Nanobody-scfv antibody fragment bispecific for epcam and CD3

Abstract

The present invention relates to a bispecific antibody fragment in which a nanobody that specifically binds to EpCAM is connected through a flexible peptide linker ((GGGGS)n) to a single-chain antibody fragment (scFv) that specifically binds to CD3, and to a robust bispecific antibody platform that has high target specificity and penetration ability in solid cancer tissues, secures regulation of binding ability and immune safety, can be efficiently produced in an E. coli expression system, and maintains stable antigen-binding affinity and excellent antitumor activity even under tumor microenvironment conditions.

Description

Nanobody-SCFV antibody fragment dual-specific for EPCAM and CD3

[0001] The present invention relates to a bispecific antibody technology, and more particularly, to an antibody fragment having bispecificity for EpCAM and CD3, and in which a nanobody and a single-chain variable fragment (scFv) are linked by a linker.

[0002]

[0003] Immunotherapy for the treatment of tumors has made remarkable progress in recent years. In particular, bispecific antibodies have emerged as a key therapeutic platform that simultaneously target tumor antigens and immune cell surface antigens to induce T cell-mediated cell killing. Bispecific antibodies promote physical binding (immune synapse) between immune cells and tumor cells without requiring a separate antigen presentation process, directly activating the patient's immune system and eliminating cancer cells.

[0004] The most representative bispecific antibody structure developed to date is the bispecific T cell engager (BiTE) scFv / scFv structure, primarily developed for the treatment of hematological malignancies. However, this structure has the following technical limitations.

[0005] First, scFv, a structure artificially linked by a heavy chain variable domain (VH) and a light chain variable domain (VL), has low folding stability and tends to form aggregates due to hydrophobic interactions during expression. This limits productivity and quality consistency.

[0006] Second, the total molecular weight is around 50-60 kDa, which limits its penetration into the interstitial space of solid tumor tissue and the vascular endothelial barrier, and it is difficult to secure sufficient tumor penetration.

[0007] Third, if the antigen-binding affinity of scFv for CD3 is excessive, serious immunotoxicity such as cytokine release syndrome (CRS) due to overactivation of T cells may occur, and a technology to control binding affinity to prevent this is essential.

[0008] In addition, solid tumor tissues have difficulty in penetrating therapeutic antibodies due to the dense extracellular matrix (ECM) and vascular barrier, and many therapeutic proteins exhibit structural instability in the acidified tumor microenvironment (TME), which reduces the therapeutic effect.

[0009] As an alternative to overcome these technical limitations, nanobodies, which possess small size and a stable structure, can be utilized as antibody domains. Nanobodies, as single-domain antibodies (variable heavy chain domains (VHHs),) offer a compact structure, excellent folding stability, and high tissue penetration.

[0010] The present invention aims to overcome the limitations of the prior art as described above, and has developed a bispecific antibody fragment that can provide high permeability and specificity for solid tumor target antigens such as EpCAM, and suppress immunotoxicity by therapeutically appropriately controlling binding affinity to CD3.

[0011] In particular, the antibody fragment of the present invention is designed to maintain stable antigen binding affinity even under the acidic conditions (pH 5.6-6.7) of the tumor microenvironment (TME), and is configured to enable high-yield production using an Escherichia coli (E. coli) expression system. Such nanobody-scFv bispecific antibody fragment and anticancer treatment technology comprising the same effectively improve the technical limitations of existing bispecific antibody structures, while providing a new platform technology that can simultaneously secure therapeutic efficacy, productivity, and safety.

[0012] The present invention relates to a bispecific antibody fragment comprising a nanobody specifically binding to EpCAM and CD3 and a single-chain antibody fragment (scFv) linked by a linker, and a pharmaceutical composition for anticancer treatment comprising the same, and more particularly, to a bispecific antibody platform technology capable of simultaneously targeting EpCAM on the surface of solid cancer cells and CD3 on the surface of immune cells, thereby inducing the formation of an immune synapse between tumor cells and T cells, and promoting tumor cell death through T cell activation.

[0013] The antibody fragment of the present invention was designed to overcome the technical problems of conventional scFv / scFv-based bispecific antibodies, such as insufficient folding stability, aggregate formation, and limited penetration into solid tumor tissues. In particular, a nanobody was adopted as the EpCAM binding domain to maximize tissue penetration, and an scFv with adjustable therapeutic binding affinity was applied to the CD3 binding domain to minimize the risk of T cell hyperactivation and immunotoxicity (cytokine release syndrome, CRS). In addition, optimization was achieved by introducing point mutations in the antigen-binding region (CDR) to adjust the binding affinity for the antigen to a therapeutically suitable level and reduce nonspecific binding.

[0014] In addition, the antibody fragment of the present invention has the characteristic of maintaining or increasing antigen binding affinity even under acidified conditions (pH 5.6 to 6.7) of the tumor microenvironment (TME), and provides the advantage of being able to be produced in high yield and high purity through an E. coli expression system.

[0015] The present invention aims to overcome the limitations of existing bispecific antibody technology through structural and functional improvements as described above, and to provide effective immune cell-mediated therapeutic effects against solid cancers and other tumors having CD3-positive targets.

[0016]

[0017] The technical problems to be solved by the present invention are not limited to the technical problems mentioned above, and other technical problems not mentioned can be clearly understood by a person having ordinary skill in the technical field to which the present invention belongs from the description below.

[0018]

[0019] As an embodiment for achieving the above technical task, the present invention provides a bispecific antibody fragment in which a nanobody specifically binding to EpCAM and a single-chain antibody fragment (scFv) specifically binding to CD3 are connected by a linker.

[0020] In addition, as an embodiment of the present invention, the linker in the bispecific antibody fragment may include a (GGGGS)n sequence, and more specifically, may include a (GGGGS)₃ sequence.

[0021] In addition, as another embodiment of the present invention, the antibody fragment may have a characteristic in which antigen binding affinity to EpCAM and CD3 is maintained or increased even in a slightly acidic (pH 5.6 to 6.7) tumor microenvironment.

[0022] In addition, as an embodiment of the present invention, a pharmaceutical composition for preventing or treating cancer comprising the bispecific antibody fragment is provided.

[0023] As another example, the cancer may include one or more selected from the group consisting of liver cancer, colon cancer, pancreatic cancer, breast cancer, stomach cancer, lung cancer, prostate cancer, ovarian cancer, cervical cancer, esophageal cancer, and bladder cancer.

[0024]

[0025] The bispecific antibody fragment according to the present invention can dual-specifically bind to EpCAM and CD3, thereby inducing physical binding (immune synapse formation) between solid tumor cells and T cells, and effectively promoting T cell activation and tumor cell death. In particular, by adopting a miniaturized nanobody as the EpCAM binding domain, high permeability and antigen binding affinity can be maintained even in densely packed areas between cells in solid tumor tissues, and by applying an scFv with adjustable binding affinity as the CD3 binding domain, the risk of immunotoxicity (cytokine release syndrome, CRS) can be minimized.

[0026] The antibody fragment of the present invention exhibits the characteristic of maintaining or increasing antigen binding affinity to EpCAM and CD3 even in an acidified tumor microenvironment (pH 5.6 to 6.7), and can provide an improved therapeutic effect in a solid tumor environment.

[0027] In addition, the antibody fragment of the present invention can be produced in a high yield in a water-soluble form through an E. coli expression system, which can provide excellent advantages in terms of reducing production costs and mass production.

[0028]

[0029] 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 detailed description of the present invention or the composition of the invention described in the claims.

[0030]

[0031] Figure 1 is a schematic diagram schematically showing the structural configuration of a bispecific antibody fragment in which the EpCAM nanobody and CD3 scFv of the present invention are connected by a linker.

[0032] FIG. 2 is a graph showing the results of measuring the binding affinity (Kd) of antibody fragments manufactured through Examples 1 (FIG. 2a), 2 (FIG. 2b), and 3 (FIG. 2c) of the present invention to hEpCAM. The hEpCAM antigen binding ability according to the concentration of the antibody fragment was evaluated by the ELISA method. The X-axis represents the antibody concentration, the Y-axis represents the absorbance (OD450nm), and the Kd values ​​were measured to be approximately 4.94 nM, 4.68 nM, and 5.36 nM, respectively. The Kd values ​​were measured using the formula f = Bmax * abs(x) / (Kd + abs(x)).

[0033] FIG. 3 is a graph showing the results of measuring the binding affinity (Kd) of antibody fragments according to Example 1 (FIG. 3a), Example 2 (FIG. 3b), and Example 3 (FIG. 3c) of the present invention for the hCD3 antigen. The binding affinity to the CD3 antigen was evaluated by the ELISA method according to the antibody concentration. The X-axis represents the antibody concentration (Concentration, M), and the Y-axis represents the absorbance (OD450 nm). The Kd value was measured to be about 4.39 nM for the antibody fragment according to Example 1, about 83.7 nM for the antibody fragment according to Example 2, and about 38.2 nM for the antibody fragment according to Example 3.

