KR20130057959A - A fusion monoclonal antibody comprising an anti-HER2 monoclonal antibody and IL-2 and a composition for treating cancer comprising the same - Google Patents

Abstract

PURPOSE: A fusion monoclonal antibody is provided to enhance antibody-dependent cytotoxicity and to effectively treat cancer by activation of immunocytes around cancer cells by IL-2(interleukin-2). CONSTITUTION: A fusion monoclonal antibody contains IL-2 which is specifically conjugate to HER2(cancer cell surface antigen). An antibody which is specific to HER2 is IgG 1 type or scFv(single-chain variable fragment) type. The antibody which is specific to HER2 is trastuzumab. A polynucleotide encodes the fusion monoclonal antibody. A pharmaceutical composition for preventing or treating cancer contains the fusion monoclonal antibody. The composition additionally contains a pharmaceutically acceptable carrier, excipient, or diluent.

Description

A fusion monoclonal antibody comprising HER2 antibody and IL-2, and pharmaceutical composition comprising the same}

The present invention relates to a fusion monoclonal antibody comprising IL-2 linked to an anti-HER2 monoclonal antibody and a composition for treating cancer comprising the same. More specifically, the present invention provides an anti-HER2 monoclonal antibody capable of specifically binding to HER2, a cancer cell surface antigen, and a fusion monoclonal antibody in which IL-2 is coupled to increase the activity of immune cells. A polynucleotide encoding, an expression vector containing the polynucleotide, a transformant into which the expression vector is introduced, a method for producing the fusion monoclonal antibody using the transformant, and the fusion monoclonal antibody as an active ingredient It relates to a composition for treating cancer comprising.

HER2 (Human Epidermal Growth Factor Receptor 2, ErbB2) is a member of the transmembrane receptor of the epidermal growth factor receptor (EGFR) family. Overexpression of HER2 has been observed in approximately 20 human breast cancers, which has a close impact on tumor-invasive growth and poor clinical outcome.

Trastuzumab (CAS 180288-69-1, huMAb4D5-8, rhuMAb HER2, trade name: Herceptin, from Genentech) is an extracellular of the human epidermal growth factor receptor 2 protein HER2 in cell-based assays. A recombinant DNA-derived humanized, IgG1 kappa monoclonal antibody version of murine HER2 antibody that selectively binds to domains with high affinity. Trastuzumab contains a human backbone region involving the complementarity determining regions of murine antibodies that bind HER2. Since trastuzumab binds to the HER2 antigen, it inhibits the growth of cancer cells by blocking cell division signals by HER2. Trastuzumab has been found to inhibit the proliferation of human tumor cells overexpressing HER2 and is a mediator of antibody-dependent cellular cytotoxicity (ADCC). Although the development of trastuzumab has markedly outperformed chemotherapy alone in patients with HER2-positive tumors, almost all patients with HER2-positive metastatic breast cancer (MBC) have disease despite all currently available therapies. This is a progressing state. Opportunities remain to improve for MBC patients, but despite trastuzumab's various mechanisms of action, many patients treated with trastuzumab did not respond at all or stopped responding after a period of therapeutic benefit. . Some HER2-positive tumors failed to respond to trastuzumab, and the majority of patients with tumors eventually developed the disease. There is a significant clinical need to develop additional HER2-induced cancer therapies for patients with HER2-overexpressing tumors or other diseases associated with HER2 expression that either do not respond to trastuzumab treatment or respond at poor levels. In addition, the rate of overexpression of HER2 in breast cancer patients is known to be about 25%, and now it is reported that about 75% of patients who do not express HER2 or underexpression does not show a therapeutic effect on trastuzumab, There is an urgent need to develop new breast cancer antibody therapeutics that will surpass trastuzumab.

IL-2, on the other hand, is an immune-promoting cytokine that is secreted when antigens invade the body and promotes a T cell-mediated immune response. Proliferation and differentiation of T cells, antibody production of B cells, and NK cell activation It protects our body from external invasive substances by promoting it. Recombinant IL-2 has been developed by Chiron Corporation as an agent for the treatment of malignant melanoma and renal cell cancer using the function of this immune stimulation, but it is used in excess (several hundred mg / kg) There have been reports of fatal side effects that cause vascular damage and hepatotoxicity. In order to prevent these side effects and develop more effective therapeutic agents for the treatment of cancer, methods for concentrating the efficacy of IL-2 on the site of cancer development have been developed.

For example, US 7,462,350 discloses a fusion protein in which a non-IL-2 moiety, such as an antibody, a portion of an antibody, albumin, and the like, and a mutant IL-2 moiety mutated to increase serum half-life are fused, and US 7,851,599 shows a cancer. Disclosed is a composition for treating cancer comprising a combination of a fusion protein fused with IL-2 and a gemcitabine, which is a cancer treatment agent, in which a part of an antibody specific for the EDb-fibronectin domain known as a marker is an active ingredient. However, the effect of a fusion protein comprising a mutant IL-2 moiety can only be seen when including a mutated IL-2 moiety, but cannot be applied when a normal IL-2 moiety is included. The fusion protein containing the antibody against the B-fibronectin domain may have the effect of focusing the efficacy of IL-2 on the site of cancer development, but the antibody itself does not show any anticancer effect.

Accordingly, the present inventors have made intensive efforts to develop a therapeutic agent exhibiting an improved anticancer effect. As a result, we have prepared a new type of fusion monoclonal antibody in which IL-2 is fused to anti-HER2 monoclonal antibody, trastuzumab, and increased anticancer effect. Confirmed to complete the present invention.

One object of the present invention is to provide a fusion monoclonal antibody in which IL-2 is linked to an antibody that specifically binds to HER2, a cancer cell surface antigen.

Another object of the present invention is to provide a polynucleotide encoding the fusion monoclonal antibody.

Still another object of the present invention is to provide an expression vector comprising the polynucleotide.

Still another object of the present invention is to provide a transformant into which the expression vector is introduced.

Still another object of the present invention is to provide a method for preparing the fusion monoclonal antibody using the transformant.

Still another object of the present invention is to provide a composition for preventing or treating cancer comprising the fusion monoclonal antibody as an active ingredient.

As one aspect for achieving the above object, the present invention provides a fusion monoclonal antibody in which IL-2 (interleukin-2) is linked to an antibody that specifically binds to Human Epidermal Growth Factor Receptor 2 (HER2), which is a cancer cell surface antigen do.

As used herein, the term “fused monoclonal antibody” refers to a form in which all or part of a monoclonal antibody and other peptides are linked to each other and does not exist naturally and is an artificially produced monoclonal gene which is genetic engineering or any method. Means an antibody. For the purposes of the present invention, the fusion monoclonal antibody means a fusion protein in a form in which an antibody specifically binding to HER2, a cancer cell surface antigen, and IL-2 are directly or indirectly linked through a linker, but are not limited thereto. . In this case, the site where IL-2 is directly or indirectly linked through a linker is not particularly limited thereto, but may be an Fc moiety, Fab ′, F (ab ′) 2, Fab, Fv, and the like, and the fusion monoclonal Antibodies stimulate the immune cells and at the same time specifically bind to HER2, a cancer cell surface antigen. In other words, the fusion monoclonal antibody acts on cancer cells overexpressing HER2 to show anticancer activity by the HER2 antibody, as well as to promote the immune response by IL-2, thereby producing antibody-dependent cell cytotoxicity, ADCC) is also imported. Therefore, the fusion monoclonal antibody has the advantage that can be usefully used in the treatment of diseases, such as cancer that has the property of overexpressing HER2.

The term "antibody that specifically binds HER2" may be used in combination with "anti-HER2 antibody". This includes any antibody that specifically binds to the cancer cell surface antigen HER2 (Human Epidermal Growth Factor Receptor 2) as an antigen.

