EA043079B1 - ANTI-MSLN-ANTIBODY AND PHARMACEUTICAL COMPOSITION FOR THE TREATMENT OF ONCOLOGICAL DISEASES CONTAINING IT - Google Patents

Description

Among the various causes of death, there is often death due to cancer, which accounts for the second largest proportion of deaths. Various attempts have been made in the past to treat cancer. Currently, in relation to methods of treating oncological diseases, they are reduced to the introduction of an antitumor agent, radiation therapy or surgery. However, such treatments may be effective in the early stages of a malignant disease and have little therapeutic effect in the terminal stages of cancer, when the disease has spread to other tissues, or when it recurs.

Recently, in order to find a treatment for oncological diseases without the manifestation of side effects, studies of targeted antitumor agents using antibodies have been actively conducted. Typically, Herceptin, an anti-HER2 antibody, is used to treat breast cancer, and Avastin, an antibody against vascular endothelial growth factor (VEGF), is used to treat colorectal cancer. A key factor in the development of targeted antitumor agents using antibodies is the development of antibodies against surface membrane proteins that are overexpressed in tumor cells.

Meanwhile, mesothelin (MSLN) is a precursor polypeptide with a molecular weight of 69 to 71 kDa, and is a glycoprotein located on the surface of the cells of the mesothelial layer of the abdominal, pleural and pericardial cavities. However, to date, the function of MSLN has not been clearly elucidated, it has been reported that overexpression of MSLN is observed in mesothelioma, ovarian cancer, pancreatic cancer, gastric cancer, lung cancer, and endometrial cancer, which are cancer cells or tumor cells. In addition, studies have shown that chimeric mouse/human anti-MSLN antibodies inhibit the growth of tumor cells when tumor cells overexpressing MSLN are exposed to such antibodies (Hassan R et al., Cancer Immun., 19; 7: 20, 2007).

Accordingly, mouse antibodies have been developed that specifically bind to MSLNs. However, clinical trials have not been successful due to the lack of cross-reactivity between heterologous individuals. In addition, antibodies produced in patients treated with mouse antibodies or chimeric antibodies have problems with high toxicity or reduced therapeutic efficacy.

Therefore, there is a need to develop an anti-MSLN antibody that can be cross-reactive with humans while having high affinity for MSLN.

Technical problem

The present invention has been made to solve the above problems of the prior art. It is an object of the present invention to provide an antibody having a high binding affinity to mesothelin (MSLN) and a pharmaceutical composition having a high efficacy in the treatment of oncological diseases using it.

However, the problem to be solved by the present invention is not limited to the above problems, and other problems that have not been mentioned will become clear to those skilled in the art from the following description of the invention.

Solution

In an aspect of the present invention, there is provided an antibody comprising a light chain variable region (VL region) consisting of a sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 1 and a heavy chain variable region (VH region) consisting of from a sequence having at least 80% identity with the amino acid sequence of any of SEQ ID NOs: 3-5.

In yet another aspect of the present invention, a polynucleotide is provided that encodes a light chain variable region (VL region) and a heavy chain variable region (VH region) of an antibody.

In yet another aspect of the present invention, an expression vector is provided that contains a polynucleotide.

In yet another aspect of the present invention, a host cell transformed with an expression vector is provided.

In another aspect of the present invention, there is provided a method for producing an antibody that specifically binds to MSLN, comprising the step of culturing a host cell.

In yet another aspect of the present invention, a pharmaceutical composition for the prevention or treatment of cancer is provided, comprising an antibody or fragment thereof.

Advantageous Effects of the Invention

Due to its high affinity and specificity for MSLN, the anti-MSLN antibody of the present invention can be effectively used in the prevention or treatment of cancer.

It should be understood that the effect of the present invention is not limited to the effects described above, and it includes all effects that result from the configuration of the invention described in the Detailed Description section or the claims of the present invention.

Brief description of the figures

In FIG. 1 shows the results obtained in an affinity assay of the chimeric mouse/human IgG1 antibody MI323 for human pancreatic tumor cells (AsPC1) expressing MSLN.

In FIG. 2 shows the results obtained in an affinity assay of the chimeric mouse/human IgG1 antibody MI323 for human pancreatic tumor cells (Capan2) expressing MSLN.

In FIG. 3 shows the results obtained in an affinity assay of the chimeric mouse/human IgG1 antibody MI323 for human pleural mesothelioma (H226) expressing MSLN.

In FIG. 4 shows the results obtained in an affinity assay of the chimeric mouse/human IgG1 antibody MI323 for human pancreatic tumor cells (PL45) expressing MSLN.

In FIG. 5 shows the results obtained in an affinity assay of the humanized IgG1 antibody MI323 for human pancreatic tumor cells (AsPC1) expressing MSLN.

In FIG. 6 shows the results obtained in an affinity assay of the humanized IgG1 antibody MI323 for human pancreatic tumor cells (Capan2) expressing MSLN.

