Conjugate of modified fusion protein and scfv, and use thereof

Abstract

The present disclosure relates to a conjugate of a multi-specific modified fusion protein and scFv that binds to an antigen of a binding scFv, a vascular endothelial growth factor, and a placental growth factor, and to a use thereof. The conjugate, according to one embodiment of the present disclosure, can exhibit excellent productivity and stability. The conjugate, according to one embodiment of the present disclosure, can exhibit excellent blood stability. The conjugate, according to one embodiment of the present disclosure, can exhibit excellent anti-tumor, anti-fibrosis, and / or immune cell activation effects.

Description

Conjugate of modified fusion protein and scFv and use thereof

[0001] The present disclosure relates to a conjugate of a modified fusion protein and scFv that binds to an antigen of vascular endothelial growth factor, placental growth factor and the scFv to which it is bound, and uses thereof.

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

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

[0004] The development of drugs that target angiogenesis inhibition by removing vascular endothelial growth factor, a key factor in tumorigenesis, has continued from the past to the present. A representative example is bevacizumab, a monoclonal antibody targeting VEGF-A (Stacker, et al., 2013. Chinese Journal of Cancer Research. 32(6): pp. 297-302.; Kazazi-Hyseni, et al., 2010. Oncologist. 15(8): pp. 819-825; DrugBank Accession Number: DB00112). Monoclonal antibody drugs have a mechanism of action that binds to a single vascular endothelial growth factor, and since multiple growth factors are involved in cancer formation, they have been ineffective drugs for demonstrating remarkable efficacy through monoclonal antibodies. Bevacizumab, on the other hand, has the side effect of increasing the expression of other growth factors that are not targeted by the target through intracellular compensatory mechanisms, leading to the development of resistance (Bagley, et al., 2011. Clinical cancer research 17(5): pp. 976-988; Lieu, et al., 2013. Plos One 8: e77117; Cutsem, et al., 2020 Clinical cancer research 26(3): pp. 717-725). For this reason, the need for the development of drugs that simultaneously target cancer-related angiogenic growth factors and cancer cells has gradually expanded.

[0005] To overcome the shortcomings of monoclonal antibody drugs, multi-targeting drugs for vascular endothelial growth factors have been developed, and representative protein drugs include VEGF-Trap (Ciombor, et al., 2013, Clinical cancer research. 19 (8): pp. 1920-1925. DrugBank Accession Number: DB08885) and VEGF-Grab (Lee et al., 2015. Mol. Cancer. Ther. 14 (2): pp. 470-479.). Both of these drugs mimic parts of decoy receptors, and VEGF-Trap is a heterologous protein form of VEGFR1 D2-VEGFR2 D3, while VEGF-Grab is a recombinant fusion protein in which the heterologous protein form of VEGFR1 D2-D3 is fused to the Fc of immunoglobulin. Efforts to improve the properties of these multi-targeting fusion proteins for vascular endothelial growth factors have been ongoing.

[0006] Meanwhile, one could consider conjugating additional antigen-targeting single-chain variable fragments (scFvs) to these fusion proteins, including scFvs targeting programmed cell death ligand 1 (PD-L1), which is overexpressed in many cancer tissues (Dong, et al., 2002. Nat. Med 8:787-9). The programmed cell death 1 (PD-1) receptor and its ligand, programmed cell death ligand 1 (PD-L1), are immune checkpoint proteins implicated in the suppression of immune responses associated with chronic infections, pregnancy, tissue allografts, autoimmune diseases, and cancer. PD-L1 regulates immune responses by binding to the inhibitory receptor PD-1, which is expressed on the surface of T cells, B cells, and monocytes. PD-L1 also negatively regulates T cell function through interaction with another receptor, B7-1. Formation of PD-L1 / PD-1 and PD-L1 / B7-1 complexes negatively regulates T-cell receptor signaling, resulting in subsequent downregulation of T-cell activation and inhibition of anti-tumor immune activity.

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

[0008] Therefore, there has been a need for the development of a conjugate that has excellent anticancer effects while having high physical and chemical stability that can satisfy high productivity at the level of protein therapeutics in terms of commercial-scale production, while enabling selective targeting based on high binding affinity for target substances in cancer cells and possessing increased in-vivo duration.

[0009] Improving the physical properties of proteins maximizes production efficiency by increasing the physical and chemical stability of proteins during expression and purification, reduces non-specific binding, aggregation, and degradation when proteins are introduced into the body, thereby achieving high in vivo persistence, and in the case of anticancer drugs, increases selective targeting of cancer cells, thereby demonstrating excellent anticancer effects.

[0010] The inventors of the present disclosure developed a modified fusion protein comprising VEGFR1 domain D2 and domain D3 by introducing amino acid mutations in specific regions of domain D3, length control and mutation of a linker, and disulfide bonds within the fusion protein, and developed a conjugate combining a PD-L1 targeting scFv with the modified fusion protein, and confirmed that the conjugate exhibited excellent productivity, stability, and anticancer efficacy.

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

[0012] 2. One embodiment of the "fusion protein" of the present disclosure may be directed to a modified fusion protein comprising a VEGFR1 extracellular domain, a linker and a multimerization domain, wherein the VEGFR1 extracellular domain comprises an immunoglobulin (Ig)-like domain D2 and an Ig-like domain D3 of VEGFR1, and a linker is present between the Ig-like domain D3 and the multimerization domain, and has one or more of the following characteristics (a) to (c): (a) a substitution of one or more amino acids among K241E, L243S, R244V, and H246E on the β1-β2 loop of domain D3, an amino acid substitution of L258A, L258S, or L258D on the β2-β3 loop of domain D3, and an amino acid substitution of one or more amino acids among K300G, Q302T, and K304S on the β5-β6 loop of domain D3; Wherein the β1-β2 loop of domain D3 comprises amino acid residues T236 to T247 of the amino acid sequence of VEGFR 1 (e.g., the amino acid sequence of VEGFR1 in Table 2; SEQ ID NO: 41), the β2-β3 loop comprises amino acid residues T256 to V262 of the amino acid sequence of VEGFR 1, and the β5-β6 loop comprises amino acid residues D299 to L308 of the amino acid sequence of VEGFR 1; (b) the linker has a length of about 14 to about 35 amino acids; and (c) a disulfide bond is present in the fusion protein, and one of the amino acid residues present at positions -2, -1, 0, +1, +2, and +3 relative to L243 and Y329 of domain D3 on the β1-β2 loop of domain D3, particularly the 331 amino acid residue present at position +2, is substituted with cysteine.

[0013] 3. In one embodiment, the characteristic (a) of the modified fusion protein may include amino acid substitutions K241E, L243S, R244V, and H246E on the β1-β2 loop of domain D3; amino acid substitutions L258A, L258S, or L258D on the β2-β3 loop of domain D3; and amino acid substitutions K300G, Q302T, and K304S on the β5-β6 loop of domain D3. With respect to characteristic (a), the substitutions may be amino acids that decrease the net pI of the protein, amino acids that have a negatively charged side chain, or amino acids that have a negatively electrostatic side chain compared to the residue prior to the substitution.

[0014] 4. In one embodiment, the modified fusion protein may be one in which the N-terminus of domain D2 begins with the amino acid sequence EF.

[0015] 5. In one embodiment, the modified fusion protein, with respect to feature (b), wherein the linker comprises a GS repeat sequence, wherein the GS repeat sequence may be an amino acid sequence of 2 to 35 amino acids in length consisting solely of G and S.

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

[0017] 7. In one embodiment, the modified fusion protein comprises, with respect to characteristic (b), an amino acid sequence of a hinge region derived from an immunoglobulin, wherein the amino acid sequence of the hinge region may be modified.

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

[0019] 9. In one embodiment, the modified fusion protein may have an amino acid mutation in a papain recognition site or a glycosylation site present in a hinge region derived from an immunoglobulin, with respect to feature (b). Specifically, the amino acid mutation in the papain recognition site may be a substitution of one or more amino acid residues present in the papain recognition site with alanine, serine, tyrosine, proline, or threonine, or an insertion of 1 to 10 amino acid sequences comprising at least one amino acid having an aromatic carbon or a cyclic carbon in a side chain into the papain recognition site, and the amino acid mutation in the glycosylation site may be a substitution of a serine or threonine present in the hinge region derived from an immunoglobulin by (i) deleting it, or (ii) substituting it with an amino acid other than serine, asparagine, or threonine.

[0020] 10. In one embodiment, the modified fusion protein may comprise, with respect to property (b), a linker comprising (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, in that order from the N-terminus to the C-terminus, wherein the amino acid sequence of the hinge region may be modified.

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

[0022] 12. In one embodiment, the modified fusion protein comprises (a) amino acid substitutions K241E, L243S, R244V, and H246E on the β1-β2 loop of domain D3, amino acid substitutions L258A, L258S, or L258D on the β2-β3 loop of domain D3, and amino acid substitutions K300G, Q302T, and K304S on the β5-β6 loop of domain D3; (b) the linker is an amino acid sequence selected from the group consisting of CSSGDATPTSPPSP, CSKVDKKVEPKSSDTPPTCPPCP, CSGGGGSAEPKAGDATPPTCPPCP, CSGGGGSGGGGSGGGGSAESKYGPPCPPCP, CSNTGSGGEEKKKEKEKEEQEERSSDTPPTCPPCP, CSNTGSGGEEKKKEKEKEEQEERSCDTPPTCPPCP, CGSSGGSSGEPKSDATPTCPPCP, and CSKVDKKVEPKSSDKTYTCPPCP; (c) the amino acid residue at the +2 position based on L243 and Y329 of domain D3 may be substituted 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 amino acid sequences C61 to C75 of Table 2 (SEQ ID NOs: 44 to 58).

[0024] 14. In one embodiment, the multimerization domain can be (a) an Fc region of an immunoglobulin; (b) a CH3 region of IgG1 or IgG4; (c) a CH2 and CH3 region of IgG1 or IgG4; (d) an Fc region of an immunoglobulin comprising an amino acid sequence having at least 85% identity to the amino acid sequence of C88 of Table 2 (SEQ ID NO: 59); or (e) an 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 multimerization domain may comprise or consist of an Fc portion of IgG1 consisting of an amino acid sequence of SEQ ID NO: 60. Meanwhile, the multimerization domain may have one or more of i) a T20Q amino acid substitution, a D126E amino acid substitution, a L128M amino acid substitution, a M198L amino acid substitution, and a K217 amino acid deletion with respect to SEQ ID NO: 60, or ii) a L4A amino acid substitution, a L5A amino acid substitution, a H38Q amino acid substitution, a K44Q amino acid substitution, a Y66F amino acid substitution, an A97G amino acid substitution, an A100S amino acid substitution, a P101S amino acid substitution, a R125Q amino acid substitution, a D126E amino acid substitution, a L128M amino acid substitution, a K179R amino acid substitution, a Q189E amino acid substitution, a P215L amino acid substitution, and a K217 amino acid deletion with respect to SEQ ID NO: 60.

[0026] 16. In one embodiment, the modified fusion protein may be in a dimeric or multimeric form.

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

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

[0029] 19. One embodiment of the present disclosure may be directed to a nucleic acid molecule encoding a complex of a modified fusion protein and scFv according to one embodiment of the present disclosure.

[0030] 20. One embodiment of the present disclosure may relate to a host cell comprising a nucleotide sequence encoding a complex of a modified fusion protein and scFv according to one embodiment of the present disclosure.

[0031] 21. One embodiment of the present disclosure may relate to a vector comprising a nucleotide sequence encoding a complex of a modified fusion protein and scFv according to one embodiment of the present disclosure.

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

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

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

[0035] 26. One embodiment of the present disclosure may relate to a method for preventing or treating at least one selected from the group consisting of chronic infection, tissue allograft, autoimmune disease, inflammatory disease, neoplastic disease, cancer, angiogenesis-related disease, or ocular disease, by administering to a subject in need thereof a recombinant viral vector comprising a nucleotide sequence encoding a combination of a modified fusion protein and scFv according to one embodiment of the present disclosure and / or a combination of a modified fusion protein and scFv according to one embodiment of the present disclosure.

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

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

[0038] The conjugate of the modified fusion protein and anti-PD-L1 scFv according to one embodiment of the present disclosure may exhibit excellent blood stability.

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

[0040] The complex of the modified fusion protein and the anti-PD-L1 scFv according to one embodiment of the present disclosure can exhibit excellent simultaneous binding affinity for the antigen of each of the modified fusion protein and the scFv.

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

[0042] The conjugate of the modified fusion protein and the anti-PD-L1 scFv according to one embodiment of the present disclosure may exhibit acidic properties and an increased proportion of charge variants in the acidic and neutral regions of the pI distribution. Accordingly, the modified fusion protein may exhibit improved nonspecific interactions with the matrix and cell surface in the body.

[0043] A combination of a modified fusion protein and an anti-PD-L1 scFv according to one embodiment of the present disclosure can exhibit excellent anticancer effects.

[0044] According to one embodiment of the present disclosure, the conjugate of the modified fusion protein and the anti-PD-L1 scFv can inhibit the activity of CD141-expressing fibroblasts (Cancer-associated fibroblasts, CAFs) by lowering the level of PlGF in the tumor microenvironment (TME), thereby inhibiting fibrosis. This reduces vascular pressure and normalizes blood vessels along with lowering the level of VEGF-A. Normalized blood vessels and improved peritumoral fibrosis can increase drug accessibility and increase immune cell infiltration, thereby inhibiting tumor growth.

[0045] The complex of the modified fusion protein according to one embodiment of the present disclosure and an anti-PD-L1 scFv may exhibit anti-tumor, anti-fibrotic and / or immune cell activation effects.

[0046] Figure 1 is a 2D structure diagram showing the form in which anti-PD-L1 scFv is bound to the D2-D3 domain of modified VEGFR1 (modified fusion protein of the present disclosure).

[0047] Figures 2a to 2o are graphs showing the productivity and purity of a complex of an anti-PD-L1 scFv and a modified fusion protein.

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

[0049] Figures 4a to 4k are graphs showing the binding affinity of a conjugate of an anti-PD-L1 scFv and a modified fusion protein that binds to vascular endothelial growth factor, placental growth factor, and PD-L1 to VEGF-A, PlGF, and PD-L1 (control group: prototype conjugate based on anti-PD-L1 scFv and VEGF-Grab).

[0050] Figures 5a to 5f are graphs showing the simultaneous binding affinity of a complex of an anti-PD-L1 scFv and a modified fusion protein that binds to vascular endothelial growth factor, placental growth factor, and PD-L1.

[0051] Figures 6a to 6o are graphs showing the signal transduction inhibitory effect on VEGF-A and PD-L1 at the cellular level of a conjugate (PB203) of the present disclosure's anti-PD-L1 scFv that binds to vascular endothelial growth factor, placental growth factor and PD-L1 and a modified fusion protein (control: VEGF-Grab / PB101; H-12C18; H-30D01; Tecentriq; anti-PD-1 antibody; modified fusion protein / backbone / PB102).

[0052] Figures 7a to 7r are graphs measuring the isoelectric point of a combination according to the present disclosure (control group: PB101 / PD20A01).

[0053] Figures 8a to 8d are graphs analyzing the thermal stability of the composite according to the present disclosure.

[0054] Figures 9a and 9b are PK analysis graphs of a combination according to the present disclosure.

[0055] Figure 10 is a graph showing the tumor growth inhibitory efficacy of a conjugate of the present disclosure's anti-PD-L1 scFv and a modified fusion protein that binds to vascular endothelial growth factor, placental growth factor, and PD-L1 in a mouse model.

[0056] The various embodiments or examples described in this document are exemplified for the purpose of clearly explaining the technical idea of ​​the present disclosure and are not intended to be limited to specific embodiments. The technical idea of ​​the present disclosure includes various modifications, equivalents, alternatives, and embodiments or examples selectively combined from all or part of each embodiment or example described in this document.

[0057] All technical and scientific terms used in this disclosure, unless otherwise defined, have the meaning commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0058] The singular forms used in this document may include the plural form unless the context clearly indicates otherwise, and the same applies to the singular forms set forth in the claims.

[0059] I. Definition

[0060] In this disclosure, the term "about" can indicate a typical error range for each value, as is well known to those skilled in the art. In the context of a numerical value or range described in this disclosure, this can mean ±20%, ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% of the numerical value or range stated or claimed in one embodiment. In the case of a nucleotide or amino acid sequence length, "about" can indicate that the nucleic acid or protein is not limited to the number of nucleotides or amino acids described, and several nucleotides or amino acids may be added or removed from either terminus, provided that such addition or removal does not impair functional activity.

[0061] Expressions such as “including,” “comprising,” “having,” and the like, as used in this disclosure, should be understood as open-ended terms that imply the possibility of including other embodiments in a similar manner to “comprising,” unless otherwise stated in the phrase or sentence in which the expression is included.

[0062] The term "and / or" as used herein may mean any one or more of the items, any combination of the items, or all of the items associated with the term.

[0063] The term "amino acid" as used herein may mean any naturally occurring L-α amino acid. This definition may include norleucine (Nle), ornithine, and homocysteine.

[0064] The term "variant" or "amino acid variation" as used herein may mean a substitution, insertion, deletion, or a combination thereof in an amino acid sequence as compared to a reference (e.g., native sequence) polypeptide or protein, and the term "variant" may mean a molecule having some difference in amino acid sequence as compared to a reference polypeptide or protein due to such variation.

[0065] Additionally, the scope of variants of the present disclosure may include proteins or fragments or derivatives thereof that exhibit the same or similar biological activity and derivatives that are separately modified during or after translation, for example, by glycosylation, proteolytic cleavage, linkage to antibody molecules or other cellular ligands, etc.

[0066] The term "carriers" as used herein may include pharmaceutically acceptable carriers, excipients, or stabilizers that are nontoxic to cells or mammals at the dosages and concentrations employed. Often, the pharmaceutically acceptable carrier is an aqueous pH buffered solution. Examples of pharmaceutically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or TWEEN. ® , polyethylene glycol (PEG) and PLURONICS ® Nonionic surfactants such as, but not limited to,

[0067] The term "effective amount," as used herein, may refer to an amount sufficient to produce a beneficial or desired clinical or biochemical result. The effective amount may be administered one or more times. An effective amount may refer to an amount sufficient to alleviate, improve, stabilize, slow, or delay the progression of a disease state.

[0068] The term "ligand" as used herein may refer to a molecule (e.g., VEGF-A, -B, -C, -D, and PlGF) that binds with high affinity to a modified fusion protein. In another context, "ligand" may refer to any molecule, agent, or compound that specifically binds, either covalently or transiently, to a molecule, such as a polypeptide or protein.

