Novel vascularization inhibitor and use thereof

Abstract

The present invention relates to an antibody fragment specifically binding to vascular endothelial growth factor (VEGF) and, more specifically, to a dimeric antibody fragment comprising a structure in which two antibody fragments are dimerized by a SARAH domain, and expressing high binding affinity and excellent biological activity for VEGF. The antibody of the present invention inhibits VEGF-induced proliferation of human vascular endothelial cells (HUVEC) and blocks VEGFR2-mediated signaling, and thus effectively inhibits angiogenesis.

Description

Novel angiogenesis inhibitors and their uses

[0001] The present invention relates to a pharmaceutical composition for inhibiting angiogenesis comprising a VEGF-targeted antibody fragment and the use thereof.

[0002]

[0003] Tumor cells developing resistance to chemotherapy drugs is becoming a serious problem in the field of clinical oncology. In fact, this is one of the main reasons why most forms of human cancer still exhibit resistance to chemotherapy, despite advancements in this field.

[0004] Another cancer treatment method is the use of "immunotoxins," which deliver toxins to tumor cells using anti-tumor cell antibodies. However, like chemotherapy, immunotoxin therapy has serious drawbacks when used on solid tumors. For example, antigen-negative or antigen-deficient cells can survive, causing the tumor to re-colonize or progress to a metastatic stage.

[0005] Another reason solid tumors exhibit resistance to antibody-based therapies is that macromolecular agents, such as antibodies and immunotoxins, generally cannot be absorbed into the tumor body. Both interstitial pressure and physical diffusion distance within the tumor are factors that significantly limit this form of therapy. Consequently, solid tumors, which account for more than 90% of all human cancers, become resistant to antibody and immunotoxin therapies.

[0006] A more recent treatment method involves targeting the vasculature of solid tumors. Targeting the blood vessels of tumor cells rather than the tumor cells themselves has the advantage of preventing the development of resistant tumor cells and allowing easy access to the target cells. Additionally, since many tumor cells rely on a single blood vessel for oxygen and nutrient supply, destroying the vessel amplifies the anti-tumor effect. Examples of vasculature-targeting methods are described in U.S. Patents No. 5,855,866 and No. 5,965,132, which specifically describe the targeted delivery of anti-cell agents and toxins to markers in the tumor vasculature.

[0007] Another effective method among methods targeting blood vessels is to target coagulant factors to markers expressed or absorbed within the tumor vascular system {cf. Huang et al., 1997; U.S. Patents No. 5,877,289, 6,004,555 and 6,093,399}. The method of delivering coagulant factors to the tumor vascular system instead of toxins has the advantage of reducing immunogenicity and the risk of adverse effects from toxins. As described in U.S. Patent No. 5,877,289, preferred coagulant factors for use in the aforementioned tumor-specific "coagulant ligand" are the major initiator of blood coagulation, tissue factor (TF), and truncated versions of human coagulation-inducing proteins.

[0008] While methods for specifically delivering toxins and coagulation factors to tumor blood vessels represent a significant advance in tumor treatment, certain peripheral tumor cells can withstand intratumoral destruction caused by such therapies. Therefore, anti-angiogenic methods are used in conjunction with the tumor destruction methods described in U.S. Patents No. 5,855,866 and No. 5,877,289.

[0009] Anti-angiogenic tumor therapies are generally based on inhibiting the proliferation of blood vessels growing from the distal ends of solid tumors. These therapies are typically used to reduce the risk of micrometastasis or to inhibit further growth of solid tumors following more conventional treatments (e.g., surgery or chemotherapy).

[0010] Angiogenesis is the development of new blood vessels from existing blood vessels and / or circulating endothelial stem cells {cf. Asahara et al., 1997; Springer et al., 1998; Folkman and Shing, 1992}. Angiogenesis plays a key role in many physiological processes (e.g., embryogenesis, wound healing, and menstruation). Furthermore, angiogenesis is important in certain pathological phenomena. In addition to its role in the growth and metastasis of solid tumors, other distinct conditions involving angiogenic components include arthritis, psoriasis, and diabetic retinopathy {cf. Hanahan and Folkman, 1996; Fidler and Ellis, 1994}.

[0011] Angiogenesis is regulated in normal and malignant tissues through the balance of angiogenic stimulants and inhibitors produced in target tissues and distant sites {References: Fidler et al., 1998; McNamara et al., 1998}. Vascular endothelial growth factor-A [VEGF, also known as vascular permeability factor (VPF)] is a major stimulant of angiogenesis.

[0012] Although VEGF plays an important role in normal physiological angiogenesis and tissue regeneration processes, in certain pathological conditions it is overexpressed and contributes to the onset and progression of diseases by inducing abnormal neovascularization. For example, eye diseases such as age-related macular degeneration (AMD), diabetic retinopathy (DR), and central retinal vein occlusion (CRVO) are closely related to the pathological activity of VEGF, and abnormal neovascularization acts as a major pathological mechanism in these diseases.

[0013] For this reason, protein-based therapeutics targeting VEGF, such as antibody-based therapies, have been actively developed. Currently, VEGF inhibitory antibodies used mainly consist of full-length antibodies, scFvs (short-chain antibody fragments), and bispecific antibodies, and some are composed of fusion proteins containing an Fc domain. However, these antibody-based therapies have the disadvantage of being difficult to mass-produce in microbial expression systems and high-cost due to their large molecular weight and complex structure. Furthermore, Fc domain-based dimerization strategies carry the potential to induce non-specific immune responses mediated by immune cells, and there is room for improvement, particularly in terms of safety, for ophthalmic diseases that require repeated administration.

[0014] To overcome these limitations, the inventors prepared a new type of VEGF-targeted antibody fragment that induces natural dimerization by fusing the SARAH (Salvador / RASSF / Hippo) domain, known to perform self-dimerization functions in cell signaling proteins such as MST1 and RASSF, with the antibody fragment. The antibody fragment of the present invention has a structure in which two VEGF-specific antibody fragments composed of monomers are dimerized via the SARAH domain, and can simultaneously secure various advantages such as structural stability, high binding affinity, low immunogenicity, and excellent productivity without the need for a separate chemical linker or Fc domain.

[0015] Conventional anti-angiogenesis antibodies have primarily been developed in the form of full-length antibodies or fusion proteins containing Fc domains, and these are used as therapeutic agents to inhibit angiogenesis by binding to VEGF. However, these antibodies have limitations in that they can only be produced in cell-based expression systems due to their large molecular weight and complex structure, and the production process is complex and costly. Furthermore, the presence of the Fc domain can lead to non-specific binding and activation of immune cells, which may pose safety concerns when used as a treatment for chronic diseases, such as eye diseases, that require repeated administration. To address these issues, single-domain antibody fragments such as nanobodies have been proposed as alternatives; however, nanobodies in a monomeric state have the disadvantage of limited therapeutic efficacy in terms of binding affinity and half-life, as well as a lack of persistence in the body.

[0016] Accordingly, the present invention aims to provide an antibody fragment capable of realizing a stable dimer structure and improving binding affinity by providing two antibody fragments that specifically bind to VEGF as a dimerized structure through the SARAH (Salvador / RASSF / Hippo) domain. The antibody fragment according to the present invention can achieve dimerization without a separate chemical linker or Fc domain by utilizing the self-dimerization characteristics of the SARAH domain, thereby minimizing non-specific stimulation of immune cells and maintaining low immunogenicity. Furthermore, by enabling the efficient production of an antibody fragment composed of a single polypeptide sequence in a microbial expression system, it offers the advantages of being suitable for mass production and ensuring process efficiency.

[0017] In addition, the antibody fragment of the present invention effectively blocks the interaction between VEGF and the VEGF receptor (VEGFR), thereby inhibiting angiogenesis and can be usefully applied to the prevention or treatment of neovascular eye diseases such as macular degeneration, diabetic retinopathy, and central retinal vein occlusion caused by pathological angiogenesis. Accordingly, the present invention aims to provide a new type of dimeric antibody fragment that improves antigen specificity, binding affinity, structural stability, productivity, and safety.