In FIG. 7 shows the results obtained in the affinity assay of the humanized IgG1 antibody MI323 for human pleural mesothelioma (H226) expressing MSLN.

In FIG. 8 shows the results obtained in an affinity assay of the humanized IgG1 antibody MI323 for human pancreatic tumor cells (PL45) expressing MSLN.

Detailed description of the invention

Hereinafter, the present invention will be described in detail.

In an aspect of the present invention, there is provided an antibody comprising a light chain variable region (VL region) consisting of a sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 1 and a heavy chain variable region (VH region) consisting of from a sequence having at least 80% identity with the amino acid sequence of any of SEQ ID NOs: 3-5.

The light chain variable region may consist of an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95%, and most preferably at least 99% identity with the amino acid sequence of SEQ ID NO: 1.

The heavy chain variable region may consist of an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95%, and most preferably at least 99% identity with the amino acid sequence of any of SEQ ID NO: 3 -5.

An antibody containing a light chain variable region and a heavy chain variable region can specifically bind to mesothelin (MSLN). Here, the MSLN to which the antibody binds may be a human MSLN. That is, the antibody can specifically bind to the human MSLN or tumor cells associated with it.

As used herein, the term MSLN may refer to a concept that collectively refers to MSLN itself and any of its variants, isotypes, and paralogs that are present in an animal, and preferably in humans and monkeys. In addition, as used here, the term human MSLN refers to MSLN derived from humans. As used here, the term murine MSLN refers to an MSLN derived from a mouse.

As used herein, the term antibody refers to an immunoglobulin (Ig) molecule that reacts immunologically with a particular antigen, i. a protein molecule that functions as a receptor that specifically recognizes an antigen. In addition, the antibody may be a complete antibody or an antibody fragment.

In the light and heavy chain variable regions, certain amino acids may be substituted, inserted and/or deleted, as long as the properties relevant to the object of the present invention, such as affinity and specificity for MSLN, are retained. For example, conservative amino acid substitutions may occur in the light and/or heavy chain variable regions. Conservative substitution means replacing the original amino acid sequence with another amino acid residue that has similar properties.

For example, lysine, arginine, and histidine have similar properties in that they have a basic side chain, and aspartic acid and glutamic acid have similar properties in that they have an acidic side chain. In addition, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine and tryptophan have similar properties in that they contain an uncharged polar side

- 2 043079 chain; alanine, valine, leucine, threonine, isoleucine, proline, phenylalanine and methionine have similar properties in that they contain a non-polar side chain; and tyrosine, phenylalanine, tryptophan and histidine have similar properties in that they contain an aromatic side chain.

Therefore, it will be apparent to those skilled in the art that amino acid substitutions in a group of amino acids having similar properties as described above will not cause any significant change in properties. For this reason, antibodies that have undergone changes caused by a conservative substitution in the variable region are also included in the scope of the present invention, if such antibodies retain the properties of the antibody of the present invention.

On the other hand, the antibody can specifically bind to tumor cells expressing MSLN, specifically to the surface of tumor cells, through specific binding to MSLN. Here, MSLN-expressing tumor cells may include, but are not limited to, human tumor cells.

The variable regions of the light and heavy chains of an antibody may consist of complementarity determining regions (CDRs) and framework regions (FRs). Typically, CDRs provide specificity for binding to specific antigens, and FRs function to form an antibody fold, to maintain CDR binding, or the like.

The antibody may be an antibody that has as backbone the amino acid sequence of the CDR of the existing mouse anti-MSLN antibody MI323 and the amino acid sequence of the FR and constant region (Fc) of a human antibody similar to MI323 in which some of the amino acid residues of the CDR and FR portions are in form, being substituted, so that these residues can specifically bind to human.

The antibody may contain a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 9; A light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 10; Light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 11; A heavy chain CDR1 comprising the amino acid sequence of any sequence selected from the group consisting of SEQ ID NOs: 12-14; A heavy chain CDR2 comprising the amino acid sequence of any sequence selected from the group consisting of SEQ ID NOs: 15-17; and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 18.

In one embodiment, the antibody may comprise a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 9; A light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 10; Light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 11; A heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 12; A heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 15; and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 18.

The antibody may contain a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 9; A light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 10; Light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 11; A heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 13; A heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 16; and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 18.

The antibody may contain a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 9; A light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 10; Light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 11; A heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 14; A heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 17; and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 18.

Therefore, the antibody may be a humanized antibody that specifically binds to a human MSLN. As used herein, the term humanized antibody refers to a chimeric antibody that contains the minimum sequence derived from the immunoglobulin of a non-human antibody, such as a mouse antibody, and may mean an antibody in which all parts, except for the sequence corresponding to the hypervariable region, replaced by their human counterparts.

In addition, the term hypervariable region (HVR) refers to a region of a variable domain that exhibits hypervariability or forms a structurally defined loop in the sequence of an antibody. Among the definitions that identify it, the definition of complementarity determining region (CDR) according to the Kabat system is most often used to classify regions based on sequence variability.

