Bispecific antibody specifically binding to vista and MSLN and uses thereof

Abstract

The present invention relates to a bispecific antibody that specifically binds to VISTA and MSLN, and uses thereof. The bispecific antibody exhibits significant immuno-oncological activity and ADCC activity compared to a monospecific antibody, degrades target proteins through internalization activity, and possesses tumor-targeting ability, and thus can be advantageously used for the prevention or treatment of various cancers.

Description

Bispecific antibodies specifically binding to VISTA and MSLN and uses thereof

[0001] The present invention relates to a bispecific antibody that specifically binds to VISTA and MSLN and uses thereof.

[0002]

[0003] In the environment where cancer exists, various immune cells (T cells, NK cells, Tregs, myeloid-derived suppressor cells, dendritic cells, M2 macrophages, M1 macrophages, etc.) and normal cells form a tumor microenvironment (TME) together with cancer cells. Within this environment, cancer creates an environment that suppresses the patient's innate or adaptive immunity, thereby preventing the patient's immune system from attacking the cancer cells. Therefore, methods to kill cancer cells, such as blocking negative immune checkpoints such as PD-1, are being studied as a method to remove cancer from the TME environment.

[0004] Programmed death-1 (PD-1) and its ligands PD-L1 / PD-L2 are another immune negative checkpoint axis, and the PD-1 pathway impairs T cell responses and promotes Foxp3+ Treg induction in the periphery, thereby downregulating tumor-specific immune responses. Therefore, blocking the PD-L1 / PD-1 pathway together with other immunotherapies suppresses tumor progression. Meanwhile, VISTA (V-domain Ig suppressor of T cell activation) is a novel negative checkpoint ligand with homology to PD-L1 and exhibits a distinct expression pattern that suppresses T cell activation (Xing Huang et al., Journal of Hematology & Oncology, 2020;13(83)).

[0005] MSLN (mesothelin) is a 69-71 kDa precursor polypeptide and glycoprotein expressed on the cell surface, where it facilitates cell-to-cell adhesion and signal transmission. While low in normal tissues, overexpression has been observed in several solid tumors, including mesothelioma, pancreatic cancer, and ovarian cancer. Research targeting MSLN as an anticancer agent is ongoing.

[0006] Accordingly, the present inventors developed a bispecific antibody that specifically binds to the two proteins through correlation analysis of VISTA and MSLN expression, and confirmed that it has a more remarkable cancer prevention or treatment effect than a single antibody, thereby completing the present invention.

[0007]

[0008] An object of the present invention is to provide a bispecific antibody comprising a first antigen binding domain that specifically binds to VISTA (V-domain immunoglobulin suppressor of T cell activation); and a second antigen binding domain that specifically binds to MSLN (Mesothelin).

[0009] Another object of the present invention is to provide an isolated nucleic acid molecule encoding the bispecific antibody.

[0010] Another object of the present invention is to provide a recombinant expression vector comprising the nucleic acid molecule.

[0011] Another object of the present invention is to provide a host cell transformed with the recombinant expression vector.

[0012] Another object of the present invention is to provide a pharmaceutical composition comprising the bispecific antibody and a pharmaceutically acceptable carrier.

[0013] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer, comprising the bispecific antibody; the recombinant expression vector; or the host cell as an active ingredient.

[0014] Another object of the present invention is to provide a pharmaceutical composition for combination administration for the prevention or treatment of cancer, comprising the bispecific antibody and CAR-NK (chimeric antigen receptor natural killer) cells as active ingredients.

[0015] Another object of the present invention is to provide a pharmaceutical composition for combination administration for the prevention or treatment of cancer, comprising as active ingredients a first component comprising an antibody or an antigen-binding fragment thereof that specifically binds to VISTA; and a second component comprising an antibody or an antigen-binding fragment thereof that specifically binds to MSLN.

[0016]

[0017] To achieve the above purpose, the present invention provides a bispecific antibody comprising a first antigen binding domain that specifically binds to VISTA (V-domain immunoglobulin suppressor of T cell activation); and a second antigen binding domain that specifically binds to MSLN (Mesothelin).

[0018] In one embodiment of the present invention, the bispecific antibody may comprise an Fc domain.

[0019] In one embodiment of the present invention, the Fc domain may be selected from the group consisting of human IgG1, IgG2, IgG3 and IgG4, but is not limited thereto.

[0020] In one embodiment of the present invention, the first antigen binding domain and the second antigen binding domain may each be independently selected from the group consisting of a Fab fragment, a Fab' fragment, an F(ab')2 fragment, an Fv fragment, a disulfide-linked Fv (dsFv), a single-chain Fv (scFv), a single-chain Fab (scFab), a diabody, a minibody, and combinations thereof, but are not limited thereto.

[0021] In one embodiment of the present invention, the bispecific antibody may be, but is not limited to, a two-in-one antibody or a dual-functional Fab (DAF).

[0022] In one embodiment of the present invention, the bispecific antibody may be included as part of a multispecific antibody.

[0023] In one embodiment of the present invention, either the first antigen binding domain or the second antigen binding domain may be linked directly or via a peptide linker to the N-terminus of the Fc domain, and wherein the first antigen binding domain may be linked directly or via a peptide linker to the N-terminus or C-terminus of the second antigen binding domain.

[0024] In one embodiment of the present invention, the first antigen binding domain and the second antigen binding domain may each be independently linked to the N-terminus of the Fc domain, either directly or via a peptide linker, wherein the first antigen binding domain is a Fab fragment and the second antigen binding domain is a scFv fragment; or the first antigen binding domain is a scFv fragment and the second antigen binding domain is a Fab fragment.

[0025] In one embodiment of the present invention, the bispecific antibody may be an afucosylated bispecific antibody.

[0026] In one embodiment of the present invention, the first antigen binding domain may include a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 comprising an amino acid sequence of SEQ ID NO: 2, and a heavy chain CDR3 comprising an amino acid sequence of SEQ ID NO: 3; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence of SEQ ID NO: 4, a light chain CDR2 comprising an amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 comprising an amino acid sequence of SEQ ID NO: 6.

[0027] In one embodiment of the present invention, the second antigen binding domain may include a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence of SEQ ID NO: 7, a heavy chain CDR2 comprising an amino acid sequence of SEQ ID NO: 8, and a heavy chain CDR3 comprising an amino acid sequence of SEQ ID NO: 9; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence of SEQ ID NO: 10, a light chain CDR2 comprising an amino acid sequence of SEQ ID NO: 11, and a light chain CDR3 comprising an amino acid sequence of SEQ ID NO: 12.

[0028] In one embodiment of the present invention, the first antigen binding domain may include a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 13 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 14.

[0029] In one embodiment of the present invention, the second antigen binding domain may include a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 15 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 16.

[0030] In one embodiment of the present invention, the bispecific antibody may have immuno-anticancer activity, may have ADCC (Antibody-Dependent Cellular Cytotoxicity) activity, may degrade a target protein through internalization activity, and may have tumor-targeting ability.

[0031] The present invention also provides an isolated nucleic acid molecule encoding the bispecific antibody.

[0032] Additionally, the present invention provides a recombinant expression vector comprising the nucleic acid molecule.

[0033] In addition, the present invention provides a host cell transformed with the recombinant expression vector.

[0034] In addition, the present invention provides a pharmaceutical composition comprising the bispecific antibody and a pharmaceutically acceptable carrier.

[0035] In addition, the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising the bispecific antibody; the recombinant expression vector; or the host cell as an active ingredient.