[0034] Figure 4 is a graph showing the results of flow cytometry analyzing the ability of antibody fragments according to Examples 1 to 3 of the present invention to bind to cells expressing antigens. The analysis was performed on CD3-expressing Jurkat cells (Figure 4a) and EpCAM-expressing cancer cell lines (MCF7, SW480, HCC827, Hep3B, NCI-H358; Figures 4b to 4f). The X-axis represents fluorescence intensity (FITC-A), and the Y-axis represents the number of cells. As a result of the analysis, it was confirmed that the antibody fragments according to Examples 1, 2, and 3 specifically bind to both CD3-expressing Jurkat cells and EpCAM-expressing cancer cell lines, and the fluorescence intensity was clearly increased compared to the control group (untreated), proving the binding ability of the antibody fragments to the target antigen.

[0035] Figure 5 is a graph showing the results of analyzing the apoptotic effect by treating human PBMC and human cancer cell lines (Hep3B, NCI-H358, SW480) with the antibody fragment of Example 2 of the present invention. The X-axis represents the log value of antibody concentration (Conc. Log [M]), and the Y-axis represents cell viability (Cell Viability, %). As a result of the analysis, all antibody fragments induced apoptosis in a concentration-dependent manner in cancer cell lines, and the EC50 values ​​were measured as 211.8 nM in Hep3B cells, 54.48 nM in NCI-H358 cells, and 216.9 nM in SW480 cells. In the control group, PBMC, no apoptosis was observed due to antibody treatment, confirming the selective anticancer efficacy of the antibody fragment.

[0036] Figure 6 is a graph showing the results of analyzing the cell killing effect by treating human PBMC and human cancer cell lines (Hep3B, NCI-H358, SW480) with antibody fragments to which point mutations for CD3 binding affinity control were applied according to Examples 2 and 4 and 5 of the present invention. The X-axis represents the log value of antibody concentration (Conc. Log [M]), and the Y-axis represents cell viability (Cell Viability, %). As a result of the analysis, the concentration-dependent cell killing effect was maintained even in the antibody fragment containing the point mutation (Y101F or K187R) in the CD3 CDR region compared to the antibody fragment without mutation (WT). In all cancer cell lines (Hep3B, NCI-H358, SW480), the point mutation antibody fragment showed equivalent or improved cell killing activity compared to the WT, and no cell killing was observed in PBMC, demonstrating selective anticancer effect and immune safety.

[0037] Figure 7 is a graph showing the change in tumor volume of antibody fragments according to Examples 1 to 3 of the present invention in a BALB / c nude mouse model. The X-axis represents the treatment period (Day), and the Y-axis represents the tumor volume (Tumor volume, mm³). After transplanting tumor cells (NCI-H358), the antibody fragment or control group (PBS) was administered, and the tumor volume was measured for a certain period of time. As a result of the analysis, the tumor volume continuously increased in the control group, whereas the increase in tumor volume was suppressed in the antibody fragment administration groups according to Examples 1, 2, and 3, and in particular, the highest tumor inhibition effect was observed in the antibody fragment according to Example 3.

[0038] Figure 8 shows the tumor weight (Figure 8a) and the photograph of the extracted tumor tissue (Figure 8b) after administration of the antibody fragment according to Examples 1 to 3 of the present invention in a BALB / c nude mouse model. In the graph on the left, the X-axis represents the administration group (Control, Example 2, Example 1, Example 3), and the Y-axis represents the tumor weight (tumor weight, g). After transplanting tumor cells (NCI-H358), the antibody fragment or the control group (PBS) was administered, and after the treatment was completed, the tumor was extracted and the weight was measured. As a result of the analysis, the tumor weight was reduced in all antibody fragment administration groups according to Examples 1, 2, and 3 compared to the control group. In the photograph on the right, the reduction in the size and weight of the extracted tumor tissue in each group is visually confirmed, and in particular, the most prominent tumor inhibition effect was observed in the antibody fragment administration groups according to Examples 1 and 3.

[0039] Figure 9 is a graph showing the change in tumor size of antibody fragments according to Examples 1 to 3 of the present invention in a humanized mice model. The X-axis represents the day of antibody fragment administration (ALB101 injection day), and the Y-axis represents the tumor volume (Tumor volume, mm³). After transplanting tumor cells (NCI-H358), the antibody fragment or control group (PBS) was administered together with human PBMC, and the tumor volume was measured for a certain period of time. As a result of the analysis, the tumor volume continuously increased in the control group, whereas the tumor volume decreased in the antibody fragment administration groups according to Examples 1, 2, and 3, and in particular, the antibody fragment according to Example 3 showed the highest tumor inhibition effect.

[0040] Figure 10 is a graph showing the results of measuring the change in mouse body weight after administration of antibody fragments according to Examples 1 to 3 of the present invention in a BALB / c nude mouse model. The X-axis represents the treatment period (Day), and the Y-axis represents the mouse body weight (mouse body weight, g). The change in body weight was monitored for a certain period of time after administration of the antibody fragment or the control group (Control, PBS). As a result of the analysis, weight gain was observed in all groups, and in the antibody fragment administration groups according to Examples 1, 2, and 3, no weight loss was observed, but rather weight gain was confirmed. This suggests that the antibody fragment has low toxicity and excellent biosafety.

[0041] Fig. 11 is a graph showing the results of ELISA analyzing the antigen binding capacity according to the pH change of antibody fragments according to Example 1 (Fig. 11(a), (d)), Example 2 (Fig. 11(b), (e)), and Example 3 (Fig. 11(c), (f)) of the present invention. The upper three graphs show the binding capacity to EpCAM, and the lower three graphs show the binding capacity to CD3, respectively. The X-axis represents the treatment conditions (BLANK, 2nd, pH 5.8, pH 6.5, pH 7.4), and the Y-axis represents the absorbance (OD450 nm). The analysis results showed that the binding capacity to EpCAM and CD3 antigens was maintained not only at normal pH (7.4) but also at a weakly acidic pH simulating the tumor microenvironment, and in the antibody fragments according to Examples 1 and 3, a tendency for the binding capacity to increase under weakly acidic conditions was observed. These results suggest that the antibody fragment of the present invention maintains stable binding capacity even in an acidic environment such as a tumor microenvironment, and provides sustained therapeutic efficacy.

[0042] Figure 12 is a graph showing the results of comparative analysis of the cytotoxicity of antibody fragments and scFv-scFv control groups according to Examples 2 and 3 of the present invention in normal cells and tumor cells. The X-axis represents antibody concentration (ug / ml), and the Y-axis represents cell viability (%). As a result of the analysis, the scFv-scFv control group exhibited cytotoxicity in both normal cells and tumor cells, whereas the antibody fragment of the present invention exhibited almost no cytotoxicity in normal cells, while similar or slightly lower cytotoxicity was observed in tumor cells compared to the control group, demonstrating selective anticancer effects and immune safety.

[0043]

[0044] The present invention relates to a bispecific antibody fragment comprising a nanobody specifically binding to the EpCAM antigen and a single-chain antibody fragment (scFv) specifically binding to the CD3 antigen, linked by a flexible linker. The antibody fragment of the present invention can have the effect of inducing tumor cell death by activating T cells by inducing the formation of an immune synapse between tumor cells (EpCAM-positive) and immune cells (CD3-positive).

[0045] Preferably, a nanobody with excellent structural stability and tissue penetration is adopted as the EpCAM binding domain, an scFv with controllable binding affinity is adopted as the CD3 binding domain, and a (GGGGS)n (n=1~3) linker consisting of a Gly₄Ser repeat sequence is inserted between the two domains, thereby preventing steric interference between antibody domains and enabling independent antigen binding.

[0046] The antibody fragment of the present invention can be produced in high yield as a soluble protein using an Escherichia coli (E. coli) expression system, and is purified using the IMAC method by attaching a His-tag. Furthermore, by using a buffer solution containing arginine to ensure antibody formulation stability, antigen binding affinity can be maintained without aggregation even during long-term storage.

[0047] The above bispecific antibody fragment exhibits a property of maintaining or increasing binding affinity for EpCAM and CD3 antigens not only at normal pH but also in the tumor microenvironment of pH 5.6 to 6.7, and can exhibit anticancer effects that significantly reduce tumor size in solid tumors in in vitro and in vivo model experiments. In particular, CDR mutations (point mutations) for modulating CD3 binding affinity can maintain high tumor-killing capacity while preventing immune cell hyperactivation.

[0048] In this way, the antibody fragment of the present invention simultaneously secures antigen specificity, tissue penetration, productivity, stability, and immunological safety, and can be effectively applied to various cancer types, including solid cancer.

[0049]

[0050] Hereinafter, the present invention will be described in more detail with specific examples. However, the following examples are provided as examples to ensure that those skilled in the art can sufficiently convey the spirit of the present invention.

[0051] Therefore, the present invention is not limited to the embodiments presented below and may be embodied in other forms. The embodiments presented below are described only to clarify the idea of ​​the present invention, and the present invention is not limited thereto.

[0052]

[0053] definition

[0054] Expressions such as "including," "comprising," "having," and the like used herein 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.

[0055] 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.

[0056] In this specification, "nanobody" may mean an antibody fragment comprising a single heavy chain variable region (VHH) antibody domain derived from camelids or the like, and which is miniaturized compared to general antibodies to provide high penetrating power and stability.

[0057] In this specification, “scFv (single-chain variable fragment)” or “single-chain antibody fragment” may refer to an antibody fragment having an antigen recognition function, which is a structure in which a variable heavy chain (VH) and a variable light chain (VL) of an antibody are connected by a linker.