[0018]

[0019] To achieve the above objective, the present invention provides a dimeric antibody fragment comprising a structure in which two antibody fragments that specifically bind to vascular endothelial growth factor (VEGF) are dimeric by a SARAH domain.

[0020] In one embodiment, the present invention provides a dimeric antibody fragment characterized in that each antibody fragment constituting the dimeric antibody fragment comprises the amino acid sequence described in SEQ ID NO. 1.

[0021] In another embodiment, the present invention provides a dimeric antibody fragment characterized in that each antibody fragment constituting the dimeric antibody fragment comprises the amino acid sequence described in SEQ ID NO. 2.

[0022] In another embodiment, the present invention provides a dimeric antibody fragment characterized in that each antibody fragment constituting the dimeric antibody fragment is expressed by the nucleic acid sequence described in SEQ ID NO. 3.

[0023] As another embodiment, a dimeric antibody fragment is provided, characterized in that each antibody fragment constituting the dimeric antibody fragment comprises an Fh8 fusion domain at the N-terminus.

[0024] As another embodiment, a dimeric antibody fragment is provided, characterized by additionally including a TEV protease cleavage site at the C-terminus of the Fh8 fusion domain.

[0025] As another embodiment, a dimeric antibody fragment is provided, characterized in that each antibody fragment constituting the dimeric antibody fragment includes a 6x His tag at the C-terminus.

[0026] In another embodiment, the dimeric antibody fragment is provided, characterized by inhibiting angiogenesis by inhibiting the proliferation of vascular endothelial cells or by blocking VEGFR2-mediated signaling.

[0027] In another embodiment, a pharmaceutical composition comprising a dimeric antibody fragment is provided, wherein the composition is characterized by being used for the prevention or treatment of diabetic retinopathy, diabetic macular edema, age-related macular degeneration, retinal neovascularization, central retinal vein occlusion, branched retinal vein occlusion, polyparyular choroidal angiopathy, choroidal neovascularization, colorectal cancer, lung cancer, breast cancer, ovarian cancer, cervical cancer, liver cancer, kidney cancer, or brain tumor, wherein the dimeric antibody fragment comprises a structure in which two antibody fragments that specifically bind to vascular endothelial growth factor (VEGF) are dimeric via a SARAH domain.

[0028]

[0029] In the present invention, the binding strength and structural stability of antibody fragments can be simultaneously improved by dimerizing two antibody fragments that specifically bind to vascular endothelial growth factor (VEGF) through the SARAH (Salvador / RASSF / Hippo) domain. The SARAH domain is an α-helix domain that induces self-dimerization in intracellular signaling proteins. In the present invention, natural dimerization between antibody fragments was induced by utilizing the physical binding properties of this domain, and a functional dimer structure was stably realized without introducing a separate chemical linker or Fc domain.

[0030] Due to these structural features, the antibody fragment of the present invention has improved binding affinity for VEGF compared to the monomeric antibody fragment, and can more effectively inhibit the interaction between VEGF and the VEGF receptor (VEGFR). In particular, the inhibitory effect on angiogenesis through the blockade of VEGF-VEGFR signaling has been experimentally proven through in vitro luciferase reporter analysis, HUVEC proliferation inhibition tests, and in vivo hydrogel-based angiogenesis induction models.

[0031] Furthermore, the antibody fragment of the present invention consists of a single polypeptide, exhibiting high expression efficiency in an E. coli-based microbial expression system. Additionally, by utilizing TEV protease after purification, it can provide excellent process characteristics in terms of productivity and purity. This process efficiency offers the advantage of significantly reducing production costs compared to conventional full-length antibodies or Fc fusion protein-based antibodies.

[0032] Furthermore, since the antibody fragment of the present invention does not contain an Fc domain, non-specific stimulation of immune cells is suppressed, and low immunogenicity suitable for treating chronic eye diseases requiring repeated administration can be secured. In addition, structurally, it has a small molecular weight and excellent penetration, so it has potential for use in non-invasive administration methods such as eye drops or topical ophthalmic agents in terms of future formulation development.

[0033] Accordingly, the present invention provides improved technical effects in terms of the structural stability, target binding affinity, production efficiency, immunological safety, and pharmaceutical applicability of the antibody fragment, and can provide a novel antibody-based therapeutic agent that can be effectively utilized for the prevention or treatment of neovascular eye diseases such as macular degeneration, diabetic retinopathy, and central retinal vein occlusion.

[0034]

[0035] The effects of the present invention are not limited to the effects described above, and should be understood to include all effects that can be inferred from the configuration of the invention described in the detailed description of the invention or the claims.

[0036]

[0037] Figure 1 is a schematic diagram of the VCB-1502 antibody fragment of the present invention.

[0038] Figure 2 shows the plasmid map of the pET-21a(+) expression vector in which the VCB-1502 antibody fragment gene of the present invention is cloned. The inserted gene was inserted into the NdeI and XhoI restriction enzyme sites, and the vector was designed with a structure capable of IPTG-induced expression under the T7 promoter.

[0039] Figure 3 shows the results of SDS-PAGE analysis of protein expression in E. coli transgenic strains cloned with the VCB-1502 antibody fragment gene depending on whether IPTG was treated. Con is the non-transgenic control, and #3 and #7 represent the respective transgenic clones. When IPTG was treated (+O), a distinct protein band was observed around 30 kDa, confirming that the expression of the VCB-1502 antibody fragment was induced.

[0040] Figure 4 is a graph showing the binding affinity of the VCB-1502 antibody fragment to the VEGF antigen measured by ELISA analysis. A concentration-dependent binding pattern was observed, with the OD450 value gradually increasing according to antibody fragment concentrations of 1, 5, and 10 μg / mL, which demonstrates that the VCB-1502 antibody fragment specifically binds to VEGF.

[0041] Figure 5 shows the concentration-response curve calculated by analyzing the binding affinity of the VCB-1502 antibody fragment to VEGF. Through this experiment, the EC50 value was approximately 9.289 × 10⁻⁶. -9 It was calculated as M, which indicates that the corresponding antibody fragment has a high functional affinity for VEGF.

[0042] Figure 6 is a graph showing the measured binding constant (Kd) of the VCB-1502 antibody fragment to VEGF. As a result of analyzing the binding reaction at different antibody concentrations based on an OD of 450 nm, the Kd value was approximately 9.475 × 10⁻⁶. -9 It was confirmed to be M, which shows that the VCB-1502 antibody fragment has a high affinity for VEGF.

[0043] Figure 7 is a graph evaluating the inhibitory effect of the VCB-1502 antibody fragment on the proliferation of human umbilical vein endothelial cells (HUVEC) induced by VEGF165. Cell proliferation induced by VEGF was inhibited in treatment groups at various concentrations of the antibody fragment, and an inhibitory effect similar to that of the control antibody, Avastin, was observed.

[0044] Figure 8 is a graph showing the VEGFR2 signaling pathway activated by VEGF165 using a luciferase reporter assay. When VCB-1502 antibody fragments were treated at concentrations of 0.25–10 μg / mL, the luciferase expression level (RLU) showed a concentration-dependent decreasing trend, and an inhibitory effect similar to or superior to that of the control drug Avastin (10 μg / mL) was confirmed.

[0045] Figure 9 is a graph showing the change in body weight over 7 days in each group (G1–G5) administered VCB-1502 antibody fragment or Avastin in combination after injecting VEGF and hydrogel subcutaneously into mice. No significant weight loss was observed in any of the groups, and in vivo safety was maintained in the G3 and G4 groups administered VCB-1502 antibody fragment.

[0046] Figure 10 shows external images of the in vivo sites of each group (G1–G5) in which VCB-1502 antibody fragments or Avastin were administered in combination after inducing angiogenesis by injecting VEGF and hydrogel subcutaneously into mice. In the G2 group, which was administered only VEGF, subcutaneous blood vessels were clearly formed, whereas in the G3 and G4 groups, which were administered VCB-1502 antibody fragments, angiogenesis was significantly reduced.