[0036] In one embodiment of the present invention, the cancer may be selected from the group consisting of malignant mesothelioma, pancreatic cancer, ovarian cancer, cholangiocarcinoma, biliary tract cancer, breast cancer, kidney cancer, stomach cancer, liver cancer, lung cancer, colorectal cancer, skin cancer, bladder cancer, testicular cancer, uterine cancer, cervical cancer, thyroid cancer, head and neck cancer, prostate cancer, non-small cell lung cancer, neuroblastoma, brain cancer, colon cancer, squamous cell carcinoma, melanoma, myeloma, lymphoma, and leukemia, but is not limited thereto.

[0037] In addition, the present invention provides a pharmaceutical composition for combination administration for the prevention or treatment of cancer, comprising the bispecific antibody and CAR-NK (chimeric antigen receptor natural killer) cells as active ingredients.

[0038] In addition, the present invention provides a pharmaceutical composition for combination administration for the prevention or treatment of cancer, comprising as active ingredients a first component comprising an antibody or an antigen-binding fragment thereof that specifically binds to VISTA; and a second component comprising an antibody or an antigen-binding fragment thereof that specifically binds to MSLN.

[0039] In one embodiment of the present invention, the first component and the second component may be formulated and administered simultaneously or sequentially.

[0040]

[0041] The bispecific antibody according to the present invention has remarkable immunostimulatory activity and ADCC activity compared to single antibodies, degrades target proteins through internalization activity, and has tumor targeting ability, so it can be usefully used for the prevention or treatment of various cancers.

[0042]

[0043] Figure 1 shows the results of analyzing the transcriptome-level expression correlation of VISTA and MSLN using the TCGA-Meso database.

[0044] Figure 2 shows the results of analyzing the transcript level expression correlation of VISTA and MSLN using the GEO database (GSE163722).

[0045] Figure 3 shows the results of analyzing the transcript level expression correlation of VISTA and MSLN using the GEO database (GSE51024).

[0046] Figure 4 shows the results of analyzing the expression patterns of VISTA and MSLN proteins in FFPE blocks of patients with epithelial-type malignant mesothelioma using immunohistochemistry.

[0047] Figure 5 shows the results of analyzing the expression patterns of VISTA and MSLN proteins in FFPE blocks of patients with sarcomatoid malignant mesothelioma using immunohistochemistry.

[0048] Figure 6 shows the results of confirming the expression levels of VISTA and MSLN in various malignant mesothelioma cell lines through FACS.

[0049] Figure 7 shows the results of confirming the expression levels of VISTA and MSLN in various ovarian cancer cell lines through FACS.

[0050] Figure 8 shows the results of confirming the expression levels of VISTA and MSLN in various pancreatic cancer cell lines through FACS.

[0051] Figure 9 shows the results of confirming the expression and purification of bispecific antibodies by form.

[0052] Figure 10 shows the results of confirming the binding ability of a bispecific antibody to a cell line according to pH.

[0053] Figure 11 shows the results of confirming the binding ability of a bispecific antibody to an antigen through immunoprecipitation.

[0054] Figure 12 shows the results of confirming the inhibitory ability of a bispecific antibody against an antigen.

[0055] Figure 13 shows the results of confirming the neutralizing ability of a bispecific antibody to a binding ligand according to pH.

[0056] Figure 14 shows the results of confirming the immune activation ability of a bispecific antibody through an SEB assay using human peripheral blood mononuclear cells (PBMC).

[0057] Figure 15 shows the results of confirming the ADCC efficacy of a bispecific antibody through an ADCC assay using the NK92MI-CD16a cell line.

[0058] Figure 16 shows the results of confirming the ADCC efficacy of a bispecific antibody through an ADCC assay using an improved NK cell line.

[0059] Figure 17a shows the results of confirming ADCC activity by co-administration of VISTA antibody (fixed) and various MSLN antibodies, and Figure 17b shows the results of confirming ADCC activity by co-administration of MSLN antibody (fixed) and various VISTA antibodies.

[0060] Figure 18 shows the results confirming the ADCC efficacy of a non-fucosylated bispecific antibody.

[0061] Figure 19a shows the results of confirming the internalization characteristics of a bispecific antibody through a ZAP assay, and Figure 19b shows the results of confirming the degree of target protein degradation through internalization of a bispecific antibody through a degradation assay.

[0062] Figure 20 shows the results of confirming tumor targeting ability by detecting CF750 fluorescence of a bispecific antibody in the NCI-H226 xenograft model.

[0063]

[0064] Hereinafter, the present invention will be described in detail.

[0065] The terms used in this invention have been selected from widely used, common terms, taking into account the functionality of the invention. However, these terms may vary depending on the intentions of those skilled in the art or the emergence of new technologies. Furthermore, in certain cases, terms may be arbitrarily selected, and in such cases, their meanings will be described in detail in the description of the relevant embodiments. Therefore, the terms used in this invention should not be defined simply as names, but rather based on their meanings and the overall content of the invention.

[0066] When the present invention is said to “include” a certain component or a certain step, this does not mean that other components or other steps are excluded, but rather that other components or other steps may be further included, unless specifically stated otherwise.

[0067]

[0068] The present invention provides a bispecific antibody that specifically binds to VISTA and MSLN, comprising a first antigen binding domain that specifically binds to VISTA (V-domain immunoglobulin suppressor of T cell activation); and a second antigen binding domain that specifically binds to MSLN (Mesothelin).

[0069] As used herein, the term "antibody" refers to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen, and includes antibody fragments (e.g., Fab, F(ab')2, and Fv), as well as single monoclonal antibodies (including agonist and antagonist antibodies), antibody compositions having polyepitopic specificity, as long as they exhibit the desired biological activity. In some embodiments, the antibody can be a monoclonal, polyclonal, chimeric, single-chain, tetra-specific or bi-agonist, simian, human, and humanized antibody, as well as active fragments thereof. Examples of active fragments of molecules that bind to known antigens include Fab, F(ab')2, scFv, and Fv fragments, including the products of Fab immunoglobulin expression libraries and epitope-binding fragments of any of the antibodies and fragments mentioned above. In some embodiments, an antibody may comprise an immunoglobulin molecule and an immunologically active portion of an immunoglobulin molecule, i.e., a molecule containing a binding site that immunospecifically binds to an antigen. The immunoglobulin may be of any type (IgG, IgM, IgD, IgE, IgA, and IgY) or class (IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or subclass of an immunoglobulin molecule. In one embodiment, the antibody may be a whole antibody or any antigen-binding fragment derived from a whole antibody. A typical antibody typically refers to a heterotetrameric protein comprising two heavy (H) chains and two light (L) chains. Each heavy chain is comprised of a heavy chain variable region (VH) and a heavy chain constant region. Each light chain is comprised of a light chain variable region (VL) and a light chain constant region. The VH and VL regions can be further subdivided into domains of hypervariable complementarity determining regions (CDRs) and more conserved regions called framework regions (FRs).Each variable domain (VH or VL) typically consists of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Within the variable regions of the light and heavy chains are binding regions that interact with antigen.

[0070] The term "bispecific antibody" of the present invention is an antibody having two different antigen binding sites and is a type of multispecific antibody.

[0071] In the present invention, the bispecific antibody may include an Fc domain, and the Fc domain may be selected from the group consisting of human IgG1, IgG2, IgG3, and IgG4, but is not limited thereto.