An antibody fragment may also be used, provided that the antibody fragment retains the function of the antibody. An antibody or antibody fragment may include, but is not limited to, single chain antibodies, diabodies, tribodies, tetrabodies, Fab fragments, F(ab') ₂ fragments, Fd, scFv, domain antibodies, minibodies, scAb, IgD antibodies, IgE antibodies, IgM antibodies, IgG1 antibodies, IgG2 antibodies, IgG3 antibodies, IgG4 antibodies, derivatives of antibody constant regions, artificial antibodies based on

- 3 043079 protein scaffolds and the like, which retain the function of binding to MSLN.

The antibody can also be used in the form of an antibody-drug conjugate (ADC) obtained by linking the antibody to an antitumor drug that is effective in inhibiting tumor cell proliferation. As used herein, the term antineoplastic includes the prophylactic and therapeutic effect on a malignant tumor, and prevention means any action aimed at inhibiting or delaying the development of oncological diseases. In addition, treatment means any action aimed at reducing or curatively changing the symptoms of oncological diseases.

The drug that can be used in the antibody-drug conjugate includes any compound having a cytotoxic or cytostatic effect and a moiety or functional group of the compound. Examples of the drug include microtubulin polymerization inhibitors, meiosis inhibitors, RNA polymerase inhibitors, topoisomerase inhibitors, DNA intercalators, DNA alkylators, ribosome inhibitors, siRNA, shRNA, siRNA, radioisotopes, and toxins, among which at least one compound can be used.

The drug may include, but is not limited to, maytansinoid, auristatin, dolastatin, trichothecene, CC1065 (NSC 298223), calicheamicin, taxane, anthracycline, methotrexate, adriamycin, vindesine, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomy qing C, chlorambucil, daunorubicin, daunomycin, etoposide, teniposide, carminomycin, aminopterin, dactinomycin, mitomycins, bleomycins, esperamycins, other enediine antibiotics, 5fluorouracil, other nitrogen mustards and their stereoisomers, isosteres, homologues or derivatives, cisplatin and cisplatin homologues, other interca lator enzymes and fragments thereof, such as nucleases, antibiotics, toxins (enzymatically active toxins or small molecular weight toxins of bacterial, fungal, plant or animal origin) and various antitumor or anticancer agents such as cisplatin, CPT-11, paclitaxel and docetaxel.

In addition, the radioisotope (radionuclide) includes ³ H, ¹⁴ C, ³² P, ³⁵ S, ³⁶ Cl, ⁵¹ Cr, ⁵⁷ Co, ⁵⁸ Co, ⁵⁹ Fe, ⁹⁰ Y, ¹²⁵ I, ¹³¹ I, ¹⁸⁶ Re and the like. You can also use miRNA (siRNA), siPHK, shRNA and the like, which can inhibit the expression of certain oncogenes.

The binding of an anti-MSLN antibody to a drug is preferably achieved by conjugation using a functional group such as the thiol group of an amino acid residue such as lysine or cysteine in the antibody. If desired, the conjugation can also be carried out in a linker-mediated form, which is commonly used. You can also use a linker based on maleimide or iodoacetamide.

When a drug is conjugated to an antibody or fragment thereof, the drug may be conjugated to a C-terminal site opposite the antigen-binding site to reduce the effect on the ability or specificity of the antibody or fragment to bind to MSLNs and the like. When a complete antibody is used rather than a fragment thereof, the drug can be conjugated to the Fc region.

In addition, the antibody can also be used as a therapeutic agent based on a chimeric antigen receptor (CAR) containing it. Examples of such a therapeutic agent preferably include, but are not limited to, chimeric antigen receptor T cell (CAR T cell) or natural killer chimeric antigen receptor (CAR NK cell) therapeutic agents.

The antibody can also be used in the form of a bispecific antibody comprising an anti-MSLN antibody. A bispecific antibody is an antibody that has the ability to bind to two antigens simultaneously and can generally be in a form in which pairs of heavy and light chains that bind to different antigens are attached to each other.

In addition, the bispecific antibody is available in a form such as a bispecific single chain antibody, wherein single chain antibody (scFv) fragments in which VL and VH are linked to each other via a short linker peptide are linked in the form of scFv1-scFv2(-Fc), a dual antibody based on a single domain antibody (sdAb) using VH, and a bispecific antibody generated using BiTE technology (see http://www.micromet.de) from Micromet, Germany.

The bispecific antibody may be in the form in which the anti-MSLN antibody is linked to an antibody or fragment thereof that has the ability to bind to a target molecule specific for an immunoeffective cell. The target molecule specific for the immunoeffective cell may preferably be selected from, but not limited to, TCR/CD3, CD16 (FcyRIIIa), CD44, CD56, CD69, CD64 (FcyRI), CD89, and CD11b/CD18 (CR3).

In yet another aspect of the present invention, there is provided a polynucleotide encoding a light chain variable region (VL region) and a heavy chain variable region (VH region) of an antibody of the present invention, and an expression vector containing the same.