[0069] The term "subject" or "object" as used herein may refer to a mammal. Mammals include, but are not limited to, domestic animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In one embodiment, the subject or object may be a human.

[0070] The term "pharmaceutically acceptable carrier and / or diluent" as used herein may include, but is not limited to, all solvents, dispersion media, coating agents, antibacterial and antifungal agents, isotonic agents and adsorption delaying agents.

[0071] An embodiment of the present disclosure described in this disclosure is understood to include "comprising," "consisting of," and / or "consisting essentially of" an embodiment.

[0072] II. Modified fusion proteins and modified fusion protein components

[0073] 1. Vascular endothelial growth factor receptor (VEGFR)

[0074] In one embodiment, the modified fusion protein of the present disclosure may comprise a VEGFR1 extracellular domain as a component. VEGFR1, also known as fms-related tyrosine kinase (FLT-1), is encoded by the FLT1 gene. The amino acid sequence of VEGFR1 is provided in Table 2 as the amino acid sequence of VEGFR1 and can be found under UniProtKB. #P17948. In one embodiment, VEGFR1 is derived from a mammal, such as a human. VEGFR1 has seven immunoglobulin (Ig)-like domains, numbered 1, 2, 3, 4, 5, 6, and 7 from the N-terminus to the C-terminus of the extracellular domain, as are VEGFR2 and VEGFR3.

[0075] In one embodiment, the VEGFR1 extracellular domain may comprise an Ig-like domain D2 and an Ig-like domain D3. As used herein, the term "Ig-like domain D2" of VEGFR1 refers to the second Ig-like domain found at the N-terminus of the extracellular region of VEGFR1, and the term "Ig-like domain D3" of VEGFR refers to the third Ig-like domain found at the N-terminus of the extracellular region of VEGFR1, but the term encompasses variants within the range where the functionality of these domains (e.g., binding to VEGF ligands and / or inhibition of VEGFR pathway activation) is maintained. Since the Ig-like domains D2 and D3 of VEGFR1 may differ in the amino acid sequence of proteins exhibiting activity depending on the species, the term is not limited to the origin or sequence thereof, and may include wild-type or active variants thereof.

[0076] In one embodiment, the modified fusion protein of the present disclosure may additionally comprise another VEGFR extracellular domain in addition to the Ig-like domain D2 and Ig-like domain D3 of VEGFR1.

[0077] Without being bound by theory, the present disclosure believes that the extracellular domain of VEGFR inhibits activation of the VEGF pathway by binding to a VEGF ligand, thereby blocking the interaction of the VEGF ligand with the VEGFR. Furthermore, without being bound by theory, the present disclosure believes that the extracellular domain of VEGFR can bind to VEGFR to achieve dominant negative inhibition of the VEGF signaling pathway. In one embodiment, the extracellular domain of VEGFR can bind to one or more VEGF ligands selected from the group consisting of VEGF-A, VEGF-B, VEGF-C, VEGF-D, and PlGF.

[0078] In one embodiment, the VEGFR extracellular domain may or may not comprise a signal peptide that acts as a signal sequence for secretion of the VEGFR extracellular domain or a modified fusion protein comprising the VEGFR extracellular domain from a host cell. Such a signal peptide may be operably linked to a nucleic acid encoding a protein of interest (e.g., the extracellular domain of VEGFR1).

[0079] 2. Linker

[0080] Components of the modified fusion protein (e.g., the extracellular domain or multimerization domain of VEGFR1) can be linked by a linking moiety, such as a peptide linker. Such a linker increases the flexibility of the fusion protein components without significantly disrupting the structure of each functional component within the fusion protein.

[0081] In one embodiment, a linker can be used to connect the C-terminal end of the VEGFR1 extracellular domain (e.g., the C-terminus of the Ig-like domain D3) to the N-terminal end of a multimerization domain (e.g., an IgG1 Fc region).

[0082] In one embodiment, the linker may comprise an amino acid sequence of a hinge region derived from an immunoglobulin. The hinge region derived from an immunoglobulin may comprise a hinge region present in the N-terminal region of the Fc region of the immunoglobulin. In one embodiment, the hinge region derived from an immunoglobulin may be derived from human IgD or IgG.

[0083] 3. Multimerization domain

[0084] The present disclosure provides a multimerization domain (e.g., the Fc region of an immunoglobulin) that can be a component of any modified fusion protein. A multimerization domain is a portion of a multimeric protein that promotes the association of subunits to form, for example, dimers, trimers, tetramers, etc. The term "multimerization domain" as used herein can be used to refer to a dimerization domain, a trimerization domain, a tetramerization domain, etc. A fusion protein comprising a multimerization domain can interact with another fusion protein comprising a multimerization domain to form a fusion protein multimer (e.g., a fusion protein dimer). For example, an IgG Fc region is a dimerization domain that can be fused with the extracellular domain of VEGFR1 as described herein. A fusion protein comprising the extracellular domain of VEGFR1 and an IgG Fc region can dimerize with another fusion protein comprising an IgG Fc region to generate a fusion protein dimer capable of simultaneously binding to VEGF ligand and PlGF ligand.

[0085] In one embodiment, the Fc region may be partially glycosylated or entirely non-glycosylated.

[0086] In one embodiment, the Fc region can have a T20Q amino acid substitution, a D126E amino acid substitution, a L128M amino acid substitution, a M198L amino acid substitution, and / or a K217 amino acid deletion based on SEQ ID NO: 60. These amino acid mutations can increase the half-life of the fusion protein through interaction with FcRn. The amino acid mutations can be identically present in other immunoglobulin Fc regions having a sequence corresponding to the amino acid sequence of SEQ ID NO: 60, and the modified fusion protein of the present disclosure can include other immunoglobulin Fc regions having the amino acid mutations as a multimerization domain component.

[0087] In one embodiment, the Fc region can have an L4A amino acid substitution, an L5A amino acid substitution, an H38Q amino acid substitution, a K44Q amino acid substitution, a Y66F amino acid substitution, an A97G amino acid substitution, an A100S amino acid substitution, a P101S amino acid substitution, an R125Q amino acid substitution, a D126E amino acid substitution, an L128M amino acid substitution, a K179R amino acid substitution, a Q189E amino acid substitution, a P215L amino acid substitution and / or a K217 amino acid deletion based on SEQ ID NO: 60. These amino acid mutations can eliminate the effector function of the Fc region. The amino acid mutations can be identically present in other immunoglobulin Fc regions having a sequence corresponding to the amino acid sequence of SEQ ID NO: 60, and the modified fusion protein of the present disclosure can comprise another immunoglobulin Fc region having the amino acid mutations as a multimerization domain component.

[0088] Below, the characteristics of the modified fusion protein of the present disclosure are described.

[0089] 4. Surface charge transfer transition characteristics

[0090] In one embodiment, the modified fusion protein of the present disclosure may have surface charge transfer mutation properties. In the present disclosure, a modified fusion protein having surface charge transfer mutation properties may be used interchangeably as a "surface charge transfer mutation" or a "surface charge mutation."

[0091] In one embodiment, the surface charge transfer mutation characteristic of the modified fusion protein may be one in which the amino acid substitutions K241E, L243S, R244V, and H246E on the β1-β2 loop of the Ig-like domain D3, L258A, L258S, or L258D on the β2-β3 loop of the domain D3, and / or K300G, Q302T, and K304S on the β5-β6 loop of the domain D3 are substituted with amino acids that decrease the net pI of the protein, amino acids that have a negatively charged side chain, or amino acids that have an electro-negative side chain. In one embodiment, an amino acid having a side chain with a negative electrostatic charge may mean that the amino acid residue or molecule after substitution will have a negative electrostatic charge, depending on the properties of the adjacent amino acid residue or molecule, and may refer to a relative concept that arises from the distribution of electrons within the molecule. For example, an amino acid residue containing a hydroxyl (-OH) group may become electrostatically negative due to the unshared electron pair present on the oxygen atom, and this phenomenon may reduce the net pI of the protein.

[0092] In one embodiment, the β1-β2 loop of the Ig-like domain D3 can comprise amino acid residues T236 to T247 of the amino acid sequence of VEGFR 1 of Table 2. In one embodiment, the β2-β3 loop of the Ig-like domain D3 can comprise amino acid residues T256 to V262 of the amino acid sequence of VEGFR 1 of Table 2. In one embodiment, the β5-β6 loop of the Ig-like domain D3 can comprise amino acid residues D299 to L308 of the amino acid sequence of VEGFR 1 of Table 2. In the present disclosure, the β1-β2 loop can be referred to as "Site 1," the β2-β3 loop can be referred to as "Site 2," and the β5-β6 loop can be referred to as "Site 3." The inventors of the present disclosure confirmed that the amino acid residues present in the loop are exposed to the surface of domain D3, and achieved surface charge stabilization and structural stabilization of the modified fusion protein through mutation of the amino acid residues present in the loop.

[0093] In one embodiment, the amino acid residues present at Site 1 and Site 3 influence each other through interactions between amino acid residues due to their structural positions, thereby allowing double, triple or quadruple amino acid mutations (e.g., amino acid substitutions) to be introduced at each Site.

[0094] In one embodiment, the three loops should be understood as encompassing amino acid sequence variants within a range that maintains their function. For example, in one embodiment, the β1-β2 loop, the β2-β3 loop, and the β5-β6 loop may comprise amino acid sequences that exhibit at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequences TPRPVKLLRGHT, TPLNTRV, and DKMQNKDKGL, respectively.

[0095] In one embodiment, the linker mutation characteristic of the modified fusion protein may be one in which one or more amino acid residues on the β1-β2 loop of the Ig-like domain D3, and one or more amino acid residues on the β5-β6 loop of the domain D3, are substituted with an amino acid that reduces the net pI of the protein, an amino acid that has a negatively charged side chain, or an amino acid that has a negatively electrostatic side chain.

[0096] 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 the present disclosure may be produced by inserting the nucleic acid sequence or nucleic acid molecule encoding it into vectors of various origins. In this case, depending on the type or origin of the vector into which the nucleic acid sequence or nucleic acid molecule is inserted, the N-terminus of the Ig-like domain D2 may not start with G132, but may start from an amino acid sequence derived from the vector (e.g., amino acid sequence EF), resulting in a mutation in the amino acid sequence of the N-terminus of domain D2. However, such mutation of the N-terminal sequence due to the vector-derived amino acid sequence may not affect the productivity or properties of the modified fusion protein of the present disclosure.

[0097] 5. Linker mutation characteristics

[0098] In one embodiment, the modified fusion protein of the present disclosure may have linker mutation characteristics. In the present disclosure, a modified fusion protein having linker mutation characteristics may be used interchangeably as a "linker variant." The modified fusion protein of the present disclosure may, due to the linker mutation characteristics, (i) closely mimic the structure of domains D2 and D3 of the native VEGFR1 extracellular domain in which they bind to a ligand, (ii) have a length that reaches the dimerization site of domain D4, which is a structural characteristic that domain D3 of the native VEGFR1 extracellular domain must secure for smooth ligand binding, and (iii) have excellent structural stability of the linker itself.

[0099] In one embodiment, the linker mutation characteristic may be from about 14 to about 35 amino acids in length.

[0100] In one embodiment, the linker variant features the linker may comprise a GS repeat sequence.

[0101] In a linker mutation characteristic 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.

[0102] In one embodiment, the linker variant comprises an amino acid sequence of a hinge region derived from an immunoglobulin. In one embodiment, the amino acid sequence of the hinge region may be modified.

[0103] In the present disclosure, the linker may refer to the entire region located between the C-terminus of VEGFR1 domain D3 and the Fc region of human immunoglobulin produced by papain digestion, from P243 located at the N-terminal region of the CH2 region (A244-K360) contained in the Fc to the C-terminus of the domain D3. Here, the amino acid positions of the Fc region and the CH2 region contained in the Fc are according to the Kabat numbering (Kabat et. al., Sequences of Proteins of Immunological Interest 5 th Ed., US Department of Health and Human Services, NIH Publication No. 91-3242, 1991). In one embodiment, the linker may comprise a portion of a human immunoglobulin-derived region, wherein the portion may be an amino acid sequence of CH1 (K218-V223) or a hinge region (E226-P243) or a portion of such an amino acid sequence.

[0104] In the present disclosure, when the linker comprises an amino acid sequence of a hinge region derived from an immunoglobulin, the characteristics of the included hinge region are intact. For example, the hinge region of the Fc region has a cleavage site for various proteases, and the hinge region can induce oligomerization by O-glycosylation occurring at Ser / Thr amino acids after introduction into a fusion protein (Song et al., 2020. Comput Struct Biotechnol J. 18: pp. 3925-3935.). In the case of a modified fusion protein having the linker mutation characteristics of the present disclosure, the amino acid sequence of the hinge region derived from an immunoglobulin is modified to achieve excellent structural stability of the linker itself and improve its physical properties.

[0105] In one embodiment, the linker variant characteristic of the hinge region derived from an immunoglobulin may include a sequence derived from the CH1 region, an upper hinge, and / or a core hinge. In one embodiment, some amino acid sequences derived from the CH1 region of the immunoglobulin may form a β-strand or a β-sheet. In one embodiment, the hinge region derived from the immunoglobulin may be derived from human IgD, IgG, or a combination thereof.

[0106] In one embodiment, when the hinge region is derived from IgG1, the amino acid sequence KTHT of the papain recognition site having the mutation may be modified to an amino acid sequence selected from the group consisting of KTYT, TPP, ATPT, and ATPPTCP.

[0107] In one embodiment, the linker variant characteristic comprises (i) an amino acid sequence selected from the group consisting of GS, GSSG, (GSSG)2, GGGGS, GS(GGGGS) and GS(GGGGS)3, and (ii) a hinge region derived from an immunoglobulin selected from the group consisting of human IgG1, IgG4 or IgD / G1, in that order from the N-terminus to the C-terminus, wherein the amino acid sequence of the hinge region may be modified.

[0108] In the present disclosure, the hinge region derived from IgD / G1 may refer to a combination of hinge sequences of IgD and IgG1, and specifically, the hinge region derived from IgD / G1 may be NTGSGGEEKKKEKEKEEQEERSSDKTHTCPPCP (SEQ ID NO: 76). In one embodiment, the hinge sequence of IgD may be NTGSGGEEKKKEKEKEEQEERSS (SEQ ID NO: 77), NTGRGGEEKKKEKEKEEQEER (SEQ ID NO: 78), or a variant thereof. In one embodiment, the hinge sequence of IgG1 may be DKTHTCPPCP (SEQ ID NO: 79) or a variant thereof.

[0109] In one embodiment, the linker variant characteristic may be an amino acid sequence selected from the group consisting of CSSGDATPTSPPSP (SEQ ID NO: 68), CSKVDKKVEPKSSDTPPTCPPCP (SEQ ID NO: 69), CSGGGGSAEPKAGDATPPTCPPCP (SEQ ID NO: 70), CSGGGGSGGGGSGGGGSAESKYGPPCPPCP (SEQ ID NO: 71), CSNTGSGGEEKKKEKEKEEQEERSSDTPPTCPPCP (SEQ ID NO: 72), CSNTGSGGEEKKKEKEKEEQEERSCDTPPTCPPCP (SEQ ID NO: 73), CGSSGGSSGEPKSDATPTCPPCP (SEQ ID NO: 74), and CSKVDKKVEPKSSDKTYTCPPCP (SEQ ID NO: 75).

[0110] 6. Disulfide bond mutation characteristics

[0111] In one embodiment, the modified fusion protein of the present disclosure may have a disulfide bond mutation characteristic. In the present disclosure, a modified fusion protein having such a disulfide bond mutation characteristic may be interchangeably referred to as a "disulfide bond mutation." Since the modified fusion protein of the present disclosure is a fusion of heterologous proteins, it is expected that stability will be reduced at the site where different types of proteins bind. Therefore, the disulfide bond mutation characteristic was introduced to prevent physical cleavage of the modified fusion protein and improve its stability.

[0112] To introduce disulfide bond mutation characteristics, candidate amino acid residues were selected using a 3D structure energy prediction program (foldx in YASARA, Schymkowitz et al., 2005., Nucleic Acids Research. 33:W382-388), and it was confirmed that the characteristics of the modified fusion protein (e.g., productivity, physical properties, and binding affinity to ligand) due to disulfide bonds were improved by substituting these amino acid residues with cysteine ​​(see Example 2).

[0113] In one embodiment, for the disulfide bond mutation characteristic, (i) one of the amino acid residues on the β1-β2 loop of the Ig-like domain D3 or one of the amino acid residues on the β5-β6 loop, and (ii) the amino acid residue at the +2 position relative to Y329 of the Ig-like domain D3 can be substituted with cysteine. By such substitution, the modified fusion protein of the present disclosure can have a disulfide bond within the protein. In one embodiment, the selection of the amino acid residues present at the positions described in (i) and (ii) above for the introduction of the disulfide bond mutation characteristic can be determined according to the criteria (1) to (3) above.

[0114] 7. Fusion proteins

[0115] In one embodiment of the present disclosure, a modified fusion protein capable of simultaneously binding a VEGF ligand and a PlGF ligand may be provided. In one embodiment, the modified fusion protein may comprise a first binding specificity for a VEGF ligand and a second binding specificity for a PlGF ligand. Wherein the VEGF ligand may be one or more of VEGF-A, VEGF-B, VEGF-C, and VEGF-D, and specifically may be VEGF-A.

[0116] In the present disclosure, a fusion protein comprising D2 and D3 of VEGFR1 based on the VEGFR1 wild-type protein used as a control may be referred to as PB101 (a fusion protein comprising D2 of VEGFR1 and D3 in which 1 to 3 amino acids are glycosylated based on the VEGFR1 wild-type protein) or VEGF-Grab.

[0117] In one embodiment, the modified fusion protein may comprise a VEGFR1 extracellular domain, a linker, and a multimerization domain. In one embodiment, the modified fusion protein may comprise components arranged in the following order from the N-terminus to the C-terminus: the VEGFR1 extracellular domain, the linker, and the multimerization domain. In one embodiment, the VEGFR1 extracellular domain may comprise the Ig-like domain D2 and the Ig-like domain D3 of VEGFR1.

[0118] In one embodiment, the modified fusion protein may have (a) surface charge transfer properties, (b) linker mutation properties, and (c) disulfide bond mutation properties.