The HER2 protein is one of proteins belonging to the epidermal growth factor receptor (EGFR, ErbB) family and may be used in combination with Neu, ErbB-2, CD340 or p185. Overexpression of these proteins is known to play a very important role in the development and progression of cancer, especially breast cancer. In addition to breast cancer, other cancers such as ovarian cancer overexpress HER2 and are associated with poor prognosis in various cancers such as breast cancer, ovarian cancer or gastric cancer. Therefore, identifying a substance that inhibits the biological activity of the HER2 is very important in the treatment of cancer. Antibodies that specifically bind to HER2 of the present invention can target the antibody and IL-2 linked to the antibody to tissues overexpressing HER2, such as breast cancer tissues, and thus can be usefully used in the treatment of cancer.

The antibody that specifically binds to HER2 may be in the form of a monoclonal antibody, but is not limited thereto. Antibodies that specifically bind to HER2 for the purposes of the present invention include any protein that exhibits anticancer effect by binding to HER2 protein and inhibiting its biological activity, for example, a therapeutic antibody that targets HER2. It may be trastuzumab or pertuzumab, more preferably trastuzumab. The antibody that binds to HER2 comprises a heavy chain amino acid sequence comprising the amino acid sequence of SEQ ID NO: 3 and a light chain amino acid sequence comprising the amino acid sequence of SEQ ID NO: 4, or an amino acid sequence encoded by the polynucleotide of SEQ ID NO: 5 It may be a monoclonal antibody comprising a light chain amino acid sequence D having a heavy chain amino acid sequence and an amino acid sequence encoded by a polynucleotide of SEQ ID NO: 6. Trastuzumab, also called Herceptin, is a humanized antibody against HER2 developed by Genentech, Inc., which is known as a therapeutic agent for HER2 / neu antibody mainly expressed in breast cancer cells.

In addition, the antibody specifically binding to HER2 is 70% or more, preferably 80% or more, more preferably not only the protein comprising the heavy chain amino acid sequence of SEQ ID NO: 3 and the amino acid sequence of SEQ ID NO: 4 Is an amino acid sequence of at least 90%, even more preferably at least 95% and most preferably at least 98% of the amino acid sequence having a biological activity that substantially binds to the HER2 protein so that some sequences are deleted, modified, substituted or added It is obvious that protein variants having an amino acid sequence are also included within the scope of the present invention, and may include, for example and without limitation, amino acid sequences having internal or terminal additions, deletions or substitutions, or rearranged sequences.

In addition, the antibody that specifically binds to HER2 may be in the form of a whole antibody or antibody fragment. As used herein, the term "antibody fragment" includes antigen-binding forms of an antibody, including fragments having antigen-binding ability, such as Fab ', F (ab') 2, Fab, scFv, dsFv and rIgG. . In one embodiment of the present invention, Herceptin in the IgG form, trastuzumab, was used as an antibody that specifically binds to HER2.

In addition, the antibody fragment may be a single-chain variable fragment (scFv) domain capable of binding to HER2. The term scFv of the present invention refers to the minimum antibody fragment having a complete antigen-recognition and antigen-binding site, comprising the V _H and V _L domains of an antibody, wherein this domain is present in a single polypeptide chain.

As used herein, the term “antibody” refers to a protein molecule that acts as a receptor that specifically recognizes an antigen, including an immunoglobulin molecule that is immunologically reactive with a specific antigen, including polyclonal antibodies, monoclonal antibodies, It includes both whole antibodies and antibody fragments. Preferably, it may be, but is not limited to, a monoclonal antibody. The term also includes chimeric antibodies (eg, humanized murine antibodies) and bivalent or bispecific molecules (eg, bispecific antibodies). , Diabodies, triabodies, and tetrabodies. The total antibody is a structure having two full length light chains and two full length heavy chains, each of which is linked by heavy and disulfide bonds. The total antibody includes IgA, IgD, IgE, IgM, IgT, IgY and IgG, and IgG is isotype, including IgG1, IgG2, IgG3 and IgG4. Preferably for the purposes of the present invention may be in the form of IgG, more preferably may be IgG1, but is not limited thereto. The antibody fragment refers to a fragment having an antigen binding function, and includes Fab, Fab ', F (ab') 2, Fv and the like. The Fab has one antigen binding site in a structure having a variable region of the light and heavy chains, a constant region of the light chain, and a first constant region of the heavy chain (CH1 domain). Fab 'differs from Fab in that it has a hinge region that contains at least one cysteine residue at the C-terminus of the heavy chain CH1 domain. The F (ab ') 2 antibody is produced when the cysteine residue of the hinge region of the Fab' forms a disulfide bond. Fv (variable fragment) means a minimum antibody fragment having only the heavy chain variable region and light chain variable region. Double-chain Fv (dsFv) is a disulfide bond is linked to the heavy chain variable region and light chain variable region, and short-chain Fv (scFv) is generally covalently linked to the variable region of the heavy chain and the light chain through a peptide linker. Such an antibody fragment can be obtained using a protein hydrolyzing enzyme (for example, an F (ab ') 2 fragment can be obtained by cutting a whole antibody into papain and obtaining a Fab and digesting with pepsin) Can be produced through recombinant DNA technology.

As used herein, the term "monoclonal antibody" means an antibody molecule of a single molecular composition obtained from substantially the same antibody population, wherein said monoclonal antibody exhibits single binding specificity and affinity for a particular epitope. For the purposes of the present invention, the monoclonal antibody may be a monoclonal antibody that can specifically bind to HER2, which is a cancer cell surface antigen. The antibody capable of binding to HER2, a cancer cell surface antigen of the present invention, can not only concentrate the fusion monoclonal antibody of the present invention to cancer cells expressing HER2, but also bind to HER2 and have anticancer activity by itself. . In addition, it is possible to maximize cancer cell dependent anticancer activity by stimulating immune cells by IL-2 linked to the antibody concentrated on cancer cells. The monoclonal antibody is not particularly limited thereto, but is preferably a natural antibody such as IgG, IgM, IgA, IgE, IgD, IgT, IgY, single chain antibody; Variant antibodies in which a portion of the native antibody is mutated to lower the dissociation constant of the antibody to the antigen, to reduce the rejection of the antibody in the body, such as a humanized antibody, or to enhance the stability of the antibody; It may be a recombinant antibody or the like configured to include a portion of the natural antibody, more preferably, a natural antibody such as IgG, mutated antibody, modified directly to lower the dissociation constant value of the antibody to the antigen, binding directly to the antigen And a recombinant antibody including a complementarity determining region (CDR). More preferably, the IgG may include various isotypes, but most preferably may be in IgG1 form.

The fusion monoclonal antibody of the present invention has a form in which IL-2 is linked to the antibody specifically binding to HER2, and has the advantage of concentrating IL-2 on cancer cells overexpressing HER2.

