CONJUGATES OF MODIFIED FUSION PROTEINS AND SCFv AND USES THEREOF

Abstract

The present disclosure relates to a conjugate of a modified multispecific fusion protein and scFv that binds to an antigen of a binding scFv, a vascular endothelial growth factor and a placental growth factor, and uses thereof. Conjugates according to embodiments of the present disclosure may also exhibit excellent yield and stability. A conjugate according to an embodiment of the present disclosure may exhibit excellent blood stability. The conjugates according to embodiments of the present disclosure may exhibit excellent anti-tumor, anti-fibrosis, and / or immune cell activation effects.

Description

Technical Field

[0001] This disclosure relates to conjugates of modified fusion proteins and single-chain variable fragments (scFv) and their uses therein, said conjugates binding to vascular endothelial growth factor, placental growth factor and antigens that bind to scFv. Background Technology

[0002] Vascular endothelial growth factor (VEGF) is a signaling protein that plays an important role in angiogenesis and angiogenesis. Its general functions include the formation of new blood vessels during embryonic development, the formation of new blood vessels in muscles after injury or exercise, and the formation of new blood vessels that bypass blocked vessels; however, abnormal increases in VEGF are closely associated with tumor development and metastasis (Carmeliet, P., Jain, RK, 2000. Nature 407: 249–257).

[0003] VEGF is classified into five types: VEGF-A, VEGF-B, VEGF-C, VEGF-D, and placental growth factor (PlGF), which are known to contribute to angiogenesis (Carmeliet, P., Jain, RK, 2000. *Nature* 407: pp. 249–257; Kuwano M et al., 2001. *Intern Med* 40: pp. 565–572). On the other hand, three types of VEGF receptors exist: VEGF R1, R2, and R3, and the binding affinity of the ligands varies depending on the type of receptor. Among these receptors, VEGFR-1 / -2 is known to be involved in angiogenesis (Veikkola et al., 2000. *Cancer Research* 60: pp. 203–212).

[0004] Researchers have long sought to develop drugs that inhibit angiogenesis by removing vascular endothelial growth factor (VEGF), a key factor in tumorigenesis. Bevacizumab (a monoclonal antibody targeting VEGF-A) is a representative example (Stacker et al., 2013. Chinese Journal of Cancer Research. 32(6): 297-302; Kazazi-Hyseni et al., 2010. Oncologist 15(8): 819-825; DrugBank accession number: DB00112). The mechanism of action of monoclonal antibody drugs involves binding to a single VEGF; however, because multiple growth factors are involved in carcinogenesis, monoclonal antibodies are not always effective drugs with significant therapeutic effects. Bevacizumab actually induces increased expression of other types of non-targeted growth factors through intracellular compensatory mechanisms, thereby leading to drug resistance (Bagley et al., 2011. Clinical Cancer Research 17(5): 976-988; Lieu et al., 2013. PLoS One 8: e77117; Cutsem et al., 2020. Clinical Cancer Research 26(3): 717-725). For these reasons, there is a growing need to develop drugs that can simultaneously target multiple angiogenic growth factors associated with carcinogenesis and also have targeting functions for cancer cells.

[0005] To overcome the limitations of monoclonal antibody drugs, drugs targeting multiple vascular endothelial growth factors have been developed. Representative protein drugs are VEGF-Trap (Ciombor et al., 2013. Clinical Cancer Research 19(8): 1920-1925; DrugBank accession number: DB08885) and VEGF-Grab (Lee et al., 2015. Molecular Cancer Therapy 14(2): 470–479). Both drugs mimic a portion of the decoy receptor, with VEGF-Trap being a heterologous protein in the form of VEGFR1 D2–VEGFR2 D3 and VEGF-Grab being a homologous protein in the form of VEGFR1 D2–D3. Each drug is a recombinant fusion protein fused to the Fc of an immunoglobulin. Efforts have been made to improve the physicochemical properties of these fusion proteins targeting multiple vascular endothelial growth factors.

[0006] Additional antigen-targeting single-chain variable fragments (scFvs) could be considered for binding to such fusion proteins, and among these, scFvs targeting programmed cell death protein ligand 1 (PD-L1) could be considered, which is overexpressed in many cancer tissues (Dong et al., 2002. *Nature Medicine* 8: 787–789). The PD-1 receptor and its ligand (i.e., PD-L1) are immune checkpoint proteins associated with the suppression of immune system responses related to chronic infection, pregnancy, tissue allogeneic transplantation, autoimmune diseases, and cancer. PD-L1 modulates immune responses by binding to the inhibitory receptor PD-1 expressed on the surface of T cells, B cells, and monocytes. PD-L1 also negatively regulates T cell function through its interaction with another receptor, B7-1. 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 anti-tumor immune activity.

[0007] Cancer tissues that overexpress PD-L1 include melanoma, squamous cell carcinoma of the skin (CSCC), squamous cell carcinoma of the head and neck (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 is a need to develop a conjugate that exhibits excellent anticancer effects, selectively targets cancer cells based on high binding affinity, has increased in vivo residence time, achieves high yield at the protein therapeutic level in the context of commercial-scale production, and possesses physical and chemical stability. Summary of the Invention

[0009] Technical issues The improved physicochemical properties of proteins can maximize production efficiency by enhancing their physical and chemical stability during expression and purification, and reduce non-specific binding, aggregation, and degradation when introduced into the body, thus achieving high persistence in vivo. In the case of anticancer agents, this improvement increases selective targeting of cancer cells, resulting in superior anticancer effects.

[0010] The inventors of this disclosure have developed modified fusion proteins by introducing amino acid variations, linker length adjustments and variations, or disulfide bonds in specific regions of domain D3 of a fusion protein containing VEGFR1 domains D2 and D3. Furthermore, the inventors have developed a conjugate of scFv targeting PD-L1 binding to the modified fusion protein and demonstrated that such conjugates exhibit excellent yield, stability, and anticancer efficacy.

[0011] Technical solutions 1. An embodiment of this disclosure may be a conjugate of a PD-L1-targeting scFv (anti-PD-L1 scFv) bound to a VEGFR domain. The conjugate can inhibit vascular endothelial cell differentiation by binding the scFv to a target expressed on the surface of cancer cells, and simultaneously binding to VEGF and PlGF near the cancer cells. This inhibits cancer growth by selectively suppressing angiogenesis around cancer cells. An embodiment of this disclosure includes a PD-L1-targeting scFv that binds to a fusion protein comprising a VEGFR1 (vascular endothelial growth factor receptor 1) extracellular domain, a linker, and a polymerizing domain, and the conjugate is characterized by the fusion of the PD-L1-targeting scFv with the N-terminus of the fusion protein. In embodiments of this disclosure, the PD-L1-targeting scFv may specifically be an atezolizumab scFv (the atezolizumab scFv is an anti-PD-L1 scFv) or a separately developed anti-PD-L1 scFv (A167 scFv; SEQ ID NO: 40).

[0012] 2. Embodiments of the "fusion protein" of this disclosure may relate to a modified fusion protein comprising a VEGFR1 extracellular domain, a linker, and a polymerizing domain, wherein the VEGFR1 extracellular domain comprises an immunoglobulin (Ig)-like domain D2 and an Ig-like domain D3 of VEGFR1, the linker is located between the Ig-like domain D3 and the polymerizing domain, and the modified fusion protein has one or more of the following properties (a) to (c): (a) binding One or more amino acid substitutions selected from K241E, L243S, R244V, and H246E on the β1-β2 ring of domain D3; amino acid substitutions of L258A, L258S, or L258D on the β2-β3 ring of domain D3; and one or more amino acid substitutions selected from K300G, Q302T, and K304S on the β5-β6 ring of domain D3, wherein the β1-β2 ring of domain D3 includes the VEGFR1 amino acid sequence (e.g., the VEGFR1 amino acid sequence in Table 2; SEQ ID NO: ... (a) The linker is of a length of about 14 to about 35 amino acids; and (b) the fusion protein contains amino acid residues T236 to T247 of the VEGFR1 amino acid sequence in the β2-β3 ring, wherein the β5-β6 ring comprises amino acid residues D299 to L308 of the VEGFR1 amino acid sequence; and (c) the fusion protein contains disulfide bonds, and in the amino acid residues of the β1-β2 ring of the domain D3, one amino acid residue L243 and one amino acid residue at positions -2, -1, 0, +1, +2 and +3 relative to Y329 of the domain D3, particularly residue 331 at position +2, is replaced by a cysteine ​​residue.

[0013] 3. In one embodiment, characteristic (a) of the modified fusion protein may include: amino acid substitutions of K241E, L243S, R244V, and H246E on the β1-β2 ring of domain D3; amino acid substitutions of L258A, L258S, or L258D on the β2-β3 ring of domain D3; and amino acid substitutions of K300G, Q302T, and K304S on the β5-β6 ring of domain D3. Regarding characteristic (a), the substitutions may be amino acid substitutions, wherein the amino acids, compared to the residues before substitution, can reduce the protein's net pI, have negatively charged side chains, or have side chains with electrostatic negative charges.

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

[0015] 5. In one embodiment, with respect to characteristic (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 consisting only of G and S with a length of 2 to 35.

[0016] 6. In one embodiment, one or more glycine (G) residues of the GS repeat sequence included in the linker of the modified fusion protein may be replaced by cysteine ​​(C).

[0017] 7. In one embodiment, regarding characteristic (b), the linker of the modified fusion protein may include an amino acid sequence derived from the hinge region of an immunoglobulin, and the amino acid sequence of the hinge region may be modified.

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

[0019] 9. In one embodiment, regarding characteristic (b), the modified fusion protein may have amino acid variations at the papain recognition site or glycosylation site present in the hinge region of the immunoglobulin-derived protein. Specifically, the amino acid variation at the papain recognition site may be the substitution of one or more amino acid residues present at the papain recognition site with alanine, serine, tyrosine, proline, or threonine, or the insertion of an amino acid sequence of 1 to 10 amino acids, including at least one amino acid having an aromatic or cyclic carbon in its side chain, at the papain recognition site. The amino acid variation at the glycosylation site may be (i) the deletion of serine or threonine present in the hinge region of the immunoglobulin-derived protein, or (ii) the substitution of serine or threonine present in the hinge region of the immunoglobulin-derived protein with an amino acid other than serine, asparagine, or threonine.

[0020] 10. In one embodiment, regarding characteristic (b), the linker of the modified fusion protein may sequentially comprise from the N-terminus to the C-terminus: (i) an amino acid sequence selected from the group consisting of: 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, wherein the amino acid sequence of the hinge region may be modified.

[0021] 11. In one embodiment, regarding characteristic (b), the linker of the modified fusion protein may be an amino acid sequence selected from the group consisting of: CSSGDATPTSPPSP (SEQ ID NO: 68), CSKVDKKVEPKSSDTPPTCPPCP (SEQ ID NO: 69), CSGGGGSAEPKAG DATPPTCPPCP (SEQ ID NO: 70), CSGGGGSGGGGSGGGSAESKYGPPCPPCP (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 may include: (a) amino acid substitutions of K241E, L243S, R244V, and H246E on the β1-β2 ring of domain D3; amino acid substitutions of L258A, L258S, or L258D on the β2-β3 ring of domain D3; and amino acid substitutions of K300G, Q302T, and K304S on the β5-β6 ring of domain D3; (b) a linker, the linker being an amino acid sequence selected from the group consisting of: CSSGDATPTSPPSP, CSKVDKKV EPKSSDTPPTCPPCP, CSGGGGSAEPKAGDATPPTCPPCP, CSGGGGSGGGGSGGGSAESKYGPPCPPCP, CSNTGSGGEEKKKEKEKEEQEERSSDTPPTCPPC P, CSNTGSGGEEKKKEKEKEEQEERSCDTPPTCPPCP, CGSSGGSSGEPKSDA TPTCPPCP and CSKVDKKVEPKSSDKTYTCPPCP; and (c) replacing the amino acid residues at the +2 position of L243 and Y329 relative to domain D3 with cysteine.

[0023] 13. In one embodiment, the modified fusion protein may be composed of an amino acid sequence selected from the group consisting of the amino acid sequences of C61 to C75 (SEQ ID NO: 44 to 58) of Table 2.

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

[0025] 15. In one embodiment, the polymerizing domain may include or be composed of the Fc region of IgG1, the Fc region being composed of the amino acid sequence shown in SEQ ID NO: 60. The polymerizing domain may have one or more of the following: (i) T20Q amino acid substitution, D126E amino acid substitution, L128M amino acid substitution, M198L amino acid substitution and K217 deletion relative to SEQ ID NO: 60; or (ii) 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 deletion.

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

[0027] 17. Embodiments of this disclosure relate to conjugates of modified fusion proteins and scFv according to embodiments of this disclosure, wherein the scFv may be atezolizumab scFv or A167 scFv that binds to PD-L1 (Table 9; SEQ ID NO: 39 or 40).

[0028] 18. Embodiments of this disclosure may relate to a pharmaceutical composition for the prevention or treatment of chronic infections, tissue allogeneic transplantation, autoimmune diseases, inflammatory diseases, neoplastic diseases, cancer, angiogenesis-related diseases, or eye diseases, said pharmaceutical composition comprising a modified fusion protein and a conjugate of scFv according to embodiments of this disclosure as an active ingredient.