[0072] The bispecific antibodies of the present invention can be prepared using any bispecific antibody format or technique. For example, an antibody or fragment thereof having a first antigen-binding specificity can be functionally linked (e.g., by chemical coupling, genetic fusion, non-covalent association, or otherwise) to one or more other molecular entities, such as another antibody or antibody fragment having a second antigen-binding specificity, to produce a bispecific antibody. Specific exemplary bispecific formats that may be used in the context of the present invention include, but are not limited to, scFv-based or diabody bispecific formats, IgG-scFv fusions, dual variable domain (DVD)-Ig, quadromas, knob-into-hole, common light chains (e.g., common light chains with knob-into-holes, etc.), CrossMabs, CrossFabs, IgG1 / IgG2, two-in-one antibodies, dual-acting Fabs (DAFs), and Mab2 bispecific formats.

[0073] In the present invention, the first antigen binding domain and the second antigen binding domain may each independently be an antigen binding fragment selected from the group consisting of a Fab fragment, a Fab' fragment, an F(ab')2 fragment, an Fv fragment, a disulfide-linked Fv (dsFv), a single-chain Fv (scFv), a single-chain Fab (scFab), a diabody, a minibody, and a combination thereof, but are not limited thereto.

[0074] In the present invention, the bispecific antibody may be a two-in-one antibody or a dual-functional Fab (DAF), but is not limited thereto.

[0075] In the present invention, the bispecific antibody may be included as part of a multispecific antibody. A "multispecific antibody" is an antibody having multiple different antigen-binding sites, which can bind to multiple different antigens or epitopes simultaneously or sequentially.

[0076] The term "antigen binding fragment" or "antibody fragment" above includes a portion of an antibody that lacks some amino acids compared to the full-length chain, but is still capable of specifically binding to an antigen. Such fragments may be considered biologically active in that they specifically bind to a target antigen or can compete with other antibodies or antigen-binding fragments for binding to a specific epitope. In one aspect, such fragments include at least one CDR present in a full-length light or heavy chain, and in some embodiments, they include shorter heavy and / or light chains, or portions thereof. Such biologically active fragments may be produced by recombinant DNA techniques, or may be produced, for example, by enzymatic or chemical cleavage of an intact antibody.

[0077] In the present invention, the bispecific antibody may include a signal peptide for its production.

[0078] The term "signal peptide" refers to a short peptide present at the N-terminus of newly synthesized proteins that are sorted into the secretory pathway. Such signal peptides are well characterized in the art and are typically known to contain 16 to 30 amino acid residues, although they may contain more or fewer. A typical signal peptide consists of three regions: a basic N-terminal region, a central hydrophobic region, and a more polar C-terminal region. The central hydrophobic region contains 4 to 12 hydrophobic residues that anchor the signal sequence through the membrane lipid bilayer during the movement of the immature polypeptide. After initiation, the signal sequence is cleaved within the lumen of the ER by cellular enzymes commonly known as signal peptidases.

[0079] In the present invention, the signal peptide may be composed of an amino acid sequence of SEQ ID NO: 17 (METDTLLLWVLLLWVPGSTG) or may include another signal sequence known in the art (e.g., see Watson et al, Nucleic Acid Research 1984 12:5145-5164).

[0080] In the present invention, either the first antigen binding domain or the second antigen binding domain may be linked directly or via a peptide linker to the N-terminus of the Fc domain, and at this time, the first antigen binding domain may be linked directly or via a peptide linker to the N-terminus or C-terminus of the second antigen binding domain. That is, the bispecific antibody of the present invention may be in the form of IgG x scFv.

[0081] The term "peptide linker" of the present invention may include 1 to 100 amino acids, specifically 2 to 50 amino acids, and any kind of amino acid may be included without limitation. The peptide linker may include, for example, Gly, Asn, and / or Ser residues, and may also include neutral amino acids such as Thr and / or Ala. Amino acid sequences suitable for the peptide linker may be known in the relevant art. Meanwhile, the length of the peptide linker may be determined in various ways within a range that does not affect the function of the fusion protein. For example, a peptide linker may be formed that includes a total of about 1 to about 100, about 2 to about 50, or about 5 to about 25 amino acids selected from the group consisting of Gly, Asn, Ser, Thr, and Ala. In one specific embodiment, the peptide linker can be represented by (GmS1)n (m, l and n are independently integers from about 1 to about 70, specifically integers from about 1 to about 64). Preferably, the peptide linker can be comprised of the amino acid sequence of SEQ ID NO: 18 (GGGGSGGGGSGGGGS, (GGGGS)3) or SEQ ID NO: 19 (GGGSGGGSGGGSGGGS, (GGGS)4), but is not limited thereto.

[0082] In the present invention, the first antigen binding domain and the second antigen binding domain may each be independently linked to the N-terminus of the Fc domain directly or via a peptide linker, wherein the first antigen binding domain may be a Fab fragment and the second antigen binding domain may be a scFv fragment; or the first antigen binding domain may be a scFv fragment and the second antigen binding domain may be a Fab fragment.

[0083] In the present invention, the bispecific antibody may be an afucosylated bispecific antibody.

[0084] In the present invention, the first antigen binding domain may include a heavy chain variable region including a heavy chain CDR1 comprising an amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 including an amino acid sequence of SEQ ID NO: 2, and a heavy chain CDR3 including an amino acid sequence of SEQ ID NO: 3; and a light chain variable region including a light chain CDR1 including an amino acid sequence of SEQ ID NO: 4, a light chain CDR2 including an amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 including an amino acid sequence of SEQ ID NO: 6.

[0085] In the present invention, the second antigen binding domain may include a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence of SEQ ID NO: 7, a heavy chain CDR2 comprising an amino acid sequence of SEQ ID NO: 8, and a heavy chain CDR3 comprising an amino acid sequence of SEQ ID NO: 9; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence of SEQ ID NO: 10, a light chain CDR2 comprising an amino acid sequence of SEQ ID NO: 11, and a light chain CDR3 comprising an amino acid sequence of SEQ ID NO: 12.

[0086] In the present invention, the first antigen binding domain may include a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 13 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 14.

[0087] In the present invention, the second antigen binding domain may include a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 15 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 16.

[0088] In the present invention, the bispecific antibody may have immuno-anticancer activity, may have ADCC (Antibody-Dependent Cellular Cytotoxicity) activity, may degrade a target protein through internalization activity, and may have tumor-targeting ability.

[0089] The present invention also provides an isolated nucleic acid molecule encoding the bispecific antibody.

[0090] Additionally, the present invention provides a recombinant expression vector comprising the nucleic acid molecule.

[0091] The term "vector" in the present invention is understood as a nucleic acid means comprising a nucleotide sequence that can be introduced into a host cell and recombined and integrated into the host cell genome, or can be autonomously replicated as an episome. The vector includes linear nucleic acids, plasmids, phagemids, cosmids, RNA vectors, viral vectors, and analogs thereof. Examples of viral vectors include, but are not limited to, retroviruses, adenoviruses, and adeno-associated viruses.

[0092] In addition, the present invention provides a host cell transformed with the recombinant expression vector.

[0093] In the present invention, a suitable host cell can be transformed or transfected with the DNA sequence of the present invention and used for expression and / or secretion of the target protein. Currently preferred host cells that can be used in the present invention include immortal hybridoma cells, NS / 0 myeloma cells, 293 cells, Chinese hamster ovary (CHO) cells, HeLa cells, CapT cells (human amniotic fluid derived cells), and COS cells.

[0094] The terms "transformation" and "transfection" of the present invention refer to the introduction of a nucleic acid (e.g., a vector) into a cell by various techniques known in the art.

[0095]

[0096] <Pharmaceutical Composition>

[0097] In addition, the present invention provides a pharmaceutical composition comprising the bispecific antibody and a pharmaceutically acceptable carrier.

[0098] In addition, the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising the bispecific antibody; the recombinant expression vector; or the host cell as an active ingredient.