[0047] Figure 11 is an image taken after collecting and photographing the angiogenic tissue (plugs) formed within the hydrogel injected subcutaneously into mice after 7 days. In the control group (G2) treated only with VEGF, angiogenic tissue with a strong hemoglobin color was observed, whereas in the G3 and G4 groups administered with VCB-1502 antibody fragments, angiogenesis within the tissue was inhibited and the tissue was pale, and an inhibitory effect similar to that of the Avastin-treated group (G5) was confirmed.

[0048]

[0049] The present invention will be explained in more detail below with specific examples. However, the embodiments described below are provided as examples to ensure that the concept of the present invention is sufficiently conveyed to those skilled in the art.

[0050] Accordingly, the present invention is not limited to the embodiments presented below and may be embodied in other forms, and the embodiments presented below are described merely to clarify the concept of the present invention and are not limited thereto.

[0051]

[0052] definition

[0053] In this specification, expressions such as "comprising," "comprising," "having," etc., should be understood as open-ended terms implying the possibility of including other embodiments in a manner similar to "comprising," unless otherwise stated in the phrase or sentence containing such expressions.

[0054] In this specification, "and / or" may mean any one or more of the items, any combination of the items, or all of the items in relation to the term.

[0055] In this specification, "nanobody" may refer to an antibody fragment comprising a camel or a single heavy chain variable region (VHH) antibody domain derived therefrom, which is miniaturized compared to a conventional antibody and provides high penetration power and stability.

[0056] In this specification, “VEGF (Vascular Endothelial Growth Factor)” refers to a growth factor protein that induces the survival, proliferation, migration, and angiogenesis of vascular endothelial cells, and among them, VEGF-A may mean a major factor mediating pathological angiogenesis.

[0057] In this specification, “VEGFR (Vascular Endothelial Growth Factor Receptor)” refers to a receptor protein that binds to VEGF and induces signal transduction, and among them, VEGFR2 may refer to a receptor that mediates a major pathway related to angiogenesis.

[0058] In this specification, the term “SARAH(Salvador / RASSF / Hippo) domain” may refer to a protein binding domain with an α-helix structure found in MST1, RASSF1A, Salvador, etc., and may mean a protein sequence having structural features capable of forming a protein complex by inducing autodimerization or heterodimerization.

[0059] In this specification, “VCB-1502 antibody fragment” may refer to the core recombinant protein of the present invention having a structure in which two antibody fragments that specifically bind to VEGF are dimerized through a SARAH domain.

[0060] In this specification, “VHH” may refer to a single-domain antibody derived from a heavy-chain antibody of camels, which has antigen-binding ability solely through the heavy-chain variable domain (VH) among the variable domains of a general antibody.

[0061] In this specification, “HUVEC (Human Umbilical Vein Endothelial Cell)” refers to a vascular endothelial cell derived from the human umbilical vein, which may be a cell line widely used in experiments related to angiogenesis.

[0062] In this specification, “angiogenesis” may refer to a physiological or pathological biological process in which new blood vessels are formed from existing blood vessels.

[0063] In this specification, the term “luciferase reporter assay” may refer to a biological analysis technique that measures the luminescence response of a luciferase enzyme to quantify the degree of specific gene expression.

[0064] In this specification, the term “dimeric antibody fragment” may refer to a protein complex formed by two antibody fragments being non-covalently bound by a self-dimerization domain. The dimeric antibody fragment of the present invention may refer to a protein complex formed by two antibody fragments that specifically bind to vascular endothelial growth factor (VEGF) being non-covalently bound by the self-dimerization characteristics of the SARAH (Salvador / RASSF / Hippo) domain. In this specification, each monomeric antibody fragment constituting the dimeric antibody fragment may include a nanobody composed of a sequence capable of specifically binding to VEGF, and more preferably may include a SARAH domain, an Fh8 fusion domain, a TEV protease cleavage site, and a 6x His tag.

[0065]

[0066] The present invention will be described in detail below.

[0067]

[0068] The present invention relates to a dimeric antibody fragment that improves structural stability and antigen binding affinity by connecting two antibody fragments through a domain having self-dimerizing properties, based on an antibody fragment that specifically binds to Vascular Endothelial Growth Factor (VEGF).

[0069] The dimeric antibody fragment of the present invention can be configured as shown in FIG. 1.

[0070] In particular, the present invention is designed so that the protein itself can form a stable dimeric structure without separate external processing by fusing the SARAH (Salvador / RASSF / Hippo) domain to the C-terminus of a monomer nanobody and utilizing the unique self-dimerization ability of this domain, thereby providing clear structural and functional differentiation from existing antibody design technologies.

[0071] Existing dimerization antibody technologies have primarily induced multimerization based on leucine zippers or Fc domains, or artificially induced connections between protein domains through flexible linker sequences. However, such methods involve unstable design structures or include Fc regions that can cause non-specific binding with immune cells, which can lead to safety issues in the treatment of ophthalmic diseases requiring repeated administration, and can also impose a biological burden in terms of expression and purification efficiency.

[0072] In contrast, the present invention utilizes the natural dimerization properties of the SARAH domain to induce alignment between antibody fragments without a separate chemical linker, thereby enabling dimer formation to be structurally clearer and more reproducible. Specifically, by utilizing the α-helix-based self-binding properties of the SARAH domain, predetermined binding angle and distance conditions between antibody fragments are spontaneously formed, thereby maximizing the structural reproducibility and binding affinity of bivalent binding to VEGF targets. Furthermore, since the SARAH domain is a non-immunogenic α-helix protein that does not induce non-specific binding to immune cells, it provides excellent biological safety and low immunoreactivity, even in cases such as ophthalmic diseases requiring repeated administration.

[0073] Furthermore, since dimerization is implemented at the gene level using a single open reading frame (ORF), cloning is simple, it is suitable for E. coli-based high-efficiency expression systems, and process advantages in terms of productivity and purity compared to Fc-based antibodies can be secured.

[0074] The above SARAH domain is an α-helix-based protein binding motif found in intracellular signaling proteins such as MST1, MST2, RASSF1A, and Salvador, and possesses a complementary binding surface within a single domain that induces dimerization; this characteristic enables stable bimolecule interactions even upon fusion with antibody fragments. In the present invention, by designing the SARAH domain to be fused to the C-terminus of each antibody fragment, the two protein molecules are induced to self-dimerize via the SARAH domain to form a physical dimer. Accordingly, not only is the binding affinity of the antibody enhanced, but the total molecular weight can also be maintained relatively small while preserving the dimer structure, thereby simultaneously improving tissue penetration and drug delivery efficiency.

[0075] The antibody fragment used in the present invention may be composed of a single-domain antibody (VHH), i.e., a nanobody, and is characterized by containing a sequence that specifically binds to VEGF. Nanobodies have a structure much smaller than traditional antibodies and possess excellent biological properties in terms of stability, expression, and tissue permeability. However, because general nanobodies have a monomeric structure, they have limitations in terms of antigen affinity, and there was a disadvantage of low efficiency when repeated administration is required due to their short half-life in the body. In contrast, the nanobody of the present invention is dimerized through the SARAH domain, allowing two antibody fragments to bind to the antigen simultaneously; consequently, the binding affinity is increased, and the binding affinity for VEGF can be increased.

[0076] The above dimeric antibody fragment may have a sequence capable of specifically binding to VEGF.

[0077] Specifically, each monomeric antibody fragment constituting the above dimeric antibody fragment may include a nanobody composed of a sequence capable of specifically binding to VEGF, a sardo domain, an Fh8 fusion domain, a TEV protease cleavage site, and a 6x His tag, and the nanobody composed of a sequence capable of specifically binding to VEGF may be composed of SEQ ID NO. 1.