A polynucleotide that encodes for the heavy chain variable region of an antibody or antibody fragment, i. gene, can be easily obtained by those skilled in the art from the amino acid after

- 4 043079 anti-MSLN antibodies.

As used herein, the term expression vector refers to a recombinant vector capable of expressing a target protein in a host cell and means a gene construct that contains essential regulatory elements operably linked thereto for expression of the inserted gene. The gene encoding the anti-MSLN antibody may be inserted into a separate vector or may be used in the form of an insert into the same vector.

In particular, a polynucleotide that encodes the amino acid sequence of an anti-MSLN antibody can be used in the form of an insert in a separate or the same vector, and a polynucleotide that encodes a heavy chain or its variable region can be used in the form of an insert in a separate or the same vector. same vector.

As used herein, the term operably linked means that a nucleic acid expression control sequence and a nucleic acid sequence encoding a desired protein are operably linked to perform the desired function. Operable linkage to a recombinant vector can be achieved using genetic recombination techniques well known in the art, and site-specific DNA cleavage and ligation can be easily achieved using enzymes and the like well known in the art.

Expression vectors suitable for the production of anti-MSLN antibodies may contain signal sequences for membrane or secretion targeting in addition to regulatory expression elements such as promoters, initiation codons, termination codons, polyadenylation signals, and enhancers. Start codons and termination codons are generally considered to be part of the nucleotide sequence encoding the immunogenic target protein. Such codons must be functional in the individual when the gene construct is introduced and must be in reading frame with the coding sequence. In general, promoters may be constitutive or inducible. The promoter may include, but is not limited to, prokaryotic promoters such as lac, tac, T3 and T7, simian virus 40 (SV40) promoters, mouse mammary tumor virus (MMTV) promoters, human immunodeficiency virus (HIV) promoters, e.g. HIV long terminal repeat (LTR), Moloney murine leukemia virus promoters, cytomegalovirus (CMV) promoters, Epstein-Barr virus (EBV) promoters, Rous sarcoma virus (RSV) promoters, and P-actin promoters, eukaryotic promoters of human hemoglobin, human muscle tissue creatine, human metallothionein, and the like.

The expression vector may further contain a selectable marker that allows selection of host cells containing it. The selectable marker is used to select cells transformed with the vector. As a selectable marker, markers that confer a selectable phenotype such as drug resistance, auxotrophy, resistance to cytotoxic agents, or expression of surface proteins can be used. In the medium treated with the selective agent, only cells expressing the selectable marker survive, allowing the selection of transformed cells. In addition, when the vector is a replicable expression vector, then such a vector may contain an origin of replication, which is a specific nucleic acid sequence from which replication is initiated.

As a recombinant expression vector for inserting a foreign gene, various forms of vectors such as plasmids, viruses, and cosmids can be used. The type of recombinant vector is not specifically limited, as long as the vector functions to express the desired gene and produce the desired protein in various host cells, including prokaryotic and/or eukaryotic cells. The vector may preferably be a vector capable of producing a large amount of foreign protein, which is in a form similar to its natural state, while having a promoter with high activity and high expression ability.

Various host/expression vector combinations can be used to express the anti-MSLN antibody. An expression vector suitable for eukaryotic hosts includes, but is not limited to, expression control sequences derived from SV40, bovine papillomavirus, adenovirus, adeno-associated virus, cytomegalovirus, and retrovirus. An expression vector that can be used in bacterial hosts includes Escherichia coli derived bacterial plasmids such as pET, pRSET, pBluescript, pGEX2T, pUC vector, colE1, pCR1, pBR322, pMB9 and their derivatives; plasmids having a wide range of hosts such as RP4; DNA phages, exemplified by the wide range of lambda phage derivatives such as λgt10, λgt11 and NM989; and other DNA phages such as M13 and filamentous single stranded DNA phages. An expression vector suitable for yeast cells may include 2 micron plasmids and their derivatives. A vector suitable for insect cells may be pVL941.

In yet another aspect of the present invention, there is provided a host cell transformed with an expression vector in accordance with the present invention. An expression vector can be inserted into a host cell to form a transformant. A suitable host cell for the vector may include prokaryotic cells such as Escherichia coli, Bacillus subtilis, Streptomyces sp., Pseudo

- 5 043079 monas sp., Proteus mirabilis or Staphylococcus sp. In addition, the host cell may include eukaryotic cells, including lower eukaryotic cells from fungi such as Aspergillus sp., yeasts such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces sp. and Neurospora crassa, and other lower eukaryotes as well as higher eukaryotic cells such as insect cells. In addition, the host cell can also be derived from plants or mammals. Preferably, the host cell that can be used includes, but is not limited to, green monkey kidney cells (COS7 cells), NSO cells (mouse myeloma cells), SP2/0 cells (mouse myeloma cells), other myeloma cells, Chinese ovary cells hamster (CHO) cells, W138 cells (human diploid cell culture), baby hamster kidney (BHK) cells, MDCK cells, HuT 78, HEK293 cells and the like, wherein CHO cells are preferred.

