Epcam and CD3-bispecific antibody fragment having affinity-modulated mutant

Abstract

The present invention relates to a bispecific antibody fragment in which a nanobody specifically binding to EpCAM is connected by a flexible peptide linker ((GGGGS)n) to a single-chain antibody fragment (scFv) specifically binding to CD3, and is an antibody platform technology for anticancer therapy, wherein a selective point mutation is introduced into the antigen-binding site (CDR) of the antibody, thereby modulating binding affinity for an antigen and immunological safety. The design is such that the EpCAM-binding domain has maximized accessibility and selective binding ability to solid cancer cells by having utilized a nanobody having excellent tissue penetration, and the CD3-binding domain can inhibit T-cell overactivation and immunotoxicity (CRS) by having utilized scFv, the affinity of which can be modulated based on the therapeutic purpose. In particular, the affinity of antibody fragments to EpCAM and CD3 antigens is stably maintained or improved even under weakly acidic conditions (pH 5.6 to 6.7) of the tumor microenvironment, indicating that the antibody fragments can exhibit excellent anticancer effects even in solid cancer environments.

Description

EPCAM and CD3 bispecific antibody fragments with avidity-modulating mutations

[0001] The present invention relates to the field of bispecific antibody technology, and more particularly, to a bispecific antibody fragment in which binding affinity-modulating mutations are introduced into antibody fragments that specifically bind to EpCAM and CD3 antigens, respectively, and to an anticancer therapeutic composition comprising the same. In particular, the present invention relates to a bispecific antibody fragment that introduces mutations capable of fine-tuning binding affinity into the antigen-binding portion of the antibody, thereby maintaining target binding ability for tumor cells and immune cells while minimizing the possibility of excessive immune activation and immunotoxicity, and maintaining stable antigen-binding affinity even under the special conditions of the tumor microenvironment (TME).

[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 including 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]

[0013] 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.

[0014] 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.

[0015] 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 a high-yield, high-purity, water-soluble form through an E. coli expression system.

[0016] 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.

[0017]

[0018] 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.

[0019]

[0020] 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. The antibody fragment of the present invention may include a structure in which a point mutation is introduced into at least one antigen-binding region (CDR) of the nanobody and the single-chain antibody fragment.

[0021] In addition, as an embodiment of the present invention, the linker in the bispecific antibody fragment may include a repeating sequence consisting of Gly-Gly-Gly-Gly-Ser, and more specifically, may include a (GGGGS)₃ sequence.

[0022] As another example, the antibody fragment may include one or more of the following mutations: a mutation in which the 101st amino acid, Tyrosine (Y), in a CD3-binding single-chain antibody fragment, is substituted with Phenylalanine (F) or Alanine (A), a mutation in which the 180th amino acid, Arginine (R), is substituted with Alanine (A) or Lysine (K), a mutation in which the 187th amino acid, Lysine (K), is substituted with Arginine (R), Alanine (A), or Glutamic acid (E).

[0023] As another example, the antibody fragment may comprise one or more of the following point mutations introduced into the EpCAM binding nanobody: a mutation in which the 46th amino acid, Glutamic acid (E), is substituted with Alanine (A), a mutation in which the 109th amino acid, Glutamine (Q), is substituted with Alanine (A), and a mutation in which the 115th amino acid, Arginine (R), is substituted with Alanine (A).

[0024] As another example, the antibody fragment may comprise a structure in which a Y101F or K187R single mutation is introduced into the CD3 domain.

[0025] As another example, the antibody fragment may comprise a structure that simultaneously includes the E46A mutation of the EpCAM domain and the Y101F mutation of the CD3 domain.

[0026] As another embodiment, the present invention relates to a pharmaceutical composition for preventing or treating cancer, comprising the bispecific antibody fragment as an active ingredient, wherein 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.

[0027]

[0028] 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 apoptosis. In particular, the EpCAM binding domain adopts a nanobody with small molecular weight and excellent penetrability, thereby maintaining high accessibility and binding affinity even in densely packed areas (such as tight junctions) between cells in tumor tissues. In addition, the binding affinity to antigen can be enhanced through the introduction of point mutations.

[0029] Furthermore, by applying a single-chain antibody fragment (scFv) as the CD3-binding domain and introducing point mutations in the antigen-binding region (CDR) of the domain, binding affinity can be adjusted to a therapeutically appropriate level. This suppresses excessive binding to CD3 and minimizes the potential for immunotoxicity (cytokine release syndrome, CRS) due to T cell overactivation.

[0030] The antibody fragment of the present invention can further enhance tumor cell selectivity and binding stability by introducing optimized mutations to improve antigen binding affinity into the EpCAM nanobody.

[0031] In addition, these antibody fragments maintain or even increase antigen binding affinity to EpCAM and CD3 even in an acidified tumor microenvironment (pH 5.6 to 6.7), so stable antibody activity is possible even in a solid tumor environment and can provide improved therapeutic efficacy.

[0032]

[0033] 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.

[0034]

[0035] 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.

[0036] Figure 2 is a graph showing the results of measuring the binding affinity (Kd) for hEpCAM of antibody fragments manufactured through Examples 1, 2, and 5 of the present invention. 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)).

[0037] Figure 3 is a graph showing the results of measuring the binding affinity (Kd) of antibody fragments according to Examples 1, 2, and 5 of the present invention to 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, nM), and the Y-axis represents the absorbance (OD450 nm). The Kd value

[0038] The antibody fragment according to Example 1 was measured at about 4.39 nM, the antibody fragment according to Example 2 was measured at about 83.7 nM, and the antibody fragment according to Example 5 was measured at about 38.2 nM.

[0039] Figure 4 is a graph showing the results of flow cytometry analyzing the ability of antibody fragments according to Examples 1, 2, and 5 of the present invention to bind to cells expressing antigens. The analysis was performed on CD3-expressing Jurkat cells (A) and EpCAM-expressing cancer cell lines (MCF7, SW480, HCC827, Hep3B, NCI-H358; B to F). 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 5 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.