[0119] (a) amino acid substitutions of K241E, L243S, R244V, and H246E on the β1-β2 loop of domain D3, amino acid substitutions of L258A, L258S, or L258D on the β2-β3 loop of domain D3, and / or amino acid substitutions of K300G, Q302T, and K304S on the β5-β6 loop of domain D3;

[0120] (b) the linker is an amino acid sequence selected from the group consisting of CSSGDATPTSPPSP (SEQ ID NO: 68), CSKVDKKVEPKSSDTPPTCPPCP (SEQ ID NO: 69), CSGGGGSAEPKAGDATPPTCPPCP (SEQ ID NO: 70), CSGGGGSGGGGSGGGGSAESKYGPPCPPCP (SEQ ID NO: 71), CSNTGSGGEEKKKEKEKEEQEERSSDTPPTCPPCP (SEQ ID NO: 72), CSNTGSGGEEKKKEKEKEEQEERSCDTPPTCPPCP (SEQ ID NO: 73), CGSSGGSSGEPKSDATPTCPPCP (SEQ ID NO: 74), and CSKVDKKVEPKSSDKTYTCPPCP (SEQ ID NO: 75),

[0121] (c) The L243 amino acid residue on the β1-β2 loop of domain D3 is substituted with cysteine, and the amino acid residue at position +2 relative to Y329 of domain D3 is substituted with cysteine.

[0122] In one embodiment, the modified fusion protein may comprise an amino acid sequence selected from the group consisting of amino acid sequences C61 to C75 of Table 2. In one embodiment, the modified fusion protein may comprise an amino acid sequence selected from the group consisting of amino acid sequences C61 to C75 of Table 2.

[0123] In one embodiment, the modified fusion protein can comprise at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to an amino acid sequence selected from the group consisting of amino acid sequences C61 to C75 of Table 2. Such variants can include amino acid mutations (e.g., conservative amino acid substitutions) that occur in other amino acid sequences, while still including or maintaining the amino acid mutations that occur in the properties of the modified fusion proteins of the present disclosure.

[0124] In one embodiment, the components of the modified fusion protein of the present disclosure (VEGFR1 extracellular domain, linker and multimerization domain) can have post-translational modifications including, for example, glycosylation, sialylation, acetylation and phosphorylation.

[0125] In one embodiment, the modified fusion protein of the present disclosure may comprise conservative amino acid substitutions at positions other than those at which amino acid mutations occur in the properties described herein. Such conservative amino acid substitutions may be introduced into any of the components of the modified fusion protein of the present disclosure (e.g., the extracellular domain, linker, and multimerization domain of VEGFR1).

[0126] In one embodiment, conservative substitutions that may be included in the modified fusion proteins of the present disclosure may be as set forth in Table 0 below. Such conservative substitutions in the modified fusion proteins of the present disclosure may result in desired activities, such as maintained / improved target binding affinity, reduced immunogenicity, or improved serum / thermal stability.

[0127] Examples of original residue-conservative substitutions ResiduesAla (A)Val; Leu; IleArg (R)Lys; Gln; AsnAsn (N)Gln; Lys; Asp; Lys; ArgAsp (D)Glu; AsnCys (C)Ser; AlaGln (Q)Asn; GluGlu (E)Asp; GlnGly (G)AlaHis (H)Asn; Gln; Lys; ArgIle (I)Leu; Val; Met; Ala; Phe; NleLeu (L)Nle; Ile; Val; Met; Ala; PheLys (K)Arg; Gln; AsnMet (M)Leu; Phe; IlePhe (F)Trp; Leu; Val; Ile; Ala; TyrPro (P)AlaSer (S)ThrThr (T)Val; SerTrp(W)Tyr; PheTyr (Y)Trp; Phe; Thr; SerVal (V)Ile; Leu; Met; Phe; Ala; Nle

[0128] In one embodiment, the modified fusion protein of the present disclosure may be as disclosed in C61 to C75 of Table 2.

[0129] 8. Conjugate of fusion protein and scFv

[0130] In one embodiment of the present disclosure, a conjugate of a modified fusion protein according to an embodiment of the present disclosure and an scFv may be provided. In one embodiment, the scFv may be an scFv targeting PD-L1, and in particular, may be an atezolizumab scFv or an A167 scFv that binds to PD-L1 as disclosed in Table 9. In the present disclosure, the conjugate of a modified fusion protein according to an embodiment of the present disclosure and an scFv targeting PD-L1 may be referred to as PB203.

[0131] In one embodiment, the conjugate of the modified fusion protein of the present disclosure and the scFv may be as set forth in SEQ ID NOs: 1 to 32 of Table 9.

[0132] In one embodiment, the conjugate can comprise at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to an amino acid sequence selected from the group consisting of amino acid sequences of Table 9. Such variants can include amino acid mutations (e.g., conservative amino acid substitutions) that occur in other amino acid sequences, while still including or maintaining the amino acid mutations that occur in the characteristics of the conjugates of the present disclosure.

[0133] In one embodiment, the components of the complex of the present disclosure (scFv, VEGFR1 extracellular domain, linker and multimerization domain) can have post-translational modifications including, for example, glycosylation, sialylation, acetylation and phosphorylation.

[0134] In one embodiment, the conjugate of the present disclosure may comprise conservative amino acid substitutions at positions other than those at which amino acid mutations occur in the characteristics described herein. Such conservative amino acid substitutions may be introduced into any of the components of the modified fusion protein of the present disclosure (e.g., scFv, the extracellular domain of VEGFR1, the linker, and the multimerization domain).

[0135] In one embodiment, the conjugate of the present disclosure may comprise a signal peptide or signal sequence for protein secretion from a cell. For example, the conjugate of the present 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 one that is recognized and processed by a eukaryotic host cell (i.e., cleaved by a signal peptide hydrolase). Information regarding signal peptides is well known in the art and is provided, for example, but not limited to, in Korean Patent No. 10-2228921. In one embodiment, the signal peptide may be a human interleukin signal sequence. In one embodiment, the signal peptide may be composed of an amino acid sequence of MVSYWDTGVLLCALLSCLLLTGSSSG (tPA), MEFGLSWVFLVALFRGVQC (H7), MKWVTFISLLFLFSSAYS (human serum albumin), MGWSCIILFLVATATGVHS (mouse Ig heavy chain), MDWTWRVFCLLAVAPGAHS (human Ig heavy chain), or MYRMQLLSCIALSLALVTNS (human interleukin-2), but any signal peptide that can be used by a person skilled in the art for the production of a fusion protein may be used without being suggested.

[0136] In one embodiment, the conjugate of the present disclosure has an activity of about 1 mg / ml, 500 ng / ml, 300 ng / ml, 100 ng / ml, 70 ng / ml, 50 ng / ml, 45 ng / ml, 40 ng / ml, 35 ng / ml, 30 ng / ml, 25 ng / ml, 20 ng / ml, 10 ng / ml, 5 ng / ml, 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, IC less than or equal to 5 pg / ml or 1 pg / mL (inclusive, including any value between these numbers) 50 can have

[0137] In one embodiment, the complex of the present disclosure has an activity against a binding partner (e.g., VEGF, PD-L1 and / or PlGF) at a concentration of 1.0 mM, 500 μM, 100 μM, 50 μM, 25 μM, 10 μM, 5 μM, 1 μM, 900 nM, 800 nM, 700 nM, 600 nM, 500 nM, 400 nM, 350 nM, 300 nM, 250 nM, 200 nM, 150 nM, 100 nM, 95 nM, 90 nM, 85 nM, 80 nM, 75 nM, 70 nM, 65 nM, 60 nM, 55 nM, 50 nM, 45 nM, 40 nM, 35 It may have a Kd less than any of nM, 30 nM, 25 nM, 20 nM, 15 nM, 10 nM, 5 nM, 1 nM, 900 pM, 800 pM, 700 pM, 600 pM, 500 pM, 400 pM, 300 pM, 200 pM, 100 pM, 50 pM, 25 pM, 12.5 pM, 6.25 pM, 5 pM, 4 pM, 3 pM, 1 pM, 0.5 pM, 0.1 pM, 0.05 pM or 0.01 pM (inclusive).

[0138] III. Nucleic Acids, Vectors, and Host Cells

[0139] nucleic acids

[0140] In one embodiment of the present disclosure, an isolated nucleic acid encoding any of the modified fusion proteins and scFvs of the present disclosure, such as an anti-PD-L1 scFv, a VEGFR1 extracellular domain and / or a multimerization domain, and a conjugate component of the modified fusion protein and scFv, may be provided. Nucleic acids encoding mammalian VEGFRs are described for all receptor types. Exemplary nucleic acid sequences, including but not limited to, can be found in U.S. Patent No. 7,928,072 and WO2006 / 113277. mRNA encoding human VEGFR1 and VEGFR2 can be found under GenBank Accession Nos. NM_002019.4 and NM_002253.2, respectively.

[0141] An isolated nucleic acid encoding a multimerization domain (e.g., an Fc region) may be provided in the present disclosure.

[0142] An isolated nucleic acid encoding a modified fusion protein of the present disclosure may be provided.

[0143] The isolated nucleic acid sequence encoding a modified fusion protein or a component of a fusion protein of the present disclosure (e.g., the extracellular domain or multimerization domain of VEGFR1) may further comprise a nucleic acid sequence encoding a linker.

[0144] In one embodiment, the isolated nucleic acid may further comprise a sequence encoding a signal peptide that serves as a signal sequence for secretion of the modified fusion protein from the host cell. In one embodiment, the isolated nucleic acid may not comprise a sequence encoding a signal peptide.

[0145] Isolated nucleic acid molecules encoding the modified fusion protein or a component of the fusion protein (e.g., VEGFR1 extracellular domain, linker or multimerization domain) of the present disclosure may be in the form of RNA, such as mRNA, hnRNA, tRNA or any other form, or in the form of DNA, including but not limited to cDNA and genomic DNA obtained by cloning or produced synthetically, or any combination thereof. Such isolated nucleic acid molecules may be prepared by a variety of methods known in the art (see, e.g., Molecular Cloning: A Laboratory Manual (Sambrook et al., 4 th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2012) and Current Protocols in Molecular Biology (FM Ausubel, et al. eds., 2003).

[0146] vector

[0147] The present disclosure may, in one embodiment, relate to a nucleic acid delivery vehicle or use thereof for introducing one or more nucleic acid sequences encoding a complex or a component of a complex into a cell for expressing said protein. The nucleic acid sequence may be a sequence of said isolated nucleic acid.

[0148] In one embodiment, examples of nucleic acid delivery vehicles include liposomes, biocompatible polymers (including natural and synthetic polymers); lipoproteins; polypeptides; polysaccharides; lipopolysaccharides; artificial viral envelopes; metal particles; and bacteria, viruses such as baculovirus, adenovirus, and retrovirus, bacteriophages, cosmids, plasmids, fungal vectors, and other recombinant vehicles commonly used in the art for expression in various eukaryotic and prokaryotic hosts. In one embodiment, the nucleic acid delivery vehicle can be an expression vector, such as a plasmid. In one embodiment, the nucleic acid sequence comprised in the expression vector can be operably linked to an expression control sequence.

[0149] In one embodiment, the vector may include any elements necessary for establishing the typical functions of an expression vector, such as a promoter, ribosome binding element, terminator, enhancer, selection marker, and origin of replication. The promoter may be a constitutive, inducible, or repressible promoter. Exemplary promoters are provided in, but are not limited to, Korean Patent No. 10-0659477.

[0150] Many expression vectors (e.g., derived from bacterial, yeast, plant, or mammalian cells) capable of delivering nucleic acids into cells are known in the art and can be used in the present disclosure to produce conjugates or conjugate components within cells. For example, E. coli can be transformed with a plasmid engineered to contain a nucleic acid encoding the conjugate, such as pBR322 (Mandel et al., J. Mol. Biol., 1970, 53:154), to produce a modified conjugate. The expressed conjugate or conjugate component can be harvested from the cells and purified according to conventional techniques known in the art and described in the present disclosure.

[0151] In one embodiment, an expression vector comprising a nucleic acid encoding the above-described conjugate and capable of replication in a bacterial or eukaryotic host can be used to transfect a host, thereby directly expressing the nucleic acid for production of the conjugate, which can be recovered in a biologically active form. In the present disclosure, the biologically active form includes a form capable of binding to a VEGF ligand, PD-L1, or PlGF ligand.

[0152] In one embodiment, an expression vector comprising a nucleic acid molecule encoding a conjugate or conjugate component can be identified by at least three common methods, including but not limited to: (a) DNA-DNA hybridization, (b) the presence or absence of a "marker" gene function, and (c) expression of the inserted sequence. Specific details of these three methods are provided in, but are not limited to, Korean Patent No. 10-0659477 or 10-2228921.

[0153] host cell

[0154] In one embodiment of the present disclosure, a host cell comprising the expression vector encoding the conjugate of the present disclosure may be provided. Such a host cell is suitable for expressing the conjugate of the present disclosure and may correspond to a host-vector system for producing the conjugate. In one embodiment, the host cell may be used to produce a viral particle (e.g., a recombinant viral vector).

[0155] The term "host cell" as used herein may include a cell that is or can be a recipient for the vector(s) of the present disclosure and its progeny. Due to natural, accidental, or sophisticated mutations, the progeny may not necessarily be completely identical (in morphology or genome of the entire DNA complement) to the original parent cell. In one embodiment, the host cell may be, but is not limited to, a bacterial cell such as E. coli, a yeast cell such as Pichia pastoris, an insect cell such as Spodoptera frugiperda, or a mammalian cell such as a COS, HEK, or CHO cell.

[0156] In one embodiment, the complex of the present disclosure can be expressed transiently, constitutively, or permanently in a host cell.

[0157] In one embodiment of the present disclosure, a method for producing a conjugate of the present disclosure is provided by culturing the host cell or host-vector system cells under conditions that allow for the production of the conjugate, and then recovering the produced conjugate. Conjugates useful in the practice of the present disclosure can be produced by expression in a prokaryotic or eukaryotic expression system. Methods for culturing host cells or producing conjugates from host cells are well known in the art, and are disclosed, for example, in Korean Patent No. 10-2228921, but are not limited thereto.

[0158] In one embodiment, the conjugate of the present disclosure produced from the host cell can be purified and identified through various methods. Methods for purifying and identifying the conjugate produced from the host cell are well known in the art and are disclosed, for example, in Korean Patent No. 10-2228921, but are not limited thereto. For further purification of the conjugate, many purification methods can be used, including, but not limited to, conventional ion exchange chromatography, affinity chromatography, differential sugar chromatography, hydrophobic interaction chromatography, reversed phase chromatography, or gel filtration.

[0159] IV. Viral particles and methods for producing viral particles

[0160] In one embodiment of the present disclosure, a viral particle (or virion) comprising a nucleic acid encoding a combination of the present disclosure may be provided.

[0161] Viral vectors can be used to deliver nucleic acids encoding a complex or a component of a complex for protein expression in target cells within a specific target tissue (e.g., a diseased tissue). Many types of viruses are known, and many are being studied for the purpose of delivering nucleic acids to target cells. Exogenous nucleic acids can be inserted into vectors, such as adenoviruses, partially deleted adenoviruses, completely deleted adenoviruses, adeno-associated viruses (AAV), retroviruses, lentiviruses, etc., for delivery into cells.

[0162] In one embodiment, the cells are present within the subject, and the virus can be delivered intravenously, intramuscularly, intravenously, or via other routes of administration. In one embodiment, the viral vector can include those derived from adenovirus, adeno-associated virus (AAV), and retrovirus (including lentivirus, e.g., human immunodeficiency virus (HIV)). For exemplary viral vectors, see, but are not limited to, U.S. Patent No. 7,928,072 and WO2006 / 113277, which are incorporated herein by reference in their entirety. Viral particles comprising nucleic acids encoding a conjugate and methods for producing the same are well known in the art, and are described, for example, but not limited to, in Korean Patent No. 10-2228921.

[0163] IV. Treatment using conjugates and virus particles

[0164] One embodiment of the present disclosure may be directed to a pharmaceutical composition for use in preventing, ameliorating or treating a disease associated with a VEGF ligand, a VEGF receptor or PD-L1 or at least one symptom of such a disease, comprising a combination of the present disclosure as an active ingredient.

[0165] One embodiment of the present disclosure may be directed to a method of preventing, ameliorating or treating a disease or at least one symptom of a disease associated with a VEGF ligand, a VEGF receptor or PD-L1, comprising administering a combination of the present disclosure.

[0166] One embodiment of the present disclosure may be directed to a use of a combination of the present disclosure for preventing, ameliorating or treating a disease associated with a VEGF ligand, a VEGF receptor or PD-L1, or at least one symptom of such a disease.

[0167] One embodiment of the present disclosure may be directed to a combination of the present disclosure or a pharmaceutical composition comprising the same for use in a method for preventing, ameliorating or treating a disease associated with a VEGF ligand, a VEGF receptor or PD-L1 or at least one of the symptoms of such a disease.

[0168] In one embodiment, the combination of the present disclosure can be used in the prevention, amelioration, or treatment of a disease or symptom associated with VEGF ligand, VEGF receptor, or PD-L1, or at least one of the symptoms of such a disease, in the form of a nucleic acid encoding the same, as a nucleic acid delivery vehicle or viral particle (e.g., a recombinant viral vector). In one embodiment, the nucleic acid delivery vehicle or viral particle can be applied to gene therapy for use in the prevention, amelioration, or treatment of a disease or symptom associated with VEGF ligand, VEGF receptor, or PD-L1. In one embodiment, any gene therapy method available in the art can be used according to the present disclosure. In one embodiment, the nucleic acid is introduced into a cell prior to in vivo administration of the final recombinant cell. Cells into which nucleic acids are introduced for gene therapy purposes include all available cell types, including, but not limited to, epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes, blood cells (e.g., T-lymphocytes, B-lymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, or granulocytes), various stem cells or progenitor cells, such as hematopoietic stem or progenitor cells obtained from bone marrow, umbilical cord blood, peripheral blood, fetal liver, etc. In one embodiment, the cells used for gene therapy may be autologous cells of the patient.

[0169] In one embodiment, the binding entity can bind to a VEGF protein, PlGF, and / or a PD-L1 protein. In one embodiment, the binding entity can 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, VEGF-D, PlGF, and PD-L1; (b) blocking binding of a VEGF family protein to a VEGF receptor and / or binding of a PD-L1 protein to a PD-1 receptor; (c) inhibiting activation of the VEGF signaling pathway and / or the PD-L1 signaling pathway; (d) preventing, ameliorating, and / or treating a disease, such as a chronic infection, tissue allograft, ocular disease, autoimmune disease, inflammatory disease, neoplasm-forming disease, or cancer; (e) attenuating, preventing, or preventing neoplasm or cancer growth; (f) inhibiting neoplasm or cancer metastasis. The activity of the conjugate can be measured in vivo and / or in vitro.