The term "Interleukin 2 (IL-2)" of the present invention is expressed in Th1 cells and its receptors are CD25 / IL2RA, CD122 / IL2RB and CD132 / IL2RG, which promote immune responses and are involved in the immune system. It refers to a 15.5kD size cytokine capable of activating various cells such as T cells, B cells, monocytes, and the like and acting as a growth factor of the T cells. In the present invention, IL-2 is used as a fusion partner to form a fusion monoclonal antibody of the present invention by binding to a monoclonal antibody that can specifically bind to the HER2, wherein IL-2 is used as IL- As long as it exhibits the activity of 2, it may be a whole, a fragment, a mutein, or the like, but preferably, IL-2 may be the whole, and most preferably, an IL-2 or a sequence comprising the amino acid sequence of SEQ ID NO: 7 IL-2 expressed from the polynucleotide sequence of No. 8, but is not limited thereto. The IL-2 is not only the amino acid sequence of SEQ ID NO: 7, but 70% or more, preferably 80% or more, more preferably 90% or more, even more preferably 95% or more, most preferably 98 Any amino acid sequence having at least% similarity as a protein having a biological activity capable of substantially acting on the IL-2 receptor to promote immune action is included, without limitation, and as a sequence having similarity, substantially binds to IL-2, and the like. As a protein capable of enhancing an immune response, it is obvious that protein variants having amino acid sequences deleted, modified, substituted, or added to some sequences are also included in the scope of the present invention. Examples may include, but are not limited to, amino acid sequences internally or terminally added, deleted or substituted, or rearranged in sequence. In addition, the polynucleotide encoding the IL-2 protein may comprise a polynucleotide sequence set forth in SEQ ID NO: 8, the activity that the protein encoded by the polynucleotide can act on the IL-2 receptor to enhance the immune response Polynucleotide sequences which exhibit at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 98% similarity with the sequence if present. It is obvious that it is included in the scope of.

As used herein, the term "immunocyte" refers to a cell that makes an antibody, and may generally be a B cell, a natural killer cell (NK cell), or a T cell. For the purposes of the present invention, the immune cells may refer to natural killer cells or T cells that act on antibody dependent cytotoxicity (ADCC) mechanisms. The IL-2 may act on immune cells such as T cells to bring about development or proliferation of immune cells, thereby activating an immune response. The activation of this immune response can remove cancer cells. Therefore, the fusion monoclonal antibody of the present invention in which the IL-2 is linked to an antibody specific for HER2 can be used for the treatment of cancer by removing cancer cells through activation of the immune cells.

The anti-HER2 antibody and IL-2 contained in the fusion monoclonal antibody of the present invention may be directly bound or may be bound via a linker. The method of directly binding the anti-HER2 antibody and IL-2 is not particularly limited thereto, and preferably, the two proteins may be bound by using a peptide bond, a disulfide bond, or the like.

As used herein, the term "linker" basically refers to two different fusion partners (e.g., biological polymers, etc.) that are hydrogen bonds, electrostatic interactions, van der Waals forces, disulfide bonds, salt bridges, It refers to a linker that can be linked using hydrophobic interactions, covalent bonds, etc., preferably to at least one disulfide bond under physiological conditions or other standard peptide conditions (eg, peptide purification conditions, peptide storage conditions). A hinge that can have at least one cysteine that can participate and, in addition to simply connecting each fusion partner, serves to provide a certain amount of spacing between the fusion partners or to provide flexibility or rigidity to the fusion. It can also play the role of). In the present invention, the linker is not particularly limited thereto, but is preferably a polypeptide capable of linking the anti-HER2 antibody with IL-2 or a poly-linkable gene connecting the gene encoding the anti-HER2 antibody with the gene encoding IL-2. It may be a polypeptide encoded by a nucleotide, more preferably a peptidic linker capable of connecting the C-terminus of the Fc region of the anti-HER2 antibody and the N-terminus of IL-2, more preferably GGGGS motif may be a peptidic linker consisting of an amino acid sequence of the form repeated three times, the GGGGS motif may be repeated 1 to 10 times, most preferably the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 It is preferred that the amino acid sequence is encoded by the nucleotide sequence of.

Linker peptide: GGGGSGGGGSGGGGS (SEQ ID NO: 1)

Linker Nucleotide: GGTGGAGGTGGCAGCGGTGGTGGCGGCAGTCCCGGTGGCGGCTCC (SEQ ID NO: 2)

The fusion monoclonal antibody is not particularly limited thereto, but a fusion monoclonal antibody in a form in which all or a portion of the anti-HER2 antibody and all or a portion of IL-2 are linked to each other; Or a fusion monoclonal antibody in a form in which all or a portion of the anti-HER2 antibody and all or a portion of IL-2 are linked by a peptide linker.

Preferably, the fusion monoclonal antibody comprises a fusion monoclonal antibody in a form in which all or part of the heavy chain of the anti-HER2 antibody and all or part of IL-2 are linked; Fused monoclonal antibodies in a form in which all or part of the light chain of the anti-HER2 antibody is linked to all or part of IL-2; Fused monoclonal antibodies in the form in which all or part of the heavy chain of the anti-HER2 antibody and all or part of the IL-2 are linked by a peptide linker; Fused monoclonal antibodies in the form in which all or part of the light chain of the anti-HER2 antibody and all or part of the IL-2 are linked by a peptide linker; Or a combination thereof.

More preferably, the fusion monoclonal antibody may comprise a fusion monoclonal antibody in a form in which all or part of the C-terminus of the heavy chain of the anti-HER2 antibody and N-terminus of all or part of the IL-2 are linked; A fusion monoclonal antibody in a form in which the C-terminus of all or part of the light chain of an anti HER2 antibody and the N-terminus of all or a portion of IL-2 are linked; Fused monoclonal antibodies in the form wherein the C-terminus of all or part of the heavy chain of the anti HER2 antibody and the N-terminus of all or part of the IL-2 are linked with a peptide linker; Fused monoclonal antibodies in the form wherein the C-terminus of all or part of the light chain of the anti HER2 antibody and the N-terminus of all or part of the IL-2 are linked by a peptide linker; Or a combination thereof.

Most preferably, the fusion monoclonal antibody is a fusion single in a form in which all or part of the C-terminus of the anti-HER2 antibody heavy chain and all or part of the N-terminus of IL-2 are linked by a peptide linker (SEQ ID NO: 9). Can be a cloned antibody.

In this case, the dissociation constant value (K _D value) for the cancer cell surface HER2 antigen of the fusion monoclonal antibody provided in the present invention is not particularly limited thereto, but is preferably 100 nM or less.

According to an embodiment of the present invention, the present inventors prepared a fusion monoclonal antibody of Herceptin _IL-2 in the form of IgG_IL-2 (Example 1), and expressed on HER2 expressed on the surface of breast cancer cell line BT474 by FACS method. It was confirmed that Herceptin (HD201) and the fusion monoclonal antibody (HD201_IL-2) that bound IL-2 to this antibody bind to HER2 of BT474 cells similarly to HD201 antibody that did not bind IL-2 (Example 2 ). In addition, as a result of confirming IL-2 dependent cell proliferation using a CTLL-2 cell line, it was confirmed that IL-2 of the fusion antibody had cell proliferation ability and IL-2 activity (Example 3). In addition, the anti-cancer effect was confirmed by transplanting Herceptin-resistant MCF-7 cell line to nude mice, while the fusion antibody showed significant tumor suppression ability, while Herceptin-administered group, IL-2 alone group, Herceptin and IL-2 combination group were significant. It was confirmed that the tumor suppressive effect was not shown (Example 4). In addition, similar effects were confirmed in experiments in which the Herceptin-resistant JIMT-1 cell line was transplanted (Example 5). These results were confirmed in vivo that the fusion monoclonal antibody of the present invention had a significant anticancer effect than the simple administration of Herceptin (HD201) and IL-2 in combination. Furthermore, even in nude mice transplanted with BT474 cell line, which is a Herceptin-sensitive cell line, it was confirmed that the fusion monoclonal antibody of the present invention exhibited an excellent anticancer effect (Example 6), and the present invention was not only used for Herceptin-resistant cancer cells but also for cancer cells having sensitivity to Herceptin. Of fusion monoclonal antibodies showed similar or greater cancer therapeutic effects than Herceptin. In addition, when fusion monoclonal antibodies were prepared in various isotype antibody formats, all isotype antibodies had anti-cancer effects, but in particular, fusion monoclonal antibodies based on the human IgG1 isotype format were confirmed to have excellent anti-cancer effects ( Example 7).