[0029] 19. Embodiments of this disclosure may relate to a nucleic acid molecule that encodes a modified fusion protein and scFv conjugate according to embodiments of this disclosure.

[0030] 20. Embodiments of this disclosure may relate to a host cell comprising a nucleotide sequence encoding a modified fusion protein and a conjugate of scFv according to embodiments of this disclosure.

[0031] 21. Embodiments of this disclosure may relate to a vector comprising a nucleotide sequence encoding a modified fusion protein and a conjugate of scFv according to embodiments of this disclosure.

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

[0033] 24. Embodiments of this disclosure may relate to a pharmaceutical composition for delivering a viral vector to a subject, the pharmaceutical composition comprising a recombinant viral vector according to embodiments of this disclosure, wherein a fusion protein encoded by the recombinant viral vector and a conjugate of scFv are expressed in the subject, the pharmaceutical composition being used for the prevention or treatment of chronic infections, tissue allogeneic transplantation, autoimmune diseases, inflammatory diseases, neoplastic diseases, cancer, angiogenesis-related diseases, or eye diseases.

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

[0035] 26. Embodiments of this disclosure may relate to a method for preventing or treating one or more of the following by administering to a subject in need a modified fusion protein and scFv conjugate according to embodiments of this disclosure and / or a recombinant viral vector containing a nucleotide sequence encoding a modified fusion protein and scFv conjugate according to embodiments of this disclosure: chronic infection, tissue allogeneic transplantation, autoimmune disease, inflammatory disease, neoplastic disease, cancer, angiogenesis-related disease, or eye disease.

[0036] 27. Embodiments of this disclosure may relate to the use of modified fusion protein and scFv conjugates according to embodiments of this disclosure and / or recombinant viral vectors containing nucleotide sequences encoding modified fusion protein and scFv conjugates according to embodiments of this disclosure for the prevention or treatment of one or more of the following: chronic infection, tissue allogeneic transplantation, autoimmune diseases, inflammatory diseases, neoplastic diseases, cancer, angiogenesis-related diseases, or eye diseases.

[0037] Beneficial effects The modified fusion protein and the anti-PD-L1 scFv conjugate according to embodiments of this disclosure can exhibit excellent yield, physical stability and chemical stability.

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

[0039] The modified fusion protein and the anti-PD-L1 scFv conjugate according to embodiments of this disclosure can exhibit excellent binding affinity for VEGF, PlGF and PD-L1.

[0040] The modified fusion protein and the anti-PD-L1 scFv conjugate according to embodiments of this disclosure can simultaneously exhibit excellent binding affinity for both the antigen of the modified fusion protein and the antigen of the scFv.

[0041] The modified fusion protein and the anti-PD-L1 scFv conjugate according to embodiments of the present disclosure can exhibit excellent inhibitory effects on the corresponding antigens of the modified fusion protein and the corresponding antigens of the scFv.

[0042] The modified fusion protein and anti-PD-L1 scFv conjugate according to embodiments of this disclosure can have acidic properties and can exhibit an enhanced charge variant ratio in the acidic and neutral regions of the pI distribution. Therefore, the modified fusion protein can have improved non-specific interactions with the matrix and cell surface in vivo.

[0043] The modified fusion protein and the anti-PD-L1 scFv conjugate according to embodiments of this disclosure can exhibit excellent anti-cancer activity.

[0044] The modified fusion protein and anti-PD-L1 scFv conjugate according to embodiments of this disclosure can inhibit fibrosis by reducing PlGF levels in the tumor microenvironment (TME) and thus suppressing activation of CD141-expressing cancer-associated fibroblasts (CAFs). This reduces vascular compression and, together with the reduction of VEGF-A levels, normalizes blood vessels. The improvement in normalized blood vessels and peritumoral fibrosis increases drug accessibility and enhances immune cell infiltration, thereby exhibiting an inhibitory effect on tumor growth.

[0045] The modified fusion protein and the anti-PD-L1 scFv conjugate according to embodiments of this disclosure can exhibit anti-tumor, anti-fibrotic, and / or immune cell activation effects. Attached Figure Description

[0046] Figure 1 This is a schematic diagram showing the binding of anti-PD-L1 scFv to the modified VEGFR1 D2-D3 domains (the modified fusion protein of this disclosure) in a 2D structure.

[0047] Figures 2a to 2o show the yield and purity of the conjugates of anti-PD-L1 scFv and the modified fusion protein.

[0048] Figures 3a to 3c show the serum stability of conjugates of anti-PD-L1scFv and modified fusion proteins that bind to vascular endothelial growth factor, placental growth factor and PD-L1 (control: fusion proteins based on VEGF-Grab (H-12C08 or prototype) and PB101 (VEGF-Grab).

[0049] Figures 4a to 4k show the binding affinity of anti-PD-L1 scFv and its modified fusion protein conjugates to VEGF-A, PlGF, and PD-L1 (control: prototype conjugates based on anti-PD-L1 scFv and VEGF-Grab).

[0050] Figures 5a to 5f illustrate the simultaneous binding affinity of anti-PD-L1scFv and modified fusion protein conjugates to multiple targets, which bind to vascular endothelial growth factor, placental growth factor, and PD-L1.

[0051] Figures 6a to 6o are diagrams at the cellular level illustrating the inhibitory effect of the presently disclosed anti-PD-L1 scFv and modified fusion protein conjugate (PB203) bound to vascular endothelial growth factor, placental growth factor and PD-L1 on VEGF-A and PD-L1 signal transduction (controls: VEGF-Grab / PB101; H-12C18; H-30D01; Tecentriq; anti-PD-1 antibody; modified fusion protein / backbone / PB102).

[0052] Figures 7a to 7r are graphs showing the isoelectric point measurements of the conjugates according to this disclosure (reference: PB101 / PD20A01).

[0053] Figures 8a to 8d are graphs showing the thermal stability analysis of the conjugates according to this disclosure.

[0054] Figures 9a and 9b are PK analysis diagrams of the conjugates according to this disclosure.

[0055] Figure 10 This is a graph illustrating the tumor growth inhibition efficacy of the presently disclosed anti-PD-L1 scFv and modified fusion protein conjugates, which bind to vascular endothelial growth factor, placental growth factor, and PD-L1, in a mouse model. Detailed Implementation

[0056] The various implementations or embodiments described herein are merely exemplary and illustrative, and do not limit the technical concept of this disclosure. The technical concept of this disclosure encompasses various modifications, equivalents, and alternatives to each implementation or embodiment described herein, as well as selective combinations of all or part of individual implementations or embodiments.

[0057] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0058] Unless the context otherwise requires, the use of the singular in this document may include the plural, and this also applies to the singular expressions listed in the claims.

[0059] I. Definition In this disclosure, the term "about" can refer to a typical range of error for a particular value listed, as is well known to those skilled in the art. When used in the context of the numerical values ​​or ranges described in this disclosure, the term can mean ±20%, ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% of the numerical values ​​or ranges listed or described in a particular embodiment or claim. Regarding the length of a nucleotide or amino acid sequence, "about" can indicate that the nucleic acid or protein is not limited to the number of nucleotides or amino acids listed, and can include cases where several nucleotides or amino acids are added to or removed from either end, provided that functional activity is not impaired.

[0060] As used herein, expressions such as “comprising,” “including,” and “having” should be interpreted as open-ended terms implying the possibility of including other implementations in a manner similar to “comprising,” unless otherwise stated in the phrase or sentence containing these expressions.

[0061] As used herein, with respect to items associated with a term, the term “and / or” may mean any one or more items in a project, any combination of items, or all items.

[0062] As used herein, the term "amino acid" can refer to all naturally occurring L-α-amino acids. This definition is intended to include ortholeucine (Nle), ornithine, and homocysteine.

[0063] As used herein, the term “variation” or “amino acid variation” may refer to a substitution, insertion, deletion or combination thereof in the amino acid sequence of a reference (e.g., native sequence) polypeptide or protein, and the term “variant” may refer to a molecule whose amino acid sequence differs from that of a reference polypeptide or protein due to such variation.

[0064] The range of variants in this article also includes proteins or fragments or derivatives thereof that exhibit the same or similar biological activities, as well as derivatives that have been modified in various ways during or after translation, such as by glycosylation, proteolytic cleavage, or linkage with antibody molecules or other cellular ligands.

[0065] As used herein, the term "carrier" can include pharmaceutically acceptable carriers, excipients, or stabilizers that are non-toxic to cells or mammals at the doses and concentrations employed. Typically, pharmaceutically acceptable carriers are pH-buffered aqueous solutions. Examples of pharmaceutically acceptable carriers include buffers such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid; low molecular weight (less than about 10 residues) peptides; 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 ions; and / or nonionic surfactants such as TWEEN®, polyethylene glycol (PEG), and PLURONICS®, but not limited to these.

[0066] As used herein, the term "effective amount" can refer to an amount sufficient to achieve a beneficial or desired clinical or biochemical outcome. An effective amount can be administered as a single or multiple dose. An effective amount can also refer to an amount sufficient to reduce, improve, stabilize, slow, or delay the progression of a disease state.

[0067] As used herein, the term "ligand" can refer to a molecule (e.g., VEGF-A, VEGF-B, VEGF-C, VEGF-D, and PlGF) that binds with high affinity to a modified fusion protein. In other contexts, "ligand" can refer to any molecule or agent, or compound, that can specifically bind covalently or transiently to a molecule, such as a polypeptide or protein.

[0068] As used herein, the terms "individual" or "subject" may refer to a mammal. Such mammals include, but are not limited to, livestock (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., human 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.

[0069] As used herein, the term “pharmaceutically acceptable carrier and / or diluent” may include, but is not limited to, any and all solvents, dispersion media, coating agents, antibacterial and antifungal agents, isotonic agents, and adsorption delay agents.

[0070] The embodiments described herein should be understood to encompass "comprising", "consisting of", and / or "substantially consisting of".

[0071] II. Modified fusion protein and its components 1. Vascular endothelial growth factor receptor (VEGFR) In one embodiment, the modified fusion protein of this disclosure may include a VEGFR1 extracellular domain as a component. VEGFR1 is also known as fms-related tyrosine kinase (FLT-1) and is composed of... FLT1 The gene encodes VEGFR1. The amino acid sequence of VEGFR1 is shown in Table 2, which can be found in 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 sequentially from the N-terminus to the C-terminus of the extracellular region; the same applies to VEGFR2 and VEGFR3.

[0072] In one embodiment, the extracellular domain of VEGFR1 may include Ig-like domains D2 and D3. As used herein, the term "Ig-like domain D2" for VEGFR1 refers to a second Ig-like domain found at the N-terminus of the extracellular domain of VEGFR1, and "Ig-like domain D3" for VEGFR1 refers to a third Ig-like domain found at the N-terminus of the extracellular domain of VEGFR1; however, these terms cover the corresponding variants as long as the function of these domains (e.g., binding VEGF ligands and / or inhibiting activation of the VEGFR pathway) is maintained. Because the amino acid sequences of active proteins can differ between species, the Ig-like domains D2 and D3 of VEGFR1 are not limited by their origin or sequence and may include wild-type or active variants thereof.

[0073] In one embodiment, the modified fusion protein of this disclosure may further include other VEGFR extracellular domains in addition to the Ig-like domains D2 and D3 of VEGFR1.

[0074] While it is undesirable to be bound by theory, it is generally believed that the extracellular domain of VEGFR inhibits the activation of the VEGF pathway by binding to VEGF ligands, thereby blocking the interaction between VEGF ligands and VEGFR. Furthermore, while it is undesirable to be bound by theory, it is generally believed that the extracellular domain of VEGFR can bind to VEGFR to achieve a dominant-negative inhibition of the VEGF signaling pathway. In one embodiment, the extracellular domain of said VEGFR is capable of binding to one or more VEGF ligands selected from the group consisting of VEGF-A, VEGF-B, VEGF-C, VEGF-D, and P1GF.

[0075] In one embodiment, the VEGFR extracellular domain may or may not contain a signal peptide, which may serve as a signal sequence secreted from a host cell as the VEGFR extracellular domain or a modified fusion protein containing the same domain. The signal peptide may be operatively linked to a nucleic acid encoding a protein of interest (e.g., the extracellular domain of VEGFR1).

[0076] 2. Connector The components of a modified fusion protein (e.g., the extracellular or polymerized domains of VEGFR1) can be linked by linker portions (such as peptide linkers). These linkers increase the flexibility of the fusion protein components without significantly interfering with the structure of each functional component within the fusion protein.

[0077] In one embodiment, the linker can be used to link the C-terminus of the extracellular domain of VEGFR1 (e.g., the C-terminus of the Ig-like domain D3) to the N-terminus of the polymerized domain (e.g., the IgG1 Fc region).

[0078] In one embodiment, the linker may include an amino acid sequence derived from the hinge region of an immunoglobulin. The hinge region derived from the immunoglobulin may include a hinge region located in the N-terminal region of the Fc region of an immunoglobulin. In one embodiment, the hinge region derived from the immunoglobulin may be derived from human IgD or IgG.

