Modified fusion protein and scFv conjugate and its applications

Abstract

This disclosure relates to conjugates of vascular endothelial growth factor, placental growth factor, and scFv antigens, as well as their uses. Conjugates according to one embodiment of this disclosure may exhibit excellent productivity and stability. Conjugates according to one embodiment of this disclosure may exhibit excellent blood stability. Conjugates according to one embodiment of this disclosure may exhibit excellent antitumor, antifibrotic, and / or immune cell activation effects.

Description

[Technical Field]

[0001] This disclosure relates to a modified fusion protein and scFv conjugate that binds to vascular endothelial growth factor, placental growth factor, and the antigen of the conjugated scFv, and to its uses. [Background technology]

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

[0003] VEGF is classified into five types: VEGF-A, -B, -C, -D, and PlGF (placenta growth factor), and these are known to contribute to angiogenesis (Carmeliet, Py Jain, RK. 2000. Nature 407: pp. 249-257; Kuwano M, et al. 2001. Intern Med 40: pp. 565-572). In contrast, there are three types of VEGF receptors: VEGF R1, R2, and R3. The type of ligand that binds to them and the degree of binding affinity differ depending on the type of receptor, and among them, VEGFR-1 / -2 is known to be involved in angiogenesis (Veikkola, et al., 2000. Cancer research 60: pp. 203-212).

[0004] The development of drugs that target angiogenesis by removing vascular endothelial growth factor, a key component in tumorigenesis, has continued from the past to the present. A representative example is bevacizumab (Stacker, et.al., 2013. Chinese Journal of Cancer Research. 32(6):pp.297-302.; Kazazi-Hyseni, et.al., 2010. Oncologist. 15(8):pp.819-825; DrugBank Accession Number: DB00112), a monoclonal antibody drug targeting VEGF-A. Although monoclonal antibody drugs have a mechanism of action that binds to a single vascular endothelial growth factor, multiple growth factors are involved in canceration, making it difficult to achieve significant efficacy with monoclonal antibodies alone. Bevacizumab, on the other hand, increased the expression rate of other forms of growth factors that were not targeted through intracellular compensatory mechanisms, leading to the side effect of developing resistance (Bagley, et.al., 2011. Clinical cancer research 17(5):pp.976-988; Lieu, et.al., 2013. Plos One 8:e77117; Cutsem, et.al., 2020 Clinical cancer research 26(3):pp.717-725). For these reasons, the need for developing drugs that multiple-target vascular growth factors related to carcinogenesis while simultaneously having a targeting function against cancer cells has been steadily increasing.

[0005] To overcome the shortcomings of monoclonal antibody drugs, drugs that multitarget vascular endothelial growth factor (VEGF) have been developed. Representative protein drugs include VEGF-Trap (Ciombor, et al., 2013, Clinical Cancer Research. 19(8): pp. 1920-1925. DrugBank Accession Number: DB08885) and VEGF-Grab (Lee, et al., 2015, Mol. Cancer. Ther. 14(2): pp. 470-479). Both mimic parts of the decoy receptor; VEGF-Trap is a recombinant fusion protein fused with immunoglobulin Fc in the form of heterologous VEGFR1 D2-VEGFR2 D3 proteins, while VEGF-Grab is a recombinant fusion protein fused with immunoglobulin Fc in the form of homologous VEGFR1 D2-D3 proteins. Efforts have been made to improve the physical properties of such fusion proteins that multitarget vascular endothelial growth factor.

[0006] On the other hand, it is possible to consider attaching additional antigen-targeting scFvs (single-chain variable fragments) to such fusion proteins, and among these, scFvs targeting programmed cell death protein ligand 1 (PD-L1), which is overexpressed in many cancer tissues, could be investigated (Dong, et al., 2002. Nat. Med 8:787-9). The programmed cell death 1 (PD-1) receptor and its ligand, programmed cell death ligand 1 (PD-L1), are immune barrier proteins involved in suppressing immune system responses associated with chronic infection, pregnancy, tissue allografts, autoimmune diseases, and cancer. PD-L1 modulates immune responses by binding to the inhibitory receptor PD-1, which is expressed on the surface of T cells, B cells, and mononuclear cells. PD-L1 also interacts with another receptor, B7-1, negatively regulating T cell function. The formation of PD-L1 / PD-1 and PD-L1 / B7-1 complexes negatively regulates T-cell receptor signaling, leading to subsequent downregulation of T-cell activation and suppression of antitumor immune activity.

[0007] Cancer tissues that overexpress PD-L1 include melanoma, cutaneous squamous cell carcinoma (CSCC), head and neck squamous cell carcinoma (HNSCC), non-small cell lung cancer, kidney cancer, head and neck cancer, thyroid cancer, colon cancer, liver cancer, ovarian cancer, breast cancer, and pancreatic cancer.

[0008] Therefore, there has been a need to develop conjugates that exhibit excellent anticancer effects while possessing not only the ability to selectively target target substances within cancer cells based on their high binding affinity and increased duration of action in the body, but also the physical and chemical stability to meet the high productivity levels of protein therapeutics required for commercial production. [Overview of the Initiative] [Problems that the invention aims to solve]

[0009] Improving the physical properties of proteins increases their physical and chemical stability during expression and purification, maximizing production efficiency. It also reduces nonspecific binding, aggregation, and degradation during introduction into the body, leading to greater persistence in the body. In the case of anticancer drugs, it increases selective targeting of cancer cells, resulting in superior anticancer effects.

[0010] The inventors of this disclosure have developed a modified fusion protein containing VEGFR1 domains D2 and D3 by performing amino acid mutations in a specific region of domain D3, modifying and mutating the linker length, and introducing disulfide bonds into the fusion protein. They have also developed a conjugate by attaching a PD-L1 targeted scFv to such a modified fusion protein and have confirmed that such a conjugate exhibits excellent productivity, stability, and anticancer efficacy. [Means for solving the problem]

[0011] 1. One embodiment of the present disclosure may be a conjugate in which a PD-L1-targeted scFv (anti-PD-L1 scFv) is bound to a VEGFR domain. The conjugate can bind to a target expressed on the surface of cancer cells via the scFv and simultaneously bind to VEGF and PlGF around the cancer cells, thereby inhibiting the differentiation of vascular endothelial cells. This selectively suppresses angiogenesis around cancer cells, leading to the inhibition of cancer growth. The conjugate in one embodiment of the present disclosure may be a conjugate containing a PD-L1-targeted scFv to a fusion protein comprising a VEGFR1 (vascular endothelial growth factor receptor 1) extracellular domain, a linker, and a multimerization domain, characterized in that the PD-L1-targeted scFv is fused to the N-terminus of the fusion protein. In one embodiment of the present disclosure, the PD-L1 targeted scFv may specifically be an anti-PD-L1 scFv such as atezolizumab scFv or a separately developed anti-PD-L1 scFv (A167 scFv; SEQ ID NO: 40).

[0012] 2. One embodiment of the “fusion protein” 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 the immunoglobulin (Ig)-like domain D2 and Ig-like domain D3 of VEGFR1, and the linker is located between the Ig-like domain D3 and the multimerization domain, and having one or more of the following characteristics (a) to (c): (a) substitution of one or more amino acids from K241E, L243S, R244V, and H246E on the β1-β2 loop of domain D3, amino acid substitution of L258A, L258S, or L258D on the β2-β3 loop of domain D3, and one or more amino acid substitutions from K300G, Q302T, and K304S on the β5-β6 loop of domain D3 The result is that the β1-β2 loop of domain D3 contains amino acid residues T236-T247 of the amino acid sequence of VEGFR1 (e.g., amino acid sequence of VEGFR1 in Table 2; Sequence ID No. 41), the β2-β3 loop contains amino acid residues T256-V262 of the amino acid sequence of VEGFR1, and the β5-β6 loop contains amino acid residues D299-L308 of the amino acid sequence of VEGFR1; (b) the linker has an amino acid length of approximately 14 to 35; and (c) a disulfide bond is present in the fusion protein, and one amino acid residue located at positions -2, -1, 0, +1, +2, and +3 relative to L243 and Y329 of domain D3, in particular the 331 amino acid residue at position +2, is substituted with cysteine.

[0013] 3. In one embodiment, the property (a) of the modified fusion protein may include amino acid substitutions of K241E, L243S, R244V, and H246E on the β1-β2 loop of domain D3; amino acid substitutions of L258A, L258S, or L258D on the β2-β3 loop of domain D3; and amino acid substitutions of K300G, Q302T, and K304S on the β5-β6 loop of domain D3. In relation to property (a), the substitutions may be such that the amino acid is substituted with an amino acid that reduces the net pI of the protein, an amino acid with a negatively charged side chain, or an amino acid that would have an electrostatic negative charge, compared to the residue before substitution.

[0014] 4. In one embodiment, the modified fusion protein may have an N-terminus of domain D2 starting with the amino acid sequence EF.

[0015] 5. In one embodiment, in relation to property (b), the linker of the modified fusion protein may contain a GS repeat sequence, and the GS repeat sequence may be an amino acid sequence with a length of 2 to 35 composed of only G and S.

[0016] 6. In one embodiment, one or more glycines (G) contained in the GS repeat sequence included in the linker of the modified fusion protein may be substituted with cysteine (C).

[0017] 7. In one embodiment, in relation to property (b), the linker of the modified fusion protein may contain an amino acid sequence of a hinge region derived from an immunoglobulin, and the amino acid sequence of the hinge region may be modified.

[0018] 8. In one embodiment, the hinge region derived from the immunoglobulin may be derived from human IgD and / or IgG.

[0019] 9. In one embodiment, the modified fusion protein may have an amino acid mutation at a papain recognition site or a glycosylation site present in the hinge region derived from an immunoglobulin in relation to characteristic (b). Specifically, the amino acid mutation at the papain recognition site may be to substitute one or more of the amino acid residues present at the papain recognition site with alanine, serine, tyrosine, proline, or threonine, or to insert a 1- to 10-amino acid sequence containing at least one amino acid having an aromatic carbon or a cyclic carbon in the side chain into the papain recognition site. The amino acid mutation at the glycosylation site may be to (i) delete serine or threonine present in the hinge region derived from an immunoglobulin, or (ii) substitute serine, asparagine, or threonine with an amino acid other than serine, asparagine, or threonine.

[0020] 10. In one embodiment, the modified fusion protein may, in relation to characteristic (b), contain, in order from the N-terminus to the C-terminus, an amino acid sequence selected from the group consisting of (i) CS, CSSG, CS(GGGGS), CS(GGGGS)3, C(GSSG)2, GS, GSSG, (GSSG)2, GGGGS, (GGGGS)4, GS(GGGGS), and GS(GGGGS)3, and (ii) a hinge region derived from an immunoglobulin selected from the group consisting of human IgG1, IgG4, or IgD / G1, and the amino acid sequence of the hinge region may be modified.

[0021] 11. In one embodiment, the modified fusion protein may, in relation to characteristic (b), have an amino acid sequence selected from the group consisting of CSSGDATPTSPPSP (SEQ ID NO: 68), CSKVDKKVEPKSSDTPPTCPPCP (SEQ ID NO: 69), CSGGGGSAEPKAGDATPPTCPPCP (SEQ ID NO: 70), CSGGGGSGGGGSGGGGSAESKYGPPCPPCP (SEQ ID NO: 71), CSNTGSGGEEKKKEKEKEEQEERSSDTPPTCPPCP (SEQ ID NO: 72), CSNTGSGGEEKKKEKEKEEQEERSCDTPPTCPPCP (SEQ ID NO: 73), CGSSGGSSGEPKSDATPTCPPCP (SEQ ID NO: 74), and CSKVDKKVEPKSSDKTYTCPPCP (SEQ ID NO: 75).

[0022] 12. 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) the linker comprises CSSGDATPTSPPSP, CSKVDKKVEPKSSDTPPTCPPCP, CSGGGGSAEPKAGDATPPTC (c) The amino acid sequence is selected from the group consisting of PPCP, CSGGGGSGGGGSGGGGSAESKYGPPCPPCP, CSNTGSGGEEKKKEKEKEEQEERSSDTPPTCPPCP, CSNTGSGGEEKKKEKEKEEQEERSCDTPPTCPPCP, CGSSGGSSGEPKSDATPTCPPCP, and CSKVDKKVEPKSSDKTYTCPPCP; (c) The amino acid residue located at +2 positions relative to L243 and Y329 in domain D3 may be substituted with cysteine.

[0023] 13. In one embodiment, the modified fusion protein may consist of an amino acid sequence selected from the group consisting of amino acid sequences C61-C75 (SEQ ID NOs. 44-58) in Table 2.

[0024] 14. In one embodiment, the polymerizing domain may be (a) the Fc site of an immunoglobulin; (b) the CH3 site of IgG1 or IgG4; (c) the CH2 and CH3 sites of IgG1 or IgG4; (d) the Fc site of an immunoglobulin containing an amino acid sequence having at least 85% identity with the C88 amino acid sequence of Table 2 (SEQ ID NO: 59); or (e) the Fc site of an immunoglobulin containing an amino acid sequence of the Fc region (e.g., SEQ ID NO: 60).

[0025] 15. In one embodiment, the polymerizing domain may include (or be composed of) the Fc site of IgG1 consisting of the amino acid sequence of SEQ ID NO: 60. On the other hand, the polymerizing domain may have i) one or more of the following amino acid substitutions: T20Q, D126E, L128M, M198L, and K217, relative to SEQ ID NO: 60; or ii) one or more of the following amino acid substitutions: L4A, L5A, H38Q, K44Q, Y66F, A97G, A100S, P101S, R125Q, D126E, L128M, K179R, Q189E, P215L, and K217.

[0026] 16. In one embodiment, the modified fusion protein may be in the form of a dimer or a polymer.

[0027] 17. One embodiment of the present disclosure relates to a conjugate of a modified fusion protein and an scFv relating to an embodiment of the present disclosure, wherein the scFv may be atezolizumab scFv or A167 scFv (Table 9; SEQ ID NO: 39 or 40) that binds to PD-L1.