[0170] In one embodiment, the pharmaceutical composition may include a pharmaceutically acceptable carrier and / or diluent. In one embodiment, the pharmaceutically acceptable carrier may be a sterile liquid, such as water and oil, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, and the like. Saline and aqueous dextrose, polyethylene glycol (PEG), and glycerol solutions may also be used as liquid carriers, particularly for injectable solutions. Such pharmaceutical compositions may further include additional ingredients, such as preservatives, buffers, isotonic agents, antioxidants and stabilizers, nonionic wetting or clarifying agents, thickening agents, and the like. The pharmaceutical compositions of the present disclosure may be packaged in single unit dose or multi-dose form. Such compositions are generally formulated as sterile and substantially isotonic solutions. In addition, the compositions may be formulated to have osmotic values ​​suitable for the aqueous humor of the eye and ocular tissues. These osmolality values ​​typically range from about 200 to 400 milliosmoles per kilogram of water (mOsm / kg), or about 300 mOsm / kg. The retina is thought to have an osmolality of about 283 mOsm / kg.

[0171] In one embodiment, the pharmaceutical composition may be a parenteral composition. It may be advantageous to formulate the parenteral composition in a dosage unit form for uniformity of dosage and ease of administration.

[0172] disease or illness

[0173] In one embodiment, the disease associated with VEGF ligand, VEGF receptor or PD-L1 may include, but is not limited to, chronic infection, tissue allograft, autoimmune disease (e.g., rheumatoid arthritis, multiple sclerosis, immune-mediated recurrent miscarriage or systemic lupus erythematosus), inflammatory disease (e.g., inflammatory arthritis, osteoarthritis, or psoriasis), neoplastic disease, cancer (e.g., breast cancer, lung cancer, gastric cancer, pancreatic cancer or leukemia), disease associated with angiogenesis (e.g., atherosclerosis) or ocular disease (e.g., age-related macular degeneration, choroidal neovascularization, or uveitis).

[0174] In one embodiment, the cancer is prostate cancer, urethral cancer, penile cancer, breast cancer, lung cancer, esophageal cancer, small intestine cancer, colon cancer, rectal cancer, colon cancer, liver cancer, urinary tract cancer (e.g., bladder cancer), kidney cancer, lung cancer (e.g., non-small cell lung cancer), ovarian cancer, cervical cancer, endometrial cancer, vaginal carcinoma, vulvar carcinoma, pancreatic cancer, stomach cancer, endocrine cancer, head and neck cancer, thyroid cancer, parathyroid cancer, thyroid cancer, adrenal cancer, skin cancer (e.g., melanoma, cutaneous squamous cell carcinoma (CSCC), squamous cell carcinoma of the head and neck (HNSCC)), Hodgkin's disease, bone cancer, hematopoietic cancer of the lymph or bone marrow, chronic or acute leukemia, head and neck cancer, nasopharyngeal carcinoma (NPC), glioblastoma, teratocarcinoma, neuroblastoma, adenocarcinoma, mesenchymal origin Cancers (e.g., fibrosarcoma or rhabdomyosarcoma), soft tissue sarcomas and carcinomas, choriocarcinoma, hepatoblastoma, central nervous system (CNS) tumors, primary CNS lymphoma, spinal cord tumors, brainstem gliomas, pituitary adenomas, Karposi's sarcoma, or Wilm's tumor may include, but are not limited to. The combinations of the present disclosure may act as anti-PD-L1 and anti-angiogenic agents or anti-VEGF and anti-PD-L1 agents to ameliorate, prevent, and / or treat cancer or symptoms of cancer.

[0175] Method of administration and dosage

[0176] In one embodiment of the present disclosure, a method for delivering an effective amount of a conjugate to a subject may be provided. The conjugate may be delivered to the subject within a composition. The conjugate may also be delivered to the subject by a nucleic acid delivery vehicle or viral particle (e.g., a recombinant viral vector) comprising a nucleic acid encoding the conjugate. In one embodiment of the present disclosure, a composition comprising the conjugate or a nucleic acid delivery vehicle or viral particle (e.g., a recombinant viral vector) comprising a nucleic acid encoding the conjugate may be provided.

[0177] In one embodiment, the compositions of the present disclosure can be administered to a subject by any route, including but not limited to: intravenously (e.g., by an infusion pump), intraperitoneally, intraocularly, intraarterially, intrapulmonaryly, orally, inhalationally, intravesically, intramuscularly, intratracheally, subcutaneously, intraocularly, intrathecally, transdermally, transpleurally, intraarterially, topically, inhalatively (e.g., as a mist of a spray), mucosally (e.g., via the nasal mucosa), subcutaneously, transdermally, gastrointestinal, intraarticularly, intracapsularly, intraventricularly, intracranially, intraurethrally, intrahepatically, and intratumorally. In one embodiment, the compositions of the present disclosure can be administered by any conventional route, such as, for example, by infusion injection or bolus injection, absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal mucosa, intestinal mucosa, etc.), and may be administered together with other biologically active ingredients. Administration may be systemic or local. In one embodiment, the compositions of the present disclosure may be introduced into the central nervous system by any suitable route, including intracerebroventricular injection and intraspinal injection, wherein intracerebroventricular injection may be achieved by an intracerebroventricular catheter, for example, a catheter attached to a reservoir such as an Ommaya reservoir.

[0178] In one embodiment, the optimal effective dose of the conjugate, the nucleic acid delivery vehicle encoding the conjugate, or a composition comprising the conjugate can be determined empirically and may vary depending on the type and severity of the disease, the route of administration, the progression of the disease and the health of the subject, and the weight and body area of ​​the subject. Such determinations are within the skill of those skilled in the art.

[0179] In one embodiment, the combination may be administered at, for example, about 0.05 ng to about 20 mg per kg of body weight per day. In one embodiment, the dosage of the complex is 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 at most about 20.0 mg / kg, at most about 17.0 mg / kg, at most about 14.0 mg / kg, at most about 11.0 mg / kg, at most about 9.0 mg / kg, at most about 7.0 mg / kg, at most about 5.5 mg / kg, at most about 5.0 The dosage of the combination may be about 4.5 mg / kg or less, about 4.0 mg / kg or less, about 3.5 mg / kg or less, about 3.0 mg / kg or less, about 2.5 mg / kg or less, about 2.0 mg / kg or less, about 1.5 mg / kg or less, or about 1.0 mg / kg or less. In one embodiment, the dosage of the combination 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.

[0180] In one embodiment, the amount of nucleic acid delivery vehicle (e.g., a recombinant viral vector) comprising a nucleic acid encoding a conjugate protein of the present disclosure is about 10 4 About 1014 The DNAse particle resistance (drp) titer of the drp can be administered to the subject. In one embodiment, the amount of the nucleic acid delivery vehicle comprising the nucleic acid encoding the modified conjugate protein is about 10 per dose. 5 About 10 13 , about 10 6 About 10 12 , about 10 7 About 10 11 , about 10 8 About 10 10 , about 10 9 About 10 10 , about 10 10 About 10 11 , or about 10 11 About 10 12 It can be administered to individuals through drp.

[0181] In one embodiment, a composition comprising a conjugate protein of the present disclosure or a nucleic acid delivery vehicle encoding the same may be administered as a single daily dose, or the total daily dose may be administered in divided doses of 2, 3, or 4 times daily. In one embodiment, a composition comprising a conjugate protein of the present disclosure may be administered 6 times a week, 5 times a week, 4 times a week, 3 times a week, 2 times a week, once a week, once every 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 vehicle comprising a nucleic acid encoding a conjugate protein of the present disclosure can 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 every year.

[0182] Diagnosis or detection

[0183] The conjugate protein of the present disclosure may be labeled with a detectable label, such as a radioisotope, fluorescent label, toxin label, enzyme label, chemiluminescent label, or nuclear magnetic resonance contrast agent, to confirm ligand-receptor binding interactions. Assay systems applicable to chimeric molecules are also contemplated. Such detectable labels are widely known in the art.

[0184] V. Manufactured Goods and Kits

[0185] In one embodiment, the present disclosure may relate to an article of manufacture or kit comprising a conjugate protein of the present disclosure, a recombinant viral vector expressing the same, or a composition comprising the same, within suitable packaging. In one embodiment, such suitable packaging is well known in the art and may include, but is not limited to, vials (e.g., sealed vials), containers, ampoules, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. Such articles of manufacture may additionally be sterilized and / or sealed.

[0186] In one embodiment, the kit can further comprise instructions for using the composition for the purposes described herein. In one embodiment, the kit can further comprise other components desirable from a commercial and user standpoint, such as other buffers, diluents, filters, needles, syringes, and package inserts (PI) comprising instructions for performing any of the methods described herein. For example, in one embodiment, the kit can comprise (i) a conjugate protein described herein and / or a recombinant viral vector encoding a conjugate protein described herein, (ii) a pharmaceutically acceptable carrier, and (iii) one or more of the following: a buffer, a diluent, a filter, a needle, a syringe, and a package insert comprising instructions for performing the administration.

[0187] Hereinafter, the configuration and effects of the present disclosure will be described in more detail with examples. However, these examples are provided solely for illustrative purposes to aid understanding of the present disclosure, and the scope and range of the present disclosure are not limited by the examples below.

[0188] Example 1 - Preparation of expression vectors

[0189] In the example below, the coding sequence region (CDS) of the designed conjugate was codon optimized into a form suitable for CHO production cells, and then the conjugate was manufactured in the final pcDNA3.1(+) vector (Invitrogen) through gene synthesis (Geneuniversal).

[0190] Example 2 - Design of modified fusion proteins

[0191] Example 2.1 - Preparation and characterization of surface charge transition variants

[0192] To obtain a fusion protein comprising a VEGFR receptor structure with increased physical properties, the inventors of the present disclosure analyzed the surface charge distribution of the protein using the structure containing the entire extracellular domain of VEGFR1 (PDB entry. 5t89) as a template and derived regions with concentrated positive charges and sites with hydrophobic surface charges.

[0193] Through this, it was confirmed that the side chains of amino acid residues present in the β1-β2 loop (named Site 1) corresponding to amino acid residues T236 to T247 in the VEGFR1 domain D3, the β2-β3 loop (named Site 2) corresponding to amino acid residues T256 to V262, and the β5-β6 loop (named Site 3) corresponding to amino acid residues D299 to L308 are exposed to the surface of the fusion protein. An attempt was made to achieve surface charge and structural stabilization of the fusion protein by mutation of amino acid residues located in the flexible loop exposed to the surface. For example, an attempt was made to substitute amino acid residues exposed on the surface with amino acid residues present at homologous positions in VEGFR2. Additionally, amino acid residues at Site 2 are exposed on the protein surface on the side that binds the ligand (VEGF or PlGF), and mutations at that position affect the binding affinity for the ligand or solubility due to the exposure of surface residues.

[0194] Example 2.2. - Preparation and characterization of linker variants

[0195] In the case of human VEGF receptor, binding to the ligand VEGF or PlGF occurs through interaction with immunoglobulin-like domains D2 and D3. In the case of a fusion protein using domains D2 and D3, domains D2 and D3 have an open structure before binding to the ligand, and form a closed structure upon binding to the ligand. Depending on the binding length of domain D3 and the multimerization domain, if the structures of domains D2 and D3 do not secure enough space to bind to the ligand, or if interference occurs between the structures, the binding affinity of the fusion protein to the ligand may decrease. Therefore, the binding length and binding method of domain D3 and the multimerization domain in a fusion protein can affect the structure of the fusion protein that binds to the ligand and the binding affinity for the ligand.

[0196] Therefore, in order to mimic and secure the structural characteristics of the native human VEGF receptor, the inventors of the present invention changed the length and type of linker in the modified fusion protein of the present disclosure. Specifically, a linker comprising a polypeptide of Gly-Ser repeat sequences, an amino acid sequence of a hinge region derived from IgG1, IgG4, and IgD, and a mutant sequence of these hinges was introduced. Through this, the tension formed in the loop (linker) between domain D3 and the multimerization domain (Fc region) upon ligand binding was reduced, thereby increasing physical stability.

[0197] In addition, amino acid sequences derived from the hinge region of immunoglobulins have cleavage sites for various proteases due to their own characteristics (Vlasak and Ionescu. 2011. Mabs 3: pp. 253-263), and after introduction into a fusion protein, glycosylation, such as O-glycosylation, may occur, resulting in oligomerization (Song et. al. 2020. Computational and Structural Biotechnology Journal 18: pp. 3925-3935). In order to improve the in vivo stability of the modified fusion protein of the present disclosure, the inventors attempted to further modify the amino acid sequence at cleavage sites by proteases, such as cleavage sites for papain, and to modify the Ser or Thr amino acid residues where glycosylation occurs. Additionally, to increase the structural flexibility of the linker, attempts were made to substitute cysteines present in sequences derived from the core hinge of the Fc hinge region (e.g., CPPCP). Modified fusion proteins according to one embodiment of the present disclosure exhibit excellent productivity and physical properties, as well as excellent binding properties to ligands.

[0198] Example 2.3. - Preparation and characterization of disulfide bond variants

[0199] Since fusion proteins are conjugates of heterologous proteins, nonspecific cleavage at the binding site is expected to reduce stability. The terminus of domain D3 forms a loop, potentially lacking secondary structure. Furthermore, since this is where the linkage with the heterologous protein, the multimerization domain, begins, stability is expected to be low. By introducing a covalent disulfide bond, we aimed to induce interdomain junctions near the site of heterologous protein binding, thereby preventing physical cleavage of the protein and improving stability.

[0200] The potential sites for disulfide bond introduction in the fusion protein were identified through 3D structure-based analysis. Among the residues located in the variable loop adjacent to the terminal region of domain D3, those residues with the side chain oriented toward the linker and the distance between the terminal atom of the side chain and the Y329 Cα atom was within 6 Å were selected as mutable residues for disulfide bond formation. After securing the 3D structure of the fusion protein through a structural modeling program (Phyre2, Swiss model), a structural model with the disulfide bond introduced was obtained through in silico mutation of the introduction site. Afterwards, the energy level change of the fusion protein due to the introduction of the disulfide bond was predicted through a 3D structure energy prediction program (foldx in YASARA, Schymkowitz et al. 2005. Nucleic Acids Research. 33:W382-388).

[0201] In the fusion protein, amino acid residues on the β1-β2 loop or the β5-β6 loop of domain D3 were selected as positions that are physically close to the end of domain D3 and can form a disulfide bond, and in the case of the end of domain D3, an amino acid residue at the +2 position based on Y329 was selected.

[0202] Example 2.4. - Preparation and characterization of combination variants

[0203] Combination mutants of fusion proteins having the characteristics of mutants exhibiting excellent characteristics of Examples 2.1 to 2.3 were prepared as shown in Table 2, and the characteristics of each mutant are as shown in Table 1.

[0204]

[0205] Table 1 lists the identification codes, amino acid sequences, and sequence numbers of the proteins described in this disclosure.