[0079] 3. Polymerized structural domains This disclosure provides a multimerizing domain (e.g., the Fc region of an immunoglobulin), which can be a component of any modified fusion protein. The multimerizing domain is part of a multimeric protein that promotes subunit association to form, for example, dimers, trimers, tetramers, etc. As used herein, the term "multimerizing domain" can refer to a dimerizing domain, a trimerizing domain, a tetramerizing domain, etc. A fusion protein containing a multimerizing domain can interact with another fusion protein containing a multimerizing domain to produce a fusion protein multimer (e.g., a fusion protein dimer). For example, the IgG Fc region is a dimerizing domain that can fuse with the extracellular domain of VEGFR1 as described herein. A fusion protein containing the extracellular domain of VEGFR1 and the IgG Fc region can dimerize with another fusion protein containing the IgG Fc region to form a fusion protein dimer that can simultaneously bind to VEGF ligands and PlGF ligands.

[0080] In one implementation, the Fc region may be partially glycosylated or completely unglycosylated.

[0081] In one embodiment, the Fc region may have amino acid substitutions based on SEQ ID NO: 60, such as T20Q, D126E, L128M, M198L, and / or the deletion of amino acid K217. These amino acid variations can enhance the half-life of the fusion protein through interaction with FcRn. These amino acid variations can occur in the same manner in other immunoglobulin Fc regions having sequences corresponding to the amino acid sequence of SEQ ID NO: 60, and the modified fusion protein of this disclosure may include another immunoglobulin Fc region with amino acid variations as a polymerizing domain component.

[0082] In one embodiment, the Fc region may have amino acid substitutions based on SEQ ID NO: 60, including L4A, L5A, H38Q, K44Q, Y66F, A97G, A100S, P101S, R125Q, D126E, L128M, K179R, Q189E, P215L, and / or the deletion of K217. This amino acid variation eliminates the effector function of the Fc region. The amino acid variation can also be present in other immunoglobulin Fc regions having sequences corresponding to the amino acid sequence shown in SEQ ID NO: 60, and the modified fusion protein of this disclosure may include another immunoglobulin Fc region having the aforementioned amino acid variation as a polymerizing domain component.

[0083] The properties of the modified fusion protein according to this disclosure are described below.

[0084] 4. Characteristics of surface charge transition variation In one embodiment, the modified fusion protein of this disclosure may possess the property of surface charge transition variation. In this disclosure, the modified fusion protein possessing the property of surface charge transition variation may be used interchangeably with "surface charge transition variant" or "surface charge variant".

[0085] In the surface charge transition variation characteristic according to one embodiment, the modified fusion protein may undergo amino acid substitution via the substitution of K241E, L243S, R244V, and H246E on the β1-β2 ring of the Ig-like domain D3, the substitution of L258A, L258S, or L258D on the β2-β3 ring of the domain D3, and / or the substitution of K300G, Q302T, and K304S on the β5-β6 ring of the domain D3. These amino acids reduce the protein's net pI, have negatively charged side chains, or have electrostatically negatively charged side chains. In one embodiment, an amino acid with an electronegative side chain may refer to an amino acid that becomes electronegative after substitution, depending on the properties of adjacent amino acid residues or the molecule, and may refer to a relative concept arising from the intramolecular electron distribution. For example, an amino acid residue containing a hydroxyl group (-OH) may become electronegative due to the lone pair of electrons present on the oxygen atom, and this phenomenon can reduce the protein's net pI.

[0086] In one embodiment, the β1-β2 loop of the Ig-like domain D3 may include amino acid residues T236 to T247 of the VEGFR1 amino acid sequence in Table 2. In one embodiment, the β2-β3 loop of the Ig-like domain D3 may include amino acid residues T256 to V262 of the VEGFR1 amino acid sequence in Table 2. In one embodiment, the β5-β6 loop of the Ig-like domain D3 may include amino acid residues D299 to L308 of the VEGFR1 amino acid sequence in Table 2. In this disclosure, the β1-β2 loop may be referred to as "site 1", the β2-β3 loop may be referred to as "site 2", and the β5-β6 loop may be referred to as "site 3". The inventors of this disclosure have determined that amino acid residues present in the loops are exposed on the surface of domain D3, and that surface charge and structural stability of the modified fusion protein have been achieved through variations in the amino acid residues present in the loops.

[0087] In one embodiment, the amino acid residues present at sites 1 and 3 influence each other through interactions between amino acid residues due to their structural positions, thus allowing the introduction of double, triple, or quadruple amino acid variations (e.g., amino acid substitutions) at each of the sites.

[0088] In one embodiment, the three rings should be understood to encompass amino acid sequence variants that maintain their function. For example, in one embodiment, the β1-β2 ring, the β2-β3 ring, and the β5-β6 ring may respectively comprise amino acid sequences having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequences TPRPVKLLRGHT, TPLNTRV, and DKMQNKDKGL.

[0089] In the linker variation characteristics according to one embodiment, one or more amino acid residues on the β1-β2 ring of the Ig-like domain D3 and one or more amino acid residues on the β5-β6 ring of the domain D3 of the modified fusion protein may have the following substitutions: amino acids that reduce the net pI of the protein, amino acids with negatively charged side chains, or amino acids with electronegative side chains.

[0090] 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 can be generated by inserting a nucleic acid sequence or nucleic acid molecule encoding the fusion protein into a vector from various sources. In this case, depending on the type or source of the vector into which the nucleic acid sequence or molecule is inserted, variations may occur in the amino acid sequence at the N-terminus of the Ig-like domain D2, where the N-terminus begins with an amino acid sequence derived from the vector (e.g., amino acid sequence EF) instead of G132. However, such variations in the N-terminal sequence due to the vector-derived amino acid sequence may not affect the yield or physicochemical properties of the modified fusion protein of this disclosure.

[0091] 5. Linker variation characteristics In one embodiment, the modified fusion protein of this disclosure may have a linker variant as a characteristic. In this disclosure, the modified fusion protein having the linker variant characteristic is interchangeable with "linker variant". Due to the linker variant characteristic, (i) the modified fusion protein of this disclosure can closely mimic the structure of ligand binding of domains D2 and D3 in the natural VEGFR1 extracellular domain, (ii) domain D3 in the natural VEGFR1 extracellular domain can have a length extending to the dimer-forming site of domain D4 as a structural characteristic required to ensure successful ligand binding, and (iii) the linker itself can have excellent structural stability.

[0092] In the linker variation characteristics according to one embodiment, the length of the linker can be from about 14 to about 35 amino acids.

[0093] In the linker variation characteristics according to one embodiment, the linker may include a GS repeat sequence.

[0094] In the linker variation characteristics according to 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.

[0095] In the linker variation characteristics according to one embodiment, the linker may include an amino acid sequence derived from the hinge region of an immunoglobulin. In one embodiment, the amino acid sequence of the hinge region may be modified.

[0096] In this disclosure, the linker is located between the C-terminus of the VEGFR1 domain D3 and the Fc region of the human immunoglobulin produced by papain digestion, and may refer to the entire region extending between P243 at the N-terminus of the CH2 region (A244-K360) and the C-terminus of domain D3. The positions of amino acid residues in the Fc region and the CH2 region included in the Fc region follow the Kabat numbering scheme (Kabat et al., Immunological Protein Sequence, 5th Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, 1991). In one embodiment, the linker may include certain regions derived from the human immunoglobulin, and said regions may be an amino acid sequence of CH1 (K218-V223) or the hinge region (E226-P243), or a portion of such an amino acid sequence.

[0097] In this disclosure, when a linker contains an amino acid sequence derived from the hinge region of an immunoglobulin, the properties of the included hinge region are retained in the linker. For example, the hinge region of the Fc region has cleavage sites for various proteases and can induce oligomerization via O-glycosylation on Ser / Thr amino acids after introduction into the fusion protein (Song et al., 2020. *Computational Struct Biotechnol J.* 18: pp. 3925-3935). In modified fusion proteins having the linker variation properties of this disclosure, the amino acid sequence of the immunoglobulin-derived hinge region is modified to achieve excellent structural stability of the linker itself and improve its physicochemical properties.

[0098] In one embodiment of the linker variation characteristics, the immunoglobulin-derived hinge region may include sequences derived from the CH1 region, the upper hinge, and / or the core hinge. In one embodiment, certain amino acid sequences of the immunoglobulin-derived CH1 region may form a β-sheet or β-sheet flap. In one embodiment, the immunoglobulin-derived hinge region may be derived from human IgD, IgG, or a combination thereof.

[0099] In one embodiment, when the hinge region is derived from IgG1, the amino acid sequence KTHT of the papain recognition site with the variant can be modified to an amino acid sequence selected from the group consisting of: KTYT, TPP, ATPT, and ATPPTCP.

[0100] In the linker variation characteristics according to one embodiment, the linker may include, from the N-terminus to the C-terminus, (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, and the amino acid sequence of the hinge region may be modified.

[0101] In this disclosure, the hinge region derived from IgD / G1 can refer to a combination of the hinge sequences of IgD and IgG1. Specifically, the hinge region derived from IgD / G1 can be NTGSGGEEKKKEKEKEEQE ERSSDKTHTCPPCP (SEQ ID NO: 76). In one embodiment, the hinge sequence of IgD can be NTGSGGEEKKKEKEKEEQEERSS (SEQ ID NO: 77), NTGRGGEEKKKEKEKEEQEER (SEQ ID NO: 78), or a variant thereof. In one embodiment, the hinge sequence of IgG1 can be DKTHTCPPCP (SEQ ID NO: 79), or a variant thereof.

[0102] In the linker variation characteristics according to one embodiment, the linker may be an amino acid sequence selected from the group consisting of: CSSGDATPTSPPSP (SEQ ID NO: 68), CSKVDKKVE PKSSDTPPTCPPCP (SEQ ID NO: 69), CSGGGGSAEPKAGDATPPTCPPCP (SEQ ID NO: 70), CSGGGGSGGGGSGGGSAESKYGPPCPPCP (SEQ ID NO: 71), CSNTGSGGEEKKKEKEKEEQEERSSDTPPTCPPCP (SEQ ID NO: 72), CSNTGSGGEEKKKEKEKEEQEERSCDTPPTCPPCP (SEQ ID NO: 73), CGSS GGSSGEPKSDATPTCPPCP (SEQ ID NO: 74) and CSGGGGSAEPKAGDATPP TCPPCPPCP (SEQ ID NO: 75).

[0103] 6. Disulfide bond variation characteristics In one embodiment, the modified fusion protein of this disclosure may possess the property of disulfide bond variation. In this disclosure, the modified fusion protein possessing the disulfide bond variation property is interchangeable with "disulfide bond variant." Since the modified fusion protein of this disclosure is a fusion of heterologous proteins, its stability is expected to decrease at the sites where different types of proteins are linked. Therefore, the disulfide bond variation property is introduced to prevent physical cleavage of the modified fusion protein and improve its stability.

[0104] To introduce disulfide bond variation properties, candidate amino acid residues were selected using a tertiary structure energy prediction program (FoldX, Schymkowitz et al. in YASARA, 2005, Nucleic Acids Research, 33: W382-388), and these amino acid residues were substituted with cysteine ​​residues to determine whether disulfide bond formation could improve the properties of the modified fusion protein (e.g., yield, physicochemical properties, ligand binding) (see Example 2).

[0105] In one embodiment, for the disulfide bond variation characteristic, (i) one amino acid residue on the β1-β2 ring of the Ig-like domain D3 or one amino acid residue on the β5-β6 ring of the Ig-like domain D3 and (ii) the amino acid residue at position +2 relative to Y329 of the Ig-like domain D3 can be replaced by a cysteine ​​residue. Through this substitution, the modified fusion protein of this disclosure can have disulfide bonds in the protein. In one embodiment, the amino acid residues present at the positions described in (i) and (ii) above for introducing the disulfide bond variation characteristic can be selected according to the above criteria (1) to (3).

[0106] 7. Fusion proteins In one embodiment of this disclosure, a modified fusion protein capable of simultaneously binding to both VEGF ligands and PlGF ligands can be provided. In one embodiment, the modified fusion protein may have a first binding specificity for VEGF ligands and a second binding specificity for PlGF ligands. Here, the VEGF ligand may be one or more of VEGF-A, VEGF-B, VEGF-C, and VEGF-D, and may specifically be VEGF-A.

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

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

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

[0110] (a) The amino acid substitutions K241E, L243S, R244V and H246E are introduced on the β1-β2 ring of the domain D3; the amino acid substitutions L258A, L258S or L258D are introduced on the β2-β3 ring of the domain D3; and / or the amino acid substitutions K300G, Q302T and K304S are introduced on the β5-β6 ring of the domain D3; (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), CSNTGSGGEEKKKEKEEQEERSSDTPPTCPPCP (SEQ ID NO: 72), CSNTGSGGEEKKKEKEKEEQEERSCDTPPTCPPCP (SEQ ID NO: 73), CGSSGGSSGEPKSDATPTCPPCP (SEQ ID NO: 74) and CSKVDKKVEPKSSDKTYTCPPCP (SEQ IDNO: 75); and (c) The L243 amino acid residue on the β1-β2 ring of the domain D3 is replaced by cysteine, and the amino acid residue at position +2 relative to Y329 of the domain D3 is replaced by cysteine.