[0028] 18. One embodiment of the present disclosure may relate to a pharmaceutical composition for the prevention or treatment of chronic infection, tissue allograft, autoimmune disease, inflammatory disease, neoplastic disease, cancer, angiogenesis-related disease, or ocular disease, comprising a modified fusion protein and scFv conjugate according to one embodiment of the present disclosure as an active ingredient.

[0029] 19. One embodiment of the present disclosure may relate to a nucleic acid molecule that encodes a modified fusion protein and scFv conjugate relating to one embodiment of the present disclosure.

[0030] 20. One embodiment of the present disclosure may relate to a host cell containing a nucleotide sequence that encodes a modified fusion protein and scFv conjugate relating to one embodiment of the present disclosure.

[0031] 21. One embodiment of the present disclosure may relate to a vector comprising a nucleotide sequence that encrypts a conjugate of a modified fusion protein and scFv relating to one embodiment of the present disclosure.

[0032] 22. In one embodiment, the vector may be a recombinant viral vector.

[0033] 24. One embodiment of the present disclosure may relate to a pharmaceutical composition for delivering a viral vector to a target body, comprising a recombinant viral vector according to one embodiment of the present disclosure, wherein a conjugate of a fusion protein encoded by the recombinant viral vector and scFv is expressed in the target body, and is for the prevention or treatment of chronic infection, tissue allograft, autoimmune disease, inflammatory disease, neoplastic disease, cancer, angiogenesis-related disease, or eye disease.

[0034] 25. In one embodiment, the recombinant viral vector may be a recombinant adeno-related viral vector.

[0035] 26. One embodiment of the present disclosure may be a method for administering to a subject requiring a modified fusion protein-scFv conjugate and / or a recombinant viral vector containing a nucleotide sequence encoding the modified fusion protein-scFv conjugate according to one embodiment of the present disclosure, to prevent or treat one or more selected from the group consisting of chronic infection, tissue allograft, autoimmune disease, inflammatory disease, neoplastic disease, cancer, angiogenesis-related disease, or ocular disease.

[0036] 27. One embodiment of the present disclosure may relate to the use of a recombinant viral vector comprising a modified fusion protein-scFv conjugate according to one embodiment of the present disclosure and / or a nucleotide sequence encoding the modified fusion protein-scFv conjugate according to one embodiment of the present disclosure for the prevention or treatment of one or more conditions selected from the group consisting of chronic infection, tissue allografts, autoimmune diseases, inflammatory diseases, neoplastic diseases, cancer, angiogenesis-related diseases, or ocular diseases. [Effects of the Invention]

[0037] A modified fusion protein and anti-PD-L1 scFv conjugate according to one embodiment of this disclosure can exhibit excellent productivity, physical stability, and chemical stability.

[0038] A modified fusion protein and anti-PD-L1 scFv conjugate according to one embodiment of this disclosure can exhibit excellent blood stability.

[0039] A modified fusion protein and anti-PD-L1 scFv conjugate according to one embodiment of this disclosure can exhibit excellent binding affinity to VEGF, PlGF, and PD-L1.

[0040] A conjugate of a modified fusion protein and anti-PD-L1 scFv according to one embodiment of this disclosure can exhibit excellent co-binding affinity to the respective antigens of the modified fusion protein and scFv.

[0041] A conjugate of a modified fusion protein and anti-PD-L1 scFv according to one embodiment of this disclosure can exhibit excellent inhibitory effects against the respective antigens of the modified fusion protein and scFv.

[0042] The modified fusion protein and anti-PD-L1 scFv conjugate according to one embodiment of this disclosure may have acidic properties and exhibit an increased ratio of charge variants in the acidic and neutral regions of the pI distribution. As a result, the modified fusion protein may exhibit an effect of improving nonspecific interactions with the matrix and cell surface in the body.

[0043] A modified fusion protein and anti-PD-L1 scFv conjugate according to one embodiment of this disclosure can exhibit excellent anticancer effects.

[0044] The modified fusion protein and anti-PD-L1 scFv conjugate according to one embodiment of this disclosure can inhibit the activity of cancer-associated fibroblasts (CAFs) that express CD141 by lowering PlGF levels in the tumor microenvironment (TME), thereby inhibiting the formation of fibrous tissue. This reduces pressure on blood vessels, lowers VEGF-A levels, and normalizes blood vessels. The normalized blood vessels and improved peri-cancer fibrosis can enhance drug access and increase immune cell infiltration, thereby suppressing tumor growth.

[0045] A modified fusion protein and anti-PD-L1 scFv conjugate according to one embodiment of this disclosure may exhibit antitumor, antifibrotic, and / or immune cell activation effects. [Brief explanation of the drawing]

[0046] [Figure 1]Figure 1 shows the 2D structure of the modified VEGFR1 D2-D3 domain (the modified fusion protein of this disclosure) bound to anti-PD-L1 scFv. [Figure 2] Figures 2A to 2O are graphs showing the productivity and purity of the conjugate of anti-PD-L1 scFv and the modified fusion protein. [Figure 3] Figures 3A to 3C are graphs showing the serum stability of conjugates of vascular endothelial growth factor, placental growth factor, and PD-L1, specifically modified fusion proteins of anti-PD-L1 scFv (control group: VEGF-Grab-based fusion protein (H-12C08 or prototype) and PB101 (VEGF-Grab)). [Figure 4] Figures 4A to 4K are graphs showing the binding affinity of conjugates of vascular endothelial growth factor, placental growth factor, and PD-L1, as well as a modified fusion protein of anti-PD-L1 scFv, to VEGF-A, PlGF, and PD-L1 (control group: prototype conjugate based on anti-PD-L1 scFv and VEGF-Grab). [Figure 5] Figures 5A to 5F are graphs showing the co-binding affinity of conjugates of vascular endothelial growth factor, placental growth factor, and PD-L1, as well as a modified anti-PD-L1 scFv fusion protein, to their respective targets. [Figure 6] Figures 6A to 6O are graphs showing the cellular-level signaling inhibitory effects on VEGF-A and PD-L1 of the conjugate (PB203) of the anti-PD-L1 scFv and modified fusion protein described herein, which binds to vascular endothelial growth factor, placental growth factor, and PD-L1 (control group: VEGF-Grab / PB101; H-12C18; H-30D01; Tecentriq; anti-PD-1 antibody; modified fusion protein / backbone / PB102). [Figure 7] Figures 7A to 7R are graphs showing the isoelectric points of the composites according to this disclosure (control group: PB101 / PD20A01). [Figure 8] Figures 8A to 8D are graphs analyzing the thermal stability of the composite according to this disclosure. [Figure 9]Figures 9A and 9B are PK analysis graphs of the composite according to this disclosure. [Figure 10] Figure 10 is a graph showing the tumor growth inhibitory efficacy of the conjugate of the anti-PD-L1 scFv and modified fusion protein described herein, which binds to vascular endothelial growth factor, placental growth factor, and PD-L1, in a mouse model. [Modes for carrying out the invention]

[0047] The various embodiments or examples described herein are illustrative for the purpose of clearly illustrating the technical concept of the Disclosure and are not intended to limit to any particular embodiment. The technical concept of the Disclosure includes various modifications, equivalents, alternatives, and embodiments or examples selectively combined from all or part of each embodiment or example described herein.

[0048] All technical and scientific terms used in this disclosure have meanings that would be generally understood by a person with ordinary skill in the art to which this disclosure pertains, unless otherwise defined.

[0049] Unless otherwise specified in the context, singular expressions used in this document may imply plural meanings, and this also applies to singular expressions used in the claims.

[0050] I. Definition

[0051] In this disclosure, the term “about” may indicate a normal range of error for each value, as is widely known to skilled technicians in the art. This may mean that, in the context of any numerical value or range described in this disclosure, it may mean ±20%, ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% of the numerical value or range mentioned or claimed in one embodiment. In terms of the length of a nucleotide or amino acid sequence, “about” may indicate that the nucleic acid or protein is not limited to the number of nucleotides or amino acids described, and may include several nucleotides or amino acids added to or removed from both ends, provided that these do not impair functional activity.

[0052] Expressions such as "includes," "equipped with," and "possess" used in this disclosure should be understood as open-ended terms that implicitly include other embodiments in a manner similar to "includes," unless otherwise specifically stated in the phrase or sentence containing such expression.

[0053] As used in this disclosure, the terms "and / or" may mean one or more of the items, any combination of the items, or all of the items in the items related to the term.

[0054] As used in this disclosure, the term "amino acid" can mean all naturally occurring L-α amino acids. This definition may include norleucine (Nle), ornithine, and homocysteine.

[0055] As used in this disclosure, the terms “mutation” or “amino acid mutation” can mean a substitution, insertion, deletion, or combination thereof of an amino acid sequence compared to a reference (e.g., native sequence) polypeptide or protein, and the term “variant” can mean a molecule that, as a result of such mutation, has any difference in its amino acid sequence compared to a reference polypeptide or protein.

[0056] Furthermore, the scope of variants in this disclosure may include proteins or fragments or derivatives thereof that exhibit the same or similar biological activity, as well as derivatives that have been otherwise modified during or after translation, for example, by glycation, cleavage by protein hydrolysis, or linkage to antibody molecules or other cellular ligands.

[0057] As used in this disclosure, the term "carriers" may include pharmaceutically acceptable carriers, excipients, or stabilizers that are nontoxic to cells or mammals at the applied dose and concentration. Often, such pharmaceutically acceptable carriers are aqueous pH buffer solutions. Examples of pharmaceutically acceptable carriers may include, but are not limited to, buffers such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; 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®.

[0058] As used in this disclosure, the term “effective dose” can mean an amount sufficient to produce a beneficial or desired clinical or biochemical outcome. An effective dose may be administered once or more times. An effective dose can mean an amount sufficient to alleviate, improve, stabilize, delay, or inhibit the progression of a disease state.

[0059] As used in this disclosure, the term “ligand” may mean a molecule that binds with high affinity to a modified fusion protein (e.g., VEGF-A, -B, -C, -D, and PlGF). In other contexts, “ligand” may mean any molecule, formulation, or compound that covalently or transiently specifically binds to a molecule such as a polypeptide or protein.

[0060] As used in this disclosure, the terms “individual” or “subject” may mean a mammal. Mammals include, but are not limited to, domestic animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In one embodiment, the individual or subject may be a human.

[0061] As used in this disclosure, the term “pharmaceutically acceptable carrier and / or diluent” may include, but is not limited to, any solvent, dispersion agent, coating agent, antimicrobial agent and antifungal agent, isoform formulation and adsorption retardant.

[0062] Any mode of implementation of the Disclosure described herein is understood to include "including," "consisting of," and / or "essentially consisting of."

[0063] II. Modified Fusion Proteins and Modified Fusion Protein Components

[0064] 1.Vascular endothelial growth factor receptor (VEGFR)

[0065] In one embodiment, the modified fusion protein of this disclosure may include the extracellular domain of VEGFR1 as a component. VEGFR1 is also known as fms-related tyrosine kinase (FLT-1) and is encoded by the FLT1 gene. The amino acid sequence of VEGFR1 is presented as the amino acid sequence of VEGFR1 in Table 2 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 to the C-terminus of the extracellular region, and this is also the case for VEGFR2 and VEGFR3.

[0066] In one embodiment, the extracellular domain of VEGFR1 may include Ig-like domains D2 and D3. As used in this disclosure, "Ig-like domain D2 of VEGFR1" refers to the second Ig-like domain located at the N-terminus of the extracellular region of VEGFR1, and "Ig-like domain D3 of VEGFR" refers to the third Ig-like domain located at the N-terminus of the extracellular region of VEGFR1, but the concept includes variants within a range that maintains the functionality of these domains (e.g., binding to VEGF ligands and / or inhibition of VEGFR pathway activation). Because there may be differences in the amino acid sequences of active proteins depending on the species, the Ig-like domains D2 and D3 of VEGFR1 may include their wild type or active variants, and are not limited to their origin or sequence.

[0067] In one embodiment, the modified fusion protein of the present disclosure may further include other VEGFR extracellular domains in addition to the Ig-like domains D2 and D3 of VEGFR1.

[0068] Without being bound by theory, this disclosure suggests that the extracellular domain of VEGFR inhibits the activation of the VEGF pathway by binding to a VEGF ligand, thereby blocking the interaction between the VEGF ligand and VEGFR. Also without being bound by theory, this disclosure suggests that the extracellular domain of VEGFR may bind to VEGFR for dominant-negative inhibition of the VEGF signaling pathway. In one embodiment, the extracellular domain of 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.

[0069] In one embodiment, the extracellular domain of VEGFR may or may not include a signal peptide that acts as a signal sequence for the secretion of the extracellular domain of VEGFR or a modified fusion protein containing it from a host cell. Such a signal peptide may be operably ligated to a nucleic acid that encodes the protein of interest (e.g., the extracellular domain of VEGFR1).

[0070] 2. Linker

[0071] The components of the modified fusion protein (e.g., the extracellular domain or multimerization domain of VEGFR1) may be linked by linking moieties such as peptide linkers. Such linkers increase the flexibility of the fusion protein components and do not significantly interfere with the structure of each functional component within the fusion protein.

[0072] In one embodiment, the linker may be used to ligate the C-terminus of the VEGFR1 extracellular domain (e.g., the C-terminus of the Ig-like domain D3) to the N-terminus of the multimerization domain (e.g., the IgG1Fc region).

[0073] In one embodiment, the linker may include an amino acid sequence of a hinge region derived from an immunoglobulin. The hinge region derived from an immunoglobulin may include a hinge region located in the N-terminal region of the Fc site of the immunoglobulin. In one embodiment, the hinge region derived from an immunoglobulin may be derived from human IgD or IgG.