As used herein, the term transformation into host cells is intended to include any method of introducing a nucleic acid into an organism, cell, tissue or organ, and such transformation can be performed using standard techniques known in the art, selected depending on the type of cell - owner. In particular, electroporation, protoplast fusion, calcium phosphate (CaPO ₄ ) precipitation, calcium chloride (CaCl ₂ ) precipitation, silicon carbide fiber agitation, Agrobacterium mediated transformation, PEG, dextran sulfate, lipofectamine mediated transformation or drying/inhibition can be used. or the like. However, the present invention is not limited to these methods.

In yet another aspect of the present invention, there is provided a method for producing an antibody that specifically binds to MSLN, comprising culturing a host cell. In particular, the method for producing an antibody may include the steps of: inserting into a vector a nucleotide sequence encoding an anti-MSLN antibody to construct a recombinant vector; transformation of the host cell with a recombinant vector and culture; and separating and purifying the humanized antibody from the cultured transformant.

Humanized antibodies can be produced in large quantities by culturing a transformant expressing the recombinant vector in a nutrient medium, and the culture medium and conditions can be appropriately selected from those known in the art, depending on the host cell type. During culturing, conditions such as temperature, medium pH, and culturing time can be appropriately adjusted to suit cell growth and bulk protein production.

Recombinantly produced anti-MSLN antibodies as described above can be isolated from the medium or cell lysate. When the antibody is in membrane-bound form, the antibody can be isolated from the membrane using an appropriate surfactant solution (eg, Triton-X 100) or by enzymatic digestion. Cells used to express humanized antibodies can be disrupted using various physical and chemical methods such as freeze-thaw cycles, sonication, mechanical disruption, or the use of cell lysing agents, and separation and purification can be performed using conventional biochemical separation techniques. . The biochemical separation method that can be used includes, but is not limited to, electrophoresis, centrifugation, gel filtration, precipitation, dialysis, chromatography (ion exchange chromatography, affinity chromatography, immunoabsorption chromatography, size exclusion chromatography, or the like), isoelectric focusing, and the like.

In yet another aspect of the present invention, a pharmaceutical composition for the prevention or treatment of oncological diseases is provided, comprising an antibody of the present invention or a fragment thereof.

The type of cancer that can be treated with the pharmaceutical composition can include both solid cancer and blood cancer, and preferably can include, but is not limited to, any type of tumor that expresses MSLN, such as mesothelioma, pancreatic cancer, ovarian cancer, stomach cancer, lung cancer, or endometrial cancer.

The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier. As a pharmaceutically acceptable carrier for oral administration, a binder, a lubricant, a disintegrant, an excipient, a solubilizer, a dispersant, a stabilizer, a suspending agent, a pigment, a fragrance, and the like can be used; a buffer, a preservative, an anesthetic, a solubilizer, an isotonic agent, a stabilizer, and the like can be used in the injectable mixture; and a base, excipient, lubricant, preservative and the like can be used for topical application.

The composition of the pharmaceutical composition of the present invention can be prepared in various ways by mixing with a pharmaceutically acceptable carrier, as described above. For example, for oral administration, the pharmaceutical composition may be in the form of tablets, lozenges, capsules, elixirs, suspensions, syrups, cachets, or the like. For injection, the pharmaceutical composition may be formulated as single dose ampoules or

- 6 043079 forms with multiple doses.

In addition, the pharmaceutical composition may contain a surfactant that can increase the permeability of the membrane. Such surfactants may be derived from steroids or may include cationic lipids such as N-[1-(2,3dioleoyl)propyl-N,N,N-trimethylammonium chloride (DOTMA) or various compounds such as cholesterol hemisuccinate and phosphatidylglycerol. However, the surfactant is not limited to this.

In yet another aspect of the present invention, there is provided a method for treating a cancer or inhibiting the growth of a cancer, comprising administering a pharmaceutical composition to an individual. The pharmaceutical composition containing the anti-MSLN antibody can be administered in a pharmaceutically effective amount to affect tumor cells or their metastases or to inhibit the growth of a malignant tumor. The effective amount may vary depending on various factors such as the type of cancer, age of the patient, body weight, nature and severity of symptoms, type of current therapy, number of treatments, dosage form and route of administration, and can be readily determined by those skilled in the art. .

The pharmaceutical composition may be administered together or sequentially with the above pharmacological or physiological components, and may also be administered in combination with additional conventional therapeutic agents, in which case the pharmaceutical composition may be administered sequentially or simultaneously with conventional therapeutic agents. Such administration may be single or multiple. In view of all of the above factors, it is important to administer an amount that is the minimum amount to provide the maximum effect without causing side effects, and such an amount can be easily determined by one skilled in the art.

As used here, the term subject refers to a mammal, preferably a human, suffering from or at risk of developing a pathological condition or disease that can be alleviated, suppressed or treated by the administration of a pharmaceutical composition.