[0078] Thus, the sequence of the monomer antibody fragment containing the above nanobody may include SEQ ID NO. 1, and more specifically, may include SEQ ID NO. 2.

[0079] A sequence such as SEQ ID NO. 2 above can be expressed by a single gene sequence encoded by a single open reading frame, and the gene sequence is described in SEQ ID NO. 3.

[0080]

[0081] Sequence No. Name Sequence Sequence No. 1 Nanobody (n-ter) composed of a sequence capable of specifically binding to VEGF SQVQLQESGGGSLQAGASLRLSCAASGFAYSTYSMGWFRQVSGKEREGVATINSGTFRLWYTDSVKGSFTISRDNAKNMLYLQMNSLKPEDTAIYYCAARAWSPYSSTVDAGD FRYWGQGTQVTVSS(c-ter) Sequence No. 2 Amino acids of the monomers forming the final VCB-1502 dimeric antibody fragment Sequence (n-ter)MPSVQEVEKLLHVLDRNGDGKVSAEELKAFADDSKCPLDSNKIKAFIKEHDKNKDGKLDLKELVSILSSSSSNNNNNNNNNNEFENLYFQGSQVQLQESGGGSLQAGASLRLSCAASGFAYSTYSMGWFRQVSGKEREGVATINSGTFRLWYTDSVKGSFTISRDNAKNMLYLQMNSLKPEDTAIYYCAARAWSPYSSTVDAGDFRYWGQGTQVTVSSSDYEFLKSWTVEDLQKRLLALDPMMEQEIEEIRQKYQSKRQPILDAIEAKLEHHHHHH(c-ter) Sequence No. 3 final VCB-1502 nucleic acid of the monomer forming the dimeric antibody fragment서열5'-ATGCCGAGCGTGCAGGAAGTGGAAAAACTGCTGCATGTGCTGGATCGCAATGGCGATGGCAAAGTGAGCGCCGAAGAACTGAAAGCGTTTGCCGATGATAGCAAATGCCCGCTGGACAGCAATAAAATTAAAGCCTTTATTAAAGAACATGATAAAAATAAAGATGGCAAACTGGATCTGAAAGAACTGGTGAGCATTCTGAGCTCAAGCAGCAGTAATAACAATAATAATAATAACAATAATAATGAATTTGAAAATCTGTATTTTCAGGGATCACAGGTGCAGCTGCAGGAAAGTGGCGGCGGCAGCCTGCAGGCCGGCGCAAGCCTGCGCCTGAGCTGCGCCGCCAGTGGTTTTGCGTATAGCACCTATAGCATGGGTTGGTTTCGCCAGGTGAGCGGCAAAGAACGCGAAGGCGTGGCGACCATTAATAGCGGCACCTTTCGCCTGTGGTATACCGATTCAGTGAAAGGCAGCTTTACCATTAGCCGTGATAATGCCAAAAATATGCTGTATCTGCAGATGAATAGCCTGAAACCGGAAGATACCGCCATTTATTATTGCGCGGCCCGCGCCTGGAGCCCGTATTCGAGCACCGTGGATGCGGGCGATTTTCGCTATTGGGGCCAGGGCACCCAGGTGACCGTGAGCAGTAGCGATTATGAATTTCTGAAAAGCTGGACCGTGGAAGATCTGCAGAAACGCCTGCTGGCCCTGGATCCGATGATGGAACAGGAAATTGAAGAAATTCGTCAGAAATATCAGTCGAAACGTCAGCCGATTCTGGATGCCATTGAAGCAAAACTCGAGCACCACCACCACCACCACTGA-3'

[0082] As described above, the nanobody portion of the antibody fragment is designed to bind to a specific target site of VEGF, and the SARAH domain is fused to the end of the corresponding sequence. The structure of the antibody fragment consists of a continuous arrangement of the antibody fragment nanobody + SARAH domain + His-tag, and can be expressed using a single open readout frame (ORF) without a separate linker. The structure of the final antibody fragment may be a dimeric antibody fragment, in which the antibody fragment is bound by the SARAH domain. This structure not only enables high-efficiency expression in an E. coli-based microbial expression system, but also allows the fused Fh8 tag to be removed via TEV protease, which is advantageous for ensuring purity after purification and the stability of the final formulation.

[0083]

[0084] In addition, the antibody fragment of the present invention can structurally block the possibility of non-specific activation of immune cells mediated by the Fcγ receptor (FcγR) by not including an Fc domain.

[0085] Existing Fc-based dimerizing antibodies have the potential to induce unnecessary immune responses as their Fc domain binds to FcγR on immune cells, and there was a possibility of immunological side effects or resistance induction, particularly in treatments requiring repeated or high-dose administration.

[0086] In contrast, the present invention can provide a non-immunogenic platform structure capable of forming a stable dimeric structure without an Fc domain by introducing a SARAH domain as a means for antibody dimerization, thereby enabling therapeutic efficacy without stimulating immune cells.

[0087] These characteristics are particularly important factors in treatments for chronic ophthalmic diseases requiring repeated administration, such as macular degeneration, diabetic retinopathy, and central retinal vein occlusion, and the antibody fragment of the present invention can serve as a next-generation antibody structure that simultaneously satisfies high safety, excellent tolerability, and low immunogenicity.

[0088] Furthermore, the removal of the Fc domain contributes to the miniaturization of the antibody structure and the simplification of the expression system, which can provide advantageous benefits for production process efficiency and expanded formulation diversity.

[0089] Consequently, the dimeric antibody fragment according to the present invention is structurally differentiated from existing antibody technology in that it utilizes a fixed dimerization method using a SARAH domain, functionally it can improve VEGF binding affinity and angiogenesis inhibition, and in terms of production and purification, it can realize expression efficiency and process simplification due to its single-sequence structure. These technical advantages can stand in clear contrast to existing multimer formation technologies or Fc-based antibody designs and can be utilized as a new platform for developing high-performance antibody-based therapeutics.

[0090]

[0091] Next, the SARAH (Salvador / RASSF / Hippo) domain, which is an important technical component of the present invention, will be described in more detail. The SARAH domain is a protein binding domain commonly found in a group of proteins constituting the intracellular Hippo signaling pathway, and is characterized by forming an α-helix structure consisting of about 40 to 50 amino acids. This domain has the function of forming protein complexes through mutual binding in MST1 (Mammalian Ste20-like kinase 1), MST2, RASSF1A (Ras association domain family member 1), Salvador (SAV1), etc., and can provide a binding surface specialized for self-dimerization or heterodimerization.

[0092] The SARAH domain is a binding module that originally serves as a platform for signal transduction between proteins within cells, but in this invention, such natural binding characteristics are applied to the dimerization structure of antibody fragments. Specifically, SARAH domains are fused to the C-terminuses of two identical antibody fragments, and by naturally inducing self-binding between SARAH domains after protein expression, a dimer structure can be stably formed without a separate chemical linker. This method can be clearly distinguished from flexible peptide linkers or leucine zipper domains commonly used in existing antibody technologies in terms of structural stability and biological compatibility.

[0093] In particular, the SARAH domain can be implemented as a genetically encoded protein sequence and can induce dimerization without a separate post-translational modification process, so the self-dimerized structure can be maintained even after protein purification during the production process. In the configuration of the present invention, since the SARAH domain exists in a fixed state at the end of the antibody fragment, the alignment of the binding surface is maintained consistently, thereby ensuring geometric stability that allows the two antibody fragments to simultaneously access the VEGF target. This can be considered an important structural feature that distinguishes it from existing technologies using atypical flexible linkers.

[0094] The dimeric antibody fragment according to the present invention is expressed as a single polypeptide, but has a structure in which the expressed protein self-difuses into two molecules to function as a functional dimer. Specifically, two monomer molecules are non-covalently bound through the SARAH domain, thereby ultimately exhibiting biological activity as a dimeric protein complex. This structure is a rare method in general antibody design, and it is considered to have very high potential as a therapeutic development platform due to the fact that the domain contributing to dimerization does not induce an immune response, and the overall structure is short and expressive.