[0058] In this specification, the term "bispecific antibody fragment" refers to an antibody fragment that specifically binds to two different antigens (EpCAM and CD3), and in the present invention, may mean a structure in which a nanobody and scFv are connected by a linker.

[0059] In this specification, the term "linker" refers to an amino acid repeat sequence that minimizes spatial interference between antibody domains and enables each domain to independently bind to an antigen, and in the present invention, may refer to a structure including a Gly₄Ser repeat sequence ((GGGGS)n).

[0060] In this specification, “tumor microenvironment (TME)” refers to an environment composed of extracellular matrix, immune cells, blood vessels, and signaling molecules surrounding tumor tissue, and may generally have the characteristics of hypoxia and weak acidity (pH 5.6 to 6.7).

[0061] In this specification, “antigen binding affinity (Kd)” refers to a dissociation constant that numerically represents the binding strength between an antibody and an antigen, and a lower Kd value may mean a higher binding strength to the antigen.

[0062] In this specification, “point mutation” may mean a mutation design that increases or decreases antigen binding ability for therapeutic purposes by replacing or modifying a specific amino acid in an antibody domain (such as CDR).

[0063] In this specification, “anticancer efficacy” or “cell killing efficacy” may mean the function of the target antibody fragment to reduce the survival rate of tumor cells or cancer cells or induce apoptosis.

[0064] In this specification, “immunosafety” may mean a property of a target antibody fragment that minimizes side effects such as overactivation of immune cells or cytokine release syndrome (CRS) while maintaining therapeutic effect.

[0065] In this specification, "BALB / c-Foxn1 nu / Arc-Gem nude mouse" refers to an immunodeficient mouse that lacks a thymus and T cells due to the introduction of a Foxn1 gene mutation into the BALB / c strain. Such mice rarely show rejection reactions when transplanted with foreign cells or tumor tissues, making them suitable for xenograft experiments with human cells or tumor cells. In the present invention, they can be used to evaluate the anticancer effect and safety of antibody fragments by transplanting human tumor cells (such as NCI-H358).

[0066]

[0067] Meanwhile, unless otherwise defined, the technical and scientific terms used herein have meanings commonly understood by those of ordinary skill in the art to which this invention pertains, and are terms defined in consideration of their functions in the present invention, which may vary depending on the intent or custom of the user or operator. Therefore, the definitions of these terms should be determined based on the contents throughout this specification, and in the following description, explanations of well-known functions and configurations that may unnecessarily obscure the gist of the present invention will be omitted.

[0068]

[0069] Hereinafter, the present invention will be described in detail.

[0070]

[0071] The present invention relates to a bispecific antibody fragment in which a nanobody specifically binding to EpCAM and a single-chain antibody fragment (scFv) specifically binding to CD3 are connected by a linker, and more specifically, to a bispecific antibody platform technology that can recognize EpCAM expressed on the surface of tumor cells and simultaneously recognize CD3 present on the surface of immune cells, thereby inducing physical binding between tumor cells and T cells and inducing death of tumor cells through T cell activation.

[0072] Most existing EpCAM-CD3 bispecific antibody structures have been constructed in the scFv / scFv format, most notably the BiTE (bispecific T cell engager) structure. However, these structures have the following technical limitations.

[0073] First, scFv is a structure in which VH and VL domains are artificially linked, and has low folding stability, is likely to form aggregates due to hydrophobic interactions during expression, and is limited in terms of productivity and quality consistency.

[0074] In addition, the molecular weight of the entire antibody was approximately 50 to 60 kDa based on the BiTE structure, which limited its penetration into the interstitial space of solid tumor tissue and the vascular endothelial barrier, and it was difficult to secure sufficient tumor penetration.

[0075] Accordingly, in order to improve structural stability and tissue penetration as described above, the present invention studied a method of adopting a nanobody structure to replace scFv.

[0076] The above research results confirmed that significantly superior effects could be achieved when adopting a nanobody compared to an antibody fragment in the form of scFv for EpCAM.

[0077] Specifically, EpCAM is a tumor-associated cell surface antigen that is overexpressed in various solid cancers, and is particularly highly expressed in liver cancer, colon cancer, pancreatic cancer, and breast cancer, and is involved in cell-to-cell adhesion and migration of tumor cells.

[0078] The intracellular expression location of EpCAM, as described above, is mainly limited to densely packed areas between cells, such as tight junctions and adherens junctions, creating a structural environment that is difficult for antibodies to physically access.

[0079] Therefore, antibodies targeting EpCAM must be able to penetrate deep into tumor tissues through a miniaturized structure, and at the same time, must maintain binding affinity and binding strength to be expected to have an effective anticancer effect.

[0080] However, scFv or full-length IgG antibodies have molecular weights of approximately 25–50 kDa and 150 kDa, respectively, and have limitations in penetrating the vascular endothelial barrier and extracellular matrix (ECM) in solid tumor tissues to access EpCAM on the surface of cancer cells.

[0081] Accordingly, the present invention adopted a nanobody as the domain binding to the EpCAM antigen. Nanobodies are small antibody fragments composed of a single heavy chain variable region (VHH). With a very small molecular weight of approximately 12-15 kDa, they exhibit superior intercellular permeability and intratissue diffusion compared to conventional antibodies.

[0082] In addition, it has the advantages of excellent folding stability due to its single domain structure, little structural denaturation even under acidic and high temperature conditions, and does not cause protein aggregation, resulting in excellent expression efficiency and ease of purification.

[0083] In particular, the EpCAM nanobody adopted in the present invention, in addition to a simple size advantage, exhibited a characteristic in which antigen binding affinity was maintained or even increased even under conditions of decreased pH in the tumor microenvironment (TME), thereby obtaining the advantage of being able to function stably even in the acidic environment within solid tumors.

[0084] In addition, since nanobodies are composed of a single genetic sequence, they have the industrial advantage of enabling high-yield production of antibody fragments through an E. coli expression system, and efficient purification and quality control can be performed without a complex recombinant antibody expression platform.

[0085] Therefore, the present invention can overcome the limitations of penetration and expression efficiency of conventional scFv-based structures by adopting a nanobody capable of maintaining a miniaturized structure and high binding affinity for a target antigen with low accessibility in tissues, such as EpCAM, as an antibody component, and can provide a structural advantage that can simultaneously improve antigen targeting precision and therapeutic efficacy in tumor tissues.

[0086] However, experiments using nanobodies as antibody-binding domains for CD3 have resulted in a number of problems.

[0087] Specifically, CD3, a component of the TCR complex present on the surface of T cells, is a key mediator that induces T cell activation in response to external stimuli. In particular, CD3 is known as a sensitive receptor capable of inducing a strong T cell response without costimulation, along with antigen-specific signaling. Therefore, the binding strength and binding characteristics of antibodies or antibody fragments to CD3 significantly impact the safety and efficacy of immune responses.

[0088] When adopting a bispecific antibody structure using a nanobody domain for CD3 as described above, a problem occurred in which excessive receptor agonist effects were induced on T cells.

[0089] Specifically, because nanobodies are structurally single-domain, binding to antigens induces cluster formation of these receptors, unintentionally triggering persistent and strong T cell activation signals. This mechanism increases the likelihood of unnecessary systemic immune cell activation and cytokine storms, posing a significant risk factor for patient safety, and has been a significant drawback in the adoption of nanobodies.

[0090] Therefore, in the present invention, scFv, rather than nanobody, was selected as the antibody-binding domain for CD3, and by performing structure-based prediction and alanine scanning of the CDR sequences within the scFv, mutation design capable of controlling binding affinity was performed, thereby effectively suppressing the risk of immunotoxicity due to overactivation while inducing appropriate T cell activation. This ensured the controllability of immune responses and therapeutic safety in the development of bispecific antibodies targeting sensitive immune receptors such as CD3.

[0091] Based on these experimental results, the present inventors developed a bispecific antibody fragment in which a nanobody that specifically binds to EpCAM and a single-chain antibody fragment (scFv) that specifically binds to CD3 are connected by a linker.

[0092]

[0093] The bispecific antibody fragment of the present invention as described above has the following integrated advantages due to the complementary technical characteristics of each component.

[0094] First, bispecific antibodies that simultaneously target EpCAM and CD3 induce direct immune synapse formation between tumor cells and T cells, thereby providing a foundation for T cells to identify and attack tumor cells without separate antigen presentation or immune cooperation. In particular, the structure of the present invention is designed to independently maintain binding affinity for EpCAM and CD3, while inducing a strong immune response only when both targets are present simultaneously, thereby securing a balance between target specificity and therapeutic efficacy.

[0095] At this time, the nanobody adopted as the EpCAM binding domain has a smaller molecular weight than conventional scFv or IgG antibodies, and its single-domain structure allows it to effectively access EpCAM present in densely packed regions between cells (e.g., tight junctions, adherens junctions). This enables accurate recognition of EpCAM targets within tumor tissues, which were difficult to bind with conventional antibodies, and enables effective antigen binding even when binding between tumor cells is maintained.

