Fusion protein comprising an anti-methotrexate antibody, an anti-CD3 antibody or an anti-EGFR antibody, bispecific or trispecific antibody comprising the same, and use thereof

Abstract

The present invention relates to a fusion protein containing a fragment of an anti-mesothelin antibody, an anti-CD3 antibody, or an anti-EGFR antibody, a bispecific antibody specific for mesothelin and CD3, and a trispecific antibody specific for mesothelin, CD3, and EGFR, and uses thereof. The bispecific or trispecific antibodies described in the present invention can be prepared with high yield and purity and have excellent tumoricidal and growth-inhibitory effects, making them effective for cancer treatment.

Description

Technical Field

[0001] The present invention relates to a fusion protein containing an anti - mesothelin antibody, an anti - CD3 antibody or an anti - EGFR antibody, a bispecific antibody specific for mesothelin and CD3, a trispecific antibody specific for mesothelin, CD3 and EGFR, and their use.

[0002] [Background Art] In cancer treatment, monoclonal antibodies as the first cancer immunotherapy show excellent effects not only on solid cancers but also on blood cancers, but still have limitations. Regarding this, techniques for manipulating the Fc region of antibodies to enhance the antibody - dependent cell - mediated cytotoxicity (ADCC) effect of antibodies, and techniques for developing bispecific or trispecific antibodies so that one antibody can access multiple targets have been introduced.

[0003] A bispecific antibody is an antibody that can bind to two different antibodies simultaneously, and immune cells such as T cells can be manipulated so that they are toxic only to specific target cells such as cancer cells and not to other normal cells. Such bispecific antibodies can exert the maximum cancer treatment effect while minimizing side effects. Therefore, several bispecific antibody formats have been developed, and their suitability for immunotherapy via T cells has been reported.

[0004] However, when co - expressed during the production of bispecific antibodies, there is a problem that the heavy and light chains of antibodies with different specificities are mispaired, resulting in the generation of a large number of non - functional by - products.

[0005] Therefore, in order to solve this problem, the development of a technology for producing the desired multispecific antibody construct in a clinically sufficient amount and purity is required.

[0006] [Disclosure of the Invention] [Technical Problem] As a result of intensive research to solve the problems of existing bispecific antibodies, the present inventors have developed a fusion protein in which the heavy chain and light chain of an antibody are linked via a linker and contained as a single chain, and a bispecific or trispecific antibody containing the same. In addition, the present inventors have found that these fusion proteins and antibodies solve the problem of mispairing and have a high killing effect on tumor cells, thereby completing the present invention.

[0007] As a result, an object of the present invention is to provide a fusion protein containing an anti-mesothelin antibody, an anti-CD3 antibody, or an anti-EGFR antibody, a bispecific or trispecific antibody containing the same, and a method for treating cancer using the same.

[0008] [Means for Solving the Problems] In order to solve the above problems, in one aspect of the present invention, a first fusion protein in a single-chain form containing an antibody that specifically binds to mesothelin and an antibody that specifically binds to CD3 is provided.

[0009] In another aspect of the present invention, a second fusion protein in a single-chain form containing an antibody that specifically binds to mesothelin is provided.

[0010] In yet another aspect of the present invention, a third fusion protein in a single-chain form containing an antibody that specifically binds to EGFR is provided.

[0011] In still another aspect of the present invention, a bispecific antibody containing the first fusion protein and the second fusion protein is provided.

[0012] In still another aspect of the present invention, a trispecific antibody containing the first fusion protein and the third fusion protein is provided.

[0013] In still another aspect of the present invention, a pharmaceutical composition for treating cancer containing a fusion protein, a bispecific antibody, or a trispecific antibody as an active ingredient is provided.

[0014] In yet another aspect of the present invention, the use of a fusion protein, bispecific antibody, or trispecific antibody is provided.

[0015] In yet another aspect of the present invention, a method for treating cancer is provided, which includes the step of administering to an individual a pharmaceutical composition for treating cancer in a therapeutically effective amount.

[0016] [Advantages of the Invention] The fusion protein of the present invention and the bispecific antibody or trispecific antibody containing the same can be produced in high yield and high purity, and have excellent anti-tumor and growth inhibitory effects, so they can be effectively used for cancer treatment. [Brief Description of the Drawings]

[0017]

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BEST MODE FOR CARRYING OUT THE INVENTION

[0018] In one aspect of the present invention, Structural Formula 1 [Structural Formula 1] N’-A-B-L1-C-D-L2-E-F-L3-G-H-L4-I-C’ (In the formula, N’ is the N-terminus of the fusion protein, C’ is the C-terminus of the fusion protein, A is the light chain variable domain of an antibody that specifically binds to mesothelin, and the domain includes the light chain CDR1 of SEQ ID NO: 25, the light chain CDR2 of SEQ ID NO: 26, and the light chain CDR3 of SEQ ID NO: 27, B is the light chain constant domain of the antibody, C is the heavy chain variable domain of an antibody that specifically binds to mesothelin, and the domain includes the heavy chain CDR1 of SEQ ID NO: 28, the heavy chain CDR2 of SEQ ID NO: 29, and the heavy chain CDR3 of SEQ ID NO: 30, D is the CH1 domain in the heavy chain constant domain of the antibody, E is the light chain variable domain of an antibody that specifically binds to CD3, and the domain includes the light chain CDR1 of SEQ ID NO: 31, the light chain CDR2 of SEQ ID NO: 32, and the light chain CDR3 of SEQ ID NO: 33, F is the light chain constant domain of the antibody, G is the heavy chain variable domain of an antibody that specifically binds to CD3, and the domain includes the heavy chain CDR1 of SEQ ID NO: 34, the heavy chain CDR2 of SEQ ID NO: 35, and the heavy chain CDR3 of SEQ ID NO: 36, H is the CH1 domain in the heavy chain constant domain of the antibody, I is the Fc region or a variant thereof, and each of L1, L2, L3, and L4 is a peptide linker) A first fusion protein having the above is provided.

[0019] In the present invention, each of L1, L2, L3, and L4 may be a peptide linker consisting of 1 to 50 amino acids.

[0020] In the present invention, L1 and L3 may be peptide linkers consisting of the amino acid sequence of SEQ ID NO: 43.

[0021] In the present invention, L2 may be a peptide linker consisting of the amino acid sequence of SEQ ID NO: 44.

[0022] In the present invention, L4 may be a peptide linker consisting of the amino acid sequence of SEQ ID NO: 45.

[0023] In the present invention, L4 may be the hinge region of an antibody.

[0024] In the present invention, A may be the variable domain of the light chain of an antibody that specifically binds to mesothelin. The domain consists of the amino acid sequence of SEQ ID NO: 7, or consists of an amino acid sequence having at least 95%, preferably at least 99% homology to the amino acid sequence of SEQ ID NO: 7.

[0025] In the present invention, B may be the constant domain of the light chain of an IgG, IgA, IgM, IgD, or IgE antibody (preferably an antibody that specifically binds to mesothelin). The domain consists of the amino acid sequence of SEQ ID NO: 46, or consists of an amino acid sequence having at least 95%, preferably at least 99% homology to the amino acid sequence of SEQ ID NO: 46.

[0026] In the present invention, C may be the variable domain of the heavy chain of an antibody that specifically binds to mesothelin. The domain consists of the amino acid sequence of SEQ ID NO: 9, or consists of an amino acid sequence having at least 95%, preferably at least 99% homology to the amino acid sequence of SEQ ID NO: 9.