[0040] 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 antibody fragments according to Example 1 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 fragments.

[0041] 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 were applied according to Examples 1, 3, and 4 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 cell killing activity equivalent to or enhanced compared to the WT, and no cell killing was observed in PBMC, demonstrating selective anticancer effect and immune safety.

[0042] Figure 7 is a graph showing the change in tumor volume of antibody fragments according to Examples 1, 2, and 5 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.

[0043] Figure 8 shows tumor weights and photographs of extracted tumor tissues after administration of antibody fragments according to Examples 1, 2, and 5 of the present invention in a BALB / c nude mouse model. In the left graph, the X-axis represents the administration group (Control, Example 2, Example 1, Example 5), and the Y-axis represents the tumor weight (tumor weight, g). After transplanting tumor cells (NCI-H358), antibody fragments or the control group (PBS) were administered, and after the treatment was completed, the tumors were extracted and their weights were measured. As a result of the analysis, compared to the control group, the tumor weights were reduced in all antibody fragment administration groups according to Examples 1, 2, and 5. In the right photograph, the reduction in the size and weight of the extracted tumor tissues in each group is visually confirmed, and in particular, the most significant tumor inhibition effect was observed in the antibody fragment administration groups according to Examples 1 and 5.

[0044] Figure 9 is a graph showing the change in tumor size of antibody fragments according to Examples 1, 2, and 5 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 5, and in particular, the antibody fragment according to Example 5 showed the highest tumor inhibition effect.

[0045] 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, 2, and 5 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, an increase in body weight was observed in all groups, and in the antibody fragment administration groups according to Examples 1, 2, and 5, no decrease in body weight was observed, but rather an increase in body weight was confirmed. This suggests that the antibody fragment has low toxicity and excellent biosafety.

[0046] Fig. 11 is a graph showing the results of ELISA analyzing the antigen binding capacity according to the pH change of the antibody fragments according to Example 1 (Fig. 11(a), (d)), Example 2 (Fig. 11(b), (e)), and Example 5 (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 shows the treatment conditions (BLANK, 2nd, pH 5.8, pH 6.5, pH 7.4), and the Y-axis shows 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 5, 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.

[0047] Figure 12 shows the SDS-PAGE results analyzing the expression of proteins with point mutations (E46A, Q109A, R115A) introduced into the EpCAM nanobody. All mutant proteins appear as a single band at approximately 15-20 kDa, confirming stable expression despite the mutations.

[0048] Figure 13 shows the SDS-PAGE results analyzing the expression of proteins with point mutations (Y101A, Y101F, R180A, R180K, K187A, K187R) introduced into CD3 scFv. All mutant proteins appear as a single band at approximately 25-39 kDa, confirming that they are stably expressed despite the mutations.

[0049] Figure 14 shows SDS-PAGE results analyzing various clones for the Y101F and K187R mutants. Similar expression patterns were observed across multiple clones, suggesting that these mutants exhibit superior expression and reproducibility.

[0050] Figure 15 is a graph showing the results of ELISA analyzing the antigen binding affinity of the EpCAM nanobody point mutant of the present invention to the EpCAM antigen. The X-axis represents antibody concentration (μg / ml), and the Y-axis represents absorbance (OD₄50nm). The changes in the binding affinity of the mutants were confirmed compared to the wild-type.

[0051] Figure 16 is a graph showing the results of ELISA analyzing the antigen-binding ability of the antibody fragment of the present invention to the CD3 antigen. The X-axis represents antibody concentration (nM), the Y-axis represents absorbance (OD₄50nm), and the Kd value of each antibody was derived to confirm the difference in binding ability. The names of the CD3 binding domain mutations of the antibody fragments shown in the graph are as follows from the left: CD3 WT, CD3 Y101A, CD3 Y101F, CD3 R180A, CD3 R180K, CD3 K187A, CD3 K187R.

[0052] Figure 17 is a graph showing the results of comparative analysis of cytotoxicity of antibody fragments according to Example 2 or 5 of the present invention and scFv-scFv control groups 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.

[0053]

[0054] The present invention relates to a bispecific antibody fragment comprising a nanobody that specifically binds to EpCAM and a single-chain antibody fragment (scFv) that binds to CD3, linked by a linker. In particular, it is designed to suppress T cell hyperactivation and modulate anticancer efficacy by inducing point mutations at specific amino acid residues in the CD3 binding domain.

[0055] In a preferred embodiment of the present invention, an antibody fragment having at least one point mutation introduced into the amino acid sequence of at least one of a nanobody and a single-chain antibody fragment to control antigen binding affinity was prepared, and the antibody fragment was produced in high yield in an Escherichia coli expression system and purified using an IMAC method. The antibody fragment selectively acts on EpCAM-positive tumor cells and can induce cytotoxicity mediated by CD3+ T cells.

[0056]

[0057] 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.

[0058] 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.

[0059]

[0060] definition

[0061] 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.

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

[0063] 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.

[0064] 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.

[0065] 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.

[0066] 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).

[0067] 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).

[0068] 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.

[0069] 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).

[0070] 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.

[0071] 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.

[0072] 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).

[0073] In this specification, “PatchDock” may refer to a program that predicts docking interactions and complex formation between molecules, such as protein-protein and protein-ligand. The program analyzes the 3D structure of two molecules to identify possible binding modes, thereby predicting how and where each molecule interacts.

[0074] In this specification, "Alanine scanning" may refer to a program that evaluates the contribution of specific amino acid residues in proteins to function or interactions by replacing them. The program identifies amino acids that play a key role in protein-protein interactions, replaces them with alanine, and investigates protein function. By evaluating the strength or presence of interactions, it is possible to determine which residues contribute to the interactions.

[0075]

[0076] 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.

[0077]

[0078] Hereinafter, the present invention will be described in detail.