[0099] In the present invention, the cancer may be selected from the group consisting of malignant mesothelioma, pancreatic cancer, ovarian cancer, cholangiocarcinoma, biliary tract cancer, breast cancer, kidney cancer, stomach cancer, liver cancer, lung cancer, colorectal cancer, skin cancer, bladder cancer, testicular cancer, uterine cancer, cervical cancer, thyroid cancer, head and neck cancer, prostate cancer, non-small cell lung cancer, neuroblastoma, brain cancer, colon cancer, squamous cell carcinoma, melanoma, myeloma, lymphoma, and leukemia, but is not limited thereto.

[0100] In the composition for preventing or treating cancer of the present invention, the active ingredient may be included in any amount (effective amount) depending on the intended use, formulation, mixing purpose, etc., as long as it can exhibit anticancer activity. Typically, the effective amount will be determined within the range of 0.001 wt% to 20.0 wt% based on the total weight of the composition. Here, the "effective amount" refers to the amount of the active ingredient capable of inducing an anticancer effect. Such an effective amount can be experimentally determined within the normal capabilities of a person skilled in the art.

[0101] The pharmaceutical composition according to the present invention may be formulated into a suitable form with a pharmaceutically acceptable carrier and may additionally contain excipients or diluents. The term "pharmaceutically acceptable" as used herein refers to a non-toxic composition that is physiologically acceptable and, when administered to humans, typically does not cause allergic reactions, such as gastrointestinal upset or dizziness, or similar reactions.

[0102] Pharmaceutically acceptable carriers may further include, for example, carriers for oral administration or carriers for parenteral administration. Carriers for oral administration may include lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, etc. In addition, various drug delivery materials used for oral administration of peptide formulations may be included. In addition, carriers for parenteral administration may include water, suitable oils, saline solution, aqueous glucose, and glycols, etc., and may further include stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium bisulfite, sodium sulfite, or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben, and chlorobutanol. In addition to the above components, the pharmaceutical composition of the present invention may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, etc. Other pharmaceutically acceptable carriers and formulations may be found in reference to the following literature (Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995).

[0103] The composition of the present invention can be administered to mammals, including humans, by any method. For example, it can be administered orally or parenterally. Parenteral administration methods include, but are not limited to, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, rectal, mucosal delivery, or administration in the form of eye drops, and mucosal delivery, transdermal, topical, or eye drop administration is preferred.

[0104] The pharmaceutical composition of the present invention can be formulated as a preparation for oral or parenteral administration, depending on the administration route as described above, and is preferably formulated as a preparation for parenteral administration. For example, in the case of preparations for parenteral administration, they can be formulated in the form of injections, creams, lotions, ointments for external use, oils, moisturizers, gels, aerosols, nasal inhalants, and eye drops by methods known in the art. These formulations are described in a literature (Remington's Pharmaceutical Science, 19th ed., Mack Publishing Company, Easton, PA, 1995), which is a generally known prescription manual in all fields of pharmaceutical chemistry.

[0105] The total effective amount of the composition of the present invention can be administered to a patient as a single dose, or can be administered by a fractionated treatment protocol in which multiple doses are administered over a long period of time. The pharmaceutical composition of the present invention may vary the content of the active ingredient depending on the severity of the disease. Preferably, the preferred total dosage of the pharmaceutical composition of the present invention may be about 0.01 ㎍ to 10,000 mg per 1 kg of patient body weight per day, and most preferably 0.1 ㎍ to 500 mg. However, since the dosage of the pharmaceutical composition is determined by taking into consideration various factors such as the formulation method, administration route, and number of treatments, as well as the patient's age, weight, health status, sex, severity of the disease, diet, and excretion rate, a person having ordinary skill in the art will be able to determine an appropriate effective dosage of the composition of the present invention considering these points. The pharmaceutical composition according to the present invention is not particularly limited in its formulation, administration route, and administration method as long as it exhibits the effects of the present invention.

[0106] In addition, the present invention provides a pharmaceutical composition for combination administration for the prevention or treatment of cancer, comprising the bispecific antibody and CAR-NK (chimeric antigen receptor natural killer) cells as active ingredients.

[0107] In the present invention, the CAR-NK cell may be a cell into which a mutated Fc receptor has been introduced, or may be a cell line with enhanced ADCC, but is not limited thereto.

[0108] In the present invention, the bispecific antibody and CAR-NK cells may be formulated separately and administered simultaneously or sequentially.

[0109] In addition, the present invention provides a pharmaceutical composition for combination administration for the prevention or treatment of cancer, comprising as active ingredients a first component comprising an antibody or an antigen-binding fragment thereof that specifically binds to VISTA (V-domain immunoglobulin suppressor of T cell activation); and a second component comprising an antibody or an antigen-binding fragment thereof that specifically binds to MSLN (Mesothelin).

[0110] In the present invention, the first component and the second component may be formulated and administered simultaneously or sequentially.

[0111]

[0112] Treatment Methods

[0113] In addition, the present invention provides a method for preventing or treating cancer, comprising administering to a subject a therapeutically effective amount of the bispecific antibody; a therapeutically effective amount of the recombinant expression vector; or a therapeutically effective amount of the host cell.

[0114] In addition, the present invention provides a method for preventing or treating cancer, comprising administering to a subject a pharmaceutical composition comprising a therapeutically effective amount of the bispecific antibody; and a therapeutically effective amount of CAR-NK (chimeric antigen receptor natural killer) cells.

[0115] In addition, the present invention provides a method for preventing or treating cancer, comprising administering to a subject a pharmaceutical composition comprising a first component comprising a therapeutically effective amount of an antibody or an antigen-binding fragment thereof that specifically binds to VISTA (V-domain immunoglobulin suppressor of T cell activation); and a second component comprising a therapeutically effective amount of an antibody or an antigen-binding fragment thereof that specifically binds to MSLN (Mesothelin).

[0116] The above therapeutically effective amount is preferably applied differently depending on various factors including the type and degree of the response to be achieved, the specific composition including whether other agents are used in some cases, the age, body weight, general health, sex and diet of the subject, the time of administration, the route of administration and the secretion rate of the composition, the treatment period, drugs used together or simultaneously with the specific composition, and similar factors well known in the medical field. Therefore, the effective amount of the composition suitable for the purpose of the present invention is preferably determined in consideration of the above-mentioned matters.

[0117] The above object is applicable to any mammal, which includes not only humans and primates, but also livestock such as cows, pigs, sheep, horses, dogs and cats.

[0118]

[0119] Hereinafter, the present invention will be described in more detail through examples. These examples are intended to more specifically illustrate the present invention, and the scope of the present invention is not limited to these examples.

[0120]

[0121] Example 1. Expression correlation analysis of VISTA and MSLN

[0122] To analyze the correlation between the transcript (mRNA) level expression of VISTA (V-domain immunoglobulin suppressor of T cell activation) and MSLN (Mesothelin), the TCGA-Meso database and the GEO database were used. Briefly, the transcript expression level correlation of VISTA and MSLN was analyzed using the Spearman correlation method in three databases: TCGA-Meso (number of patients: 87), GSE163722 (number of patients: 131), and GSE51024 (number of patients: 55). As a result, the correlation was 0.81, 0.78, and 0.61 in TCGA-Meso, GSE163722, and GSE51024, respectively, confirming that VISTA and MSLN have a strong positive correlation at the transcript level (Figs. 1 to 3).