[0074] 3. Multimerization domain

[0075] This disclosure provides a multimerizing domain (e.g., an Fc site of an immunoglobulin) that can be a component of any modified fusion protein. A multimerizing domain is a portion of a multimeric protein that facilitates the association of subunits, forming, for example, dimers, trimers, tetramers, etc. The term “multimerizing domain” as used in this disclosure may be used to refer to dimerizing domains, trimerizing domains, tetramerizing domains, etc. A fusion protein containing a multimerizing domain can interact with other fusion proteins containing multimerizing domains to produce a fusion protein multimer (e.g., a fusion protein dimer). For example, the IgG Fc site is a dimerizing domain that can fuse with the extracellular domain of VEGFR1 as described in this disclosure. A fusion protein containing the extracellular domain of VEGFR1 and the IgG Fc site can dimerize with yet another fusion protein containing the IgG Fc site to produce a fusion protein dimer that can simultaneously bind to VEGF ligand and PlGF ligand.

[0076] In one embodiment, the Fc site may be in a form in which part of the protein is glycated, or in which the entire protein is not glycated.

[0077] In one embodiment, the Fc site may have a T20Q amino acid substitution, a D126E amino acid substitution, an L128M amino acid substitution, an M198L amino acid substitution, and / or a K217 amino acid deletion, relative to SEQ ID NO: 60. Such amino acid mutations can increase the half-life of the fusion protein through interaction with FcRn. The amino acid mutations may appear identically in other immunoglobulin Fc sites having sequences corresponding to the amino acid sequence of SEQ ID NO: 60, and the modified fusion protein of this disclosure may include other immunoglobulin Fc sites having the amino acid mutations as a polymerizing domain component.

[0078] In one embodiment, the Fc site may have L4A amino acid substitution, L5A amino acid substitution, H38Q amino acid substitution, K44Q amino acid substitution, Y66F amino acid substitution, A97G amino acid substitution, A100S amino acid substitution, P101S amino acid substitution, R125Q amino acid substitution, D126E amino acid substitution, L128M amino acid substitution, K179R amino acid substitution, Q189E amino acid substitution, P215L amino acid substitution, and / or K217 amino acid deletion, relative to SEQ ID NO: 60. Such amino acid mutations may eliminate the effector function of the Fc site. The amino acid mutations may similarly appear in other immunoglobulin Fc sites having sequences corresponding to the amino acid sequence of SEQ ID NO: 60, and the modified fusion protein of this disclosure may include other immunoglobulin Fc sites having the amino acid mutations as components of the polymerizing domain.

[0079] The following describes the properties of the modified fusion protein of this disclosure.

[0080] 4. Surface charge transfer mutation characteristics

[0081] In one embodiment, the modified fusion protein of this disclosure may have surface charge transfer mutation properties. In this disclosure, the modified fusion protein having surface charge transfer mutation properties may be used interchangeably with "surface charge transfer variant" or "surface charge variant".

[0082] In the surface charge transfer mutation characteristics according to one embodiment, the deformed fusion protein may be substituted with amino acids that decrease the net pI of the protein, amino acids whose side chains become negatively charged, or amino acids whose side chains become electrostatically negative, by substitution of the K241E, L243S, R244V, and H246E amino acids on the β1-β2 loop of domain D3, substitution of the L258A, L258S, or L258D amino acids on the β2-β3 loop of domain D3, and / or substitution of the K300G, Q302T, and K304S amino acids on the β5-β6 loop of domain D3. In one embodiment, amino acids whose side chains become electrostatically negative can mean those that, after substitution, become electrostatically negative due to the properties of adjacent amino acid residues or molecules, and can represent a relative concept arising from the electron distribution within the molecule. For example, amino acid residues containing hydroxyl (-OH) can become electrostatically negatively charged due to the lone pair of electrons on the oxygen atom, and this phenomenon can reduce the net pI of the protein.

[0083] In one embodiment, the β1-β2 loop of Ig-like domain D3 may contain amino acid residues T236-T247 of the amino acid sequence of VEGFR1 shown in Table 2. In one embodiment, the β2-β3 loop of Ig-like domain D3 may contain amino acid residues T256-V262 of the amino acid sequence of VEGFR1 shown in Table 2. In one embodiment, the β5-β6 loop of Ig-like domain D3 may contain amino acid residues D299-L308 of the amino acid sequence of VEGFR1 shown in Table 2. In this 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 this disclosure have confirmed that the amino acid residues present in the loops are exposed on the surface of domain D3, and have achieved surface charge stabilization and structural stabilization of the deformed fusion protein by mutation of the amino acid residues present in the loops.

[0084] In one embodiment, amino acid residues present at Site 1 and Site 3 influence each other through interactions between amino acid residues due to their structural positions, thereby potentially introducing double, triple, or quadruple amino acid mutations (e.g., amino acid substitutions) at each site.

[0085] In one embodiment, the three loops should be understood as a concept encompassing amino acid sequence variants within a range that maintains their function. For example, in one embodiment, the β1-β2 loop, the β2-β3 loop, and the β5-β6 loop may each contain amino acid sequences 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 with respect to the amino acid sequences TPRPVKLLRGHT, TPLNTRV, and DKMQNKDKGL, respectively.

[0086] In the linker mutation characteristics according to one embodiment, the deformed fusion protein may be one or more amino acid residues on the β1-β2 loop of the Ig-like domain D3, and one or more amino acid residues on the β5-β6 loop of domain D3, substituted with amino acids that decrease the net pI of the protein, amino acids whose side chains become negatively charged, or amino acids whose side chains become electrostatically negatively charged.

[0087] In one embodiment, the N-terminus of the Ig-like domain D2 of the modified fusion protein may be further modified. The modified fusion protein of this disclosure may be produced by inserting a nucleic acid sequence or nucleic acid molecule that encodes it into a vector of diverse 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 begin at G132, but rather begin with an amino acid sequence derived from the vector (e.g., amino acid sequence EF), and a mutation may appear in the amino acid sequence at the N-terminus of domain D2. However, such a mutation in the N-terminus due to the vector-derived amino acid sequence does not affect the productivity or physical properties of the modified fusion protein of this disclosure.

[0088] 5. Linker mutation characteristics

[0089] In one embodiment, the modified fusion protein of the present disclosure may have linker mutation properties. In the present disclosure, the modified fusion protein having linker mutation properties may be used interchangeably with a “linker variant”. Due to the linker mutation properties, the modified fusion protein of the present disclosure can (i) similarly mimic the structure in which domains D2 and D3 of the native VEGFR1 extracellular domain bind to a ligand, (ii) have a length to the dimerization site of domain D4 as a structural characteristic that should be ensured for smooth ligand binding of domain D3 of the native VEGFR1 extracellular domain, and (iii) the linker itself can have excellent structural stability.

[0090] In one embodiment of the linker mutation characteristics, the linker may have an amino acid length of approximately 14 to approximately 35.

[0091] In the linker variant characteristics according to one embodiment, the linker may include a GS repeat sequence.

[0092] In the linker mutation characteristics of one embodiment, the GS repeat sequence may be selected from the group consisting of GS, GSSG, (GSSG)2, GGGGS, (GGGGS)3, GS(GGGGS), and GS(GGGGS)3, but is not limited thereto.

[0093] In one embodiment of the linker mutation characteristics, the linker may include an amino acid sequence of a hinge region derived from immunoglobulin. In one embodiment, the amino acid sequence of the hinge region may be modified.

[0094] In this disclosure, the linker is a region located between the C-terminus of the VEGFR1 domain D3 and the Fc region of human immunoglobulin produced by papain degradation, and can refer to the entire region from P243, located at the N-terminus of the CH2 region (A244-K360) contained in Fc, to the C-terminus of domain D3. Here, the amino acid positions of the Fc region and the CH2 region contained in Fc follow Kabat numbering (Kabat et.al., Sequences of 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 portion of a region derived from human immunoglobulin, which may be the amino acid sequence of CH1 (K218-V223) or a hinge region (E226-P243) or a portion of such an amino acid sequence.

[0095] In this disclosure, if the linker contains the amino acid sequence of a hinge region derived from immunoglobulin, it will retain the characteristics of the included hinge region. For example, the hinge region at the Fc site has cleavage sites for various proteases, and after the hinge region is introduced into the fusion protein, it may induce oligomerization by O-glycosylation occurring at the Ser / Thr amino acids (Song et al., 2020. Comput Struct Biotechnol J.18:pp.3925-3935). In the case of the modified fusion protein having the linker mutation characteristics of this disclosure, the amino acid sequence of the hinge region derived from immunoglobulin is modified to achieve excellent structural stability of the linker itself and to improve its physical properties.

[0096] In one embodiment of the linker mutation characteristics, the hinge region derived from immunoglobulin may include a sequence derived from the 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 immunoglobulin may form a beta-strand or a beta-sheet. In one embodiment, the hinge region derived from immunoglobulin may be derived from human IgD, IgG, or a combination thereof.

[0097] In one embodiment, if the hinge region is derived from IgG1, the amino acid sequence KTHT of the mutated papain recognition site may be modified to an amino acid sequence selected from the group consisting of KTYT, TPP, ATPT, and ATPPTCP.

[0098] In the linker mutation characteristics according to one embodiment, the linker may include (i) an amino acid sequence selected from the group consisting of GS, GSSG, (GSSG)2, GGGGS, 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 order from the N-terminus to the C-terminus, and the amino acid sequence of the hinge region may be modified.

[0099] In this disclosure, the hinge region derived from IgD / G1 can mean a combination of hinge sequences of IgD and IgG1, and specifically, the hinge region derived from IgD / G1 may be NTGSGGEEKKKEKEKEEQEERSSDKTHTCPPCP (SEQ ID NO: 76). In one embodiment, the hinge sequence of IgD may be NTGSGGEEKKKEKEKEEQEERSS (SEQ ID NO: 77), NTGRGGEEKKKEKEKEEQEER (SEQ ID NO: 78), or a variant thereof. In one embodiment, the hinge sequence of IgG1 may be DKTHTCPPCP (SEQ ID NO: 79) or a variant thereof.

[0100] In the linker mutation characteristics according to one embodiment, the linker may be an amino acid sequence selected from the group consisting of CSSGDATPTSPPSP (SEQ ID NO: 68), CSKVDKKVEPKSSDTPPTCPPCP (SEQ ID NO: 69), CSGGGGSAEPKAGDATPPTCPPCP (SEQ ID NO: 70), CSGGGGSGGGGSGGGGSAESKYGPPCPPCP (SEQ ID NO: 71), CSNTGSGGEEKKKEKEKEEQEERSSDTPPTCPPCP (SEQ ID NO: 72), CSNTGSGGEEKKKEKEKEEQEERSCDTPPTCPPCP (SEQ ID NO: 73), CGSSGGSSGEPKSDATPTCPPCP (SEQ ID NO: 74), and CSKVDKKVEPKSSDKTYTCPPCP (SEQ ID NO: 75).

[0101] 6. Disulfide bond mutation characteristics

[0102] In one embodiment, the modified fusion protein of this disclosure may have disulfide-binding mutant properties. In this disclosure, a modified fusion protein having such disulfide-binding mutant properties may be used interchangeably with a "disulfide-binding mutant." Since the modified fusion protein of this disclosure is a fusion of heterologous proteins, it is expected that its stability will be reduced at the site where different types of proteins bind. Therefore, disulfide-binding mutant properties were introduced to prevent physical cleavage of the modified fusion protein and to improve its stability.

[0103] To introduce disulfide bond mutation characteristics, candidate amino acid residues were selected using a tertiary structure energy prediction program (foldx in YASARA, Schymkowitz et al., 2005, Nucleic Acids Research. 33:W382-388). By substituting these amino acid residues with cysteine, it was confirmed that the properties of the disulfide bond-modified fusion protein (e.g., productivity, physical properties, ligand binding affinity) were improved (see Example 2).

[0104] In one embodiment, for disulfide bond mutation properties, (i) any one amino acid residue on the β1-β2 loop or any one amino acid residue on the β5-β6 loop of Ig-like domain D3 and (ii) an amino acid residue located at +2 position relative to Y329 of Ig-like domain D3 may be substituted with cysteine. Such substitutions allow the modified fusion protein of this disclosure to have a disulfide bond within the protein. In one embodiment, for the introduction of disulfide bond mutation properties, the selection of the amino acid residues located at the positions described in (i) and (ii) above may be determined according to the above criteria (1) to (3).

[0105] 7. Fusion Protein

[0106] 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 include a first binding specificity to the VEGF ligand and a second binding specificity to the PlGF ligand. Here, the VEGF ligand may be one or more of VEGF-A, VEGF-B, VEGF-C, and VEGF-D, and more specifically, VEGF-A.

[0107] In this disclosure, a fusion protein containing VEGFR1 D2 and D3 based on the VEGFR1 wild-type protein used as a control group may be referred to as PB101 (a fusion protein containing VEGFR1 D2 and D3 with 1 to 3 glycosylated amino acids based on the VEGFR1 wild-type protein) or VEGF-Grab.

[0108] In one embodiment, the modified fusion protein may include the VEGFR1 extracellular domain, a linker, and a multimerization domain. In one embodiment, the modified fusion protein may include components arranged in the order of the VEGFR1 extracellular domain, a linker, and a multimerization domain from the N-terminus to the C-terminus. In one embodiment, the VEGFR1 extracellular domain may include the VEGFR1 Ig-like domains D2 and D3.

[0109] In one embodiment, the modified fusion protein may have (a) surface charge transfer properties, (b) linker mutation properties, and (c) disulfide bond mutation properties.