[0079]

[0080] 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.

[0081] 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.

[0082] 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.

[0083] 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.

[0084] 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.

[0085] 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.

[0086] 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.

[0087] 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.

[0088] 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.

[0089] 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.

[0090] 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.

[0091] 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.

[0092] 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.

[0093] 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.

[0094] 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.

[0095] However, experiments using nanobodies as antibody-binding domains for CD3 have resulted in a number of problems.

[0096] 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.

[0097] 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.

[0098] 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.

[0099] 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.

[0100] 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.

[0101]

[0102] The bispecific antibody fragment of the present invention as described above has the following integrated advantages due to the technical characteristics of each component working in a complementary manner.

[0103] 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.

[0104] 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.

[0105] 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.

[0106] 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.

[0107] 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.

[0108] 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.

[0109] 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.

[0110] 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.

[0111] 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.

[0112] Furthermore, the linker's length (n value) can be precisely adjusted according to design conditions, thereby providing optimal distances and degrees of freedom between antibody domains, thereby securing molecular spacing suitable for immunological synapse formation. In particular, the present invention applied repeating units ranging from n=1 to 35.

[0113] 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.

[0114] Furthermore, the linker also favorably enhanced the folding stability of the expressed protein, preventing protein aggregation. These characteristics enable high-purity, high-yield production in an Escherichia coli (E. coli)-based expression system, and may also benefit the industrial productivity and quality control of antibody products.

[0115] 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.

[0116] 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.

[0117] 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.

[0118] 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.

[0119] 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.

[0120] 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.

[0121] 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.

[0122] 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.

[0123] 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.

[0124] 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.

[0125] 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.

[0126] 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.

[0127] 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.

[0128] 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).

[0129] 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.

[0130] 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 showed experimental results that their antigen binding affinity was maintained or even increased even under conditions of pH 5.6 to 6.7, preferably pH 5.8 to 6.5.

[0131] 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.

[0132] 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.

[0133] 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.

[0134] 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.

[0135] Specifically, when the antibody fragment of the present invention was administered to a mouse model transplanted with a tumor cell line expressing EpCAM and injected with immune cells, PBMC, a tumor size inhibition effect of up to 80% or more was observed compared to the control group. This tumor inhibition effect was superior to the case of using a conventional scFv / scFv-based bispecific antibody, and in particular, the binding affinity and immune cell induction function of the antibody were stably maintained even in the slightly acidic environment within the tumor, showing a continuous therapeutic effect.

[0136] 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.

[0137] 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.

[0138] 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.

[0139]

[0140] 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).

[0141] 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.

[0142] Accordingly, in order to optimize the antigen binding ability of the bispecific antibody fragment of the present invention, a precise structure-based analysis was performed on the antigen binding site (Complementarity Determining Region, CDR) of the nanobody and single-chain antibody fragment (scFv).

[0143] CDRs are key regions that determine the antigen-binding specificity and affinity of antibody domains, and are generally divided into CDR1, CDR2, and CDR3. In the present invention, amino acid residues that play a critical role in antigen binding were selected for each CDR region of an EpCAM-specific nanobody and a CD3-specific scFv through in silico structural prediction, antigen-antibody binding simulation (docking), and alanine scanning analysis.

[0144] For the scFv domain for CD3, the CDR-H3 and CDR-L3 regions were predicted to have a significant impact on antigen binding affinity, and mutagenesis experiments were designed targeting residues such as Tyrosine (Y), Arginine (R), and Lysine (K) located in these regions. In particular, CD3 is a sensitive immune activating receptor, so excessive binding affinity can cause immunotoxicity such as T cell overactivation and cytokine release syndrome (CRS), and therefore a strategy to finely lower or control the binding affinity to the antigen is necessary.

[0145] In the present invention, in order to adjust the antigen binding properties of an antibody fragment that dually specifically binds to EpCAM and CD3 to suit therapeutic purposes, structure-based point mutation design was performed on the antigen binding site (complementary determining region, CDR) of the antibody domain.

[0146] For nanobody domains for EpCAM, the length and charge distribution of the CDR3 region are known to affect antigen penetration and binding stability, and mutations aimed at enhancing binding affinity have been introduced to enhance selective binding to EpCAM antigens densely packed on the tumor cell surface.

[0147] Through this, the present invention defines specific amino acids capable of controlling binding affinity for each domain, and designs them to enable binding affinity control by replacing Glutamic acid (E), Arginine (R), Tyrosine (Y), etc. with Alanine (A), Phenylalanine (F), Lysine (K), etc.

[0148] Afterwards, through point mutation induction experiments, we confirmed an antibody fragment that satisfies both therapeutic efficacy and safety by demonstrating actual changes in binding affinity and immune cell activation effects.

[0149] For example, the following mutations can be introduced into the nanobody domain for EpCAM: E46A mutation in which the 46th glutamic acid (E) is substituted with an alanine (A), Q109A mutation in which the 109th glutamine (Q) is substituted with an alanine (A), and R115A mutation in which the 115th arginine (R) is substituted with an alanine (A).

[0150] In the present invention, in order to confirm the expression of the EpCAM nanobody point mutant protein, it was confirmed whether the antibody protein, in which point mutations (E46A, Q109A, R115A) were introduced into the EpCAM binding nanobody domain, were normally expressed through an E. coli expression system. The gene of each mutant was inserted into a pET-based vector and IPTG-induced expression was performed using the BL21 (DE3) E. coli strain.

[0151] The expressed protein was obtained in the form of inclusion bodies after centrifugation, and the obtained protein was analyzed by SDS-PAGE and confirmed as a single band with a molecular weight of approximately 15–20 kDa (Fig. 12). This indicates that all of the nanobody variants maintain structural stability and are expressed normally despite their mutations.

[0152] Through these results, it can be confirmed that the mutation design of the EpCAM nanobody domain proposed in the present invention does not adversely affect the expression of the antibody fragment.