As another aspect, the present invention provides a polynucleotide encoding the fusion monoclonal antibody, an expression vector comprising the polynucleotide and the expression vector is introduced into a host cell transformed to express the fusion monoclonal antibody Provide a sieve.

The polynucleotide encoding the fusion monoclonal antibody provided in the present invention is not particularly limited, but all or a portion of the gene encoding the anti-HER2 antibody and all or a portion of the gene encoding IL-2 is linked. Polynucleotides; Alternatively, all or a portion of the gene encoding the anti-HER2 antibody and all or a portion of the gene encoding IL-2 may be a polynucleotide in a form linked by a polynucleotide encoding a peptide linker.

Preferably, the polynucleotide is a polynucleotide in a form in which all or a portion of the gene encoding the heavy chain of the anti-HER2 antibody and all or a portion of the gene encoding IL-2 are linked; Polynucleotides in a form in which all or part of a gene encoding a light chain of an anti-HER2 antibody is linked to all or a part of a gene encoding IL-2; Polynucleotides in a form in which all or a portion of a gene encoding a heavy chain of an anti-HER2 antibody and all or a portion of a gene encoding IL-2 are linked by a polynucleotide encoding a peptide linker; Polynucleotides in a form in which all or part of a gene encoding a light chain of an anti-HER2 antibody and all or a part of a gene encoding IL-2 are linked by a polynucleotide encoding a peptide linker; Or a combination thereof.

More preferably, the polynucleotide is a polynucleotide in a form in which the 3'-terminus of all or a portion of the gene encoding the heavy chain of the anti-HER2 antibody is linked to the 5'-terminus of all or a portion of the gene encoding IL-2. ; A polynucleotide having a 3'-terminus of all or a portion of a gene encoding a light chain of an anti-HER2 antibody and a 5'-terminus of all or a portion of a gene encoding IL-2 of IL-2; A polynucleotide in a form in which the 3'-terminus of all or a portion of the gene encoding the heavy chain of the anti-HER2 antibody and the 5'-terminus of all or a portion of the gene encoding IL-2 are linked by a polynucleotide encoding a peptide linker; A polynucleotide in a form in which the 3'-terminus of all or a portion of the gene encoding the light chain of the anti HER2 antibody and the 5'-terminus of the all or part of the gene encoding IL-2 are linked by a polynucleotide encoding a peptide linker; Or a combination thereof.

Most preferably, the polynucleotide is a 3'-terminus of all or a portion of a gene encoding a heavy chain of an anti-HER2 antibody and a 5'-terminus of all or a portion of a gene encoding IL-2 encodes a peptide linker. It may be a polynucleotide in a form linked by polynucleotides.

Expression vectors comprising the polynucleotide encoding the fusion monoclonal antibody provided in the present invention is not particularly limited to, but is not limited to mammalian cells (eg, human, monkey, rabbit, rat, hamster, mouse cells, etc.), plants May be a vector capable of replicating and / or expressing the polynucleotide in eukaryotic or prokaryotic cells, including cells, yeast cells, insect cells or bacterial cells (e.g., E. coli), preferably in a host cell. The polynucleotide is operably linked to an appropriate promoter for expression and may be a vector comprising at least one selection marker, more preferably phage, plasmid, cosmid, mini-chromosome, virus, retrovirus The polynucleotide may be introduced into a vector or the like.

An expression vector comprising a polynucleotide encoding the fusion monoclonal antibody is an expression vector containing all of the polynucleotides encoding a heavy or light chain of the fusion monoclonal antibody or an expression containing both polynucleotides encoding a heavy or light chain It may be a vector.

The transformant into which the expression vector of the present invention is introduced is not particularly limited, but bacterial cells such as Escherichia coli, Streptomyces, Salmonella typhimurium transformed with the expression vector introduced therein; Yeast cells; Fungal cells such as Pichia pastoris; Insect cells such as Drosophila and Spodoptera Sf9 cells; CHO (Chinese hamster ovary cells), SP2 / 0 (mouse myeloma), human lymphoblastoid, COS, NSO (mouse myeloma), 293T, Bowmanella cells, HT-1080, BHK Animal hamster kidney cells, HEK (human embryonic kidney cells), and PERC.6 (human retinal cells); Or plant cells. According to one embodiment of the present invention, CHO-S cells were used as host cells (Example 1).

As used herein, the term "introduction" refers to a method of delivering a vector comprising a polynucleotide encoding a fusion monoclonal antibody to a host cell. Such introduction may be accomplished by methods such as calcium phosphate-DNA coprecipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposomal fusion, lipofectamine and protoplast fusion Can be carried out by various methods known in the art. Transfection also means that an object is transferred into a cell using viral particles by means of infection. In addition, vectors can be introduced into host cells by gene bombardment or the like. Introduction in the present invention can be used in combination with transformation.

As another aspect, the present invention provides a method for producing the fusion monoclonal antibody. The fusion monoclonal antibody is as described above.

Specifically, the method for producing a fusion monoclonal antibody of the present invention comprises the steps of (i) preparing a polynucleotide encoding the fusion monoclonal antibody; (ii) cloning the prepared polynucleotide to prepare an expression vector; (iii) preparing a transformant by introducing the prepared expression vector into a host cell; And, (iv) culturing the transformant and recovering the fused monoclonal antibody therefrom.

In this case, the polynucleotide is not particularly limited thereto, but a combination of a polynucleotide encoding a heavy or light chain fused with IL-2 and a polynucleotide encoding a heavy or light chain capable of forming an antibody together with the heavy or light chain. Can be.

In addition, the expression vector may be two expression vectors each comprising a combination of the polynucleotides, or a bisisttronic vector including both the polynucleotides of the combination.

In addition, the heavy or light chain may be linked directly to IL-2 or through a linker peptide. At this time, the linker peptide is not particularly limited thereto, but may be a peptide consisting of the amino acid sequence of GGGGS, which may be repeated 1 to 10 times, preferably consisting of the amino acid sequence of SEQ ID NO: 1, the linker peptide Polynucleotide encoding the is not particularly limited to, but may be composed of a polynucleotide sequence of SEQ ID NO: 2.

According to another embodiment of producing said fusion monoclonal antibody, the method of preparing the fusion monoclonal antibody of the present invention comprises (i) a heavy or light chain of an antibody comprising a domain capable of specifically binding to cancer cell surface antigen HER2. Preparing a polynucleotide encoding each; (ii) preparing a polynucleotide encoding human IL-2; (iii) the 3'-terminus of the polynucleotide encoding the prepared heavy chain and the 5'-terminus of the polynucleotide encoding the human IL-2 obtained above are linked using a polynucleotide encoding a linker peptide. Preparing a fusion polynucleotide encoding the fusion polypeptide; (iv) producing a respective expression vector by cloning the polynucleotide and fusion polynucleotide encoding the prepared light chain, respectively; (v) preparing a transformant by introducing each of the prepared expression vectors into one host cell at the same time; And, (vi) culturing the transformant and recovering the fused monoclonal antibody therefrom.

According to an embodiment of the present invention, the present inventors obtain genes encoding the heavy and light chains of Herceptin (HD201), which are antibodies that bind to the HER2 antigen, and genes encoding human IL-2, respectively. The 3'-end of the gene encoding the heavy chain and the 5'-end of the gene encoding human IL-2 were joined with a polynucleotide encoding a linker polypeptide to obtain a fusion polynucleotide. Then, the gene encoding the light chain and the fusion polynucleotide were cloned, respectively, to obtain respective expression vectors, and the obtained expression vectors were introduced into one host cell to obtain a transformant. Finally, the transformant was cultured to produce a desired fusion monoclonal antibody, which was purified from the culture to prepare a fusion monoclonal antibody (HD201_IL-2) (Example 1).