[0123] Next, the expression patterns of VISTA and MSLN proteins were analyzed in formalin-fixed paraffin-embedded (FFPE) blocks from patients with epithelioid malignant pleural mesothelioma (Epithelioid MPM) or sarcomatoid malignant pleural mesothelioma (Sarcomatoid MPM) using immunohistochemistry. Briefly, staining was performed using VISTA (D1L2G, Cell Signaling Technology #64953) at a 1:50 dilution and MSLN (D9R5G, Cell Signaling Technology #99966) at a 1:100 dilution.

[0124] As a result, it was confirmed that VISTA and MSLN proteins were expressed at the same location in the tissues of patients with epithelioid malignant mesothelioma and sarcomatoid malignant mesothelioma, and that there was a strong positive correlation in the protein expression level (Figs. 4 and 5).

[0125]

[0126] Example 2. Analysis of VISTA and MSLN expression levels in cancer cell lines

[0127] The cell surface protein expression levels of VISTA and MSLN were determined among various human mesothelioma, ovarian cancer, and pancreatic cancer cell lines. NCI-H226, NCI-H2452, NCI-H2052, MSTO-211H, NCI-H28, and MeT-5A cell lines were used as human mesothelioma cell lines. TOV112D, OVCAR4, A2780BM, OV90, IGROV1, CAOV3, TOV21G, SKOV3, and OVCAR3 cell lines were used as human ovarian cancer cell lines, and AsPC-1, BxPC3, CAPAN-1, CAPAN-2, Mia-paca2, and PANC-1 cell lines were used as human pancreatic cancer cell lines. Briefly, single cells were dissociated using cell dissociation buffer (Gibco, 13151-014), and 2.5 × 10 5 Cells were seeded individually and reacted with VISTA antibody (MIH65, BD Biosciences) and MSLN antibody (REA1057, Miltenyi Biotec) on ice for 1 h. After the reaction, the cells were washed with 1% FBS / PBS and analyzed using a BD FACS Lyrics flow cytometer.

[0128] As a result, among the malignant mesothelioma cell lines, co-expression of VISTA and MSLN was confirmed in the NCI-H226 and NCI-H2052 cell lines (Fig. 6), among the ovarian cancer cell lines, co-expression of VISTA and MSLN was confirmed in the OVCAR3 cell line, and among the pancreatic cancer cell lines, co-expression of VISTA and MSLN was confirmed in the AxPC-1, CAPAN-2, Mia-paca2, and PANC-1 cell lines (Fig. 8).

[0129]

[0130] Example 3. Production of bispecific antibodies

[0131] From the above results, it was found that VISTA and MSLN expression had a strong positive correlation at the transcript level and protein expression level, and by confirming the simultaneous expression of VISTA and MSLN in various cancer cell lines, we attempted to produce a bispecific antibody that can specifically bind to VISTA and MSLN proteins.

[0132] Specifically, as shown in FIG. 9, a bispecific antibody binding to VISTA and MSLN is produced in a form including a first antigen-binding domain that specifically binds to VISTA and a second antigen-binding domain that specifically binds to MSLN, wherein the first antigen-binding domain may be a complete antibody and the second antigen-binding domain may be an antigen-binding fragment, and wherein the first antigen-binding domain is directly linked to the C-terminus of the second antigen-binding domain or via a peptide linker (see Table 1 below).

[0133]

[0134] BsAb 1 : scFv(MSLN: LMB-100) + mAb(Vista: 4A2)HC : scFv VH(LMB100)-Linker-scFv VL(LMB100)-Linker-HC(4A2)-Fc region (서열번호 20)METDTLLLWVLLLWVPGSTG (signal peptide)QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYTMNWVRQAPGQGLEWMGLITPYNGASSYNQKFRGKATMTVDTSTSTVYMELSSLRSEDTAVYYCARGGYDGRGFDYWGQGTLVTVSS (scFv VH(LMB100))GGGGSGGGGSGGGGS (Linker)DIQMTQSPSSLSASVGDRVTITCSASSSVSYMHWYQQKSGKAPKLLIYDTSKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSKHPLTFGQGTKLEIK (scFv VL(LMB100))GGGSGGGSGGGSGGGS (Linker)EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYAMSWVRQAPGKGLEWVSEISHSGTTNYNPSLKSRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRAIWGFDYWGQGTLVTVSS (HC(4A2))ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (Fc region)LC : LC(4A2)-Fc region (서열번호 21)METDTLLLWVLLLWVPGSTG (signalpeptide)QSVLTQPPSASGTPGQRVTISCSRENIGSRSVHWYQQLPGTAPKLLIYDNNERASGVPNRFSGSKSGTSASLAISGLRSEDEADYYCQAWDSSTAVFGGGTKLTVL (LC(4A2))RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (Fc region)

[0135]

[0136] In this case, information on the CDR sequences and variable region sequences for the first antigen binding domain (anti-VISTA) and the second antigen binding domain (anti-MSLN) is as shown in Tables 2 and 3 below.

[0137]

[0138] antibodyCDR-H1CDR-H2CDR-H3CDR-L1CDR-L2CDR-L3anti-VISTA(4A2)NYAMS(SEQ ID NO: 1)EISHSGTTNYNPSLKS(SEQ ID NO: 2)TRAIWGFDY(SEQ ID NO: 3)SRENIGSRSVH(SEQ ID NO: 4)DNNERAS(SEQ ID NO: 5)QAWDSSTAV(SEQ ID NO: 6)anti-MSLN(LMB-100)GYTMN(SEQ ID NO: 7)LITPYNGASSYNQKF(SEQ ID NO: 8)RGGYDGRGFDY(SEQ ID NO: 9)SASSSVSYMH(SEQ ID NO: 10)DTSKLAS(SEQ ID NO: 11)QQWSKHPLT(SEQ ID NO: 12)

[0139]

[0140] antibodyHC (heavy chain variable region)LC (light chain variable region)anti-VISTA(4A2)EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYAMSWVRQAPGKGLEWVSEISHSGTTNYNPSLKSRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRAIWGFDYWGQGTLVTVSS (SEQ ID NO: 13)QSVLTQPPSASGTPGQRVTISCSRENIGSRSVHWYQQLPGTAPKLLIYDNNERASGVPNRFSGSKSGTSASLAISGLRSEDEADYYCQAWDSSTAVFGGGTKLTVL (SEQ ID NO: 14) anti-MSLN(LMB-100)QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYTMNWVRQAPGQGLEWMGLITPYNGASSYNQKFRGKATMTVDTSTSTVYMELSSLRSEDTAVYYCARGGYDGRGFDYWGQGTLVTVSS (SEQ ID NO: 15) DIQMTQSPSSLSASVGDRVTITCSASSSVSYMHWYQQKSGKAPKLLIYDTSKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSKHPLTFGQGTKLEIK (SEQ ID NO: 16)

[0141]

[0142] Additionally, a bispecific antibody Bis 2 was produced in which the first antigen-binding domain is an antigen-binding fragment and the second antigen-binding domain is a complete antibody, wherein the first antigen-binding domain is linked directly to the N-terminus of the second antigen-binding domain or via a peptide linker (see Table 4 below).