[0095] In addition, the SARAH domain is composed of a soluble α-helix protein, which has a low likelihood of forming aggregates during expression and purification processes, can maintain its structure stably in various expression systems, and is designed to induce structure formation independently even when fused with antibody fragments. This design approach is intended to ensure that the binding interface of the SARAH domain does not interfere with the antibody binding site, and by allowing the binding function of SARAH and the VEGF recognition function of the antibody to be maintained independently without interference, the advantages of a modularized structure can be secured.

[0096] In conventional technologies, binding structures were primarily implemented by utilizing the Fc domain of IgG or through non-specific chemical conjugation (crosslinking) to induce multimerization between antibodies. However, the Fc domain has a high probability of binding to Fc receptors on immune cells, which can induce unexpected immune activation and raise concerns about the development of immunological resistance upon repeated administration. In this invention, by using the SARAH domain, the possibility of inducing such non-specific immune responses is eliminated, and by securing a stable binding structure between antibody fragments, an antibody structure that satisfies both safety and efficacy can be realized.

[0097] In this regard, the present invention has clear structural and functional differentiation from existing antibody dimerization technologies and can provide improved technological effects, particularly in terms of high-affinity binding to VEGF, expression and purification efficiency, and biological safety. Furthermore, since the structure of the present invention can be platformized in the future through combinations of various antigen-specific nanobodies and SARAH domains, it can be utilized as a foundational technology to secure expandability of application to various targets other than VEGF.

[0098]

[0099] As described above, the dimeric antibody fragment according to the present invention may be in the form in which a monomeric antibody fragment is bound by a SARAH domain, and the monomeric antibody fragment constituting the dimeric antibody fragment may be composed of a single amino acid sequence. The dimeric antibody fragment may include a sequence corresponding to SEQ ID NO. 1, more specifically SEQ ID NO. 2, and the sequence of the antibody fragment may be structured to include a nanobody domain of the antibody fragment located at the N-terminus, a SARAH domain for self-disjunction induction located in the middle, and a C-terminal His-tag for purification.

[0100] The above antibody fragment comprises a sequence of a nanobody designed to specifically bind to VEGF. The sequence is as described in SEQ ID NO. 1, and the amino acid arrangement of the sequence has unique complementarity with respect to the binding site with VEGF. Although the present invention directly presents a specific amino acid sequence of the antibody fragment, the technical configuration of the present invention is not limited to a specific sequence, and amino acid substitution, insertion, or deletion may be permitted within the scope of maintaining the binding characteristics of the antibody fragment. For example, conservative substitution within the sequence may be selectively applied for the purpose of improving stability, solubility, or expression efficiency without affecting antigen binding affinity.

[0101] Likewise, the SARAH domain can also undergo amino acid substitution within the range capable of performing the self-disjunction function. Since the SARAH domain structurally forms a binding surface that induces self-disjunction in the form of an α-helix, modifications to specific residues of the domain may not impair the function as long as the physicochemical properties of this binding surface are maintained. Therefore, the technical concept of the present invention is not limited simply to SEQ ID NOs. 1 to 3, but can be interpreted to include functional equivalents as described above.

[0102] The above-mentioned protein can be expressed using recombinant DNA technology, and the present invention experimentally confirmed that production is possible using an E. coli expression system. Specifically, the target protein could be produced in high yield by cloning an expression cassette containing a sequence composed of an antibody fragment and a SARAH domain into a pET-based vector and then inducing expression with IPTG. Since the antibody fragment of the present invention is structurally a small protein based on a single sequence, efficient expression is possible in an E. coli system without using a complex eukaryotic cell-based expression system. This can be considered a significant advantage in terms of process efficiency compared to conventional full-length antibodies or multi-domain antibodies.

[0103] In the purification step, affinity chromatography based on a Ni-NTA column using a His-tag attached to the C-terminus can be applied, and if necessary, a fusion tag (such as Fh8) attached to the N-terminus can be selectively removed by TEV protease treatment. The TEV cleavage site is inserted between the antibody fragment and the fusion tag during the vector design stage, and by treating with the TEV enzyme after protein expression, unnecessary sequences can be removed from the final target protein, thereby ensuring safety and purity as a therapeutic protein. Since the purification process is simple and efficient, it can be advantageous for lowering production costs while ensuring pharmaceutical-grade quality.

[0104] Furthermore, because the present invention is based on a structurally short and stable protein, it is capable of expansion into various formulations and possesses physical properties applicable to topical administration formulations such as eye drops. Due to the protein's small molecular weight and high structural stability, it exhibits excellent tissue penetration and allows for flexible subsequent drug design to extend the in vivo half-life. Additionally, since the entire sequence consists of human-derived antibody sequences or non-immunogenic sequences, the low likelihood of inducing an immune response even with repeated administration is a significant advantage from a drug development perspective.

[0105]

[0106] As described above, the dimeric antibody fragment according to the present invention inhibits the activation of VEGFR2 (KDR), the major receptor of VEGF-A, by binding to VEGF-A with high affinity, and inhibits the proliferation, migration, and angiogenesis of vascular endothelial cells by blocking downstream signaling chain reactions (e.g., Src, Akt, ERK, etc.). This process is initiated by directly blocking the binding between VEGF-A and VEGFR2, and the dimeric antibody fragment of the present invention has structural and geometric advantages in that two binding domains are arranged at a specific direction and distance through the SARAH domain, enabling simultaneous access to and binding to the bivalent binding site of VEGF.

[0107] This is a distinguishing feature from existing single-link antibodies or anti-VEGF antibodies having non-dimerized structures. For example, bevacizumab (Avastin) is an antibody based on a whole IgG structure, and ranibizumab (Lucentis) is a single antibody fragment structure designed based on a Fab fragment. In contrast, the dimer antibody fragment of the present invention is based on a nanobody structure and is designed as an independent antibody platform that spontaneously dimerizes by the SARAH domain.

[0108] This structure not only offers production process advantages such as expression efficiency, ease of purification, and stability, but also possesses safety advantages as it is a non-Fc-based structure that can reduce the possibility of inducing non-specific immune responses induced by Fc receptors. Furthermore, the SARAH domain-based dimer structure can secure mechanistic efficiency by maintaining the effective binding angle and distance conditions of the antibody upon VEGF binding, thereby precisely blocking the interaction between VEGF-A and VEGFR2 and effectively blocking the resulting angiogenesis signaling.

[0109] Therefore, the antibody fragment of the present invention has clear differentiation from existing antibodies in terms of structural design and mechanism of action, and provides a technical basis that can serve as a new therapeutic alternative for VEGF-A-mediated diseases.

[0110]

[0111] In addition, the dimeric antibody fragment of the present invention is a biological agent that effectively inhibits angiogenesis by specifically binding to vascular endothelial growth factor (VEGF) and blocking the VEGFR2-mediated signaling pathway, and can be used for the prevention or treatment of various neovascular ophthalmic diseases including diabetic retinopathy, diabetic macular edema, age-related macular degeneration, retinal neovascularization disease, central retinal vein occlusion disease, branched retinal vein occlusion disease, polyparyular choroidal angiopathy, choroidal neovascularization disease, colorectal cancer, lung cancer, breast cancer, ovarian cancer, cervical cancer, liver cancer, kidney cancer, or brain tumor.

[0112] The dimeric antibody fragment of the present invention may be formulated with a pharmaceutically acceptable carrier or adjuvant to be produced in the form of an intravitreal injection, ophthalmic drop, or topical application, and the formulation may include pharmaceutical additives such as stabilizers, buffers, preservatives, and equal volumes.

[0113] These compositions can be selectively applied depending on the patient's disease state and the purpose of administration, and the antidimeric antibody fragment of the present invention has the advantage of being safe to use even during repeated administration due to its SARAH domain-based structural stability and low immunogenicity.