[0027] In the present invention, D may be the CH1 domain in the constant domain of the heavy chain of an IgG, IgA, IgM, IgD, or IgE antibody (preferably an antibody that specifically binds to mesothelin). The domain consists of the amino acid sequence of SEQ ID NO: 47, or consists of an amino acid sequence having at least 95%, preferably at least 99% homology to the amino acid sequence of SEQ ID NO: 47.

[0028] In the present invention, E may be the variable domain of the light chain of an antibody that specifically binds to CD3, and the domain consists of the amino acid sequence of SEQ ID NO: 13, or consists of an amino acid sequence having at least 95%, preferably at least 99% homology to the amino acid sequence of SEQ ID NO: 13.

[0029] In the present invention, F may be the constant domain of the light chain of an IgG, IgA, IgM, IgD, or IgE antibody (preferably an antibody that specifically binds to CD3), and the domain consists of the amino acid sequence of SEQ ID NO: 46, or consists of an amino acid sequence having at least 95%, preferably at least 99% homology to the amino acid sequence of SEQ ID NO: 46.

[0030] In the present invention, G may be the variable domain of the heavy chain of an antibody that specifically binds to CD3, and the domain consists of the amino acid sequence of SEQ ID NO: 15, or consists of an amino acid sequence having at least 95%, preferably at least 99% homology to the amino acid sequence of SEQ ID NO: 15.

[0031] In the present invention, H may be the CH1 domain in the constant domain of the heavy chain of an IgG, IgA, IgM, IgD, or IgE antibody (preferably an antibody that specifically binds to CD3), and the domain consists of the amino acid sequence of SEQ ID NO: 47, or consists of an amino acid sequence having at least 95%, preferably at least 99% homology to the amino acid sequence of SEQ ID NO: 47.

[0032] In the present invention, I may be the Fc region of an IgG, IgA, IgM, IgD, or IgE antibody or a variant thereof, and the region or variant consists of the amino acid sequence of SEQ ID NO: 48 or consists of an amino acid sequence having at least 95%, preferably at least 99% homology to the amino acid sequence of SEQ ID NO: 48. Further, I may be obtained by substitution of some amino acids in the CH3 domain of the heavy chain constant domain of the antibody. That is, the amino acid sequence of SEQ ID NO: 48 may be substituted (Y119C, K130E, and K179W), and a knob structure may be formed in the CH3 domain of the heavy chain constant domain of the antibody, or the amino acid sequence of SEQ ID NO: 48 may be substituted (Q117R, S124C, D169V, and F175T), and a hole structure may be formed therein.

[0033] In the present invention, the first fusion protein may consist of the amino acid sequence of SEQ ID NO: 1 or may consist of an amino acid sequence having at least 90%, at least 95%, preferably at least 99% homology to the amino acid sequence of SEQ ID NO: 1.

[0034] In one embodiment of the present invention, structural formula 2 [Structural formula 2] N’-A-B-L1-C-D-L5-J-C’ (In the formula, N’ is the N-terminus of the fusion protein, C’ is the C-terminus of the fusion protein, A is the light chain variable domain of an antibody that specifically binds to mesothelin, and the domain includes the light chain CDR1 of SEQ ID NO: 25, the light chain CDR2 of SEQ ID NO: 26, and the light chain CDR3 of SEQ ID NO: 27, B is the light chain constant domain of the antibody, C is the heavy chain variable domain of an antibody that specifically binds to mesothelin, and the domain includes the heavy chain CDR1 of SEQ ID NO: 28, the heavy chain CDR2 of SEQ ID NO: 29, and the heavy chain CDR3 of SEQ ID NO: 30, D is the CH1 domain in the heavy chain constant domain of the antibody, J is the Fc region or a fragment thereof, Each of L1 and L5 is a peptide linker) A second fusion protein having is provided.

[0035] In the present invention, each of L1 and L5 may be a peptide linker consisting of 1 to 50 amino acids.

[0036] In the present invention, L1 may be a peptide linker consisting of the amino acid sequence of SEQ ID NO: 43.

[0037] In the present invention, L5 may be a peptide linker consisting of the amino acid sequence of SEQ ID NO: 45.

[0038] In the present invention, L5 may be the hinge region of an antibody.

[0039] In the present invention, the second fusion protein may consist of the amino acid sequence of SEQ ID NO: 3, or may consist of an amino acid sequence having at least 90%, at least 95%, preferably at least 99% homology to the amino acid sequence of SEQ ID NO: 3.

[0040] J may be the Fc region or a variant thereof of an IgG, IgA, IgM, IgD, or IgE antibody, and the region or variant consists of the amino acid sequence of SEQ ID NO: 48, or consists of an amino acid sequence having at least 95%, preferably at least 99% homology to the amino acid sequence of SEQ ID NO: 48. Further, J may be obtained by substitution of some amino acids in the CH3 domain of the heavy chain constant domain of the antibody. That is, the amino acid sequence of SEQ ID NO: 48 undergoes substitution (Y119C, K130E, and K179W), and a knob structure may be formed in the CH3 domain of the heavy chain constant domain of the antibody, or the amino acid sequence of SEQ ID NO: 48 undergoes substitution (Q117R, S124C, D169V, and F175T), and a hole structure may be formed therein.

[0041] In one aspect of the present invention, Structural Formula 3 [Structural Formula 3] N'-K-M-L6-N-O-L7-P-C' (In the formula, N' is the N-terminus of the fusion protein, C' is the C-terminus of the fusion protein, K is the light chain variable domain of an antibody that specifically binds to EGFR, and the domain includes the light chain CDR1 of SEQ ID NO: 37, the light chain CDR2 of SEQ ID NO: 38, and the light chain CDR3 of SEQ ID NO: 39, M is the light chain constant domain of the antibody, N is the heavy chain variable domain of an antibody that specifically binds to EGFR, and the domain includes the heavy chain CDR1 of SEQ ID NO: 40, the heavy chain CDR2 of SEQ ID NO: 41, and the heavy chain CDR3 of SEQ ID NO: 42, O is CH1 in the heavy chain constant domain of the antibody, P is an Fc region or a fragment thereof, and each of L6 and L7 is a peptide linker). A third fusion protein having the same is provided.

[0042] In the present invention, each of L6 and L7 may be a peptide linker consisting of 1 to 50 amino acids.

[0043] In the present invention, L6 may be a peptide linker consisting of the amino acid sequence of SEQ ID NO: 43.

[0044] In the present invention, L7 may be a peptide linker consisting of the amino acid sequence of SEQ ID NO: 45.

[0045] In the present invention, L7 may be the hinge region of the antibody.

[0046] In the present invention, the third fusion protein may consist of the amino acid sequence of SEQ ID NO: 5.

[0047] In the present invention, K may be the light chain variable domain of an antibody that specifically binds to EGFR, and the domain consists of the amino acid sequence of SEQ ID NO: 17, or consists of an amino acid sequence having at least 95%, preferably at least 99% homology to the amino acid sequence of SEQ ID NO: 17.

[0048] In the present invention, M may be the light chain constant domain of an IgG, IgA, IgM, IgD, or IgE antibody (preferably an antibody that specifically binds to EGFR), and the domain consists of the amino acid sequence of SEQ ID NO: 46, or consists of an amino acid sequence having at least 95%, preferably at least 99% homology to the amino acid sequence of SEQ ID NO: 46.

[0049] In the present invention, N may be the heavy chain variable domain of an antibody that specifically binds to EGFR, and the domain consists of the amino acid sequence of SEQ ID NO: 19, or consists of an amino acid sequence having at least 95%, preferably at least 99% homology to the amino acid sequence of SEQ ID NO: 19.