[0110] (a) comprising amino acid substitutions of K241E, L243S, R244V, and H246E on the β1-β2 loop of domain D3, amino acid substitutions of L258A, L258S, or L258D on the β2-β3 loop of domain D3, and / or amino acid substitutions of K300G, Q302T, and K304S on the β5-β6 loop of domain D3;

[0111] (b) The linker is an amino acid sequence selected from the group consisting of CSSGDATPTSPPSP (SEQ ID NO: 68), CSKVDKKVEPKSSDTPPTCPPCP (SEQ ID NO: 69), CSGGGGSAEPKAGDATPPTCPPCP (SEQ ID NO: 70), CSGGGGSGGGGSGGGGSAESKYGPPCPPCP (SEQ ID NO: 71), CSNTGSGGEEKKKEKEKEEQEERSSDTPPTCPPCP (SEQ ID NO: 72), CSNTGSGGEEKKKEKEKEEQEERSCDTPPTCPPCP (SEQ ID NO: 73), CGSSGGSSGEPKSDATPTCPPCP (SEQ ID NO: 74), and CSKVDKKVEPKSSDKTYTCPPCP (SEQ ID NO: 75).

[0112] (c) The L243 amino acid residue on the β1-β2 loop of domain D3 is replaced with cysteine, and the amino acid residue located +2 positions relative to Y329 in domain D3 is replaced with cysteine.

[0113] In one embodiment, the modified fusion protein may consist of an amino acid sequence selected from the group consisting of amino acid sequences C61 to C75 in Table 2.

[0114] In one embodiment, the modified fusion protein may contain at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with respect to an amino acid sequence selected from the group consisting of C61-C75 amino acid sequences in Table 2. Such variants may include amino acid mutations (e.g., conservative amino acid substitutions) that appear in other amino acid sequences, while retaining or including the amino acid mutations that appear in the properties of the modified fusion protein of this disclosure.

[0115] In one embodiment, the components of the modified fusion protein of this disclosure (VEGFR1 extracellular domain, linker, and multimerization domain) may have post-translational modifications including, for example, glycosylation, sialylation, acetylation, and phosphorylation.

[0116] In one embodiment, the modified fusion protein of this disclosure may include conserved amino acid substitutions at positions other than those of amino acid mutations that appear in the properties described herein. Such conserved amino acid substitutions may be introduced into any of the components of the modified fusion protein of this disclosure (e.g., the extracellular domain, linker, and multimerization domain of VEGFR1).

[0117] In one embodiment, the conservative substitutions that may be included in the modified fusion protein of the present disclosure are as shown in Table 0 below. By such conservative substitutions, the modified fusion protein of the present disclosure can be made to have desired activities, such as maintained / improved target binding affinity, reduced immunogenicity, or improved serum stability / thermal stability.

[0118] [Table 1]

[0119] In one embodiment, the modified fusion protein of this disclosure may be one of those disclosed in Table 2, C61-C75.

[0120] 8. Fusion protein and scFv conjugate

[0121] In one embodiment of the present disclosure, a conjugate of a modified fusion protein and an scFv according to an embodiment of the present disclosure may be provided. In one embodiment, the scFv may be a PD-L1-targeting scFv, and in particular may be an atezolizumab scFv or A167 scFv that binds to PD-L1 as disclosed in Table 9. In this disclosure, the conjugate of a PD-L1-targeting scFv and a modified fusion protein according to one embodiment of the present disclosure may be referred to as PB203.

[0122] In one embodiment, the modified fusion protein and scFv conjugate of the present disclosure may be those disclosed in SEQ ID NOs: 1-32 of Table 9.

[0123] In one embodiment, the conjugate may contain at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with respect to an amino acid sequence selected from the group consisting of the amino acid sequences of Table 9. In such variants, the conjugate may include amino acid mutations that appear in other amino acid sequences (e.g., conservative amino acid substitutions) while retaining or including the amino acid mutations that appear in the properties of the conjugate of this disclosure.

[0124] In one embodiment, the components of the conjugate of the present disclosure (scFv, VEGFR1 extracellular domain, linker, and multimerization domain) may harbor post-translational modifications, including, for example, glycosylation, sialylation, acetylation, and phosphorylation.

[0125] In one embodiment, the conjugate of the Disclosure may include a conserved amino acid substitution at a position other than the amino acid mutation site that appears in the properties described herein. Such a conserved amino acid substitution may be introduced into any of the components of the modified fusion protein of the Disclosure (e.g., scFv, the extracellular domain of VEGFR1, the linker, and the multimerization domain).

[0126] In one embodiment, the conjugate of the present disclosure may include a signal peptide or signal sequence for protein secretion from a cell. For example, the conjugate of the present disclosure may further include a peptide of a different origin, specifically a signal sequence or other peptide having a cleavage site specific to the N-terminus of a mature fusion protein. In one embodiment, the signal sequence of a different origin may be recognized and processed by a eukaryotic host cell (i.e., cleaved by a signal peptide hydrolase). The content of signal peptides is well known in the art and is presented, for example, in Korean Patent Registration No. 10-2228921, but is not limited thereto. In one embodiment, the signal peptide may be a human interleukin signal sequence. In one embodiment, the signal peptide may consist of an amino acid sequence such as MVSYWDTGVLLCALLSCLLLTGSSSG(tPA), MEFGLSWVFLVALFRGVQC(H7), MKWVTFISLLFLFSSAYS(human serum albumin), MGWSCIILFLVATATGVHS(mouse Ig heavy chain), MDWTWRVFCLLAVAPGAHS(human Ig heavy chain), or MYRMQLLSCIALSLALVTNS(human interleukin-2), but any signal peptide that can be used by a normal technician for the production of a fusion protein is acceptable.

[0127] In one embodiment, the conjugates of the present disclosure may have an IC50 of approximately 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, 1 ng / ml, 100 pg / ml, 70 pg / ml, 50 pg / mL, 45 pg / ml, 40 pg / ml, 35 pg / ml, 30 pg / ml, 25 pg / ml, 20 pg / ml, 10 pg / ml, 5 pg / ml, or 1 pg / mL (inclusive, including any value between these numbers) or less for inhibition of ligand activity (e.g., inhibition of VEGF activity, PD-L1 activity, or PlGF activity).

[0128] In one embodiment, the compound of the present disclosure is bonded to a binding partner (e.g., VEGF, PD-L1, and / or PlGF) in concentrations 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, 5 It may have a Kd smaller than any of the following: 0nM, 45nM, 40nM, 35nM, 30nM, 25nM, 20nM, 15nM, 10nM, 5nM, 1nM, 900pM, 800pM, 700pM, 600pM, 500pM, 400pM, 300pM, 200pM, 100pM, 50pM, 25pM, 12.5pM, 6.25pM, 5pM, 4pM, 3pM, 1pM, 0.5pM, 0.1pM, 0.05pM, or 0.01pM (inclusive, including any value between these numbers).

[0129] III. Nucleic acids, vectors, and host cells

[0130] nucleic acid

[0131] In one embodiment of the present disclosure, isolated nucleic acids that encode components of any modified fusion protein and scFv, and components of the modified fusion protein and scFv conjugate, such as anti-PD-L1 scFv, the VEGFR1 extracellular domain, and / or the multimerization domain, may be provided. Nucleic acids encoding mammalian VEGFR are described for all receptor types. Exemplary nucleic acid sequences, but not limited to these, can be found in U.S. Patent No. 7,928,072 and WO2006 / 113277. mRNAs encoding human VEGFR1 and VEGFR2 can be found in GenBank accession numbers NM_002019.4 and NM_002253.2, respectively.

[0132] Isolated nucleic acids that encrypt a multimerization domain (e.g., an Fc site) may be provided in this disclosure.

[0133] Isolated nucleic acids for encoding the modified fusion proteins of this disclosure may be provided.

[0134] The isolated nucleic acid sequence that encodes the modified fusion protein or a component of the fusion protein (e.g., the extracellular domain or multimerization domain of VEGFR1) of the Disclosure may further include a nucleic acid sequence that encodes a linker.

[0135] In one embodiment, the isolated nucleic acid may further contain a sequence that encodes a signal peptide, which acts as a signal sequence for secreting a modified fusion protein from a host cell. In one embodiment, the isolated nucleic acid does not need to contain a sequence that encodes a signal peptide.

[0136] The isolated nucleic acid molecules encoding the modified fusion protein or components of the fusion protein (e.g., the VEGFR1 extracellular domain, linker, or multimerization domain) of the Disclosure may be in the form of RNA, e.g., 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 prepared by various methods known in the art (see: Molecular Cloning: A Laboratory Manual (Sambrook et al., 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2012) and Current Protocols in Molecular Biology (FMAusubel, et al. eds., 2003)).

[0137] vector

[0138] In one embodiment, this disclosure may relate to a nucleic acid delivery vehicle or use thereof for introducing one or more nucleic acid sequences that encode a conjugate or components of a conjugate into a cell for expressing the protein. The nucleic acid sequences may be sequences of the isolated nucleic acids.

[0139] In one embodiment, examples of nucleic acid delivery vehicles may be liposomes, biocompatible polymers (including natural and synthetic polymers); lipid proteins; polypeptides; polysaccharides; lipid polysaccharides; artificial viral envelopes; metal particles; and bacteria, viruses, e.g., baculovirus, adenovirus and retrovirus, bacteriophages, cosmids, plasmids, fungal vectors, and other recombinant vehicles commonly used in the art, described for expression in various eukaryotic and prokaryotic hosts. In one embodiment, the nucleic acid delivery vehicle may be an expression vector, e.g., a plasmid. In one embodiment, the nucleic acid sequence contained in the expression vector may be operatively ligated to an expression regulatory sequence.

[0140] In one embodiment, the vector may include any elements for establishing the normal function of the expression vector, such as a promoter, a ribosome-binding element, a terminator, an enhancer, a selection marker, and a replication origin. The promoter may be a structural, inducible, or repressive promoter. Exemplary promoters are presented in Korean Registered Patent No. 10-0659477, but are not limited thereto.

[0141] Numerous expression vectors capable of delivering nucleic acids to cells (e.g., derived from bacterial, yeast, plant, or mammalian cells) are known in the art and may be used in this disclosure to generate conjugates or components of conjugates within cells. For example, E. coli, when transformed with a plasmid engineered to contain the nucleic acid encoding the conjugate, such as pBR322 (Mandel et al., J. Mol. Biol., 1970, 53:154), can be used to generate a modified conjugate. The expressed conjugate or components of the conjugate may be harvested from cells and purified by conventional techniques known in the art and described in this disclosure.

[0142] In one embodiment, an expression vector comprising a nucleic acid encoding the above-described conjugate, which can be replicated in a bacterial or eukaryotic host, is used to transfect the host, thereby allowing the nucleic acid to be directly expressed for the production of a conjugate that can be recovered in a biologically active form. In this disclosure, the biologically active form includes a form that can bind to a VEGF ligand, PD-L1, or PlGF ligand.

[0143] In one embodiment, an expression vector comprising a conjugate or a nucleic acid molecule encoding a component of the conjugate can be confirmed by at least three common methods: (a) DNA-DNA hybridization, (b) the presence or absence of "marker" gene function, and (c) the expression of the inserted sequence. The specific details of these three methods are presented in Korean Registered Patent No. 10-0659477 or No. 10-2228921, but are not limited thereto.

[0144] host cell

[0145] In one embodiment of the present disclosure, a host cell containing the expression vector for encoding the conjugate of the present disclosure may be provided. Such a host cell may be suitable for expression of the conjugate of the present disclosure and may constitute a host-vector system for producing the conjugate. In one embodiment, the host cell may be used to generate viral particles (e.g., recombinant viral vectors).

[0146] As used in this disclosure, the term “host cell” may include cells that are or may be recipients of a vector and its progeny. Due to natural, accidental, or elaborate mutations, the progeny may not necessarily be completely identical to the original parent cell (in morphology or in the genome of the whole DNA complement). In one embodiment, the host cell may 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.

[0147] In one embodiment, the conjugates of the present disclosure may be expressed transiently, constitutively, or permanently in host cells.

[0148] In one embodiment of the present disclosure, a method for producing the conjugates of the present disclosure can be provided, which involves culturing the host cells or host-vector cells under conditions that enable the production of conjugates, and then recovering the produced conjugates. Conjugates useful for the implementation of the present disclosure can be produced by expression in a prokaryotic or eukaryotic expression system. Methods for culturing host cells or producing conjugates from host cells are well known in the art and are presented, for example, in Korean Patent Registration No. 10-2228921, but are not limited thereto.

[0149] In one embodiment, the conjugates of the present disclosure produced from the host cells may be purified and confirmed by a variety of methods. Methods for purifying and confirming conjugates produced from host cells are well known in the art and are, for example, presented in Korean Patent Registration No. 10-2228921, but are not limited thereto. Many purification methods may be used for the further purification of the conjugates, including, but are not limited to, conventional ion exchange chromatography, affinity chromatography, different sugar chromatography, hydrophobic interaction chromatography, reversed-phase chromatography, or gel filtration.

[0150] IV. Viral particles and methods for generating viral particles

[0151] In one embodiment of the present disclosure, a viral particle (or virion) containing nucleic acid for encrypting the combination of the present disclosure may be provided.

[0152] Viral vectors may be used to deliver nucleic acids that encode a conjugate or components of a conjugate for protein expression in target cells within a specific target tissue (e.g., a diseased tissue). Many types of viruses are known, and many are being 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, completely deleted adenoviruses, adeno-associated viruses (AAVs), retroviruses, lentiviruses, etc., for delivery to cells.

[0153] In one embodiment, the cells are present within an individual, and the virus may be transmitted intravenously, intramuscularly, intraportally, or through other routes of administration. In one embodiment, the viral vector may include those derived from adenoviruses, adeno-associated viruses (AAVs), and retroviruses (including lentiviruses, e.g., human immunodeficiency virus (HIV)). Exemplary viral vectors are, but are not limited to, U.S. Patent Nos. 7,928,072 and WO2006 / 113277, which are included in their entirety in this application. Viral particles containing nucleic acids that encode conjugates and methods for producing them are well known in the art, for example, as presented in Korean Registered Patent No. 10-2228921, but are not limited to these.

[0154] IV. Treatment methods using conjugates and virus particles

[0155] One embodiment of the present disclosure may relate to a pharmaceutical composition for use in the prevention, improvement, or treatment of a disease related to a VEGF ligand, a VEGF receptor, or PD-L1, or at least one symptom of such a disease, comprising the conjugate of the present disclosure as an active ingredient.