[0096] Furthermore, nanobodies have the advantages of rapid and stable structural folding and can be produced in a water-soluble form without forming inclusion bodies upon expression, enabling high-yield production even in the Escherichia coli system. In fact, the nanobodies implemented in the present invention showed an increase in yield of approximately 10-25 times or more compared to existing scFv-based structures, and high-purity purification was possible without protein aggregation.

[0097] Meanwhile, the scFv adopted as the CD3 binding domain may have the characteristic of being able to fine-tune the CDR sequence to suppress excessive activation of CD3, which is sensitive as an immune receptor, and to induce an appropriate immune response. In one embodiment of the present invention, the CDR sequence within the scFv was designed to be able to control the antigen binding affinity through structure-based prediction and alanine scanning techniques, thereby effectively suppressing side effects such as cytokine release syndrome (CRS) due to T cell overactivation. Through this, the present invention can be configured with a structure that can secure immune safety while maintaining the efficiency of anticancer action utilizing T cells.

[0098] Meanwhile, in order to enable the implementation of such a bispecific structure, it is important to select an appropriate linker connecting the nanobody and scFv domains.

[0099] Since nanobodies and scFvs are each structurally independent antigen recognition domains, sufficient distance and flexibility are required for the two domains to function without steric interference.

[0100] Additionally, the presence of a linker can contribute to improving expression efficiency and ease of purification by increasing the folding stability of the entire protein and preventing aggregate formation during expression.

[0101] In the present invention, a flexible peptide linker comprising a repeating amino acid sequence ((GGGGS)n) consisting of Gly-Gly-Gly-Gly-Ser was applied. This linker provides the following technical roles and advantages.

[0102] First, the linker minimizes spatial interference between antibody domains and provides structural flexibility, allowing each antibody fragment to fold and bind independently. Glycine, the smallest amino acid, offers a high degree of freedom and rotational potential when repetitively arranged, maximizing the flexibility of the entire linker. Serine, on the other hand, provides structural stability through hydrogen bond formation.

[0103] This (GGGGS)n linker structure enables the two binding domains to bind to their respective antigens simultaneously without steric interference in a bispecific antibody structure in which tumor cells and immune cells exist on different cell surfaces and each target antigen has a non-fixed spatial arrangement. As a result of actual experiments, it was confirmed that the antibody fragment applying the (GGGGS)n linker according to the present invention had high dual binding efficiency in cells in which EpCAM and CD3 were simultaneously expressed, and the T cell-induced cell killing effect was improved compared to the existing scFv / scFv-based bispecific antibody.

[0104] Furthermore, the linker's length (n value) can be precisely adjusted according to design conditions, thereby providing an optimal distance and degree of freedom between antibody domains, thereby securing molecular spacing suitable for immunological synapse formation. In particular, the present invention employed repeating units with n=1 to 3.

[0105] In the above range, the antibody was able to efficiently bind to both antigens simultaneously under physical binding conditions between immune cells and tumor cells, and the most preferable repeating unit was confirmed to be 3.

[0106] As described above, the flexible linker based on (GGGGS)n according to the present invention is designed to enable the nanobody that specifically binds to EpCAM and the scFv that specifically binds to CD3 to be connected in the most physically and functionally effective manner, and enables not only the interaction between antibody fragments but also the physically flexible orientation when the antibody binds near the cell membrane, thereby enabling the formation of an efficient immune synapse between tumor cells and immune cells. This structural flexibility can play an important role in enhancing binding efficiency and cell killing ability by allowing each antibody domain to be freely arranged according to the antibody-antigen binding conditions on the cell surface.

[0107] In conclusion, the nanobody-scFv antibody fragment dual-specific for EpCAM and CD3 of the present invention can provide a technical configuration that can simultaneously secure structural completeness and functional excellence as a bispecific antibody platform through a nanobody that can maximize tissue penetration and target specificity for EpCAM, an scFv that ensures controllable binding affinity for CD3 and immunological safety, and a (GGGGS)n-based linker structure that enables flexible structural connection between the two antibody fragments.

[0108] The bispecific antibody fragment of the present invention manufactured as described above may have various functionalities in addition to structural stability, antigen binding ability, immune cell induction activity, and productivity.

[0109] First, to confirm the functionality of the antibody fragment, antigen-binding and bispecific binding capacities were evaluated using cell lines expressing EpCAM and CD3, respectively. Flow cytometry analysis revealed that the bispecific antibody fragment of the present invention exhibited high binding capacities to both EpCAM and CD3.

[0110] In addition, to evaluate tumor cell killing ability and general cell toxicity, an in vitro cell killing experiment was performed by co-culturing EpCAM-positive tumor cells with PBMCs or general cells with PBMCs. As a result, it was confirmed that the antibody fragment treatment group of the present invention showed similar cell killing ability in EpCAM-positive tumor cells compared to the existing scFv / scFv-based bispecific antibody, while the toxicity in general cells tended to be significantly lower. Through this, it was possible to prove that it minimizes the risk of T cell hyperactivation and immunotoxicity (cytokine release syndrome, CRS), which are side effects of the existing scFv / scFv-based bispecific antibody.

[0111] Meanwhile, the bispecific antibody fragment of the present invention can maintain structural stability and functional efficacy even during long-term storage and transportation. Specifically, the produced antibody fragment was stored at -80°C for more than 3 months, and its physical stability (aggregation, degradation, etc.) and functional stability (antigen binding capacity) were periodically analyzed. As a result, no protein aggregation was observed, and the antigen binding capacity remained stable without significant decrease compared to the initial storage level.

[0112] Furthermore, for the bispecific antibody fragment, buffer composition optimization can be performed to ensure formulation stability. In the present invention, the solubility, stability, and biological activity of the antibody fragment were evaluated in buffer solutions under various conditions. In particular, it was confirmed that a buffer composition containing arginine showed the best results in maintaining the physical stability and function of the antibody. Arginine can suppress nonspecific interactions between proteins and prevent aggregation and precipitation.

[0113] Specifically, buffer optimization experiments demonstrated that antibody fragments in buffer solutions containing arginine maintained their antigen-binding capacity and immune cell induction function stably throughout storage without physical denaturation or aggregation. This stability can enhance quality maintenance during mass production, long-term storage, and distribution of antibody therapeutics, as well as their potential for clinical application.

[0114] This formulation stability provides quality maintenance and long-term storage convenience during commercial production and distribution, and is particularly important for ensuring consistent efficacy and safety of the product after clinical and market launch.

[0115] Therefore, the bispecific antibody fragment according to the present invention is an anticancer therapeutic platform with excellent functional characteristics that satisfy all of antigen-specific binding ability, T cell inducing activity, structural stability, and formulation stability, and can be effectively applied to the treatment of various cancers, including solid cancers.

[0116] Meanwhile, the bispecific antibody fragment of the present invention possesses significant advantages over existing bispecific antibody structures in terms of structural stability and productivity. In particular, the bispecific antibody fragment, composed of an EpCAM nanobody and a CD3 scFv, was designed to be produced with high efficiency using an Escherichia coli (E. coli) expression system.

[0117] Existing bispecific antibodies mainly rely on expression systems using mammalian cells (such as CHO cells), which have complex production processes, high production costs, and long culture periods, limiting mass production.

[0118] The E. coli expression system of the present invention, compared to mammalian cells, has a shorter culture time and produces a higher amount of protein per unit volume, enabling both rapid production cycles and cost-effectiveness. These productivity advantages enable time and cost savings during anticancer drug development and clinical trials, and can also provide competitiveness during subsequent product development and market launch.

[0119] Meanwhile, the bispecific antibody fragment of the present invention also exhibited excellent performance in terms of binding efficiency and stability in the tumor microenvironment (TME).

[0120] Unlike normal tissue, solid tumors are known to form an acidic microenvironment (pH 5.6 to 6.7) with hypoxia. In this environment, many protein therapeutics exhibit structural instability or reduced antigen binding affinity, resulting in reduced therapeutic efficacy. Indeed, while existing bispecific antibody structures (particularly scFv / scFv-based ones) remain stable under neutral (pH 7.2 to 7.4) conditions, cases have been reported where antigen binding affinity is reduced or protein denaturation occurs under the slightly acidic conditions found within tumors.

[0121] Accordingly, in the present invention, the acidic environment adaptability of EpCAM-binding nanobodies and CD3-binding scFv domains was evaluated and optimized. In particular, nanobodies have high tolerance to pH changes due to their single-domain structure and high folding stability, and the EpCAM nanobodies adopted in the present invention exhibited the characteristic of maintaining or even increasing antigen binding affinity even under conditions of pH 5.6 to 6.7, preferably pH 5.8 to 6.5, as experimental results.

[0122] For the CD3 scFv, structural stability in acidic environments was secured through alanine scanning and structural optimization of the CDR sequence. As a result, the bispecific antibody fragment of the present invention was able to stably maintain dual binding efficiency to EpCAM and CD3 antigens not only under neutral pH conditions but also in the acidic environment of solid tumor tissues.

[0123] This acidic environmental adaptability allows the target specificity and efficacy of the therapeutic agent to be maintained even within solid tumors, and in particular, it provides therapeutic selectivity by selectively exhibiting high binding affinity only in tumor tissues while minimizing nonspecific binding in normal tissues.