[0143]

[0144] BsAb 2 : scFv(Vista: 4A2) + mAb(MSLN: LMB-100)HC : scFv VH(4A2)-Linker-scFv VL(4A2)-Linker-HC(LMB-100)-Fc region (서열번호 22)METDTLLLWVLLLWVPGSTG (signal peptide)EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYAMSWVRQAPGKGLEWVSEISHSGTTNYNPSLKSRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRAIWGFDYWGQGTLVTVSS (scFv VH(4A2))GGGGSGGGGSGGGGS (Linker)QSVLTQPPSASGTPGQRVTISCSRENIGSRSVHWYQQLPGTAPKLLIYDNNERASGVPNRFSGSKSGTSASLAISGLRSEDEADYYCQAWDSSTAVFGGGTKLTVL (scFv VL(4A2))GGGSGGGSGGGSGGGS (Linker)QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYTMNWVRQAPGQGLEWMGLITPYNGASSYNQKFRGKATMTVDTSTSTVYMELSSLRSEDTAVYYCARGGYDGRGFDYWGQGTLVTVSS (HC(LMB-100))ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (Fc region)LC : LC(LMB-100)-Fc region (서열번호 23)METDTLLLWVLLLWVPGSTG (signalpeptide)DIQMTQSPSSLSASVGDRVTITCSASSSVSYMHWYQQKSGKAPKLLIYDTSKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSKHPLTFGQGTKLEIK (LC(LMB-100))RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (Fc region)

[0145]

[0146] In addition, the first antigen-binding domain and the second antigen-binding domain may be independently linked to the Fc domain as antigen-binding fragments, and in this case, a bispecific antibody Bis 3 in which the first antigen-binding domain is a Fab fragment and the second antigen-binding domain is a scFv fragment was produced (see Table 5 below), and a bispecific antibody Bis 4 in which the first antigen-binding domain is a scFv fragment and the second antigen-binding domain is a Fab fragment was produced (see Table 6 below).

[0147]

[0148] BsAb 3 : scFv(LMB-100)-Fc(hole) + mAb(4A2)-Fc(knob)HC(knob) : HC(4A2)-Fc region (서열번호 24)METDTLLLWVLLLWVPGSTG (signal peptide)EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYAMSWVRQAPGKGLEWVSEISHSGTTNYNPSLKSRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRAIWGFDYWGQGTLVTVSS (HC(4A2))ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (Fc region)HC(hole) : scFv VH(LMB100)-Linker-scFv VL(LMB100)-Fc region (서열번호 25)METDTLLLWVLLLWVPGSTG (signal peptide)QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYTMNWVRQAPGQGLEWMGLITPYNGASSYNQKFRGKATMTVDTSTSTVYMELSSLRSEDTAVYYCARGGYDGRGFDYWGQGTLVTVSS (scFv VH(LMB100))GGGGSGGGGSGGGGS (Linker)DIQMTQSPSSLSASVGDRVTITCSASSSVSYMHWYQQKSGKAPKLLIYDTSKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSKHPLTFGQGTKLEIK (scFvVL(LMB100))EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (Fc region)LC : LC(4A2)-Fc region (서열번호 21)METDTLLLWVLLLWVPGSTG (signal peptide)QSVLTQPPSASGTPGQRVTISCSRENIGSRSVHWYQQLPGTAPKLLIYDNNERASGVPNRFSGSKSGTSASLAISGLRSEDEADYYCQAWDSSTAVFGGGTKLTVL (LC(4A2))RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (Fc region)

[0149]

[0150] BsAb 4 : scFv(4A2)-Fc(hole) + mAb(LMB-100)-Fc(knob)HC(knob) : HC(LMB-100)-Fc region (서열번호 26)METDTLLLWVLLLWVPGSTG (signal peptide)QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYTMNWVRQAPGQGLEWMGLITPYNGASSYNQKFRGKATMTVDTSTSTVYMELSSLRSEDTAVYYCARGGYDGRGFDYWGQGTLVTVSS (HC(LMB-100))ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (Fc region)HC(hole) : scFv VH(4A2)-Linker-scFv VL(4A2)-Fc region (서열번호 27)METDTLLLWVLLLWVPGSTG (signal peptide)EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYAMSWVRQAPGKGLEWVSEISHSGTTNYNPSLKSRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRAIWGFDYWGQGTLVTVSS (scFv VH(4A2))GGGGSGGGGSGGGGS (Linker)QSVLTQPPSASGTPGQRVTISCSRENIGSRSVHWYQQLPGTAPKLLIYDNNERASGVPNRFSGSKSGTSASLAISGLRSEDEADYYCQAWDSSTAVFGGGTKLTVL (scFvVL(4A2))EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (Fc region)LC : LC(LMB-100)-Fc region (서열번호 23)METDTLLLWVLLLWVPGSTG (signal peptide)DIQMTQSPSSLSASVGDRVTITCSASSSVSYMHWYQQKSGKAPKLLIYDTSKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSKHPLTFGQGTKLEIK (LC(LMB-100))RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (Fc region)

[0151]

[0152] The above four types of bispecific antibodies were expressed and purified in the ExpiCHO expression system. Protein A resin (Mabselect SuRe, 11-0026-01 AD, GE Healthcare Life Sciences) was used for purification, and equilibration buffer (20 mM Sodium Phosphate, 150 mM NaCl, pH 7.2), wash buffer (35 mM Sodium Phosphate, 500 mM NaCl, pH 7.2), and elution buffer (0.1 M Sodium Citrate, pH 3.6) were used. Purification was performed using AKTA™ avant with equilibration buffer 2 times the column volume, wash buffer 5 times the column volume, and elution buffer 5 times the column volume. During elution, 1 / 5 of a pH 8.0 Tris-HCl solution was added for neutralization. After buffer exchange with PBS twice using a filtration membrane (CelluSep, 1430-45), the solution was concentrated using a centrifugal filter (Amicon Ultra-15, UFC905024, Merck).

[0153] IgG antibody proteins produced under reducing and non-reducing conditions, respectively, were confirmed using a conventional SDS-PAGE technique, and it was confirmed that both the light chain and heavy chain of each antibody were well expressed at the expected molecular weight (Fig. 9).

[0154]

[0155] Example 4. Confirmation of binding ability of bispecific antibodies to cell lines

[0156] To evaluate the binding ability of the manufactured bispecific antibody to cell lines, the binding ability of the bispecific antibody to cell lines was confirmed through flow cytometry under conditions of pH 6 as well as pH 7, which mimic the tumor microenvironment. Briefly, single cells were dissociated using cell dissociation buffer (Gibco, 13151-014), and 2.5 × 10 5Each cell was seeded, and 10 μg / ml of each antibody was added to 1% FBS / PBS adjusted to pH 7 or 6 with 1 N HCl, and reacted on ice for 1 hour. After the reaction, the cells were washed with 1% FBS / PBS, and the secondary antibody, goat anti-human IgG-FITC (109-095-098, Jackson ImmunoResearch), was treated at a ratio of 1:100 in 1% FBS / PBS adjusted to pH 7 or 6, and reacted on ice for 1 hour. After the reaction, the cells were washed with 1% FBA / PBS, and analyzed using a BD FACS Lyrics flow cytometer.

[0157] As a result, it was confirmed that the produced bispecific antibodies Bis 1 to Bis 4 showed strong cell binding ability at both pH 7 and pH 6 (Fig. 10).

[0158]

[0159] Example 5. Confirmation of antigen binding ability of bispecific antibodies

[0160] To confirm the antigen binding ability of the manufactured bispecific antibody, immunoprecipitation was performed. Briefly, the NCI-H226 cell line selected in Example 2 was dissociated using a scraper and then dissolved in PBS containing protease inhibitors and phosphatase inhibitors. The cells were physically disrupted using a sonicator, and the protein was quantified using a BCA assay. The quantified protein was bound to the antibody for 1 hour, and simultaneously blocked with Protein A beads (Sigma) using 5% skim milk for 1 hour. Afterwards, the protein and antibody solutions were bound to the blocked beads for 1 hour, and washed three times with PBS. 5X sample loading buffer containing DTT was added, and the beads were incubated at 70°C for 10 minutes to separate and reduce both the protein and antibody bound to the Protein A beads. Western blot was performed using the sample to confirm whether the bispecific antibody recognized intact VISTA and MSLN antigens using anti-human VISTA and anti-human MSLN antibodies (Cell signaling technology).

