protein by mutating the amino acid residues in the loops.

[0098] In one embodiment, due to their structural positions, the amino acid residues present at Site 1 and Site 3 influence each other through interactions between the amino acid residues, thereby allowing the introduction of double, triple, or quadruple amino acid mutations (e.g., amino acid substitutions) at each site.

[0099] In one embodiment, the amino acid residues in Site 2 are exposed on the surface of the domain D3 that binds to a VEGF or PLGF ligand, and can affect the binding strength to the ligand or the solubility due to surface residue exposure. In one embodiment, the amino acid residues in Site 2 may be substituted with amino acid residues having a smaller side chain size than the existing amino acid residues (e.g., Ala) or with amino acid residues having a polar side chain of similar size (e.g., Ser or Asp) within a range that does not significantly affect the protein structure, the binding strength to the ligand, or the solubility.

[0100] In one embodiment, the three loops should be understood as a concept that encompasses amino acid sequence variants that maintain their functions. For example, in one embodiment, the β1-β2 loop, the β2-β3 loop, and the β5-β6 loop may comprise amino acid sequences that exhibit 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 sequences TPRPVKLLRGHT, TPLNTRV, and DKMQNKDKGL, respectively.

[0101] In one embodiment of the surface charge transfer mutation characteristics, the amino acid substitution may be, but is not limited to, (i) an amino acid residue of a VEGFR2 homologous sequence, (ii) an amino acid residue that is serine, threonine, tyrosine, cysteine, asparagine, glutamine, aspartic acid, or glutamic acid, or (iii) an amino acid residue with a short side chain that is alanine or glycine.

[0102] In one embodiment of the linker mutation feature, the modified fusion protein may have one or more amino acid residues in the β1-β2 loop of Ig-like domain D3 and one or more amino acid residues in the β5-β6 loop of domain D3 substituted with amino acids that decrease the net pI of the protein, amino acids with negatively charged side chains, or amino acids with electrostatically negative side chains.

[0103] In one embodiment, the surface charge transfer mutation may include, but is not limited to, (i) one or more amino acid substitutions of K241, L243, R244, and H246 in the β1-β2 loop of Ig-like domain D3, where the amino acid residues in the β1-β2 loop may be substituted with threonine, glutamic acid, serine, or valine.

[0104] In one embodiment, the surface charge transfer mutation may include, but is not limited to, (ii) an amino acid substitution of the L258 amino acid residue in the β2-β3 loop of the Ig-like domain D3, in which the amino acid residue in the β2-β3 loop may be substituted with any one of alanine, serine, and aspartic acid.

[0105] In one embodiment, the surface charge transfer mutation may include, but is not limited to, (iii) one or more amino acid substitutions of K300, Q302, K304, and K306 in the β5-β6 loop of domain D3, where the amino acid residues in the β5-β6 loop may be substituted with any one of glycine, threonine, serine, and glutamine.

[0106] In one embodiment of the surface charge transfer mutation, the modified fusion protein may include (i) one or more amino acid substitutions of K241T, K241E, L243S, R244V, and H246E on the β1-β2 loop of domain D3, (ii) one or more amino acid substitutions of L258A, L258S, or L258D on the β2-β3 loop of domain D3, and / or (iii) one or more amino acid substitutions of K300G, Q302T, K304S, and K306Q on the β5-β6 loop of domain D3.

[0107] In one embodiment of the surface charge transfer mutation feature, the modified fusion protein may comprise (i) L243S and R244V amino acid substitutions, or (ii) K241E, L243S, R244V, and H246E amino acid substitutions on the β1-β2 loop of domain D3; L258A, L258S, or L258D amino acid substitutions on the β2-β3 loop of domain D3; and / or (i) K300G amino acid substitution, (ii) K300G, Q302T, and K304S amino acid substitutions, or (iii) K300G, Q302T, K304S, and K306Q amino acid substitutions on the β5-β6 loop of domain D3.

[0108] In one embodiment of the surface charge transfer mutation feature, the modified fusion protein may include K241E, L243S, R244V, and H246E amino acid substitutions on the β1-β2 loop of domain D3; L258A, L258S, or L258D amino acid substitutions on the β2-β3 loop of domain D3; and K300G, Q302T, and K304S amino acid substitutions on the β5-β6 loop of domain D3.

[0109] In one embodiment, the modified fusion protein may contain R238S and / or R275N amino acid substitutions in the Ig-like domain D3.

[0110] In one embodiment, the C-terminal amino acid sequence of the Ig-like domain D3 linked to the linker in the modified fusion protein is KALE based on the K331 amino acid residue, with the proviso that any one or more of the amino acids K, A, L, and E may be deleted. In one embodiment, the C-terminal amino acid sequence of the Ig-like domain D3 in the modified fusion protein may be deleted by K and A; K, A, and L; A, L, and E; or K, A, L, and E. In one embodiment, the C-terminal amino acid sequence of the Ig-like domain D3 in the modified fusion protein of the present disclosure may be D330.

[0111] In one embodiment, the N-terminus of the Ig-like domain D2 of the modified fusion protein may be further modified. The modified fusion proteins of the present disclosure may be produced by inserting the nucleic acid sequence or nucleic acid molecule encoding the fusion protein into a vector of various origins. In this case, depending on the type or origin of the vector into which the nucleic acid sequence or nucleic acid molecule is inserted, the N-terminus of the Ig-like domain D2 may not start at G132 but may start with an amino acid sequence derived from the vector (e.g., amino acid sequence EF), resulting in a mutation in the N-terminal amino acid sequence of domain D2. Furthermore, in one embodiment, the modified fusion protein of the present disclosure may further comprise the C-terminal sequence 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 the amino acid sequence SDT or the amino acid sequence EF. However, such mutation of the N-terminal sequence by a vector-derived amino acid sequence does not affect the productivity or physical properties of the modified fusion protein of the present disclosure.

[0112] 5. Linker Mutation Characteristics

[0113] In one embodiment, the modified fusion protein of the present disclosure may have a linker mutation characteristic. In the present disclosure, the modified fusion protein having a linker mutation characteristic may be used interchangeably with the term "linker mutant." Due to the linker mutation characteristic, the modified fusion protein of the present disclosure may (i) closely mimic the ligand-binding structure of domains D2 and D3 of the native VEGFR1 extracellular domain, (ii) have a length up to the dimerization site of domain D4, which is a structural characteristic that domain D3 of the native VEGFR1 extracellular domain must ensure for smooth ligand binding, and (iii) the linker itself may have excellent structural stability.

[0114] In one embodiment of the linker mutation characteristic, the linker may have a length of about 14 or more amino acids. In one embodiment, the linker may have a length of about 14 to about 70, about 14 to about 65, about 14 to about 60, about 14 to about 55, about 14 to about 50, about 14 to about 45, about 14 to about 40, about 14 to about 38, or about 14 to about 36 amino acids. In one embodiment, the linker may comprise or consist of 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 amino acids (inclusive, including any value between these numbers).

[0115] In one embodiment, the linker may comprise a GS repeat sequence. In one embodiment, the GS repeat sequence may comprise a 2-60 amino acid sequence consisting solely of G and S. Here, the N-terminal amino acid of the GS repeat sequence may be glutamic acid. In one embodiment, the GS repeat sequence may comprise or consist of 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 amino acids (inclusive, including any value between these numbers). In one embodiment, the GS repeat sequence may comprise or consist of 2-20 amino acids.

[0116] In one embodiment, the linker mutation characteristic is that the GS repeat sequence is (GS) n , (GSSG) n , (GGGGS) n、 or (GS) m (GGGGS) n where n is an integer from 1 to 10, and m is an integer from 0 to 10.

[0117] In one embodiment of the linker mutation characteristic, the GS repeat sequence may 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.

[0118] In one embodiment, the linker may comprise an amino acid sequence of a hinge region derived from an immunoglobulin. In one embodiment, the amino acid sequence of the hinge region may be modified.

[0119] In the present disclosure, the linker may refer to the entire region located between the C-terminus of VEGFR1 domain D3 and the Fc region of human immunoglobulin produced by papain digestion, from P243 located at the N-terminal end of the CH2 region (A244-K360) contained in the Fc region to the C-terminus of domain D3. Here, the amino acid positions of the Fc region and the CH2 region contained in the Fc region are according to the Kabat numbering system (Kabat et al., in *Proteins of Immunological Interest*, 5th Ed., US Department of Health and Human Services, NIH Publication No. 91-3242, 1991). In one embodiment, the linker may include a region derived from human immunoglobulin, and the region may be the amino acid sequence of CH1 (K218-V223) or the hinge region (E226-P243), or a portion of such an amino acid sequence.

[0120] In the present disclosure, when a linker contains the amino acid sequence of an immunoglobulin-derived hinge region, it retains the properties of the included hinge region. For example, the hinge region of the Fc moiety has cleavage sites for various proteases, and after the hinge region is introduced into a fusion protein, it can induce oligomerization due to O-glycosylation of Ser / Thr amino acids (Song et al., 2020. Comput Struct Biotechnol J. 18:3925-3935.). Such glycosylation in the hinge region can affect the physical properties of the protein during expression or purification, causing structural interference with the pairing of disulfide bonds in the hinge region, which occurs reversibly during the purification process, and preventing normal disulfide bond formation. The modified fusion protein with linker mutation characteristics of the present disclosure achieves excellent structural stability of the linker itself and improved physical properties by modifying the amino acid sequence of the immunoglobulin-derived hinge region.

[0121] In one embodiment, the linker variant may comprise a hinge region derived from an immunoglobulin, including a sequence derived from a CH1 region, an upper hinge, and / or a core hinge. In one embodiment, a portion of the amino acid sequence derived from the CH1 region of an immunoglobulin can form a beta-strand or a beta-sheet. In one embodiment, the hinge region derived from an immunoglobulin may be derived from human IgA, IgD, IgM, IgE, IgG, or a combination thereof.

[0122] In one embodiment, the linker mutation feature of the modified fusion protein may include an amino acid mutation in a papain recognition site or a glycosylation site present in the hinge region derived from the immunoglobulin contained in the linker.

[0123] In one embodiment of the linker mutation characteristics, the amino acid mutation in the papain recognition site may be a substitution of one or more amino acid residues present in the papain recognition site with alanine, serine, tyrosine, proline, or threonine, or an insertion into the papain recognition site of a sequence of 1 to 10 amino acids containing at least one amino acid having an aromatic carbon or cyclic carbon in its side chain.

[0124] In one embodiment, the hinge region included in the linker may be derived from IgG1. In this case, the amino acid sequence near the cleavage 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 cleavage amino acid in the papain recognition site may be substituted with proline, proline-proline, or tyrosine.

[0125] In one embodiment, when the hinge region is derived from IgG1, a sequence of 1 to 10 amino acids including at least one amino acid having an aromatic or cyclic carbon atom in its side chain may be inserted before the histidine corresponding to the cleavage amino acid. In one embodiment, the amino acid having an aromatic or cyclic carbon atom in its side chain may be proline or tyrosine. In one embodiment, the sequence inserted before the cleavage amino acid may include cysteine. If the sequence inserted into the papain recognition site includes cysteine, it may form a disulfide bond with the cysteine ​​located at the same position in the opposing monomer during dimer formation, thereby imparting rigidity to the linker and stabilizing the loop structure of the linker. In one embodiment, the sequence inserted before the cleavage amino acid may be, but is not limited to, the amino acid sequence PPTC. The sequence inserted into the papain recognition site may form hydrophobic interactions between the aromatic or cyclic carbon atom and the amino acid having an aromatic or cyclic carbon atom in its side chain, thereby stabilizing the loop structure of the linker.