[0124] In addition, the property of maintaining binding affinity suitable for the tumor microenvironment enabled the formation of effective immune synapses between immune cells and tumor cells, thereby ensuring the sustainability of T cell activation and tumor cell killing ability.

[0125] Furthermore, the bispecific antibody fragment of the present invention demonstrated excellent antigen-binding and immune cell-inducing activity in vitro, as well as in vivo anticancer efficacy. Animal model experiments using the antibody fragment of the present invention demonstrated a significant reduction in tumor size and confirmed therapeutic safety.

[0126] Specifically, when the antibody fragment of the present invention was administered to a mouse model transplanted with a tumor cell line co-expressing EpCAM and CD3, tumor size inhibition was observed to be up to 80% greater than that of the control group. Furthermore, the antibody's binding affinity and immune cell induction function were stably maintained even in the slightly acidic intratumoral environment, demonstrating a sustained therapeutic effect.

[0127] Furthermore, no weight changes or behavioral abnormalities were observed in the animal groups administered the antibody fragment of the present invention, and no significant changes were observed in general toxicity indicators (liver and kidney function values). This suggests that the antibody fragment of the present invention effectively suppresses immunotoxic side effects such as T cell hyperactivation and cytokine release syndrome (CRS), while also stably exerting anticancer efficacy.

[0128] In addition, in the pharmacodynamic evaluation of the antibody fragment of the present invention, it was confirmed that antibody accumulation in tumor tissues was stably maintained for a certain period of time after administration, and rapid metabolism and excretion occurred in non-target tissues (liver, kidney, heart, etc.), thereby simultaneously satisfying target specificity and biosafety.

[0129] These in vivo results serve as evidence to support the claim that the antibody fragment of the present invention has excellent characteristics in all aspects of structural design, productivity, functionality, and safety, and has high industrial value as a bispecific antibody platform for the treatment of various cancers, including solid cancers.

[0130] Meanwhile, in the case of the bispecific antibody fragment of the present invention, the binding affinity for the antigen can be controlled through an optimization process for the antigen binding region (CDR: Complementarity Determining Region).

[0131] As previously discussed, CD3 and EpCAM are antigens present on the surface of T cells and tumor cells, respectively, and the antigen binding strength of antibody fragments has a significant impact on therapeutic efficacy and safety.

[0132] First, CD3 is a sensitive receptor that induces immune cell activation. Excessive antibody binding affinity can lead to serious side effects, such as T cell overactivation and cytokine release syndrome (CRS). Therefore, binding affinity for CD3 must be precisely controlled to induce sufficient T cell activation while preventing toxicity caused by overactivation.

[0133] In the present invention, structure-based prediction and alanine scanning analysis were performed on the CDR sequence within scFv to select key amino acid residues contributing to antigen binding affinity, and binding affinity was finely adjusted by substituting specific amino acids.

[0134] Furthermore, because EpCAM is located in densely packed areas of solid tumor cells, excessively strong antibody binding could increase nonspecific binding to normal or nontarget cells, potentially reducing therapeutic specificity. Accordingly, the CDR sequence of the EpCAM nanobody was adjusted through alanine scanning and mutagenesis analysis to minimize nonspecific binding while maintaining selective binding to tumor cells.

[0135] In practical experiments based on the above compound, the optimized antibody fragment maintained antigen binding to EpCAM and CD3, while reducing CD3 binding compared to conventional BiTE antibodies, thereby suppressing the risk of CRS. At the same time, binding was maintained or even increased within the tumor microenvironment, ensuring the sustainability and stability of the therapeutic effect.

[0136] As described above, the CDR optimization process of the present invention can simultaneously secure therapeutic efficacy and safety by balancing the antigen binding ability and immune activation ability of the antibody fragment.

[0137] The invention can provide a pharmaceutical composition for preventing or treating cancer, comprising a bispecific antibody fragment in which a nanobody specifically binding to EpCAM and a single-chain antibody fragment (scFv) specifically binding to CD3 are linked by a linker.

[0138] In addition, in the case of the present invention, as a method for preventing or treating cancer using the bispecific antibody fragment of the present invention, a method for preventing or treating cancer can be provided by administering to a cancer patient a pharmaceutically effective amount of a bispecific antibody fragment composed of a nanobody that specifically binds to EpCAM and a single-chain antibody fragment (scFv) that specifically binds to CD3.

[0139] The cancer may include at least one selected from the group consisting of liver cancer, colon cancer, pancreatic cancer, breast cancer, stomach cancer, lung cancer, prostate cancer, ovarian cancer, cervical cancer, esophageal cancer, and bladder cancer, and the antibody fragment may be administered into a living body via intravenous injection, subcutaneous injection, or other pharmaceutically acceptable administration route.

[0140] The antibody fragment of the present invention can specifically bind to cancer cells expressing EpCAM via CD3+ T cells and induce apoptosis in these cancer cells, thereby inhibiting tumor growth, reducing the number of cancer cells, and reducing tumor size.

[0141] These preventive or therapeutic effects can be confirmed through the in vitro cell death effect, in vivo anticancer effect, and tumor inhibition results presented in Experimental Examples 3, 4, and FIG. 5 below.

[0142] As described above, the bispecific antibody fragment in which a nanobody specifically binding to EpCAM of the present invention and a single-chain antibody fragment specifically binding to CD3 are connected by a linker can be used for the treatment of cancer.

[0143]

[0144] Below, the compound of the present invention is described in detail through specific examples and experimental examples.

[0145]

[0146] Example 1. Preparation of antibody fragments containing EpCAM nanobody and CD3 scFv

[0147] Example 1-1. Sequence design of EpCAM nanobody and CD3 scFv domain

[0148] In this example, the sequences of a nanobody that specifically binds to the EpCAM antigen and a single-chain antibody fragment (scFv) that specifically binds to the CD3 antigen were designed.

[0149] The EpCAM-binding nanobody was designed based on the existing anti-EpCAM sequence. The nanobody was designed with a compact structure and high folding stability to effectively penetrate the dense intercellular spaces within tumor tissue. The specific sequence of the EpCAM-binding nanobody is shown in SEQ ID NOs: 1 and 2 below.

[0150] Sequence number 1 corresponds to the DNA sequence of the EpCAM nanobody, and sequence number 2 corresponds to the amino acid sequence of the nanobody.

[0151]

[0152] SEQ ID NO: 1 (Wild Type 3turn_EpCAM_Nanobody_DNA)5'-CAGGTGCAGCTGGTTCAGAGCGGTGGCGGTAGCGTGCAGGGTGGTGCCAGTCTGCGTCTGAGCTGTGCAGCGTCAGGTGGTGAACGTAATAATTATTGTGTGGCATGGTTTCGTCAGGCTCCGGGCAAAGAACGTGAAGTGGCAGCAATTAGCCGTGCAGCCAGCGGCGCACAGACCACCACGA AATATTATGTGGATAGTGTTAAAGGTCGTTTTACCATTAGCCAGGATACCAAAAATACCGCAACCGTTTATCTGCAGATGAATAGCCATAAACCGGAAGATACCGCGTATTGTACCGCTAAAGCAAAAATTTATCCGCCGCAGTGTACCGGTATTAGCCGTACCATTGATTATCGTGGTCAGGGTACCCAGGTGACCGTTAGCAGC-3'SEQ ID NO: 2 (Wild Type 3turn_EpCAM_Nanobody_Protein)(n-ter)QVQLVQSGGGSVQGGASLRLSCAASGGERNNYCVAWFRQAPGKEREVAAIISRAASGAQTTTKYYVDSVKGRFTISQDTKNTATVYLQMNSHKPEDTAYCTAKAKIYPPQCTGISRTIDYRGQGTQVTVSS(c-ter)

[0153] Similarly, CD3-binding single-chain antibody fragments (scFvs) were designed based on the sequence of existing CD3 antibodies. These scFvs consist of variable heavy (VH) and variable light (VL) chains linked in a continuous single chain configuration and have the property of selectively binding to the CD3 antigen, which induces T cell activation.

[0154] Sequence number 3 corresponds to the DNA sequence of the CD3-binding single-chain antibody fragment (scFv), and sequence number 4 corresponds to the amino acid sequence of the antibody fragment (scFv).

[0155]

[0156] 서열번호 3 (Wild Type_CD3_scFv_DNA)5'-GATATTAAACTGCAGCAGAGTGGCGCAGAACTGGCCCGTCCGGGTGCGAGCGTGAAAATGAGCTGCAAAACTAGCGGTTATACCTTTACACGTTATACCATGCATTGGGTTAAGCAGCGTCCAGGTCAGGGCCTGGAATGGATTGGTTATATTAATCCGAGCCGTGGCTATACCAATTATAATCAGAAATTTAAAGATAAAGCAACCCTGACCACCGATAAAAGTAGTAGCACCGCCTATATGCAGCTGAGCAGCCTGACCAGCGAAGATAGCGCAGTTTATTATTGTGCACGTTATTATGATGATCATTATTGTCTGGATTATTGGGGTCAGGGCACCACCCTGACCGTTAGCTCTGTTGAAGGTGGTAGCGGCGGTTCTGGTGGTTCAGGCGGTAGTGGTGGCGTGGATGATATTCAGCTGACCCAGTCACCGGCCATTATGAGCGCAAGTCCGGGTGAAAAAGTTACCATGACCTGTCGTGCAAGTAGCTCAGTGAGCTATATGAATTGGTATCAGCAGAAAAGCGGTACCTCACCGAAACGTTGGATTTATGATACCAGTAAAGTTGCAAGTGGCGTTCCGTACCGCTTTAGTGGCAGCGGTAGCGGCACCAGCTATTCCCTGACGATCAGCTCTATGGAAGCAGAAGATGCCGCCACCTATTATTGCCAGCAGTGGAGCAGCAATCCGCTGACCTTTGGTGCAGGTACCAAACTGGAACTGAAA-3’서열번호 4 (WildType_CD3_scFv_Protein)(n-ter)DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWG QGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK(c-ter)

[0157] Example 1-2. Synthesis of EpCAM nanobody-CD3 scFv bispecific antibody fragment gene and production of expression vector

[0158] Based on the optimized EpCAM nanobody and CD3 scFv sequences in Example 1, gene synthesis of a bispecific antibody fragment and production of an expression vector were performed.