[0156] One embodiment of the Disclosure may be a method for preventing, improving or treating a disease or at least one symptom of a disease associated with a VEGF ligand, a VEGF receptor, or PD-L1, including the step of administering a conjugate of the Disclosure.

[0157] One embodiment of the present disclosure may relate to a use for preventing, improving or treating at least one of the VEGF ligand, VEGF receptor, or PD-L1-related diseases or symptoms of such diseases of the conjugates of the present disclosure.

[0158] One embodiment of the present disclosure may relate to a conjugate of the present disclosure or a pharmaceutical composition containing the same for use in a method for preventing, improving or treating at least one of the symptoms of a disease associated with a VEGF ligand, a VEGF receptor, or PD-L1.

[0159] In one embodiment, the conjugate of the Disclosure may be used as a nucleic acid delivery vehicle or viral particle (e.g., a recombinant viral vector) in the form of a nucleic acid that encodes it, for the prevention, improvement, or treatment of at least one of the diseases or symptoms of VEGF ligands, VEGF receptors, or PD-L1-related diseases. In one embodiment, the nucleic acid delivery vehicle or viral particle may be applied to gene therapy for use in the prevention, improvement, or treatment of at least one of the diseases or symptoms of VEGF ligands, VEGF receptors, or PD-L1-related diseases. In one embodiment, any gene therapy method available in the art may be used in accordance with the Disclosure. In one embodiment, the nucleic acid is introduced into cells prior to the final in vivo administration of recombinant cells. Cells into which nucleic acids are introduced for the purpose of gene therapy may include, but are not limited to, any available cell types, such as epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes, blood cells (e.g., T lymphocytes, B lymphocytes, mononuclear cells, macrophages, neutrophils, eosinophils, megakaryocytes, or granulocytes), and various stem cells or progenitor cells (e.g., progenitor cells, such as hematopoietic stem cells 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 cells derived from the patient's own body.

[0160] In one embodiment, the conjugate may bind to VEGF protein, PlGF, and / or PD-L1 protein. In one embodiment, the conjugate may have one or more of the following properties: (a) binding to one or more proteins from the VEGF family (e.g., VEGF-A, VEGF-B, VEGF-C, VEGF-D, PlGF) and PD-L1; (b) blocking the binding of VEGF family proteins to VEGF receptors and / or the binding of PD-L1 proteins to PD-1 receptors; (c) inhibition of activation of the VEGF signaling pathway and / or the PD-L1 signaling pathway; (d) prevention, improvement, and / or treatment of diseases such as chronic infection, tissue allografts, eye diseases, autoimmune diseases, inflammatory diseases, neoplasm-forming diseases, or cancer; (e) attenuation, prevention, or prevention of neoplasm or cancer growth; (f) inhibition of neoplasm or cancer metastasis. The activity of the conjugate may be measured in vivo and / or in vitro.

[0161] In one embodiment, the pharmaceutical composition may comprise 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 petroleum, animal, plant, or synthetic sources such as peanut oil, soybean oil, or mineral oil. Saline solution and water-soluble dextrose, polyethylene glycol (PEG), and glycerol solutions may also be used as liquid carriers, particularly for injectable solutions. Such pharmaceutical compositions may further comprise additional components, such as preservatives, buffers, isotonic agents, antioxidants and stabilizers, nonionic wetting or cleaning agents, thickeners, and the like. The pharmaceutical compositions of this disclosure may be packaged in single-dose or multi-dose forms. These compositions are generally formulated as sterile and substantially isotonic solutions. The compositions may also be formulated to have an osmotic pressure suitable for aqueous solutions of the eye and ocular tissue. Such osmotic pressure values ​​are generally in the range of approximately 200 to 400 milliosmoles (mOsm / kg) per kilogram of water, or approximately 300 mOsm / kg. The retina is thought to have an osmotic pressure value of approximately 283 mOsm / kg.

[0162] In one embodiment, the pharmaceutical composition may be a parenteral composition. For uniformity of dosage and ease of administration, it may be advantageous to formulate the parenteral composition in the form of a dose unit form.

[0163] Disease or illness

[0164] In one embodiment, diseases associated with VEGF ligands, VEGF receptors, or PD-L1 may include, but are not limited to, chronic infections, allografts, autoimmune diseases (e.g., rheumatoid arthritis, multiple sclerosis, recurrent miscarriage due to immune response, or systemic lupus erythematous erythematous), inflammatory diseases (inflammatory arthritis, osteoarthritis, or psoriasis), neoplastic diseases, cancers (e.g., breast cancer, lung cancer, gastric cancer, pancreatic cancer, or leukemia), angiogenesis-related diseases (e.g., atherosclerosis), or eye diseases (e.g., age-related macular degeneration, choroidal neovascularization, or uveitis).

[0165] In one embodiment, cancers include prostate cancer, urethral cancer, penile cancer, breast cancer, lung cancer, esophageal cancer, small intestine cancer, colorectal cancer, rectal cancer, colon cancer, liver cancer, urinary tract cancer (e.g., bladder cancer), kidney cancer, lung cancer (e.g., non-small cell lung cancer), ovarian cancer, cervical cancer, endometrial cancer, vaginal cancer, vulvar cancer, pancreatic cancer, gastric cancer, endocrine cancer, head and neck cancer, thyroid cancer, parathyroid cancer, adrenal cancer, skin cancer (e.g., melanoma, cutaneous squamous cell carcinoma (CSCC), head and neck squamous cell carcinoma (HNSCC)), Hodgkin's disease, bone cancer, hematopoietic progenitor carcinoma of the lymph or bone marrow, chronic or acute leukemia, head and neck cancer, nasopharyngeal carcinoma. The conjugates of the present disclosure may include, but are not limited to, carcinoma (NPC), glioblastoma, teratocarcinoma, neuroblastoma, adenocarcinoma, mesenchymal carcinoma (e.g., fibrosarcoma or rhabdomyosarcoma), soft tissue sarcomas and carcinomas, choriocarcinoma, hepatoblastoma, central nervous system (CNS) tumors, primary central nervous system lymphomas, spinal cord tumors, brainstem gliomas, pituitary adenomas, Kaposi's sarcoma, or Wilm's tumor. The conjugates of the present disclosure may act as anti-PD-L1 and anti-angiogenic agents, or anti-VEGF and anti-PD-L1 agents, to improve, prevent, and / or treat cancer or the symptoms of cancer.

[0166] Method of administration and dosage

[0167] In one embodiment of the present disclosure, a method for delivering an effective amount of conjugate to a target object may be provided. The conjugate may be delivered to the target object within a composition. The conjugate may also be delivered to the target object by a nucleic acid delivery vehicle or viral particle (e.g., a recombinant viral vector) containing a nucleic acid that encodes the conjugate. In one embodiment of the present disclosure, a composition comprising the conjugate or a nucleic acid delivery vehicle or viral particle (e.g., a recombinant viral vector) containing a nucleic acid that encodes the conjugate may be provided.

[0168] In one embodiment, the compositions of the Disclosure may be administered to an individual by any route, but not limited to, including: intravenous (e.g., by an infusion pump), intraperitoneal, intraocular, intraarterial, intrapulmonary, oral, inhalation, intravesical, intramuscular, intratracheal, subcutaneous, intraspinal cavity, percutaneous, transpleural, topical, inhalant (e.g., mist of a spray), mucous membrane (e.g., through the nasal mucosa), gastrointestinal tract, intraarticular, intrasacral, intraventricular, intracranial, intraurethral, ​​intrahepatic, and intratumor. In one embodiment, the compositions of the Disclosure may be administered by any common route, such as infusion injection or bolus injection, absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal mucosa, intestinal mucosa, etc.), and may be administered together with other bioactive ingredients. Administration may be systemic or topical. In one embodiment, the compositions of the present disclosure may be introduced into the central nervous system by any suitable route, including intracerebroventricular injection and intraspinal injection, in which case intracerebroventricular injection may be performed by an intracerebroventricular catheter, such as a catheter attached to a reservoir, such as an Ommaya reservoir.

[0169] In one embodiment, the optimal effective amount of the conjugate, the nucleic acid delivery vehicle that encodes it, or the composition comprising them may be determined experimentally and may vary depending on the type and severity of the disease, the route of administration, the progression of the disease and the health status, the body weight and body surface area of ​​the individual. Such determination is within the scope of the ordinary skill of the art.

[0170] In one embodiment, the conjugate may be administered, for example, at a dose of approximately 0.05 ng to approximately 20 mg per kg of body weight per day. In one embodiment, the dose of the conjugate may be approximately 0.1 mg / kg or more, approximately 0.5 mg / kg or more, approximately 1.0 mg / kg or more, approximately 1.5 mg / kg or more, approximately 2.0 mg / kg or more, approximately 2.5 mg / kg or more, approximately 3.0 mg / kg or more, approximately 3.5 mg / kg or more, approximately 4.0 mg / kg or more, approximately 4.5 mg / kg or more, approximately 5.0 mg / kg or more, approximately 6.0 mg / kg or more, approximately 8.0 mg / kg or more, approximately 10.0 mg / kg or more, or approximately 15.0 mg / kg or more. The dosage may be approximately 20.0 mg / kg or less, approximately 17.0 mg / kg or less, approximately 14.0 mg / kg or less, approximately 11.0 mg / kg or less, approximately 9.0 mg / kg or less, approximately 7.0 mg / kg or less, approximately 5.5 mg / kg or less, approximately 5.0 mg / kg or less, approximately 4.5 mg / kg or less, approximately 4.0 mg / kg or less, approximately 3.5 mg / kg or less, approximately 3.0 mg / kg or less, approximately 2.5 mg / kg or less, approximately 2.0 mg / kg or less, approximately 1.5 mg / kg or less, or approximately 1.0 mg / kg or less. In one embodiment, the dosage of the conjugate may be approximately 1.0 mg / kg to approximately 10.0 mg / kg or approximately 1.0 mg / kg to approximately 5.0 mg / kg.

[0171] In one embodiment, the amount of a nucleic acid delivery vehicle (e.g., a recombinant viral vector) containing the nucleic acid encoding the conjugate protein of the Disclosure may be administered to an individual at a DNAse particle resistance (drp) titer of about 10⁴ to about 10¹⁴ drp per dose. In one embodiment, the amount of a nucleic acid delivery vehicle containing the nucleic acid encoding the deformed conjugate protein may be administered to an individual at a dose of about 10⁵ to about 10¹³, about 10⁶ to about 10¹², about 10⁷ to about 10¹¹, about 10⁸ to about 10¹⁰, about 10⁹ to about 10¹⁰, or about 10¹¹ to about 10¹² drp per dose.

[0172] In one embodiment, a composition comprising the conjugate protein of the Disclosure or a nucleic acid delivery vehicle encoding the conjugate protein thereof may be administered as a single daily dose or as a total daily dose divided into two, three, or four doses per day. In one embodiment, a composition comprising the conjugate protein of the Disclosure may be administered six times a week, five times a week, four times a week, three times a week, twice a week, once a week, every other week, every three weeks, once a month, once every two months, once every three months, once every six months, once every nine months, or once a year. In one embodiment, a composition comprising a nucleic acid delivery vehicle comprising a nucleic acid encoding the conjugate protein of the Disclosure may be administered at a lower frequency, 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 a year.

[0173] Diagnosis or detection

[0174] The conjugate proteins of this disclosure may be labeled with detectable labels such as radioisotopes, fluorescent labels, toxin labels, enzyme labels, chemiluminescent labels, or nuclear magnetic resonance contrast agents to confirm ligand-receptor binding interactions. Assay systems applicable to chimeric molecules may also be considered. Such detectable labels are widely known in the art.

[0175] V. Manufactured Articles and Kits

[0176] In one embodiment, this disclosure may relate to a manufactured article or kit comprising a conjugate protein of the Disclosure, a recombinant viral vector expressing the same, or a composition comprising the same, in appropriate packaging. In one embodiment, the appropriate packaging 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 manufactured articles may be further sterilized and / or sealed.

[0177] In one embodiment, the kit may further include instructions on how to use the composition for the purposes described herein. In one embodiment, the kit may further include other components preferred from a commercial and user standpoint, such as other 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) the conjugate protein described herein and / or a recombinant viral vector encoding the conjugate protein described herein, (ii) a pharmaceutically acceptable carrier, and (iii) one or more of the following: a package insert containing buffers, diluents, filters, needles, syringes, and instructions for performing administration.

[0178] The configuration and effects of this 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 this disclosure and are not intended to limit the scope and scope of this disclosure.

[0179] Example 1 - Production of an expression vector

[0180] In the following example, the coding sequence region (CDS) of the designed conjugate was subjected to nucleic acid codon optimization in a form suitable for CHO-producing cells, and then the conjugate was produced in the final pcDNA3.1(+) vector (Invitrogen) through gene synthesis (Geneuniversal).

[0181] Example 2 - Design of a deformed fusion protein

[0182] Example 2.1 - Production and characterization of surface charge transition mutants

[0183] To ensure a fusion protein containing the enhanced physical properties of the VEGFR receptor, the inventors of this disclosure analyzed the surface charge distribution of the protein using a structure (PDB entry.5t89) containing the entire extracellular domain of VEGFR1 as a template, and derived regions with concentrated positive charges and locations with hydrophobic surface charges.

[0184] This confirmed that the side chains of amino acid residues in the β1-β2 loop (named Site 1) corresponding to amino acid residues T236-T247, the β2-β3 loop (named Site 2) corresponding to amino acid residues T256-V262, and the β5-β6 loop (named Site 3) corresponding to amino acid residues D299-L308 in the VEGFR1 domain D3 are exposed on the surface of the fusion protein. We attempted to achieve surface charge and structural stabilization of the fusion protein by using mutations in the amino acid residues located in these surface-exposed flexible loops. For example, we attempted to replace the surface-exposed amino acid residues with amino acid residues located at homologous positions in VEGFR2. Note that the amino acid residues in Site 2 are exposed on the protein surface on the side that binds to the ligand (VEGF or PlGF), and mutations at this position affect the ligand binding strength or solubility due to surface residue exposure.