[0114]

[0115] The dimeric antibody fragment of the present invention has a structure in which two antibody fragments are dimeric via a SARAH domain to enhance binding affinity to VEGF, thereby effectively inhibiting the binding of VEGF to receptors (VEGFR1, VEGFR2, etc.). Therefore, the antibody fragment of the present invention can be usefully used for anticancer treatment by inhibiting angiogenesis in tumors such as colorectal cancer, lung cancer, breast cancer, ovarian cancer, cervical cancer, liver cancer, kidney cancer, and brain tumors, as well as in ophthalmic neovascular diseases in which VEGF-dependent neovascularization acts as a major pathological mechanism, such as diabetic retinopathy, diabetic macular edema, age-related macular degeneration, central or branched retinal vein occlusion disease, polyplic choroidal angiopathy, and choroidal neovascularization disease.

[0116] The antibody fragment according to the present invention can inhibit the proliferation, migration, and neovascularization of endothelial cells by inhibiting the biological activity of VEGF, and can effectively block abnormal blood vessel formation in tumors or the retina. In addition, the SARAH domain-mediated dimeric structure of the present invention increases the stability and half-life of the antibody fragment, thereby extending the duration of action in the body and exhibiting enhanced pharmacological activity compared to the monomeric form of the antibody fragment.

[0117] Meanwhile, the VEGF-binding dimer antibody fragment of the present invention can prevent or improve visual impairment in ophthalmic diseases such as diabetic retinopathy and macular degeneration by inhibiting neovascularization or reducing leakage. In addition, it can exhibit an anticancer effect that inhibits tumor growth and metastasis by inhibiting neovascularization around cancer cells.

[0118] Through such action, the antibody fragment dimer of the present invention can be usefully utilized as a pharmaceutical composition for the prevention or treatment of angiogenesis-related diseases, and in particular, can be utilized as a new structural platform for therapeutic agents targeting VEGF.

[0119] In addition, the present invention may provide a method for preventing or treating angiogenesis-related diseases comprising the step of administering an effective amount of the dimeric antibody fragment to a patient, and furthermore, the dimeric antibody fragment may be used for medicinal purposes for the treatment of said disease.

[0120]

[0121] The present invention will be explained in detail below through examples. However, the following examples are intended to explain the invention more specifically, and the scope of the invention is not limited by the following examples.

[0122]

[0123] Example. Preparation of a novel angiogenesis inhibitor (VCB-1502)

[0124] 1. Synthesis of VCB-1502 dimeric antibody fragment gene and vector insertion

[0125] In this embodiment, to produce VCB-1502, a novel nanobody-based dimeric antibody fragment that binds to vascular endothelial growth factor (VEGF), a dimeric antibody fragment gene was synthesized and an expression system capable of producing it in microorganisms was established. The VCB-1502 dimeric antibody fragment gene was designed to be suitable for mass production in microorganisms, and for stable production and purification, it included an Fh8 fusion region and a TEV protease cleavage site at the N-terminus, and a 6x His-tag was attached to the C-terminus.

[0126] The synthesized gene was cloned by inserting it into a pET21a(+) expression vector after restriction enzyme treatment. At this time, two pairs of polymerase chain reaction (PCR) primers were used to assemble the gene with the Fh8-TEV cleavage site-VCB-1502-histag structure.

[0127] The first reaction (PCR1) is for the insertion of NdeI-Fh8-TEV-BamHI, and the primer sequences used are shown in SEQ ID NO. 4 and SEQ ID NO. 5, respectively. The second reaction (PCR2) is for the insertion of BclI-VCB-1502-XhoI, and the primers used correspond to SEQ ID NO. 6 and SEQ ID NO. 7.

[0128] The VCB-1502 gene produced through the above procedure was transformed into a BL21(DE3) E. coli strain and expression experiments were performed, and the amino acid sequence and base sequence of the monomer constituting the designed final dimeric antibody fragment are presented in SEQ ID NO. 2 and SEQ ID NO. 3, respectively.

[0129] As shown in Fig. 2, the pET-21a(+) vector is designed to be induced by IPTG under a T7 promoter and has a structure optimized for the expression and purification of the inserted gene.

[0130]

[0131] Sequence No. Name Sequence Sequence No. 4PCR1 Forward Primer (5'NdeI_Fh8)5'-ATATATGGATCCCTGAAAATACAGATTTTCAAA-3' Sequence No. 5PCR1 Reverse Primer (3'BamHI_Fh8)5'-GATATACATATGCCGAGCGTGCAGGAAGTGGAA-3' Sequence No. 6PCR2 Forward Primer (5'BclI_VCB-1502)5'-ATATATTGATCACAGGTGCAGCTGCAGGAAAGTG-3' Sequence No. 7PCR2 Reverse Primer (3'XhoI_VCB-1502)5'-ATATATCTCGAGTTTTGCTTCAATGGCATCCAGAAT-3'

[0132] 2. VCB-1502 Antibody Expression and Purification

[0133] An expression vector containing the VCB-1502 dimeric antibody fragment gene containing SEQ ID NO. 3 was transformed into E. coli BL21(DE3) strain to induce the expression of antibody proteins. Antibody expression was performed using an induction method with isopropyl-β-D-thiogalactopyranoside (IPTG), and optimal expression conditions were established by adding IPTG at a final concentration of 1 mM and incubating at 37°C for 4 hours.

[0134] Most of the expressed proteins were produced in the form of insoluble inclusions, and the inclusions were separated by lysing the cells collected by centrifugation after culture. The inclusions were lysed under denaturation conditions containing 8 M urea, and then metal affinity chromatography (metal binding affinity purification) was performed using a His tag (6x His tag) attached as a purification label.

[0135] Purification was performed using a nickel-ion conjugated resin column (HisTrap™ FF crude). The binding buffer used contained 8 M urea, 20 mM sodium phosphate, 150 mM sodium chloride, and 20 mM imidazole, and the pH was adjusted to 9.5. After the antibody protein bound to the column, non-specific binding substances were removed using a wash buffer containing 50 mM imidazole, and the antibody protein was obtained using an elution buffer containing 500 mM imidazole. The purification process was carried out at a flow rate of 1 mL / min during the binding and elution steps and 3 mL / min during the washing step.

[0136] The purified VCB-1502 dimer antibody fragments were analyzed by polyacrylamide gel electrophoresis (SDS-PAGE) under basic conditions. As shown in Figure 3, a protein band of approximately 30 kDa was clearly identified in the IPTG-treated transgenic strains (#3, #7), indicating that the VCB-1502 antibody fragment was induced and expressed. In contrast, the band was not observed in the IPTG-untreated group and the non-transgenic control group (Con).

[0137] The dimeric antibody fragment of the present invention exhibited a purity of approximately 90% or higher, and the production yield was confirmed to be approximately 70 mg per 1 L of culture medium. The obtained antibody protein was subsequently used for efficacy evaluation after restoring its biological activity through a refolding process.

[0138]

[0139] 3. Refolding process of VCB-1502 dimeric antibody fragments

[0140] Since the purified VCB-1502 dimeric antibody fragments mostly existed in the form of insoluble inclusion bodies, a refolding process was additionally performed to restore them to a biologically active form. The inclusion bodies were completely dissolved under 8 M urea conditions, and then the proteins were induced to naturally fold into the correct tertiary structure through gradual buffer exchange.

[0141] Refolding was performed using dilution or stepwise dialysis, and redox conditions were controlled during the process to facilitate stable antibody folding. Under conditions containing appropriate reducing and oxidizing agents, the formation of disulfide bonds between protein chains was induced, and reaction times were adjusted to allow the antibodies to maintain a functional dimeric structure.

[0142] The VCB-1502 dimeric antibody fragments that underwent the refolding process were restored to a structurally stable state and were used in subsequent experiments to confirm biological activity and evaluate efficacy.