[0050] In the present invention, O may be the CH1 domain in the heavy chain constant domain of an IgG, IgA, IgM, IgD, or IgE antibody (preferably an antibody that specifically binds to EGFR), and the domain consists of the amino acid sequence of SEQ ID NO: 47, or consists of an amino acid sequence having at least 95%, preferably at least 99% homology to the amino acid sequence of SEQ ID NO: 47.

[0051] J may be the Fc region of an IgG, IgA, IgM, IgD, or IgE antibody or a variant thereof, and the region or variant consists of the amino acid sequence of SEQ ID NO: 48 or an amino acid sequence having at least 95%, preferably at least 99% homology to the amino acid sequence of SEQ ID NO: 48. Further, I may be obtained by substitution of some amino acids in the CH3 domain of the heavy chain constant domain of an IgG, IgA, IgM, IgD, or IgE antibody. That is, the amino acid sequence of SEQ ID NO: 48 may be substituted (Y119C, K130E, and K179W), and a knob structure may be formed in the CH3 domain of the heavy chain constant domain of the antibody. The amino acid sequence of SEQ ID NO: 48 may be substituted (Q117R, S124C, D169V, and F175T), and a hole structure may be formed therein.

[0052] In the present invention, the third fusion protein may consist of the amino acid sequence of SEQ ID NO: 5 or an amino acid sequence having at least 90%, at least 95%, preferably at least 99% homology to the amino acid sequence of SEQ ID NO: 5.

[0053] As used herein, the term "mesothelin (MSLN)" means wild-type MSLN present in animals, preferably humans and monkeys, and variants, isotypes, and paralogs thereof. Further, "human MSLN" refers to MSLN derived from humans. Mesothelin is a 40 kDa cell surface glycoprotein present on normal mesothelial cells and is overexpressed in several human tumors such as mesothelioma, ovarian adenocarcinoma, and pancreatic adenocarcinoma. Mesothelin has been shown to exhibit megakaryocyte colony-forming activity in the presence of interleukin 3. Mesothelin is a tumor differentiation antigen and is present at low levels in normal adult tissues such as mesothelium, but is abnormally overexpressed in a variety of human tumors such as mesothelioma, ovarian cancer, pancreatic cancer, cervical cancer, head and neck cancer, vulvar cancer, lung cancer, esophageal squamous cell carcinoma, lung adenocarcinoma, endometrial cancer, biphasic synovial sarcoma, fibromatosis round cell tumor, and gastric adenocarcinoma.

[0054] As used herein, the term "CD3" means wild-type CD3 present in animals, preferably humans and monkeys, and its variants, isotypes, and paralogs. Also, "human CD3" refers to CD3 derived from humans. CD3, an antigen molecule present on the surface of T cells, functions as a signal transduction system by binding to the T cell antigen receptor, thereby activating T cells.

[0055] As used herein, the term "EGFR" refers to a membrane protein that is an epidermal growth factor receptor and is also called ErbB-1 or HER1. Further, "human EGFR" refers to EGFR derived from humans.

[0056] As used herein, the term "antibody" refers to an immunoglobulin (Ig) molecule that immunologically reacts with a specific antigen, that is, a protein molecule that acts as a receptor specifically recognizing the antigen. Antibody is used as a concept encompassing whole antibodies and antibody fragments.

[0057] As used herein, the term "heavy chain" means both full-length heavy chains and fragments thereof, and the full-length heavy chain contains a variable domain (VH) sufficient to confer specificity for the antigen and three constant domains (CH1, CH2, CH3).

[0058] As used herein, the term "light chain" means both full-length light chains and fragments thereof, and the full-length light chain contains a variable domain (VL) sufficient to confer specificity for the antigen and a constant domain (CL).

[0059] As used herein, the term "CDR" refers to the complementarity-determining regions that are part of the variable domains of the heavy chain (VH) and light chain (VL) of an antibody.

[0060] As used herein, the term "Fc" refers to the C-terminal region of an immunoglobulin, and this region is one of the functional structural units consisting of only CH2 and CH3 among the heavy chain constant domains. Fc does not have the ability to bind to an antigen. However, Fc exhibits complement-binding activity and has a certain amino acid sequence.

[0061] In this specification, the term "hinge region" refers to a peptide that exists between CH1 and CH2 of the heavy chain. The hinge region has a highly flexible structure in which one or multiple cysteine residues are present, and the two heavy chains are linked to each other by disulfide bond(s).

[0062] In the present invention, the hinge region may contain 1 to 3 cysteine residues, for example, 1, 2, or 3 cysteine residues.

[0063] Furthermore, in the present invention, a polynucleotide encoding the above fusion protein is provided.

[0064] In the present invention, the DNA encoding the first fusion protein may consist of the nucleotide sequence of SEQ ID NO: 2, or may consist of a nucleotide sequence having at least 80%, preferably at least 90%, more preferably at least 95%, and most preferably at least 99% homology to the nucleotide sequence of SEQ ID NO: 2.

[0065] In the present invention, the DNA encoding the second fusion protein may consist of the nucleotide sequence of SEQ ID NO: 4, or may consist of a nucleotide sequence having at least 80%, preferably at least 90%, more preferably at least 95%, and most preferably at least 99% homology to the nucleotide sequence of SEQ ID NO: 4.

[0066] In the present invention, the DNA encoding the third fusion protein may consist of the nucleotide sequence of SEQ ID NO: 6, or may consist of a nucleotide sequence having at least 80%, preferably at least 90%, more preferably at least 95%, and most preferably at least 99% homology to the nucleotide sequence of SEQ ID NO: 6.

[0067] Furthermore, in the present invention, an expression vector containing the polynucleotide encoding the above fusion protein is provided.

[0068] Furthermore, the present invention provides a host cell transfected by introduction of an expression vector.

[0069] Furthermore, the present invention provides a method for producing a fusion protein, which includes the step of culturing a host cell.

[0070] In another aspect of the present invention, a bispecific antibody comprising a first fusion protein and a second fusion protein is provided.

[0071] As used herein, the term "bispecific antibody" refers to a single antibody engineered to recognize two different antigens.

[0072] The bispecific antibody of the present invention simultaneously contains an anti-MSLN antibody and an anti-CD3 antibody, and thus can specifically bind to cancer cells expressing MSLN and T cells, and induce activation of immune cells, thereby suppressing the growth or killing cancer cells expressing MSLN.

[0073] In the present invention, in order to provide a single antibody, i.e., a bispecific antibody, by the binding of a first fusion protein to a second fusion protein, each fusion protein may be substituted with some amino acid residues of the heavy chain constant domain. That is, the Fc regions of the first fusion protein and the second fusion protein may be linked to each other via a disulfide bond or a knob-into-hole structure (the knob-into-hole structure is preferred) to form a bispecific antibody.

[0074] As used herein, the term "knob-into-hole structure" refers to a structure obtained by inducing mutations in the respective CH3 domains of two different Ig heavy chains, inducing a knob structure in one Ig heavy chain CH3 domain and a hole structure in the other Ig heavy chain CH3 domain, and the two domains forming a heterodimer.

[0075] Generally, among the amino acid residues forming the knob structure, hydrophobic amino acid residues having large side chains are replaced with hydrophobic amino acid residues having small side chains, and among the amino acid residues forming the hole structure, hydrophobic amino acid residues having small side chains are replaced with hydrophobic amino acid residues having large side chains. However, the present invention is not limited thereto. Thus, when each antibody is induced to form a knob structure and a hole structure, heterodimers may be more easily formed than homodimers.