[0153] Next, the present invention sought to quantitatively analyze the effect of point mutations introduced into the EpCAM binding nanobody of the present invention on antigen binding ability.

[0154] EpCAM antigen was coated on a 96-well plate, and each nanobody variant protein was diluted to a concentration of 200 μg / ml, 100 μg / ml, 10 μg / ml, and 1 μg / ml and treated with the antigen. After the reaction, the plate was washed with PBS to remove nonspecific binding, and an anti-His HRP-conjugated antibody was treated to detect the His-tag attached to the antibody. After inducing color development using TMB substrate, the reaction was stopped, and the absorbance was measured at a wavelength of 450 nm (OD₄50 nm).

[0155] As shown in Fig. 15, the mutants showed similar or higher binding affinity than wild-type EpCAM-VHH in most concentration ranges.

[0156] Through this, it was confirmed that the binding affinity to the EpCAM antigen could be strengthened through the mutation, and among them, it was confirmed that the E46A mutation was particularly effective in increasing antigen accessibility and permeability by substituting the negatively charged glutamic acid with the hydrophobic residue alanine.

[0157] In addition, in the present invention, in order to adjust the antigen binding properties of an antibody fragment that dually specifically binds to EpCAM and CD3 to suit therapeutic purposes, a structure-based point mutation design was performed on the antigen binding site (complementary determining region, CDR) of the antibody domain.

[0158] For single-chain antibody fragment domains against CD3, a strategy to precisely lower or control binding affinity was required to prevent abnormal overactivation of T cells and the risk of developing cytokine release syndrome (CRS) due to excessive binding.

[0159] To this end, structure-based simulation, alanine scanning analysis, and amino acid substitution design were performed on the amino acids contained in the CDR region of each domain. In this process, amino acid residues predicted to have functional effects were selected, focusing on interaction factors such as charge, hydrophobicity, and hydrogen bonding that contribute to the binding interface with the antigen.

[0160] Gene sequences containing substitution mutations at selected amino acid positions were synthesized, antibody fragment proteins were expressed and purified through an E. coli expression system, and the antigen binding affinity, cell binding affinity, T cell inducing ability, and biological activity of these mutant proteins were measured to evaluate the optimal mutation combination.

[0161] Specifically, in the present invention, the expression of various point mutants (Y101A, Y101F, R180A, R180K, K187A, K187R) introduced to control the antigen binding ability of a single-chain antibody fragment (scFv) that specifically binds to the CD3 antigen was confirmed.

[0162] The gene sequence corresponding to each mutant was cloned into a pET expression vector and then transformed into BL21(DE3) E. coli cells by heat shock. 0.5 mM IPTG was then added to induce expression for 4 h. The expressed protein was analyzed by 12% SDS-PAGE after cell lysis.

[0163] As a result, as shown in Figure 13, a protein band was clearly observed at a molecular weight of approximately 30 to 35 kDa in the wild-type and each point mutant Lane, indicating that the corresponding mutant scFv protein was successfully expressed by IPTG induction.

[0164] These results confirm that the point mutations in the CD3-binding scFv antibody fragment do not adversely affect the expression and structural stability.

[0165] Next, in this experimental example, the antigen-binding affinity (Kd) of scFv antibody fragments that specifically bind to CD3 was evaluated. For the analysis, ELISA plates were coated with human CD3 (hCD3) antigen at 100 ng / well using coating buffer (Biolegend, 421701) at 4°C overnight. The plates were reacted with 1% bovine serum albumin (BSA) in wash buffer (Biolegend, 421601) at room temperature for 2 hours.

[0166] Afterwards, the plate was washed by diluting the 20X wash buffer into 1X wash buffer, and the purified ALB101 was sequentially diluted in 1% BSA blocking buffer and added to the plate (200 μl / well).

[0167] Afterwards, the plate was incubated at room temperature for 1 hour, washed with 1X wash buffer, and HRP-conjugated mouse monoclonal anti-his (1:5,000; Abcam) in 1% BSA blocking buffer was added to the plate (100 μl / well).

[0168] After this, after culturing at room temperature for 1 hour, the plate was washed, and TMB solution was mixed 1:1 and added to the plate (100 ㎕ / well), reacted for about 5-10 minutes in a light-shielded state, and 2N H2SO4 stop solution (50 ㎕ / well) was added, and the absorbance of the well was measured at 450 nm.

[0169] Based on the absorbance values ​​and antibody concentrations, the binding affinity (Kd) of each antibody fragment to the antigen was calculated using a nonlinear regression curve (simple binding model).

[0170] The results of the above binding affinity calculation are as shown in Fig. 16.

[0171] As illustrated in Figure 16, the wild-type scFv exhibited high binding affinity at high concentrations, while most mutants exhibited increasing binding affinity with increasing concentration. Among them, the Y101F and K187R mutants exhibited appropriately lower binding affinity compared to the wild-type, while maintaining gentle binding curves.

[0172] 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).

[0173] In particular, considering the expression level, clarity of the band, and reproducibility between repeated experiments, the K187R and Y101F mutant antibodies were selected as final candidates, and these two mutants were used as representative antibodies for future antigen binding affinity evaluation, cell binding ability, and in vivo activity analysis.

[0174] The above point mutations functioned as key sites that each influenced the folding stability, antigen specificity, binding affinity, and non-specific binding inhibition of the antibody domain, and the effects of these mutations were quantitatively demonstrated in actual binding assay results (WELISA, flow cytometry, cytotoxicity tests, etc.). Through this, the antibody fragment of the present invention was implemented with a structure that could simultaneously secure improved binding stability, tumor specificity, and immune cell activation regulation ability compared to the wild-type antibody.

[0175] In conclusion, the results of actual experiments based on the above compounds confirmed that the antibody fragment optimized through the above mutations can suppress the possibility of CRS occurrence by maintaining or improving the antigen binding affinity to EpCAM while lowering the binding affinity to CD3 compared to the existing BiTE antibody.