[0111] In one embodiment, the modified fusion protein may comprise an amino acid sequence selected from the group consisting of amino acid sequences selected from C61 to C75 of Table 2. In one embodiment, the modified fusion protein may consist of an amino acid sequence selected from the group consisting of amino acid sequences selected from C61 to C75 of Table 2.

[0112] In one embodiment, the modified fusion protein may have at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with an amino acid sequence selected from the group consisting of amino acid sequences from C61 to C75 of Table 2. Such variants may contain amino acid variations (e.g., conserved amino acid substitutions) in additional amino acid sequences while retaining or maintaining the amino acid variations defined in the properties of the modified fusion protein of this disclosure.

[0113] In one embodiment, the components of the modified fusion protein of this disclosure (VEGFR1 extracellular domain, linker, and polymerization domain) may have post-translational modifications, including, for example, glycosylation, sialylation, acetylation, and phosphorylation.

[0114] In one embodiment, the modified fusion protein of this disclosure may contain conserved amino acid substitutions at locations other than those defined in the characteristics described herein. These conserved amino acid substitutions may be introduced into any individual component of the modified fusion protein of this disclosure (e.g., the VEGFR1 extracellular domain, linker, and polymerization domain).

[0115] In one embodiment, the conserved substitutions that may be included in the modified fusion protein of this disclosure are shown in Table 0 below. Through such conserved substitutions, the modified fusion protein of this disclosure can obtain the desired activities, such as maintaining / enhancing binding affinity to the target, reducing immunogenicity, or improving serum stability / thermal stability.

[0116] [Table 0] In one embodiment, the modified fusion protein of this disclosure may be as disclosed in C61 to C75 of Table 2.

[0117] 8. Conjugates of fusion proteins and scFv In one embodiment of this disclosure, a conjugate of a modified fusion protein and an scFv according to embodiments of this disclosure may be provided. In one embodiment, the scFv may be a PD-L1-targeting scFv, and may specifically be an atezolizumab scFv or an 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 embodiments of this disclosure may be referred to as PB203.

[0118] In one embodiment, the modified fusion protein and scFv conjugate of this disclosure may be as disclosed in SEQ ID NO: 1 to 32 of Table 9.

[0119] In one embodiment, the conjugate may have at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with an amino acid sequence selected from the group consisting of amino acid sequences listed in Table 9. In such variations, while including or maintaining the amino acid variations shown in the properties of the conjugates of this disclosure, other amino acid variations (e.g., conserved amino acid substitutions) may be included.

[0120] In one embodiment, the components of the conjugate of this disclosure (scFv, VEGFR1 extracellular domain, linker, and polymerization domain) may have post-translational modifications, including, for example, glycosylation, sialylation, acetylation, and phosphorylation.

[0121] In one embodiment, the conjugates of this disclosure may contain conserved amino acid substitutions at positions other than the amino acid variants described herein. Such conserved amino acid substitutions may be introduced into any component of the modified fusion protein of this disclosure (e.g., scFv, the extracellular domain of VEGFR1, linkers, and polymerization domains).

[0122] In one embodiment, the conjugate of this disclosure may comprise a signal peptide or signal sequence for secreting the protein from cells. For example, the conjugate of this disclosure may further comprise a heterologous peptide, specifically a signal sequence or other peptide having a specific cleavage site at the N-terminus of the mature fusion protein. In one embodiment, the heterologous signal sequence may be a sequence that can be recognized and processed by eukaryotic host cells (i.e., cleaved by a signal peptide hydrolase). Information regarding signal peptides is well known in the art, for example, as described in Korean Patent 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 the following amino acid sequences: 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 those skilled in the art to generate fusion proteins may be used without limitation.

[0123] In one embodiment, the IC50 of the conjugates of this disclosure for inhibiting ligand activity (e.g., inhibiting VEGF activity, PD-L1 activity, or PlGF activity) 50 It can be less than or equal to about 1 mg / ml, 500 ng / ml, 300 ng / ml, 100 ng / ml, 70 ng / ml, 50 ng / ml, 45 ng / ml, 40 ng / ml, 35 ng / ml, 30 ng / ml, 25 ng / ml, 20 ng / ml, 10 ng / ml, 5 ng / ml, 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 (including the extreme values, and any value between these values).

[0124] In one embodiment, the conjugate of this disclosure may have a Kd for binding a partner (e.g., VEGF, PD-L1, and / or PlGF) that is less than any of the following: 1.0 mM, 500 μM, 100 μM, 50 μM, 25 μM, 10 μM, 5 μM, 1 μM, 900 nM, 800 nM, 700 nM, 600 nM, 500 nM, 400 nM, 350 nM, 300 nM, 250 nM, 200 nM, 150 nM, 100 nM, 95 nM, 90 nM, 85 nM, 80 nM, 75 nM, 70 nM, 65 nM, 60 nM, 55 nM, 50 nM, 45 nM, 40 nM, 35 nM, 30 nM, 25 nM, 20 nM, 15 nM, 10 nM, 5 nM, 1 nM, 900 pM, 800 pM, 700 pM, 600 pM, 500 pM, 400 pM, 300 pM, 200 pM, 100 pM, 50 pM, 25 pM, 12.5 pM, 6.25 pM, 5 pM, 4 pM, 3 pM, 1 pM, 0.5 pM, 0.1 pM, 0.05 pM, or 0.01 pM (including end values, and any value between these values).

[0125] III. Nucleic Acids, Vectors, and Host Cells Nucleic acid In one embodiment of this disclosure, isolated nucleic acids encoding the modified fusion protein, scFv, and any component of the modified fusion protein conjugate with scFv (such as anti-PD-L1 scFv, VEGFR1 extracellular domain, and / or polymerizing domain) of this disclosure may be provided. Nucleic acids encoding mammalian VEGFR have been described for all receptor types. Exemplary nucleic acid sequences can be found in, but are not limited to, U.S. Patent Nos. 7,928,072 and WO 2006 / 113277. mRNAs encoding human VEGFR1 and VEGFR2 can be found under GenBank accessions NM_002019.4 and NM_002253.2, respectively.

[0126] This disclosure may provide isolated nucleic acids encoding multi-merging domains (e.g., Fc regions).

[0127] It can provide isolated nucleic acids encoding the modified fusion proteins disclosed herein.

[0128] The isolated nucleic acid sequence encoding the modified fusion protein or a component of the fusion protein disclosed herein (e.g., the extracellular domain or polymerized domain of VEGFR1) may further include a nucleic acid sequence encoding a linker.

[0129] In one embodiment, the isolated nucleic acid may further include a sequence encoding a signal peptide, which serves as a signal sequence for secreting the modified fusion protein from a host cell. In another embodiment, the isolated nucleic acid may not include a sequence encoding a signal peptide.

[0130] The isolated nucleic acid molecules encoding the modified fusion protein or components of a fusion protein (e.g., the extracellular domain, linker, or polymerization domain of VEGFR1) disclosed herein may be in the form of RNA, such as mRNA, hnRNA, tRNA, or any other form, or in the form of DNA, including but not limited to cDNA and genomic DNA obtained by cloning or synthesized, or any combination thereof. Such isolated nucleic acid molecules can be prepared by a variety of methods known in the art (see Molecular Cloning: A Laboratory Manual). Molecular Cloning : A Laboratory Manual (Sambrook et al., 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2012) and *Laboratory Guide to Molecular Biology* (Sambrook et al., 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2012) Current Protocols in Molecular Biology (FM Ausubel et al., eds., 2003).

[0131] carrier In one embodiment, this disclosure relates to a nucleic acid delivery medium or its use for introducing one or more nucleic acid sequences encoding a conjugate or a component of a conjugate into a cell for expression of the protein. The nucleic acid sequence may be a sequence of the isolated nucleic acid described above.

[0132] In one embodiment, examples of nucleic acid delivery agents include liposomes, biocompatible polymers (including natural and synthetic polymers); lipoproteins; polypeptides; polysaccharides; lipopolysaccharides; artificial viral envelopes; metal particles; and bacterial, viral (such as baculoviruses, adenoviruses, and retroviruses), bacteriophages, granules, plasmids, fungal vectors, and other recombinant agents commonly used in the art for expression in various eukaryotic and prokaryotic hosts. In one embodiment, the nucleic acid delivery agent may be an expression vector, such as a plasmid. In one embodiment, the nucleic acid sequence contained in the expression vector may be operatively linked to an expression control sequence.

[0133] In one embodiment, the vector may contain any elements for realizing the conventional functions of the expression vector, such as a promoter, ribosome-binding element, terminator, enhancer, selection marker, and origin of replication. The promoter may be a constitutive promoter, an inducible promoter, or a repressive promoter. An exemplary promoter is provided in Korean Patent No. 10-0659477, but is not limited thereto.

[0134] Various expression vectors capable of delivering nucleic acids into cells (e.g., derived from bacterial, yeast, plant, or mammalian cells) are known in the art and can be used herein to generate conjugates or conjugate components in cells. For example, if *E. coli* ( E. coli ) were transferred into engineered nucleic acids containing coding conjugates (such as pBR322) (Mandel et al., Journal of Molecular Biology) J. Mol. Biol. According to [reference needed], 1970, 53:154), the *E. coli* can be used to produce conjugates. The expressed conjugates or conjugate components can be harvested from cells and purified according to conventional techniques known in the art and the methods described in this disclosure.

[0135] In one embodiment, an expression vector containing the nucleic acid encoding the conjugate described above and capable of replicating in a bacterial or eukaryotic host can be used to transfect the host, thereby directing the expression of the nucleic acid to produce the conjugate, which can then be recovered in a biologically active form. As used herein, the biologically active form includes forms capable of binding to VEGF ligands, PD-L1 ligands, or PlGF ligands.

[0136] In one embodiment, an expression vector containing a nucleic acid molecule encoding a conjugate or a component of a conjugate can be identified by, but is not limited to, at least three common methods: (a) DNA-DNA hybridization, (b) the presence or absence of a “marker” gene function, and (c) expression of the inserted sequence. Details of these three methods are provided in Korean Patent Nos. 10-0659477 and 10-2228921, but are not limited thereto.

[0137] host cells In one embodiment of this disclosure, a host cell containing an expression vector encoding a conjugate of this disclosure may be provided. Such host cells are suitable for expressing the conjugates of this disclosure and may constitute a host-vector system for producing said conjugates. In one embodiment, the host cell may be used to produce viral particles (e.g., recombinant viral vectors).

[0138] As used herein, the term "host cell" can include the cell that is or may be the recipient of a vector of this disclosure and its progeny cells. Due to natural, accidental, or artificial mutations, progeny cells (morphologically or in the total DNA complementary genome) are not necessarily identical to the original parent cell. In one embodiment, the host cell can be a bacterial cell (such as *Escherichia coli*), a yeast cell (such as *Pichia pastoris*), or a blastocystis jirovecii. Pichia pastoris ), insect cells (such as fall armyworm ( Spodoptera frugiperda () or mammalian cells (such as COS, HEK or CHO cells), but not limited to these.

[0139] In one embodiment, the conjugates of this disclosure can be expressed transiently, constitutively, or permanently in a host cell.

[0140] In one embodiment of this disclosure, a method for generating the conjugates of this disclosure may be provided, the method comprising growing host cells or cells of the host-vector system described above under conditions allowing for the generation of the conjugates, and then recovering the generated conjugates. The conjugates for carrying out this disclosure can be generated by expression in a prokaryotic or eukaryotic expression system. Methods for culturing host cells or generating conjugates from host cells are well known in the art, for example, as disclosed in Korean Patent No. 10-2228921, but are not limited thereto.

[0141] In one embodiment, the conjugates of this disclosure produced from the host cells described above can be purified and identified by a variety of methods. Methods for purifying and identifying conjugates produced from host cells are well known in the art, for example, as provided in Korean Patent No. 10-2228921, but are not limited thereto. For further purification of the conjugates, any number of purification methods can be used, including but not limited to conventional ion-exchange chromatography, affinity chromatography, different sugar chromatography, hydrophobic interaction chromatography, reversed-phase chromatography, or gel filtration.

[0142] IV. Viral particles and methods for generating viral particles In one embodiment of this disclosure, viral particles (or virions) comprising nucleic acids encoding conjugates of this disclosure may be provided.

[0143] Viral vectors can be used to deliver nucleic acids encoding conjugates or components of conjugates for expression of said proteins in target cells within a specific target tissue (e.g., diseased tissue). Many types of viruses are known, and numerous viruses have been studied for the purpose of delivering nucleic acids to target cells. Exogenous nucleic acids can be inserted into vectors (such as adenoviruses, partially deleted adenoviruses, completely deleted adenoviruses, adeno-associated viruses (AAVs), retroviruses, lentiviruses, etc.) for delivery to cells.

[0144] In one embodiment, the cells are located within an individual, and the virus can be delivered via intravenous, intramuscular, portal venous, or other routes of administration. In one embodiment, the viral vector may comprise a viral vector derived from adenovirus, adeno-associated virus (AAV), and retroviruses (including lentiviruses such as human immunodeficiency virus (HIV)). For exemplary viral vectors, see, but is not limited to, U.S. Patent No. 7,928,072 and WO 2006 / 113277, which are incorporated herein by reference in their entirety. Viral particles containing nucleic acids encoding conjugates and methods for generating said viral particles are well known in the art, for example, as disclosed in Korean Patent No. 10-2228921, but are not limited thereto.