[0076] In the present invention, the bispecific antibody may include a knob modification in I of the first fusion protein and a hole modification in J of the second fusion protein. Alternatively, the bispecific antibody may include a hole modification in I of the first fusion protein and a knob modification in J of the second fusion protein.

[0077] In the present invention, the bispecific antibody may be in the form of 2+1 IgG, and the heavy and light chains constituting the anti-MSLN fragment or the anti-CD3 Fab fragment are linked to each other with a linker such that each fragment is expressed as a single chain, and the anti-MSLN fragment and the anti-CD3 Fab fragment, which are single chains, may consist of two single chains obtained by linking them to each other with a peptide linker.

[0078] In another aspect of the present invention, a trispecific antibody is provided that includes a first fusion protein and a third fusion protein.

[0079] As used herein, the term "trispecific antibody" refers to a single antibody engineered to recognize three different antigens.

[0080] Since the trispecific antibody of the present invention simultaneously includes an anti-EGFR antibody, an anti-MSLN antibody, and an anti-CD3 antibody, it can specifically bind to cancer cells expressing EGFR or MSLN and T cells, and cancer cells expressing EGFR or MSLN are growth-inhibited or killed by inducing activation of immune cells.

[0081] In the present invention, in order to provide a single antibody, i.e., a trispecific antibody, by binding a first fusion protein to a third fusion protein, each fusion protein may be substituted with some amino acid residues of the heavy chain constant domain. That is, the Fc regions of the first fusion protein and the third fusion protein may be linked to each other via a disulfide bond or a knob-into-hole structure (the knob-into-hole structure is preferred) to form a trispecific antibody.

[0082] In the present invention, the trispecific antibody may include a knob modification at I of the first fusion protein and a hole modification at P of the third fusion protein. Alternatively, the trispecific antibody may include a hole modification at I of the first fusion protein and a knob modification at P of the third fusion protein.

[0083] In the present invention, the trispecific antibody may be in the form of 2+1 IgG. The heavy chain and the light chain constituting the anti-EGFR Fab fragment are linked to each other with a linker so that the fragment is expressed as a single chain. The heavy chain and the light chain constituting the anti-MSLN or anti-CD3 Fab fragment are linked to each other with a linker so that each fragment is expressed as a single chain. And it may consist of two single chains obtained by linking the anti-MSLN fragment, which is a single chain, and the anti-CD3 Fab fragment to each other with a peptide linker.

[0084] In yet another aspect of the present invention, there is provided a pharmaceutical composition for treating cancer, comprising as an active ingredient a first fusion protein, a second fusion protein, a third fusion protein, a bispecific antibody, or a trispecific antibody, and a method for treating cancer in an individual, comprising the step of administering to the individual a therapeutically effective amount of the pharmaceutical composition.

[0085] The pharmaceutical composition may further contain a pharmaceutically acceptable carrier. Examples of pharmaceutically acceptable carriers include binders, glidants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, pigments, flavors, etc. In the case of oral administration, these may be used, and buffers, preservatives, analgesics, solubilizers, isotonic agents, stabilizers, etc. may be used in the injection mixture, and bases, excipients, lubricants, preservatives, etc. may be used for topical administration.

[0086] The preparation of the pharmaceutical composition can be carried out in various ways by being mixed with a pharmaceutically acceptable carrier as described above. For example, for oral administration, the pharmaceutical composition can be formulated in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc. For injection, the pharmaceutical composition can be formulated in the form of single-dose ampoules or multi-dose forms.

[0087] The pharmaceutical composition can be administered in a pharmaceutically effective amount for treating cancer cells or their metastasis, or for suppressing the growth of cancer. The effective amount may vary depending on various factors such as the type of cancer, the age, weight, nature and severity of the patient's symptoms, the type of current treatment method, the number of treatments, the dosage form, the administration route, etc., and can be easily determined by an expert in the corresponding field.

[0088] The pharmaceutical composition may be administered together with or sequentially to the above-described pharmacological or physiological components, and may further be administered in combination with a conventional therapeutic agent. In that case, the pharmaceutical composition may be administered sequentially or simultaneously with the conventional therapeutic agent. Such administration may be a single administration or multiple administrations. Considering all of the above elements, it is important to administer an amount that is the minimum and can obtain the maximum effect without side effects, and such an amount can be easily determined by those skilled in the art.

[0089] In the present invention, the cancer can include any cancer known in the art without limitation.

[0090] In yet another aspect of the present invention, there is provided the use of a first fusion protein, a second fusion protein, a third fusion protein, a bispecific antibody, or a trispecific antibody for the manufacture of a medicament for treating cancer in an individual in need of cancer treatment.

[0091] [Embodiments of the Invention] Hereinafter, in order to facilitate the understanding of the present invention, preferred examples are provided. However, the following examples are merely given to facilitate the understanding of the present invention, and the scope of the present invention is not limited thereto.

[0092] Example 1 Production of Anti-MSLN / anti-CD3 Bispecific Antibody and Anti-EGFR / anti-MSLN / anti-CD3 Trispecific Antibody Example 1.1 Production of Antibodies Specific for MSLN, CD3, or EGFR To select an antibody specific for MSLN, mice were immunized with recombinant human MSLN, and then B cells were extracted therefrom, and various antibody genes were cloned. Using phage display, an antibody library was prepared using these genes, and an antibody that binds to recombinant human MSLN was cloned. Finally, humanization of the antibody was performed, and the HMI323 clone consisting of the amino acid sequence of SEQ ID NO: 1 was obtained, and this amino acid sequence is encoded by a gene consisting of the nucleotide sequence of SEQ ID NO: 4.

[0093] To select antibodies specific for human and monkey CD3, the mouse SP34 antibody was humanized, and antibodies that bind to CD3 with various affinities were selected. Among these, clone A07 consisting of the amino acid sequence of SEQ ID NO: 21, clone A15 consisting of the amino acid sequence of SEQ ID NO: 11, and clone E15 consisting of the amino acid sequence of SEQ ID NO: 23 were obtained, and these amino acid sequences are encoded by genes consisting of the nucleotide sequences of SEQ ID NO: 22, 12, and 24, respectively.

[0094] For the EGFR-specific antibody, cetuximab, which is a therapeutic agent for colorectal cancer, was used. An antibody consisting of the amino acid sequence of SEQ ID NO: 5 was obtained, and this amino acid sequence is encoded by a gene consisting of the nucleotide sequence of SEQ ID NO: 6.

[0095] Example 1.2 Transfection of the Antibody Expression Vector Example 1.2.1 Construction of the Antibody Expression Vector As shown in Figure 1, co-expression of the anti-MSLN antibody (HMI323) and the antibody obtained by linking anti-CD3 Fab (A15) to anti-MSLN Fab (HMI323) was performed, and the vector was constructed as follows so that the bispecific antibody (HMI323 / HMI323-A15) having a knob-into-hole structure was expressed.

[0096] Using In-fusionHD (Takara, Mountain View, CA, USA), the DNA of the anti-MSLN (HMI323) antibody sequence (SEQ ID NO: 4) or the DNA of the antibody sequence obtained by linking anti-CD3 Fab (A15) to anti-MSLN Fab (HMI323) (SEQ ID NO: 2) was inserted into each pCI vector (Promega, Madison, WI, USA), and then heat shock transformation at 42°C for 45 seconds was performed using E. coli competent cells. The transformants were seeded on LB plates containing carbenicillin and cultured in an incubator at 37°C for 14 hours. After culturing, the transformant colonies cultured on the plate were inoculated into 2 ml of LB medium containing carbenicillin and cultured in a shaking incubator at 37°C for 16 hours. Then, plasmids were extracted from the cultured transformants, and sequencing showed that the gene for antibody expression was cloned into the vector.