[0176] At the same time, the mutant antibody exhibits a characteristic in which binding affinity is maintained or even increased within the tumor microenvironment, thereby ensuring the sustainability and stability of the therapeutic effect. Therefore, the CDR optimization process of the present invention can secure both therapeutic efficacy and safety by balancing the antigen binding affinity and immune activation ability of the antibody fragment.

[0177]

[0178] As described above, the present invention relates to a bispecific antibody fragment in which a nanobody (domain antibody) that specifically binds to EpCAM (Epithelial Cell Adhesion Molecule) and a single-chain antibody fragment (single-chain variable fragment, scFv) that specifically binds to CD3 are connected by a linker, and specifically, the amino acid sequences of the nanobody and single-chain antibody fragment may be characterized in that at least one amino acid sequence among the EpCAM-binding nanobody amino acid sequence corresponding to SEQ ID NO: 2 and the CD3-binding single-chain antibody fragment amino acid sequence corresponding to SEQ ID NO: 4 is introduced with one or more point mutations for controlling antigen binding ability.

[0179] More specifically, for example, various mutant sequences such as SEQ ID NO: 7, 8, 10, 11, 12, 13 or 14 can be applied to the CD3-binding single-chain antibody fragment, and these sequences are designed to maintain or enhance binding affinity to the CD3 antigen while preventing non-specific reactions or excessive T cell activation, thereby contributing to increasing the stability and selectivity of the T cell-mediated cytotoxic response.

[0180] Meanwhile, a mutant sequence such as SEQ ID NO: 9, 15 or 16 can be applied to the EpCAM binding nanobody, and these sequences can increase binding affinity and tumor selectivity for EpCAM expressing cancer cells, thereby improving cancer cell targeting accuracy and immune cell induction efficiency.

[0181] In certain embodiments, point mutations may be introduced into both the CD3-binding single-chain antibody fragment and the EpCAM-binding nanobody, for example, the mutant sequences of SEQ ID NO: 7 and SEQ ID NO: 9, respectively. Such antibodies can be structurally optimized to maximize tumor-specific T cell activity and reduce systemic toxicity by simultaneously enhancing dual binding specificity for the antigen.

[0182] In addition, the nanobody and single-chain antibody fragment, which are components of the bispecific antibody fragment, are connected by a linker, and in the present invention, may include a repeating sequence (e.g., SEQ ID NO: 5) that enables flexible binding, or more specifically, may be composed of a linker sequence corresponding to SEQ ID NO: 6. This linker structure may be designed to secure an appropriate three-dimensional arrangement and stability while maintaining the independent functions of the two antibody fragments.

[0183] Such bispecific antibody fragments can be prepared alone as pharmaceutical compositions.

[0184] Specifically, 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, wherein the bispecific antibody fragment sequence is characterized in that at least one point mutation is introduced to control antigen binding affinity in at least one of a nanobody sequence comprising SEQ ID NO: 2 and a single-chain antibody fragment sequence comprising SEQ ID NO: 4.

[0185] The above pharmaceutical composition may be formulated as an intravenous injection, subcutaneous injection, etc. by being prepared together with a pharmacologically acceptable carrier and auxiliary ingredients. The composition of the present invention may be useful as a drug for preventing or treating various solid cancers in which EpCAM is overexpressed (liver cancer, colon cancer, pancreatic cancer, breast cancer, stomach cancer, lung cancer, prostate cancer, ovarian cancer, cervical cancer, esophageal cancer, bladder cancer, etc.).

[0186] In addition, in the case of the present invention, a method for preventing or treating cancer can be provided, which comprises administering an effective amount of a bispecific antibody fragment to a cancer patient, wherein the bispecific antibody fragment is a bispecific antibody fragment in which a nanobody (domain antibody) that specifically binds to EpCAM and a single-chain antibody fragment (scFv) that specifically binds to CD3 are connected by a linker, and at least one point mutation for controlling antigen binding ability is introduced into at least one of a nanobody sequence including SEQ ID NO: 2 and a single-chain antibody fragment sequence including SEQ ID NO: 4.

[0187] 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.

[0188] The above antibody fragment can inhibit cancer cell proliferation and reduce tumor size by activating CD3-positive T cells and inducing apoptosis of EpCAM-positive cancer cells. This therapeutic effect can be confirmed through Experimental Examples 3, 4, and 5 below.

[0189] As described above, a bispecific antibody fragment in which a nanobody (domain antibody) that specifically binds to EpCAM of the present invention and a single-chain antibody fragment (scFv) that specifically binds to CD3 are connected by a linker, wherein at least one point mutation for controlling antigen binding ability is introduced into at least one of the nanobody sequence including the following sequence number 2 and the single-chain antibody fragment sequence including the following sequence number 4, can be utilized for the purpose of treating cancer.

[0190]

[0191] Below, the compound of the present invention is described in detail through specific examples and experimental examples.

[0192]

[0193] Example 1. Preparation of antibody fragments containing EpCAM nanobody and CD3 scFv

[0194] Example 1-1. Sequence design of EpCAM nanobody and CD3 scFv domain

[0195] 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.

[0196] 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.

[0197] 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.

[0198]

[0199] 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)

[0200] Similarly, a CD3-binding single-chain antibody fragment (scFv) was designed based on the sequence of an existing CD3 antibody. The scFv comprises a variable heavy (VH) chain and a variable light (VL) chain linked in a continuous single chain, and has the property of selectively binding to the CD3 antigen, which induces T cell activation. Sequence number 3 corresponds to the DNA sequence of the CD3-binding single-chain antibody fragment (scFv), and sequence number 4 corresponds to its amino acid sequence.