[0185] Example 2.2 - Production and characterization of linker variants

[0186] In the case of human VEGF receptors, binding to ligands, VEGF or PlGF, occurs through interaction with immunoglobulin-like domains D2 and D3. In fusion proteins utilizing such domains D2 and D3, domains D2 and D3 have an open structure before binding to the ligand, but form a closed structure upon binding. Depending on the binding length between domain D3 and the multimerization domain, the structures of domains D2 and D3 may not have enough space to bind to the ligand, or interference may occur between the structures, which can lead to a decrease in the binding affinity of the fusion protein to the ligand. Therefore, the binding length and binding method between domain D3 and the multimerization domain in a fusion protein can affect the structure of the fusion protein that binds to the ligand and its binding affinity to the ligand.

[0187] Therefore, in order to mimic and ensure the structural characteristics of the innate human VEGF receptor, the inventors modified the length and type of linker in the modified fusion protein of this disclosure. Specifically, linkers were introduced that included a polypeptide of a Gly-Ser repeat sequence, amino acid sequences of hinge regions derived from IgG1, IgG4, and IgD, and variant sequences of these hinges. This was intended to reduce the tension in the loop (linker) formed between domain D3 and the polymerization domain (Fc region) upon ligand binding, thereby increasing physical stability.

[0188] Furthermore, in the case of amino acid sequences derived from the hinge region of immunoglobulins, due to their unique characteristics, they have cleavage sites for various proteases (Vlasak and Ionescu. 2011. Mabs 3: pp. 253-263), and after introduction into a fusion protein, glycosylation, such as O-glycosylation, may occur, leading to oligomerization (Song et. al. 2020. Computational and Structural Biotechnology Journal 18: pp. 3925-3935). To improve the in vivo stability of the modified fusion protein of this disclosure, the inventors attempted to further modify the amino acid sequence at the cleavage site by proteases, for example, the cleavage site for papain, thereby modifying the Ser or Thr amino acid residues that undergo glycosylation. Furthermore, in order to increase the structural flexibility of the linker, we attempted to substitute cysteine ​​present in sequences derived from the core hinge of the Fc hinge region (e.g., CPPCP). The modified fusion protein according to one embodiment of this disclosure exhibits excellent productivity and superior physical properties, as well as superior characteristics in terms of ligand binding affinity.

[0189] Example 2.3 - Production and characterization of disulfide-bonded mutants

[0190] Since fusion proteins are conjugates of heterologous proteins, a decrease in stability due to nonspecific cleavage at the binding site is expected. The terminal end of domain D3 is a loop-forming region that may lack secondary structure, and since it is the site where linkage with the heterologous multimerization domain begins, its stability is expected to be low. We attempted to improve stability by introducing a covalent disulfide bond and inducing interdomain junction near the site where heterologous protein binding occurs, thereby preventing physical cleavage of the protein.

[0191] The potential locations for disulfide bond introduction on the fusion protein were derived through tertiary structure-based analysis. Among the residues located in the variable loop adjacent to the D3 terminal region of the domain, residues whose side chains are oriented toward the linker and whose distance between the terminal atom of the side chain and the Y329 Cα atom is within 6 Å were selected as mutant residues for disulfide bond introduction. After securing the three-dimensional structure of the fusion protein through a structural modeling program (Phyre2, Swiss-model), a structural model with introduced disulfide bonds was secured through in-silico mutation of the introduction site. Subsequently, the change in the energy level of the fusion protein due to the introduction of disulfide bonds was predicted using a tertiary structure energy prediction program (FoldX in YASARA).

[0192] In the fusion protein, amino acid residues on the β1-β2 loop or β5-β6 loop of domain D3 were selected as positions that are physically close to the D3 terminus and capable of forming a disulfide bond. In the case of the D3 terminus, amino acid residues located +2 positions relative to Y329 were selected.

[0193] Example 2.4 - Production and Characterization of Combination Mutants

[0194] Combination mutants of fusion proteins possessing the characteristics of the mutants exhibiting the superior properties of Examples 2.1 to 2.3 described above were manufactured as shown in Table 2. The characteristics of each mutant are shown in Table 1.

[0195] [Table 2]

[0196] Table 1 lists the identification codes, amino acid sequences, and sequence numbers of the proteins described herein.

[0197] [Table 3] TIFF2026527576000005.tif252170 TIFF2026527576000006.tif253170 TIFF2026527576000007.tif252170 TIFF2026527576000008.tif252170 TIFF2026527576000009.tif230170

[0198] The surface charge mutation sites or disulfide bond sites described in the following examples or tables are based on the amino acid sequence of VEGF-Grab3 in Table 2, and the mutated sites and amino acid residues are listed. The physical properties and binding affinity of combination mutants with mutations introduced at different sites, produced as shown in Table 2 above, were evaluated. The physical property evaluation results for mutants produced based on VEGFR wild-type protein and the wild-type protein are shown in Table 3, and the binding affinity evaluation results are shown in Tables 4 and 5. The physical property evaluation results for mutants produced based on VEGF-Grab3 are shown in Table 6, and the binding affinity evaluation results are shown in Tables 7 and 8. Table 7 shows the relative binding affinity evaluation results for VEGF-Grab3 (Panoros Biosciences), and Table 8 shows the relative binding affinity evaluation results for VEGF-Grab3 (Samsung Biologics). In the case of relative binding affinity evaluation for VEGF-Grab3 (Panoros Biosciences), the comparison was made with a product produced under the same conditions and batch as the mutant.

[0199] [Table 4]

[0200] [Table 5]

[0201] [Table 6]

[0202] [Table 7]

[0203] [Table 8]

[0204] [Table 9]

[0205] According to the results in Tables 3 to 8, the modified fusion protein according to one embodiment of this disclosure was confirmed to exhibit excellent productivity based on the results of titer and protein elution, and to exhibit excellent physical properties based on the results of functional monomer content. Furthermore, it was confirmed that it exhibits excellent properties in terms of ligand binding affinity, for example, based on the results for kon. From these results, it was confirmed that the properties introduced during the structural design of the modified fusion protein in Examples 2.1 to 2.3 were effectively manifested, and that these properties combined to show a synergistic effect.

[0206] Example 3 - Production of the compound

[0207] A conjugate was designed by linking the C61-C75 fusion protein produced in Example 2 with atezolizumab and A167 scFv, which bind to PD-L1 (Figure 1). As can be seen in Figure 1, the fusion protein of this disclosure contains a VEGF decoy receptor including engineered VEGFR1 domains 2 and 3 that can capture VEGF-A and PlGF, and a novel functional module can be linked to its N-terminus or C-terminus. The conjugate according to this disclosure was produced by linking anti-PD-L1 scFv to the N-terminus of the fusion protein of this disclosure.

[0208] [Table 10] TIFF2026527576000017.tif252170 TIFF2026527576000018.tif252170 TIFF2026527576000019.tif252170 TIFF2026527576000020.tif252170 TIFF2026527576000021.tif252170 TIFF2026527576000022.tif252170 TIFF2026527576000023.tif251170 TIFF2026527576000024.tif252170 TIFF2026527576000025.tif253170 TIFF2026527576000026.tif252170 TIFF2026527576000027.tif252170 TIFF2026527576000028.tif195170

[0209] In the composition description section of Table 9, H and L represent the heavy and light chains of scFv; Cnn (where n is an integer) corresponds to the sequence name in Table 2 above; G4S means the GGGGS linker; the linkers that connect scFv to the fusion protein are V1, V2, and V3, with V1 having the sequence GGGGSGGGGSDT, V2 having GSAEPKAGGGGSGGGGSGGGGS, and V3 having GSGGGGSGGGGSGGGGSGGGGS; C65H means that the C65 hinge portion of the fusion protein's hinge portion was used, as shown in Table 2; G4H means that IgG4 was used instead of IgG1 in the fusion protein's hinge portion; G4Fc means the effector function. QL means an IgG4 sequence with the function removed; QL means a mutation has been introduced that enhances the binding affinity to FcRn, which is known to increase the FcRn cycle and extend the half-life; Cys means a Cys mutation has been introduced in the fusion protein (Ser->Cys at position 494 in the case of C65, and at position 493 in the case of C75); sur1 means a mutant in which a reverse mutation of some surface charge mutations has been advanced.

[0210] ExpiCHO used in the production of the designed composite TM The cells (Gibco) were used for primary expression at passage numbers between 10 and 20. ExpiCHO TM Cell subculturing was performed at intervals of 3 or 4 days. Subculturing was performed at a density of 0.3 × 10⁶ cells / mL for 3 days and at a density of 0.15 × 10⁶ cells / mL for 4 days. Cell density and viability were measured using a Countess II (Invitrogen) instrument via trypan blue staining. Primary expression of the conjugate was performed using Expifectamine provided by the manufacturer (Gibco). TM The Max Titer protocol culture method using Gibco was employed. ExpiCHO was used the day before transfection. TMThe cells were passaged at a density of 3.5×106 cells / mL, and the cell density and viability were measured again the next day. After confirming that the cell density was 7.0×106 cells / mL or higher and the cell viability was 95% or higher, ExpiCHO was added to dilute the cells to a density of 6.0×106 cells / mL. TM 25 mL of the cell culture was dispensed into a 125 mL Erlenmeyer flask, and the cells were stored in an incubator at 37 °C until just before the preparation of the transfection mixture. 20 μg of the plasmid gene was thoroughly mixed with 1 mL of OptiPro TM SFM medium, and 80 μL of Expifectamine TM CHO reagent was thoroughly mixed with 920 μL of OptiPro TM SFM medium. The Expifectamine - OptiPro TM SFM mixture was added to the previously prepared plasmid - OptiPro TM SFM and mixed well, then left at room temperature for 3 minutes. The mixture was slowly added to the previously dispensed cell culture, and then cultured in an incubator set at 37 °C, 8% CO2, and 130 rpm. When 18 - 22 hours had passed since transfection, 150 μL of Expifectamine TM CHO enhancer and 4 mL of ExpiCHO TM Feed were added to the flask during culture, and cell culture was continued in an incubator set at 32 °C, 5% CO2, and 130 rpm. When producing a larger amount of the conjugate, 200 mL of the cell culture was dispensed into a 1 L Erlenmeyer flask, and the cells were stored in an incubator at 37 °C until just before the preparation of the transfection mixture. 160 μg of the plasmid gene was thoroughly mixed with 8 mL of OptiPro TM SFM medium, and 640 μL of Expifectamine TM CHO reagent was thoroughly mixed with 7.4 mL of OptiPro TM SFM medium. The Expifectamine - OptiPro TM SFM mixture was added to the previously prepared plasmid - OptiProTM After adding it to SFM and mixing thoroughly, it was left at room temperature for 3 minutes. The mixture was slowly added to the cell culture medium that had been pre-dispensed, and then cultured in an incubator set to 37°C, 8% CO2, and 130 rpm. Expifectamine was added 18-22 hours after transfection. TM CHO enhancer 1200 μL and ExpiCHO TM 32 mL of feed was added to the culture flask, and cell culture was continued in an incubator set to 32°C, 5% CO2, and 130 rpm. Cell density and viability were measured on days 2 and 5 after transfection. On day 5, the final titer was measured using a Cedex (Roche) instrument, and the cell culture medium containing protein was collected after centrifugation at 3000 xg for 30 minutes. The collected supernatant was filtered through a 0.22 μm polyethersulfone (PES) filter and stored at 4°C for short-term storage and -80°C for long-term storage.

[0211] Example 4 - Purification of conjugated proteins (Protein A affinity chromatography and size exclusion chromatography)

[0212] Protein purification from the culture medium was performed by affinity chromatography using an AKTA avant 25 instrument equipped with an Amsphere A3 column. The culture medium, filtered through a 0.22 μm bottle-top filter, was flowed at a rate of 13.33 mL / min. To remove nonspecific binding, the column was first flushed with a buffer of 50 mM Na-Pi, 0.5 M NaCl, pH 7.0, and secondarily with a buffer of 50 mM sodium acetate, pH 4.5, each at a volume eight times the column volume. After removal of nonspecific binding, the bound proteins were eluted by flowing elution buffer of 100 mM glycine, pH 3.0 at a volume ten times the column volume, and the eluate was collected in a 50 mL tube. The collected protein was neutralized by adding 1 M Trizma® base solution (pH 11.0) at a volume of 1% of the collected protein volume, and the precipitate was removed using a 0.22 μm syringe filter. The samples before and after neutralization were quantified by UV measurement, and their purity was analyzed by SEC-HPLC. To achieve a purity of 90% or higher, the primary purified material was further subjected to size exclusion chromatography using a Superdex 200pg 26 / 600 column. First, the neutralized sample was subjected to Millipore TM Stirred cell 250 mL concentrator and Millipore TM The sample was concentrated to a volume of 13 mL or less using Discs 50 kDa. The concentrated sample was injected into a column buffered with PBS (pH 6.5), and the eluted protein was collected in a tube, its purity confirmed by SEC-HPLC, and then concentrated again. The final concentrated protein was analyzed by UV quantification, and its purity was confirmed by SEC-HPLC and SDS-PAGE analysis.