[0145] V. Treatment methods using conjugates and viral particles In one embodiment, this disclosure relates to a pharmaceutical composition for preventing, improving, or treating at least one symptom of VEGF ligand, VEGF receptor, or PD-L1-related diseases or symptoms of said diseases, said pharmaceutical composition comprising a conjugate of this disclosure as an active ingredient.

[0146] In one embodiment, this disclosure relates to a method for preventing, improving, or treating at least one of VEGF ligand, VEGF receptor, or PD-L1-related diseases or symptoms of said diseases, the method comprising administering a conjugate of this disclosure.

[0147] In one embodiment, this disclosure relates to the use of the conjugates of this disclosure for the prevention, improvement or treatment of at least one symptom of VEGF ligand, VEGF receptor or PD-L1 related diseases or symptoms of said diseases.

[0148] In one embodiment, this disclosure relates to a conjugate of the present disclosure or a pharmaceutical composition comprising said conjugate in a method for preventing, improving or treating at least one of VEGF ligand, VEGF receptor or PD-L1 related diseases or symptoms of said diseases.

[0149] In one embodiment, the conjugate, in the form of a nucleic acid encoding a conjugate of this disclosure, can be used in a nucleic acid delivery medium or viral particle (e.g., a recombinant viral vector) for the prevention, improvement, or treatment of at least one symptom of VEGF ligand, VEGF receptor, or PD-L1-related disease or symptoms of said disease. In one embodiment, the nucleic acid delivery medium or viral particle can be used in gene therapy for the prevention, improvement, or treatment of at least one symptom of VEGF ligand, VEGF receptor, or PD-L1-related disease or symptoms of said disease. In one embodiment, any gene therapy method available in the art can be used according to this disclosure. In one embodiment, the nucleic acid is introduced into the cells before in vivo administration of the resulting recombinant cells. Cells incorporating nucleic acids for gene therapy purposes encompass all available cell types and include, for example, epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes, blood cells (e.g., T lymphocytes, B lymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, or granulocytes), various stem cells or progenitor cells such as hematopoietic stem cells or progenitor cells obtained from bone marrow, umbilical cord blood, peripheral blood, or fetal liver, etc., but are not limited thereto. In one embodiment, the cells used for gene therapy may be the patient's own cells.

[0150] 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 of the VEGF family (e.g., VEGF-A, VEGF-B, VEGF-C, and VEGF-D, PlGF, and PD-L1); (b) blocking the binding of VEGF family proteins to VEGF receptors and / or the binding of PD-L1 protein to PD-1 receptors; (c) inhibiting the activation of VEGF signaling pathways and / or PD-L1 signaling pathways; (d) preventing, improving, and / or treating diseases such as chronic infections, tissue allogeneic transplantation, eye diseases, autoimmune diseases, inflammatory diseases, neoplastic diseases, or cancer; (e) attenuating, inhibiting, or preventing the growth of tumors or cancer; and (f) inhibiting the metastasis of tumors or cancer. The activity of the conjugate may be measured in vivo and / or in vitro.

[0151] In one embodiment, the pharmaceutical composition may comprise a pharmaceutically acceptable carrier and / or diluent. In one embodiment, a pharmaceutically acceptable carrier may be a sterile liquid, such as water and oil, including oils of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, etc. Physiological saline and glucose solutions, polyethylene glycol (PEG) and glycerol solutions may also be used as liquid carriers, particularly for injectable solutions. Such pharmaceutical compositions may further comprise additional ingredients such as preservatives, buffers, isotonic agents, antioxidants and stabilizers, nonionic wetting or clarifying agents, thickeners, etc. The pharmaceutical compositions of this disclosure may be packaged in single-unit doses or in multiple-dose packages. These compositions are typically formulated into sterile and substantially isotonic solutions. Furthermore, the compositions may be formulated to have an osmotic pressure compatible with the aqueous humor and ophthalmic tissues. Such osmotic pressure values ​​typically range from about 200 millimoles per kilogram of water (“mOsm / kg”) to 400 mOsm / kg, or about 300 mOsm / kg. The osmotic pressure of the retina is believed to be approximately 283 mOsm / kg.

[0152] In one embodiment, the pharmaceutical composition may be a parenteral composition. It may be advantageous to prepare the parenteral composition in dosage units to ensure dosage consistency and ease of administration.

[0153] Disease or ailment In one implementation, VEGF ligand, VEGF receptor, or PD-L1-related diseases may include, but are not limited to, chronic infections, tissue allogeneic transplantation, autoimmune diseases (e.g., rheumatoid arthritis, multiple sclerosis, recurrent miscarriage due to immune response, or systemic lupus erythematosus), 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).

[0154] In one implementation, cancers may include prostate cancer, urethral cancer, penile cancer, breast cancer, lung cancer, esophageal cancer, small bowel cancer, colorectal cancer, rectal cancer, colon cancer, liver cancer, urinary system cancers (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, stomach cancer, endocrine cancer, head and neck cancer, thyroid cancer, parathyroid cancer, thyroid gland cancer, adrenal cancer, skin cancer (e.g., melanoma, squamous cell carcinoma of the skin (CSCC), squamous cell carcinoma of the head and neck (HNSCC)), and Hodgkin's disease. Cancer, bone cancer, lymphoma or myeloid hematopoietic system cancer, chronic or acute leukemia, head and neck cancer, nasopharyngeal carcinoma (NPC), glioblastoma, teratoma, neuroblastoma, adenocarcinoma, mesenchymal-derived cancer (e.g., fibrosarcoma or rhabdomyosarcoma), soft tissue sarcoma and carcinoma, choriocarcinoma, hepatoblastoma, central nervous system (CNS) tumors, primary central nervous system lymphoma, spinal cord tumors, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, or Wilm's tumor, but not limited thereto. The conjugates disclosed herein can be used as anti-PD-L1 and anti-angiogenic agents or anti-VEGF and anti-PD-L1 agents to improve, prevent, and / or treat cancer or cancer symptoms.

[0155] Application method and dosage In one embodiment of this disclosure, a method for delivering an effective amount of the conjugate to a subject can be provided. The conjugate can be delivered to the subject in the form of a composition. The conjugate can also be delivered to the subject via a nucleic acid delivery medium or viral particles (e.g., a recombinant viral vector), wherein the nucleic acid delivery medium or viral particles contain nucleic acids encoding the conjugate. In one embodiment of this disclosure, a composition comprising the conjugate or a nucleic acid delivery medium or viral particles (e.g., a recombinant viral vector) containing nucleic acids encoding the conjugate can be provided.

[0156] In one embodiment, the compositions of this disclosure can be administered to an individual via any route, including but not limited to: intravenous (e.g., via an infusion pump), intraperitoneal, intraocular, intraarterial, intrapulmonary, oral, inhalation, intravesical, intramuscular, intratracheal, subcutaneous, intraocular, intrathecal, percutaneous, transpleural, intra-arterial, local, inhalation (e.g., as a spray mist), mucosal (e.g., via nasal mucosa), subcutaneous, percutaneous, gastrointestinal, intra-articular, intracisional, intravenous, intracranial, intraurethral, ​​intrahepatic, and intratumoral. In one embodiment, the compositions of this disclosure can be administered via any conventional route, such as by infusion or bolus injection, by absorption via the epithelial or mucosal lining (e.g., oral mucosa, rectal mucosa, intestinal mucosa, etc.), and may also be administered with other bioactive ingredients. Administration can be systemic or local. In one embodiment, the compositions of this disclosure can be introduced into the central nervous system by any suitable means, including intraventricular injection and intrathecal injection, wherein intraventricular injection can be administered via an intraventricular catheter, for example, connected to a reservoir (such as the Ommaya reservoir).

[0157] In one embodiment, the optimal effective amount of the conjugate, the nucleic acid delivery medium encoding the conjugate, or the composition containing the conjugate can be determined empirically and may depend on the type and severity of the disease, the route of administration, disease progression and health status, and the individual's weight and body surface area. Such determinations are within the skill of a person of ordinary skill in the art.

[0158] In one embodiment, the conjugate may be administered, for example, at a dose of about 0.05 ng to about 20 mg per kilogram of body weight per day. In one embodiment, the dosage of the conjugate may be at least about 0.1 mg / kg, at least about 0.5 mg / kg, at least about 1.0 mg / kg, at least about 1.5 mg / kg, at least about 2.0 mg / kg, at least about 2.5 mg / kg, at least about 3.0 mg / kg, at least about 3.5 mg / kg, at least about 4.0 mg / kg, at least about 4.5 mg / kg, at least about 5.0 mg / kg, at least about 6.0 mg / kg, at least about 8.0 mg / kg, at least about 10.0 mg / kg, at least about 15.0 mg / kg, or about 20.0 mg / kg or less, about 17.0 mg / kg or less, about 14.0 mg / kg or less, about 11.0 mg / kg or less, about 9.0 mg / kg or less, about 7.0 mg / kg or less, about 5.5 mg / kg or less, about 5.0 mg / kg or less, about 4.5 mg / kg or less, or about 4.0 mg / kg. mg / kg or less, about 3.5 mg / kg or less, about 3.0 mg / kg or less, about 2.5 mg / kg or less, about 2.0 mg / kg or less, about 1.5 mg / kg or less, or about 1.0 mg / kg or less. In one embodiment, the dosage of the conjugate may be about 1.0 mg / kg to about 10.0 mg / kg or about 1.0 mg / kg to about 5.0 mg / kg.

[0159] In one embodiment, the amount of nucleic acid delivery medium (e.g., a recombinant viral vector) containing nucleic acid encoding the conjugate disclosed herein can be delivered at approximately 10 per dose. 4 To about 10 14 The titer of DNAse particle resistance (DRP) is applied to an individual. In one embodiment, the amount of nucleic acid delivery medium containing nucleic acid encoding the conjugate can be administered at approximately 10 per dose. 5 To about 10 13 Approximately 10 6 To about 10 12 Approximately 10 7 To about 10 11 Approximately 10 8 To about 10 10 Approximately 10 9 To about 10 10 Approximately 10 10 To about 10 11 Or about 10 11 To about 10 12 DRP is applied to individuals.

[0160] In one embodiment, a composition comprising a conjugate of the present disclosure or a nucleic acid delivery medium encoding said conjugate may be administered as a single daily dose, or the total daily dose may be administered in divided doses two, three, or four times daily. In one embodiment, a composition comprising a conjugate of the present 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, once every two weeks, once 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 medium, said nucleic acid delivery medium comprising a nucleic acid encoding a conjugate of the present disclosure, may be administered less frequently, 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.

[0161] Diagnosis or testing The conjugates disclosed herein can be labeled with detectable markers, such as radioisotopes, fluorescent markers, toxin markers, enzyme markers, chemiluminescent markers, or nuclear magnetic resonance contrast agents, to confirm ligand-receptor binding interactions. Applications to detection systems for chimeric molecules are also possible. Such detectable markers are well known in the art.

[0162] VI. Products and Reagent Kits In one embodiment, this disclosure relates to an article or kit containing a conjugate of this disclosure, a recombinant viral vector expressing the conjugate, or a composition containing the above components, and packaged in a suitable manner. In one embodiment, the suitable packaging is well known in the art and includes, but is not limited to, vials (e.g., sealed vials), containers, ampoules, bottles, wide-mouth bottles, flexible packaging (e.g., sealed Mylar or plastic bags). These articles may further be sterilized and / or sealed.

[0163] 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 required from a commercial and user perspective, such as other buffers, diluents, filters, needles, syringes, and a package insert (PI) containing instructions for performing any of the methods described herein. For example, in one embodiment, the kit may contain (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) any one or more of the following: buffers, diluents, filters, needles, syringes, and a package insert containing instructions for use.

[0164] The technical features and effects of this disclosure will be described in more detail below with reference to working embodiments. However, these embodiments are provided for illustrative purposes only to aid in understanding this disclosure, and the scope of this disclosure is not limited to the following embodiments.

[0165] Example 1 – Construction of expression vector The coding sequence (CDS) region of the conjugates designed in the following examples was codon-optimized to make it suitable for CHO production cells, and the conjugates were prepared in the final pcDNA3.1(+) vector (Invitrogen) by gene synthesis (Geneuniversal).

[0166] Example 2 – Design of Modified Fusion Proteins Example 2.1 – Preparation and Characterization of Surface Charge Transformation Variants To ensure the acquisition of a fusion protein with enhanced physicochemical properties and containing VEGFR receptor components, the inventors of this disclosure used a structure containing the entire extracellular domain of VEGFR1 (PDB entry 5t89) as a template to analyze the surface charge distribution of the protein and deduced the regions of high positive charge density and the locations of hydrophobic surface charges.