As used here, the term administration means the administration of a predetermined substance to an individual by any suitable route, and the pharmaceutical composition may be administered by any route, provided that the route allows the pharmaceutical composition to reach the target tissue. Such administration may include, without limitation, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, pulmonary administration, or rectal administration. Here, in the case of oral administration, from the viewpoint that proteins undergo digestion, it may be desirable to formulate the composition for oral administration in such a way that the active agent is coated or the composition is protected from digestion in the stomach. In addition, the pharmaceutical composition can be administered using any device so that the active ingredient can migrate into its target cell.

In yet another aspect of the present invention, the use of an antibody of the present invention for the prevention or treatment of an oncological disease is provided.

In yet another aspect of the present invention, the use of an antibody of the present invention for the manufacture of a medicament for the prevention or treatment of cancer is provided.

In yet another aspect of the present invention, there is provided a method for preventing or treating an oncological disease, comprising administering an antibody of the present invention to an individual.

Best Mode for Carrying Out the Invention

Hereinafter, the present invention will be described in more detail by way of examples. The following examples are described for the purpose of illustrating the present invention, and the scope of the present invention is not limited to this.

Example 1 Preparation of Humanized Anti-MSLN Antibodies

Example 1.1. Selection of candidate antibodies for humanization.

The amino acid sequences of the light chain variable region (VL region) and the heavy chain variable region (VH region) of the murine MI323 antibody, known as the anti-MSLN antibody, were entered into a web database (IgBLAST) and then searched for the most similar human embryonic antibody sequences. As a result, the highest amino acid sequence similarity was shown between the light chain variable region of mouse antibody MI323 and Homo sapiens IGKV7-39*01 (gene name in the IMGT database) and between the heavy chain variable region of mouse antibody MI323 and Homo sapiens IGHV1-3*01 ( gene name in the IMGT database).

Example 1.2. Humanization of the light chain variable region

The amino acid sequence of the CDR of Homo sapiens IGKV7-39*01 (gene name in the IMGT database), a human embryonic antibody having a sequence most similar to the MI323 light chain variable region, was replaced with the CDR sequence of a mouse antibody

- 7 043079

MI323 to obtain a partially humanized MI323 light chain variable region.

To enhance the antigen-binding properties of the partially humanized light chain variable region MI323, amino acid residues in the CDR sequences that are believed to play an important role in providing antigen-binding properties were replaced with the same amino acid residues as in Homo sapiens IGKV7-39*01 (gene name in IMGT database). The amino acid sequence of the humanized MI323 light chain variable region thus obtained is shown in Table 1. 1 below.

As shown in Table. 1, a humanized light chain variable region of MI323 was obtained by replacing the 24th amino acid residue in the light chain variable region of the mouse MI323 antibody, lysine (K) with arginine (R). Here, the light chain variable region of the murine MI323 antibody was used as a control to compare affinity for the MSLN antigen.

Variable Table 1 Amino acid sequence SEQ Clone region (areas in bold indicate ID light chain CDR1, CDR2, CDR3 in order) DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQ NO VL-1 light chain QKPGKAPKLLIYSASYRYPGVPSRFSGSGSGTDFTTLTI SSLQPEDFATYYCQQHYSTPWTFGGGTKLEIKR 1 MI DIVMTQSHKFMSTSVGDRVSITCKASQDVSTAVAWY 323 light chain QQKPGQSPKLLIYSASYRYPGVPDRFTGSGSGTDFTFT ISSVQAEDLALYYCQQHYSTPWTFGGGTKLEIKR 2

Example 1.3. Humanization of the heavy chain variable region

The amino acid sequence of the CDR of Homo sapiens IGHV 1-3*01 (gene name in the IMGT database), a human embryonic antibody having a sequence most similar to the heavy chain variable region of MI323, was replaced with the CDR sequence of the murine MI323 antibody, to obtain a partially humanized heavy variable region MI323 chains.

To enhance the antigen-binding properties of the partially humanized MI323 heavy chain variable region, amino acid residues in the CDR and framework region (FR) sequences that are believed to play an important role in providing antigen-binding properties were replaced with the same amino acid residues as in the murine MI323 antibody. The amino acid sequence of the humanized MI323 heavy chain variable region thus obtained is shown in Table 1. 2 below.