[0097] As shown in Figure 2, co-expression of the anti-EGFR antibody (cetuximab) and the antibody obtained by linking anti-CD3 Fab (A15) to anti-MSLN Fab (HMI323) was performed, and the vector was constructed as follows so that the trispecific antibody (cetuximab / HMI323-A15) having a knob-into-hole structure was expressed.

[0098] Using In-fusionHD (Takara, Mountain View, CA, USA), the DNA of the anti-EGFR (cetuximab) antibody sequence (SEQ ID NO: 6), or the DNA of the antibody sequence (SEQ ID NO: 2) obtained by linking anti-CD3 Fab (A15) to anti-MSLN Fab (HMI323) was inserted into each pCI vector (Promega, Madison, WI, USA). Then, using Escherichia coli competent cells, heat shock transformation was performed at 42°C for 45 seconds. The transformants were seeded on LB plates containing carbenicillin and cultured in an incubator at 37°C for 14 hours. After culturing, the transformant colonies cultured on the plate were inoculated into 2 ml of LB medium containing carbenicillin and cultured in a shaking incubator at 37°C for 16 hours. Subsequently, plasmids were extracted from the cultured transformants, and sequencing showed that the gene for antibody expression was cloned into the vector.

[0099] Example 1.2.2 Transfection of the Antibody Expression Vector into Host Cells Twenty-four hours before, Expi293F™ cells (Thermo Fisher Scientific) were subcultured at a density of 2.0×10 6 cells / ml using Expi293™ Expression Medium (Thermo Fisher Scientific) in a shaking incubator at 125 ± 10 rpm, 37°C, and 8% CO2. At the time of transfection, the cell number and cell viability were measured to confirm whether the cell viability was 95% or more. Five × 10 8 cells were dispensed into a 500 mL culture flask, and then Expi293™ Expression Medium was added to adjust the final volume to 170 mL (based on 200 mL).

[0100] Using Opti-MEM I (trademark) medium (Thermo Fisher Scientific), 200 μg of the antibody expression vector was mixed to a total volume of 1,500 μl and cultured at room temperature for 5 minutes. Using Opti-MEM I (trademark) medium, 540 μl of the transfection reagent (ExpiFectamine (trademark) 293 Reagent, Thermo Fisher Scientific) was mixed to a total volume of 1,500 μl and cultured at room temperature for 5 minutes. The Opti-MEM I (trademark) medium containing the vector and the transfection reagent was gently mixed and reacted at room temperature for 20 minutes. Then, the resulting mixture was placed into a flask containing Expi293F (trademark) cells.

[0101] The culture was carried out in a shaking incubator at 37 °C and 8% CO2 at 125 ± 10 rpm for 16 - 20 hours. Then, 1 ml of the transfection enhancer I (ExpiFectamine (trademark) 293 Enhancer I, Thermo Fisher Scientific) and 10 ml of the transfection enhancer II (ExpiFectamine (trademark) 293 Enhancer II, Thermo Fisher Scientific) were added, and the culture was continued for 5 days to obtain the candidate antibody.

[0102] Example 2 Purification of Anti-MSLN / Anti-CD3 Bispecific Antibody and Anti-EGFR / Anti-MSLN / Anti-CD3 Trispecific Antibody Example 2.1 Purification Using Affinity Chromatography As an experiment to isolate only antibodies using the specific interaction between protein A and antibodies, the culture solution was centrifuged at 4000 rpm for 30 minutes, filtered through a 0.22 μm bottle-top filter, and the supernatant with cell debris removed was prepared. Subsequently, a column filled with 5 ml of Mabselect PrismA resin (GE Healthcare) was connected to an AKTA PrimePlus (GE Healthcare) and used. Then, washing with a binding buffer (Pierce) was performed, and then the supernatant was loaded at a rate of 5 ml / min. After loading all of the prepared supernatant, washing with the binding buffer (Pierce) was performed at the same rate to remove non-specific binding.

[0103] After washing, while flowing the IgG elution buffer (Pierce), the portions where the value of UV280 nm increased were fractionated into test tubes containing 5 ml of the binding buffer (Pierce) in 5 ml aliquots, and the eluate was immediately neutralized. The neutralized eluate was subjected to buffer exchange with PBS using a Zeba spin desalting column (Thermo Fisher Scientific). Example 2.2 Purification using size exclusion chromatography (SEC) As an experiment to separate only the 200 kDa target antibody among the separated antibodies by size-based separation and purification method, a Superdex 200 SEC column (GE Healthcare) was connected to an AKTA pure 150L apparatus (GE Healthcare) and used. The antibody sample that had been subjected to buffer exchange with PBS was loaded into the sample loop and connected to the AKTA pure 150L apparatus (GE Healthcare). Then, the process was carried out under a flow rate condition of 1 ml / min, and the eluted samples were fractionated into 1 ml aliquots according to the peak pattern. The fractionated eluates were subjected to SDS-PAGE (NuPAGE (registered trademark), Novex 4 - 12% Bis-Tris Gel, Invitrogen), and then stained with Coomassie blue for confirmation. Only the high-purity 200 kDa target antibody was pooled.

[0104] By doing so, the results shown in Table 1 were obtained.

[0105]

Table 1

[0106] Example 3 Measurement of Affinity of Antibodies against MSLN, CD3, and EGFR Using the Octet system, the affinities of the bispecific antibody (HMI323 / HMI323-A15) and the trispecific antibody (cetuximab / HMI323-A15) against human MSLN, human CD3, and human EGFR were measured respectively.

[0107] Specifically, recombinant human MSLN, CD3, and EGFR were prepared at 20 μg / ml with 1× kinetic buffer and plated at 200 μl / well in a 96-well plate. The plated MSLN, CD3, and EGFR were immobilized on an aminopropylsilane (APS) sensor (catalog number 18-5045, Fortebio). The bispecific antibody (HMI323 / HMI323-A15) and the trispecific antibody (cetuximab / HMI323-A15) were prepared at 100, 50, 25, 12.5, 6.25 nM with 10× kinetic buffer and plated at a concentration of 200 μl / well. The interactions of each antibody with the recombinant human MSLN (Table 2), human CD3 (Table 3), and human EGFR (Table 4) immobilized on the sensor were analyzed, and the antigen-antibody affinities were calculated. The results are shown in Tables 2 to 4 respectively.

[0108]

Table 2

[0109]

Table 3

[0110]

Table 4

[0111] From the measurement results shown in Tables 2 to 4, it was found that the bispecific antibody (HMI323 / HMI323-A15) and the trispecific antibody (cetuximab / HMI323-A15) showed excellent affinity for human MSLN, human CD3, and human EGFR.

[0112] Example 4 Analysis of the Affinity of Antibodies for Cells Expressing MSLN, EGFR, and CD3 Using flow cytometry, it was confirmed whether the bispecific antibody (HMI323 / HMI323-A15) or the trispecific antibody (cetuximab / HMI323-A15) showed affinity for specific cell lines.

[0113] Specifically, test tubes containing 100 μl of FACS buffer (2% FBS / sheath buffer) were prepared, and three types of cell lines were added to each of them, namely, the H226 cancer cell line (ATCC®, CRL-5826®) overexpressing MSLN and EGFR, the AsPC-1 cancer cell line (ATCC®, CRL-1682®) overexpressing MSLN and EGFR, and the Jurkat E6-1 T cell line (ATCC®, TIB-152®) expressing CD3, each at a concentration of 0.5×10 6 cells. Then, each test tube was treated with 1 μg of the primary antibody (HMI323 / HMI323-A15 or cetuximab / HMI323-A15). Then, it was incubated for one and a half hours in the dark at 4°C. Thereafter, 200 μl of FACS buffer was added thereto. Centrifugation was performed at 4°C and 2,000 rpm for 3 minutes, and then the supernatant was removed.