[0167] This analysis demonstrated that in the VEGFR1 domain D3, the side chains of amino acid residues in the β1-β2 loop (site 1) corresponding to amino acid residues T236 to T247, the β2-β3 loop (site 2) corresponding to amino acid residues T256 to V262, and the β5-β6 loop (site 3) corresponding to amino acid residues D299 to L308 are exposed on the surface of the fusion protein. Researchers attempted to stabilize the surface charge and structure of the fusion protein by altering the amino acid residues in the flexible loops exposed on the surface. For example, they attempted to replace the exposed amino acid residues with amino acid residues present at VEGFR2 homologous sites. Furthermore, the amino acid residues at site 2 are exposed on the protein surface on the side that binds to the ligand (VEGF or PlGF), and variations at these sites affect the binding affinity or solubility to the ligand due to the surface exposure of the residues.

[0168] Example 2.2 – Preparation and Characterization of Linker Variants The human VEGF receptor binds to ligands VEGF or PlGF via interactions with immunoglobulin-like domains D2 and D3. For fusion proteins using these domains D2 and D3, domains D2 and D3 are open before ligand binding and then form closed structures upon ligand binding. If the structure of domains D2 and D3 does not ensure sufficient space for ligand binding, or if interference occurs between constructs, the fusion protein's ligand binding affinity may be reduced. Therefore, the binding length and binding mode of domains D3 and the multimerizing domain in a fusion protein can influence the fusion protein's structure upon ligand binding and its ligand binding affinity.

[0169] Therefore, the inventors modified the length and type of linkers in the modified fusion protein of this disclosure to mimic and ensure the structural characteristics of the natural human VEGF receptor. Specifically, linkers comprising a polypeptide with a Gly-Ser repeat sequence, an amino acid sequence derived from the hinge region of IgG1, IgG4, or IgD, and variant sequences of such hinges are introduced. Such linkers are designed to increase physical stability by reducing the tension generated in the loop (linker) formed between the D3 domain and the polymerized domain (Fc region) during ligand binding.

[0170] Furthermore, the amino acid sequences of the hinge region derived from immunoglobulins possess multiple protease cleavage sites due to their inherent characteristics (Vlasak and Ionescu, 2011, *Monoclonal Antibodies (Mabs)* 3: pp. 253-263), and upon introduction into the fusion protein, they may undergo glycosylation (e.g., O-glycosylation), 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 further modified the amino acid sequences at protease cleavage sites (e.g., papain cleavage sites) and attempted to modify the glycosylated amino acid residues Ser or Thr. In addition, to increase the structural flexibility of the linker, they also attempted to replace cysteine ​​residues present in the core hinge sequence (e.g., CPCCPP) derived from the Fc hinge region. The modified fusion proteins according to embodiments of this disclosure exhibit excellent yields and superior physicochemical properties, as well as better ligand binding affinity.

[0171] Example 2.3 – Preparation and Characterization of Disulfide Bond Variants Since fusion proteins are conjugates of heterologous proteins, their stability is expected to decrease due to non-specific cleavage at the linker site. The terminal region of domain D3 forms a loop and may lack secondary structure, and because it is the site where it begins to link with the polymerized domain (a heterologous protein), it is expected to have weak stability. By introducing covalent disulfide bonds, the inventors aim to induce interdomain linkages near the linker site of the heterologous protein, thereby preventing physical breakage of the protein and improving its stability.

[0172] Based on three-dimensional structure analysis, sites in the fusion protein where disulfide bonds could be introduced were identified. Residues in the variable loop located in the terminal region of the adjacent domain D3 were selected as variant residues for disulfide bond formation, with side chains oriented towards the linker and the distance between the terminal atom of the side chain and the Cα atom of Y329 within 6 Å. After ensuring the three-dimensional structure of the fusion protein using the structural modeling program (Phyre2, Swiss Model), a structural model for introducing disulfide bonds was obtained by computer simulation of variations at the introduction sites. Subsequently, the energy level changes of the fusion protein due to the introduction of disulfide bonds were predicted using a three-dimensional structure energy prediction program (foldX in YASARA; Schymkowitz et al., 2005, Nucleic Acid Research 33: W382-388).

[0173] As a position that is physically close to the terminal region of domain D3 and can form a disulfide bond, amino acid residues on the β1-β2 or β5-β6 rings of domain D3 were selected, while for the terminal region of domain D3, amino acid residues at the +2 position relative to Y329 were selected.

[0174] Example 2.4 – Preparation and Characterization of Combinatorial Variants As shown in Table 2, combined variants of the fusion protein with the combined properties of the variants exhibiting superior properties in Examples 2.1 to 2.3 were prepared, and the properties of each variant are shown in Table 1.

[0175] [Table 1] Table 1 provides the identification codes, amino acid sequences, and SEQ ID NOs of the proteins described in this disclosure.

[0176] [Table 2] The surface charge variation positions or disulfide bond positions described in the following examples or tables refer to the positions and amino acid residues of variations based on the amino acid sequence of VEGF-Grab3 in Table 2. The physicochemical properties and binding affinity of the combined variants prepared by introducing variations at different positions as shown in Table 2 were evaluated. Table 3 shows the physicochemical property evaluation results of the variants prepared based on the VEGFR wild-type protein and the wild-type protein, and Tables 4 and 5 show the binding affinity evaluation results. Table 6 shows the physicochemical property evaluation results of the variants prepared based on VEGF-Grab3, and Tables 7 and 8 show the binding affinity evaluation results, where Table 7 shows the relative binding affinity evaluation results for VEGF-Grab3 (manufactured by Panolos Bioscience), and Table 8 shows the relative binding affinity evaluation results for VEGF-Grab3 (manufactured by Samsung Biologics). The relative binding affinity evaluations for VEGF-Grab3 (manufactured by Panolos Bioscience) were compared with those produced under the same conditions and batches as the variants.

[0177] [Table 3] [Table 4] [Table 5] [Table 6] [Table 7] [Table 8] Based on the results in Tables 3 to 8, it can be confirmed that the modified fusion proteins according to the embodiments of this disclosure exhibit excellent yields based on titer and protein elution results, and excellent physicochemical properties based on functional monomer content results. Furthermore, for example, based on the results of Kon et al., it has been confirmed that the modified fusion proteins also exhibit superior binding affinity to ligands. These results confirm that the properties introduced during the structural design of the modified fusion proteins in Examples 2.1 to 2.3 are effectively realized, and that these properties work together to exhibit a synergistic effect.

[0178] Example 3 – Formation of Conjugates Conjugates were designed by conjugating the fusion proteins C61 to C75 prepared in Example 2 above with atezolizumab that binds PD-L1 and A167 scFv. Figure 1 ).like Figure 1 As can be seen from the present disclosure, the fusion protein includes a VEGF decoy receptor comprising engineered VEGFR1 domains 2 and 3 capable of capturing VEGF-A and PlGF, and novel functional modules that can be attached to their N-terminus or C-terminus. Anti-PD-L1 scFv is attached to the N-terminus of the fusion protein of the present disclosure to prepare a conjugate according to the present disclosure.

[0179] [Table 9] In the configuration descriptions in Table 9, H and L represent the heavy and light chains of scFv; Cnn (where n is an integer) corresponds to the sequence names in Table 2 above; G4S signifies the GGGGS linker; the linkers connecting scFv and the fusion protein are V1, V2, and V3, where V1 has the sequence GGGGSGGGGSDT, V2 has the sequence GSAEPPKAGGGGSGGGGSGGGGS, and V3 has the sequence GSGGGGSGGGGSGGGGSGGGGS; C65H indicates that the hinge portion of the fusion protein uses the sequence from Table 2. C65 hinge region; G4H indicates that IgG4 is used instead of IgG1 in the hinge region of the fusion protein; G4Fc indicates that the IgG4 sequence with effector function removed; QL indicates that a mutation has been introduced to increase the binding affinity for FcRn, which is known to increase FcRn cycling and prolong half-life; Cys indicates that a Cys mutation (Ser→Cys, at position 494 for C65 and at position 493 for C75) has been introduced into the fusion protein; and sur1 indicates a variant with a partial surface charge variation (reverse mutation).

[0180] ExpiCHO for generating conjugates for design TM Cells (Gibco) were used for transient expression at passage numbers of 10 or more and 20 or fewer. ExpiCHO was administered at 3 or 4-day intervals. TM Cell passaging; for 3-day cultures, cells were cultured at a rate of 0.3 × 10⁻⁶. 6 Cells were passaged at a density of 10 cells / mL, and for 4 days of culture, cells were cultured at a density of 0.15 × 10⁶ cells / mL. 6 Cells were passaged at a density of [number] cells / mL. Cell density and viability were measured using a Countess II (Ingenieur) instrument via trypan blue staining. Maximum titer culture was performed using the maximum titer protocol and Expifectamine supplied by the manufacturer. TM Transient expression of the conjugate was performed using Gibco. The day before transfection, at a concentration of 3.5 × 10⁻⁶... 6ExpiCHO cells / mL density for passage culture TM Cells. The next day, cell density and viability were measured again to confirm a cell density of at least 7.0 × 10⁻⁶. 6 Cells / mL and cell viability of at least 95%, and by adding ExpiCHO TM The cells were diluted with expression medium to achieve a cell density of 6.0 × 10⁶. 6 Cells / mL. After aliquoting 25 mL of cell culture into 125 mL Erlenmeyer flasks, incubate the cells at 37°C until the transfection mixture is ready. Add 20 μg of plasmid DNA to 1 mL of OptiPro. TM Mix thoroughly with SFM medium and add 80 μL of Expifectamine. TM CHO reagent and 920 μL OptiPro TM Mix thoroughly with SFM medium. Add Expifectamine–OptiPro. TM SFM mixture added to pre-prepared plasmid - OptiPro TM Mix thoroughly with SFM and incubate at room temperature for 3 minutes. Slowly add the mixture to pre-allocated cell cultures and incubate in an incubator set at 37°C, 8% CO2, and 130 rpm. 18–22 hours post-transfection, add 150 μL of Expifectamine. TM CHO enhancer and 4 mL ExpiCHO TM Feed culture medium was added to the flask, and cell culture was continued in an incubator set to 32°C, 5% CO2, and 130 rpm. To generate more conjugates, 200 mL of cell culture was aliquoted into 1 L Erlenmeyer flasks, and the cells were kept in an incubator at 37°C until the transfection mixture was prepared. 160 μg of plasmid DNA was added to 8 mL of OptiPro... TM The SFM medium was thoroughly mixed, and 640 μL of Expifectamine was added. TM CHO reagent and 7.4 mL OptiPro TM Mix thoroughly with SFM medium. Add Expifectamine–OptiPro. TM SFM mixture added to pre-prepared plasmid - OptiPro TMMix thoroughly with SFM and incubate at room temperature for 3 minutes. Slowly add the mixture to pre-allocated cell cultures and incubate in an incubator set at 37°C, 8% CO2, and 130 rpm. 18–22 hours post-transfection, add 1200 μL of Expiratory Filter (EFF) to the culture medium. TM CHO enhancer and 32 mL ExpiCHO TM Feed culture medium was added to the flasks, 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 post-transfection. On day 5, after measuring the final titer using a Cedex (Roche) instrument, the culture was centrifuged at 3000 × g for 30 minutes to recover the protein-containing cell culture supernatant. The recovered supernatant was filtered through a 0.22 μm polyethersulfone (PES) filter and stored at 4°C (for short-term storage) or -80°C (for long-term storage).

[0181] Example 4 – Purification of conjugated proteins (protein A affinity chromatography and size exclusion chromatography) Proteins were purified from culture supernatant using affinity chromatography on an AKTA Avant 25 instrument equipped with an Amsphere a3 column. The culture supernatant, filtered through a 0.22 μm top-mounted filter, was loaded at a flow rate of 13.33 mL / min. To remove non-specific binding, buffers were applied sequentially in 8 column volumes: first, a buffer of 50 mM Na-Pi, 0.5 M NaCl, pH 7.0; and second, a buffer of 50 mM sodium acetate, pH 4.5. After removing non-specific binding, bound proteins were eluted by applying 10 column volumes of 100 mM glycine (pH 3.0) elution buffer, and the eluent was collected in 50 mL tubes. 1 M Trizma was added at 1% of the collected protein volume. ® The collected protein solution was neutralized with alkali (pH 11.0), and the precipitate was removed using a 0.22 μm syringe filter. Samples before and after neutralization were quantified by UV methods, and purity was analyzed by SEC-HPLC. The initially purified material was further subjected to size exclusion chromatography using a Superdex 200 pg 26 / 600 column to achieve a purity of 90% or higher. Initially, Millipore was used... TM 250 mL concentrator with stirring tank and 50 kDa Millipore TMThe neutralized sample was concentrated to a volume of 13 mL or less. The concentrated sample was loaded onto a column equilibrated with PBS at pH 6.5, and the eluted protein was collected in a tube. After confirming purity by SEC-HPLC, the sample was further concentrated. The finally concentrated protein was quantitatively analyzed by UV, and purity was confirmed by SEC-HPLC and SDS-PAGE.