As shown in Table. 2, random modifications were made to amino acid residues in the CDR and FR of the heavy chain variable region of the murine MI323 antibody, resulting in a total of 3 humanized MI323 heavy chain variable regions. Here, the heavy chain variable region of the murine MI323 antibody was used as a control to compare affinity for the MSLN antigen.

table 2

Clone Variable region Amino acid sequence (areas in bold indicate light chain CDR1, CDR2, CDR3 in order) SEQ ID NO VH-1 heavy chain QVQLVQSGAEVKKPGASVKVSCKASGYSFTSYFMH WVRQAPGQRLEWMGWIFPNGNGNTKYSQKFQGRVT ITRDTSASTAYMELSSLRSEDTAVYYCARSGGYQYYF DYWGQGTLVTVSS 3 VH-2 heavy chain QVQLVQSGAEVKKPGSSVKVSCKASGYSFTSYFISW VRQAPGQGLEWMGGIFPGSGNANYAQKFQGRVTIT ADESTSTAYMELSSLRSEDTAVYYCARSGGYQYYFD YWGQGTLVTVSS 4 VH-3 heavy chain EVQLVQSGAEVKKPGTSVKVSCKASGYSFTSYFIQW VRQAPGQGLEWIGWIFPSGNTKYSQKFQGRVTITR DTSTSTAYMELSSLRSEDTAVYYCARSGGYQYYFDY WGQGTLVTVSS 5 MI 323 heavy chain EVQLQQSGPELVKPGTSVKISCKASGYSFTSYFIQWV KQRPGQGLEWIGWIFPSGNTKYNEMFKGKATLAA DTSSSTAYMQLSSLTSEDSAVYFCARSGGYQYYFDY WGQGTSVTVSS 6

Example 1.4. Cloning of humanized anti-MSLN antibodies

Each light chain variable region gene (VL-1) as obtained above and the light chain variable region gene MI323 were inserted into the pcDNA3.4 animal cell expression vector containing the kappa light chain constant region (k-CL). In addition, each of the genes has 3 variable

- 8 043079 heavy chain regions (VH-1, VH-2, VH-3) and the MI323 heavy chain variable region gene were inserted into the pcDNA3.4 animal cell expression vector containing IgG1 constant regions (CHI, hinge, CH2, SNZ).

The corresponding specific amino acid sequences for the kappa light chain constant region and IgG1 heavy chain constant region are shown in Table 1. 3 below.

Table 3

Clone Constant domain Amino acid sequence SEQ ID NO To light chain TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSK ADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 7 IgGl heavy chain ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVT VSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCP APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVV SV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 8

Example 1.5. Transfection of humanized anti-MSLN antibodies

24 h prior to transfection, Expi293F cells at a density of 2.0x10 ⁶ cells/ml were passaged in Expi293 medium in a shaking incubator at 125±10 rpm at 37°C and 8% CO ₂ . When transfection was performed, the number of cells was measured and the cell viability was assessed to determine if the cell viability was 95% or higher.

Cells were placed at a titer of 7.5 x 10 ⁷ cells in a 125 ml culture flask and then Expi293 medium was added to bring the final volume to 25 ml (based on 30 ml). Using Opti-MEM I medium, 30 μg of the antibody-expressing vector was mixed into a total volume of 1.5 ml and incubated for 5 minutes at room temperature. For antibody vectors, a total of 3 humanized IgG1 MI323 antibodies were used, obtained by combining an expression vector for one light chain variable region and expression vectors for 3 variable heavy chain regions. The chimeric mouse/human IgG1 antibody MI323 was used as a control antibody vector.

Using Opti-MEM I medium, 80 μl of transfection reagent was mixed to a total volume of 1.5 ml and incubated for 5 minutes at room temperature. Opti-MEM I, containing the vector and transfection reagent, respectively, was gently mixed and allowed to react at room temperature for 20 min. The resulting mixture was then placed into a flask containing Expi293F cells. Incubation was carried out in a thermostat with shaking at a speed of 125±10 rpm for 16-20 h at 37°C and 8% CO ₂ . Then 1.5 ml of transfection enhancer I and 150 µl of transfection enhancer II were added and incubated for 6 days to obtain antibodies.

Example 1.6. Purification of antibodies

The incubation mixture was centrifuged at 4000 rpm for 30 min, filtered through a 0.22 μm filter, and then the cell debris was removed to obtain the supernatant. 0.2 ml of Mabselect Xtra resin was added to the column and equilibration was carried out using protein A binding buffer in a volume corresponding to 10 times the volume of the resin.

The supernatant was then loaded onto the column by gravity. After completion of the loading, the column was washed with 10 times the volume of the resin with protein A binding buffer.

Then, IgG eluting buffer was added to the column and elution was performed. The eluate was neutralized by adding 25 μl of 1.5 M Tris-Cl per 1 ml of eluate. Then the concentration of the eluate was measured at OD 280 nm. The eluent for which the concentration was measured was dialyzed with buffer exchange with PBS.

Example 2 Affinity Measurement of Humanized Anti-MSLN Antibodies for Recombinant MSLN

The Octet system was used to measure the recombinant MSLN affinity of humanized anti-MSLN antibodies (HMI323) prepared as described in Example 1. HMI323 refers to humanized MI323.

In particular, a solution of recombinant human MSLN was prepared at a concentration of 5 μg/ml in 1 x kinetic buffer and used to process a 96-well plate at a rate of 200 μl/well. MSLNs after treatment were fixed on an anti-Penta His biosensor (HISIK, Cat # 18-5121, Fortebio).