[0143]

[0144] Experimental Example 1. Evaluation of the binding ability of VCB-1502 dimeric antibody fragments to VEGF

[0145] This experimental example relates to the results of performing an enzyme immunoassay (ELISA) to confirm the binding ability of the VCB-1502 dimeric antibody fragment to human vascular endothelial growth factor (VEGF165). Through this experiment, the aim was to quantitatively evaluate whether the VCB-1502 dimeric antibody fragment specifically binds to VEGF and to demonstrate the biological effects of the invention.

[0146] In the experiment, human-derived VEGF165 antigen was first coated onto a 96-well ELISA plate, followed by treatment with VCB-1502 dimeric antibody fragments at various concentrations. Subsequently, antibody binding was measured by absorbance using a secondary antibody and substrate, and changes in binding amount according to each concentration were compared and analyzed. The VCB-1502 dimeric antibody fragment used in the experiment was prepared through purification and refolding processes in the example, and is a protein containing His-tag and Fh8 domains.

[0147] The experimental results are as shown in Figure 4.

[0148] As the concentration of the antibody fragment increased to 1, 5, and 10 μg / mL, the OD450 value increased significantly, and a tendency to show a saturation curve was confirmed above a certain concentration. Through this, the concentration-dependent binding affinity of the VCB-1502 dimeric antibody fragment to the VEGF165 antigen could be confirmed. Specifically, a saturated binding pattern was observed from a concentration of approximately 1 μg / mL (32.67 nM) or higher, and minimal binding was measured at 0.03125 μg / mL (1.02 nM).

[0149] Additionally, to quantitatively evaluate the functional affinity of the VCB-1502 antibody fragment for VEGF, concentration-response curves were constructed based on ELISA data and EC50 values ​​were calculated. As shown in Figure 5, through the increase curve of the OD450 signal according to antibody concentration, the EC50 is approximately 9.289 × 10⁻⁶. -9It was confirmed to be M, which demonstrates that the VCB-1502 antibody fragment has a high binding affinity.

[0150] In addition, the Kd value was calculated to precisely confirm the binding affinity of the VCB-1502 antibody fragment for VEGF. As shown in Fig. 6, from the binding curve based on the ELISA analysis results, Kd is approximately 9.475 × 10⁻⁶. -9 It was calculated as M, which means that stable binding is possible at nanomolar concentrations.

[0151] The above results demonstrate that the VCB-1502 dimeric antibody fragment specifically binds to human VEGF165 with high affinity, and provide evidence supporting the possibility that this dimeric antibody fragment may have a therapeutic effect in diseases related to angiogenesis, particularly diseases such as macular degeneration.

[0152]

[0153] Experimental Example 2. Evaluation of cell proliferation inhibition by VCB-1502 dimeric antibody fragment in HUVEC cells

[0154] This experiment was conducted to evaluate the effect of VCB-1502 dimeric antibody fragments on the proliferation of human umbilical vein-derived vascular endothelial cells (HUVECs) induced by VEGF165. VEGF is a representative angiogenic factor that promotes the differentiation and induces the proliferation of vascular endothelial cells, and this experiment was designed to functionally verify the VEGF blocking effect of VCB-1502 dimeric antibody fragments.

[0155] In the experiment, HUVEC cells were first seeded into a 96-well plate, and cell proliferation was induced by treating them with a specific concentration of VEGF165. Subsequently, the VEGF-treated group was treated with VCB-1502 dimeric antibody fragments prepared in the example at concentrations ranging from 0 to 10 μg / mL, while a control group treated only with VEGF and a blank group with no treatment were established, respectively. After culturing for a certain period following treatment, the degree of cell proliferation was quantified through absorbance analysis using the WST-8 reagent.

[0156] As shown in Figure 7, the experimental results showed that the VCB-1502 dimeric antibody fragment inhibited the proliferation of HUVEC cells induced by VEGF165 in a concentration-dependent manner, exhibiting an inhibitory effect similar to that of the control drug Avastin. In particular, a significant decrease in cell proliferation was observed at concentrations of 1 μg / mL or higher, and the absorbance value showed a tendency to continuously decrease as the concentration of the dimeric antibody fragment increased. These results indicate that the VCB-1502 dimeric antibody fragment can inhibit the proliferation process of vascular endothelial cells by blocking VEGF signaling.

[0157] The above results demonstrate that the VCB-1502 dimeric antibody fragment exerts a substantial inhibitory effect on the VEGF-mediated angiogenesis process even in an in vitro environment, providing evidence supporting its potential as a therapeutic agent for angiogenic diseases such as macular degeneration.

[0158]

[0159] Experimental Example 3. Evaluation of the efficacy of VCB-1502 dimeric antibody fragments for the inhibition of VEGFR2 signaling.

[0160] This experimental example was performed to evaluate whether the VCB-1502 dimeric antibody fragment can block signal transduction by inhibiting the binding between VEGF and VEGFR2 (vascular endothelial growth factor receptor 2). To this end, a reporter system in which the NFAT-response factor (NFAT-RE) and the luciferase gene were introduced into a genetically engineered HEK293 cell line expressing VEGFR2 was utilized. This system is configured so that the luciferase enzyme is expressed when intracellular signals are activated by VEGF stimulation, allowing the degree of signal transduction caused by VEGF / VEGFR2 binding to be measured by luminescence intensity.

[0161] In the experiment, HEK293 cells expressing VEGFR2 / NFAT-RE luciferase were first cultured, and then VEGF165 was administered to induce signal transduction. Subsequently, VCB-1502 dimeric antibody fragments at various concentrations prepared in the example were administered along with VEGF to confirm whether the dimeric antibody fragments inhibited signal transduction by blocking the binding of VEGF / VEGFR2. As control groups, a condition treated only with VEGF and an untreated group without the dimeric antibody fragments were established.

[0162] As shown in Fig. 8, the experimental results indicated that the VCB-1502 dimeric antibody fragment inhibited VEGF165-induced luciferase expression in a concentration-dependent manner. Specifically, a clear trend was observed where the luciferase luminescence intensity decreased as the concentration of the dimeric antibody fragment increased, suggesting that the VCB-1502 dimeric antibody fragment effectively blocks the interaction between VEGF and VEGFR2. In particular, at concentrations of 5–10 μg / mL, the inhibitory effect of the VCB-1502 dimeric antibody fragment was similar to that of Avastin, an existing anti-VEGF antibody therapeutic.

[0163] These results demonstrate that the VCB-1502 dimeric antibody fragment is a functional antibody fragment capable of effectively inhibiting the VEGFR2-mediated VEGF signaling pathway, supporting the possibility that it can inhibit angiogenesis in vivo.

[0164]

[0165] Experimental Example 4. Evaluation of the Stability and Angiogenesis Inhibitory Effect of VCB-1502 Dimeric Antibody Fragments in a Mouse Model

[0166] This experimental example was conducted on a mouse model to determine whether the VCB-1502 dimeric antibody fragment could maintain stability in an in vivo environment and inhibit VEGF-induced angiogenesis. The experiment was conducted by establishing conditions in which local angiogenesis is induced by combining vascular endothelial growth factor (VEGF165) and a hydrogel, administering the VCB-1502 dimeric antibody fragment under these conditions, and then observing the stability of the antibody fragment and the effect of inhibiting angiogenesis.

[0167] The experimental animals used in the study were 6-week-old female C57BL / 6 mice, with a total of 30 subjects. All individuals were weighed prior to the study to ensure balance between groups, and then randomly assigned to five groups. The substances administered to each group were as follows:

[0168]

[0169] G1: PBS + Hydrogel (Negative Control)

[0170] G2: VEGF165 + Hydrogel (Positive Control)

[0171] G3: VEGF165 + Hydrogel + VCB-1502 (5 mg / kg)

[0172] G4: VEGF165 + Hydrogel + VCB-1502 (25 mg / kg)

[0173] G5: VEGF165 + Hydrogel + Avastin (25 mg / kg, control group)

[0174]

[0175] In this experiment, the safety and angiogenesis inhibitory effects of VCB-1502 antibody fragments were evaluated in a mouse model in which angiogenesis was induced by subcutaneous injection of VEGF and hydrogel. As shown in Figure 9, the body weight of mice in both the control group (G1) and the treatment groups (G2–G5) showed a tendency to increase steadily during the experimental period (7 days), and no significant decrease in body weight was observed in the VCB-1502 antibody fragment treatment groups (G3, G4), confirming that the antibody fragments do not cause in vivo toxicity.