As another aspect, the present invention provides a pharmaceutical composition for preventing or treating cancer comprising the fusion monoclonal antibody.

As used herein, the term "cancer" refers to a malignant tumor caused by a commonly known cause, but the present invention is not particularly limited thereto, and includes all cancers in which HER2 is abnormally overexpressed on the surface of cancer cells. Breast cancer, stomach cancer, colon cancer, lung cancer, cervical cancer, endometrial cancer, ovarian cancer, prostate cancer, bladder cancer, pancreatic cancer, brain cancer, lymphoma, etc., more preferably breast cancer or stomach cancer, most preferably breast cancer Can be.

As used herein, the term "prevention" may mean any action that inhibits or delays the onset of cancer by administration of the composition. In addition, "treatment" means any action that improves or advantageously alters the symptoms caused by cancer by administration of the composition.

In addition, the composition for treating cancer of the present invention may further comprise an appropriate carrier, excipient or diluent conventionally used in the production of a pharmaceutical composition. The composition for treating cancer comprising the fusion monoclonal antibody of the present invention as an active ingredient can be administered orally or parenterally in the form of oral, granule, tablet, capsule, suspension, emulsion, oral formulation such as syrup or aerosol, Suppositories, and sterile injectable solutions. In the present invention, the carrier, excipient and diluent which may be contained in the composition containing the red head extract and the fractions include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, Gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient such as starch, calcium carbonate, in the red bean extract and fractions thereof. , Sucrose or lactose, gelatin and the like are mixed and prepared. In addition to simple excipients, lubricants such as magnesium styrate and talc are also used. Liquid preparations for oral use may include various excipients, such as wetting agents, sweeteners, fragrances, preservatives, etc., in addition to water and liquid paraffin, which are commonly used to include suspensions, solutions, emulsions, and syrups. have. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

According to one embodiment of the present invention, the present inventors confirmed that the fusion monoclonal antibody has an antibody-dependent cytotoxicity (ADCC) effect that is a cancer cell killing function, through which it can be used as an anticancer composition. Specifically, the anticancer treatment effect was confirmed in an animal model administered with breast cancer carcinoma MCF-7, BT474, JIMT1 cells, which supports that the fusion monoclonal antibody of the present invention can be used as an active ingredient of cancer treatment composition. .

As another aspect for achieving the above object, the present invention is a method for treating cancer comprising administering to a subject having the cancer in a pharmaceutically effective amount a composition comprising the fusion monoclonal antibody as an active ingredient To provide. In this case, the cancer treatment composition may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with a conventional therapeutic agent.

The cancer therapeutic composition administered in the method for treating cancer may be administered in the form of a liquid preparation, a powder, an aerosol, a capsule, an intravaginal tablet, a capsule, or a suppository. Routes of administration include, but are not limited to, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, endothelial administration, oral administration, topical administration, intranasal administration, pulmonary administration, rectal administration, and the like. However, upon oral administration, the peptide will be digested and the oral composition should be formulated to coat the active agent or protect it from degradation from above. In addition, the composition may be administered by any device capable of transferring the active agent to the target cell.

As used herein, the term "pharmaceutically effective amount" means an amount sufficient to treat or prevent a disease at a reasonable benefit / risk ratio applicable to medical treatment or prevention, and an effective dose level refers to the severity of the disease, the activity of the drug, Factors including the age, body weight, health, sex of the patient, sensitivity to the drug of the patient, time of administration of the composition of the invention used, route of administration and duration of treatment, the drug used in combination with or concurrently with the composition of the invention used And other factors well known in the medical arts.

The fusion monoclonal antibody of the present invention binds to HER2, a cancer cell surface antigen, and exhibits anti-cancer effects, and may cause IL-2, which promotes an immune response, to be concentrated on cancer cells, resulting in cancer cell death through enhanced immune responses. It can be used efficiently in treatment. In particular, the expression of HER2 protein can be usefully used in the treatment of breast cancer, which plays an important role in the progression of cancer.

The fusion monoclonal antibody of the present invention is strongly activated by immune cells around cancer cells by IL-2, and may exhibit increased antibody-dependent cytotoxicity than control anticancer antibody or Herceptin, a reported anti-HER2 antibody, and thus, more effective anticancer therapy. It can be widely used.

1 is a schematic diagram showing the structure of the fusion monoclonal antibody of the present invention.
Figure 2 is a graph showing the binding capacity of the anticancer antibody Herceptin (HD201), the fusion anticancer antibody (HD201_IL2) and the control antibody (IgG1) to BT474 cell surface HER2 antigen through a Fluorescence-activated cell sorting (FACS) device.
Figure 3 is a graph showing the effect of promoting the proliferation of CTLL-2 cells according to IL-2 or fusion monoclonal antibody (HD201_IL2).
Figure 4 shows tumor size derived from MCF7, a Herceptin resistant breast cancer cell line generated in nude mice treated with IL-2, Herceptin (HD201), IL-2 and Herceptin co-administration (HD201 + IL2), or fusion monoclonal antibody (HD201_IL2). The graph shows the change over time.
5 is derived from JIMT-1, a Herceptin resistant breast cancer cell line generated in nude mice treated with IL-2, Herceptin (HD201), IL-2 and Herceptin co-administration (HD201 + IL2), or fusion monoclonal antibody (HD201_IL2). The graph shows the change in tumor size over time.
6 is a graph showing the change in tumor size derived from BT474, a Herceptin-sensitive breast cancer cell line generated in nude mice treated with Herceptin (HD201) or fusion monoclonal antibody (HD201_IL2), over time.
Figure 7 shows the change in tumor size derived from MCF7, a Herceptin resistant breast cancer cell line generated in nude mice treated with fusion monoclonal antibody (HD201-IgG1_IL2) and fusion monoclonal antibody (HD201-IgG3_IL2) with different formats. It is a graph shown according to.

Hereinafter, the present invention will be described in more detail with reference to the following examples. These embodiments are only for illustrating the present invention, and the scope of the present invention is not construed as being limited by these embodiments.

Example 1: Preparation of a fusion monoclonal antibody in which Herceptin (HD201), which is an anti-HER2 antibody, and human IL-2

Genes encoding the heavy and light chains of Herceptin were used as SEQ ID NOs: 5 and 6 for the purpose of producing a fusion anticancer antibody combining the Herceptin and IL-2 in a CHO-S mammalian cell line, and the human IL-2 gene was NCBI. The gene sequence (SEQ ID NO: 8) published in the National Center for Biotechnology Information was used.

Expression of the nucleic acid encoding the fusion antibody can be carried out by conventional methods in prokaryotic or eukaryotic cells, and eukaryotic host-vector systems include a wide range of vectors for many different species. A representative vector for amplifying DNA is a pcDNA3.1 vector, which in turn was recombined to obtain a vector capable of expressing an antibody protein encoding an antibody polypeptide in a CHO-S mammalian cell line.

First, using a gene encoding GGGGSGGGGSGGGGS (SEQ ID NO: 1) as a linker, the 3 'end of the gene encoding the heavy chain region of Herceptin and the 5' end of the gene encoding human IL-2, The gene encoding the fusion peptide (SEQ ID NO: 9) in which the region coding for the heavy chain region of Herceptin and the region coding for IL-2 was connected through a linker gene was obtained and cloned into an expression vector.

In addition, the gene encoding the light chain region of Herceptin was cloned into another expression vector pcDNA3.1. Then, the expression vector obtained by the cloning was introduced together into the CHO-S mammalian cell line to obtain transformants, and the transformants were injected into cells in a volume of 200 mL per bottle in a 500 mL culture flask. Four days of incubation was performed in an incubator (SanYo, Japan) to produce fusion monoclonal antibodies. At this time, the medium used was a mixture of RPMI medium (Invitrogen Co., U.S.A.) supplemented with ultra low IgG fetal bovine serum and a medium dedicated to CHO cells (mixture ratio 1: 1).