[0126] In one embodiment, when the hinge region is derived from IgG1, the amino acid sequence KTHT of the mutated papain recognition site may be modified with an amino acid sequence selected from the group consisting of KTYT, TPP, ATPT, STYT, KAPP, and ATPPTCP.

[0127] In one embodiment of the linker mutation, the amino acid mutation at the glycosylation site may be a deletion or substitution of a serine or threonine present in the hinge region derived from an 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. Substitution of a serine or threonine amino acid can prevent O-glycosylation that may occur in the hinge region.

[0128] In one embodiment, a cysteine ​​in the core hinge may be substituted with serine or glycine, which removes a disulfide bond in the core hinge, increasing the flexibility of the linker and providing the same effect as increasing the length of the linker.

[0129] In one embodiment of the linker mutation characteristic, the linker may comprise, from the N-terminus, (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, in that order, and the amino acid sequence of the hinge region may be modified.

[0130] 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 IgD may be NTGSGGEEKKKEKEKEEQEERSS, NTGRGGEEKKKEKEKEEQEER, or a variant thereof. In one embodiment, the hinge sequence of IgG1 may be DKTHTCPPCP or a variant thereof.

[0131] In one embodiment, the linker mutation characteristics are GSSGDKTHTSPPSP, GSSGEPKSSDKTYTSPPSP, GSKVDKKVEPKSSDKTHTCPPCP, GSKVDKKVEPKSSDKTYTCPPCP, GSKVDKKVEPKSSDTPPTCPPCP, GSGGGGSGGGGSGGGGSAESKYGPPCPPCP, GSNTGSGGEEKKKEKEKEEQEERSSDKTYTCPPCP, GSNTGSGGEEKKKEKEKEEQEERSSDTPPTCPPCP, EGSSGGSSGEPKSDATPTCPPCP, EGSSGGSSGEPKSDSTYTCPPCP, GCKVDKKVEPKSSDKTYTCPPCP, CS It may be an amino acid sequence selected from the group consisting of KVDKKVEPKSSDKTYTCPPCP, GGGGSAEPKAGDKAPPGPPGP, GGGGSAEPKSSDKTYTCPPCP, GGGGSGGGGSGGGGSGGGGSAEPKSSDKTYTCPPCP, CSSGDATPTSPPSP, CSKVDKKVEPKSSDTPPTCPPCP, CSGGGGSAEPKAGDATPPTCPPCP, CSGGGGSGGGGSGGGGSAESKYGPPCPPCP, CSNTGSGGEEKKKEKEKEEQEERSSDTPPTCPPCP, CGSSGGSSGEPKSDATPTCPPCP, and CSGGGGSAEPKAGDATPPTCPPCPPCP.

[0132] 6. Disulfide bond mutation characteristics

[0133] In one embodiment, the modified fusion protein of the present disclosure may have a disulfide bond mutation characteristic. In the present disclosure, the modified fusion protein having such a disulfide bond mutation characteristic may be used interchangeably as a "disulfide bond mutant." Because the modified fusion protein of the present disclosure is a fusion of heterologous proteins, it is expected that the stability will be reduced at the binding site of the different proteins. Therefore, the disulfide bond mutation characteristic was introduced to prevent physical cleavage of the modified fusion protein and improve its stability.

[0134] To introduce disulfide bond mutations, we searched for amino acid residues suitable for disulfide bond mutations, focusing on the C-terminal region of the Ig-like domain D3, where heterologous protein attachment begins and where secondary structure may be lacking, based on the following criteria: (1) the candidate amino acid residue must be exposed on the surface of the fusion protein and capable of forming a disulfide bond; (2) it must be present in the flexible loop-linker where the heterologous protein is attached; and (3) amino acid residues at two different sites must be within sufficient distance to form a disulfide bond. Based on these criteria, we selected candidate amino acid residues using a tertiary structure energy prediction program (foldx in YASARA, Schymkowitz et al. 2005 Nucleic Acids Research. 33:W382-388). Substitution of these amino acid residues with cysteine ​​was confirmed to improve the properties (e.g., productivity, physical properties, and ligand binding) of the disulfide bond-modified fusion protein (see Example 2.3). In one embodiment, disulfide bond mutation characteristics may be introduced by substitution with cysteine ​​residues at a site in the VEGFR1 domain and at a site in the linker, thereby achieving superior stability of the covalently modified fusion protein, which is one of the stable bond forms.

[0135] In one embodiment, to introduce a disulfide bond mutation characteristic, (i) any one of the amino acid residues in the β1-β2 loop or any one of the amino acid residues in the β5-β6 loop of the Ig-like domain D3, and (ii) any one of the amino acid residues at positions -2, -1, 0, +1, +2, and +3 relative to Y329 of the Ig-like domain D3 may be substituted with cysteine. Such substitutions allow the modified fusion protein of the present disclosure to have a disulfide bond within the protein. In one embodiment, the selection of amino acid residues at the positions described in (i) and (ii) above for introducing a disulfide bond mutation characteristic may be determined by the above criteria (1) to (3).

[0136] In one embodiment, in (i) above, any one of the amino acid residues on the β1-β2 loop may be any one of the amino acid residues on the amino acid sequence of V240 to T247.

[0137] In one embodiment, due to the disulfide bond mutation characteristics, any one of the amino acid residues T241, L243, and R244 on the β1-β2 loop of Ig-like domain D3 or the amino acid residue N303 on the β5-β6 loop may be substituted with cysteine, and any one of the amino acid residues at positions 0, +2, and +3 relative to Y329 of Ig-like domain D3 may be substituted with cysteine.

[0138] In one embodiment, due to the disulfide bond mutation characteristics, the modified fusion protein of the present disclosure may have the amino acid residues T241 and +3 relative to Y329 in domain D3 substituted with cysteine; L243C and Y329C amino acid mutations; L243 and +2 relative to Y329 in domain D3 substituted with cysteine; R244C and Y329C amino acid mutations; or N303C and Y329C amino acid mutations.

[0139] 7. Fusion Proteins

[0140] In one embodiment of the present disclosure, a modified fusion protein capable of simultaneously binding to a VEGF ligand and a PLGF ligand may be provided. In one embodiment, the modified fusion protein may comprise a first binding specificity for a VEGF ligand and a second binding specificity for a PLGF ligand. The VEGF ligand may be any one or more of VEGF-A, VEGF-B, VEGF-C, and VEGF-D, and may specifically be VEGF-A.

[0141] In one embodiment, the modified fusion protein may comprise a VEGFR1 extracellular domain, a linker, and a multimerization domain. In one embodiment, the modified fusion protein may comprise components arranged in this order from N-terminus to C-terminus: a VEGFR1 extracellular domain, a linker, and a multimerization domain. In one embodiment, the VEGFR1 extracellular domain may comprise Ig-like domain D2 and Ig-like domain D3 of VEGFR1.

[0142] In one embodiment, the modified fusion protein may have one or more of the following characteristics: (a) a surface charge transfer characteristic; (b) a linker mutation characteristic; and (c) a disulfide bond mutation characteristic.

[0143] In one embodiment, the modified fusion protein may have all of the following characteristics (a), (b), and (c):

[0144] (a) comprising one or more amino acid substitutions of K241T, K241E, L243S, R244V, and H246E in the β1-β2 loop of domain D3, L258A, L258S, or L258D in the β2-β3 loop of domain D3, and / or one or more amino acid substitutions of K300G, Q302T, K304S, and K306Q in the β5-β6 loop of domain D3;

[0145] (b) If the linker is GSSGDKTHTSPPSP, GSSGEPKSSDKTYTSPPSP, GSKVDKKVEPKSSDKTHTCPPCP, GSKVDKKVEPKSSDKTYTCPPCP, GSKVDKKVEPKSSDTPPTCPPCP, GSGGGGSGGGGSGGGGSAESKYGPPCPPCP, GSNT GSGGEEKKKEKEKEEQEERSSDKTYTCPPCP, GSNTGSGGEEKKKEKEKEEQEERSSDTPPTCPCP, EGSSGGSSGEPKSDATPTCPPCP, EGSSGGSSGEPKSDSTYTCPPCP, GCKVDKKVEPKSSDKTYTCPPCP, CSKVDKKVEP an amino acid sequence selected from the group consisting of KSSDKTYTCPPCP, GGGGSAEPKAGDKAPPGPPGP, GGGGSAEPKSSDKTYTCPPCP, GGGGSGGGGSGGGGSGGGGSAEPKSSDKTYTCPPCP, CSSGDATPTSPPSP, CSKVDKKVEPKSSDTPPTCPPCP, CSGGGGSAEPKAGDATPPTCPPCP, CSGGGGSGGGGSGGGGSAESKYGPPCPPCP, CSNTGSGGEEKKKEKEKEEQEERSSDTPPTCPPCP, CGSSGGSSGEPKSDATPTCPPCP, and CSGGGGSAEPKAGDATPPTCPPCPPCP;

[0146] (c) Any 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 a cysteine, and any one of the amino acid residues at positions 0, +2, and +3 relative to Y329 of domain D3 is substituted with a cysteine.

[0147] In one embodiment, the modified fusion protein may have all of the following characteristics (a), (b), and (c):

[0148] (a) comprising (i) L243S and R244V amino acid substitutions, or (ii) K241E, L243S, R244V, and H246E amino acid substitutions on the β1-β2 loop of domain D3, L258A, L258S, or L258D amino acid substitutions on the β2-β3 loop of domain D3, and / or (i) K300G amino acid substitution, (ii) K300G, Q302T, and K304S amino acid substitutions, or (iii) K300G, Q302T, K304S, and K306Q amino acid substitutions on the β5-β6 loop of domain D3;

[0149] (b) the linker is an amino acid sequence selected from the group consisting of CSSGDATPTSPPSP, CSKVDKKVEPKSSDTPPTCPPCP, CSGGGGSAEPKAGDATPPTCPPCP, CSGGGGSGGGGSGGGGSAESKYGPPCPPCP, CSNTGSGGEEKKKEKEKEEQEERSSDTPPTCPPCP, CGSSGGSSGEPKSDATPTCPPCP, CSKVDKKVEPKSSDKTYTCPPCP, and CSGGGGSAEPKAGDATPPTCPPCPPCP;

[0150] (c) The amino acid residues at positions L243 and +2 relative to Y329 in domain D3 are substituted with cysteine.

[0151] In one embodiment, the modified fusion protein may have all of the following characteristics (a), (b), and (c):

[0152] (a) comprising K241E, L243S, R244V, and H246E amino acid substitutions on the β1-β2 loop of domain D3, L258A, L258S, or L258D amino acid substitutions on the β2-β3 loop of domain D3, and K300G, Q302T, and K304S amino acid substitutions on the β5-β6 loop of domain D3;

[0153] (b) the linker is an amino acid sequence selected from the group consisting of CSSGDATPTSPPSP, CSKVDKKVEPKSSDTPPTCPPCP, CSGGGGSAEPKAGDATPPTCPPCP, CSGGGGSGGGGSGGGGSAESKYGPPCPPCP, CSNTGSGGEEKKKEKEKEEQEERSSDTPPTCPPCP, CGSSGGSSGEPKSDATPTCPPCP, CSKVDKKVEPKSSDKTYTCPPCP, and CSGGGGSAEPKAGDATPPTCPPCPPCP;

[0154] (c) The amino acid residues at positions L243 and +2 relative to Y329 in domain D3 are substituted with cysteine.