[0201]

[0202] 서열번호 3 (Wild Type_CD3_scFv_DNA)5'-GATATTAAACTGCAGCAGAGTGGCGCAGAACTGGCCCGTCCGGGTGCGAGCGTGAAAATGAGCTGCAAAACTAGCGGTTATACCTTTACACGTTATACCATGCATTGGGTTAAGCAGCGTCCAGGTCAGGGCCTGGAATGGATTGGTTATATTAATCCGAGCCGTGGCTATACCAATTATAATCAGAAATTTAAAGATAAAGCAACCCTGACCACCGATAAAAGTAGTAGCACCGCCTATATGCAGCTGAGCAGCCTGACCAGCGAAGATAGCGCAGTTTATTATTGTGCACGTTATTATGATGATCATTATTGTCTGGATTATTGGGGTCAGGGCACCACCCTGACCGTTAGCTCTGTTGAAGGTGGTAGCGGCGGTTCTGGTGGTTCAGGCGGTAGTGGTGGCGTGGATGATATTCAGCTGACCCAGTCACCGGCCATTATGAGCGCAAGTCCGGGTGAAAAAGTTACCATGACCTGTCGTGCAAGTAGCTCAGTGAGCTATATGAATTGGTATCAGCAGAAAAGCGGTACCTCACCGAAACGTTGGATTTATGATACCAGTAAAGTTGCAAGTGGCGTTCCGTACCGCTTTAGTGGCAGCGGTAGCGGCACCAGCTATTCCCTGACGATCAGCTCTATGGAAGCAGAAGATGCCGCCACCTATTATTGCCAGCAGTGGAGCAGCAATCCGCTGACCTTTGGTGCAGGTACCAAACTGGAACTGAAA-3’서열번호 4 (WildType_CD3_scFv_Protein)(n-ter)DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWG QGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK(c-ter)

[0203] Example 1-2. EpCAM nanobody-CD3 scFv bispecific antibody fragment gene synthesis and expression vector production 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.

[0204] 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.

[0205] 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.

[0206] The linker may consist of a connecting sequence located after the C-terminus of the nanobody and before the N-terminus of the scFv.

[0207]

[0208] SEQ ID NO: 5 (n=1 linker)(n-ter)GGGGS(c-ter)SEQ ID NO: 6 (n=3 linker)(n-ter)GGGGSGGGGSGGGGS(c-ter)

[0209] 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 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. The EpCAM nanobody and CD3 scFv sequences were inserted into the 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 E. coli.

[0210]

[0211] Example 1-3. Expression and purification of antibody fragments using Escherichia coli (E. coli)

[0212] 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.

[0213] 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.

[0214] 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.

[0215] 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).

[0216] 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.

[0217]

[0218] Example 2. Controlling the length of the linker

[0219] 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.

[0220]

[0221] Example 3. Optimization design through genetic mutation

[0222] In Example 3, point mutations were introduced to the amino acid sequence at specific positions to control the antigen binding affinity and biological activity of the EpCAM nanobody and CD3 scFv antibody fragment.

[0223] 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 modulate affinity.

[0224] In this example, antibody fragment of Example 3 (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).

[0225] The amino acid sequence of the CD3 scFv of this example manufactured through the above mutation corresponds to SEQ ID NO: 7.

[0226]

[0227] sequence number 7(n-ter)DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYFDDHYCLDYWGQGTTLTVSSVE GGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK(c-ter)

[0228] Example 4.

[0229] Example 4 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).

[0230] The amino acid sequence of the CD3 scFv of this example manufactured through the above mutation corresponds to SEQ ID NO: 8.

[0231]

[0232] sequence number 8(n-ter)DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVE GGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSRVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK(c-ter)

[0233] Example 5.

[0234] Example 3 antibody fragment (ALB101-M1) was prepared in the same manner as Example 2, except that the 46th amino acid sequence of the EpCAM nanobody corresponding to sequence number 2 was mutated from Glutamic acid (E) to Alanine (A), and the 101st amino acid sequence of the CD3 scFv corresponding to sequence number 4 was mutated from Tyrosine (Y) to Phenylalanine (F).

[0235] The amino acid sequence of the EpCAM nanobody of this example manufactured through the above mutation corresponds to SEQ ID NO: 9, and the CD3 scFv amino acid sequence corresponds to SEQ ID NO: 7.

[0236]

[0237] Sequence number 9(n-ter)QVQLVQSGGGSVQGGASLRLSCAASGGERNNYCVAWFRQAPGKERAVAAISRAASGAQTTTKYYVDSVKGRFTISQDTKNTATVYLQMNSHKPEDTAYCTAKAKIYPPQCTGISRTIDYRGQGTQVTVSS(c-ter)

[0238] Experimental Example 1. Confirmation of Expression of Point Mutant Proteins

[0239] In this experimental example, the effects of various point mutations (Y101F, K187R) introduced into a single-chain antibody fragment (scFv) that specifically binds to the CD3 antigen on the expression level were confirmed.

[0240] Each point-mutated antibody fragment was cloned into a pET-based expression vector and transformed into BL21(DE3) E. coli by heat shock. Expression was then induced by the addition of IPTG (final concentration 0.5 mM) for 15 h at 20°C. Experimental groups were set up with at least four replicate clones (#1–#8) for each of the Y101F and K187R mutations.

[0241] The level of expressed antibody protein was assessed by SDS-PAGE analysis, and as shown in Figure 14, a major band corresponding to approximately 40–45 kDa was observed in both antibody fragments introducing the K187R and Y101F mutations. This was consistent with the molecular weight of the bispecific antibody fragment expressed in the E. coli system, confirming that the point mutations did not adversely affect expression and structural stability.

[0242]

[0243] Experimental Example 2. Evaluation of binding capacity at the cellular level (flow cytometry analysis)

[0244] 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.

[0245] 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 2 and Example 5 to cell surface antigens was evaluated.

[0246] 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 to 2 and Example 5, 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.

[0247] 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.

[0248] As a result of the analysis, all antibody fragments according to Examples 1 to 2 and Example 5 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).

[0249] 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.