After the incubation was completed, the culture solution was centrifuged to obtain a supernatant, which was filtered through a 0.22 vacuum filter device (Millipore, U.S.A.) to obtain a culture solution containing the desired fusion monoclonal antibody.

The obtained culture was applied to a recombinant protein-A Sepharose column (Hitrap MabSelect Sure, 5 mL, GE healthcare, U.S.A.). The fusion monoclonal antibody was purified from the culture.

Specifically, the obtained culture was loaded onto a recombinant Protein-A Sepharose column, and the column was first washed with 50 mM Tris-Cl (pH 7.5), 20 mM column volume with 100 mM NaCl, followed by a 10-fold column. Second washes with a volume of 50 mM Na-citrate (pH 5.0). The eluate (5 mM Na-citrate 10 mM NaCl, pH 3.4) was then added to elute the antibody fraction, and the eluted antibody fraction was neutralized by addition of 1M Tris-HCl, pH 8.0 buffer.

The eluted antibody fractions were analyzed by SDS-PAGE and the active fractions were collected and applied to a centrifugal concentrator (Amicon Ultra, 30,000 MWCO, Millipore, USA) and concentrated while exchanging the buffer with phosphate buffer. The concentrated antibody fraction was then sterile filtered with a 0.22 μm pore diameter syringe filter and the absorbance (A280) was measured to determine antibody concentration. 1 is a schematic diagram showing the structure of the fusion monoclonal antibody of the present invention. As shown in FIG. 1, it was confirmed that a new fusion monoclonal antibody having the form of IL-2 bound to the end of the IgG type antibody can be produced.

Example 2: Determination of binding ability of fusion antibody to HER2 using FACS instrument

Whether fusion monoclonal antibody of the present invention prepared in Example 1 binds to HER2 expressed in BT474 breast cancer cell line was confirmed.

First, 1 μg / ml of control IgG, Herceptin (HD201), or fusion monoclonal antibody (HD201_IL-2) was treated for 1 hour in 1 × 10 ⁵ BT474 cells, and the reaction was completed for 1 hour. The labeled secondary antibody (anti human IgG-FITC, Sigma) was added to further react, and the binding capacity of each antibody to HER2 was measured by FACS instrument (FIG. 2). 2 is a graph showing the result of measuring the binding force of the control antibody (IgG1), anticancer antibody (HD201) and fusion anticancer antibody (HD201_IL-2) to BT474 cell surface HER2 antigen using a FACS (Fluorescence-activated cell sorting) device to be. As shown in Figure 2, compared with the control antibody, the anticancer antibody (HD201) and fusion anticancer antibody (HD201_IL-2) showed a high binding force to the BT474 cell surface HER2 antigen, the anticancer antibody (HD201) and fusion anticancer antibody (HD201_IL-2) showed a specific binding activity to the BT474 cell surface HER2 antigen.

These results indicate that the fusion antibody of the present invention can bind to HER2 expressing cancer cells in the same way as the anticancer antibody and cause ADCC. In other words, IL-2 binding does not alter the binding properties of the actual anticancer antibody.

Example 3: In vitro IL-2 Dependent Cell Proliferation

IL-2R or fusion monoclonal antibody diluted to desired concentrations by CTLL-2 (ATCC, TIB-214) cells, cytotoxic T cell lines overexpressing IL-2R, in 96-well plates and diluted to 5 x 10 ⁴ cells (HD201_IL-2) was treated and then incubated for 72 hours in 37 incubator. Subsequently, 10 μl of WST-8 (Dojindo laboratories) was added per well and reacted for 1 hour, and then absorbance was measured at 450 nm (FIG. 3). Figure 3 is a graph showing the effect of promoting the proliferation of CTLL-2 cells according to IL-2 or fusion monoclonal antibody (HD201_IL-2). As shown in Figure 3, it was confirmed that the fusion monoclonal antibody (HD201_IL-2) has an effect of promoting the proliferation of cells to a similar degree to IL-2.

These results indicate that the fusion antibody of the present invention not only concentrates anticancer antibodies into HER2 expressing cells but also increases the proliferation of cytotoxic T cells by IL-2, thereby effectively killing cancer cells with antibody-dependent cytotoxicity. In addition, it was analyzed that the fusion antibody of the present invention is likely to exhibit higher antibody-dependent cytotoxicity than anticancer antibody alone or in combination with IL-2 in vivo.

Example 4: Anti-cancer effect confirmation experiment in anti-cancer model animal using MCF-7 cell line (Herceptin resistance)

Beta-estradiol sustained-release pallets were implanted under the back of nude mice, and after 3 days, MCF-7 cells were mixed with matrigel 1: 1 (v / v), and the mixed cells were mixed. Subcutaneous transplantation was performed on the back of the nude mouse at a concentration of 1 × 10 ⁷ cells / 200 μl / mouse. From the time when transplanted tumor becomes 200mm ³ (7 ~ 8 days after tumor transplantation) twice a week, the control group (Vehicle), anticancer antibody HD201 (100㎍), IL-2 (10㎍), HD201 (100㎍ ) And combination of IL-2 (10 μg) (HD201 + IL-2) and fusion monoclonal antibody HD201_IL-2 (110 μg), respectively, were administered intraperitoneally, and tumor size was measured twice a week (FIG. 4). ). Figure 4 shows changes in tumor size in nude mice treated with IL-2, Herceptin (HD201), IL-2 and Herceptin co-administration (HD201 + IL-2), or fusion monoclonal antibody (HD201_IL-2). The graph shows over time. In the fusion monoclonal antibody (HD201_IL-2) administration group, it was confirmed that the proliferation of the tumor was significantly suppressed after administration of the antibody. On the other hand, Herceptin (HD201) administration group, IL-2 alone administration group, Herceptin and IL-2 combination group did not show a significant tumor suppression effect.

These results indicate that the fusion antibody of the present invention not only concentrates the anticancer antibody into cells expressing HER2, but also increases the proliferation of cytotoxic T cells and macrophage activity by IL-2, thereby inhibiting cancer cells with antibody-dependent cytotoxicity. Supports effective killing Therefore, when administration of the fusion monoclonal antibody (HD201_IL-2) than administration of Herceptin (HD201) or IL-2 alone, it was confirmed that excellent cancer treatment effect even in cancer cells resistant to Herceptin. Moreover, the fusion monoclonal antibody of the present invention significantly reduced the tumor size than the combination of HD201 and IL-2, respectively, to support a significant effect as an anticancer therapeutic agent. The excellence of the effect of the fusion monoclonal antibody of the invention was confirmed.

Example  5: JIMT Anticancer Effect Test in Anticancer Model Animals Using -1 Cell Line (Herceptin Resistance)

JIMT-1 cells were mixed 1: 1 with matrigel (v / v), and the mixed cells were subcutaneously implanted at the back of nude mice at a concentration of 1 × 10 ⁷ cells / 200 μl / mouse. From the time when transplanted tumor becomes 200mm ³ (7 ~ 8 days after tumor transplantation) twice a week, the control group (Vehicle), anticancer antibody HD201 (100㎍), IL-2 (10㎍), HD201 (100㎍ ) And combination of IL-2 (10 μg) (HD201 + IL-2) and fusion monoclonal antibody HD201_IL-2 (110 μg) were administered to the abdominal cavity, respectively, and tumor size was measured twice a week (FIG. 5). ). Figure 5 shows changes in tumor size in nude mice treated with IL-2, Herceptin (HD201), IL-2 and Herceptin co-administration (HD201 + IL-2), or fusion monoclonal antibody (HD201_IL-2). The graph shows over time. As shown in FIG. 5, in the fusion monoclonal antibody (HD201_IL-2) -administered group, tumor proliferation was significantly suppressed after administration of the antibody, and significantly better tumor growth inhibition than Herceptin (HD201) alone group or Herceptin and IL-2 combination group. Was confirmed. IL-2 alone group did not show significant tumor suppression ability compared to the control group. These results indicate that the fusion monoclonal antibody of the present invention shows excellent cancer treatment effect. Moreover, it was confirmed in vivo that the fused monoclonal antibodies of the present invention in fused form than in simple combination administration showed better therapeutic effect.