[0176] Next, to confirm the angiogenesis-inhibiting effect of the VCB-1502 antibody fragment of the present invention, VEGF and hydrogel were injected subcutaneously into mice to induce angiogenesis, and then the antibody fragment was administered in combination.

[0177] As a result of the experiment, as shown in Fig. 10, a distinct pattern of angiogenesis was observed in the G2 group (VEGF165-treated group), whereas a large number of individuals in the G3, G4, and G5 groups showed clearly reduced angiogenesis. In particular, there were relatively many individuals with inhibited angiogenesis in the G4 and G5 groups.

[0178] In addition, after 7 days of observation, tissue samples were collected from the hydrogel sites of the experimental groups (G1–G5) by incising them, and the presence of neovascularization was evaluated visually. As shown in Fig. 11, vascular tissue with a strong hemoglobin was clearly observed in the G2 group treated with VEGF alone, whereas in the G3 and G4 groups treated with VCB-1502 antibody fragments, the tissues were pale and neovascularization was confirmed to be inhibited. This result directly demonstrates that the antibody fragments of the present invention effectively inhibit VEGF-mediated neovascularization.

[0179] The above results demonstrate that the VCB-1502 dimeric antibody fragment has biological activity capable of inhibiting VEGF-induced angiogenesis in vivo.

[0180]

[0181] As described above, the present invention relates to a novel dimeric antibody fragment that specifically binds to vascular endothelial growth factor (VEGF). It is characterized by being designed to form a stable dimeric structure based on self-dimerization by fusing a SARAH (Salvador / RASSF / Hippo) domain to the nanobody of the antibody fragment. The antibody fragment according to the present invention is clearly differentiated from existing anti-VEGF antibody preparations through the following effects.

[0182] Specifically, the VCB-1502 dimeric antibody fragment of the present invention is based on a small nanobody structure and is designed as a protein therapeutic capable of high-yield production through an E. coli-based expression system, exhibiting superior tissue penetration compared to conventional IgG or Fab antibodies. In particular, the antibody fragment of the present invention induces spontaneous dimerization using the SARAH domain, thereby enabling the two antibody fragments to stably bind and form a dual-arm structure against an antigen without the introduction of a separate linker. This provides a clear distinction in terms of structural stability, expression efficiency, and biological compatibility compared to existing flexible linker or leucine zipper-based dimerization technologies.

[0183] These structural features increase the avidity of the antibody and lead to improved binding affinity for VEGF. The EC50 and Kd values ​​for VEGF confirmed through actual ELISA analysis were found to be approximately 9 nM, indicating high specificity and binding affinity. Furthermore, experiments inhibiting VEGF-induced vascular endothelial cell proliferation and VEGFR2-mediated luciferase reporter analysis confirmed that the VCB-1502 antibody fragment effectively blocks VEGF signaling in a concentration-dependent manner.

[0184] In addition, in an angiogenesis model experiment using mice, the VCB-1502 antibody fragment significantly inhibited VEGF-induced angiogenesis and demonstrated in vivo efficacy similar to that of the commercial antibody agent Avastin. In vivo safety was also proven as no adverse reactions, such as changes in body weight, were observed.

[0185] As such, the antibody fragment of the present invention has technical features that differentiate it from existing anti-VEGF antibodies such as bevacizumab (Avastin) or ranibizumab (Lucentis) in terms of miniaturization, productivity, affinity, dimerization stability, or biological efficacy, and can be utilized as a platform antibody technology that can be usefully applied to the prevention or treatment of diabetic retinopathy, diabetic macular edema, age-related macular degeneration, retinal neovascularization, central retinal vein occlusion, branched retinal vein occlusion, polyparyular choroidal angiopathy, choroidal neovascularization, colorectal cancer, lung cancer, breast cancer, ovarian cancer, cervical cancer, liver cancer, kidney cancer, or brain tumor.

[0186]

[0187] The present invention has been described above with reference to its preferred embodiments. Those skilled in the art will understand that the present invention may be embodied in modified forms without departing from the essential characteristics of the invention. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the invention is defined by the claims, not by the foregoing description, and all variations within the scope of the claims should be interpreted as being included in the invention.

[0188] The scope of the present invention is defined by the claims set forth below, and all modifications or variations derived from the meaning and scope of the claims and equivalent concepts thereof should be interpreted as being included within the scope of the present invention.

Claims

1. A dimeric antibody fragment comprising two antibody fragments that specifically bind to vascular endothelial growth factor (VEGF) and are dimeric by a SARAH domain.

2. In Paragraph 1, A dimeric antibody fragment characterized in that each antibody fragment constituting the above dimeric antibody fragment comprises an amino acid sequence corresponding to SEQ ID NO.

1.

3. In Paragraph 1, A dimeric antibody fragment characterized in that each antibody fragment constituting the above dimeric antibody fragment comprises an amino acid sequence corresponding to SEQ ID NO.

2.

4. In Paragraph 3, A dimeric antibody fragment characterized in that each antibody fragment constituting the above dimeric antibody fragment is expressed by a nucleic acid sequence corresponding to SEQ ID NO.

3.

5. In Paragraph 1, A dimeric antibody fragment characterized by having an Fh8 fusion domain included at the N-terminus of each antibody fragment constituting the dimeric antibody fragment.

6. In Paragraph 5, A dimeric antibody fragment characterized by additionally including a TEV protease cleavage site at the C-terminus of the above Fh8 fusion domain.

7. In Paragraph 1, A dimeric antibody fragment characterized by including a 6x His tag at the C-terminus of each antibody fragment constituting the dimeric antibody fragment.

8. In Paragraph 1, The above dimeric antibody fragment is characterized by inhibiting angiogenesis by inhibiting the proliferation of vascular endothelial cells or by blocking VEGFR2-mediated signaling.

9. A dimeric antibody fragment comprising a structure in which two antibody fragments that specifically bind to vascular endothelial growth factor (VEGF) are dimeric by a SARAH domain, A pharmaceutical composition for the prevention or treatment of diabetic retinopathy, diabetic macular edema, age-related macular degeneration, retinal neovascularization, central retinal vein occlusion, branched retinal vein occlusion, polyparyular choroidal angiopathy, choroidal neovascularization, colorectal cancer, lung cancer, breast cancer, ovarian cancer, cervical cancer, liver cancer, kidney cancer, or brain tumor.

10. A step comprising administering an effective amount of a dimeric antibody fragment to a patient, the dimeric structure in which two antibody fragments that specifically bind to vascular endothelial growth factor (VEGF) are dimeric by a SARAH domain, Methods for the prevention or treatment of diabetic retinopathy, diabetic macular edema, age-related macular degeneration, retinal neovascularization, central retinal vein occlusion, branched retinal vein occlusion, polyparyular choroidal angiopathy, choroidal neovascularization, colorectal cancer, lung cancer, breast cancer, ovarian cancer, cervical cancer, liver cancer, kidney cancer, or brain tumor.

11. A dimeric antibody fragment having a structure in which two antibody fragments that specifically bind to vascular endothelial growth factor (VEGF) are dimeric by a SARAH domain, Use for the treatment of diabetic retinopathy, diabetic macular edema, age-related macular degeneration, retinal neovascularization, central retinal vein occlusion, branched retinal vein occlusion, polyparyular choroidal angiopathy, choroidal neovascularization, colorectal cancer, lung cancer, breast cancer, ovarian cancer, cervical cancer, liver cancer, kidney cancer, or brain tumor.

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