[0250]

[0251] Experimental Example 3. Evaluation of apoptotic activity of human cancer cell lines in vitro.

[0252] In this experimental example, the antibody fragments manufactured through Examples 2 to 4 were used to evaluate the T cell-mediated apoptosis effect in an in vitro environment.

[0253] 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).

[0254] 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).

[0255] 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.

[0256] Additionally, the efficacy of antibody fragments with point mutations introduced into the CD3 scFv region (Examples 3 and 4) 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 antibody's tumoricidal capacity was maintained despite the introduction of mutations that reduce the risk of excessive T cell activation and cytokine release syndrome (CRS).

[0257] 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).

[0258]

[0259] Experimental Example 4. In vivo evaluation of anticancer efficacy of a bispecific antibody fragment.

[0260] 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, 2, and 5.

[0261]

[0262] Experimental Example 4-1. Evaluation of anticancer efficacy in a BALB / c nude mouse model

[0263] 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 similar tumor sizes 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 5, respectively.

[0264] 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).

[0265] 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, at 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 5 showed a tumor volume reduction effect of approximately 51.64%.

[0266] Additionally, after the experiment, tumor tissues were extracted and weighed, and tumor weights were significantly reduced in the antibody fragment-treated group compared to the control group (Fig. 8). In particular, the antibody fragment-treated groups according to Examples 1 and 5 demonstrated the greatest anticancer effects, and the antibody fragment-treated group in Example 5, which had a point mutation, exhibited the greatest effect.

[0267]

[0268] Experimental Example 4-2. Evaluation of Anticancer Efficacy in a Humanized Mice Model

[0269] 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, 2, and 5. 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.

[0270] 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 5 showed an 80% decrease in tumor size (Fig. 9).

[0271]

[0272] Experimental Example 4-3. Sintering

[0273] In conclusion, the antibody fragments according to Examples 1, 2, and 5 exhibited excellent anticancer efficacy in both the BALB / c nude mouse model and the humanized mouse model, and in particular, the antibody fragment of Example 5 including a CD3 binding affinity regulatory mutation (point mutation) was confirmed to exhibit the best 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.

[0274]

[0275] Experimental Example 5. In vivo safety evaluation of bispecific antibody fragments.

[0276] In this example, animal experiments were performed using a BALB / c nude mouse model to evaluate the safety of antibody fragments manufactured through Examples 1, 2, and 5.

[0277] 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, 2, and 5 were administered under the same conditions as the anticancer efficacy evaluation in Example 4-1. Safety evaluation was performed based on body weight changes.

[0278] As a result of body weight measurement, no decrease in body weight was observed in any of the antibody treatment groups of Experimental Examples 1, 2, and 5 after antibody injection. Rather, the body weight increased by 4.03%, 3.67%, and 4.23%, respectively, indicating that there were no signs of toxicity. In particular, it was confirmed that Experimental Example 5, in which point mutation occurred, had the lowest toxicity (Fig. 10).

[0279]

[0280] Experimental Example 6. Evaluation of Antigen Binding Ability of Antibody Fragments According to pH Changes

[0281] 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, 2 and 5 of the present invention was analyzed under various pH conditions.

[0282] 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).

[0283] 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, 2, and 5 were processed, and absorbance was measured at 450 nm using a His-tag detection antibody and TMB substrate.

[0284] As a result of EpCAM antigen binding affinity analysis, the antibody fragments according to Examples 1 and 5 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.

[0285] Similar results were observed in the CD3 antigen binding assay. The binding affinity of the antibody fragments according to Examples 1 and 5 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.

[0286] These results suggest that the antibody fragments according to Examples 1 and 5 maintain stable binding affinity, or even exhibit enhanced antigen binding affinity, even under the slightly acidic conditions of the tumor microenvironment, and are highly likely to exhibit enhanced therapeutic efficacy in solid tumor environments. (Figure 11)

[0287]

[0288] Experimental Example 7. Comparative Evaluation with scFv-scFv Antibody Fragments

[0289] 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 (Hep3B, NCI-H358) with PBMC or general cells (HEK293) with PBMC.

[0290] 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 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 17)

[0291]

[0292] 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 can provide significantly superior technical effects compared to existing technologies in terms of antigen binding ability, immune cell induction activity, tumor selectivity, structural stability, and productivity.

[0293] In particular, the antibody fragment of the present invention can selectively introduce point mutations into the antigen-binding region (CDR). Through such point mutations, antigen binding affinity can be appropriately controlled, and in particular, when binding to CD3 is selectively weakened, the potential for immunotoxicity, such as immune cell hyperactivation and cytokine release syndrome (CRS), can be effectively suppressed.

[0294] A total of eight amino acid substitution mutations (Y101A, Y101F, R180A, R180K, K187A, K187R, K187E) can be made in the CD3 binding domain, and despite the above mutations, there is no problem in antibody expression, confirming that the binding affinity to CD3 can be controlled through the above mutations. In particular, it was confirmed that the Y101F or K187R mutation can appropriately weaken the CD3 binding affinity while maintaining high anticancer activity.

[0295] In addition, we were able to demonstrate that the E46A, Q109A, and R115A mutations can induce enhanced antigen binding affinity for EpCAM nanobody, and that these mutations do not interfere with antibody expression.

[0296] The amino acid sequences of CD3 scFv produced through the above Y101A, R180A, R180K, K187A and K187E mutations correspond to SEQ ID NOs: 10 to 14, respectively, and the amino acid sequences of EpCAM nanobody produced through the above Q109A and R115A mutations correspond to SEQ ID NOs: 15 and 16, respectively.