[0155] In one embodiment, the modified fusion protein may comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 10 to 70, 77 to 93, and 98. In one embodiment, the modified fusion protein may consist of an amino acid sequence selected from the group consisting of SEQ ID NOs: 10 to 70, 77 to 93, and 98.

[0156] In one embodiment, the modified fusion protein may 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 NOs: 10 to 70, 77 to 93, and 98. Such variants may contain amino acid mutations (e.g., conservative amino acid substitutions) that appear in other amino acid sequences while retaining or containing the amino acid mutations that confer the properties of the modified fusion proteins of the present disclosure.

[0157] In one embodiment, the components of the modified fusion protein of the present disclosure (VEGFR1 extracellular domain, linker, and multimerization domain) may possess post-translational modifications including, for example, glycosylation, sialylation, acetylation, and phosphorylation.

[0158] In one embodiment, the modified fusion protein of the present disclosure may contain conservative amino acid substitutions at positions other than the amino acid mutation positions exhibiting the characteristics described in the present disclosure. Such conservative amino acid substitutions may be introduced into any of the components of the modified fusion protein of the present disclosure (e.g., the extracellular domain of VEGFR1, the linker, and the multimerization domain).

[0159] In one embodiment, the modified fusion protein of the present disclosure may include a signal peptide or signal sequence for protein secretion from cells. For example, the modified fusion protein of the present disclosure may further include a heterologous peptide, specifically a signal sequence or other peptide having a specific cleavage site at the N-terminus of the mature fusion protein. In one embodiment, the heterologous signal sequence may be recognized and processed by a eukaryotic host cell (i.e., cleaved by a signal peptide hydrolase). Details regarding signal peptides are well known in the art and are provided, for example, but not limited to, in Korean Patent Registration No. 10-2228921. In one embodiment, the signal peptide may be a human interleukin signal sequence. In one embodiment, the signal peptide may consist of the 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). However, any signal peptide that can be used by a skilled artisan to prepare a fusion protein can be used without limitation.

[0160] In one embodiment of the present disclosure, a multimeric fusion protein may be provided that includes two or more modified fusion proteins of the present disclosure. Multimers (e.g., dimers, trimers, tetramers, etc.) may be formed from homogeneous fusion proteins (e.g., homomultimers) or heterogeneous fusion proteins (e.g., heteromultimers). In one embodiment, the multimeric fusion protein may include at least one modified fusion protein that includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 10 to 70, 77 to 93, and 98, or an amino acid sequence that exhibits at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 10 to 70, 77 to 93, and 98. In one embodiment, such fusion proteins are recovered as protein fusion multimers from a host cell containing a nucleic acid encoding a modified fusion protein of the present disclosure. In one embodiment, such multimeric fusion proteins may be glycosylated. For example, such multimeric proteins may be glycosylated at the extracellular domain and / or multimerization domain of the VEGFR after release from the host cell.

[0161] In one embodiment, the modified fusion proteins of the present disclosure have an IC of 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 (inclusive, including any value between these numbers) or less for inhibition of ligand activity (e.g., inhibition of VEGF activity or PLGF activity). 50 may have

[0162] In one embodiment, the modified fusion proteins of the present disclosure have a binding affinity of 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, or greater to a binding partner (e.g., VEGF and / or PLGF). , 50 nM, 45 nM, 40 nM, 35 nM, 30 nM, 25 nM, 20 nM, 15 nM, 10 nM, 5 nM, 1 nM, 900 pM, 800 pM, 700 pM, 600 pM, 500 pM, 400 pM, 300 pM, 200 pM, 100 pM, 50 pM, 25 pM, 12.5 pM, 6.25 pM, 5 pM, 4 pM, 3 pM, 1 pM, 0.5 pM, 0.1 pM, 0.05 pM or 0.01 pM (inclusive, including any value between these numbers).

[0163] III. Nucleic Acids, Vectors and Host Cells

[0164] nucleic acid

[0165] In one embodiment of the present disclosure, an isolated nucleic acid encoding any modified fusion protein component of the present disclosure, such as the VEGFR1 extracellular domain and / or multimerization domain, may be provided. Nucleic acids encoding mammalian VEGFRs have been described for all receptor types. Exemplary nucleic acid sequences can be found, but are not limited to, in U.S. Patent No. 7,928,072 and WO 2006 / 113277. mRNAs encoding human VEGFR1 and VEGFR2 can be found under GenBank accession numbers NM_002019.4 and NM_002253.2, respectively. In one embodiment, the isolated nucleic acid can encode the Ig-like domains D2 and D3 of VEGFR1 comprising the amino acid sequence of SEQ ID NO: 100. In one embodiment, the isolated nucleic acid can encode Ig-like domains D2 and D3 of VEGFR1 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 to the amino acid sequence of SEQ ID NO:100.

[0166] The present disclosure may provide an isolated nucleic acid encoding a multimerization domain (e.g., an Fc portion). In one embodiment, the isolated nucleic acid can encode a multimerization domain comprising the amino acid sequence of SEQ ID NO: 99. In some embodiments, the isolated nucleic acid can encode a multimerization domain 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 to an amino acid sequence selected from the group consisting of SEQ ID NO: 99.

[0167] Isolated nucleic acids encoding modified fusion proteins of the present disclosure may be provided. 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 NOs: 10 to 70, 77 to 93, and 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 to an amino acid sequence selected from the group consisting of SEQ ID NOs: 10 to 70, 77 to 93, and 98.

[0168] An isolated nucleic acid sequence encoding a modified fusion protein or a component of a fusion protein of the present disclosure (eg, the extracellular domain or multimerization domain of VEGFR1) may further comprise a nucleic acid sequence encoding a linker.

[0169] In one embodiment, the isolated nucleic acid may further comprise a sequence encoding a signal peptide that serves as a signal sequence for secretion of the modified fusion protein from a host cell, or may be free of a sequence encoding a signal peptide.

[0170] An isolated nucleic acid molecule encoding a modified fusion protein or a component of a fusion protein (e.g., a VEGFR1 extracellular domain, a linker, or a multimerization domain) of the present disclosure may 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 produced synthetically, or any combination thereof. Such isolated nucleic acid molecules may be produced by various methods known in the art (see, e.g., Molecular Cloning: A Laboratory Manual (Sambrook et al., 2004)). th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2012) and Current Protocols in Molecular Biology (FMAusubel, et al. eds., 2003).

[0171] vector

[0172] In one embodiment of the present disclosure, the present disclosure may relate to a nucleic acid transfer vehicle for introducing one or more nucleic acid sequences encoding a modified fusion protein or fusion protein components into a cell for expression of the protein, or the use thereof. The nucleic acid sequence may be the sequence of the isolated nucleic acid.

[0173] In one embodiment, examples of nucleic acid transfer vehicles include liposomes, biocompatible polymers (including natural and synthetic polymers); lipoproteins; polypeptides; polysaccharides; lipopolysaccharides; artificial viral envelopes; metal particles; and bacteria, viruses, such as baculoviruses, adenoviruses, and retroviruses, bacteriophages, cosmids, plasmids, fungal vectors, and other recombinant vehicles commonly used in the art, described for expression in a variety of eukaryotic and prokaryotic hosts. In one embodiment, the nucleic acid transfer 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 linked to an expression control sequence.

[0174] In one embodiment, the vector may include any elements necessary for establishing the normal function of an expression vector, such as a promoter, a ribosome binding element, a terminator, an enhancer, a selection marker, and an origin of replication. The promoter may be a constitutive, inducible, or repressible promoter. Exemplary promoters are listed in Korean Patent Registration No. 10-0659477, but are not limited thereto.

[0175] Numerous expression vectors capable of transferring nucleic acids into cells (e.g., derived from bacterial, yeast, plant, or mammalian cells) are known in the art and may be used in the present disclosure to produce modified fusion proteins or fusion protein components in cells. For example, E. coli may be transformed with a plasmid, e.g., pBR322 (Mandel et al., J. Mol. Biol., 1970, 53:154), engineered to contain nucleic acid encoding the fusion protein and used to produce the modified fusion protein. The expressed modified fusion protein or fusion protein component may be harvested from the cells and purified by conventional techniques known in the art and as described in this disclosure.

[0176] In one embodiment, an expression vector capable of replicating from a bacterial or eukaryotic host, containing a nucleic acid encoding the modified fusion protein described above, may be used to transfect the host, thereby directly expressing the nucleic acid for the production of a modified fusion protein that may be recovered in a biologically active form, which, in the present disclosure, includes a form capable of binding to a VEGF ligand or a PLGF ligand.

[0177] In one embodiment, expression vectors containing nucleic acid molecules encoding modified fusion proteins or fusion protein components can be identified by at least three common methods, including, but not limited to, (a) DNA-DNA hybridization, (b) the presence or absence of "marker" gene function, and (c) expression of the inserted sequence. Specific details of these three methods are provided in Korean Patent Registration Nos. 10-0659477 and 10-2228921, but are not limited thereto.

[0178] host cell

[0179] In one embodiment of the present disclosure, a host cell may be provided that contains the expression vector encoding the modified fusion protein of the present disclosure. Such a host cell is suitable for expressing the modified fusion protein of the present disclosure and may constitute a host-vector system for producing the modified fusion protein. In one embodiment, the host cell may be used to produce viral particles (e.g., recombinant viral vectors).

[0180] The term "host cell," as used in this disclosure, includes any cell that is or can be a donor for the vectors of this disclosure and their progeny. Due to natural, accidental, or elaborate mutations, the progeny may not necessarily be completely identical (in morphology or genome of total DNA complement) to the original parent cell. In one embodiment, the host cell can be, but is not limited to, a bacterial cell such as E. coli, a yeast cell such as Pichia pastoris, an insect cell such as Spodoptera frugiperda, or a mammalian cell such as a COS, HEK, or CHO cell.

[0181] In one embodiment, the modified fusion proteins of the present disclosure may be expressed transiently, constitutively, or permanently in a host cell.

[0182] In one embodiment of the present disclosure, a method for producing the modified fusion protein of the present disclosure can be provided by culturing the host cells or cells of the host-vector system under conditions that allow the production of a modified fusion protein, and then recovering the produced fusion protein. The modified fusion proteins useful in practicing the present disclosure can be produced by expression 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 and are disclosed, for example, but not limited to, in Korean Patent Registration No. 10-2228921.

[0183] In one embodiment, the modified fusion protein of the present disclosure produced from the host cells may be purified and characterized by various methods. Methods for purifying and characterizing fusion proteins produced from host cells are well known in the art and are disclosed, for example, but not limited to, in Korean Patent Registration No. 10-2228921. For example, the fusion protein produced from the host cells may be recovered from the cells as soluble proteins or inclusion bodies that are quantitatively extracted by dialysis with 8 M guanidinium hydrochloride. Various purification methods may be used to further purify the fusion protein, including, but not limited to, conventional ion exchange chromatography, affinity chromatography, different sugar chromatography, hydrophobic interaction chromatography, reversed-phase chromatography, or gel filtration.