[0114] Next, each test tube was treated with 0.2 μg of a fluorescently labeled secondary antibody (PE-labeled anti-human IgG) capable of specifically binding to the primary antibody in 100 μl of FACS buffer. Then, it was incubated for 30 minutes in the dark at 4°C. Thereafter, 200 μl of FACS buffer was added thereto, centrifugation was performed at 4°C and 2,000 rpm for 3 minutes, and then the supernatant was removed to obtain a sample.

[0115] To suspend the cells, 200 μl of BD Cytofix™ was added to the sample and analyzed using BD LSR Fortessa™. The EC 50 value of the binding affinity for specific cells is shown in Table 5 below.

[0116]

Table 5

[0117] From the results of the analysis shown in Figures 7 to 9, it was found that both the bispecific antibody (HMI323 / HMI323-A15) and the trispecific antibody (cetuximab / HMI323-A15) bind to three cell lines. The negative control antibody used as an irrelevant control did not bind to any of the three cell lines.

[0118] Example 5 Evaluation of the cytotoxic effect of antibodies against tumor cell lines in the presence of PBMC Example 5.1 Construction of target cell lines Two types of MSLN+ tumor cells (H226, AsPC-1) were transduced with IncuCyte® NucLight Red Lentivirus Reagent (EF-1α promoter, puromycin selection) and treated with puromycin to finally construct target cell lines that are tumor cell lines transduced with NucLight Red.

[0119] Example 5.2 Preparation of target cell lines The cells were collected with 1× trypsin-EDTA solution and centrifuged at 1,200 rpm for 5 minutes at 4°C. Then, the supernatant was removed and resuspended in cRPMI (RPMI, A10491-01 + 10% FBS + 55 μM β-ME). Next, the cell count was quantified. A suspension of each cell line was prepared and added to a 96-well plate at 10,000 cells / well, and the plate was incubated in a 37°C CO2 incubator for 1 day to prepare the target cell lines.

[0120] Example 5.3 Preparation of peripheral blood mononuclear cells The cryopreserved peripheral blood mononuclear cells (PBMCs) were rapidly thawed in a 37°C water bath and then transferred to a 50 ml conical test tube. Thawing medium (RPMI, 11875 - 093 + 10% FBS + 55 μM β - ME) was added dropwise thereto and mixed with shaking. Then, centrifugation was performed at 4°C and 1,200 rpm for 10 minutes to remove the supernatant, and the cells were resuspended in cRPMI. Subsequently, the cell count was quantified.

[0121] Example 5.4 Plating of Peripheral Blood Mononuclear Cells and Antibodies Target cells were plated in wells. After 24 hours, each of the bispecific antibody (HMI323 / HMI323 - A15) and the trispecific antibody (cetuximab / HMI323 - A15) was diluted with cRPMI and then diluted 1 / 5 starting from 20 nM (in the concentration range of 0.26 pM to 20 nM). Then, PBMCs were prepared at a ratio of PBMC:target cells = 20:1 or 10:1, suspended in cRPMI, and then added to the wells.

[0122] Example 5.5 Real - Time Cell Measurement and Analysis Using IncuCyte S3 Bright - field and red fluorescence were measured 10 - fold at 24 hours and 48 hours using IncuCyte S3 while incubating in a 37°C CO₂ incubator for 2 days. The IC 50 values of cell killing mediated by PBMCs by the antibody against MSLN - expressing tumor cells are as shown in Table 6 below, and the maximum effect (%) of cell killing mediated by PBMCs by the antibody against MSLN - expressing tumor cells is as shown in Table 7 below.

[0123]

Table 6

[0124]

Table 7

[0125] From the evaluation results shown in FIGS. 10 to 15, the cytotoxic effects of the bispecific antibody (HMI323 / HMI323-A15) and the trispecific antibody (cetuximab / HMI323-A15) against cancer cell lines (H226, AsPC-1) expressing two types of MSLN and EGFR were confirmed to be dose-dependent under the conditions of PBMC:target cells = 20:1 or 10:1. Furthermore, the bispecific antibody (irrelevant / CD3) obtained by linking an irrelevant antibody and a CD3 antibody did not induce cell death.

[0126] Example 6 Evaluation of the cytotoxic effect of antibodies against MSLN+ tumor cell lines in the presence of T cells Example 6.1 Construction of target cell lines Two types of MSLN+ tumors (H226, AsPC-1) were transduced with IncuCyte® NucLight Red Lentivirus Reagent (EF-1α promoter, puromycin selection) and treated with puromycin to finally construct target cell lines that are tumor cell lines transduced with NucLight Red.

[0127] Example 6.2 Preparation of target cell lines Cells were collected with 1× trypsin-EDTA solution and centrifuged at 4°C and 1,200 rpm for 5 minutes. Then, the supernatant was removed and resuspended in cRPMI (RPMI, A10491-01 + 10% FBS + 55 μM β-ME). Next, the cell count was quantified. Suspensions of each cell line were prepared and added to a 96-well plate at 10,000 cells / well, and the plate was incubated in a 37°C CO2 incubator for 1 day to prepare target cell lines.

[0128] Example 6.3 Preparation of proliferated T cells The cryopreserved peripheral blood mononuclear cells (PBMCs) were rapidly thawed in a 37°C water bath and then transferred to a 50 ml conical test tube. Thawing medium (RPMI, 11875 - 093 + 10% FBS + 55 μM β - ME) was added dropwise thereto and mixed while shaking. Then, centrifugation was performed at 4°C and 1,200 rpm for 10 minutes to remove the supernatant, and the cells were resuspended in MACS medium (PBS + 0.5% FBS + 2 mM EDTA). CD3 microbeads were added thereto according to the cell count, and staining was performed at 4°C for 15 minutes. Only CD3 T cells were isolated using an LS column. The isolated CD3 T cells were suspended in X - VIVO. Then, αCD3 / 28 Dynabeads (registered trademark), IL - 2 (200 U / ml, Proleukin (registered trademark)), and human plasma (5%) were added thereto, and incubation was performed in a 37°C incubator. The cell count was measured 4 days and 7 days after the start of incubation, and X - VIVO, IL - 2, and human plasma were additionally added so that the cell concentration became 1×10 6 cells / ml.

[0129] Example 6.4 Plating of T cells and antibodies Target cells were plated in wells. After 24 hours, each of the bispecific antibody (HMI323 / HMI323 - A15) and the trispecific antibody (cetuximab / HMI323 - A15) was diluted with cRPMI and then diluted 1 / 5 starting from 20 nM (in the concentration range of 0.26 pM to 20 nM). Then, T cells grown at a ratio of T cell:target cell = 10:1 or 5:1 were finally prepared, suspended in cRPMI, and then added to the wells.

[0130] Example 6.5 Real - time cell measurement and analysis using IncuCyteS3 Bright - field and red fluorescence were measured 10 - fold 24 hours and 48 hours later using IncuCyteS3 (Essenbio) while incubating in a 37°C CO2 incubator for 2 days. The IC of T - cell - mediated killing by antibodies against MSLN - expressing tumor cells 50The values are as shown in Table 8 below, and the maximum effect (%) of antibody-mediated T cell-mediated killing of MSLN-expressing tumor cells is as shown in Table 9 below.