- 9 043079

Then 3 humanized aHTu-MSLN antibodies prepared as described in Example 1 and MI323 chimeric mouse/human IgG1 clones were diluted to 50, 25, 12.5, 6.25, or 3.125 nM in x kinetic buffer and processed at 200 µl/well. For the preparation of 1 x kinetic buffer, a buffer prepared by 10-fold dilution of 10-fold kinetic buffer (ForteBio, Cat # 18-1092) with PBS was used.

The interaction between the MSLN fixed on the biosensor and the antibody at several concentrations was analyzed to calculate the antigen-antibody affinity, and the results are shown in table. 4 below.

Table 4

Clone ka(1/Ms) kd (1/s) KD (nM) MI323 4.54x105 1.30x10-5 0.0286 HMI323VL-1/HMI323VH-1 6.65x105 9.25x10-5 0.139 HMI323VL-1/HMI323VH-2 1.86x106 5.09x10-4 0.273 HMI323VL-1/HMI323VH-3 8.42x105 2.75x10-5 0.0327

As can be seen from the results presented in Table. 4, the HMI323VL-1/HMI323VH-3 combination was found to retain the closest affinity for MI323, and the other two clones were found to show slightly lower affinity compared to the MI323 antibody, but overall retain high affinity for MSLN.

Example 3 Affinity Measurement of Humanized Anti-MSLN Antibodies for MSLN Expressed on the Surface of Tumor Cells

Flow cytometry was used to identify whether the humanized antiMSLN antibodies (HMI323) prepared as described in Example 1 also showed affinity for MSLN expressed on the surface of tumor cells.

In particular, the cells of each of the H226 (mesothelioma), AsPC-1 (pancreatic cancer), Capan-2 (pancreatic cancer) and PL45 (pancreatic cancer) lines were centrifuged at 1500 rpm for 5 minutes and then three times washed with FACS buffer (PBS containing 3% FBS). Then the appropriate tumor cells at a concentration of 3x10 ⁶ cells/ml were diluted with FACS buffer and used at 100 μl to process each well of a 96-well plate.

Then, each chimeric mouse/human IgG1 antibody MI323 and IgG1 antibody HMI323VL1/HMI323VH-3 were added to each well at a concentration of 5 μg/ml, mixed with cells, and then incubated at 4°C for 1 hour. Then, centrifugation was performed at 1500 rpm. min for 5 min and then the supernatant was discarded. The procedure in which 200 μl of FACS buffer was added to this mixture to resuspend and wash the cells was repeated three times.

Then, the procedure in which the phycoerythrin (PE)-conjugated human IgG antibody was diluted 500-fold with FACS buffer and used to wash the cells was repeated three times. The washed cells were fixed using 100 μl of BD Cytofix™ and the mean fluorescence intensity (MFI) of each sample was analyzed using the LSRFortessa™ instrument (flow cytometer). The results of the analysis are shown in Fig. 1-8.

As shown in FIG. 1-8, it was found that all antibodies that bind to recombinant human MSLN also specifically bind to tumor cells expressing human MSLN.

While the embodiments have been described with the limited examples and figures provided above, it will be apparent to those skilled in the art that various changes and modifications can be made without departing from the spirit and scope of the invention. For example, the desired results can be achieved even when the described techniques are performed in a different order than the described method and/or the described components are formulated or combined in a form different from the described method, or are replaced or substituted with other components or equivalents.

Therefore, other implementations, other embodiments, and equivalents of the appended claims fall within the scope of the appended claims.

Claims (14)

1. An antibody that specifically binds to mesothelin (MSLN) containing a light chain variable region (VL region) consisting of a sequence with the amino acid sequence of SEQ ID NO: 1; and a heavy chain variable region (VH region) consisting of a sequence with an amino acid sequence of any of SEQ ID NOs: 3-5. 2. The antibody of claim 1, wherein the MSLN is a human MSLN. 3. The antibody of claim 1, wherein the antibody specifically binds to tumor cells expressing MSLNs. - 10 043079 4. The antibody of claim 3, wherein the MSLN-expressing tumor cell is a human tumor cell. 5. The antibody of claim 1, wherein the antibody is a humanized antibody. 6. A polynucleotide that encodes the light chain variable region (VL region) and the heavy chain variable region (VH region) of the antibody of claim 1. 7. An expression vector containing a polynucleotide according to claim 6. 8. Host cell transformed with the expression vector according to claim 7. 9. A method for producing an antibody that specifically binds to MSLN, comprising culturing a host cell according to claim 8. 10. Pharmaceutical composition for the prevention or treatment of cancer, containing the antibody according to claim 1 or a fragment thereof. 11. The pharmaceutical composition of claim 10 wherein the cancer is mesothelioma, pancreatic cancer, ovarian cancer, gastric cancer, lung cancer, or endometrial cancer. 12. The use of an antibody according to claim 1 for the prevention or treatment of cancer. 13. The use of an antibody according to claim 1 for the manufacture of a medicament for the prevention or treatment of cancer. 14. A method for preventing or treating an oncological disease, comprising administering an antibody according to claim 1 to an individual.