[0161] As a result, it was confirmed that the produced bispecific antibodies Bis 1 to Bis 4 selectively recognized and bound well to the native forms of VISTA and MSLN (Fig. 11).

[0162]

[0163] Example 6. Confirmation of the antigen-inhibitory ability of bispecific antibodies.

[0164] An experiment was conducted to confirm the antigen-inhibitory activity of the produced bispecific antibody. Briefly, after treating the CHO-K1 VISTA / MSLN stable cell line with the bispecific antibody or single antibody, the degree of inhibition (blocking) of VISTA and MSLN on the cell surface was compared. Briefly, single cells were dissociated using cell dissociation buffer (Gibco, 13151-014), and 2.5 × 10 5 Cells were seeded individually, and 10 μg / ml of bispecific and monospecific antibodies were added and incubated on ice for 1 hour. After incubation, the cells were washed with 1% FBS / PBS and incubated with VISTA antibody (MIH65, BD Biosciences) and MSLN antibody (REA1057, Miltenyi Biotec) for 1 hour on ice. After incubation, the cells were washed with 1% FBS / PBS and analyzed using a BD FACS Lyrics flow cytometer.

[0165] As a result, it was confirmed that the manufactured bispecific antibodies Bis 1 to Bis 4 could bind more strongly to VISTA and MSLN on the cell surface and inhibit their function compared to the group treated with a single antibody (Figure 12).

[0166]

[0167] Example 7. Confirmation of neutralizing ability of bispecific antibodies against binding ligands

[0168] To confirm the neutralizing ability of the manufactured bispecific antibody against its binding ligands, the binding neutralizing ability between the binding ligands of VISTA, PSGL-1 (pH 6) and VSIG-3 (pH 7), and the binding ligand of MSLN, CA125 (pH 6, pH 7), was confirmed by ELISA. 250 ng each of human PSGL1 protein (hPSGL1 Fc, R&D systems, Cat. No. 3345-PS), human VSIG3 protein (hVSIG3 Fc, R&D systems, Cat. No. 9229-VS), and human CA125 protein (hCA125 Fc, R&D systems, Cat. No. 5609-MU) were added to a 96-well plate and coated overnight at 4°C. Afterwards, the plates were washed three times with 0.1% PBS-T adjusted to pH 6 and pH 7 with 1 N HCl, and blocked with 50 mg / mL BSA / PBS adjusted to pH 6 and pH 7 with 1 N HCl for 2 hours at room temperature. Meanwhile, 100 nM of human VISTA-biotin and MSLN-biotin proteins and bispecific antibodies were diluted to a 1 / 3 concentration from 60 μg / mL and allowed to bind for 1 hour at room temperature. The blocked 96-well plate was washed three times with washing buffer, and 100 μL each of VISTA-antibody and MSLN-antibody binding solutions were added and allowed to bind for 2 hours at room temperature. After binding, the plates were washed three times with washing buffer, treated with Streptavidin-HRP (R&D systems) at a ratio of 1:5000, and incubated for 1 hour at room temperature. After incubation, the cells were washed three times with washing buffer, 100 μL of TMB solution (Surmodics) was added, and the reaction was allowed to proceed at room temperature for 20 minutes. 50 μL of stop solution was added, and the 450 nm wavelength value was measured using an ELISA reader.

[0169] As a result, it was confirmed that the produced bispecific antibodies Bis 1 to Bis 4 maintained the ligand neutralization ability of the single antibodies (Fig. 13).

[0170]

[0171] Example 8. Confirmation of the immune activation ability of bispecific antibodies

[0172] To confirm the immune activation ability of the manufactured bispecific antibody, a Staphylococcal enterotoxin B (SEB) activity assay was performed. Briefly, human VISTA protein (hVISTA-Fc, RND systems, Cat. No. 7126-B7) was coated on a 96-well plate at a concentration of 5 μg / ml at 4°C overnight. The next day, after washing three times with PBS, human PBMCs were seeded in a 96-well plate at a density of 2.0 × 10 5 Each cell was placed in a culture medium and treated with 100 ng / mL of SEB (Abion), 10 μg / mL of anti-VISTA antibody, and 10 μg / mL of anti-PD-1 antibody (Pembrolizumab) and incubated at 37°C for 3 days. After 3 days, 100 μL of the supernatant was used for further analysis. The amount of IL-6 secretion through immune activation was measured using ELISA (Human IL-6 DuoSet ELISA, R&D systems, Cat. No. DY206).

[0173] As a result, it was confirmed that the produced bispecific antibodies Bis 1 to Bis 4 had the ability to activate immunity by VISTA antibody (Fig. 14).

[0174]

[0175] Example 9. Confirmation of ADCC efficacy of bispecific antibodies

[0176] To confirm the ADCC (Antibody-Dependent Cellular Cytotoxicity) efficacy of the manufactured bispecific antibody, an ADCC assay was performed. Human malignant mesothelioma, pancreatic cancer, and ovarian cancer cell lines were used as target cells, and NK92MI cell lines transfected with human CD16 and an improved NK cell line were used as effector cells. After dissociating the target cells into single cells using a dissociation buffer, 2.0 × 10 were seeded in a 96-well plate. 4 The effector cells were divided into 1.0×10 5 Each bispecific antibody was diluted to a concentration of 1 / 5 from 30 μg / ml and added to target cells, followed by incubation at 37°C for 4 hours. Afterwards, cytotoxicity was confirmed by performing an LDH assay (Promega, CytoTox 96®) using 50 μL of the supernatant.

[0177] As a result, it was confirmed that the manufactured bispecific antibodies Bis 1 to Bis 4 had superior ADCC activity compared to single antibodies (Figs. 15 and 16). In particular, it was confirmed that the bispecific antibody of the present invention had a remarkable effect when administered in combination with an improved NK cell line (Fig. 16).

[0178]

[0179] Example 10. Confirmation of ADCC efficacy by combined administration of VISTA antibody and MSLN antibody.

[0180] Experiments were conducted to determine the ADCC efficacy of combinations of various VISTA antibodies and MSLN antibodies. Briefly, an ADCC assay was performed using the same method as in Example 9 using a combination of six VISTA antibodies (4D2, 4F2, 4G3, 4H2, 4A2, 4A3) and three MSLN antibodies (LMB-100, Anetumab, REA1057).

[0181] As a result, it was confirmed that the ADCC efficacy of combined administration of VISTA antibody and MSLN antibody was similar to that of bispecific antibody, and had superior ADCC activity compared to single antibody (Fig. 17).

[0182]

[0183] Example 11. Confirmation of ADCC efficacy of a nonfucosylated bispecific antibody.

[0184] After producing an afucosylated bispecific antibody from the produced bispecific antibody, an experiment was performed to confirm the ADCC (Antibody-Dependent Cellular Cytotoxicity) efficacy. A human malignant mesothelioma cell line was used as the target cell, and the NK92MI-hCD16a cell line was used as the effector cell, and the ADCC assay was performed using the same method as in Example 9. The target and effector cells were mixed in a ratio of 1:1.

[0185] As a result, it was confirmed that the produced non-fucosylated bispecific antibody (Afuco BsAb3) showed a more marked increase in ADCC activity (Fig. 18).

[0186]

[0187] Example 12. Confirmation of internalization characteristics of bispecific antibodies.