[0184] IV. Viral Particles and Methods of Producing Viral Particles

[0185] In one embodiment of the present disclosure, a viral particle (or virion) may be provided that includes a nucleic acid encoding a modified fusion protein of the present disclosure.

[0186] Viral vectors may be used to deliver nucleic acids encoding fusion proteins or fusion protein components for expression of the proteins in target cells within a specific target tissue (e.g., diseased tissue). Many types of viruses are known, and many have been studied for the purpose of delivering nucleic acids to target cells. Exogenous nucleic acids may be inserted into vectors, such as adenoviruses, partially deleted adenoviruses, fully deleted adenoviruses, adeno-associated viruses (AAVs), retroviruses, lentiviruses, etc., for delivery to cells.

[0187] In one embodiment, the cells are present in an individual, and the virus may be delivered intravenously, intramuscularly, via pancreatic islets, or by other routes of administration. In one embodiment, viral vectors may include those derived from adenovirus, adeno-associated virus (AAV), and retrovirus (including lentivirus, e.g., human immunodeficiency virus (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 entireties. Viral particles containing nucleic acids encoding fusion proteins and methods for producing the same are well known in the art and are provided, for example, but not limited to, in Korean Patent Registration No. 10-2228921.

[0188] IV. Treatment with Fusion Proteins and Viral Particles

[0189] One embodiment of the present disclosure may relate to a pharmaceutical composition comprising a modified fusion protein of the present disclosure as an active ingredient, for use in preventing, ameliorating, or treating a disease associated with a VEGF ligand or a VEGF receptor, or at least one symptom of such a disease.

[0190] One embodiment of the present disclosure may relate to a method for preventing, ameliorating, or treating a disease associated with a VEGF ligand or a VEGF receptor, or at least one symptom of such a disease, comprising administering a modified fusion protein of the present disclosure.

[0191] One embodiment of the present disclosure may relate to the use of a modified fusion protein of the present disclosure for preventing, ameliorating, or treating a disease associated with a VEGF ligand or a VEGF receptor, or at least one symptom of such a disease.

[0192] One embodiment of the present disclosure may relate to a modified fusion protein of the present disclosure or a pharmaceutical composition comprising the same for use in a method for preventing, ameliorating, or treating a disease associated with a VEGF ligand or a VEGF receptor or at least one symptom of such a disease.

[0193] In one embodiment, the modified fusion proteins of the present disclosure may be used in the form of a nucleic acid encoding the modified fusion protein via a nucleic acid transfer vehicle or viral particle (e.g., a recombinant viral vector) for the prevention, amelioration, or treatment of a disease associated with a VEGF ligand or a VEGF receptor, or at least one symptom of such a disease. In one embodiment, the nucleic acid transfer vehicle or viral particle may be used in gene therapy for the prevention, amelioration, or treatment of a disease associated with a VEGF ligand or a VEGF receptor, or at least one symptom of such a disease. In one embodiment, the nucleic acid can produce the encoded protein that mediates a therapeutic effect, i.e., the modified fusion protein of the present disclosure. Any gene therapy method available in the art can be used in accordance with the present disclosure. In one embodiment, the nucleic acid is introduced into a cell prior to final in vivo administration of the recombinant cell. Cells into which nucleic acids may be introduced for gene therapy purposes may include any available cell type, including, but not limited to, 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 or progenitor cells, such as hematopoietic stem or progenitor cells obtained from bone marrow, umbilical cord blood, peripheral blood, or fetal liver. In one embodiment, the cells used for gene therapy may be autologous to the patient.

[0194] In one embodiment, the modified fusion protein may bind to a VEGF protein and / or a PLGF protein. In one embodiment, the modified fusion protein may have any 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 activation of the VEGF signaling pathway; (d) preventing, ameliorating, and / or treating a disease such as an ocular disease, an autoimmune disease, an inflammatory disease, a neoplastic disease, or a cancer; (e) attenuating, preventing, or preventing the growth of a neoplasm or cancer; or (f) inhibiting the metastasis of a neoplasm or cancer. The activity of the modified fusion protein may be measured in vivo and / or in vitro.

[0195] 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 or oil, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, etc. Saline solutions and aqueous dextrose, polyethylene glycol (PEG), and glycerol solutions may also be liquid carriers, particularly for injectable solutions. Such pharmaceutical compositions may further include additional components, such as preservatives, buffers, isotonicity agents, antioxidants and stabilizers, non-ionic wetting or clarifying agents, viscosity enhancers, etc. The pharmaceutical compositions of the present disclosure may be packaged in single unit dosage or multi-dose form. Such compositions are generally formulated as sterile, substantially isotonic solutions. The compositions may also be formulated to possess an osmolality compatible with the aqueous humor of the eye and ophthalmic tissues. Such osmolarity may generally range from about 200 to 400 milliosmoles per kilogram of water ("mOsm / kg"), or about 300 mOsm / kg. The retina is believed to possess an osmolarity of about 283 mOsm / kg.

[0196] In one embodiment, the pharmaceutical composition may be a parenteral composition, which may be advantageously formulated in dosage unit form for uniformity of dosage and ease of administration.

[0197] illness or disease

[0198] In one embodiment, the disease associated with a VEGF ligand or a VEGF receptor may include, but is not limited to, an autoimmune disease (e.g., rheumatoid arthritis, multiple sclerosis, or systemic lupus erythematosus), an inflammatory disease (inflammatory arthritis, osteoarthritis, or psoriasis), a neoplastic disease, cancer (e.g., breast cancer, lung cancer, gastric cancer, pancreatic cancer, or leukemia), an angiogenesis-related disease (e.g., atherosclerosis), or an ophthalmic disease (e.g., age-related macular degeneration, choroidal neovascularization, or uveitis).

[0199] In one embodiment, the cancer is prostate cancer, urethral cancer, penile cancer, breast cancer, lung cancer, esophageal cancer, small intestine cancer, large intestine cancer, rectal cancer, colon cancer, liver cancer, urinary tract cancer (e.g., bladder cancer), kidney cancer, lung cancer (e.g., non-small cell lung cancer), ovarian cancer, cervical cancer, endometrial cancer, vaginal carcinoma, vulvar carcinoma, pancreatic cancer, gastric cancer, endocrine gland cancer, thyroid cancer, parathyroid cancer, adrenal gland cancer, skin cancer (e.g., melanoma), Hodgkin's disease, bone cancer, hematopoietic stem cell cancer of the lymph or bone marrow, chronic or acute leukemia, head and neck cancer, nasopharyngeal carcinoma The tumors may include, but are not limited to, NPC, glioblastoma, teratocarcinoma, neuroblastoma, adenocarcinoma, carcinoma of hepatic lobar origin (e.g., fibrosarcoma or rhabdomyosarcoma), soft tissue sarcomas and carcinomas, choriocarcinoma, hepatoblastoma, central nervous system (CNS) tumors, primary CNS lymphomas, spinal cord tumors, brain stem gliomas, pituitary adenomas, Karposi's sarcoma, or Wilms' tumor. The modified fusion proteins of the present disclosure may act as anti-angiogenic or anti-VEGF agents and may ameliorate, prevent, and / or treat cancer or cancer symptoms.

[0200] Administration method and dosage

[0201] In one embodiment of the present disclosure, a method for delivering an effective amount of a modified fusion protein to a subject may be provided. The modified fusion protein may be delivered to a subject within a composition. The modified fusion protein may also be delivered to a subject by a nucleic acid delivery vehicle or 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 may be provided that includes the modified fusion protein or a nucleic acid delivery vehicle or viral particle (e.g., a recombinant viral vector) containing a nucleic acid encoding the modified fusion protein.

[0202] In one embodiment, the compositions of the present disclosure may be administered to an individual by any route, including, but not limited to, intravenous (e.g., via an infusion pump), intraperitoneal, intraocular, intraarterial, intrapulmonary, oral, inhalation, intravesical, intramuscular, intratracheal, subcutaneous, intraocular, intraspinal, transdermal, transpleural, intraarterial, topical, inhalation (e.g., spray mist), mucosal (e.g., through the nasal mucosa), subcutaneous, transdermal, gastrointestinal, intraarticular, intravesical, intraventricular, intracranial, intraurethral, ​​intrahepatic, and intratumoral. In one embodiment, the compositions of the present disclosure may be administered by any common route, such as by infusion or bolus injection, absorption through epithelial or mucocutaneous linings (e.g., oral, rectal, and intestinal mucosa), or may be administered together with other biologically active ingredients. Administration may 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 intraspinal injection, which may be accomplished by an intraventricular catheter, e.g., a catheter attached to a reservoir such as an Ommaya reservoir.

[0203] In one embodiment, the optimal effective amount of the modified fusion protein, nucleic acid transfer vehicle encoding the same, or composition containing the same can be determined empirically and may vary depending on the type and severity of the disease, the route of administration, the progression and health of the disease, and the weight and body area of ​​the individual. Such determinations are within the skill of one of ordinary skill in the art.

[0204] In one embodiment, the modified fusion protein may be administered at, for example, about 0.05 ng to about 20 mg per kg of body weight per day. In one embodiment, the dosage of the modified fusion protein is about 0.1 mg / kg or more, about 0.5 mg / kg or more, about 1.0 mg / kg or more, about 1.5 mg / kg or more, about 2.0 mg / kg or more, about 2.5 mg / kg or more, about 3.0 mg / kg or more, about 3.5 mg / kg or more, about 4.0 mg / kg or more, about 4.5 mg / kg or more, about 5.0 mg / kg or more, about 6.0 mg / kg or more, about 8.0 mg / kg or more, about 10.0 mg / kg or more, or about 15.0 mg / kg or more. or less, or about 20.0 mg / kg or less, about 17.0 mg / kg or less, about 14.0 mg / kg or less, about 11.0 mg / kg or less, about 9.0 mg / kg or less, about 7.0 mg / kg or less, about 5.5 mg / kg or less, about 5.0 mg / kg or less, about 4.5 mg / kg or less, about 4.0 mg / kg or less, about 3.5 mg / kg or less, about 3.0 mg / kg or less, about 2.5 mg / kg or less, about 2.0 mg / kg or less, about 1.5 mg / kg or less, or about 1.0 mg / kg or less. In one embodiment, the dosage of the modified fusion protein may be about 1.0 mg / kg to about 10.0 mg / kg or about 1.0 mg / kg to about 5.0 mg / kg.

[0205] In one embodiment, the amount of nucleic acid delivery vehicle (e.g., recombinant viral vector) containing a nucleic acid encoding a modified fusion protein of the present disclosure is about 10 per dose. 4 ~about 10 14 In one embodiment, the amount of nucleic acid transfer vehicle containing the nucleic acid encoding the modified fusion protein is about 10 per dose. 5 ~about 10 13, about 10 6 ~about 10 12 , about 10 7 ~about 10 11 , about 10 8 ~about 10 10 , about 10 9 ~about 10 10 , about 10 10 ~about 10 11 , or about 10 11 ~about 10 12 It may be administered to an individual via drp.