[0159] The EpCAM nanobody and CD3 scFv sequences were connected by a flexible linker consisting of a repeating unit sequence ((GGGGS)n) of SEQ ID NO: 5.

[0160] In this example, considering the flexibility and length of the linker, a (GGGGS)₃ sequence comprising n=3 repeating units was applied as a linker consisting of SEQ ID NO: 6. This linker minimizes steric interference between antibody domains and provides structural flexibility that allows each domain to bind to an antigen independently.

[0161] The linker may consist of a connecting sequence located after the C-terminus of the nanobody and before the N-terminus of the scFv.

[0162]

[0163] SEQ ID NO: 5 (n=1 linker)(n-ter)GGGGS(c-ter)SEQ ID NO: 6 (n=3 linker)(n-ter)GGGGSGGGGSGGGGS(c-ter)

[0164] The DNA sequence synthesized using optimized codons based on the amino acid sequence of each domain was cloned into the pET-28a(+) vector (Novagen) or an equivalent expression vector. The vector was constructed to enable high-throughput expression of antibody fragments under the T7 promoter, and a His-tag (6xHis) sequence was added to the C-terminus for easy purification of the expressed protein.

[0165] The EpCAM nanobody and CD3 scFv sequences were inserted into an expression vector using NdeI and XhoI restriction enzyme cleavage sites, and the accuracy of the inserted genes was confirmed by Sanger sequencing. The expression vector constructed in this way was subsequently used for the expression and purification of antibody fragments in Escherichia coli.

[0166]

[0167] Example 1-3. Expression and purification of antibody fragments using Escherichia coli (E. coli)

[0168] Expression and purification of the antibody fragment was performed in E. coli BL21 (DE3) strain using the EpCAM nanobody-CD3 scFv bispecific antibody fragment expression vector produced in Example 1-2.

[0169] The transformed BL21(DE3) strain was cultured in LB medium containing 1% ethanol, 10 mM MgCl2, 0.08% glycerol, and Ampicillin (100 μg / mL) at 37°C with shaking. Cell density (OD 500 ) reached 0.6–0.8, IPTG (1 mM) was added to induce protein expression. After inducing expression, additional culture was performed at 37°C for 3 hours to express the protein.

[0170] After incubation, cells were collected by centrifugation at 4,000 rpm (15 min, 4°C), and the harvested cells were resuspended in buffer (10 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.1 M Dextrose, and 0.1 M D-Mannitol) and disrupted using an ultrasonicator at low temperature. Insoluble inclusion bodies were separated from the cell lysate by centrifugation (4,000 rpm for 20 min at 4°C). Insoluble inclusion bodies obtained by centrifugation were suspended in buffer (10 nM Tris-Hcl (pH 8.0), 0.1% Triton X-100, 100 ng / ml Lysozyme, 1 mM PMSF, 2 mM MgCl2) and separated into cell lysates by centrifugation (4,000 rpm for 20 min at 4 °C). The pellet was carefully resuspended in protein unfolding buffer (10 mM Sodium phosphate, 150 mM NaCl, 8 M Urea [pH 8.5], 10 mM DTT, 0.2 mM PMSF) and incubated overnight at 4 °C until most of the pellet was dissolved with gentle shaking. The dissolved sample was centrifuged at 12,000 xg at 4°C for 30 minutes, the supernatant was separated, and then filtered using a 0.45 μm filter.

[0171] Antibody fragments contained in the supernatant were purified by immobilized metal affinity chromatography (IMAC) using nickel-NTA agarose resin (Qiagen). The His-tagged antibody fragments were bound to the resin in binding buffer (20 mM sodium phosphate buffer, 150 mM NaCl, 20 mM Imidazole, 8 M urea). After that, nonspecifically bound proteins were removed using washing buffer (containing 20 mM imidazole), and the antibody fragments were eluted using elution buffer (20 mM sodium phosphate buffer, 150 mM NaCl, 500 mM Imidazole, 8 M urea).

[0172] Subsequently, refolding was performed by sequentially removing imidazole and urea present in the buffer solution containing the purified antibody fragment through a dialysis process. The antibody fragment of Example 1 (ALB101-3T) that had undergone the above refolding process was subsequently used in experiments to evaluate binding affinity and biological activity.

[0173]

[0174] Example 2. Controlling the length of the linker

[0175] Example 2 antibody fragment (ALB101-1T) was prepared in the same manner as Example 1, except that n was adjusted to 1 in (GGGGS)n and a linker corresponding to sequence number 5 was used.

[0176]

[0177] Example 3. Optimization design through genetic mutation

[0178] In Example 3, point mutations were introduced to the amino acid sequence at specific positions to control the antigen binding affinity of the EpCAM nanobody and CD3 scFv antibody fragment and to improve biological activity.

[0179] First, structure-based prediction and alanine scanning analysis were performed on the CDR (Complementarity Determining Region) sequences of the EpCAM nanobody domain and CD3 scFv domain to identify key amino acid residues contributing to antigen binding affinity. Subsequently, alanine substitutions or amino acid mutations were performed on each antibody domain to enhance affinity.

[0180] In this example, an antibody fragment (ALB101-M1) of Example 3 was prepared in the same manner as in Example 2, except that the sequence of the 46th amino acid of the EpCAM nanobody corresponding to SEQ ID NO: 2 was changed from Glutamic acid (E) to Alanine (A), and the sequence of the 101st amino acid of the CD3 scFv corresponding to SEQ ID NO: 4 was changed from Tyrosine (Y) to Phenylalanine (F).

[0181] The amino acid sequence of the EpCAM nanobody of this example manufactured through the above mutation corresponds to SEQ ID NO: 7, and the amino acid sequence of the CD3 scFv corresponds to SEQ ID NO: 8.

[0182]

[0183] Sequence number 7 (n-ter) QVQLVQSGGGSVQGGASLRLSCAASGGERNNYCVAWFRQAPGKERAVAAISRAASGAQTTTKYYVDSVKGRFTISQDTKNTATVYLQMNSHKPEDTAYCTAKAKIYPPQCTGISRTIDYRGQGTQVTVSS (c-ter) Sequence number 8(n-ter)DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYFDDHYCLDYWGQGTTLTVSSVE GGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK(c-ter)

[0184] Example 4.

[0185] Example 4 antibody fragment (ALB101-CD3(Y101F)) was prepared in the same manner as Example 2, except that the sequence of the 101st amino acid of CD3 scFv corresponding to sequence number 4 was mutated from Tyrosine (Y) to Phenylalanine (F).

[0186] The amino acid sequence of the CD3 scFv of this example manufactured through the above mutation corresponds to SEQ ID NO: 8.

[0187]

[0188] Example 5.

[0189] Example 5 antibody fragment (ALB101-CD3 (K187R)) was prepared in the same manner as Example 2, except that the sequence of the 187th amino acid of CD3 scFv corresponding to sequence number 4 was mutated from Lysine (K) to Arginine (R).

[0190] The amino acid sequence of the CD3 scFv of this example manufactured through the above mutation corresponds to SEQ ID NO: 9.

[0191]

[0192] sequence number 9(n-ter)DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVE GGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSRVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK(c-ter)

[0193] Experimental Example 1. Evaluation of Antigen Binding Ability

[0194] In this experimental example, the antigen binding affinity (Kd) of the nanobody-scFv antibody fragments that dual-specifically bind to EpCAM and CD3, manufactured through Examples 1 to 3, was evaluated. For the analysis, human EpCAM (hEpCAM) and human CD3 (hCD3) antigens were each immobilized on a 96-well plate.

[0195] ELISA plates were coated with EpCAM and CD3-ε proteins at a concentration of 100 ng / well each using coating buffer (BioLegend, product number 421701) at 4°C overnight. The plates were then incubated with a solution containing 1% bovine serum albumin (BSA) in washing buffer (BioLegend, product number 421601) at room temperature for 2 hours (blocking step).

[0196] The plate was washed with 1X dilution of BioLegend's 20X concentrated wash buffer, and then 200 μL / well of purified ALB101 antibody fragments were added to the plate, serially diluted in 1% BSA blocking buffer. The plate was incubated at room temperature for 1 hour and then washed with 1X wash buffer.