[0213] Through the above process, one or more of the following physical properties of each conjugate were measured: titer, elution amount, yield (step yield), high molecular weight species (HMWS) content, low molecular weight species (LMWS) content, and functional monomer (Monomer) content. Here, titer refers to the concentration of Fc fusion protein present in the culture medium as measured at the time of cell culture medium recovery; elution amount refers to the total amount of protein recovered after affinity chromatography, which is the primary purification step; yield is the value obtained by dividing the total amount of protein recovered after primary purification by the total amount of protein measured in the culture medium, and represents the recovery rate of protein recovered by the column during primary purification. High molecular weight species (HMWS) are protein species with a mass greater than the predicted functional unit of the protein, and low molecular weight species (LMWS) are protein species with a mass smaller than the predicted functional unit of the protein; both refer to fractions with abnormal protein folding. Functional monomers refer to fractions with the normal structure and function of the protein. Figures 2A to 2O show the productivity and purity of conjugates of anti-PD-L1 scFv and a modified fusion protein (modified VEGFR1-D2-D3). Specifically, conjugates H-12C94 to H-12C99, based on the modified fusion protein of this disclosure, showed higher purity (approximately 20% vs. approximately 90%) when purified under the same conditions compared to a control group conjugate (H-12C08; A167 scFv-HG4SL-VEGF-Grab) produced with an unmodified fusion protein. It was confirmed that conjugates produced with an unmodified fusion protein generated many high molecular weight aggregates (Figures 2C and 2D), while conjugates produced with a modified fusion protein showed a significant reduction in such aggregates (Figures 2E to 2N). This means that the physical properties of the conjugates in this disclosure are improved by including a modified fusion protein, which in turn means an improvement in yield in drug manufacturing.

[0214] Example 5 - Freeze / thaw stability test of the compound

[0215] The stability of the produced conjugates after freezing and thawing was confirmed. The conjugate protein sample was frozen by standing it at -70°C ultra-low temperature for 1 hour, then removed to room temperature and left to stand for 30 minutes to thaw. This process was repeated 5 times, and 50 µl of the protein sample was sampled after each repetition and the change in purity was confirmed by SEC-HPLC analysis (Table 10 shows the second repetition (2R), Table 11 shows the third repetition (3R)).

[0216] [Table 11]

[0217] [Table 12]

[0218] Table 10 above shows the freeze / thaw stability results from the second experiment (2R), and Table 11 shows the freeze / thaw stability results from the third experiment (3R). As can be seen in Table 10, there was no significant difference in the freeze / thaw stability of the conjugates between linkers V2 and V3, although V3 showed slightly higher stability. In the case of scFv, atezolizumab showed higher stability compared to A167, but, similar to the linkers, it was difficult to say that the difference was statistically significant. Overall, regardless of the linker and scFv, high freeze / thaw stability was observed in the conjugates based on the modified fusion proteins of this disclosure.

[0219] This was also observed in the comparative results of Fc variants using A167 scFv and V3 linkers. As shown in Table 11, it was confirmed that the purity of all variants remained within 2% even after five freeze / thaw cycles.

[0220] These results were obtained using a general buffer rather than the optimal formulation, suggesting that freeze / thaw stability may be improved through future formulation research.

[0221] Overall, the modified fusion protein-based conjugates of this disclosure exhibited high freeze / thaw stability.

[0222] Example 6 - Purity analysis of conjugated proteins using size exclusion-high performance liquid chromatography.

[0223] Analysis was performed using a Waters Arc HPLC system coupled with a Tosoh TSKgel G3000SWXL SEC column, with PBS (pH 7.4) as the mobile phase. While flowing the mobile phase at 0.5 mL / min, 30 μg of protein sample was injected into the column and detected at 280 nm for 30 minutes to measure one or more of the following: high molecular weight species (HMWS), low molecular weight species (LMWS), and functional monomer (Monomer) content.

[0224] Example 7 - Evaluation of binding affinity to VEGF-A, PlGF, and PD-L1

[0225] The binding affinity of each protein to VEGF-A and PlGF ligands was measured using an Octet® RED96e (ForteBIO) instrument, in accordance with previous literature (Kamat et al, 2017).

[0226] First, before measuring the binding strength, the AHC sensor was stabilized by immersion in 1x KB buffer (ForteBIO). The conjugate protein PB203, the substance to be analyzed, was bound to the sensor at a concentration of 10 nM, and then washed with 1x KB buffer to remove any remaining protein after binding. Subsequently, VEGF-A, PlGF, and PD-L1 were passed through the sensor at eight different concentration intervals, and the binding strength was measured. From the results of the binding strength measurements, three or more concentrations were selected that fell within the range of the reliability indicators Full X^2 ​​(≤3) and Full R^2 (≥0.95), and these were set as the Global Fit. After that, the binding strength (KD), Kon, and Kdis values ​​were calculated. The results are shown in Figures 4A to 4K. As can be seen from Figures 4A to 4K, the conjugates of this disclosure showed increased binding rates to VEGF-A and PlGF compared to conjugates produced with undeformed fusion proteins, and it was confirmed that the PD-L1 binding strength of scFv was well maintained without decrease.

[0227] However, when producing a conjugate of the modified fusion protein and an scFv targeting a specific antigen according to this disclosure, it is not possible to predict in advance whether the binding affinity of the fusion protein to VEGF and / or PlGF and the binding affinity of the conjugated scFv to the specific antigen will function without interfering with each other. In this disclosure, it has been confirmed that the binding affinity of the modified fusion protein to VEGF and PlGF and the binding affinity of the anti-PD-L1 scFv to PD-L1 are maintained without antagonism, which is an effect that cannot be easily predicted.

[0228] Example 8 - Stability evaluation in a blood-mimicking environment

[0229] To indirectly analyze the in vivo stability of PB203, the concentration of residual protein variants after specific time intervals was measured by ELISA through a reaction with rat empty serum. Based on the test execution time, PB203 was mixed with rat empty serum at 168 hours, 144 hours, 72 hours, 48 ​​hours, 24 hours, 4 hours, 2 hours, and 0 hours to prepare a final concentration of 10 μg / mL, and then stored in a 37°C incubator. On the day before the test, human VEGF-A (R&D systems) was coated onto plates by overnight incubation at 4°C, washed with 0.1% PBS-T washing buffer to remove unbound human VEGF-A, and then treated with 0.1% PBS-T containing 5% skim milk for a 1-hour blocking process at room temperature. Protein-serum mixtures prepared for different time periods were diluted 50-fold with blocking buffer, and 100 μL of each mixture was added to the wells of a plate. The mixture was then reacted at room temperature for 2 hours. After that, all the reaction solution in the wells was removed, and the plate was washed with wash buffer. The detection antibody, HRP Goat anti-human IgG Fc Cross-Absorbed Secondary antibody (Invitrogen), was reacted at room temperature for 1 hour. After washing to remove unreacted detection antibody, 100 μL of 3,3',5,5'-tetramethylbenzidine (TMB) solution (Sigma) was added to each well and reacted at room temperature for 10 minutes. The reaction was then stopped by adding stop solution (Sigma). Absorbance was measured at 450 nm using a plate reader. The remaining amount (%) was calculated based on the absorbance measured at 0 hours and compared relatively. The results are shown in Figures 3A to 3C.

[0230] As can be seen in Figures 3A and 3B, it can be confirmed that the conjugates H-12C94 to H-12C99, which are based on the modified fusion protein of this disclosure, exhibit significantly superior serum stability in a blood-mimicking environment compared to the conjugate produced with the unmodified fusion protein (H-12C08). Furthermore, as can be seen with H-32D01, H-32D02, H-32D09, and H-32D10, it was confirmed that they exhibit significantly superior serum stability in the blood-mimicking environment regardless of the use of the V2 or V3 linker and the type of scFv (Figure 3C).

[0231] Example 9 - Isoelectric Focus Analysis

[0232] The isoelectric point of the PB203 protein was analyzed using the icIEF (Image capillary isoelectric focusing) method. 40 μL of the deformed fusion protein, diluted to a concentration of 2 mg / mL, and a pre-prepared 160 μL of master mix solution (SERVALYT) were used. TM After mixing low pI marker (3.38) (ProteinSimple), high pI marker (9.5) (ProteinSimple), 1% methylcellulose (ProteinSimple), DW, 500 mM arginine (Sigma), 200 mM iminodiacetate (Sigma), and 10 M urea (Sigma), the precipitate and bubbles were removed by centrifugation, and the mixture was inserted into each well of the instrument (Maurice, ProteinSimple). 2 mL each of the OH- and H+ positions of the cartridge were filled with the anolyte solution and the cartridge was installed in the instrument. The isoelectric point was then measured according to the instrument's instructions. Since the measured pI appeared not as a single peak but as multiple peaks within a specific range, the distribution of protein pI was defined by dividing it into three intervals: acidic (pI 6-7), neutral (pI 7-8), and basic (pI < 8), and the interval with the highest proportion of distribution was compared. The results are shown in Figures 7A to 7R.

[0233] As shown in Figures 7G, ​​7M, and 7R, the H-12C94~H-12C99 conjugates based on the modified fusion protein of this disclosure show a higher acid / neutral ratio compared to the control conjugate (H-12C08;A167+VEGF-Grab) produced from the unmodified fusion protein. Furthermore, as can be seen from Figures 7M and 7R, a higher acid / neutral ratio is observed compared to the unmodified fusion protein and the conjugates produced from them, regardless of linker V2 or V3, scFv type, or presence or absence of Fc mutations. This means that the conjugates of this disclosure have a higher tendency to become cathode-positive in the in vivo environment, suggesting a lower possibility of nonspecific binding during in vivo circulation.

[0234] Example 10 - Thermal Stability Analysis

[0235] PB203 was prepared by diluting it to a final concentration of 2 mg / ml using 1x PBS buffer. 12.5 μl of protein was mixed with 2.5 μl of 8X protein thermal shift. TM Dyeing agent (Protein Thermal Shift) TM (Life Technologies) and 5.0 μl of protein thermal shift dye TM Buffer (Life Technologies) was added to prepare the final reaction mixture. 20 μl of the prepared reaction mixture was placed in a PCR tube and processed in Quanto Studio. TM Real-time PCR (QuantStudio TM After placing the samples in a tray equipped with Real-time PCR, the Tm value was measured. All analyses were performed in four replicate experiments. The results are shown in Figures 8A to 8D.

[0236] Specifically, Figure 8B shows that linkers V2 and V3 do not significantly affect thermal stability, and instead, it can vary depending on the type of scFv used. Furthermore, Figure 8D confirms that Fc mutations are also unrelated to thermal stability.

[0237] The temperature during in vivo injection is within 40°C, and in the case of protein pharmaceuticals, storage stability is evaluated at 25°C and 40°C, assuming room temperature and biological temperature. Based on these criteria, it was confirmed that the conjugates based on the modified fusion protein of this disclosure exhibit high thermal stability of 60°C or higher overall.

[0238] This indicates that the modified fusion protein-based conjugates of this disclosure possess physicochemical properties suitable for development as a drug.

[0239] Example 11 - VEGF-A inhibitory ability analysis

[0240] To quantitatively analyze the inhibitory activity against VEGF-A, a genetically modified cell line (KDR / NFAT-RE HEK293, Promega) was used, incorporating VEGFR-2 (KDR) expression and a luciferase reporter system mediated by VEGF-A / VEGFR-2 interaction. 25 μL of cells at a density of 1.6 × 10⁶ cells / mL were dispensed into a 96-well plate. Then, 25 μL of VEGF-A solution (33.3 ng / mL concentration) and 25 μL of PB203 diluted to different concentrations were added and incubated at 37°C in a 5% CO₂ incubator for 6 hours. After cooling the plate to room temperature for 15 minutes, 75 μL of Bio-Glo reagent (Promega, luciferase assay buffer + substrate mixture) was added. After incubation in a light-shielded environment for 10 minutes, luminescence was measured using a plate reader. IC50 values ​​for each substance were calculated using GraphPad Prism.

[0241] As shown in Figures 6C-6L and 6N, the conjugates based on the modified fusion protein of this disclosure were confirmed to have cellular-level VEGF-A signaling inhibitory activity comparable to that of the modified fusion protein before binding, regardless of the type of linker and scFv. Furthermore, this inhibitory activity was confirmed to be superior to that of the unmodified fusion protein, VEGF-Grab(PB101).

[0242] Example 12 - PD-1 / PD-L1 signal suppression ability analysis

[0243] To quantitatively analyze the inhibitory activity against PD-L1, genetically modified cell lines (Jurkat PD-1 / U2OS PD-L1, Eurofins Discovery) incorporating PD-1 and PD-L1 expression and a luciferase reporter system mediated by PD-1 / PD-L1 interaction were used. 40 μL of U2OS PD-L1 cells at a density of 1 × 10⁵ cells / mL were dispensed into a 96-well plate, followed by the addition of 20 μL of diluted PB203 solution and a control substance. The mixture was incubated at 37°C in a 5% CO₂ incubator for 1 hour. 40 μL of Jurkat PD-1 cells at a density of 2.4 × 10⁵ cells / mL were added to the well plate and incubated at room temperature for 2 hours. Next, 10 μL of Detection reagent 1 was added to the plate and incubated at room temperature for 15 minutes, avoiding light. Subsequently, 40 μL of Detection reagent 2 was added and incubated at room temperature for 3 hours, avoiding light. Finally, luminescence was measured using a plate reader. The IC50 values ​​for each substance were calculated by analyzing the data using GraphPad Prism.

[0244] The inhibitory effects of the conjugate of anti-PD-L1 scFv and a modified fusion protein (modified VEGFR1-D2-D3) on VEGF-A and PD-L1 are shown in Figures 6A to 6E.

[0245] From Figures 6I to 6N and 6O, it was confirmed that the conjugates based on the modified fusion proteins of this disclosure (H-32D01, H-32D02, H-32D04, H-32D06, H-32D08, H-32D09, H-32D10, etc.) possess cellular-level PD-L1 signaling inhibitory activity comparable to that of the pre-binding anti-PD-1 antibody, regardless of the type of linker and scFv.

[0246] This demonstrates that the modified fusion protein-based conjugates of this disclosure maintain their respective functions without mutual interference through structural design, despite their complex structure.

[0247] Example 13 - Evaluation of the co-binding ability of VEGF-A, PlGF, and PD-L1 ligands.

[0248] To evaluate the binding affinity based on the binding order when two or more of the VEGF-A, PlGF, and PD-L1 ligands bind, we used the Octet® RED96e (ForteBIO) instrument.