[0206] In one embodiment, a composition comprising a modified fusion protein of the present disclosure or a nucleic acid transfer vehicle encoding the same may be administered in a single daily dose, or the total daily dose may be administered in divided doses two, three, or four times daily. In one embodiment, a composition comprising a modified fusion protein of the present disclosure may be administered six times per week, five times per week, four times per week, three times per week, twice per week, once per week, once every two weeks, once every three weeks, once per month, once every two months, once every three months, once every six months, once every nine months, or once per year. In one embodiment, a composition comprising a nucleic acid transfer vehicle containing a nucleic acid encoding a modified fusion protein of the present disclosure may be administered relatively infrequently, for example, once every three months, once every four months, once every five months, once every six months, once every seven months, once every eight months, once every nine months, once every ten months, once every eleven months, or once every year.

[0207] Diagnosis or detection

[0208] The modified fusion proteins of the present disclosure may be labeled with a detectable label, such as a radioisotope, a fluorescent label, a toxin label, an enzyme label, a chemiluminescent label, or a nuclear magnetic resonance imaging agent, to confirm the ligand-receptor binding interaction. Assay systems applicable to the chimeric molecule may also be considered. Such detectable labels are widely known in the art.

[0209] V. ARTICLES OF MANUFACTURE AND KITS

[0210] One embodiment of the present disclosure may relate to an article of manufacture or kit containing a modified fusion protein of the present disclosure, a recombinant viral vector expressing the same, or a composition containing the same in suitable packaging. In one embodiment, the suitable packaging is well known in the art and may include, but is not limited to, vials (e.g., sealed vials), containers, ampoules, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), etc. Such articles of manufacture may be further sterilized and / or sealed.

[0211] In one embodiment, the kit may further include instructions for using the composition for the applications described herein. In one embodiment, the kit may further include other components that are desirable from a commercial and user perspective, such as buffers, diluents, filters, needles, syringes, and package inserts (PIs) containing instructions for performing any of the methods described herein. For example, in one embodiment, the kit may include (i) a fusion protein described herein and / or a recombinant viral vector encoding a fusion protein described herein, (ii) a pharmaceutically acceptable carrier, and (iii) any one or more of the following: buffers, diluents, filters, needles, syringes, and package inserts containing instructions for administration.

[0212] The configuration and effects of the present disclosure will be described in more detail below with reference to examples, however, these examples are provided for illustrative purposes only to aid in understanding the present disclosure and are not intended to limit the scope of the present disclosure.

[0213] [Example 1] Protein production, evaluation of physical properties, and evaluation of binding strength

[0214] Example 1.1 - Production of Expression Vectors

[0215] The coding sequence (CDS) of the modified fusion protein designed in the following examples was codon-optimized to a form suitable for CHO production cells, and the final modified fusion protein was synthesized in the pcDNA3.1(+) vector (Invitrogen) by gene synthesis (Geneuniversal). The existing fusion proteins VEGF-Grab1 and VEGF-Grab3 were also synthesized using the same method.

[0216] Example 1.2 - Production and Characterization of Modified Fusion Proteins

[0217] ExpiCHO used for protein production TM Cells (Gibco) were used for transient expression at passage numbers between 10 and 20. VEGF-Grab1 or VEGF-Grab3 were cultured and purified under the same conditions at each stage and used as a control to measure the effect of each modified fusion protein on improving the physical properties. Transient expression of VEGF-Grab1, VEGF-Grab3, and the modified fusion proteins was performed using Expifectamine provided by the manufacturer (Gibco). TM The Max Titer protocol was used with ExpiCHO (Gibco). One day before transfection, TM 3.0 x 10 cells 6 The cells were subcultured at a density of 6.0 × 10 cells / ml, and the next day, the cell density and viability were measured again. 6 ExpiCHO to give cells / ml TM The expression medium was added to dilute the cells. 25 ml of cell culture medium was dispensed into a 125 ml Erlenmeyer flask, and the cells were stored in a 37°C incubator until the transfection mixture was prepared. 25 μg of the plasmid gene was added to 1 ml of OptiPro TM Mix thoroughly with SFM medium. 80 ul of Expifectamine TM CHO cells in 920 ul of OptiPro TMAfter thoroughly mixing with SFM medium, the cells were left at room temperature for 2 minutes. TM The SFM mixture was added to the premixed plasmid-OptiPro TM After adding the mixture to SFM and mixing well, the mixture was left at room temperature for 2 minutes. After 2 minutes, the mixture was gradually added to the cell culture medium that had been dispensed in advance, and the cells were cultured in an incubator set at 37°C, 8% CO2, and 130 rpm. 18 to 22 hours after transfection, Expifectamine was added. TM CHO Enhancer 150ul and ExpiCHO TM After mixing with 4 ml of feed, the mixture was added to the flask during culture, and cell culture was continued in an incubator set at 32°C, 8% CO2, and 130 rpm. TM An additional 4 ml of feed was added. From day 6 after transfection, cell density and viability were measured using a Countess II (Invitrogen) instrument using trypan blue staining. When cell viability reached 70% to less than 80%, the final titer was measured using a Cedex (Roche) instrument, and the cell culture medium containing the protein was collected by centrifugation at 3,000 x g for 30 minutes. The collected supernatant was filtered using a 0.22 μm syringe filter, and the culture medium was stored at -80°C.

[0218] Protein purification from the culture medium was performed by affinity chromatography using an AKTA avant25 instrument equipped with a Trap Fibro PrismA column. The culture medium, filtered through a 0.22 μm syringe filter, was loaded at a rate of 16 ml per minute and washed with 15 column volumes of 50 mM Na-Pi, 50 mM NaCl, pH 7.0 buffer. To remove nonspecific binding, the column was washed with 15 column volumes of 50 mM Na-Pi, 1 M NaCl, pH 5.5 buffer, and then with 50 mM Na-OAc, pH 4.6 buffer. Bound proteins were eluted with 15 column volumes of 100 mM glycine, pH 3.0 elution buffer and collected in a deep-well block or 15 ml tube. The collected protein was neutralized by adding 1 M Trizma® base (pH 11.0) solution at 1% of the collected protein volume, and the precipitate was removed using a 0.22 μm syringe filter. The pre- and post-neutralization samples were quantified by UV measurement and analyzed by SE-HPLC to determine purity. For binding capacity measurements, samples that did not reach 90% purity were further purified by size exclusion chromatography using a Superdex200 increase 10 / 300 column. The concentrated protein was injected into a Centricon® 30 MWCO concentrator at a volume of 500 μl or less onto a column buffered 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 tube, and its purity was confirmed by SE-HPLC before further concentration. The final concentrated protein was analyzed by UV quantitation and purity was confirmed by SEC-HPLC and SDS-PAGE analysis.

[0219] Using the above procedure, the physical properties of each protein were measured, including one or more of the following: titer, elution volume, step yield, high molecular weight species (HMWS) content, low molecular weight species (LMWS) content, and functional monomer content. Here, titer refers to the concentration of Fc-fusion protein present in the cell culture medium measured at the time of collection. elution volume refers to the total amount of protein recovered after affinity chromatography (the primary purification step). Yield refers to the total amount of protein recovered after primary purification divided by the total amount of protein measured in the culture medium, and refers to the recovery rate of protein recovered by the column during primary purification. High molecular weight species (HMWS) are protein species with masses larger than the predicted functional units of the protein, and low molecular weight species (LMWS) are protein species with masses smaller than the predicted functional units of the protein. Both refer to fractions with abnormal protein folding. Functional monomer refers to fractions with normal protein structure and function.

[0220] Example 1.3 - Evaluation of binding strength to VEGF-A and PLGF

[0221] The binding affinity of each protein for VEGF-A and PLGF ligands was previously measured using an Octet® RED96e (ForteBIO) instrument according to the literature (Kamat et al., 2017).

[0222] Before measuring binding affinity, the AHC sensor was stabilized by soaking in 1x KB buffer (ForteBIO). The target analytes, VEGF-Grab1 or VEGF-Grab-3, and modified fusion proteins were injected at a concentration of 20 nM to allow binding to the sensor, followed by washing with 1x KB buffer to remove any remaining proteins. VEGF-A and PLGF were then injected at three concentrations (VEGF-A: 40, 20, and 10 nM; PLGF: 5, 2.5, and 1.25 nM) to measure binding affinity. The average binding affinity measured at two concentrations within the range of Full X^2 ​​(less than 3) and Full R^2 (more than 0.95), which are used to ensure reliability, was calculated and compared.

[0223] [Example 2] Design of modified fusion proteins

[0224] Example 2.1 - Preparation and characterization of surface charge transition mutants

[0225] To secure a fusion protein containing a VEGFR receptor construct with enhanced physical properties, the present inventors analyzed the surface charge distribution of the protein using a structure containing the entire extracellular domain of VEGFR1 (PDB entry 5t89) as a template, and identified areas with densely packed positive charges and hydrophobic surface charges (Figure 1A).

[0226] This confirmed that the side chains of amino acid residues in the β1-β2 loop (designated Site 1) corresponding to amino acid residues T236-T247 in the VEGFR1 domain D3, the β2-β3 loop (designated Site 2) corresponding to amino acid residues T256-V262, and the β5-β6 loop (designated Site 3) corresponding to amino acid residues D299-L308 in the VEGFR1 domain D3 were exposed on the surface of the fusion protein. Positions for potential mutations were selected and inferred by substituting positions derived from the surface charge distribution of the protein around the surface-exposed amino acid residues (Figures 1B and 1C). Mutations of the surface-exposed amino acid residues in the flexible loop were used to stabilize the surface charge and structure of the fusion protein. For example, we attempted to replace the surface-exposed amino acid residues with amino acid residues present at homologous positions in VEGFR2. In addition, the amino acid residues at Site 2 are exposed on the protein surface that binds to the ligand (VEGF or PLGF), and mutations at these positions affect the binding strength with the ligand or the solubility due to the exposed surface residues. Therefore, we attempted to replace the amino acid residues at Site 2 with amino acid residues having polar side chains that are smaller or similar in size to the side chains of the existing amino acid residues, within the range that does not significantly affect the protein structure or the binding strength with the ligand.

[0227] Modified fusion proteins with mutations at different positions were prepared as shown in Tables 32 and 33. The mutation positions of each mutant are shown in Table 1. The mutants were prepared according to Example 1, and their physical properties and binding strength were evaluated. The results of evaluating the physical properties of mutants prepared based on the wild-type VEGFR protein (C88) are shown in Table 2, and the results of evaluating the binding strength are shown in Tables 3 and 4. The results of evaluating the physical properties of mutants prepared based on VEGF-Grab3 are shown in Table 5, and the results of evaluating the binding strength are shown in Table 6. The results of evaluating the binding strength represent the relative binding strength to VEGF-Grab3 (manufactured by Samsung Biologics).

[0228] The surface charge mutation positions or disulfide bond positions described in the following examples or tables are those for the C10 to C87 mutants (SEQ ID NO: 10 to SEQ ID NO: 70) that are mutated based on the amino acid sequence of SEQ ID NO: 3, and those for the C97 to C114 and C119 mutants (SEQ ID NO: 77 to SEQ ID NO: 93 and SEQ ID NO: 98) that are mutated based on the amino acid sequence of SEQ ID NO: 71.