Example  6: BT474  Anti-cancer Model Animals Using Cell Line (Herceptin Sensitivity) Anticancer effect And confirmation experiment

A beta-estradiol sustained-release pallet was implanted under the back of the nude mouse, and after 3 days, BT474 cells were mixed with matrigel 1: 1 (v / v), and the mixed cells were mixed at 1 × ^{10 7.} Cells were subcutaneously implanted at the back of the nude mice at a concentration of 200 μl / mouse. From the time when the transplanted tumor becomes 200 mm ³ (7-8 days after tumor transplantation) twice a week, the control group (Vehicle), the anticancer antibody HD201 (10㎍), and the fusion monoclonal antibody HD201_IL-2 (11㎍) Were administered to the abdominal cavity, respectively, and the tumor size was measured twice a week (FIG. 6). FIG. 6 is a graph showing the change in tumor size over time in nude mice treated with Herceptin (HD201) or fusion monoclonal antibody (HD201_IL-2).

As shown in FIG. 6, in the HD201 and fusion monoclonal antibody (HD201_IL-2) -administered group, the tumor proliferation was remarkably suppressed from 10 days after the first antibody administration, and 70 at the end of the experiment compared to the control (vehicle). It was confirmed that the tumor growth inhibitory effect was more than%, HD201_IL-2 administration group showed a similar or greater efficacy than HD201 administration group.

These results indicate that not only Herceptin resistant cancer cells but also cancer cells having susceptibility to Herceptin (HD201), the fusion antibody of the present invention exhibits a similar or more cancer treatment effect than Herceptin (HD201).

Example  7: MCF Comparison of anticancer efficacy of fusion antibodies with different formats in anticancer model animals using -7 cell line

Beta-estradiol sustained-release pallets were implanted under the back of nude mice, and after 3 days, MCF-7 cells were mixed with matrigel 1: 1 (v / v), and the mixed cells were mixed. Subcutaneous transplantation was performed on the back of the nude mouse at a concentration of 1 × 10 ⁷ cells / 200 μl / mouse. From the time when the transplanted tumor becomes 200 mm ³ (7-8 days after tumor transplantation), once a week, the control group (Vehicle), HD201-IgG1_IL-2 (100 µg), a fusion monoclonal antibody based on different isotype formats ) And HD201-IgG3_IL-2 (100 μg) anticancer antibody were administered to the abdominal cavity, respectively, and tumor size was measured twice a week (FIG. 7).

Figure 7 shows nude mice administered once weekly with a fusion monoclonal antibody (HD201-IgG1_IL-2) based on the human IgG1 isotype format and a fusion monoclonal antibody (HD201-IgG3_IL-2) based on the human IgG3 isotype format. It is a graph over time obtained by measuring the change in tumor size occurred twice a week.

In the fusion monoclonal antibody (HD201-IgG1_IL-2) group, the tumor proliferation was observed up to 22 days after the antibody administration, and the tumor size was reduced to about 43% compared to the control group (Vehicle) group. When monoclonal antibody (HD201-IgG3_IL-2) was administered, there was a difference in its remarkability such as being reduced to about 72% compared to the control group (Vehicle) administration group.

Thus, once weekly administration, the fusion monoclonal antibody (HD201-IgG1_IL-2 Ab) based on the human IgG1 isotype format was further improved compared to the fusion monoclonal antibody (HD201-IgG3_IL-2 Ab) based on the IgG3 isotype format. It was confirmed that there is a tendency to show a tumor suppressor effect on the MCF-7 cells.

In general, the human half-life of IgG1 isotype is known to be longer and more stable than IgG3 isotype. Such results indicate that the fusion antibody of the present invention (HD201-IgG1_IL-2 Ab) is excellent cancer treatment even in cancer cells resistant to Herceptin. The effect was reaffirmed.

From the above description, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are to be considered in all respects as illustrative and not restrictive. The scope of the present invention should be construed to cover all modifications and variations that come within the meaning and range of equivalents of the following claims and their equivalents, rather than the foregoing detailed description.