[0206] Protein code Amino acid sequence VEGFR1 (FLT1) SEQ ID NO: 41 DLAARNILLSENNVVKICDFGLARDIYKNPDYVRKGDTRLPLKWMAPESIFDKIYSTKSDVWSYGVLLWEIFSLGGSPYPGVQMDEDFCSRLREGMRMRAPEYSTPEIYQIMLDCWHRDPKERPRFAELVEKLGDLLQANVQQDGKDYIPINAILTGNSGFTYSTPAFSEDFFKESISAPKFNSGSSDDVRYVNAFKFMSLERIKTFEELLPNATSMFDDYQGDSSTLLASPMLKRFTWTDSKPKASLKIDLRVTSKSKESGLSDVSRPSFCHSSCGHVSEGKRRFTYDHAELERKIACCSPPPDYNSVVLYSTPPIVEGF-Grab1 (a fusion protein based on the VEGFR1 wild-type protein containing D2 of VEGFR1 and D3 on which two amino acids are glycosylated) Protein) SEQ ID NO: 42EFGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVQISTPSPV TLLRGHTLVLNCTATTPLNTRVQMTWSYPDEKNKRASVRRRIDQSNSHANIFYSVLTIDKMQNKDKGLYTCRVRSGPSFKSVNTSVHIYDKALEDKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKVEGF-Grab3 (a fusion protein based on VEGFR1 containing D2 of VEGFR1 and D3 with 3 amino acids glycosylated) SEQ ID NO.43EFGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVQISTPSPVTLLRGHTLVLNCTATTPLNTRVQMTWSYPDEKNKNASVRRRIDQSNSHANIFYSVLTIDKMQNKDKGLYTCRVRSGPSFKSVNTSVHIYDKALEDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKC61서열번호 44EFGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVQISTPSPVELCVGETLVLNCTATTPANTRVQMTWSYPDEKNKNASVRRRIDQSNSHANIFYSVLTIDGMTNSDKGLYTCRVRSGPSFKSVNTSVHIYDCSSGDATPTSPPSPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGC62서열번호45EFGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVQISTPSPVELCVGETLVLNCTATTPANTRVQMTWSYPDEKNKNASVRRRIDQSNSHANIFYSVLTIDGMTNSDKGLYTCRVRSGPSFKSVNTSVHIYDCSKVDKKVEPKSSDTPPTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGC63서열번호 46EFGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVQISTPSPVELCVGETLVLNCTATTPANTRVQMTWSYPDEKNKNASVRRRIDQSNSHANIFYSVLTIDGMTNSDKGLYTCRVRSGPSFKSVNTSVHIYDCSGGGGSAEPKAGDATPPTCPPCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGC64서열번호47EFGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVQISTPSPVELCVGETLVLNCTATTPANTRVQMTWSYPDEKNKNASVRRRIDQSNSHANIFYSVLTIDGMTNSDKGLYTCRVRSGPSFKSVNTSVHIYDCSGGGGSGGGGSGGGGSAESKYGPPCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGC65서열번호 48EFGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVQISTPSPVELCVGETLVLNCTATTPANTRVQMTWSYPDEKNKNASVRRRIDQSNSHANIFYSVLTIDGMTNSDKGLYTCRVRSGPSFKSVNTSVHIYDCSNTGSGGEEKKKEKEKEEQEERSSDTPPTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGC66서열번호49EFGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVQISTPSPVELCVGETLVLNCTATTPANTRVQMTWSYPDEKNKNASVRRRIDQSNSHANIFYSVLTIDGMTNSDKGLYTCRVRSGPSFKSVNTSVHIYDCGSSGGSSGEPKSDATPTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGC67서열번호 50EFGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVQISTPSPVELCVGETLVLNCTATTPANTRVQMTWSYPDEKNKNASVRRRIDQSNSHANIFYSVLTIDGMTNSDKGLYTCRVRSGPSFKSVNTSVHIYDCSKVDKKVEPKSSDKTYTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGC68서열번호51EFGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVQISTPSPVELCVGETLVLNCTATTPSNTRVQMTWSYPDEKNKNASVRRRIDQSNSHANIFYSVLTIDGMTNSDKGLYTCRVRSGPSFKSVNTSVHIYDCSKVDKKVEPKSSDTPPTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGC69서열번호 52EFGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVQISTPSPVELCVGETLVLNCTATTPDNTRVQMTWSYPDEKNKNASVRRRIDQSNSHANIFYSVLTIDGMTNSDKGLYTCRVRSGPSFKSVNTSVHIYDCSKVDKKVEPKSSDTPPTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGC70서열번호53EFGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVQISTPSPVELCVGETLVLNCTATTPSNTRVQMTWSYPDEKNKNASVRRRIDQSNSHANIFYSVLTIDGMTNSDKGLYTCRVRSGPSFKSVNTSVHIYDCSGGGGSGGGGSGGGGSAESKYGPPCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGC71서열번호 54EFGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVQISTPSPVELCVGETLVLNCTATTPDNTRVQMTWSYPDEKNKNASVRRRIDQSNSHANIFYSVLTIDGMTNSDKGLYTCRVRSGPSFKSVNTSVHIYDCSGGGGSGGGGSGGGGSAESKYGPPCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGC72서열번호55EFGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVQISTPSPVELCVGETLVLNCTATTPSNTRVQMTWSYPDEKNKNASVRRRIDQSNSHANIFYSVLTIDGMTNSDKGLYTCRVRSGPSFKSVNTSVHIYDCGSSGGSSGEPKSDATPTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGC73서열번호 56EFGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVQISTPSPVELCVGETLVLNCTATTPDNTRVQMTWSYPDEKNKNASVRRRIDQSNSHANIFYSVLTIDGMTNSDKGLYTCRVRSGPSFKSVNTSVHIYDCGSSGGSSGEPKSDATPTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGC74서열번호57EFGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVQISTPSPVELCVGETLVLNCTATTPDNTRVQMTWSYPDEKNKNASVRRRIDQSNSHANIFYSVLTIDGMTNSDKGLYTCRVRSGPSFKSVNTSVHIYDCSSGDATPTSPPSPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGC75서열번호 58EFGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVQISTPSPVELCVGETLVLNCTATTPDNTRVQMTWSYPDEKNKNASVRRRIDQSNSHANIFYSVLTIDGMTNSDKGLYTCRVRSGPSFKSVNTSVHIYDCSNTGSGGEEKKKEKEKEEQEERSSDTPPTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGC88서열번호59EFGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVQISTPRPVKLLRGHTLVLNCTATTPLNTRVQMTWSYPDEKNKRASVRRRIDQSNSHANIFYSVLTIDKMQNKDKGLYTCRVRSGPSFKSVNTSVHIYDKALEDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGFc 영역서열번호 60APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKVEGFR1의 Ig 유사 도메인 D2 및 D3서열번호 61GRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVQISTPRPVKLLRGHTLVLNCTATTPLNTRVQMTWSYPDEKNKRASVRRRIDQSNSHANIFYSVLTIDKMQNKDKGLYTCRVRSGPSFKSVNTSVHIYDKAC08(대조군: VEGF-Grab)서열번호62GRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVQISTPSPVTLLRGHTLVLNCTATTPLNTRVQMTWSYPDEKNKNASVRRRIDQSNSHANIFYSVLTIDKMQNKDKGLYTCRVRSGPSFKSVNTSVHIYDKDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGC94서열번호 63GRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVQISTPSPVELCVGETLVLNCTATTPDNTRVQMTWSYPDEKNKNASVRRRIDQSNSHANIFYSVLTIDGMTNSDKGLYTCRVRSGPSFKSVNTSVHIYDCGSSGGSSGEPKSDATPTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGC95서열번호64GRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVQISTPSPVELCVGETLVLNCTATTPDNTRVQMTWSYPDEKNKNASVRRRIDQSNSHANIFYSVLTIDGMTNSDKGLYTCRVRSGPSFKSVNTSVHIYDCGSSGGSSGEPKSDATPTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGC96서열번호 65GRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVQISTPSPVELCVGETLVLNCTATTPDNTRVQMTWSYPDEKNKNASVRRRIDQSNSHANIFYSVLTIDGMTNSDKGLYTCRVRSGPSFKSVNTSVHIYDCSNTGSGGEEKKKEKEKEEQEERSCDTPPTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGC97서열번호66GRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVQISTPSPVELCVGETLVLNCTATTPDNTRVQMTWSYPDEKNKNASVRRRIDQSNSHANIFYSVLTIDGMTNSDKGLYTCRVRSGPSFKSVNTSVHIYDCSGGGGSGGGGSGGGGSAESKYGPPCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGC99서열번호 67GRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVQISTPSPVELCVGETLVLNCTATTPDNTRVQMTWSYPDEKNKNASVRRRIDQSNSHANIFYSVLTIDGMTNSDKGLYTCRVRSGPSFKSVNTSVHIYDCGSSGGSSGEPKSDATPTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0207] The surface charge mutation sites or disulfide bond sites described in the examples or tables below are based on the amino acid sequence of VEGF-Grab3 in Table 2, and the mutated sites and amino acid residues are described. The physical properties and binding affinity of the combination mutants having mutations introduced at different sites manufactured as in Table 2 were evaluated. The results of the physical property evaluation for the mutants manufactured based on the VEGFR-wild type protein and the wild type protein are shown in Table 3, and the results of the binding affinity evaluation are shown in Tables 4 to 5. The results of the physical property evaluation for the mutants manufactured based on VEGF-Grab3 are shown in Table 6, and the results of the binding affinity evaluation are shown in Tables 7 and 8. Table 7 shows the results of the relative binding affinity evaluation for VEGF-Grab3 (manufactured by Panolos Biosciences), and Table 8 shows the results of the relative binding affinity evaluation for VEGF-Grab3 (manufactured by Samsung BioLogics). For the evaluation of relative binding affinity for VEGF-Grab3 (manufactured by Panoros Biosciences), it was compared with that produced under the same conditions and batch as the mutant.

[0208] Protein code, titer (g / L), elution amount (mg), yield (%), HMWS (%), functional monomer content (%), LMWS (%), C650.1856.9123.136.162.91.1, C730.1435.0116.420.279.80.0

[0209] Protein Code Binding Ability to VEGF-A Binding Ability to VEGF-A Compared to VEGFR-wild type protein C88 (fold increase) KD (M) kon (1 / Ms) kdis (1 / s) fold (KD) fold (kon) fold (kdis) C6 5 8.46E-115.16E+0 5 4.37E-0 5 0.72 0.89 0.80 C7 3 1.53E-136.52E+0 5 <1.0E-07> 60.86 1.13> 100

[0210] Protein CodeBinding to PlGFBinding to PlGF compared to VEGFR-wild type protein C88 (fold increase)KD(M)kon(1 / Ms)kdis(1 / s)fold(KD)fold(kon)fold(kdis)C651.37E-102.41E+063.31E-041.491.990.75C731.68E-102.22E+063.72E-041.221.830.67

[0211] Protein Code Titer (g / L) Dissolution Amount (mg) Yield (%) HMWS (%) Functional Monomer Content (%) LMWS (%) VEGF-Grab 10.52 78.64 6.73 3.143.0 23.8 VEGF-Grab 30.35 95.45 0.55 3.94 4.31 7 C6 10.53 710 62.21 0.28 9.80 0 C6 20.23 15.98 5.711 28 8.80 0 C6 30.50 59.66 3.49 59 0.50 0 C6 40.17 16.21 21.613 78 6.30 0 C6 50.66 8.54 2.79 79 0.30 0 C6 0.21 26.29 8.120.7 79.40.0C670.4375.843.9595.10.0C680.1536.3137.34555.00.0C690.378544.42.697.40.0C700.61843.913.286.90.0C710.6987.13 3.84.695.40.0C720.4517.354.19.590.50.0C730.4087.158.33.196.90.0C740.5355.835.96.793.30.0C750.2958.292.20.499.60.0

[0212] Protein CodeBinding Ability to VEGF-A (Fold Increase)Binding Ability to PlGF (Fold Increase)fold(KD)fold(kon)fold(kdis)fold(KD)fold(kon)fold(kdis)C611.350.741.831.240.761.62C62> 1001.27> 1001.931.701.13C631.540.702.211.380.891.56C642.541.391.831.821.701.07C652.120 .722.931.830.912.00C661.461.241.183.771.253.01C671.950.942.061.030.961.07C68> 1001.83> 1001.212.430.50C69> 1001.37> 1000.831.240.67C703.011.202.521.381.301.06C713.060.993.101.430.821.75C720.950.821.151.480.702.11C73> 1001.29> 1002.100.663.18C74> 1002.09> 1001.072.060.52C750.271.380.200.882.070.42

[0213] Protein CodeBinding Ability to VEGF-A (Fold Increase)Binding Ability to PlGF (Fold Increase)fold(KD)fold(kon)fold(kdis)fold(KD)fold(kon)fold(kdis)C61< 0.010.94< 0.011.531.141.35C621.830.551.002.392.540.94C63< 0.010.99< 0.011.711.321.30C640.010.50< 0.012.262.540.89C65< 0.010.96< 0.012.261.361.66C66< 0.010.56< 0.014.671.872.50C67< 0.010.73< 0.011.271.430.89C682.650.381.001.493.630.41C690.420.510.211.031.860.56C700.010.58< 0.011.701.930.88C710.010.70< 0.011.771.221.45C72< 0.010.84< 0.011.841.051.75C731.870.541.002.600.992.64C741.171.171.001.472.540.58C75< 0.010.77< 0.011.202.560.47

[0214] According to the results in Tables 3 to 8, it can be confirmed that the modified fusion protein according to one embodiment of the present disclosure exhibits excellent productivity as shown in the results of titer and protein elution amount, and excellent physical properties as shown in the results of functional monomer content. In addition, for example, it can be confirmed that it exhibits excellent properties in binding affinity to ligands as shown in the results for kon, etc. From these results, it can be confirmed that the characteristics introduced during the structural design of the modified fusion protein in Examples 2.1 to 2.3 are effective, and these characteristics are combined to exhibit a synergistic effect.

[0215] Example 3 - Production of the conjugate

[0216] A conjugate was designed by combining the fusion protein of C61 to C75 manufactured in Example 2 with atezolizumab and A167 scFv that bind to PD-L1 (Fig. 1). As can be seen in Fig. 1, the fusion protein of the present disclosure includes a VEGF decoy receptor comprising engineered VEGFR1 domains 2 and 3 capable of capturing VEGF-A and PlGF, and a new functional module can be linked to the N-terminus or C-terminus thereof. The conjugate according to the present disclosure was prepared by combining an anti-PD-L1 scFv to the N-terminus of the fusion protein of the present disclosure.