[0229] [Table 1]

[0230] [Table 2]

[0231] [Table 3]

[0232] [Table 4]

[0233] [Table 5]

[0234] [Table 6]

[0235] The results in Tables 2 to 6 confirm that the modified fusion protein according to one embodiment of the present disclosure exhibits excellent productivity based on the titer and protein elution volume results, and excellent physical properties based on the functional monomer results. Furthermore, the results for kon, for example, confirm that the modified fusion protein also exhibits excellent properties in terms of binding strength to ligands.

[0236] Example 2.2 - Preparation and characterization of linker mutants

[0237] The human VEGF receptor binds to its ligand, VEGF or PLGF, through interaction with immunoglobulin-like domains D2 and D3 (see Figure 2A). Fusion proteins using domains D2 and D3 (e.g., VEGF-Grab3) have an open conformation between domains D2 and D3 before binding to the ligand, but then form a closed conformation upon binding to the ligand (see Figures 2B and 2C). If the bond length between domain D3 and the multimerization domain does not provide sufficient space for the structure of domains D2 and D3 to bind to the ligand, or if interference occurs between the structures, the binding avidity of the fusion protein to the ligand may be reduced. Therefore, the bond length and binding mode between domain D3 and the multimerization domain in the fusion protein may affect the structure of the fusion protein when it binds to the ligand and its binding avidity to the ligand.

[0238] In the native human VEGF receptor, the distance from the end of domain D3 to the dimerization site of domain D4 is approximately 37 Å (see Figure 2D), and identical domains exist bound to each other in the domain D4 and subsequent domains. Domain D4 and subsequent domains can be considered to have a structure similar to the multimerization domain in the fusion protein. Therefore, the present inventors modified the length and type of linker in the modified fusion protein of the present disclosure to mimic and maintain the structural characteristics of the native human VEGF receptor. Specifically, they introduced a linker consisting of a Gly-Ser repeat polypeptide and amino acid sequences of hinge regions derived from IgG1, IgG4, and IgD, as well as mutant sequences of these hinges. This was intended to reduce tension in the loop (linker) formed between domain D3 and the multimerization domain (Fc region) upon ligand binding, thereby increasing physical stability.

[0239] In addition, amino acid sequences derived from the hinge region of immunoglobulins have various protease cleavage sites due to their own properties (Vlasak and Ionescu, 2011, Mabs 3:253-263). After introduction into a fusion protein, glycosylation, such as O-glycosylation, and oligomerization may occur (Song et al., 2020, Computational and Structural Biotechnology Journal 18:3925-3935). To improve the in vivo stability of the modified fusion proteins of the present disclosure, the inventors further modified the amino acid sequence at the protease cleavage site, e.g., the papain cleavage site, and attempted to modify the Ser or Thr amino acid residue at which glycosylation occurs. Furthermore, to increase the structural flexibility of the linker, the inventors attempted to replace cysteines present in the core hinge-derived sequence of the Fc hinge region (e.g., CPPCP). In the present disclosure, an exemplary scheme for designing the introduced linker is shown in FIG.

[0240] The modified fusion proteins with linkers introduced were prepared as shown in Tables 32 and 33, and the linker characteristics of each variant are shown in Table 7. In Table 7, the amino acids in bold and underlined indicate the amino acid residues at the positions where the mutations were made.

[0241] Mutants were prepared according to Example 1, and their physical properties and binding strength were evaluated. The results of evaluating the physical properties of mutants prepared based on the wild-type VEGFR protein are shown in Table 8, and the results of evaluating binding strength are shown in Tables 9 and 10. The results of evaluating the physical properties of mutants prepared based on VEGF-Grab3 are shown in Table 11, and the results of evaluating binding strength are shown in Tables 12 and 13. Table 12 shows the results of evaluating the relative binding strength to VEGF-Grab3 (Panoros Biosciences), and Table 13 shows the results of evaluating the relative binding strength to VEGF-Grab3 (Samsung Biologics). The relative binding strength to C88 or VEGF-Grab3 (Panoros Biosciences) was compared with that produced under the same conditions and in the same batch as the mutants.

[0242] [Table 7]

[0243] [Table 8]

[0244] [Table 9]

[0245] [Table 10]

[0246] [Table 11]

[0247] [Table 12]

[0248] [Table 13]

[0249] The results in Tables 8 to 13 confirm that the modified fusion protein according to one embodiment of the present disclosure exhibits excellent productivity based on the titer and protein elution volume results, and excellent physical properties based on the functional monomer results. Furthermore, the results for, for example, kon, confirm that the modified fusion protein also exhibits excellent properties in terms of binding strength to ligands.

[0250] On the other hand, it was confirmed that mutants (C115 to C118) having linker sequences outside the linker length of the linker mutants according to one embodiment of the present disclosure had significantly reduced titers or functional monomer ratios compared to the modified fusion proteins of the present disclosure (e.g., the C108 mutant).

[0251] Example 2.3 - Preparation and Characterization of Disulfide Bond Mutants

[0252] Because fusion proteins are conjugated heterologous proteins, it is expected that stability will decrease due to nonspecific cleavage at the binding site. The end of domain D3 forms a loop, and because it lacks secondary structure, it is expected to have weak stability because it is the site where binding to the heterologous protein multimerization domain begins. By introducing a covalent disulfide bond, the researchers attempted to induce interdomain binding near the site where heterologous protein binding occurs, preventing physical cleavage of the protein and improving stability.

[0253] The potential site for disulfide bond introduction in the fusion protein was determined by tertiary structure analysis. Residues located in the variable loop adjacent to the terminal region of domain D3, with their side chains pointing toward the linker and within 6 Å of the Y329Cα atom, were selected as potential disulfide bond mutation residues. The 3D structure of the fusion protein was determined using a structural modeling program (Phyre2, Swissmodel), and a structural model with the disulfide bond introduced was obtained by in-silico mutation of the insertion site. The energy level change of the fusion protein due to the introduction of the disulfide bond was then predicted using a tertiary structure energy prediction program (foldx in YASARA, Schymkowitz et al. 2005 Nucleic Acids Research. 33:W382-388).

[0254] The amino acid residues in the β1-β2 loop or the β5-β6 loop of domain D3 were selected as positions that are physically close to the end of domain D3 in the fusion protein and can form disulfide bonds.The amino acid residues at positions -2, -1, 0, +1, +2, and +3 relative to Y329 were selected as the end of domain D3.

[0255] Modified fusion proteins with introduced disulfide bonds were prepared as shown in Tables 32 and 33, and the disulfide bond characteristics of each mutant are shown in Table 14. The energy values ​​of the mutants calculated using a tertiary structure energy prediction program are shown in Table 15. Mutants were prepared according to Example 1, and their physical properties and binding avidity were evaluated. The physical property evaluation results for mutants prepared based on the wild-type VEGFR protein are shown in Tables 16, and the binding avidity evaluation results are shown in Tables 17 and 18. The physical property evaluation results for mutants prepared based on VEGF-Grab3 are shown in Tables 19, and the binding avidity evaluation results are shown in Tables 20 and 21. Table 20 shows the results of the relative binding avidity evaluation to VEGF-Grab3 (Panoros Biosciences), and Table 21 shows the results of the relative binding avidity evaluation to VEGF-Grab3 (Samsung Biologics). The relative binding avidity evaluation to VEGF-Grab3 (Panoros Biosciences) was compared with that produced under the same conditions and configuration as the mutants.

[0256] [Table 14]

[0257] [Table 15]

[0258] [Table 16]

[0259] [Table 17]

[0260] [Table 18]

[0261] [Table 19]

[0262] [Table 20]

[0263] [Table 21]

[0264] The results in Tables 16 to 21 confirm that the modified fusion protein according to one embodiment of the present disclosure exhibits excellent productivity based on the titer and protein elution volume results, and excellent physical properties based on the functional monomer results. These results can also be confirmed by the calculation results of the energy levels of the structural model. Table 15 shows that the energy values ​​of the fusion protein structure with the introduced disulfide bond are equal to or lower than the energy values ​​of the structure without the introduced disulfide bond, indicating that the introduction of the disulfide bond ensures structural stability. Furthermore, the results for, for example, kon confirm that the modified fusion protein also exhibits excellent properties in terms of binding strength to ligands.

[0265] Example 2.4 - Preparation and characterization of combinatorial mutants

[0266] Combination mutants were prepared by combining the properties of mutants exhibiting relatively superior properties from the mutants obtained from the results of Examples 2.1 to 2.3 above. The properties of each mutant are shown in Table 22. Mutants were prepared according to Example 1, and their physical properties and binding strength were evaluated. The results of evaluating the physical properties of mutants prepared based on the VEGFR wild-type protein are shown in Table 23, and the results of evaluating the binding strength are shown in Tables 24 and 25 (here, for comparison, the VEGFR wild-type protein (C88), C65, and C73 mutants were also evaluated). The results of evaluating the physical properties of mutants prepared based on VEGF-Grab3 are shown in Table 26, and the results of evaluating the binding strength are shown in Tables 27 and 28. Table 27 shows the results of evaluating the binding strength relative to VEGF-Grab3 (Panoros Biosciences), and Table 28 shows the results of evaluating the binding strength relative to VEGF-Grab3 (Samsung Biologics). The relative binding strength to VEGF-Grab3 (Panoros Biosciences) was evaluated by comparing with that produced under the same conditions and arrangement as the mutant.

[0267] [Table 22] TIFF2025530910000024.tif83165

[0268] [Table 23]

[0269] [Table 24]

[0270] [Table 25]

[0271] [Table 26]

[0272] [Table 27]

[0273] [Table 28]

[0274] The results in Tables 23 to 28 confirm that the modified fusion proteins according to one embodiment of the present disclosure exhibit excellent productivity based on the titer and protein elution volume results, and excellent physical properties based on the functional monomer results. Furthermore, the results for, for example, KON confirm that the proteins also exhibit excellent properties in terms of binding strength to ligands. These results confirm that the properties introduced during the structural design of the modified fusion proteins in Examples 2.1 to 2.3 are effectively manifested, and that the combination of these properties produces a synergistic effect.

[0275] Example 2.5 - Production and Characterization of Other Mutants

[0276] The DKA amino acid sequence located at the D3 end of the fusion protein is the structural site where the linkage to the multimerization domain begins. Because it forms a loop when linked to the linker, it is a physically weak site, making it highly susceptible to cleavage during protein expression and purification. The presence of physically weak sites can lead to the generation of fragments during protein expression and purification. Therefore, in Examples 2.1 to 2.3 above, modified fusion proteins were prepared by deleting the KA at the D3 end of the domain from most of the sequences. To confirm that the deletion of the amino acid sequence at the D3 end of the domain does not affect the improved physical properties of the modified fusion proteins of the present disclosure, deletion mutants of the KALE amino acid sequence at the linker-linked C-terminus of the D3 domain were prepared and their physical properties were examined. Specifically, a mutant (C89) in which the KA amino acid was deleted based on the VEGFR wild-type protein (C88) and mutants (C90 and C91) in which the KA and KAL were deleted based on the VEGF-Grab3 were prepared.

[0277] When producing a fusion protein containing the VEGFR1 extracellular domain, mutations may occur in the N-terminal sequence of domain D2 depending on the type or characteristics of the expression vector. To confirm that such mutations do not affect the physical properties of the fusion protein, mutants with amino acid mutations at the N-terminus of domain D2 were produced. Specifically, since all of the mutants in Examples 2.1 to 2.3 contain the vector-derived EF amino acid sequence at the N-terminus of domain D2, mutants containing the EF amino acid sequence (C4 and C5), mutants lacking the EF amino acid sequence (C8 and C9), and mutants beginning with SDT and containing the native VEGFR1 sequence at the N-terminus (C6 and C7) were produced.