[0249] First, before measuring the binding strength, the AHC sensor was stabilized by immersion in 1x KB buffer (ForteBIO). The substance to be analyzed, PB203 protein, was bound to the sensor at a concentration of 100 nM, and then washed with 1x KB buffer to remove any remaining protein after binding. Subsequently, VEGF-A, PlGF, and PD-L1 were bound at 30 nM (high concentration), and then different types of binding proteins (e.g., VEGF-A → PD-L1, PlGF → PD-L1, PD-L1 → VEGF-A, PD-L1 → PlGF) were bound at eight concentration intervals (30, 15, 7.5, 3.75, 1.875, 0.937, 0.468, 0 nM) while the previously bound protein was still present (30 nM), and the signals were measured. From the results of the binding affinity measurements, four or more concentration intervals were identified in which the indicators for ensuring reliability, Full X^2 ​​(≤3) and Full R^2 (≥0.95), were within the specified range. Binding affinity and other indicators were calculated through global fit for these intervals. As a result, the co-binding affinity of the anti-PD-L1 scFv and the modified VEGFR1-D2-D3 fusion protein conjugates to their targets is shown in Figures 5A to 5F. Measurement of the antigen's co-binding affinity confirmed that it was maintained with almost no difference even when the type of antigen was different. Specifically, as shown in Figures 5A and 5B, it was confirmed that the binding affinity of the modified fusion protein-based conjugates to PD-L1 was similarly maintained regardless of whether the ligand was VEGF-A or PlGF. Furthermore, as can be seen in Figures 5C to 5F, the modified fusion protein-based conjugates maintain a good level of binding affinity to a single ligand, regardless of the binding order or type of the target ligand. This demonstrates that the modified fusion protein-based conjugates of this disclosure maintain their respective functions without mutual interference through structural design, despite their complex structure.

[0250] Example 14 - Pharmacokinetic evaluation after single intravenous administration in SD rats

[0251] Seven-week-old male SD rats were acclimatized for one week before being used in the study. PB203 was administered intravenously as a single dose at a concentration of 3 mpk (mg / kg). Blood was collected via the jugular vein at different time points (0, 0.083, 2, 8, 24, 48, 96, 120, 168, and 336 hours), and the plasma was separated.

[0252] The day before the experiment, human PD-L1 (R&D systems) was coated onto plates by overnight incubation at 4°C. This was then washed with 0.1% PBS-T washing buffer to remove any unbound human PD-L1. Finally, a blocking process was performed at room temperature for 1 hour using 0.1% PBS-T containing 5% skim milk. The prepared plasma samples were then diluted 20-fold with blocking buffer, and 100 μL were placed in each well of the plate. The mixture was reacted at room temperature for 2 hours. Afterward, all reaction solution was removed from the wells, and the plates were washed with washing buffer. The detection antibody, HRP Goat anti-human IgG Fc Cross-Absorbed Secondary antibody (Invitrogen), was reacted at room temperature for 1 hour. After washing to remove unreacted detection antibody, 100 μL of 3,3',5,5'-tetramethylbenzidine (TMB) solution (Sigma) was added in increments and reacted at room temperature for 10 minutes. The reaction was then stopped with stop solution (Sigma). Absorbance was measured at 450 nm using a plate reader. The quantitative standard substance was PB203, prepared by serial dilution at concentrations from 62.5 to 0.48. A standard curve was drawn based on the measured absorbance (OD450), and the concentration of PB203 in plasma was calculated using the derived equation and quantitatively evaluated. Finally, the PK parameter was calculated using the time-dependent concentration results. First, to investigate the pharmacokinetics of scFv, a V3 linker was used in common, and H-32D02 and H-32D10 conjugated with either A167 or atezolizumab scFv were evaluated. As shown in Figure 9A, the A167 conjugate showed relatively superior pharmacokinetics in the body, and Fc variants were created for it. Four Fc variants, including wild-type Fc, were produced and evaluated for the A167 conjugate. As can be seen in Figure 9B, the H-32D04, H-32D06, and H-32D08 conjugates showed superior pharmacokinetics in the body compared to the H-32D02 conjugate.The pharmacokinetics of these three conjugates were similar.

[0253] Example 15 - Anti-cancer efficacy test in a mouse model

[0254] Genetically modified mouse models B6 / JGpt-Cd274tm1(hCD274) / Gpt were subcutaneously transplanted with the genetically modified cell line MC-38-hPD-L1, and the mice were divided into groups when the tumor size reached approximately 100 mm³. Subsequently, the candidate substance PB203 and the control drug were administered intraperitoneally at a dose of 10 mpk three times a week, and the tumor size and weight were measured over a three-week period. This study was conducted in compliance with animal experiment ethics guidelines. The results are shown in Figure 10. As shown in Figure 10, among the conjugates of this disclosure, the Fc wild-type and ADCC / CDC activation-inhibited Fc showed tumor growth inhibition (TGI) that was approximately 10% or more superior to that of the existing commercial antibody Tecentriq, and the other two Fc mutants also showed high levels of tumor growth inhibition. In particular, in the case of H-32D04, complete regression was observed in approximately 40% of individuals, which is a superior result compared to the commercial antibody Tecentriq, in which complete remission was not observed at all.

[0255] From the above description, a person ordinary in the art to which this disclosure pertains will understand that the technical idea or essential features of this disclosure may be implemented in other specific forms without alteration. In this regard, the embodiments described above should be understood to be illustrative and not limiting in any respect. The scope of this disclosure should be interpreted as including any modified or altered forms derived from the meaning and scope of the claims and their equivalent concepts, which are described later in the detailed description above.

Claims

1. A conjugate comprising a fusion protein containing a VEGFR1 (vascular endothelial growth factor receptor 1) extracellular domain, a linker, and a multimerization domain, and a programmed cell death protein ligand 1 (PD-L1) targeted scFv, characterized in that the PD-L1 targeted scFv is fused to the N-terminus of the fusion protein.

2. The aforementioned fusion protein comprises a VEGFR1 extracellular domain, a linker, and a multimerization domain. The VEGFR1 extracellular domain includes the immunoglobulin (Ig)-like domain D2 and Ig-like domain D3 of VEGFR1, and the linker is located between the Ig-like domain D3 and the multimerization domain. The conjugate according to claim 1 is a modified fusion protein having one or more of the following characteristics (a) to (c): (a) Domain D3 having one or more amino acid substitutions of K241E, L243S, R244V, and H246E in the β1-β2 loop, L258A, L258S, or L258D in the β2-β3 loop, and one or more amino acid substitutions of K300G, Q302T, and K304S in the β5-β6 loop, wherein the β1-β2 loop of domain D3 contains amino acid residues T236-T247 of the amino acid sequence of VEGFR1 including SEQ ID NO: 41, the β2-β3 loop contains amino acid residues T256-V262 of the amino acid sequence of VEGFR1, and the β5-β6 loop contains amino acid residues D299-L308 of the amino acid sequence of VEGFR1; (b) The linker has a length of approximately 14 to 35 amino acids; and (c) A disulfide bond is present in the fusion protein, and one of the amino acid residues located at positions -2, -1, 0, +1, +2, and +3 relative to L243 and Y329 of domain D3 is substituted with cysteine.

3. The characteristic (a) of the aforementioned fusion protein is, The conjugate according to claim 2, comprising: 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 K300G, Q302T, and K304S amino acid substitutions in the β5-β6 loop of domain D3.

4. The conjugate according to claim 2, wherein the modified fusion protein has the N-terminus of domain D2 beginning with amino acid sequence EF.

5. The conjugate according to claim 2, wherein in the characteristics (b) of the modified fusion protein, the linker comprises a glycine-serine (GS) repeat sequence, and the GS repeat sequence is a sequence of 2 to 35 amino acids in length consisting of glycine (G) and serine (S).

6. The conjugate according to claim 5, wherein one or more glycine (G) molecules in the GS repeat sequence are substituted with cysteine ​​(C).

7. The conjugate according to claim 2, wherein the characteristic (b) of the modified fusion protein includes an amino acid sequence of a hinge region derived from immunoglobulin, and the amino acid sequence of the hinge region is deformable.

8. The conjugate according to claim 7, wherein the hinge region derived from the immunoglobulin is derived from human IgD and / or IgG.

9. The conjugate according to claim 7, wherein the characteristic (b) of the modified fusion protein has an amino acid mutation in the papain recognition site or glycosylation site located in the hinge region derived from immunoglobulin.

10. The compound according to claim 2, wherein in the characteristics (b) of the modified fusion protein, the linker contains, in order from the N-terminus to the C-terminus, an amino acid sequence selected from the group consisting of (i) CS, CSSG, CS(GGGGGS), CS(GGGGGS)3, C(GSSG)2, GS, GSSG, (GSSG)2, GGGGS, (GGGGGS)4, GS(GGGGGS), and GS(GGGGGS)3, and (ii) a hinge region derived from an immunoglobulin selected from the group consisting of human IgG1, IgG4, or IgD / G1, and the amino acid sequence of the hinge region is deformable.

11. The conjugate according to claim 2, wherein in the characteristics (b) of the modified fusion protein, the linker is an amino acid sequence selected from the group consisting of CSSGDATPSPPS, CSKVDKKVEPKSSSDTPTPCPPCP, CSGGGGSAEPKAGDATPTCPCPP, CSGGGGGGSGGGGGSAGGGGSAESKKYGPCPPPPCP, CSNTGSGGEEKKKEKEKEQUEERSSDTPTPTCPPCP, CSNTGSGGGEEKKKEKEKEKEQUEERSSCDTTPTPTCPPCP, CGSGGGSGEPKKSDATPTCPPCP, and CSKVDKKVEPKSSSDKTYTCPPCP.

12. 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, CSKVDKKVEPKSSSDTPTPCPPPP, CSGGGGGSAEPKAGDATPTPCPPPP, CSGGG The conjugate according to claim 2, wherein the amino acid sequence is selected from the group consisting of GSGGGGGGGGGSAESKYGPPCPPP, CSNTGSGGEEKKKKEKEKEQUEERSSDTPPTTCPPPP, CSNTGSGGEEKKKKEKEKEQUEERSSCDTTPTPTCPPPP, CGSGGGGSGEPKSDATTPTCPPPP, and CSKVDKKVEPKSSSDKTYTCPCPPP; (c) the amino acid residue located at the +2 position relative to L243 and Y329 of domain D3 is substituted with cysteine.

13. The conjugate according to claim 2, wherein the modified fusion protein is composed of an amino acid sequence selected from the group consisting of any one of the amino acid sequences of SEQ ID NOs: 44 to 58.

14. The conjugate according to claim 2, wherein the polymerizing domain is (a) an Fc site of immunoglobulin; (b) a CH3 site of IgG1 or IgG4; (c) CH2 and CH3 sites of IgG1 or IgG4; (d) an Fc site of immunoglobulin containing an amino acid sequence having at least 85% identity with the amino acid sequence of SEQ ID NO: 59; or (e) an Fc site of immunoglobulin containing the amino acid sequence of SEQ ID NO:

60.

15. The conjugate according to claim 14, wherein the polymerizing domain includes an IgG1Fc site consisting of the amino acid sequence of SEQ ID NO:

60.

16. The aforementioned polymerizing domain corresponds to Sequence ID No. 60, i) Having one or more of the following: T20Q amino acid substitution, D126E amino acid substitution, L128M amino acid substitution, M198L amino acid substitution, and K217 amino acid deletion, ii) The conjugate according to claim 2, having one or more of the following: L4A amino acid substitution, L5A amino acid substitution, H38Q amino acid substitution, K44Q amino acid substitution, Y66F amino acid substitution, A97G amino acid substitution, A100S amino acid substitution, P101S amino acid substitution, R125Q amino acid substitution, D126E amino acid substitution, L128M amino acid substitution, K179R amino acid substitution, Q189E amino acid substitution, P215L amino acid substitution, and K217 amino acid deletion.

17. The conjugate according to claim 2, wherein the modified fusion protein is in the form of a dimer or a polymer.

18. The conjugate according to claim 1, wherein the PD-L1-targeted scFv is atezolizumab scFv or an anti-PD-L1 scFv containing the amino acid sequence of SEQ ID NO:

40.

19. A pharmaceutical composition for the prevention or treatment of chronic infection, tissue allograft, autoimmune disease, inflammatory disease, neoplastic disease, cancer, angiogenesis-related disease, or eye disease, comprising the conjugate described in any one of claims 1 to 18 as an active ingredient.

20. A nucleic acid molecule for encrypting the compound according to any one of claims 1 to 18.

21. A host cell comprising a nucleotide sequence that encrypts the conjugate according to any one of claims 1 to 18.

22. A vector comprising a nucleotide sequence for encrypting the conjugate according to any one of claims 1 to 18.

23. The vector according to claim 22, which is a recombinant viral vector.

24. A pharmaceutical composition for delivering a viral vector to a target body, comprising the recombinant viral vector described in claim 23, wherein the fusion protein encoded by the recombinant viral vector is expressed in the target body, and the pharmaceutical composition is for the prevention or treatment of autoimmune diseases, inflammatory diseases, neoplastic diseases, cancer, angiogenesis-related diseases, or eye diseases.

25. The pharmaceutical composition according to claim 24, wherein the recombinant viral vector is a recombinant adeno-associated virus vector.

26. A method for preventing or treating one or more conditions selected from the group consisting of chronic infection, tissue allograft, autoimmune disease, inflammatory disease, neoplastic disease, cancer, angiogenesis-related disease, or ocular disease, by administering to a subject requiring the conjugate described in any one of claims 1 to 18 and / or the recombinant viral vector of claim 23.

27. Uses of the conjugate described in any one of claims 1 to 18 and / or the recombinant viral vector described in claim 23 for the prevention or treatment of one or more conditions selected from the group consisting of chronic infection, tissue allograft, autoimmune disease, inflammatory disease, neoplastic disease, cancer, angiogenesis-related disease, or ocular disease.

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