[0297]

[0298] Sequence number, mutation type, amino acid sequence, SEQ ID NO: 10 CD3, Y101A (n-ter), DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYADDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK (c-ter), SEQ ID NO: 11 CD3 R180A(n-ter)DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVE GGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKAWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK(c-ter)SEQ ID NO. 12CD3 R180K(n-ter)DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVE GGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKKWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK(c-ter)SEQ ID NO. 13CD3K187A(n-ter)DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSAVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK(c-ter)서열번호 14CD3 K187E(n-ter)DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSEVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK(c-ter)서열번호 15EpCAM Q109A(n-ter)QVQLVQSGGGSVQGGASLRLSCAASGGERNNYCVAWFRQAPGKEREVAAISRAASGAQTTTKYYVDSVKGRFTISQDTKNTATVYLQMNSHKPEDTAYCTAKAKIYPPACTGISRTIDYRGQGTQVTVSS(c-ter)서열번호 16EpCAM R115A(n-ter)QVQLVQSGGGSVQGGASLRLSCAASGGERNNYCVAWFRQAPGKEREVAAISRAASGAQTTTKYYVDSVKGRFTISQDTKNTATVYLQMNSHKPEDTAYCTAKAKIYPPQCTGISATIDYRGQGTQVTVSS(c-ter)

[0299] In particular, the antibody fragment containing both the E46A mutation of the EpCAM nanobody and the Y101F mutation of the CD3 scFv simultaneously exhibited the best binding stability, cell killing efficacy, and immunotoxicity inhibition effect in both in vitro cell experiments and in vivo animal models, and showed the characteristic of maintaining or increasing binding strength even under the acidic conditions of the tumor microenvironment, and was evaluated as the most desirable structure in terms of therapeutic efficiency and safety. In addition, the antibody fragment of the present invention minimizes steric interference between the nanobody and scFv domains through a linker composed of a Gly-Gly-Gly-Gly-Ser repeat sequence ((GGGGS)n), and enables each binding domain to act independently on the antigen, thereby enabling effective immune synapse formation between immune cells and tumor cells. At this time, the most desirable repeat sequence was found to be 3.

[0300] In vitro testing demonstrated that the antibody fragment of the present invention exhibited high antigen-binding affinity and T-cell-induced cytotoxicity. Furthermore, in vivo animal models demonstrated superior tumor suppression and immunotoxicity inhibition compared to existing bispecific antibodies. In particular, the composite variant described in Example 5 demonstrated both the highest tumor suppression and biosafety, and was identified as an optimal structure with high potential for industrial application.

[0301] In conclusion, the bispecific antibody fragment of the present invention is a bispecific antibody platform for the treatment of solid cancer that satisfies structural stability, target specificity, immunological safety, production efficiency, and long-term stability, and it was confirmed that it has technological excellence that can simultaneously solve two tasks: anticancer efficacy and minimization of side effects.

[0302]

[0303] The bispecific antibody fragment according to the present invention is useful as an immunotherapy that maintains anticancer activity while minimizing immunotoxicity through modulation of CD3 binding affinity. In particular, it can be developed into an immunotherapy that simultaneously targets EpCAM and CD3 for various cancer types, including solid tumors. Furthermore, its E. coli-based production process enables low-cost, high-efficiency industrial production.

[0304] Therefore, the present invention has wide industrial applicability in the fields of antibody drugs and immuno-oncology drugs.

Claims

1. A bispecific antibody fragment consisting of a nanobody that specifically binds to EpCAM and a single-chain antibody fragment (scFv) that specifically binds to CD3. The above nanobody comprises an amino acid sequence corresponding to sequence number 2, The above single chain antibody fragment comprises an amino acid sequence corresponding to sequence number 4, A bispecific antibody fragment characterized in that at least one amino acid sequence among the above nanobody and single-chain antibody fragments has one or more point mutations introduced to control antigen binding affinity.

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 comprises a sequence of SEQ ID NO:

6.

4. In paragraph 1, A point mutation was introduced into the CD3-binding single-chain antibody fragment corresponding to sequence number 4. A bispecific antibody fragment characterized by comprising an amino acid sequence corresponding to sequence number 7, 8, 10, 11, 12, 13 or 14.

5. In paragraph 1, A point mutation was introduced into the EpCAM binding nanobody corresponding to sequence number 2. A bispecific antibody fragment characterized by comprising an amino acid sequence corresponding to sequence number 9, 15 or 16.

6. In paragraph 1, A point mutation was introduced into the CD3-binding single-chain antibody fragment corresponding to sequence number 4. A bispecific antibody fragment characterized by comprising an amino acid sequence corresponding to sequence number 7 or 8.

7. In paragraph 1, A point mutation was introduced into the EpCAM binding nanobody corresponding to sequence number 2 and the CD3 binding single-chain antibody fragment corresponding to sequence number 4. A bispecific antibody fragment characterized by comprising amino acid sequences corresponding to sequence numbers 7 and 9.

8. A bispecific antibody fragment comprising a nanobody that specifically binds to EpCAM and a single-chain antibody fragment (scFv) that specifically binds to CD3, which are connected by a linker, A pharmaceutical composition for preventing or treating cancer, characterized in that at least one of the above nanobody and single-chain antibody fragments has one or more point mutations introduced to control antigen binding affinity.

9. In paragraph 8, 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.

10. A bispecific antibody fragment in which a nanobody (domain antibody) that specifically binds to EpCAM and a single-chain antibody fragment (scFv) that specifically binds to CD3 are connected by a linker. A bispecific antibody fragment characterized in that at least one point mutation is introduced to control antigen binding affinity in at least one of a nanobody sequence comprising sequence number 2 and a single-chain antibody fragment sequence comprising sequence number 4. A method for preventing or treating cancer, comprising administering an effective amount to a cancer patient.

11. A bispecific antibody fragment in which a nanobody (domain antibody) that specifically binds to EpCAM and a single-chain antibody fragment (scFv) that specifically binds to CD3 are connected by a linker. A bispecific antibody fragment characterized in that at least one point mutation is introduced to control antigen binding affinity in at least one of a nanobody sequence comprising sequence number 2 and a single-chain antibody fragment sequence comprising sequence number 4. For use in the treatment of cancer.

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