[0278] In the multimerization domain used to prepare the fusion protein, mutants were prepared to confirm that the D126E amino acid substitution, L128M amino acid substitution, and / or K217 amino acid deletion based on SEQ ID NO: 99 did not affect the physical properties or efficacy of the fusion protein. Specifically, a mutant (C95) with D126E and L128M amino acid substitutions and a mutant (C96) with K217 amino acid deletion based on VEGF-Grab3 were prepared.

[0279] Modified fusion proteins containing the above additional mutations were prepared as shown in Table 33. The mutants were prepared according to Example 1, and their physical properties, serum stability, and VEGF-A inhibitory activity were evaluated. Physical 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 activity was evaluated according to the method described in Example 6. The evaluation results are shown in Tables 29 to 31 and Figure 4.

[0280] [Table 29]

[0281] [Table 30]

[0282] [Table 31]

[0283] The results in Tables 29 to 31 confirm that mutations at the end of domain D3, mutations at the N-terminus of domain D2, and mutations in the multimerization domain do not affect the improved physical properties, serum stability, or VEGF-A inhibitory ability of the modified fusion protein.

[0284] The properties of the mutants in Examples 2.1 to 2.4 above are summarized in Table 32 below.

[0285] [Table 32] TIFF2025530910000035.tif244165 TIFF2025530910000036.tif240165 TIFF2025530910000037.tif127165

[0286] Table 33 lists the identification codes, amino acid sequences, and SEQ ID NOs for the proteins described in this disclosure.

[0287] [Table 33] TIFF2025530910000039.tif244167 TIFF2025530910000040.tif244167 TIFF2025530910000041.tif244167 TIFF2025530910000042.tif244167 TIFF2025530910000043.tif245167 TIFF2025530910000044.tif244167 TIFF2025530910000045.tif244167 TIFF2025530910000046.tif244167 TIFF2025530910000047.tif245167 TIFF2025530910000048.tif245167 TIFF2025530910000049.tif244167 TIFF2025530910000050.tif244167 TIFF2025530910000051.tif244167 TIFF2025530910000052.tif244167 TIFF2025530910000053.tif245167 TIFF2025530910000054.tif166167

[0288] [Example 3] Stability evaluation in a blood-mimicking environment

[0289] To indirectly analyze the in vivo stability of the modified fusion proteins of the present disclosure, the concentration of the protein variants remaining after a specific time period was measured by ELISA using a reaction with rat reference blood. At 168, 96, 48, 24, 4, 2, 1, and 0 hours before the experimental run, VEGF-Grab3, the modified fusion proteins, and Zaltrap (SANOFI; SEQ ID NO: 101) were mixed with rat reference blood to a final concentration of 10 μg / ml and stored in a 37°C incubator. On the day before the experiment, human VEGF-A (R&D Systems) was coated onto a plate after overnight incubation at 4°C. The plate was washed with 0.1% PBS-T wash buffer to remove insufficiently bound human VEGF-A, followed by a 1-hour blocking treatment with 5% skim milk in 0.1% PBS-T at room temperature. Standards used for concentration comparison were prepared by serial dilution of VEGF-Grab3 and Zaltrap at concentrations ranging from 50 to 0.39 μg / ml. The standard serial dilutions and the protein-serum mixtures prepared for each time point were diluted 200-fold in blocking buffer and then added in 100 μl portions to plate wells. The wells were incubated for 2 hours at room temperature. The reaction solution was then removed, and the plate was washed with wash buffer. The detection antibody, HRP Goat Anti-Human IgG Fc Cross-Absorbed Secondary Antibody (Invitrogen), was incubated for 1 hour at room temperature. Unreacted detection antibody was then removed by washing, and 100 μl of 3,3',5,5'-tetramethylbenzidine (TMB) solution (Sigma) was added to each well. The incubation was continued for 10 minutes at room temperature, followed by the addition of stop solution (Sigma). The absorbance was measured at 450 nm using a plate reader.A standard curve was plotted based on the absorbance of the measured standards (VEGF-Grab3 and Zaltrap), and the remaining amount of modified fusion protein was calculated and compared. From the calculated results, the concentration at 168 hours (7 days) was calculated relative to the concentration at time 0, and the relative absorbance % is shown in Tables 34, 35, and Figure 5.

[0290] [Table 34]

[0291] [Table 35]

[0292] According to Table 34 and Figure 5, the modified fusion proteins of the present disclosure had superior serum stability compared to the wild-type VEGFR protein. The C112 mutant, into which a disulfide bond was introduced, exhibited significantly superior serum stability (6.7%) compared to the C109 mutant, which has the same linker sequence but no disulfide bond introduced. The C115, C116, and C118 mutants, which have linker sequences outside the linker length of the linker mutant according to one embodiment of the present disclosure, did not exhibit superior serum stability compared to the wild-type VEGFR protein.

[0293] According to Table 35, the modified fusion proteins of the present disclosure exhibited serum stability of greater than approximately 97%, demonstrating serum stability comparable to or superior to that of the commercially available pharmaceutical, Zaltrap®. No protein degradation patterns were observed in any of the proteins during the experimental period (7 days). These results confirm that the modified fusion proteins of the present disclosure have excellent in vivo stability, comparable to that of pharmaceuticals compared to Zaltrap®. It can be indirectly predicted that the modified fusion proteins of the present disclosure may improve the duration of efficacy of the substance upon in vivo administration, which may affect efficacy.

[0294] [Example 4] Isoelectric point analysis

[0295] The isoelectric points of VEGF-Grab3 and the modified fusion proteins of the present disclosure were analyzed using an image capillary isoelectric focusing (iclEF) system. 40 μl of the modified fusion protein diluted to a concentration of 2 mg / ml was added to 160 μl of a pre-prepared master mix solution (SERVALYT). TM A mixture of low pI marker (5.85) (ProteinSimple), high pI marker (9.99) (ProteinSimple), 1% methylcellulose (ProteinSimple), DW, 500 mM arginine (Sigma), 200 mM iminodiacetic acid (Sigma), and 10 M urea (Sigma) was mixed and centrifuged to remove any precipitates or air bubbles. The mixture was then inserted into each well of the instrument (Maurice, ProteinSimple). Two ml of anode solution (Catholyte solution) and cathode solution (Anolyte solution) were added to the OH- and H+ positions of the cartridge, respectively, and the cartridge was then inserted into the instrument. The isoelectric point was then measured according to the instrument's instructions. Since the measured pI was not a single peak but multiple peaks in a specific range, the protein pI distribution was defined as three intervals: acidic (pI 6-7), neutral (pI 7-8), and basic (pI 8<), and the intervals with the highest distribution ratios were compared. The results are shown in Table 36, Figures 6 and 7.

[0296] [Table 36]

[0297] As shown in Table 36, Figures 6 and 7, the existing substance VEGF-Grab3 was measured to have a higher pI over a wider range than Zaltrap. The highest proportion of charge variants was found in the basic range (pI < 8.0), in contrast to Zaltrap, which had its highest distribution in the acidic range (pI 6.0-7.0). The modified fusion protein of the present disclosure showed a decreased (C62) or absent (C61, C65, C71, C72, C73) distribution rate in the basic range, and the range with the highest distribution shifted to the acidic range. Therefore, the modified fusion protein of the present disclosure is expected to have reduced nonspecific interactions with the matrix and cell surface. Furthermore, this reduced nonspecific interaction may improve the intracellular recycling of the modified fusion protein of the present disclosure, thereby improving the in vivo half-life and efficacy of the modified fusion protein of the present disclosure.

[0298] [Example 5] Thermal stability analysis

[0299] VEGF-Grab3 and modified fusion proteins of the present disclosure (C62, C72, C73) were prepared by diluting with 1x PBS buffer to a final concentration of 2mg / ml. 12.5µl of protein was diluted with 2.5µl of 8x protein thermal shift buffer. TM Protein Thermal Shift™ dye (Life Technologies) and 5.0 μl Protein Thermal Shift TM Buffer (Life Technologies) was added to prepare the final reaction solution. 20 μl of the prepared reaction solution was placed in a PCR tube and analyzed using QuantStudio. TM Real-time PCR (QuantStudio TM After arranging the proteins in trays on a real-time PCR system, the Tm values ​​were measured. All analyses were performed in quadruplicate. The melting curve plots and Tm1, Tm2, ​​and Tm3 values ​​for each protein are shown in Figure 8 and Table 37.

[0300] [Table 37]

[0301] As shown in Figure 8 and Table 37, the existing VEGF-Grab3 melting point was observed to have three intervals: Tm1 = 41.83°C, Tm2 = 48.50°C, and Tm3 = 67.55°C. The modified fusion proteins (C62, C72, and C73) of the present disclosure exhibited a single Tm point, with Tms increased by a minimum of 11°C and a maximum of 20°C compared to the initial Tm of VEGF-Grab3. This confirms that the modified fusion proteins of the present disclosure possess excellent thermal stability.

[0302] [Example 6] VEGF-A suppressive ability analysis

[0303] To quantitatively analyze the inhibitory effect of VEGF-A, we used a genetically engineered cell line (KDR / NFAT-RE HEK293, Promega) that expresses VEGFR-2 (KDR) and has a luciferase reporter system for VEGF-A / VEGFR-2 interaction. 6 25 μl of cells at a density of 1000 cells / ml were dispensed into a 96-well plate, and 25 μl of VEGF-A solution (33.3 ng / ml concentration) and 25 μl of VEGF-Grab3 or modified fusion proteins of the present disclosure diluted at different concentrations were added and incubated for 6 hours at 37°C in a 5% CO2 incubator. The plate was then cooled at room temperature for 15 minutes, after which 75 μl of Bio-Glo reagent (Promega, luciferase assay buffer + substrate mixture) was added. After 10 minutes of incubation in the dark, luminescence was measured using a plate reader. Analysis was performed using GraphPad Prism to determine the IC for each substance. 50 The values ​​were calculated, and the results are shown in Tables 38 to 41 below and Figure 9. In Figure 9C, the graphs for VEGFA stimulation and VEGF stimulation represent the results when only VEGFA or VEGF was treated without any mutant treatment.

[0304] The data in Tables 38 to 41 each show values ​​for proteins produced under the same conditions and configurations, and therefore show values ​​for VEGF-Grab3 (Panol Biosciences), which was used as a control.

[0305] [Table 38]

[0306] [Table 39]

[0307] [Table 40]

[0308] [Table 41]

[0309] According to Figure 9 and Tables 38 to 41, both VEGF-Grab3 and the modified fusion proteins of the present disclosure exhibited low levels of IC 50 The mutants C115 and C116, which have linker sequences outside the linker length of the linker mutant according to one embodiment of the present disclosure, did not exhibit superior VEGF-A inhibitory ability compared to the wild-type VEGFR protein.

[0310] From the above description, it will be understood by those skilled in the art to which the present disclosure pertains that the present disclosure may be embodied in other specific forms without changing the technical spirit or essential features of the present disclosure. In this regard, it should be understood that the above-described embodiments are illustrative in all respects and are not limiting. The scope of the present disclosure should be interpreted as including all modifications and variations derived from the meaning and scope of the appended claims and their equivalents, rather than the above detailed description.