[0182] Through the above process, one or more of the following physicochemical parameters are measured for each conjugate: titer, elution volume, step yield, high molecular weight species (HMWS) content, low molecular weight species (LMWS) content, and functional monomer content. Here, "titer" refers to the concentration of Fc fusion protein present in the culture supernatant as measured at the time of harvesting the cell culture; "elution volume" refers to the total amount of protein recovered after the preliminary purification step (affinity chromatography); and "yield" is the value obtained by dividing the total amount of protein recovered after preliminary purification by the total amount of protein measured in the culture supernatant, and indicates the recovery rate of protein recovered by column chromatography during preliminary purification. HMWS refers to protein species with a mass greater than the predicted functional unit of the protein, and LMWS refers to protein species with a mass less than the predicted functional unit of the protein; in both cases, they refer to components with anomalous protein folding. Functional monomers refer to fractions with normal protein structure and function. Figures 2a to 2o show the yields and purity of the anti-PD-L1 scFv and the modified fusion protein (modified VEGFR1-D2-D3) conjugates. Specifically, conjugates H-12C94 to H-12C99 (which are conjugates based on the modified fusion protein of this disclosure) exhibited higher purity (approximately 20% vs. approximately 90%) when purified under the same conditions compared to the control conjugates (H-12C08; A167 scFv-HG4SL-VEGF-Grab) prepared using the unmodified fusion protein. It was confirmed that the conjugates prepared using the unmodified fusion protein produced a large number of high-molecular-weight aggregates (Figures 2c and 2d), while such aggregates were significantly reduced in the conjugates prepared using the modified fusion protein (Figures 2e to 2n). This indicates that the physicochemical properties of the conjugates of this disclosure are improved by incorporating the modified fusion protein, which in turn suggests an increased yield for drug manufacturing.

[0183] Example 5 – Freezing / Thaw Stability Test of Conjugate The freeze / thaw stability of the resulting conjugates was assessed. To confirm the stability after freeze / thaw, the conjugate protein samples were frozen by standing in an ultra-low temperature freezer at -70°C for 1 hour, and then thawed by standing at room temperature for 30 minutes. This freeze / thaw procedure was repeated five times. In each cycle, 50 μL of protein sample was collected and analyzed by SEC-HPLC to confirm changes in purity (Table 10: Round 2 (2R); Table 11: Round 3 (3R)).

[0184] [Table 10] [Table 11] Table 10 shows the freeze / thaw stability results obtained in the second round of experiments (2R), and Table 11 shows the freeze / thaw stability results obtained in the third round of experiments (3R). As shown in Table 10, there was no significant difference in the freeze / thaw stability of the conjugates between linkers V2 and V3, but V3 showed slightly higher stability. For scFv, atezolizumab showed relatively higher stability than A167; however, as with the linkers, this difference is difficult to consider meaningful. Overall, high freeze / thaw stability was observed for conjugates based on the modified fusion proteins of this disclosure, regardless of the linker and scFv.

[0185] This was also observed in the comparison results using the Fc variants with A167 scFv and V3 linkers. As shown in Table 11, for all variants, the reduction in purity was confirmed to be within 2%, even after five freeze / thaw cycles.

[0186] The results were obtained in a standard buffer solution, rather than in an optimized formulation, suggesting that freeze / thaw stability could be further improved through future formulation studies.

[0187] Overall, the conjugates of the modified fusion proteins based on this disclosure exhibit high freeze / thaw stability.

[0188] Example 6 – Purity analysis of conjugate proteins using size exclusion high-performance liquid chromatography The purity of conjugated proteins was analyzed by connecting a Tosoh TSKgel G3000SWXL SEC column to a Waters Arc HPLC system with PBS (pH 7.4) as the mobile phase. A 30 μg protein sample was injected into the column while the mobile phase flowed at 0.5 mL / min, and detection was performed at 280 nm for 30 min to measure one or more of the following: high molecular weight species (HMWS) content, low molecular weight species (LMWS) content, and functional monomer content.

[0189] Example 7 – Evaluation of binding affinity for VEGF-A, PlGF and PD-L1 Based on the above reference (Kamat et al., 2017), Octet was used. ® The RED96e (ForteBIO) instrument measures the binding affinity of each protein to VEGF-A and PlGF ligands.

[0190] First, the AHC sensor was equilibrated by immersion in 1×KB buffer (Forte Biosciences) before binding measurements. After binding the analyte conjugate protein PB203 to the sensor at a concentration of 10 nM, the sensor was washed with 1×KB buffer to remove residual protein. Subsequently, VEGF-A, PlGF, and PD-L1 were each flowed through the sensor at eight different concentration points to measure binding. Based on the binding results, at least three concentrations satisfying Full X were selected and set. 2 (≤3) and FullR 2 A global fit was performed at a concentration of (≥0.95) (which is an indicator to ensure reliability), and binding affinity (KD) as well as Kon and Kdis values ​​were calculated. The results are shown in Figures 4a to 4k. As can be seen in Figures 4a to 4k, the conjugates of this disclosure demonstrate increased association rates with VEGF-A and PlGF compared to conjugates prepared using unmodified fusion proteins, and the binding of scFv to PD-L1 is well maintained without reduction.

[0191] However, when preparing conjugates of the modified fusion protein and the scFv targeting a specific antigen as disclosed herein, it is impossible to predict in advance whether the binding of the fusion protein to VEGF and / or PlGF and the binding of the scFv to the specific antigen will function without interference. In this disclosure, it has been demonstrated that the binding of the modified fusion protein to VEGF and PlGF and the binding of the anti-PD-L1 scFv to PD-L1 are maintained without antagonism, an effect that is difficult to predict.

[0192] Example 8 – Stability assessment in a blood simulation environment To indirectly analyze the in vivo stability of PB203, the concentration of residual protein variants was measured by ELISA after a certain time period by reacting with pooled serum collected from rats. PB203 was mixed with rat pooled serum to a final concentration of 10 μg / mL and stored at 37°C at 168, 144, 72, 48, 24, 4, 2, and 0 hours before the start of the assay. The day before the assay, the ELISA plates were coated with human VEGF-A (R&D Systems) and incubated overnight at 4°C. The plates were washed with 0.1% PBS-T wash buffer to remove insufficiently bound human VEGF-A, and then blocked with 0.1% PBS-T containing 5% skim milk at room temperature for 1 hour. The protein-serum mixture prepared at each time point was diluted 50-fold in blocking buffer, and 100 μL was added to each well and reacted at room temperature for 2 hours. Afterward, the reaction solution in the wells was removed, and the ELISA plate was washed with washing buffer. The detection antibody HRP goat anti-human IgG Fc cross-adsorption secondary antibody (Ingenium Biotech) was reacted at room temperature for 1 hour, followed by washing to remove unreacted detection antibody. Then, 100 μL of 3,3',5,5'-tetramethylbenzidine (TMB) solution (Sigma) was added and reacted at room temperature for 10 minutes, and the reaction was terminated by adding stop solution (Sigma). The absorbance was measured at 450 nm using a microplate reader. The remaining amount (%) was calculated relative to the absorbance at 0 hours and compared. The results are shown in Figures 3a to 3c.

[0193] As can be seen in Figures 3a and 3b, H-12C94 to H-12C99 (which are conjugates based on the modified fusion protein of this disclosure) exhibit significantly superior serum stability in a blood-simulated environment compared to the conjugate prepared using the unmodified fusion protein (H-12C08). Furthermore, as can be seen in H-32D01, H-32D02, H-32D09, and H-32D10, significantly superior serum stability in a blood-simulated environment is demonstrated regardless of whether the V2 or V3 linker is used, and regardless of the type of scFv (Figure 3c).

[0194] Example 9 – Isoelectric Point Analysis The isoelectric point (pI) of PB203 protein was analyzed using an icIEF (image capillary isoelectric focusing) system. Forty μL of the modified fusion protein diluted to a concentration of 2 mg / mL was mixed with 160 μL of a pre-prepared master mixture (SERVALYT). TMThe following mixture was prepared: a low pI marker (3.38) (ProteinSimple), a high pI marker (9.5) (ProteinSimple), 1% methylcellulose (ProteinSimple), DW, 500 mM arginine (Sigma), 200 mM iminodiacetic acid (Sigma), and 10 M urea (Sigma). After centrifugation to remove precipitate and air bubbles, the mixture was loaded into each well of an instrument (Maurice, ProteinSimple). Two mL of the cathode electrolyte solution and anolyte solution were placed in the OH- ion exchange chamber of the cartridge. - and H + The location was determined, and the cartridge was installed on the instrument. Subsequently, the pI was measured according to the instrument's instruction manual. Since the measured pI exhibits multi-peak rather than single-peak behavior within a specific range, the pI distribution of the protein was defined by dividing it into three regions: acidic (pI 6–7), neutral (pI 7–8), and basic (pI > 8), with comparisons showing the regions with the highest proportions. The results are shown in Figures 7a to 7r.

[0195] As can be seen in Figures 7g, 7m, and 7r, the conjugates H-12C94 to H-12C99 based on the modified fusion protein of this disclosure exhibit an increased acid / neutral ratio compared to the control conjugates (H-12C08; A167 + VEGF-Grab) prepared using the unmodified fusion protein. Furthermore, as shown in Figures 7m and 7r, the increased acid / neutral ratio is confirmed compared to the unmodified fusion protein and the conjugates prepared therefrom, regardless of whether linker V2 or V3 is used, regardless of the type of scFv, and regardless of the presence of Fc variants. This suggests that the conjugates of this disclosure tend to carry a more negative charge in the in vivo environment, thus indicating a reduced likelihood of nonspecific binding during in vivo circulation.

[0196] Example 10 – Thermal Stability Analysis PB203 was prepared by diluting to a final concentration of 2 mg / mL in 1× PBS buffer. 2.5 μL of 8× Protein Thermal Shift™ dye (Life Technologies) and 5.0 μL of Protein Thermal Shift were added to 12.5 μL of protein. TM Buffer solution (Life Technologies) was used to prepare the final reaction mixture. 20 μL of the prepared reaction mixture was loaded into a PCR tube and placed in QuantStudio. TMThe samples were placed on the tray of a real-time PCR instrument, and the melting temperature (Tm) was measured. All analyses were performed independently in quadruplicate. The results are shown in Figures 8a through 8d.

[0197] Specifically, Figure 8b shows that the effects of connectors V2 and V3 on thermal stability are not significant, while thermal stability varies depending on the scFv used. Furthermore, Figure 8d confirms that Fc variation is independent of thermal stability.

[0198] Because the temperature during in vivo administration is below 40°C, the storage stability of protein therapeutics is typically evaluated at 25°C and 40°C (assuming room temperature and physiological temperature). Based on these criteria, conjugates of the modified fusion proteins according to this disclosure have demonstrated high overall thermal stability at 60°C or higher.

[0199] This demonstrates that the conjugates of the modified fusion proteins based on this disclosure possess physicochemical properties suitable for pharmaceutical development.

[0200] Example 11 – Analysis of VEGF-A inhibitory activity To quantify the inhibitory activity against VEGF-A, a genetically engineered cell line (KDR / NFAT-REHEK293, Promega) was used, incorporating a luciferase reporter system based on VEGFR-2 (KDR) expression and VEGF-A / VEGFR-2 interaction. 25 μL of a 1.6 × 10⁻⁶ ppm solution was used. 6 Cells were aliquoted at 100 cells / mL into 96-well plates, followed by the addition of 25 μL of VEGF-A solution (33.3 ng / mL) and 25 μL of PB203 diluted to different concentrations. The plates were incubated at 37°C for 6 hours in a 5% CO2 incubator. The plates were then cooled to room temperature for 15 minutes, and 75 μL of LBiO-Glo reagent (Promega, luciferase assay buffer + substrate mixture) was added. After incubation in the dark for 10 minutes, the luminescence intensity was measured using a microplate reader. Data were analyzed using GraphPad Prism to calculate the IC50 for each substance. 50 value.

[0201] As shown in Figures 6c to 6l and Figure 6n, the conjugates based on the modified fusion protein of this disclosure exhibit cellular-level VEGF-A signal inhibition comparable to that of the unconjugated modified fusion protein, regardless of the type of linker or scFv used. Furthermore, this inhibitory activity was demonstrated to be superior to that of the unmodified fusion protein VEGF-Grab (PB101).

[0202] Example 12 – Analysis of PD-1 / PD-L1 signaling inhibitory activity To quantify the inhibitory activity against PD-L1, a genetically engineered cell line (Jurkat PD-1 / U2OS PD-L1, Eurofins Discovery) was used. This cell line incorporates a luciferase reporter system based on PD-1 and PD-L1 expression and PD-1 / PD-L1 interaction. Forty μL of a 1 × 10⁻⁶ ppm solution was used. 5 U2OSPD-L1 cells / mL were aliquoted into 96-well plates, followed by the addition of 20 μL of diluted PB203 and control material, and incubated at 37°C for 1 hour in a 5% CO2 incubator. Then, 40 μL of a 2.4 × 10⁻⁶ mcg solution was added. 5 Jurkat PD-1 cells / mL were additionally aliquoted into 96-well plates and incubated at room temperature for 2 hours. Afterward, 10 μL of assay reagent 1 was added to each well and incubated in the dark at room temperature for 15 minutes. Subsequently, 40 μL of assay reagent 2 was added and incubated in the dark at room temperature for 3 hours. Finally, the luminescence intensity was measured using a microplate reader. The data were analyzed using a GraphPad Prism to calculate the IC50 for each substance. 50 value.