<110> HANWHA CHEMICAL CORPORATION <120> A fusion monoclonal antibody comprising HER2 antibody and IL-2,          and pharmaceutical composition comprising the same <130> PA121032 / KR <150> KR10-2011-0123826 <151> 2011-11-24 <160> 9 <170> Kopatentin 1.71 <210> 1 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> linker peptide <400> 1 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser   1 5 10 15 <210> 2 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> linker nucleotide <400> 2 ggtggaggtg gcagcggtgg tggcggcagt cccggtggcg gctcc 45 <210> 3 <211> 450 <212> PRT <213> Artificial Sequence <220> <223> HD201 heavy chain peptide <400> 3 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr              20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val      50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr  65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln             100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Ser Ser Val         115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala     130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val                 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro             180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys         195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp     210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile                 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu             260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His         275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg     290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu                 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr             340 345 350 Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu         355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp     370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp                 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His             420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro         435 440 445 Gly lys     450 <210> 4 <211> 215 <212> PRT <213> Artificial Sequence <220> <223> HD201 light chain peptide <400> 4 Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val   1 5 10 15 Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr              20 25 30 Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu          35 40 45 Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser      50 55 60 Gly Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln  65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro                  85 90 95 Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala             100 105 110 Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser         115 120 125 Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu     130 135 140 Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser 145 150 155 160 Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu                 165 170 175 Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val             180 185 190 Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys         195 200 205 Ser Phe Asn Arg Gly Glu Cys     210 215 <210> 5 <211> 1353 <212> DNA <213> Artificial Sequence <220> <223> HD201 heavy chain nucleotide <400> 5 gaggtgcagc tggtggagtc cggcggcggc ctggtgcagc ccggcggctc cctgcggctg 60 tcctgcgccg cctccggctt caacatcaag gacacctaca tccactgggt gcggcaggcc 120 cccggcaagg gcctggagtg ggtggcccgg atctacccca ccaacggcta cacccggtac 180 gccgactccg tgaagggccg gttcaccatc tccgccgaca cctccaagaa caccgcctac 240 ctgcagatga actccctgcg ggccgaggac accgccgtgt actactgctc ccggtggggc 300 ggcgacggct tctacgccat ggactactgg ggccagggca ccctggtgac cgtgtcctcc 360 gcctccacca agggcccctc cgtgttcccc ctggccccct cctccaagtc cacctccggc 420 ggcaccgccg ccctgggctg cctggtgaag gactacttcc ccgagcccgt gaccgtgtcc 480 tggaactccg gcgccctgac ctccggcgtg cacaccttcc ccgccgtgct gcagtcctcc 540 ggcctgtact ccctgtcctc cgtggtgacc gtgccctcct cctccctggg cacccagacc 600 tacatctgca acgtgaacca caagccctcc aacaccaagg tggacaagaa ggtggagccc 660 aagtcctgcg acaagaccca cacctgcccc ccctgccccg cccccgagct gctgggcggc 720 ccctccgtgt tcctgttccc ccccaagccc aaggacaccc tgatgatctc ccggaccccc 780 gaggtgacct gcgtggtggt ggacgtgtcc cacgaggacc ccgaggtgaa gttcaactgg 840 tacgtggacg gcgtggaggt gcacaacgcc aagaccaagc cccgggagga gcagtacaac 900 tccacctacc gggtggtgtc cgtgctgacc gtgctgcacc aggactggct gaacggcaag 960 gagtacaagt gcaaggtgtc caacaaggcc ctgcccgccc ccatcgagaa gaccatctcc 1020 aaggccaagg gccagccccg ggagccccag gtgtacaccc tgcccccctc ccgggaggag 1080 atgaccaaga accaggtgtc cctgacctgc ctggtgaagg gcttctaccc ctccgacatc 1140 gccgtggagt gggagtccaa cggccagccc gagaacaact acaagaccac cccccccgtg 1200 ctggactccg acggctcctt cttcctgtac tccaagctga ccgtggacaa gtcccggtgg 1260 cagcagggca acgtgttctc ctgctccgtg atgcacgagg ccctgcacaa ccactacacc 1320 cagaagtccc tgtccctgtc ccccggcaag tga 1353 <210> 6 <211> 648 <212> DNA <213> Artificial Sequence <220> <223> HD201 light chain nucleotide <400> 6 tccgacatcc agatgaccca gtccccctcc tccctgtccg cctccgtggg cgaccgggtg 60 accatcacct gccgggcctc ccaggacgtg aacaccgccg tggcctggta ccagcagaag 120 cccggcaagg cccccaagct gctgatctac tccgcctcct tcctgtactc cggcgtgccc 180 tcccggttct ccggctcccg gtccggcacc gacttcaccc tgaccatctc ctccctgcag 240 cccgaggact tcgccaccta ctactgccag cagcactaca ccaccccccc caccttcggc 300 cagggcacca aggtggagat caagcggacc gtggccgccc cctccgtgtt catcttcccc 360 ccctccgacg agcagctgaa gtccggcacc gcctccgtgg tgtgcctgct gaacaacttc 420 tacccccggg aggccaaggt gcagtggaag gtggacaacg ccctgcagtc cggcaactcc 480 caggagtccg tgaccgagca ggactccaag gactccacct actccctgtc ctccaccctg 540 accctgtcca aggccgacta cgagaagcac aaggtgtacg cctgcgaggt gacccaccag 600 ggcctgtcct cccccgtgac caagtccttc aaccggggcg agtgctga 648 <210> 7 <211> 134 <212> PRT <213> Homo sapiens <400> 7 Ala Pro Thr Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His   1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys              20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys          35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys      50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu  65 70 75 80 Arg Pro As Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu                  85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala             100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile         115 120 125 Ile Ser Thr Leu Thr Ala     130 <210> 8 <211> 407 <212> DNA <213> Homo sapiens <400> 8 gcccccacct cctcctccac caagaagacc cagctgcagc tggagcacct gctgctggac 60 ctgcagatga tcctgaacgg catcaacaac tacaagaacc ccaagctgac ccggatgctg 120 accttcaagt tctacatgcc caagaaggcc accgagctga agcacctgca gtgcctggag 180 gaggagctga agcccctgga ggaggtgctg aacctggccc agtccaagaa cttccacctg 240 cggccccggg acctgatctc caacatcaac gtgatcgtgc tggagctgaa gggctccgag 300 accaccttca tgtgcgagta cgccgacgag accgccacca tcgtggagtt cctgaaccgg 360 tggatcacct tctgccagtc catcatctcc accctgaccg cggccgc 407 <210> 9 <211> 599 <212> PRT <213> Artificial Sequence <220> <223> HD201_IL-2 heavy chain peptide <400> 9 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr              20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val      50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr  65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln             100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Ser Ser Val         115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala     130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val                 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro             180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys         195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp     210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile                 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu             260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His         275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg     290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu                 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr             340 345 350 Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu         355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp     370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp                 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His             420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro         435 440 445 Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly     450 455 460 Ser Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu 465 470 475 480 His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr                 485 490 495 Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro             500 505 510 Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu         515 520 525 Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His     530 535 540 Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu 545 550 555 560 Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr                 565 570 575 Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser             580 585 590 Ile Ile Ser Thr Leu Thr Ala         595

Claims (23)

A fusion monoclonal antibody in which IL-2 (Interleukin-2) is linked to an antibody that specifically binds to HER2, a cancer cell surface antigen.
The fusion monoclonal antibody of claim 1, wherein the antibody that specifically binds to HER2, the cancer cell surface antigen, is in IgG form.
The fusion monoclonal antibody of claim 2, wherein the IgG is in IgG1 form.
The fusion monoclonal antibody of claim 1, wherein the antibody that specifically binds to HER2 is in the form of a single-chain variable fragment (scFv).
The fusion monoclonal antibody of claim 1, wherein the antibody specifically binding to HER2 is trastuzumab.
The fusion monoclonal antibody of claim 1, wherein the IL-2 is linked to the C-terminus of the antibody Fc region that specifically binds to HER2.
The fusion monoclonal antibody of claim 1, wherein the antibody specifically binding to HER2 and IL-2 are linked by a linker.
8. The fusion monoclonal antibody of claim 7, wherein said linker is a peptide linker.
The fusion monoclonal antibody of claim 8, wherein the peptide linker consists of the amino acid sequence of SEQ ID NO: 1.
The method of claim 1, wherein the dissociation constant (K _D for cancer cell surface antigens) A fusion monoclonal antibody having an affinity with a value of 100 nM or less.
The method of claim 1, wherein the antibody specifically binding to HER2 comprises a heavy chain amino acid sequence comprising the amino acid sequence of SEQ ID NO: 3 and a light chain amino acid sequence comprising the amino acid sequence of SEQ ID NO: 4, Fusion monoclonal antibodies.
The fusion monoclonal antibody of claim 1, wherein the IL-2 comprises the amino acid sequence of SEQ ID NO. 7.
The fusion monoclonal antibody of claim 1, comprising a peptide consisting of the amino acid sequence of SEQ ID NO. 9.
A polynucleotide encoding the fusion monoclonal antibody of any one of claims 1 to 13.
The polynucleotide of claim 14, wherein all or a portion of a gene encoding an antibody specifically binding to HER2 and a polynucleotide in a form in which all or a portion of a gene encoding IL-2 are linked to each other; Or a polynucleotide in which all or a portion of the gene encoding the antibody and all or a portion of the gene encoding IL-2 are linked by a polynucleotide encoding a peptide linker.
The polynucleotide of claim 14, wherein the gene encoding the antibody comprises a polynucleotide sequence of SEQ ID NOs: 5 and 6. 15.
The polynucleotide of claim 14, wherein the gene encoding IL-2 comprises a polynucleotide sequence of SEQ ID NO: 8. 16.
An expression vector comprising the polynucleotide of claim 14.
A transformant transformed by introducing an expression vector of claim 18 into a host cell.
(i) preparing a polynucleotide encoding the fusion monoclonal antibody of any one of claims 1 to 13;
(ii) cloning the prepared polynucleotide to prepare an expression vector;
(iii) preparing a transformant by introducing the prepared expression vector into a host cell; And
(iv) culturing the transformant and recovering the fused monoclonal antibody therefrom, the method of producing the fused monoclonal antibody of any one of claims 1 to 13.
A pharmaceutical composition for preventing or treating cancer, comprising the fusion monoclonal antibody of any one of claims 1 to 13.
The composition of claim 21 further comprising a pharmaceutically acceptable carrier, excipient or diluent.
The composition of claim 21, wherein the cancer is breast cancer, stomach cancer, colon cancer, lung cancer, cervical cancer, endometrial cancer, ovarian cancer, prostate cancer, bladder cancer, pancreatic cancer, brain cancer or lymphoma.

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