[0217] Composition sequence: PD-L1 scFv (atezolizumab) SEQ ID NO: 39EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEI PD-L1 scFv (A167 scFv) SEQ ID NO: 40QVQLQESGPGLVKPSETLSITCTVSGFSLSNYDISWIRQPPGKGLEWLGVIWTGGATNYNPALKSRLTISRDNSKNQVSLKMSSVTAADTAVYYCVRDSNYRYDEPFTYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSEIVLTQSPDTLSVTPKEKVTLTCRASQSIGTNIHWFQQKPGQSPKLLIKYASESISGVPSRFSGSGSGTDFTLTINSVEAEDAATYYCQQSNSWPYTFGQGTKLEIKRCode name (composition description)Sequence H-12C08 (Control: A167HG4SL-VEGF-Grab)Sequence number1QVQLQESGPGLVKPSETLSITCTVSGFSLSNYDISWIRQPPGKGLEWLGVIWTGGATNYNPALKSRLTISRDNSKNQVSLKMSSVTAADTAVYYCVRDSNYRYDEPFTYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSEIVLTQSPDTLSVTPKEKVTLTCRASQSIGTNIHWFQQKPGQSPKLLIKYASESISGVPSRFSGSGSGTDFTLTINSVEAEDAATYYCQQSNSWPYTFGQGTKLEIKRTVGGGGSEFGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVQISTPSPVTLLRGHTLVLNCTATTPLNTRVQMTWSYPDEKNKNASVRRRIDQSNSHANIFYSVLTIDKMQNKDKGLYTCRVRSGPSFKSVNTSVHIYDKALEDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKH-12C09 (대조군: A167LG4SH- VEGF-Grab)서열번호2EIVLTQSPDTLSVTPKEKVTLTCRASQSIGTNIHWFQQKPGQSPKLLIKYASESISGVPSRFSGSGSGTDFTLTINSVEAEDAATYYCQQSNSWPYTFGQGTKLEIKRTVGGGGSGGGGSGGGGSGGGGSGGGGSQVQLQESGPGLVKPSETLSITCTVSGFSLSNYDISWIRQPPGKGLEWLGVIWTGGATNYNPALKSRLTISRDNSKNQVSLKMSSVTAADTAVYYCVRDSNYRYDEPFTYWGQGTLVTVSSGGGGSEFGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVQISTPSPVTLLRGHTLVLNCTATTPLNTRVQMTWSYPDEKNKNASVRRRIDQSNSHANIFYSVLTIDKMQNKDKGLYTCRVRSGPSFKSVNTSVHIYDKALEDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKH-12C18 (대조군: A167-3(G4S)-Fc)서열번호3QVQLQESGPGLVKPSETLSITCTVSGFSLSNYDISWIRQPPGKGLEWLGVIWTGGATNYNPALKSRLTISRDNSKNQVSLKMSSVTAADTAVYYCVRDSNYRYDEPFTYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSEIVLTQSPDTLSVTPKEKVTLTCRASQSIGTNIHWFQQKPGQSPKLLIKYASESISGVPSRFSGSGSGTDFTLTINSVEAEDAATYYCQQSNSWPYTFGQGTKLEIKRTVGGGGSGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKH-12C21 (대조군: VEGF-Grab-3(G4S)-A167)서열번호4EFGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVQISTPSPVTLLRGHTLVLNCTATTPLNTRVQMTWSYPDEKNKNASVRRRIDQSNSHANIFYSVLTIDKMQNKDKGLYTCRVRSGPSFKSVNTSVHIYDKALEDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSQVQLQESGPGLVKPSETLSITCTVSGFSLSNYDISWIRQPPGKGLEWLGVIWTGGATNYNPALKSRLTISRDNSKNQVSLKMSSVTAADTAVYYCVRDSNYRYDEPFTYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSEIVLTQSPDTLSVTPKEKVTLTCRASQSIGTNIHWFQQKPGQSPKLLIKYASESISGVPSRFSGSGSGTDFTLTINSVEAEDAATYYCQQSNSWPYTFGQGTKLEIKRTVH-12C70 (A167-V3-C66_sur1)서열번호5QVQLQESGPGLVKPSETLSITCTVSGFSLSNYDISWIRQPPGKGLEWLGVIWTGGATNYNPALKSRLTISRDNSKNQVSLKMSSVTAADTAVYYCVRDSNYRYDEPFTYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSEIVLTQSPDTLSVTPKEKVTLTCRASQSIGTNIHWFQQKPGQSPKLLIKYASESISGVPSRFSGSGSGTDFTLTINSVEAEDAATYYCQQSNSWPYTFGQGTKLEIKRGSGGGGSGGGGSGGGGSGGGGSGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVQISTPSPVTLCRGHTLVLNCTATTPANTRVQMTWSYPDEKNKNASVRRRIDQSNSHANIFYSVLTIDGMTNSDQGLYTCRVRSGPSFKSVNTSVHIYDCGSSGGSSGEPKSDATPTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGH-12C71 (A167-V1-C66_sur1)서열번호6QVQLQESGPGLVKPSETLSITCTVSGFSLSNYDISWIRQPPGKGLEWLGVIWTGGATNYNPALKSRLTISRDNSKNQVSLKMSSVTAADTAVYYCVRDSNYRYDEPFTYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSEIVLTQSPDTLSVTPKEKVTLTCRASQSIGTNIHWFQQKPGQSPKLLIKYASESISGVPSRFSGSGSGTDFTLTINSVEAEDAATYYCQQSNSWPYTFGQGTKLEIKRGGGGSGGGGSDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVQISTPSPVTLCRGHTLVLNCTATTPANTRVQMTWSYPDEKNKNASVRRRIDQSNSHANIFYSVLTIDGMTNSDQGLYTCRVRSGPSFKSVNTSVHIYDCGSSGGSSGEPKSDATPTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGH-12C72 (A167-V3-C65(Cys)_sur1)서열번호7QVQLQESGPGLVKPSETLSITCTVSGFSLSNYDISWIRQPPGKGLEWLGVIWTGGATNYNPALKSRLTISRDNSKNQVSLKMSSVTAADTAVYYCVRDSNYRYDEPFTYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSEIVLTQSPDTLSVTPKEKVTLTCRASQSIGTNIHWFQQKPGQSPKLLIKYASESISGVPSRFSGSGSGTDFTLTINSVEAEDAATYYCQQSNSWPYTFGQGTKLEIKRGSGGGGSGGGGSGGGGSGGGGSGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVQISTPSPVTLCRGHTLVLNCTATTPANTRVQMTWSYPDEKNKNASVRRRIDQSNSHANIFYSVLTIDGMTNSDQGLYTCRVRSGPSFKSVNTSVHIYDCSNTGSGGEEKKKEKEKEEQEERSCDTPPTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGH-12C73 (A167-V3-C64_sur1)서열번호8QVQLQESGPGLVKPSETLSITCTVSGFSLSNYDISWIRQPPGKGLEWLGVIWTGGATNYNPALKSRLTISRDNSKNQVSLKMSSVTAADTAVYYCVRDSNYRYDEPFTYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSEIVLTQSPDTLSVTPKEKVTLTCRASQSIGTNIHWFQQKPGQSPKLLIKYASESISGVPSRFSGSGSGTDFTLTINSVEAEDAATYYCQQSNSWPYTFGQGTKLEIKRGSGGGGSGGGGSGGGGSGGGGSGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVQISTPSPVTLCRGHTLVLNCTATTPANTRVQMTWSYPDEKNKNASVRRRIDQSNSHANIFYSVLTIDGMTNSDQGLYTCRVRSGPSFKSVNTSVHIYDCSGGGGSGGGGSGGGGSAESKYGPPCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGH-12C74 (A167-V1-C61_sur1)서열번호9QVQLQESGPGLVKPSETLSITCTVSGFSLSNYDISWIRQPPGKGLEWLGVIWTGGATNYNPALKSRLTISRDNSKNQVSLKMSSVTAADTAVYYCVRDSNYRYDEPFTYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSEIVLTQSPDTLSVTPKEKVTLTCRASQSIGTNIHWFQQKPGQSPKLLIKYASESISGVPSRFSGSGSGTDFTLTINSVEAEDAATYYCQQSNSWPYTFGQGTKLEIKRGGGGSGGGGSDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVQISTPSPVTLCRGHTLVLNCTATTPANTRVQMTWSYPDEKNKNASVRRRIDQSNSHANIFYSVLTIDGMTNSDQGLYTCRVRSGPSFKSVNTSVHIYDCSSGDATPTSPPSPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGH-12C75 (A167-V2-C66_sur1)서열번호10QVQLQESGPGLVKPSETLSITCTVSGFSLSNYDISWIRQPPGKGLEWLGVIWTGGATNYNPALKSRLTISRDNSKNQVSLKMSSVTAADTAVYYCVRDSNYRYDEPFTYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSEIVLTQSPDTLSVTPKEKVTLTCRASQSIGTNIHWFQQKPGQSPKLLIKYASESISGVPSRFSGSGSGTDFTLTINSVEAEDAATYYCQQSNSWPYTFGQGTKLEIKRGSAEPKAGGGGSGGGGSGGGGSGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVQISTPSPVTLCRGHTLVLNCTATTPANTRVQMTWSYPDEKNKNASVRRRIDQSNSHANIFYSVLTIDGMTNSDQGLYTCRVRSGPSFKSVNTSVHIYDCGSSGGSSGEPKSDATPTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGH-12C88 (A167-V3-C66)서열번호11QVQLQESGPGLVKPSETLSITCTVSGFSLSNYDISWIRQPPGKGLEWLGVIWTGGATNYNPALKSRLTISRDNSKNQVSLKMSSVTAADTAVYYCVRDSNYRYDEPFTYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSEIVLTQSPDTLSVTPKEKVTLTCRASQSIGTNIHWFQQKPGQSPKLLIKYASESISGVPSRFSGSGSGTDFTLTINSVEAEDAATYYCQQSNSWPYTFGQGTKLEIKRGSGGGGSGGGGSGGGGSGGGGSGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVQISTPSPVELCVGETLVLNCTATTPANTRVQMTWSYPDEKNKNASVRRRIDQSNSHANIFYSVLTIDGMTNSDKGLYTCRVRSGPSFKSVNTSVHIYDCGSSGGSSGEPKSDATPTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGH-12C89 (A167-V1-C66)서열번호12QVQLQESGPGLVKPSETLSITCTVSGFSLSNYDISWIRQPPGKGLEWLGVIWTGGATNYNPALKSRLTISRDNSKNQVSLKMSSVTAADTAVYYCVRDSNYRYDEPFTYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSEIVLTQSPDTLSVTPKEKVTLTCRASQSIGTNIHWFQQKPGQSPKLLIKYASESISGVPSRFSGSGSGTDFTLTINSVEAEDAATYYCQQSNSWPYTFGQGTKLEIKRGGGGSGGGGSDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVQISTPSPVELCVGETLVLNCTATTPANTRVQMTWSYPDEKNKNASVRRRIDQSNSHANIFYSVLTIDGMTNSDKGLYTCRVRSGPSFKSVNTSVHIYDCGSSGGSSGEPKSDATPTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGH-12C90 (A167-V3-C65(Cys))서열번호13QVQLQESGPGLVKPSETLSITCTVSGFSLSNYDISWIRQPPGKGLEWLGVIWTGGATNYNPALKSRLTISRDNSKNQVSLKMSSVTAADTAVYYCVRDSNYRYDEPFTYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSEIVLTQSPDTLSVTPKEKVTLTCRASQSIGTNIHWFQQKPGQSPKLLIKYASESISGVPSRFSGSGSGTDFTLTINSVEAEDAATYYCQQSNSWPYTFGQGTKLEIKRGSGGGGSGGGGSGGGGSGGGGSGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVQISTPSPVELCVGETLVLNCTATTPANTRVQMTWSYPDEKNKNASVRRRIDQSNSHANIFYSVLTIDGMTNSDKGLYTCRVRSGPSFKSVNTSVHIYDCSNTGSGGEEKKKEKEKEEQEERSCDTPPTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGH-12C91 (A167-V3-C64)서열번호14QVQLQESGPGLVKPSETLSITCTVSGFSLSNYDISWIRQPPGKGLEWLGVIWTGGATNYNPALKSRLTISRDNSKNQVSLKMSSVTAADTAVYYCVRDSNYRYDEPFTYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSEIVLTQSPDTLSVTPKEKVTLTCRASQSIGTNIHWFQQKPGQSPKLLIKYASESISGVPSRFSGSGSGTDFTLTINSVEAEDAATYYCQQSNSWPYTFGQGTKLEIKRGSGGGGSGGGGSGGGGSGGGGSGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVQISTPSPVELCVGETLVLNCTATTPANTRVQMTWSYPDEKNKNASVRRRIDQSNSHANIFYSVLTIDGMTNSDKGLYTCRVRSGPSFKSVNTSVHIYDCSGGGGSGGGGSGGGGSAESKYGPPCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGH-12C92 (A167-V1-C61)서열번호15QVQLQESGPGLVKPSETLSITCTVSGFSLSNYDISWIRQPPGKGLEWLGVIWTGGATNYNPALKSRLTISRDNSKNQVSLKMSSVTAADTAVYYCVRDSNYRYDEPFTYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSEIVLTQSPDTLSVTPKEKVTLTCRASQSIGTNIHWFQQKPGQSPKLLIKYASESISGVPSRFSGSGSGTDFTLTINSVEAEDAATYYCQQSNSWPYTFGQGTKLEIKRGGGGSGGGGSDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVQISTPSPVELCVGETLVLNCTATTPANTRVQMTWSYPDEKNKNASVRRRIDQSNSHANIFYSVLTIDGMTNSDKGLYTCRVRSGPSFKSVNTSVHIYDCSSGDATPTSPPSPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGH-12C93 (A167-V2-C66)서열번호16QVQLQESGPGLVKPSETLSITCTVSGFSLSNYDISWIRQPPGKGLEWLGVIWTGGATNYNPALKSRLTISRDNSKNQVSLKMSSVTAADTAVYYCVRDSNYRYDEPFTYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSEIVLTQSPDTLSVTPKEKVTLTCRASQSIGTNIHWFQQKPGQSPKLLIKYASESISGVPSRFSGSGSGTDFTLTINSVEAEDAATYYCQQSNSWPYTFGQGTKLEIKRGSAEPKAGGGGSGGGGSGGGGSGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVQISTPSPVELCVGETLVLNCTATTPANTRVQMTWSYPDEKNKNASVRRRIDQSNSHANIFYSVLTIDGMTNSDKGLYTCRVRSGPSFKSVNTSVHIYDCGSSGGSSGEPKSDATPTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGH-12C94 (A167-V3-C73)서열번호17QVQLQESGPGLVKPSETLSITCTVSGFSLSNYDISWIRQPPGKGLEWLGVIWTGGATNYNPALKSRLTISRDNSKNQVSLKMSSVTAADTAVYYCVRDSNYRYDEPFTYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSEIVLTQSPDTLSVTPKEKVTLTCRASQSIGTNIHWFQQKPGQSPKLLIKYASESISGVPSRFSGSGSGTDFTLTINSVEAEDAATYYCQQSNSWPYTFGQGTKLEIKRGSGGGGSGGGGSGGGGSGGGGSGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVQISTPSPVELCVGETLVLNCTATTPDNTRVQMTWSYPDEKNKNASVRRRIDQSNSHANIFYSVLTIDGMTNSDKGLYTCRVRSGPSFKSVNTSVHIYDCGSSGGSSGEPKSDATPTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGH-12C95 (A167-V1-C73)서열번호18QVQLQESGPGLVKPSETLSITCTVSGFSLSNYDISWIRQPPGKGLEWLGVIWTGGATNYNPALKSRLTISRDNSKNQVSLKMSSVTAADTAVYYCVRDSNYRYDEPFTYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSEIVLTQSPDTLSVTPKEKVTLTCRASQSIGTNIHWFQQKPGQSPKLLIKYASESISGVPSRFSGSGSGTDFTLTINSVEAEDAATYYCQQSNSWPYTFGQGTKLEIKRGGGGSGGGGSDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVQISTPSPVELCVGETLVLNCTATTPDNTRVQMTWSYPDEKNKNASVRRRIDQSNSHANIFYSVLTIDGMTNSDKGLYTCRVRSGPSFKSVNTSVHIYDCGSSGGSSGEPKSDATPTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGH-12C96 (A167-V3-C75(Cys))서열번호19QVQLQESGPGLVKPSETLSITCTVSGFSLSNYDISWIRQPPGKGLEWLGVIWTGGATNYNPALKSRLTISRDNSKNQVSLKMSSVTAADTAVYYCVRDSNYRYDEPFTYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSEIVLTQSPDTLSVTPKEKVTLTCRASQSIGTNIHWFQQKPGQSPKLLIKYASESISGVPSRFSGSGSGTDFTLTINSVEAEDAATYYCQQSNSWPYTFGQGTKLEIKRGSGGGGSGGGGSGGGGSGGGGSGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVQISTPSPVELCVGETLVLNCTATTPDNTRVQMTWSYPDEKNKNASVRRRIDQSNSHANIFYSVLTIDGMTNSDKGLYTCRVRSGPSFKSVNTSVHIYDCSNTGSGGEEKKKEKEKEEQEERSCDTPPTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGH-12C97 (A167-V3-C71)서열번호20QVQLQESGPGLVKPSETLSITCTVSGFSLSNYDISWIRQPPGKGLEWLGVIWTGGATNYNPALKSRLTISRDNSKNQVSLKMSSVTAADTAVYYCVRDSNYRYDEPFTYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSEIVLTQSPDTLSVTPKEKVTLTCRASQSIGTNIHWFQQKPGQSPKLLIKYASESISGVPSRFSGSGSGTDFTLTINSVEAEDAATYYCQQSNSWPYTFGQGTKLEIKRGSGGGGSGGGGSGGGGSGGGGSGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVQISTPSPVELCVGETLVLNCTATTPDNTRVQMTWSYPDEKNKNASVRRRIDQSNSHANIFYSVLTIDGMTNSDKGLYTCRVRSGPSFKSVNTSVHIYDCSGGGGSGGGGSGGGGSAESKYGPPCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGH-12C98 (A167-V1-C74)서열번호21QVQLQESGPGLVKPSETLSITCTVSGFSLSNYDISWIRQPPGKGLEWLGVIWTGGATNYNPALKSRLTISRDNSKNQVSLKMSSVTAADTAVYYCVRDSNYRYDEPFTYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSEIVLTQSPDTLSVTPKEKVTLTCRASQSIGTNIHWFQQKPGQSPKLLIKYASESISGVPSRFSGSGSGTDFTLTINSVEAEDAATYYCQQSNSWPYTFGQGTKLEIKRGGGGSGGGGSDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVQISTPSPVELCVGETLVLNCTATTPDNTRVQMTWSYPDEKNKNASVRRRIDQSNSHANIFYSVLTIDGMTNSDKGLYTCRVRSGPSFKSVNTSVHIYDCSSGDATPTSPPSPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGH-12C99 (A167-V2-C73)서열번호22QVQLQESGPGLVKPSETLSITCTVSGFSLSNYDISWIRQPPGKGLEWLGVIWTGGATNYNPALKSRLTISRDNSKNQVSLKMSSVTAADTAVYYCVRDSNYRYDEPFTYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSEIVLTQSPDTLSVTPKEKVTLTCRASQSIGTNIHWFQQKPGQSPKLLIKYASESISGVPSRFSGSGSGTDFTLTINSVEAEDAATYYCQQSNSWPYTFGQGTKLEIKRGSAEPKAGGGGSGGGGSGGGGSGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVQISTPSPVELCVGETLVLNCTATTPDNTRVQMTWSYPDEKNKNASVRRRIDQSNSHANIFYSVLTIDGMTNSDKGLYTCRVRSGPSFKSVNTSVHIYDCGSSGGSSGEPKSDATPTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGH-32D01 (A167-V2-C65)서열번호23QVQLQESGPGLVKPSETLSITCTVSGFSLSNYDISWIRQPPGKGLEWLGVIWTGGATNYNPALKSRLTISRDNSKNQVSLKMSSVTAADTAVYYCVRDSNYRYDEPFTYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSEIVLTQSPDTLSVTPKEKVTLTCRASQSIGTNIHWFQQKPGQSPKLLIKYASESISGVPSRFSGSGSGTDFTLTINSVEAEDAATYYCQQSNSWPYTFGQGTKLEIKRGSAEPKAGGGGSGGGGSGGGGSGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVQISTPSPVELCVGETLVLNCTATTPANTRVQMTWSYPDEKNKNASVRRRIDQSNSHANIFYSVLTIDGMTNSDKGLYTCRVRSGPSFKSVNTSVHIYDCSNTGSGGEEKKKEKEKEEQEERSSDTPPTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGH-32D02 (A167-V3-C65)서열번호24QVQLQESGPGLVKPSETLSITCTVSGFSLSNYDISWIRQPPGKGLEWLGVIWTGGATNYNPALKSRLTISRDNSKNQVSLKMSSVTAADTAVYYCVRDSNYRYDEPFTYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSEIVLTQSPDTLSVTPKEKVTLTCRASQSIGTNIHWFQQKPGQSPKLLIKYASESISGVPSRFSGSGSGTDFTLTINSVEAEDAATYYCQQSNSWPYTFGQGTKLEIKRGSGGGGSGGGGSGGGGSGGGGSGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVQISTPSPVELCVGETLVLNCTATTPANTRVQMTWSYPDEKNKNASVRRRIDQSNSHANIFYSVLTIDGMTNSDKGLYTCRVRSGPSFKSVNTSVHIYDCSNTGSGGEEKKKEKEKEEQEERSSDTPPTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGH-32D03 (A167-V2-G4H-G4Fc)서열번호25QVQLQESGPGLVKPSETLSITCTVSGFSLSNYDISWIRQPPGKGLEWLGVIWTGGATNYNPALKSRLTISRDNSKNQVSLKMSSVTAADTAVYYCVRDSNYRYDEPFTYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSEIVLTQSPDTLSVTPKEKVTLTCRASQSIGTNIHWFQQKPGQSPKLLIKYASESISGVPSRFSGSGSGTDFTLTINSVEAEDAATYYCQQSNSWPYTFGQGTKLEIKRGSAEPKAGGGGSGGGGSGGGGSGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVQISTPSPVELCVGETLVLNCTATTPANTRVQMTWSYPDEKNKNASVRRRIDQSNSHANIFYSVLTIDGMTNSDKGLYTCRVRSGPSFKSVNTSVHIYDCSGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGH-32D04 (A167-V3-G4H-G4Fc)서열번호26QVQLQESGPGLVKPSETLSITCTVSGFSLSNYDISWIRQPPGKGLEWLGVIWTGGATNYNPALKSRLTISRDNSKNQVSLKMSSVTAADTAVYYCVRDSNYRYDEPFTYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSEIVLTQSPDTLSVTPKEKVTLTCRASQSIGTNIHWFQQKPGQSPKLLIKYASESISGVPSRFSGSGSGTDFTLTINSVEAEDAATYYCQQSNSWPYTFGQGTKLEIKRGSGGGGSGGGGSGGGGSGGGGSGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVQISTPSPVELCVGETLVLNCTATTPANTRVQMTWSYPDEKNKNASVRRRIDQSNSHANIFYSVLTIDGMTNSDKGLYTCRVRSGPSFKSVNTSVHIYDCSGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGH-32D05 (A167-V2-C65H-G4Fc)서열번호27QVQLQESGPGLVKPSETLSITCTVSGFSLSNYDISWIRQPPGKGLEWLGVIWTGGATNYNPALKSRLTISRDNSKNQVSLKMSSVTAADTAVYYCVRDSNYRYDEPFTYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSEIVLTQSPDTLSVTPKEKVTLTCRASQSIGTNIHWFQQKPGQSPKLLIKYASESISGVPSRFSGSGSGTDFTLTINSVEAEDAATYYCQQSNSWPYTFGQGTKLEIKRGSAEPKAGGGGSGGGGSGGGGSGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVQISTPSPVELCVGETLVLNCTATTPANTRVQMTWSYPDEKNKNASVRRRIDQSNSHANIFYSVLTIDGMTNSDKGLYTCRVRSGPSFKSVNTSVHIYDCSNTGSGGEEKKKEKEKEEQEERSSDTPPTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGH-32D06 (A167-V3-C65H-G4Fc)서열번호28QVQLQESGPGLVKPSETLSITCTVSGFSLSNYDISWIRQPPGKGLEWLGVIWTGGATNYNPALKSRLTISRDNSKNQVSLKMSSVTAADTAVYYCVRDSNYRYDEPFTYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSEIVLTQSPDTLSVTPKEKVTLTCRASQSIGTNIHWFQQKPGQSPKLLIKYASESISGVPSRFSGSGSGTDFTLTINSVEAEDAATYYCQQSNSWPYTFGQGTKLEIKRGSGGGGSGGGGSGGGGSGGGGSGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVQISTPSPVELCVGETLVLNCTATTPANTRVQMTWSYPDEKNKNASVRRRIDQSNSHANIFYSVLTIDGMTNSDKGLYTCRVRSGPSFKSVNTSVHIYDCSNTGSGGEEKKKEKEKEEQEERSSDTPPTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGH-32D07 (A167-V2-C65H-G4Fc+QL)서열번호29QVQLQESGPGLVKPSETLSITCTVSGFSLSNYDISWIRQPPGKGLEWLGVIWTGGATNYNPALKSRLTISRDNSKNQVSLKMSSVTAADTAVYYCVRDSNYRYDEPFTYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSEIVLTQSPDTLSVTPKEKVTLTCRASQSIGTNIHWFQQKPGQSPKLLIKYASESISGVPSRFSGSGSGTDFTLTINSVEAEDAATYYCQQSNSWPYTFGQGTKLEIKRGSAEPKAGGGGSGGGGSGGGGSGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVQISTPSPVELCVGETLVLNCTATTPANTRVQMTWSYPDEKNKNASVRRRIDQSNSHANIFYSVLTIDGMTNSDKGLYTCRVRSGPSFKSVNTSVHIYDCSNTGSGGEEKKKEKEKEEQEERSSDTPPTCPPCPAPEAAGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVLHEALHNHYTQKSLSLSLGH-32D08 (A167-V3-C65H-G4Fc+QL)서열번호30QVQLQESGPGLVKPSETLSITCTVSGFSLSNYDISWIRQPPGKGLEWLGVIWTGGATNYNPALKSRLTISRDNSKNQVSLKMSSVTAADTAVYYCVRDSNYRYDEPFTYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSEIVLTQSPDTLSVTPKEKVTLTCRASQSIGTNIHWFQQKPGQSPKLLIKYASESISGVPSRFSGSGSGTDFTLTINSVEAEDAATYYCQQSNSWPYTFGQGTKLEIKRGSGGGGSGGGGSGGGGSGGGGSGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVQISTPSPVELCVGETLVLNCTATTPANTRVQMTWSYPDEKNKNASVRRRIDQSNSHANIFYSVLTIDGMTNSDKGLYTCRVRSGPSFKSVNTSVHIYDCSNTGSGGEEKKKEKEKEEQEERSSDTPPTCPPCPAPEAAGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVLHEALHNHYTQKSLSLSLGH-32D09 (Ate-V2-C65)서열번호31EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKRGSAEPKAGGGGSGGGGSGGGGSGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVQISTPSPVELCVGETLVLNCTATTPANTRVQMTWSYPDEKNKNASVRRRIDQSNSHANIFYSVLTIDGMTNSDKGLYTCRVRSGPSFKSVNTSVHIYDCSNTGSGGEEKKKEKEKEEQEERSSDTPPTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGH-32D10 (Ate-V3-C65)서열번호32EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKRGSGGGGSGGGGSGGGGSGGGGSGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVQISTPSPVELCVGETLVLNCTATTPANTRVQMTWSYPDEKNKNASVRRRIDQSNSHANIFYSVLTIDGMTNSDKGLYTCRVRSGPSFKSVNTSVHIYDCSNTGSGGEEKKKEKEKEEQEERSSDTPPTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGH-32D11 (Ate-V2-G4H-G4Fc+QL)서열번호33EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKRGSAEPKAGGGGSGGGGSGGGGSGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVQISTPSPVELCVGETLVLNCTATTPANTRVQMTWSYPDEKNKNASVRRRIDQSNSHANIFYSVLTIDGMTNSDKGLYTCRVRSGPSFKSVNTSVHIYDCSGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVLHEALHNHYTQKSLSLSLGH-32D12 (Ate-V3-G4H-G4Fc+QL)서열번호34EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKRGSGGGGSGGGGSGGGGSGGGGSGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVQISTPSPVELCVGETLVLNCTATTPANTRVQMTWSYPDEKNKNASVRRRIDQSNSHANIFYSVLTIDGMTNSDKGLYTCRVRSGPSFKSVNTSVHIYDCSGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVLHEALHNHYTQKSLSLSLGH-32D13 (Ate-V2-C65H-G4Fc+QL)서열번호35EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKRGSAEPKAGGGGSGGGGSGGGGSGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVQISTPSPVELCVGETLVLNCTATTPANTRVQMTWSYPDEKNKNASVRRRIDQSNSHANIFYSVLTIDGMTNSDKGLYTCRVRSGPSFKSVNTSVHIYDCSNTGSGGEEKKKEKEKEEQEERSSDTPPTCPPCPAPEAAGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVLHEALHNHYTQKSLSLSLGH-32D14 (Ate-V3-C65H-G4Fc+QL)서열번호36EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKRGSGGGGSGGGGSGGGGSGGGGSGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVQISTPSPVELCVGETLVLNCTATTPANTRVQMTWSYPDEKNKNASVRRRIDQSNSHANIFYSVLTIDGMTNSDKGLYTCRVRSGPSFKSVNTSVHIYDCSNTGSGGEEKKKEKEKEEQEERSSDTPPTCPPCPAPEAAGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVLHEALHNHYTQKSLSLSLGH-12C18 (A167-3(G4S)-Fc)서열번호 37- Full 항체가 아닌 scFv-Fc 결합대조군QVQLQESGPGLVKPSETLSITCTVSGFSLSNYDISWIRQPPGKGLEWLGVIWTGGATNYNPALKSRLTISRDNSKNQVSLKMSSVTAADTAVYYCVRDSNYRYDEPFTYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSEIVLTQSPDTLSVTPKEKVTLTCRASQSIGTNIHWFQQKPGQSPKLLIKYASESISGVPSRFSGSGSGTDFTLTINSVEAEDAATYYCQQSNSWPYTFGQGTKLEIKRTVGGGGSGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKH-30D01 (Ate-IgG1 Fc)서열번호 38- Full 항체가 아닌 scFv-Fc 결합대조군EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKRCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0218] * In the composition description part of Table 9, H and L represent the heavy and light chains of scFv; Cnn (n is an integer) corresponds to the sequence designation in Table 2 above; G4S represents a GGGGS linker; the linkers connecting scFv and the fusion protein are V1, V2, and V3, with V1 having a sequence of GGGGSGGGGSDT, V2 having a sequence of GSAEPKAGGGGSGGGGSGGGGS, and V3 having a sequence of GSGGGGSGGGGSGGGGSGGGGS; C65H represents that the C65 hinge portion of Table 2 is used among the hinge portions of the fusion protein; G4H represents that IgG4 is used instead of IgG1 among the hinge portions of the fusion protein; G4Fc represents an IgG4 sequence with the effector function removed; QL represents that a mutation is introduced to increase the binding affinity to FcRn, which is known to increase the half-life by increasing the FcRn cycle; Cys refers to a fusion protein that has introduced a Cys mutation (Ser -> Cys at position 494 for C65 and 493 for C75); sur1 refers to a mutant that has undergone back mutation of some surface charge mutations.