[0131]

Table 8

[0132]

Table 9

[0133] From the evaluation results shown in FIGS. 16 to 19, the cytotoxic effects of the bispecific antibody (HMI323 / HMI323-A15) and the trispecific antibody (cetuximab / HMI323-A15) against cancer cell lines (H226, AsPC-1) expressing two types of MSLN and EGFR were confirmed to be dose-dependent under the conditions of T cell:target cell = 10:1 or 5:1. Furthermore, the bispecific antibody (irrelevant / CD3) obtained by linking an irrelevant antibody and a CD3 antibody did not induce cell death.

[0134] Example 7 Analysis of the tumor growth inhibitory effect of antibodies in mice To confirm the tumor growth inhibitory effects of the bispecific antibody (HMI323 / HMI323-A15) and the trispecific antibody (cetuximab / HMI323-A15), which are being developed as T cell-inducing antibodies, these antibodies were administered to tumor xenograft model mice.

[0135] Example 7.1 Establishment of a cell line xenograft model Mice transferred to the animal breeding room were given an acclimation period of approximately one week before the start of the experiment. H226 tumor cell line (1×10 7 / 200 μL PBS) or AsPC-1 tumor cell line (5×10 6 / 200 μL of PBS) was subcutaneously injected into the axilla of the mice. On the 5th day after tumor cell injection, purified and activated human T cells were intraperitoneally injected at a ratio of T cell:tumor cell = 2:1. On the 7th day after tumor cell injection, the mice were divided into 4 groups (5 mice per group), and statistical analysis was performed. As a result, it was confirmed that there was no significant difference in tumor size among the groups.

[0136] Example 7.2 Administration of Antibody From 2 days after T cell injection, PBS or antibody (HMI323 / HMI323 - A15 or cetuximab / HMI323 - A15) was intraperitoneally administered at a concentration of 3 mg / kg twice a week (a total of 4 times).

[0137] Example 7.3 Measurement of Tumor Size From the day of grouping until the end point, the tumor size (tumor volume (mm 3 ) = short axis × long axis × height × 0.5) was measured twice a week. The lengths of the short axis, long axis, and height were measured by the same tester using a ruler.

[0138] Example 7.4 Calculation of Tumor Growth Rate (RTV%), Relative Tumor Growth Rate (T / C%), and Tumor Growth Inhibition Rate (Inhibitory Effect) The tumor growth rate (relative tumor volume (RTV)%) was calculated using the V1 / V0 method. V1 represents the volume at the time of measuring the experimental effect, and V0 represents the volume at the start of the experiment. The relative tumor growth rate (T / C%) of each group was obtained by dividing the average tumor growth rate of each group by the average tumor growth rate of the PBS group (the average tumor growth rate obtained at the time of measurement) and multiplying the resulting value by 100. The tumor growth inhibition rate (inhibitory effect) was calculated as the value obtained by subtracting the relative tumor growth rate from 100. For statistical analysis, one - way ANOVA or two - way ANOVA was used according to the type of experiment, and a p - value less than 0.05 was judged to be statistically significant.

[0139] Example 7.5 Immunohistochemistry One week after treating tumor-bearing mice with an antibody (HMI323 / HMI323-A15 or cetuximab / HMI323-A15), tumor samples were removed and fixed with formalin. Subsequently, the samples were infiltrated with paraffin and embedded, and then paraffin sections were obtained using a microtome. The paraffin sections were deparaffinized, hydrated, and washed, and then stained with human CD3 (anti-CD3, Abcam) or human CD8 (anti-CD8, Abcam). The tissue sections were counterstained with Mayer's hematoxylin (Dako) and observed under an Olympus BX51 microscope.

[0140] Example 7.6 End of Experiment and Results The mice were euthanized by cervical dislocation or CO2. As a result, when the anti-cetuximab / HMI323-A15 or HMI323 / HMI323-A15 antibody was intraperitoneally administered to model mice xenografted with the mesothelin-overexpressing lung adenocarcinoma cell line H226 at a concentration of 3 mg / kg for a total of 4 times, both antibody administration groups showed an excellent tumor growth inhibitory effect compared to the PBS administration group (Figures 20 and 21).

[0141] Immunohistochemical staining showed that the infiltration of immune T cells into the tumor tissue (stained darkly) was higher in the antibody (cetuximab / HMI323-A15 or HMI323 / HMI323-A15) administration group than in the PBS group (Figure 22). As shown in Table 10 below, the anti-cetuximab / HMI323-A15 antibody that binds to mesothelin, EGFR, and CD3 showed a 100% tumor growth inhibitory effect from day 20 after tumor xenotransplantation, and the anti-HMI323 / HMI323-A15 antibody that binds to mesothelin and CD3 showed a 100% tumor growth inhibitory effect from day 25 after tumor xenotransplantation.

[0142]

Table 10

[0143] In addition, immunohistochemical staining showed that the infiltration of immune T cells (stained dark) into tumor tissues was higher in the antibody (cetuximab / HMI323-A15 or HMI323 / HMI323-A15) administration groups than in the PBS group (Fig. 25). As shown in Table 11 below, a 100% tumor growth inhibitory effect was observed from day 20 after tumor xenografting with cetuximab / HMI323-A15, an antibody that binds to mesothelin, EGFR, and CD3, and from day 28 after tumor xenografting with HMI323 / HMI323-A15, an antibody that binds to mesothelin and CD3.

[0144]

Table 11

[0145] Example 8 PK Analysis of Antibodies in Mice To obtain the pharmacokinetic (PK) data of cetuximab / HMI323-A15 or HMI323 / HMI323-A15, which are being developed as T cell-inducing antibodies, the PK parameters of the antibodies in mouse serum were analyzed.

[0146] Example 8.1 Obtaining Test Solution (Mouse Serum) Cetuximab / HMI323-A15 or HMI323 / HMI323-A15 antibodies were injected into the tail vein of mice at a concentration of 3 mg / kg. After antibody injection, blood was collected from the retro-orbital plexus at 5 minutes, 1 hour, 4 hours, 8 hours, 24 hours, 48 hours, 72 hours, 120 hours, 168 hours, 240 hours, 336 hours, 504 hours, and 672 hours. 100 μL of blood was collected from more than 3 animals in each group. After allowing the blood to clot at room temperature for about 20 - 30 minutes, it was centrifuged at 10,000 rpm for 10 minutes to separate the serum.

[0147] Example 8.2 ELISA Assay Anti-human Fab (50 ng / 100 μL / well) was added to the plate and reacted overnight at 4°C. Then, the remaining solution was completely removed. 1% BSA / PBS solution was added at 200 μL / well and reacted at room temperature for 1 hour. Then, the remaining solution was completely removed. The standard solution (standard antibody) was adjusted to 1 μg / mL using 1% serum / 1% BSA / PBS solution and then prepared by diluting it to 1 / 2. The test solution was prepared by diluting 100-fold with 1% BSA / PBS solution.

[0148] The prepared standard solution and test solution were dispensed 100 μL each into two wells for each concentration and reacted at room temperature for 1 hour. After the reaction, 300 μL of PBST (0.05% Tween 20) solution was dispensed per well and washed a total of 3 times. Anti-human Fc-HRP was diluted 5000-fold with 1% BSA / PBST solution and then dispensed 100 μL each into each well. It was reacted at room temperature for 1 hour. After the reaction, 300 μL of PBST (0.05% Tween 20) solution was dispensed per well and washed a total of 3 times. Before the experiment, the TMB peroxidase substrate solution was returned to room temperature and dispensed 100 μL each into each well. It was reacted at room temperature for 30 minutes. 100 μL of TMB stop solution was dispensed into each well, gently stirred for better mixing, and then the absorbance was measured at a wavelength of 450 nm.