[0203] The inhibitory effects of the anti-PD-L1 scFv conjugate and the modified fusion protein (modified VEGFR1-D2-D3) on VEGF-A and PD-L1 are shown in Figures 6a to 6e.

[0204] As shown in Figures 6i to 6n and Figure 6o, the conjugates of the modified fusion proteins based on this disclosure (H-32D01, H-32D02, H-32D04, H-32D06, H-32D08, H-32D09, H-32D10, etc.) exhibit cellular-level PD-L1 signal inhibition comparable to that of the anti-PD-1 antibody before conjugation, regardless of the type of linker or scFv used.

[0205] This demonstrates that although the structures of the modified fusion protein conjugates based on this disclosure are more complex, the conjugates retain their respective functions through structural design without interfering with each other.

[0206] Example 13 – Evaluation of simultaneous binding ability with VEGF-A, PlGF and PD-L1 ligands When bound to two or more ligands from VEGF-A, PlGF, and PD-L1, Octet was used to evaluate binding affinity based on binding sequence. ® RED96e instrument (Forte Biosciences).

[0207] Prior to the binding assay, the AHC sensor was equilibrated in 1x KB buffer (Forte Biosciences). The analyte protein PB203 was immobilized on the sensor at a concentration of 100 nM and then washed with 1x KB buffer to remove unbound protein. Subsequently, VEGF-A, PlGF, or PD-L1 were bound at 30 nM (high concentration), and then ligands different from the previously bound ligand (e.g., VEGF-A → PD-L1, PlGF → PD-L1, PD-L1 → VEGF-A, PD-L1 → PlGF) were introduced at eight concentration points (30, 15, 7.5, 3.75, 1.875, 0.937, 0.468, and 0 nM) in the presence of the previously bound ligand (30 nM), and the binding signal was measured. From the binding results, four or more ligands meeting the reliability criteria (Full X) were selected. 2 (3 or smaller) and Full R 2 Concentration points (0.95 or greater) were determined, and binding affinity and other parameters were calculated through global fitting. Figures 5a to 5f show the obtained simultaneous binding affinity of the anti-PD-L1 scFv and the modified VEGFR1-D2-D3 fusion protein conjugates to the target. As a result of measuring simultaneous antigen-binding affinity, it was confirmed that the affinity was maintained even when the antigen type changed, with very small differences. Specifically, as shown in Figures 5a and 5b, the conjugates based on the modified fusion protein of this disclosure maintained similar binding affinity to PD-L1 whether binding to VEGF-A or PlGF. Furthermore, as shown in Figures 5c to 5f, the conjugates based on the modified fusion protein of this disclosure maintained binding affinity comparable to that observed when binding to a single ligand, regardless of the order or type of ligand binding. This demonstrates that although the structures of the conjugates based on the modified fusion protein of this disclosure are more complex, the conjugates maintain their respective functions through structural design without mutual interference.

[0208] Example 14 – Pharmacokinetic evaluation after a single intravenous administration in SD rats One week prior to the study, seven-week-old male Sprague–Dawley (SD) rats were acclimated and then used in the experiments. PB203 was administered as a single intravenous dose of 3 mpk. Blood samples were collected via jugular vein at specified time points (0, 0.083, 2, 8, 24, 48, 96, 120, 168, and 336 hours), followed by plasma separation.

[0209] The day before the assay, the ELISA plate was coated with human PD-L1 (R&D Systems) and incubated overnight at 4°C. After washing with 0.1% PBS-T wash buffer to remove unbound human PD-L1, it was blocked with 0.1% PBS-T containing 5% skim milk for 1 hour at room temperature. The prepared plasma sample was diluted 20-fold with blocking buffer, and 100 μL of each sample was added to each well and incubated at room temperature for 2 hours. The reaction solution was then removed, and the ELISA plate was washed with wash buffer. The detection antibody HRP goat anti-human IgG Fc cross-adsorption secondary antibody (Ingenium) was reacted at room temperature for 1 hour, followed by washing to remove unreacted detection antibody. Then, 100 μL of 3,3',5,5'-tetramethylbenzidine (TMB) solution (Sigma) was added and reacted at room temperature for 10 minutes, after which stop solution (Sigma) was added to terminate the reaction. The absorbance was measured at 450 nm using an ELISA reader. Quantitative standards for PB203 were prepared using serial dilutions from 62.5 to 0.48, and standard curves were generated based on measured absorbance (OD450). Plasma concentrations of PB203 were calculated using derived equations via inverse calculation, and PK parameters were finally calculated using time-dependent concentration results. First, to validate pharmacokinetic characteristics according to scFv type, H-32D02 and H-32D10, which typically use linker V3 and are conjugated to A167 or atezolizumab scFv, respectively, were evaluated. As shown in Figure 9a, the A167 conjugate exhibited superior in vivo pharmacokinetic behavior, and its Fc variants were subsequently prepared. Four Fc variants of the A167 conjugate containing wild-type Fc were prepared and evaluated. As shown in Figure 9b, the H-32D04, H-32D06, and H-32D08 conjugates exhibited superior in vivo pharmacokinetic characteristics compared to the H-32D02 conjugate. The pharmacokinetic characteristics of these three conjugates are similar to each other.

[0210] Example 15 – Study on antitumor efficacy in a mouse model Genetically engineered mice (B6 / JGpt-Cd274tm1(hCD274) / Gpt) were subcutaneously implanted with the genetically modified cell line MC-38-hPD-L1, and the tumor volume reached approximately 100 mm. 3 Animals were randomly assigned to groups. Subsequently, PB203 candidate material and control drug were administered intraperitoneally three times weekly at a dose of 10 mpk, and tumor volume and weight were measured over three weeks. The study was conducted in accordance with animal ethics regulations. Results were... Figure 10 As shown in the image. Figure 10As shown, among the conjugates disclosed herein, the conjugates containing wild-type Fc and Fc variants with inhibitory ADCC / CDC activation exhibited approximately 10% or more superior tumor growth inhibition (TGI) compared to the commercially available antibody Tecentriq, and the other two Fc variants also showed high levels of tumor growth inhibition. Specifically, H-32D04 demonstrated complete remission in approximately 40% of subjects, a superior result compared to Tecentriq, where no complete remission was observed.

[0211] Based on the foregoing description, those skilled in the art will understand that this disclosure may be implemented in other specific forms without departing from its technical spirit or essential characteristics. In this regard, the above embodiments should be understood in all respects as illustrative rather than restrictive. The scope of this disclosure should not be construed from the foregoing detailed description, but rather from the meaning and scope of the claims set forth below, and all variations or modifications derived from their equivalents should be included within the scope of this disclosure.

Claims

1. A conjugate comprising: an scFv targeting Programmed Cell Death Protein Ligand 1 (PD-L1); and a fusion protein comprising a VEGFR1 (vascular endothelial growth factor receptor 1) extracellular domain, a linker, and a multimerization domain, wherein the scFv targeting PD-L1 is fused to the N-terminus of the fusion protein.

2. The conjugate of claim 1, wherein the fusion protein comprises a VEGFR1 extracellular domain, a linker, and a multimerization domain, wherein the VEGFR1 extracellular domain includes immunoglobulin (Ig) -like domain D2 and Ig-like domain D3 of VEGFR1, the linker is between the Ig-like domain D3 and the multimerization domain, and the fusion protein is a modified fusion protein having one or more of the following properties (a) to (c): (a) one or more amino acid substitutions on the b1-b2 loop of domain D3 selected from K241E, L243S, R244V, and H246E; the amino acid substitution L258A, L258S, or L258D on the b2-b3 loop of domain D3; and one or more amino acid substitutions on the b5-b6 loop of domain D3 selected from K300G, Q302T, and K304S, wherein the b1-b2 loop of domain D3 includes amino acid residues T236 to T247 of the amino acid sequence of VEGFR1 comprising SEQ ID NO: 41, the b2-b3 loop includes amino acid residues T256 to V262 of the amino acid sequence of VEGFR1, and the b5-b6 loop includes amino acid residues D299 to L308 of the amino acid sequence of VEGFR1; (b) the linker is from about 14 to about 35 amino acids in length; and (c) a disulfide bond is present in the fusion protein, and one of the amino acid residues L243 and the amino acid residues located at positions -2, -1, 0, +1, +2, and +3 relative to Y329 of domain D3 is substituted with cysteine in the amino acid residues on the b1-b2 loop of domain D3.

3. The conjugate of claim 2, wherein the property (a) of the fusion protein includes: the amino acid substitutions K241E, L243S, R244V, and H246E on the b1-b2 loop of domain D3; the amino acid substitution L258A, L258S, or L258D on the b2-b3 loop of domain D3; and the amino acid substitutions K300G, Q302T, and K304S on the b5-b6 loop of domain D3.

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

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

6. The conjugate of claim 5, wherein one or more glycine (G) residues included in the GS repeat sequence is substituted with cysteine (C).

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

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

9. The conjugate of claim 7, wherein in property (b) of the modified fusion protein, a papain recognition site or a glycosylation site present in the hinge region derived from an immunoglobulin has an amino acid variation.

10. The conjugate of claim 2, wherein in property (b) of the modified fusion protein, the linker comprises, in order from N-terminus to C-terminus: (i) an amino acid sequence selected from the group consisting of 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, wherein the amino acid sequence of the hinge region is modifiable.

11. The conjugate of claim 2, wherein in property (b) of the modified fusion protein, the linker is an amino acid sequence selected from the group consisting of CSSGDATPTSPPSP, CSKVDKKVEPKSSDTPPTCPPCP, CSGGGGSAEPKAGDATPPTCPPCP, CS GGGGSGGGGSGGGGSAESKYGPPCPPCP, CSNTGSGGEEKKKEKEKEEQEERSSDTPPTCPPCP, CSNTGSGGEEKKKEKEKEEQEERSCDTPPTCPPCP, CGSSGGSSGEPKSDATPTCPPCP, and CSKVDKKVEPKSSDKTYTCPPCP.

12. The conjugate of claim 2, wherein the modified fusion protein: (a) comprises the amino acid substitutions K241E, L243S, R244V, and H246E on the b1-b2 loop of the domain D3, the amino acid substitution L258A, L258S, or L258D on the b2-b3 loop of the domain D3, and the amino acid substitutions K300G, Q302T, and K304S on the b5-b6 loop of the domain D3; (b) has a linker that is an amino acid sequence selected from the group consisting of: CSSGDATPTSPPSP, CSKVDKKVEPKSSDTPPTCPPCP, CSGGGGSAEPKAGDATPPTCPPCP, CSGGGSGGGGSGGGGSAESKYGPPCPPCP, CSNTGSGGEEKKKEKEKEEQEERSSDTPPTCPPCP, CSNTGSGGEEKKKEKEKEEQEERSCDTPPTCPPCP, CGSSGGSSGEPKSDATPTCPPCP, and CSKVDKKVEPKSSDKTYTCP PCP; and (c) comprises a cysteine that replaces the amino acid residue at position +2 relative to Y329 of domain D3.

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

14. The conjugate of claim 2, wherein the multimerization domain is: (a) an Fc region of an immunoglobulin; (b) a CH3 region of IgGl or IgG4; (c) a CH2 and CH3 region of IgGl or IgG4; (d) an Fc region of an immunoglobulin comprising an amino acid sequence that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 59; or (e) an Fc region of an immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO:

60.

15. The conjugate of claim 14, wherein the multimerization domain comprises an IgGl Fc region consisting of the amino acid sequence set forth in SEQ ID NO:

60.

16. The conjugate of claim 2, wherein the multimerization domain has the following based on SEQ ID NO: 60: (i) one or more selected from the group consisting of: T20Q amino acid substitution, D126E amino acid substitution, L128M amino acid substitution, M198L amino acid substitution, and K217 amino acid deletion, or (ii) one or more selected from the group consisting of: 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 of claim 2, wherein the modified fusion protein is in a dimeric or multimeric form.

18. The conjugate of claim 1, wherein the scFv targeting PD-L1 is an atezolizumab scFv or an anti-PD-Ll scFv comprising the amino acid sequence set forth in SEQ ID NO:

40.

19. A pharmaceutical composition for preventing or treating a chronic infection, a tissue allograft, an autoimmune disease, an inflammatory disease, a neoplastic disease, a cancer, an angiogenesis-related disease, or an ocular disease, the pharmaceutical composition comprising as an active ingredient the conjugate according to any one of claims 1 to 18.

20. A nucleic acid molecule encoding the conjugate according to any one of claims 1 to 18.

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

22. A vector comprising a nucleotide sequence encoding 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 subject, the pharmaceutical composition comprising the recombinant viral vector according to claim 23, wherein the fusion protein encoded by the recombinant viral vector is expressed in the subject in vivo, and the pharmaceutical composition is for preventing or treating an autoimmune disease, an inflammatory disease, a neoplastic disease, a cancer, an angiogenesis-related disease, or an ocular disease.

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

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

27. Use of the conjugate according to any one of claims 1 to 18 and / or the recombinant viral vector according to claim 23 for preventing or treating one or more selected from the group consisting of a chronic infection, a tissue allograft, an autoimmune disease, an inflammatory disease, a neoplastic disease, a cancer, an angiogenesis-related disease, and an ocular disease.

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