[0219] ExpiCHO used to produce designed composites TM Cells (Gibco) were used for transient expression at passage numbers 10 to 20. ExpiCHO TM Cell passage was performed at 3- or 4-day intervals, and 0.3 x 10 cells were cultured for 3 days. 6 Cells were subcultured at a density of 10 cells / ml, and after 4 days of culture, the number reached 0.15 x 10 6 Cells were subcultured at a density of 10 cells / ml. Cell density and viability were measured using a Countess II (Invitrogen) device via trypan blue staining. Transient expression of the complex was measured using Expifectamine provided by the manufacturer (Gibco). TMThe Max Titer protocol culture method using (Gibco) was used. ExpiCHO was used the day before transfection. TM 3.5 x 10 cells 6 Subculture at a density of 10 cells / ml, and measure the cell density and viability again the next day to determine that the cell density was 7.0 x 10 6 After confirming that the cell density is 6.0 x 10 cells / ml or more and the cell viability is 95% or more, 6 ExpiCHO to cells / ml TM The cells were diluted by adding expression medium. 25 ml of cell culture medium was dispensed into a 125 ml Erlenmeyer flask and stored in a 37°C incubator until the preparation of the transfection mixture. 20 μg of plasmid gene was added to 1 ml of OptiPro TM It was mixed well with SFM medium and 80 μl of Expifectamine TM Add 920 μl of CHO reagent to OptiPro TM It was mixed well with SFM medium. Expifectamine-OptiPro TM Plasmid-OptiPro prepared in advance with SFM mixture TM After adding to SFM and mixing well, it was left at room temperature for 3 minutes. The mixture was slowly added to the pre-dispensed cell culture medium and cultured in an incubator set at 37°C, 8% CO2, and 130 rpm. At 18-22 hours after transfection, Expifectamine TM CHO Enhancer 150 μl and ExpiCHO TM4 ml of feed was added to the culture flask and cell culture was continued in an incubator set at 32°C, 5% CO2, and 130 rpm. When producing a larger amount of conjugates, 200 ml of cell culture was dispensed into a 1 l Erlenmeyer flask and the cells were stored in an incubator at 37°C until the preparation of the transfection mixture. 160 μg of plasmid gene was added to 8 ml of OptiPro TM It was mixed well with SFM medium and 640 μl of Expifectamine TM Add 7.4 ml of CHO reagent to OptiPro TM It was mixed well with SFM medium. Expifectamine-OptiPro TM Plasmid-OptiPro prepared in advance with SFM mixture TM After adding to SFM and mixing well, it was left at room temperature for 3 minutes. The mixture was slowly added to the pre-dispensed cell culture medium and cultured in an incubator set at 37°C, 8% CO2, and 130 rpm. At 18-22 hours after transfection, Expifectamine TM CHO Enhancer 1200 μl and ExpiCHO TM 32 ml of feed was added to the culture flask, and cell culture was continued in an incubator set at 32°C, 5% CO2, and 130 rpm. Cell density and viability were measured on days 2 and 5 after transfection, and on day 5, the final titer was measured using Cedex (Roche) equipment, and then the cell culture solution containing protein was recovered by centrifugation at 3000 xg for 30 minutes. The recovered supernatant was filtered through a 0.22 μm polyethersulfone (PES) filter and stored at 4°C for short-term storage and at -80°C for long-term storage.

[0220] Example 4 - Purification of the conjugate protein (Protein A affinity chromatography and size exclusion chromatography)

[0221] Protein purification from the culture medium was performed by affinity chromatography using an AKTA avant25 instrument equipped with an Amsphere a3 column. The culture medium filtered through a 0.22 μm bottle-top filter was passed through a flow rate of 13.33 ml / min, and 8 column volumes were passed through the buffer condition of 50 mM Na-Pi, 0.5 M NaCl, pH 7.0, and 50 mM sodium acetate, pH 4.5, respectively, to remove nonspecific binding. After removing nonspecific binding, 10 column volumes of the elution buffer condition of 100 mM glycine, pH 3.0 were passed through the elution buffer to elute the bound proteins, and the eluate was collected in a 50 ml tube. The collected proteins were added to a 1 M Trizma ® The protein solution was neutralized by adding 1% of the collected protein volume of a base (pH 11.0) solution, and the precipitate was removed using a 0.22 μm syringe filter. The samples before and after neutralization were quantified by UV measurement, and the purity was analyzed by SEC-HPLC. The primary purified material was additionally subjected to size exclusion chromatography using a Superdex200pg 26 / 600 column to ensure a purity of 90% or more. The neutralized sample was then purified using a Millipore TM Studcell 250 mL concentrator and Millipore TM Discs 50 kDa were used to concentrate the sample to a volume of 13 mL or less. The concentrated sample was injected into a column buffered with PBS, pH 6.5, and the eluted protein was collected in a tube and its purity was confirmed by SEC-HPLC before concentration. The final concentrated protein was analyzed by UV quantitation, and its purity was confirmed by SEC-HPLC and SDS-PAGE analysis.

[0222] Through the above process, one or more of the following properties of each conjugate were measured: titer, elution amount, yield (step yield), content of high molecular weight species (HMWS), content of low molecular weight species (LMWS), and content of functional monomers. Here, titer refers to the concentration of the Fc-fusion protein present in the culture medium measured when the cell culture medium was recovered, elution amount refers to the total amount of protein recovered after performing affinity chromatography, which is the first purification step, and yield refers to the recovery rate of the protein recovered by the column during the first purification, which is calculated by dividing the total amount of protein recovered after the first purification by the total amount of protein measured in the culture medium. High molecular weight species (HMWS) are protein species with a mass larger than the predicted functional unit of the protein, and low molecular weight species (LMWS) are protein species with a mass smaller than the predicted functional unit of the protein. In both cases, they refer to fractions with abnormal protein folding. Functional monomers refer to fractions with the structure and function of a normal protein. Figures 2a to 2o show the productivity and purity of the conjugates of anti-PD-L1 scFv and modified fusion proteins (modified VEGFR1-D2-D3). That is, it was confirmed that the purity of H-12C94 to H-12C99, which are conjugates based on the modified fusion proteins of the present disclosure, was higher than that of the control conjugate (H-12C08; A167 scFv-HG4SL-VEGF-Grab) prepared with the unmodified fusion protein when purified under the same conditions (purity of about 20% vs. about 90%). It was confirmed that many high-molecular-weight aggregates were generated in the conjugate prepared with the unmodified fusion protein (Figures 2c and 2d), whereas it was confirmed that the aggregates were significantly reduced in the conjugates prepared with the modified fusion proteins (Figures 2e to 2n).This means that the conjugate of the present disclosure has improved properties by including a modified fusion protein, which in turn means improved yield in drug manufacturing.

[0223] Example 5 - Freeze / thaw stability test of the composite

[0224] The stability of the produced conjugate after freezing / thawing was confirmed. The conjugate protein sample was frozen for one hour in a -70℃ ultra-low temperature freezer, then taken out to room temperature and thawed for 30 minutes. This process was repeated five times. For each cycle, 50 μl of the protein sample was sampled and analyzed by SEC-HPLC to confirm changes in purity (Table 10: 2nd cycle (2R); Table 11: 3rd cycle (3R)).

[0225] Protein Code Control 1st F / T 2nd F / T 3rd F / T 4th F / T 5th F / TH-32D0192.0186.5483.9283.6583.1183.48H-32D0294.7889.6488.3687.8787.5987.56H-32D0996.1796.4695.0994.5294.2293.81H-32D1096.2995.0594.0193.6293.492.94

[0226] Protein Code Control Group 1st F / T 2nd F / T 3rd F / T 4th F / T 5th F / TH-32D0294.0393.7393.2493.0692.9692.84H-32D0493.5792.7892.289291.8191.79H-32D0693.2492.692.4592.1892.0491.98H-32D0895.8994.994.594.0593.9193.82

[0227] Table 10 above shows the freeze / thaw stability results from the second experiment (2R), and Table 11 shows the freeze / thaw stability results from the third experiment (3R). As shown in Table 10, there was no significant difference in the freeze / thaw stability of the conjugates depending on the linkers V2 and V3, but V3 showed slightly higher stability. In the case of scFv, atezolizumab showed relatively higher stability than A167, but as with the linker, it was difficult to see a significant difference. Overall, high freeze / thaw stability was observed in conjugates based on the modified fusion proteins of the present disclosure regardless of the linker and scFv.

[0228] This was also evident in the comparative results of Fc variants borrowing the A167 scFv and V3 linkers. As shown in Table 11, purity was confirmed to decrease by less than 2% for all variants even after five freeze / thaw cycles.

[0229] The above results were tested in a typical buffer rather than an optimal formulation, suggesting the possibility of improving freeze / thaw stability through future formulation studies.

[0230] In summary, the conjugates based on the modified fusion proteins of the present disclosure showed high freeze / thaw stability.

[0231] Example 6 - Purity analysis of conjugate proteins using size exclusion-high performance liquid chromatography techniques.

[0232] Waters Analysis was performed using a Tosoh TSKgel G3000SWXL SEC column connected to an Arc HPLC system, and PBS, pH 7.4, was used as the mobile phase. 30 μg of protein sample was injected into the column while flowing the mobile phase at 0.5 mL / min, and detected at 280 nm for 30 min to measure at least one of the content of high molecular weight species (HMWS), low molecular weight species (LMWS), and functional monomers.

[0233] Example 7 - Evaluation of binding affinity to VEGF-A, PlGF, and PD-L1

[0234] The binding affinity of each protein for VEGF-A and PlGF ligands was determined using Octet according to previous literature (Kamat et al, 2017). ® Measurements were made using a RED96e (ForteBIO) device.

[0235] First, the AHC sensor was stabilized by immersing it in 1x KB buffer (ForteBIO) before measuring the binding affinity. The substance to be analyzed, the conjugate protein PB203, was bound to the sensor at a concentration of 10 nM, and then washed with 1X KB buffer to remove the protein remaining after binding. After that, the binding affinity was measured by flowing VEGF-A, PlGF, and PD-L1 in 8 concentration ranges each. Among the results of the binding affinity measurement, three or more concentrations that fall within the range of Full X^2 ​​(3 or less) and Full R^2 (0.95 or more), which are indices for ensuring reliability, were selected and set as Global fit, and then the binding affinity (KD) and Kon, Kdis values ​​were calculated. The results are shown in Figures 4a to 4k. As can be seen in the drawings 4a to 4k, the conjugate of the present disclosure was confirmed to have an increased binding rate to VEGF-A and PlGF compared to a conjugate prepared with an unmodified fusion protein, and it can be confirmed that the PD-L1 binding affinity of the scFv is well maintained without any decrease.

[0236] However, when preparing a complex of the modified fusion protein of the present disclosure and an scFv targeting a specific antigen, it is not possible to predict in advance whether the binding affinity of the fusion protein to VEGF and / or PlGF and the binding affinity of the scFv to the specific antigen will function without interference with each other. In the present disclosure, it was confirmed that the binding affinity of the modified fusion protein to VEGF and PlGF and the binding affinity of the anti-PD-L1 scFv to PD-L1 were maintained without antagonizing each other, which is an effect that could not have been easily predicted.

[0237] Example 8 - Stability Evaluation in a Blood-Mimulated Environment

[0238] To indirectly analyze the in vivo stability of PB203, the concentration of protein variants remaining after a specific time was measured through ELISA through a reaction with the blank serum collected from rats. PB203 was mixed with the rat blank serum at 168 hours, 144 hours, 72 hours, 48 ​​hours, 24 hours, 4 hours, 2 hours, and 0 hours based on the test execution time, and the final concentration was prepared as 10 μg / ml, and then stored in a 37 ℃ incubator. The plate was coated with human VEGF-A (R&D systems) through overnight incubation at 4 ℃ the day before the test, and this was washed with 0.1% PBS-T washing buffer to remove human VEGF-A that was not sufficiently bound, and then the blocking process was performed for 1 hour at room temperature by treating with 0.1% PBS-T containing 5% skim milk. The protein-serum mixture prepared for each time point was diluted 50-fold in blocking buffer, and 100 μl was added to each well of the plate and incubated at room temperature for 2 hours. Afterwards, all the reaction solution in the well was removed, and the plate was washed with washing buffer. After incubation with the detection antibody, HRP Goat anti-human IgG Fc Cross-Absorbed Secondary antibody (Invitrogen) at room temperature for 1 hour, unreacted detection antibody was removed through washing, and 100 μl of 3,3',5,5'tetramethylbenzidine (TMB) solution (Sigma) was added at each time point, incubated at room temperature for 10 minutes, and then the reaction was stopped by adding stop solution (Sigma). The absorbance was measured at 450 nm using a plate reader. The residual amount (%) was calculated based on the measured absorbance at 0 hour and compared relatively. The results are shown in Figures 3a to 3c.

[0239] As can be seen in FIGS. 3a and 3b, it can be confirmed that the conjugates H-12C94 to H-12C99 based on the modified fusion proteins of the present disclosure exhibit significantly superior serum stability in a blood-mimetic environment compared to the conjugate (H-12C08) prepared with the unmodified fusion protein. In addition, as can be seen in H-32D01, H-32D02, H-32D09, and H-32D10, it was confirmed that they exhibit significantly superior serum stability in the corresponding blood-mimetic environment regardless of the use of the V2 linker or the V3 linker and the type of scFv (FIG. 3c).