[0149] Cetuximab / HMI323-A15, an antibody that binds to mesothelin, EGFR, and CD3, or HMI323 / HMI323-A15, an antibody that binds to mesothelin and CD3, was injected into the tail vein of nude mice at a dose of 3 mg / kg. Then, blood samples were collected 5 minutes, 1 hour, 4 hours, 8 hours, 24 hours, 48 hours, 72 hours, 96 hours, 168 hours, 240 hours, 336 hours, 504 hours, and 672 hours after injection, and the antibody concentration in mouse serum was quantified by ELISA. The results are shown in Figures 26 and 27.

[0150] Based on the ELISA results, PK parameters were analyzed. As a result, it was confirmed that the half-life of the cetuximab / HMI323-A15 antibody was approximately 207.94 hours and that of the HMI323 / HMI323-A15 antibody was approximately 202.58 hours (Tables 12 and 13). These half-lives are within the range of the half-lives of typical IgG-type bispecific antibodies. Furthermore, the extrapolated value of the area under the curve (AUC) was 20% or less, and thus the reliability of the calculated PK parameters was ensured.

[0151] The results of the PK parameters of cetuximab / HMI323-A15 are as shown in Table 12 below, and the results of the PK parameters of HMI323 / HMI323-A15 are as shown in Table 13.

[0152]

Table 12

[0153]

Table 13

Claims

**Claim 1**: A bispecific antibody comprising the fusion protein of Structural Formula 1 and the fusion protein of Structural Formula 2, wherein: [Structural Formula 1] N’-A-B-L1-C-D-L2-E-F-L3-G-H-L4-I-C’ (In the formula, N’ is the N-terminus of the fusion protein, C’ is the C-terminus of the fusion protein, A is the light chain variable domain of an antibody that specifically binds to mesothelin, and said domain comprises the light chain CDR1 of SEQ ID NO: 25, the light chain CDR2 of SEQ ID NO: 26, and the light chain CDR3 of SEQ ID NO: 27, B is the light chain constant domain of said antibody, C is the heavy chain variable domain of said antibody that specifically binds to mesothelin, and said domain comprises the heavy chain CDR1 of SEQ ID NO: 28, the heavy chain CDR2 of SEQ ID NO: 29, and the heavy chain CDR3 of SEQ ID NO: 30, D is the CH1 domain in the heavy chain constant domain of said antibody, E is the light chain variable domain of an antibody that specifically binds to CD3, and said domain comprises the light chain CDR1 of SEQ ID NO: 31, the light chain CDR2 of SEQ ID NO: 32, and the light chain CDR3 of SEQ ID NO: 33, F is the light chain constant domain of said antibody, G is the heavy chain variable domain of said antibody that specifically binds to CD3, and said domain comprises the heavy chain CDR1 of SEQ ID NO: 34, the heavy chain CDR2 of SEQ ID NO: 35, and the heavy chain CDR3 of SEQ ID NO: 36, H is the CH1 domain in the heavy chain constant domain of said antibody, I is the Fc region or a variant thereof, and each of L1, L2, L3, and L4 is a peptide linker) and [Structural Formula 2] N’-A-B-L1-C-D-L5-J-C’ (In the formula, N’ is the N-terminus of the fusion protein, C’ is the C-terminus of the fusion protein, A is the light chain variable domain of an antibody that specifically binds to mesothelin, and said domain comprises the light chain CDR1 of SEQ ID NO: 25, the light chain CDR2 of SEQ ID NO: 26, and the light chain CDR3 of SEQ ID NO: 27, B is the light chain constant domain of said antibody, C is the heavy chain variable domain of said antibody that specifically binds to mesothelin, and said domain comprises the heavy chain CDR1 of SEQ ID NO: 28, the heavy chain CDR2 of SEQ ID NO: 29, and the heavy chain CDR3 of SEQ ID NO: 30, D is the CH1 domain in the heavy chain constant domain of said antibody, J is the Fc region or a fragment thereof, and each of L1 and L5 is a peptide linker) is a bispecific antibody. **Claim 2** The knob modification is included in I of the structural formula 1, and the hole modification is included in J of the structural formula 2; or, the hole modification is included in I of the structural formula 1, and the knob modification is included in J of the structural formula 2, the bispecific antibody according to claim 1.

3. A trispecific antibody comprising a fusion protein of structural formula 1, and a fusion protein of structural formula 3, wherein [Structural formula 1] N'-A-B-L1-C-D-L2-E-F-L3-G-H-L4-I-C' (wherein N' is the N-terminus of the fusion protein, C' is the C-terminus of the fusion protein, A is the light chain variable domain of an antibody that specifically binds to mesothelin, and the domain includes the light chain CDR1 of SEQ ID NO: 25, the light chain CDR2 of SEQ ID NO: 26, and the light chain CDR3 of SEQ ID NO: 27, B is the light chain constant domain of the antibody, C is the heavy chain variable domain of the antibody that specifically binds to mesothelin, and the domain includes the heavy chain CDR1 of SEQ ID NO: 28, the heavy chain CDR2 of SEQ ID NO: 29, and the heavy chain CDR3 of SEQ ID NO: 30, D is the CH1 domain in the heavy chain constant domain of the antibody, E is the light chain variable domain of an antibody that specifically binds to CD3, and the domain includes the light chain CDR1 of SEQ ID NO: 31, the light chain CDR2 of SEQ ID NO: 32, and the light chain CDR3 of SEQ ID NO: 33, F is the light chain constant domain of the antibody, G is the heavy chain variable domain of the antibody that specifically binds to CD3, and the domain includes the heavy chain CDR1 of SEQ ID NO: 34, the heavy chain CDR2 of SEQ ID NO: 35, and the heavy chain CDR3 of SEQ ID NO: 36, H is the CH1 domain in the heavy chain constant domain of the antibody, I is the Fc region or a variant thereof, and each of L1, L2, L3, and L4 is a peptide linker) and [Structural formula 3] N'-K-M-L6-N-O-L7-P-C' (wherein N' is the N-terminus of the fusion protein, C' is the C-terminus of the fusion protein, K is the light chain variable domain of an antibody that specifically binds to EGFR, and the domain includes the light chain CDR1 of SEQ ID NO: 37, the light chain CDR2 of SEQ ID NO: 38, and the light chain CDR3 of SEQ ID NO: 39, M is the light chain constant domain of the antibody, N is the heavy chain variable domain of the antibody that specifically binds to EGFR, and the domain includes the heavy chain CDR1 of SEQ ID NO: 40, the heavy chain CDR2 of SEQ ID NO: 41, and the heavy chain CDR3 of SEQ ID NO: 42, O is the CH1 domain in the heavy chain constant domain of the antibody, P is the Fc region or a fragment thereof, each of L6 and L7 is a peptide linker) A trispecific antibody. **Claim 4** The knob modification is included in I of Structural Formula 1, and the hole modification is included in P of Structural Formula 3; or, the hole modification is included in I of Structural Formula 1, and the knob modification is included in P of Structural Formula 3. The trispecific antibody according to claim 3. **Claim 5** A pharmaceutical composition for treating cancer, comprising, as an active ingredient, the bispecific antibody according to claim 1, or the trispecific antibody according to claim 3, A pharmaceutical composition. **Claim 6** Use of the following antibody for the manufacture of a medicament for treating cancer in an individual in need of cancer treatment, the bispecific antibody according to claim 1, or the trispecific antibody according to claim 3, Use.

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