Claims

1. comprising a VEGFR1 extracellular domain, a linker, and a multimerization domain; the VEGFR1 extracellular domain comprises an immunoglobulin (Ig)-like domain D2 and an Ig-like domain D3 of VEGFR1, and a linker is present between the Ig-like domain D3 and the multimerization domain; A modified fusion protein having any one or more of the following 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, or with an amino acid whose side chain carries a negative charge, or with an amino acid whose side chain carries an electrostatic negative charge; wherein the β1-β2 loop of domain D3 comprises amino acid residues T236 to T247 of the amino acid sequence of SEQ ID NO: 1, the β2-β3 loop comprises amino acid residues T256 to V262 of the amino acid sequence of SEQ ID NO: 1, and the β5-β6 loop comprises amino acid residues D299 to L308 of the amino acid sequence of 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 any one of the amino acid residues on the β1-β2 loop or any one of the amino acid residues on the β5-β6 loop of domain D3 and any one of the amino acid residues at positions −2, −1, 0, +1, +2, and +3 relative to Y329 of domain D3 is substituted with a cysteine.

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

3. 2. The modified fusion protein of claim 1, wherein property (a) is that 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 have been substituted with an amino acid that reduces the net pI of the protein, an amino acid whose side chain carries a negative charge, or an amino acid whose side chain carries an electrostatic negative charge.

4. The modified fusion protein of claim 3, wherein property (a) is that one or more amino acid residues on the β2-β3 loop of domain D3 have been replaced with an amino acid residue having a side chain smaller in size than the existing amino acid residue or an amino acid residue having a polar side chain of similar size.

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

6. Characteristic (a) is one or more amino acid substitutions of K241T, K241E, L243S, R244V, and H246E on the β1-β2 loop of domain D3; an amino acid substitution of L258A, L258S, or L258D on the β2-β3 loop of domain D3, and / or The modified fusion protein of claim 5, which comprises one or more amino acid substitutions of K300G, Q302T, K304S, and K306Q on the β5-β6 loop of domain D3.

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

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

9. 2. The modified fusion protein of claim 1, comprising R238S and R275N amino acid substitutions in domain D3.

10. The modified fusion protein of claim 1, wherein the C-terminal amino acid sequence of domain D3 linked to the linker is KALE based on the K331 amino acid residue, and in this case, any one or more of the amino acids K, A, L, and E can be deleted.

11. The modified fusion protein of claim 1, wherein the N-terminus of domain D2 begins with the amino acid sequence SDT or the amino acid sequence EF.

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

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

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

15. The GS repeat sequence is GS, GSSG, (GSSG) ２ , GGGGS, (GGGGS) ４ , GS(GGGGS) and GS(GGGGS) ３ 15. The modified fusion protein of claim 14, which is selected from the group consisting of:

16. 2. The modified fusion protein of claim 1, wherein in property (b), the linker comprises an amino acid sequence of a hinge region derived from an immunoglobulin, and the amino acid sequence of the hinge region can be modified.

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

19. 17. The modified fusion protein of claim 16, wherein the fusion protein has an amino acid mutation at a papain recognition site or a glycosylation site present in a hinge region derived from an immunoglobulin, in property (b).

20. In the characteristic (b), the amino acid mutation in the papain recognition site is performed by substituting any one or more amino acid residues present in the papain recognition site with alanine, serine, tyrosine, proline, or threonine, or by inserting into the papain recognition site a sequence of 1 to 10 amino acids containing at least one amino acid having an aromatic carbon or a cyclic carbon in its side chain; The modified fusion protein of claim 19, wherein the amino acid mutation in the glycosylation site is either (i) a deletion of a serine or threonine present in the hinge region derived from an immunoglobulin, or (ii) a substitution with an amino acid other than serine, asparagine, or threonine.

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

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

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

24. In property (b), The linker is selected from the group consisting of GSSGDKTHTSPPSP, GSSGEPKSSDKTYTSPPPSP, GSKVDKKVEPKSSDKTHTCPPCP, GSKVDKKVEPKSSDKTYTCPPCP, GSKVDKKVEPKSSDTPPTCPPCP, GSGGGGSGGGGGSGGGGGSAESKYGPPCPPPCP, and GSNTGSGGEE. KKKEKEKEEQEERSSDKTYTCPPCP, GSNTGSGGEEKKKEKEKEEQEERSSDTPPTCPPCP, EGSSGGSSGEPKSDA TPTCPPCP,EGSSGGSSGEPKSDSTYTCPPCP,GCKVDKKVEPKSSDKTYTCPPCP,CSKVDKKVEPKSSDKTYTCPP CP, GGGGSAEPKAGDKAPPGPPGP, GGGGSAEPKSSDKTYTCPPCP, GGGGSGGGGSGGGGSGGGSAEPKSSDKTY TCPPCP,CSSGDATPTSPPSP,CSKVDKKVEPKSSDTPPTCPPCP,CSGGGGSAEPKAGDATPPTCPPCP,CSGGGGS 2. The modified fusion protein of claim 1, which has an amino acid sequence selected from the group consisting of GGGGSGGGGSAESKYGPPCPPPCP, CSNTGSGGEEKKKEKEKEEQEERSSDTPPTCPPCP, CGSSGGSSGEPKSDATPTCPPCP, and CSGGGGSAEPKAGDATPPTCPPCPPPCP.

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

26. In property (c), any 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; The modified fusion protein of claim 1, wherein any one of the amino acid residues at positions 0, +2, and +3 relative to Y329 in domain D3 is substituted with cysteine.

27. In property (c), T241 and the amino acid residue at position +3 relative to Y329 in domain D3 are substituted with cysteine; harboring L243C and Y329C amino acid mutations; L243 and the amino acid residue at position +2 relative to Y329 in domain D3 are substituted with cysteine; or having R244C and Y329C amino acid mutations; 27. The modified fusion protein of claim 26, having N303C and Y329C amino acid mutations.

28. (a) one or more amino acid substitutions of K241T, K241E, L243S, R244V, and H246E on the β1-β2 loop of domain D3; an amino acid substitution of L258A, L258S, or L258D on the β2-β3 loop of domain D3, and / or comprising any one or more amino acid substitutions of K300G, Q302T, K304S, and K306Q on the β5-β6 loop of domain D3; (b) Linkers are GSSGDKTHTSPPPSP, GSSGEPKSSDKTYTSPPPSP, GSKVDKKVEPKSSDKTHTCPPCP, GSKVDKKVEPKSSDKTYTCPPCP, GSKVDKKVEPKSSDTPPTCPPCP, GSGGGGSGGGGGSGGGGGSAESKYGPPCPPPCP, GSNT GSGGEEKKKEKEKEEQEERSSDKTYTCPPCP, GSNTGSGGEEKKKEKEKEEQEERSSDTPPTCPPCP, EGSSGGS SGEPKSDATPTCPPCP, EGSSGGSSGEPKSDSTYTCPPCP, GCKVDKKVEPKSSDKTYTCPPCP, CSKVDKKVEP KSSDKTYTCPPCP, GGGGSAEPKAGDKAPPGPPGP, GGGGSAEPKSSDKTYTCPPCP, GGGGSGGGGGSGGGGSGG GGSAEPKSSDKTYTCPPCP, CSSGDATPTSPPSP, CSKVDKKVEPKSSDTPPTCPPCP, CSGGGGSAEPKAGDAT an amino acid sequence selected from the group consisting of PPTCPPCP, CSGGGGSGGGGGSGGGGGSAESKYGPPCPPPCP, CSNTGSGGEEKKKEKEKEEQEERSSDTPPTCPPCP, CGSSGGSSGEPKSDATPTCPPCP, and CSGGGGSAEPKAGDATPPTCPPCPPPCP; (c) any 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 of domain D3 is substituted with cysteine, and any one of the amino acid residues at positions 0, +2, and +3 relative to Y329 of domain D3 is substituted with cysteine; The modified fusion protein of claim 1, which has all of the above properties (a) to (c).

29. (a) (i) L243S and R244V amino acid substitutions, or (ii) K241E, L243S, R244V, and H246E amino acid substitutions on the β1-β2 loop of domain D3; an L258A, L258S, or L258D amino acid substitution on the β2-β3 loop of domain D3, and / or comprising (i) a K300G amino acid substitution, (ii) a K300G, Q302T, and K304S amino acid substitution, or (iii) a K300G, Q302T, K304S, and K306Q amino acid substitution 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, CSGGGGSGGGGSGGGGGSAESKYGPPCPPPCP, CSNTGSGGEEEKKKEKEKEEQEERSSDTPPTCPPCP, CGSSGGSSGEPKSDATPTCPPCP, CSKVDKKVEPKSSDKTYTCPPCP, and CSGGGGSAEPKAGDATPPTCPPCPPCP; (c) L243 and the amino acid residue at position +2 relative to Y329 in domain D3 are substituted with cysteine; The modified fusion protein of claim 1, which has all of the above properties (a) to (c).

30. (a) K241E, L243S, R244V, and H246E amino acid substitutions on the β1-β2 loop of domain D3; an L258A, L258S, or L258D amino acid substitution on the β2-β3 loop of domain D3, and comprising K300G, Q302T, and K304S amino acid substitutions 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, CSGGGGSGGGGSGGGGGSAESKYGPPCPPPCP, CSNTGSGGEEEKKKEKEKEEQEERSSDTPPTCPPCP, CGSSGGSSGEPKSDATPTCPPCP, CSKVDKKVEPKSSDKTYTCPPCP, and CSGGGGSAEPKAGDATPPTCPPCPPCP; (c) L243 and the amino acid residue at position +2 relative to Y329 in domain D3 are substituted with cysteine; The modified fusion protein of claim 1, which has all of the above properties (a) to (c).

31. 2. The modified fusion protein of claim 1, 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.

32. the multimerization domain is (a) the Fc portion of an immunoglobulin; (b) the CH3 site of IgG1, IgG2, IgG3, or IgG4; (c) CH2 and CH3 sites of IgG1, IgG2, IgG3, or IgG4; (d) an Fc portion of an immunoglobulin comprising an amino acid sequence having at least 85% identity to SEQ ID NO: 99; or (e) The modified fusion protein of claim 1, which is an Fc portion of an immunoglobulin comprising the amino acid sequence of SEQ ID NO:

99.

33. the multimerization domain is an Fc portion of an immunoglobulin; The modified fusion protein of claim 1, starting from the lower hinge region of the Fc portion.

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

99.

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

99.

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

37. A pharmaceutical composition for the prevention or treatment of an autoimmune disease, an inflammatory disease, a neoplastic disease, a cancer, an angiogenesis-related disease, or an ocular disease, comprising the modified fusion protein of any one of claims 1 to 35 as an active ingredient.

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

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

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

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

42. 42. A pharmaceutical composition for delivering a viral vector to a subject, comprising the recombinant viral vector of claim 41, wherein a fusion protein encoded by the recombinant viral vector is expressed in the subject, and the pharmaceutical composition is for the prevention or treatment of an autoimmune disease, an inflammatory disease, a neoplastic disease, a cancer, an angiogenesis-related disease, or an eye disease.

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

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