[0240] Example 9 - Isoelectric Point Analysis

[0241] The isoelectric point of PB203 protein was analyzed using the image capillary isoelectric focusing (iclEF) method. 40 μl of the modified fusion protein diluted to a concentration of 2 mg / ml and 160 μl of the pre-prepared master mix solution (SERVALYT TM+ Low pI marker (3.38) (ProteinSimple) + High pI marker (9.5) (ProteinSimple) + 1% methyl cellulose (ProteinSimple) + DW + 500 mM arginine (Sigma) + 200 mM iminodiacetic acid (Sigma) + 10 M urea (Sigma)) were mixed, centrifuged to remove precipitates and air bubbles, and then inserted into each well of the device (Maurice, ProteinSimple). 2 ml each of cathode solution (Catholyte solution) and cathode solution (Anolyte solution) were added to the OH- and H+ positions of the cartridge, and the cartridge was mounted on the device. The isoelectric point was then measured according to the device instructions. Since the measured pI appears as multiple peaks within a specific range rather than a single peak, the pI distribution of the protein was defined by dividing it into three sections: acidic (pI 6-7), neutral (pI 7-8), and basic (pI 8 <), and the section with the highest ratio of distribution was compared. The results are shown in Figures 7a to 7r.

[0242] As can be seen in FIGS. 7g, 7m, and 7r, the conjugates based on the modified fusion proteins of the present disclosure, H-12C94 to H-12C99, have a higher acid / neutral ratio compared to the control conjugate (H-12C08; A167+VEGF-Grab) prepared with an unmodified fusion protein. In addition, as can be seen in FIGS. 7m and 7r, the acid / neutral ratio is higher compared to the unmodified fusion protein and the conjugates prepared therefrom, regardless of the type of linker V2 or V3, scFv, or Fc mutation. This means that the conjugates of the present disclosure have a higher tendency to exhibit a negative polarity in the in vivo environment, suggesting that the possibility of nonspecific binding during in vivo circulation is lowered.

[0243] Example 10 - Thermal Stability Analysis

[0244] PB203 was prepared by diluting to a final concentration of 2 mg / ml using 1x PBS buffer. 2.5 μl of 8X Protein Thermal Shift was added to 12.5 μl of protein. TM Die (Protein Thermal Shift) TM dye)(Life Technologies) and 5.0 μl protein thermal shift TM The final reaction solution was prepared by adding buffer (Life Technologies). 20 μl of the prepared reaction solution was placed in a PCR tube and placed in QuantStudio. TM After aligning the samples on the tray of a real-time PCR (QuantStudio Real-time PCR) device, Tm values ​​were measured. All analyses were performed in four replicates. The results are shown in Figures 8a to 8d.

[0245] Specifically, Figure 8b confirms that the thermal stability of linkers V2 and V3 is not significantly affected, but rather can vary depending on the scFv used. Furthermore, Figure 8d confirms that Fc mutations are unrelated to thermal stability.

[0246] The temperature at the time of bioinjection is within 40°C, and for protein pharmaceuticals, storage stability is evaluated at 25°C and 40°C, assuming room temperature and body temperature. Based on the above criteria, it was confirmed that the conjugates based on the modified fusion protein of the present disclosure exhibited high thermal stability above 60°C overall.

[0247] This demonstrates that conjugates based on the modified fusion proteins of the present disclosure have physicochemical properties suitable for development as drugs.

[0248] Example 11 - VEGF-A inhibitory activity analysis

[0249] To quantitatively analyze the inhibitory activity against VEGF-A, a genetically engineered cell line (KDR / NFAT-RE HEK293, Promega) with a luciferase reporter system for the expression of VEGFR-2 (KDR) and VEGF-A / VEGFR-2 interaction was used. 1.6 x10 6 After dispensing 25 μl of cells at a density of 1 cell / ml into a 96-well plate, 25 μl of VEGF-A solution (33.3 ng / ml concentration) and 25 μl of PB203 diluted to different concentrations were incubated for 6 hours in a 37°C, 5% CO2 incubator. After cooling the plate to room temperature for 15 minutes, 75 μl of Bio-Glo reagent (Promega, luciferase assay buffer + substrate mixture) was added. After reacting for 10 minutes in a light-blocked state, luminescence was measured using a plate reader. The IC for each substance was analyzed using GraphPad Prism. 50 The value was calculated.

[0250] As can be seen in FIGS. 6C to 6L and 6N, the conjugates based on the modified fusion proteins of the present disclosure were confirmed to have a similar degree of cellular-level VEGF-A signal transduction inhibition ability as the modified fusion proteins before conjugation, regardless of the type of linker and scFv. In addition, the inhibitory ability was confirmed to be superior compared to the unmodified fusion protein, VEGF-Grab (PB101).

[0251] Example 12 - Analysis of PD-1 / PD-L1 signal inhibition ability

[0252] To quantitatively analyze the inhibitory effect on PD-L1, a genetically engineered cell line (Jurkat PD-1 / U2OS PD-L1, Eurofins Discovery) with a luciferase reporter system for PD-1 and PD-L1 expression and PD-1 / PD-L1 interaction was used. 1x10 5After dispensing 40 μl of U2OS PD-L1 cells at a density of 2.4 x 10 cells / ml into a 96-well plate, 20 μl of PB203 dilution solution and control substances were added, and the cells were incubated for 1 hour in a 37°C, 5% CO2 incubator. 5 40 μl of Jurkat PD-1 cells at a density of 1 cell / ml were additionally dispensed into a 96-well plate and incubated at room temperature for 2 hours. 10 μl of Detection reagent 1 was then added to the plate and incubated at room temperature for 15 minutes, avoiding light. Next, 40 μl of Detection reagent 2 was added and incubated at room temperature for 3 hours, avoiding light. Finally, luminescence was measured using a plate reader. The IC for each substance was analyzed using GraphPad Prism. 50 The value was calculated.

[0253] The inhibitory effects of the complex of anti-PD-L1 scFv and modified fusion protein (modified VEGFR1-D2-D3) on VEGF-A and PD-L1 are shown in Figures 6a to 6e.

[0254] As can be seen in FIGS. 6i to 6n and 6o, the conjugates based on the modified fusion proteins of the present disclosure (H-32D01, H-32D02, H-32D04, H-32D06, H-32D08, H-32D09, H-32D10, etc.) were confirmed to have a similar degree of cellular level PD-L1 signal transduction inhibition ability as the pre-conjugated anti-PD-1 antibody, regardless of the type of linker and scFv.

[0255] This demonstrates that the complexes based on the modified fusion proteins of the present disclosure maintain their respective functions without interference with each other through structural design despite their complex structures.

[0256] Example 13 - Evaluation of simultaneous binding capacity of VEGF-A, PlGF, and PD-L1 ligands

[0257] When two or more types of VEGF-A, PlGF, and PD-L1 ligands are combined, Octet was used to evaluate the binding affinity according to the binding order. ® The RED96e (ForteBIO) device was used.

[0258] First, the AHC sensor was stabilized by immersing it in 1x KB buffer (ForteBIO) before measuring the binding strength. The substance to be analyzed, PB203 protein, was bound to the sensor at a concentration of 100 nM, and then washed with 1x KB buffer to remove the protein remaining after binding. Afterwards, VEGF-A, PlGF, and PD-L1 were bound at 30 nM (high concentration), and then different types of previously bound proteins (e.g., VEGF-A → PD-L1, PlGF → PD-L1, PD-L1 → VEGF-A, PD-L1 → PlGF) were bound at 8 concentration ranges (30, 15, 7.5, 3.75, 1.875, 0.937, 0.468, 0 nM) in the state where the previous proteins were included (30 nM), and the signals were measured. Among the results of the binding affinity measurement, to ensure reliability, four or more concentration intervals were identified where Full X^2 ​​(3 or less) and Full R^2 (0.95 or more) were within the range, and the binding affinity and other indices were calculated through a global fit for these. As a result, the simultaneous binding affinity of the complex of anti-PD-L1 scFv and modified VEGFR1-D2-D3 fusion protein to the target is shown in Figs. 5a to 5f. As a result of measuring the simultaneous binding affinity of the antigen, it can be confirmed that it is maintained with almost no difference even when the antigen type is changed. Specifically, as shown in Figs. 5a and 5b, it was confirmed that the binding affinity to PD-L1 was similarly maintained regardless of which ligand among VEGF-A and PlGF the complex based on the modified fusion protein of the present disclosure is bound to. Furthermore, as can be seen in FIGS. 5C to 5F, the conjugates based on the modified fusion proteins of the present disclosure demonstrate excellent levels of binding affinity when bound to a single ligand, regardless of the binding order or type of the target ligands. This demonstrates that the conjugates based on the modified fusion proteins of the present disclosure maintain their respective functions without interfering with each other through structural design, despite their complex structures.

[0259] Example 14 - Pharmacokinetic evaluation through single intravenous administration in SD rats

[0260] Seven-week-old male SD rats were used for the test after one week of acclimatization, and PB203 was administered intravenously once at a concentration of 3 mcg. Blood samples were collected through the jugular vein at 0, 0.083, 2, 8, 24, 48, 96, 120, 168, and 336 h, and plasma was separated.

[0261] The day before the test, human PD-L1 (R&D systems) was coated on the plate through overnight incubation at 4℃, and then washed with 0.1% PBS-T washing buffer to remove human PD-L1 that was not sufficiently bound. Then, 0.1% PBS-T containing 5% skim milk was treated to perform a blocking process at room temperature for 1 hour. The prepared plasma sample was diluted 20-fold in blocking buffer, and 100 μl was added to each well of the plate and reacted for 2 hours at room temperature. Afterwards, all reaction solutions in the wells were removed, and the plate was washed using washing buffer. After reacting with the detection antibody, HRP Goat anti-human IgG Fc Cross-Absorbed Secondary antibody (Invitrogen), at room temperature for 1 hour, unreacted detection antibody was removed by washing, and then 100 μl of 3,3',5,5'tetramethylbenzidine (TMB) solution (Sigma) was added, reacted for 10 minutes at room temperature, and then the reaction was stopped by adding stop solution (Sigma). The absorbance was measured at 450 nm using a plate reader. The quantitative standard substance was prepared using the serial dilution method with the PB203 substance at a concentration of 62.5 to 0.48, and a standard curve was drawn based on the measured absorbance (OD450), and the PB203 concentration in plasma was back-calculated using the derived equation for quantitative evaluation. Finally, the PK parameter was calculated using the results of the concentration over time. First, to verify the pharmacokinetics according to scFv, H-32D02 and H-32D10, which share the V3 linker and are bound to A167 or atezolizumab scFv, respectively, were evaluated.As shown in Fig. 9a, the A167 conjugate showed superior in vivo pharmacokinetic patterns, and Fc variants for it were prepared. Four Fc variants including the native Fc for the A167 conjugate were prepared and evaluated, and as can be seen in Fig. 9b, the H-32D04, H-32D06, and H-32D08 conjugates showed superior in vivo pharmacokinetic patterns compared to the H-32D02 conjugate. The in vivo pharmacokinetic patterns among the three conjugates showed similar levels.

[0262] Example 15 - Anticancer efficacy test in a mouse model

[0263] The genetically modified cell line MC-38-hPD-L1 was subcutaneously transplanted into the genetically modified mouse B6 / JGpt-Cd274tm1 (hCD274) / Gpt model, and the tumor size was approximately 100 mm. 3 When the target was reached, the group was separated. Afterwards, the PB203 candidate substance and the control drug were administered intraperitoneally three times a week at a dose of 10 mpk, and the tumor size and weight were measured for 3 weeks. This test was performed in compliance with the Animal Experiment Ethics Regulation. The results are shown in Fig. 10. As shown in Fig. 10, among the conjugates of the present disclosure, the Fc native type and ADCC / CDC activation inhibitory Fc showed excellent tumor growth inhibition (TGI) of about 10% or more compared to the existing commercial antibody Tecentriq, and the other two Fc variants also showed high levels of tumor growth inhibition. In particular, in the case of H-32D04, complete regression was observed in about 40% of the subjects, which is an excellent result compared to the commercial antibody Tecentriq, in which complete regression was not observed at all.

[0264] From the above description, those skilled in the art will understand that the present disclosure can be implemented in other specific forms without altering the technical spirit or essential characteristics of the present disclosure. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present disclosure should be interpreted as encompassing all changes or modifications derived from the meaning and scope of the claims described below, and their equivalent concepts, rather than the detailed description above.

Claims

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

2. In the first paragraph, the fusion protein comprises a VEGFR1 extracellular domain, a linker, and a multimerization domain, The above VEGFR1 extracellular domain comprises an immunoglobulin (Ig)-like domain D2 and an Ig-like domain D3 of VEGFR1, and a linker exists between the Ig-like domain D3 and the multimerization domain, A conjugate, which is a modified fusion protein having one or more of the following characteristics (a) to (c): (a) one or more amino acid substitutions of K241E, L243S, R244V, and H246E on the β1-β2 loop of domain D3, an amino acid substitution of L258A, L258S, or L258D on the β2-β3 loop of domain D3, and one or more amino acid substitutions of K300G, Q302T, and K304S on the β5-β6 loop of domain D3, wherein the β1-β2 loop of domain D3 comprises amino acid residues T236 to T247 of the amino acid sequence of VEGFR 1 comprising SEQ ID NO: 41, the β2-β3 loop comprises amino acid residues T256 to V262 of the amino acid sequence of VEGFR 1, and the β5-β6 loop comprises amino acid residues D299 to L308 of the amino acid sequence of VEGFR 1; (b) the linker has a length of about 14 to about 35 amino acids; and (c) A disulfide bond exists within the fusion protein, and one of the amino acid residues at positions -2, -1, 0, +1, +2, and +3 based on L243 and Y329 of domain D3 among the amino acid residues on the β1-β2 loop of domain D3 is substituted with cysteine.

3. In the second paragraph, the characteristic (a) of the fusion protein is, A complex comprising amino acid substitutions K241E, L243S, R244V, and H246E in the β1-β2 loop of domain D3; amino acid substitutions L258A, L258S, or L258D in the β2-β3 loop of domain D3; and amino acid substitutions K300G, Q302T, and K304S in the β5-β6 loop of domain D3.

4. In the second paragraph, the modified fusion protein is a complex in which the N-terminus of domain D2 begins with the amino acid sequence EF.

5. In the second paragraph, in the characteristic (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. A conjugate in the fifth paragraph, wherein at least one glycine (G) included in the GS repeat sequence is substituted with cysteine ​​(C).

7. In the second paragraph, a conjugate comprising an amino acid sequence of a hinge region derived from an immunoglobulin in the characteristic (b) of the modified fusion protein, wherein the amino acid sequence of the hinge region can be modified.

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

9. In the 7th paragraph, a complex having an amino acid mutation in a papain recognition site or glycosylation site present in the hinge region derived from immunoglobulin, in the characteristic (b) of the modified fusion protein.

10. In the second paragraph, in the characteristic (b) of the modified fusion protein, the linker comprises (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, in order from the N-terminus to the C-terminus, wherein the amino acid sequence of the hinge region can be modified.

11. In the second paragraph, in the characteristic (b) of the modified fusion protein, the linker is an amino acid sequence selected from the group consisting of CSSGDATPTSPPSP, CSKVDKKVEPKSSDTPPTCPPCP, CSGGGGSAEPKAGDATPPTCPPCP, CSGGGGSGGGGSGGGGSAESKYGPPCPPCP, CSNTGSGGEEKKKEKEKEEQEERSSDTPPTCPPCP, CSNTGSGGEEKKKEKEKEEQEERSCDTPPTCPPCP, CGSSGGSSGEPKSDATPTCPPCP, and CSKVDKKVEPKSSDKTYTCPPCP.

12. In the second paragraph, the modified fusion protein comprises (a) amino acid substitutions K241E, L243S, R244V, and H246E on the β1-β2 loop of domain D3, amino acid substitutions L258A, L258S, or L258D on the β2-β3 loop of domain D3, and amino acid substitutions K300G, Q302T, and K304S on the β5-β6 loop of domain D3; (b) a conjugate wherein the linker is an amino acid sequence selected from the group consisting of CSSGDATPTSPPSP, CSKVDKKVEPKSSDTPPTCPPCP, CSGGGGSAEPKAGDATPPTCPPCP, CSGGGGSGGGGSGGGGSAESKYGPPCPPCP, CSNTGSGGEEKKKEKEKEEQEERSSDTPPTCPPCP, CSNTGSGGEEKKKEKEKEEQEERSCDTPPTCPPCP, CGSSGGSSGEPKSDATPTCPPCP, and CSKVDKKVEPKSSDKTYTCPPCP; and (c) an amino acid residue at position +2 relative to L243 and Y329 of domain D3 is substituted with cysteine.

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

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

60.

15. A complex according to claim 14, wherein the multimerization domain comprises an IgG1 Fc region consisting of an amino acid sequence of SEQ ID NO:

60.

16. In the second paragraph, the multimerization domain is as per SEQ ID NO:

60. i) has one or more of the following amino acid substitutions: T20Q, D126E, L128M, M198L, and K217; or ii) A conjugate having at least one of an L4A amino acid substitution, an L5A amino acid substitution, an H38Q amino acid substitution, a K44Q amino acid substitution, a Y66F amino acid substitution, an A97G amino acid substitution, an A100S amino acid substitution, a P101S amino acid substitution, an R125Q amino acid substitution, a D126E amino acid substitution, an L128M amino acid substitution, a K179R amino acid substitution, a Q189E amino acid substitution, a P215L amino acid substitution, and a K217 amino acid deletion.

17. In the second paragraph, the modified fusion protein is a complex in the form of a dimer or multimer.

18. A complex according to claim 1, wherein the PD-L1 targeting scFv is an atezolizumab scFv or an anti-PD-L1 scFv comprising an amino acid sequence of SEQ ID NO:

40.

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

20. A nucleic acid molecule encoding a combination of any one of claims 1 to 18.

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

22. A vector comprising a nucleotide sequence encoding a combination of any one of claims 1 to 18.

23. A vector in claim 22, which is a recombinant viral vector.

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

25. A 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 at least one selected from the group consisting of chronic infection, tissue allograft, autoimmune disease, inflammatory disease, neoplastic disease, cancer, angiogenesis-related disease, or ocular disease, by administering to a subject in need thereof a combination of any one of claims 1 to 18 and / or a recombinant viral vector of claim 23.

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

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