[0197] Next, 100 μl / well of HRP (peroxidase)-conjugated anti-His tag mouse monoclonal antibody (Abcam, dilution 1:5,000) in 1% BSA blocking buffer was added. After incubation for 1 hour at room temperature, the plate was washed, and 100 μl / well of TMB substrate solution was added, mixed in a 1:1 ratio, and the reaction was allowed to proceed for approximately 5 to 10 minutes in a light-blocking state.

[0198] After the reaction, the reaction was stopped by adding 50 ㎕ / well of 2 N sulfuric acid (H₂SO₄) termination solution, and then the absorbance (OD) was measured at a wavelength of 450 nm.

[0199] Based on the measured absorbance values ​​and antibody concentration data, the antigen binding affinity (Kd) of each antibody fragment was calculated through nonlinear regression analysis (simple binding model).

[0200] The results of the above binding affinity calculation are as shown in Figures 2 and 3.

[0201] As a result of the experiment, the Kd value of the Example 2 antibody for the hEpCAM antigen was measured to be approximately 4.68 nM, the Example 1 antibody to be approximately 4.94 nM, and the Example 3 antibody to be approximately 5.36 nM. (Fig. 2) This confirmed that all three antibodies maintained excellent binding affinity for the EpCAM antigen.

[0202] In addition, the Kd values ​​for the hCD3 antigen were measured to be about 83.7 nM for the antibody of Example 2, about 4.39 nM for the antibody of Example 1, and about 38.2 nM for the antibody of Example 3 (Fig. 3).

[0203] Through this, it was confirmed that the binding affinity for CD3 can be controlled by introducing mutations and adjusting the CDR sequence, and by appropriately weakening the binding affinity for excessive CD3 antigen, it can contribute to optimizing the antibody's immune cell activation ability and reducing the risk of excessive T cell activation and cytokine release syndrome (CRS).

[0204] In conclusion, it can be confirmed that the antibody fragment of the present invention has excellent or controlled binding affinity for EpCAM and CD3 antigens, respectively, thereby providing structural and functional advantages that can improve side effects and enhance therapeutic efficacy compared to existing bispecific antibodies.

[0205]

[0206] Experimental Example 2. Evaluation of binding capacity at the cellular level (flow cytometry analysis)

[0207] To evaluate the dual binding ability of the bispecific antibody fragment of the present invention at the cellular level, flow cytometry analysis was performed on cell lines expressing EpCAM and CD3 antigens, respectively.

[0208] Specifically, in this experimental example, the binding ability of the nanobody-scFv antibody fragments that dual-specifically bind to EpCAM and CD3 manufactured through Examples 1 to 3 to cell surface antigens was evaluated.

[0209] First, Jurkat T cells expressing the CD3 antigen and five cancer cell lines (MCF7, SW480, HCC827, Hep3B, and NCI-H358) expressing the EpCAM antigen were prepared. Each cell line was washed with FACS buffer (DPBS, 1% BSA, 0.02% NaN3, 1 mM EDTA), and then treated with the same concentration of antibody fragments according to Examples 1-3, and an antibody-antigen binding reaction was performed at 4°C for 2 hours. Unbound antibodies were removed by washing with FACS buffer, and anti-IgG Alexa488 antibody was added to detect the attached antibody fragments, and the cells were washed after the reaction.

[0210] Fluorescently labeled cells were subjected to flow cytometry analysis using a BD FACSVerse™ Flow Cytometer (BD Biosciences, San Jose, CA, USA, Catalog No. 651154), and the mean fluorescence intensity (MFI) of each cell was measured.

[0211] As a result of the analysis, all antibody fragments according to Examples 1 to 3 exhibited concentration-dependent binding signals for Jurkat cells expressing CD3 antigen and MCF7, SW480, HCC827, Hep3B, and NCI-H358 cells expressing EpCAM antigen. In contrast, no significant fluorescence signal was observed in the unlabeled control group and the antibody-untreated cell control group (Fig. 4).

[0212] In particular, all antibody fragments showed distinct antigen binding signals for the target cell line, thereby confirming that the bispecific antibody fragments of the present invention effectively exhibit EpCAM and CD3 bispecificity on the actual cell surface.

[0213]

[0214] Experimental Example 3. Evaluation of apoptotic activity of human cancer cell lines in vitro.

[0215] In this experimental example, the antibody fragments prepared through Examples 2, 4, or 5 were used to evaluate the T cell-mediated apoptosis effect in an in vitro environment.

[0216] Specifically, experiments were conducted on human lung cancer cell lines (NCI-H358), hepatoma cell lines (HEP3B), colon cancer cell lines (SW480), and normal peripheral blood mononuclear cells (PBMCs).

[0217] After co-culturing each cancer cell line and PBMC, the antibody fragment of Example 2 was treated at various concentrations, and cell viability was measured using the WST-8 assay. As a result, it was confirmed that, compared to the PBMC-only treatment group, the antibody fragment of the present invention induced a concentration-dependent cell death in the group in which cancer cells and PBMC were co-cultured (Fig. 5).

[0218] In particular, EC in NCI-H358 cell line 50 The lowest value was 54.48 nM, indicating high apoptotic activity, and EC values ​​of 211.8 nM and 216.9 nM were observed in HEP3B and SW480, respectively. 50 Differential activity was observed depending on the cell line, indicating the value.

[0219] Additionally, the efficacy of antibody fragments with point mutations introduced into the CD3 scFv region (Examples 4 and 5) was comparatively evaluated. Similar to the antibody in Example 2, these mutant antibody fragments induced a dose-dependent apoptosis in cancer cells (Fig. 6). Specifically, in a group co-cultured with PBMCs and cancer cells, the mutant antibodies also exhibited a potent apoptotic effect, demonstrating that the tumoricidal capacity of the antibody was maintained despite the introduction of mutations that reduce the risk of excessive T cell activation and cytokine release syndrome (CRS).

[0220] In conclusion, these results suggest that the antibody fragment of the present invention can effectively induce T cell activation and tumor cell apoptosis through dual specific binding to EpCAM and CD3, and it was confirmed that high anticancer activity was maintained despite regulation of binding affinity to CD3 (point mutation).

[0221]

[0222] Experimental Example 4. In vivo evaluation of anticancer efficacy of a bispecific antibody fragment.

[0223] In this experimental example, experiments were performed in two animal models to evaluate the in vivo anticancer efficacy of the antibody fragments manufactured through Examples 1 to 3.

[0224]

[0225] Experimental Example 4-1. Evaluation of anticancer efficacy in a BALB / c nude mouse model

[0226] First, a tumor xenograft model was established using a human lung cancer cell line (NCI-H358) in BALB / c-Foxn1 nu / Arc-Gem nude mice. 8 × 10 cells were injected into each of 16 mice. 6 NCI-H358 cells were injected subcutaneously (sc) into the rats, and 14 days later, 12 rats with tumors of similar size were selected and randomly assigned to 4 groups of 3 rats each. Each group was treated with PBS (control), the antibody fragment of Example 1, the antibody fragment of Example 2, and the antibody fragment of Example 3, respectively.

[0227] Antibody fragments were injected intraperitoneally (ip) at a dose of 12 μg / 200 μL, and administered four times over a period of one week. Tumor volumes were then measured every three days, and the tumor volume (W 2 × L) / 2 was calculated using the formula (W: short axis length, L: long axis length).

[0228] As a result, in the control group, tumor volume continued to increase, but in the group administered antibody fragments, tumor volume increase was significantly suppressed (Fig. 7). Specifically, on Day 12, compared to the control group, the antibody fragment treatment group according to Example 2 showed a tumor volume reduction effect of approximately 26.77%, the antibody fragment treatment group according to Example 1 showed a tumor volume reduction effect of approximately 47.53%, and the antibody fragment treatment group according to Example 3 showed a tumor volume reduction effect of approximately 51.64%.

[0229] Additionally, after the experiment, tumor tissues were removed and weighed. The antibody fragment-treated group showed a significant reduction in tumor weight compared to the control group (Fig. 8). In particular, the antibody fragment-treated groups according to Examples 1 and 3 demonstrated the greatest anticancer effects.

[0230]

[0231] Experimental Example 4-2. Evaluation of Anticancer Efficacy in a Humanized Mice Model

[0232] Next, the anticancer efficacy was verified in a humanized mouse model. 5×10 6 A xenograft model was created by subcutaneously injecting NCI-H358 cells, and tumor size was measured 11 days later. Afterwards, each mouse was injected with human PBMC (5 × 10 6 ) was injected into the tail vein, and then treated with PBS or antibody fragments according to Examples 1 to 3. The antibody fragments were administered repeatedly daily for 4 days starting from 1 day after the first administration at a dose of 20 μg / 200 μL.

[0233] After treatment, tumor size was measured daily, and in the control group, tumor size continued to increase. On the other hand, the antibody fragment treatment group according to Example 2 showed a 69% decrease in tumor size compared to the control group, the antibody fragment according to Example 1 showed a 77% decrease in tumor size, and the antibody fragment according to Example 3 showed an 80% decrease in tumor size (Fig. 9).