[0188] To confirm the internalization characteristics of the manufactured bispecific antibody, an internalization assay was performed using an internalization assay kit (ZAP antibody internalization kit, Advanced Targeting Systems, Cat. No. IT-22). Briefly, 3.0 × 10 NCI-H226 cells were seeded in a 96-well plate. 3The cells were divided into individual cells and incubated overnight. Using saporin as a positive control, the bispecific antibody and anti-human IgG-saporin were treated together on the NCI-H226 cell line. After incubation for 3 days, the 450 nm wavelength value was measured using an ELISA reader (Fig. 19a).

[0189] Next, the degree of target protein degradation through internalization of the bispecific antibody was confirmed using a degradation assay. Briefly, NCI-H226 cells were treated with 10 nM of the bispecific antibody and incubated for 1 day. NCI-H226 cells were dissociated using a scraper and resuspended in PBS containing protease inhibitors and phosphatase inhibitors. Proteins were quantified using a BCA assay, and changes in VISTA and MSLN protein expression levels through internalization and degradation were confirmed using a Western blot assay (Fig. 19b).

[0190] As a result, it was confirmed that the bispecific antibody had increased internalization activity compared to single antibody and combination administration (Figure 19).

[0191]

[0192] Example 13. Confirmation of in vivo tumor targeting ability of bispecific antibodies.

[0193] An experiment was performed to confirm the in vivo tumor-targeting ability of the manufactured bispecific antibody. Briefly, the NCI-H226 cell line was injected into immunodeficient nude mice at a density of 3.0 × 10 per mouse. 6 Tumors were induced by subcutaneous injection into each dog. Tumor size was 200 mm. 3 When this was achieved, the bispecific antibody was injected into mice by detecting CF750 fluorescence, and the tumor targeting ability was confirmed through the IVIS spectrum in vivo imaging system.

[0194] As a result, it was confirmed that the produced bispecific antibody had superior tumor targeting ability compared to when a single antibody was administered (Figure 20).

[0195]

[0196] The foregoing description of the present invention is for illustrative purposes only, and those skilled in the art will readily appreciate that the present invention can be readily modified into other specific forms without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive. For example, each component described as a single entity may be implemented in a distributed manner, and similarly, components described as distributed may be implemented in a combined manner.

[0197] The scope of the present invention is indicated by the claims set forth below, and all changes or modifications derived from the meaning and scope of the claims and their equivalent concepts should be interpreted as being included in the scope of the present invention.

Claims

1. A bispecific antibody comprising a first antigen-binding domain that specifically binds to VISTA (V-domain immunoglobulin suppressor of T cell activation); and a second antigen-binding domain that specifically binds to MSLN (Mesothelin).

2. In paragraph 1, A bispecific antibody comprising an Fc domain.

3. In paragraph 2, A bispecific antibody, wherein the Fc domain is selected from the group consisting of human IgG1, IgG2, IgG3 and IgG4.

4. In paragraph 1, A bispecific antibody, wherein the first antigen binding domain and the second antigen binding domain are each independently selected from the group consisting of a Fab fragment, a Fab' fragment, an F(ab')2 fragment, an Fv fragment, a disulfide-linked Fv (dsFv), a single-chain Fv (scFv), a single-chain Fab (scFab), a diabody, a minibody, and combinations thereof.

5. In paragraph 1, A bispecific antibody, wherein the bispecific antibody is a two-in-one antibody or a dual-functional Fab (DAF).

6. In paragraph 1, A bispecific antibody, wherein the bispecific antibody is included as part of a multispecific antibody.

7. In paragraph 1, A bispecific antibody, wherein either the first antigen-binding domain or the second antigen-binding domain is linked directly or via a peptide linker to the N-terminus of the Fc domain.

8. In paragraph 7, A bispecific antibody, wherein the first antigen binding domain is linked directly or via a peptide linker to the N-terminus or C-terminus of the second antigen binding domain.

9. In paragraph 1, A bispecific antibody, wherein the first antigen-binding domain and the second antigen-binding domain are each independently linked to the N-terminus of the Fc domain, either directly or via a peptide linker.

10. In paragraph 9, A bispecific antibody wherein the first antigen-binding domain is a Fab fragment and the second antigen-binding domain is a scFv fragment.

11. In paragraph 9, A bispecific antibody wherein the first antigen-binding domain is a scFv fragment and the second antigen-binding domain is a Fab fragment.

12. In paragraph 1, A bispecific antibody, wherein the bispecific antibody is an afucosylated bispecific antibody.

13. In paragraph 1, The first antigen binding domain comprises a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 comprising an amino acid sequence of SEQ ID NO: 2, and a heavy chain CDR3 comprising an amino acid sequence of SEQ ID NO: 3; and A bispecific antibody comprising a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:

6.

14. In paragraph 1, The second antigen binding domain comprises a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence of SEQ ID NO: 7, a heavy chain CDR2 comprising an amino acid sequence of SEQ ID NO: 8, and a heavy chain CDR3 comprising an amino acid sequence of SEQ ID NO: 9; and A bispecific antibody comprising a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 10, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:

12.

15. In paragraph 1, A bispecific antibody, wherein the first antigen binding domain comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 13 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:

14.

16. In paragraph 1, A bispecific antibody, wherein the second antigen binding domain comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 15 and a light chain variable region comprising an amino acid sequence of SEQ ID NO:

16.

17. In paragraph 1, The above bispecific antibody is a bispecific antibody having immuno-oncology activity.

18. In paragraph 1, The above bispecific antibody is a bispecific antibody having ADCC (Antibody-Dependent Cellular Cytotoxicity) activity.

19. In paragraph 1, The above bispecific antibody is a bispecific antibody that degrades a target protein through internalization activity.

20. In paragraph 1, The above bispecific antibody is a bispecific antibody having tumor targeting ability.

21. An isolated nucleic acid molecule encoding a bispecific antibody according to any one of claims 1 to 20.

22. A recombinant expression vector comprising a nucleic acid molecule according to claim 21.

23. A host cell transformed with a recombinant expression vector according to Article 22.

24. A pharmaceutical composition comprising a bispecific antibody according to any one of claims 1 to 20 and a pharmaceutically acceptable carrier.

25. A pharmaceutical composition for preventing or treating cancer, comprising as an active ingredient a bispecific antibody according to any one of claims 1 to 20; a recombinant expression vector according to claim 22; or a host cell according to claim 23.

26. In paragraph 25, A pharmaceutical composition, wherein the cancer is selected from the group consisting of malignant mesothelioma, pancreatic cancer, ovarian cancer, cholangiocarcinoma, bile duct cancer, breast cancer, kidney cancer, stomach cancer, liver cancer, lung cancer, colorectal cancer, skin cancer, bladder cancer, testicular cancer, uterine cancer, cervical cancer, thyroid cancer, head and neck cancer, prostate cancer, non-small cell lung cancer, neuroblastoma, brain cancer, colon cancer, squamous cell carcinoma, melanoma, myeloma, lymphoma, and leukemia.

27. A pharmaceutical composition for combination administration for the prevention or treatment of cancer, comprising a bispecific antibody according to any one of claims 1 to 20; and a CAR-NK (chimeric antigen receptor natural killer) cell as an active ingredient.

28. A pharmaceutical composition for combination administration for the prevention or treatment of cancer, comprising as active ingredients a first component comprising an antibody or an antigen-binding fragment thereof that specifically binds to VISTA (V-domain immunoglobulin suppressor of T cell activation); and a second component comprising an antibody or an antigen-binding fragment thereof that specifically binds to MSLN (Mesothelin).

29. In paragraph 28, A pharmaceutical composition wherein the first component and the second component are each formulated and administered simultaneously or sequentially.

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