[0234]

[0235] Experimental Example 4-3. Sintering

[0236] The antibody fragments according to Examples 1 to 3 exhibited excellent anticancer efficacy in both the BALB / c nude mouse model and the humanized mouse model, and in particular, it was confirmed that the antibody fragment of Example 3 including a CD3 binding affinity regulatory mutation (point mutation) exhibited the most excellent tumor inhibition effect. Through this, it was confirmed that the antibody fragment of the present invention effectively exhibits EpCAM and CD3 bispecificity and simultaneously implements high anticancer efficacy and immunotoxicity regulation properties in an in vivo environment.

[0237]

[0238] Experimental Example 5. In vivo safety evaluation of bispecific antibody fragments.

[0239] In this experimental example, animal testing was performed using a BALB / c nude mouse model to evaluate the safety of the antibody fragments manufactured through Examples 1 to 3.

[0240] Specifically, a tumor transplantation model was established by injecting NCI-H358 cells into BALB / c-Foxn1 nu / Arc-Gem nude mice, and the antibody fragments according to Examples 1 to 3 were administered under the same conditions as the anticancer efficacy evaluation in Example 4-1. Safety evaluation was performed based on body weight changes.

[0241] As a result of body weight measurement, no weight loss was observed in any of the antibody treatment groups in Experimental Examples 1 to 3 after antibody injection. Rather, the weight increased by 4.03%, 3.67%, and 4.23%, respectively, indicating that there were no signs of toxicity (Fig. 10).

[0242]

[0243] Experimental Example 6. Evaluation of Antigen Binding Ability of Antibody Fragments According to pH Changes

[0244] In order to evaluate whether the bispecific antibody fragment of the present invention maintains antigen binding ability even under the slightly acidic conditions of the tumor microenvironment (TME), the antigen binding ability of the antibody fragments of Examples 1 to 3 of the present invention was analyzed under various pH conditions.

[0245] The tumor microenvironment (TME) is generally more acidic than normal tissue, with a pH of approximately 5.6 to 6.7. Accordingly, in this experimental example, the binding affinity of antibody fragments to EpCAM and CD3 antigens was compared and analyzed under the slightly acidic conditions of the tumor microenvironment (pH 5.8 and 6.5) and the normal neutral conditions (pH 7.4).

[0246] Specifically, to perform ELISA analysis, EpCAM or CD3 antigens were immobilized on a 96-well plate under each pH condition. Subsequently, antibody fragments according to Examples 1 to 3 were processed, and absorbance was measured at 450 nm using a His-tag detection antibody and TMB substrate.

[0247] As a result of EpCAM antigen binding affinity analysis, the antibody fragments according to Examples 1 and 3 showed a tendency for slightly increased binding affinity under slightly acidic conditions of pH 5.8 to 6.5 compared to pH 7.4. On the other hand, the antibody fragment according to Example 2 was confirmed to have a slightly decreased binding affinity under slightly acidic conditions.

[0248] Similar results were observed in the CD3 antigen binding assay. The binding affinity of the antibody fragments according to Examples 1 and 3 tended to increase at pH 5.8 to 6.5, while the binding affinity of the antibody fragment according to Example 2 tended to remain constant.

[0249] These results suggest that the antibody fragments according to Examples 1 and 3 maintain stable binding affinity even under the slightly acidic conditions of the tumor microenvironment, or even exhibit enhanced antigen binding affinity, and are highly likely to exhibit enhanced therapeutic efficacy in solid tumor environments. (Figure 11)

[0250]

[0251] Experimental Example 7. Comparative Evaluation with scFv-scFv Antibody Fragments

[0252] To compare the tumor cell killing ability and general cell toxicity with scFv-scFv form of BiTE (solitomab, HY-P99592, MedChemExpress, USA), an in vitro cell killing experiment was performed by co-culturing EpCAM-positive tumor cells (NCI-H358) with PBMC or general cells (HEK293) with PBMC.

[0253] As a result, it was confirmed that the antibody fragment treatment group of the present invention showed a similar cell killing ability in EpCAM-positive tumor cells compared to the existing scFv / scFv-based bispecific antibody, while showing a tendency for significantly lower toxicity in normal cells. Through this, it was confirmed that the bispecific antibody comprising the nanobody specifically binding to EpCAM of the present invention and the single-chain antibody fragment (scFv) specifically binding to CD3 can minimize the risk of T cell hyperactivation and immunotoxicity (cytokine release syndrome, CRS), which are one of the side effects of the existing scFv / scFv-based bispecific antibody. (Figure 12)

[0254]

[0255] As can be confirmed through the above examples, the EpCAM and CD3 bispecific antibody fragment according to the present invention has a structure in which a nanobody specifically binding to EpCAM and a single-chain antibody fragment (scFv) specifically binding to CD3 are connected by a flexible linker, and the bispecific antibody fragment can provide a technical effect that is significantly superior to existing technologies in terms of antigen binding ability, immune cell induction activity, and stability.

[0256] Specifically, the nanobody for EpCAM binding has a small structure that can effectively penetrate the dense intercellular space of tumor tissue, and exhibits the characteristic of maintaining or even increasing high antigen binding affinity even in the acidic environment inside solid tumors, thereby overcoming the limitations of existing scFv or IgG-based antibodies. In addition, the CD3-binding scFv was able to induce sufficient T cell activation while suppressing excessive immune responses and side effects by optimizing binding affinity through structure-based prediction and alanine scanning.

[0257] In particular, the two antibody fragments can function independently without steric interference through a linker composed of Gly-Gly-Gly-Gly-Ser repeating sequences ((GGGGS)n), and the flexibility and optimal length of the linker enabled the formation of effective immune synapses between immune cells and tumor cells.

[0258] In the case of the above bispecific antibody fragment, high antigen binding affinity and T cell-induced cytotoxicity were confirmed in in vitro tests, and in vivo animal model experiments also demonstrated superior tumor size inhibition and immunotoxicity inhibition effects compared to existing bispecific antibodies.

[0259] In conclusion, it can be confirmed that the bispecific antibody fragment of the present invention has very high industrial value as a bispecific antibody platform for treating solid cancers that simultaneously secures structural stability, target specificity, immunological safety, production efficiency, and long-term stability.

[0260]

[0261] The EpCAM and CD3 bispecific antibody fragment according to the present invention can be industrially utilized as an anticancer treatment for various cancers, including solid tumors. In particular, the antibody fragment, as a bispecific antibody platform technology that simultaneously recognizes a tumor antigen (EpCAM) and an immune cell marker antigen (CD3), kills tumor cells via T cells, potentially providing an innovative therapeutic approach that overcomes the limitations of existing antibody therapeutics.

[0262] In addition, since the antibody fragment of the present invention can be produced in high yield through an E. coli expression system, the production cost is lower than that of existing mammalian cell-based antibody production methods, and the production process is simple, making it easy for mass production and commercialization.

[0263] EpCAM is a target overexpressed in various solid cancers, including liver cancer, colon cancer, lung cancer, breast cancer, and pancreatic cancer. Since the therapeutic effect of the present invention has been verified in these cancer types, it has great potential for industrial application in the development of antibody drugs for cancer treatment and the antibody-based immunotherapy market.

[0264] Therefore, the bispecific antibody fragment of the present invention can be widely used in the biopharmaceutical industry, especially in the field of immuno-oncology drug development, and can be clinically and commercially applied in the future as an intravenous or subcutaneous administration formulation.

Claims

1. A bispecific antibody fragment in which a nanobody that specifically binds to EpCAM and a single-chain antibody fragment (scFv) that specifically binds to CD3 are connected by a linker.

2. In paragraph 1, A bispecific antibody fragment, characterized in that the linker comprises a repeating unit sequence of SEQ ID NO:

5.

3. In paragraph 2, A bispecific antibody fragment, characterized in that the linker is composed of sequence number 6.

4. In paragraph 1, A bispecific antibody fragment characterized in that the above antibody fragment is produced through an E. coli expression system.

5. In paragraph 1, The above antibody fragment is a bispecific antibody fragment characterized in that the antigen binding affinity for EpCAM and CD3 is maintained or increased in a tumor microenvironment of pH 5.6 to 6.

7.

6. A pharmaceutical composition for preventing or treating cancer, comprising a bispecific antibody fragment in which a nanobody specifically binding to EpCAM and a single-chain antibody fragment (scFv) specifically binding to CD3 are connected by a linker.

7. In paragraph 6, A pharmaceutical composition for preventing or treating cancer, characterized in that the cancer comprises at least one selected from the group consisting of liver cancer, colon cancer, pancreatic cancer, breast cancer, stomach cancer, lung cancer, prostate cancer, ovarian cancer, cervical cancer, esophageal cancer, and bladder cancer.

8. A method for preventing or treating cancer, comprising administering to a cancer patient an effective amount of a bispecific antibody fragment in which a nanobody that specifically binds to EpCAM and a single-chain antibody fragment that specifically binds to CD3 are linked by a linker.

9. In paragraph 8, A method for preventing or treating cancer, characterized in that the cancer comprises at least one selected from the group consisting of liver cancer, colon cancer, pancreatic cancer, breast cancer, stomach cancer, lung cancer, prostate cancer, ovarian cancer, cervical cancer, esophageal cancer, and bladder cancer.

10. Use of a bispecific antibody fragment comprising a nanobody specifically binding to EpCAM and a single-chain antibody fragment specifically binding to CD3 linked by a linker for the treatment of cancer.

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