RU2575612C2 - Anti-mesothelin immunoconjugates and use thereof - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to immunology and biotechnology. What is described is an immunoconjugate for treating mesothelin-related tumours. The conjugate consists of a human or humanised antibody, or its functional fragment containing an antigen-binding site fused with DM4 cytotoxic agent through SPDB linker. The antibody or its functional fragment show invariant binding to mesothelin, i.e. binding to various tumour cells expressing mesothelin of various forms. The antigen-binding site contains 3 respective light chain CDR-domains and 3 respective heavy chain CDR-domains. There are described a pharmaceutical composition and method of treating mesothelin-related tumours using the immunoconjugate. What is also disclosed is a method for producing the immunoconjugate involving DM4 cytotoxic agent attachment to the antibody through SPDB linker.

EFFECT: using the invention provides the immunoconjugate based on the new antibody able to bind invariantly a mesothelin epitope not cancelled by CA125 that can find application in therapy of tumours.

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Description

The present invention provides immunoconjugates comprising an antibody or fragment thereof specific for a mesothelin protein and a medicament. Compositions of such immunoconjugates can be used in the treatment, prophylaxis, or diagnosis of mesothelin-related disorders, such as cancer.

BACKGROUND OF THE INVENTION

The appearance of cancer is usually associated with aging, according to which 65% of new cases of cancer are reported for patients aged 65 years or more. Cancer is second only to heart disease among the most common causes of death in the United States. Indeed, the American Cancer Society has estimated that one in four people in the United States will die from cancer, provided that the existing mortality rate remains unchanged. Only in the USA in 2008, 1,437,180 new cases of cancer and 565,650 deaths from it are expected.

Antibody-based treatment is very effective in various types of cancer, including solid tumors. For example, HERCEPTIN® has been used successfully to treat breast cancer. A key part of the successful development of antibody-based treatment is the isolation of antibodies from cell surface proteins, which are preferably expressed on tumor cells. The polypeptide, the precursor of mesothelin, is a glucophosphatidylinositol (GPI), an anchor protein with a glucosylated cell surface that splits into a 30 kDa N-terminus hidden polypeptide and a 40 kDa C-terminus polypeptide, while taking a GPI-anchored form bound to the membrane ( Chang, K. and I. Pastan, Proc. Natl. Acad. Sci. USA, (1996) 93 (1): 136), and which is called mesothelin. Mesothelin is preferably expressed from certain tumor cells, in particular from mesotheliomatic cells, pancreatic tumor cells and ovarian tumor cells, while its expression in normal tissue is limited, which makes it the object of study in the development of tumor treatment (Argani, P. et al ., Clin. Cancer Res. (2001) 7 (12): 3862; Hassan, R., et al., Dm. Cancer Res. (2004) 10 (12 Pt 1): 3937). The function of mesothelin is not known, and no reproductive, hematological or anatomical abnormalities were found in mice that lacked the expression of the mesothelin gene (Bera, T.K. and I. Pastan, Mol. Cell. Biol. (2000) 20 (8) ): 2902).

Antibody-based targeted treatment of mesothelin-expressing cancer cells has been proposed for the treatment of cancer of the lungs, ovaries, and pancreas. Mab K1 was the first membrane-bound mesothelin polypeptide antibody and has been described (Chang, K., et al., Int. J. Cancer, (1992) 50 (3): 373). Mab K1 was obtained by immunizing mice. Due to the small similarity and low absorption of the antibody, an immunotoxin consisting of Mab K1, which is attached to a chemically shortened modified form of Pseudomonas exotoxin A, was not suitable for clinical development. (2000) 23 (4): 473; Hassan, R., et al., Clin. Cancer Res. (2004) 10 (12 Pt 1): 3937). Subsequently, single-chain antibodies with a higher level of similarity were obtained, including SSl- (dsFv) -PE38, which showed the ability to kill tumor cells under artificial conditions (Hassan, R., et al., Clin. Cancer Res. (2002) 8 ( 11): 3520), as well as efficacy in a murine model of human tumors expressing mesothelin (Fan, D., et al., Mol. Cancer Ther. (2002) 1 (8): 595). These facts confirm that the study of mesothelin will help in the development of immunotherapy for the treatment of various types of cancer. It was found that SSl- (dsFv) -PE38 is capable of rapid blood purification, and attempts have been made to increase molecular weight by attaching polyethylene glycol to a fusion protein (Filpula, D., et al., Bioconjugate Chem. (2007) 18 (3) : 773).

MS-1, MS-2 and MS-3 are mesothelin-binding antibodies that induce immune effector activity on the surface of cells due to their human IgG1 isotype and are absorbed by cells expressing mesothelin (WO 2006/099141 A2). One of the antibodies, the unconjugated, chimeric (mouse / human) IgG1 anti-mesothelin antibody MORAb 009, is currently undergoing a clinical trial to identify therapeutic effects in the treatment of pancreatic cancer. The condition for the action of MORAb 009 is the implementation of immune effector functions, such as ADCC (antibody-dependent cellular cytotoxicity) and blocking function.

New treatments with improved ability to fight aggressive cancers such as ovarian, pancreatic, and lung cancers are needed and will give an advantage in science. Thus, the present invention provides novel conjugate compositions that will be useful in the treatment, prevention and / or diagnosis of mesothelin-related disorders, for example, cancer.

SUMMARY OF THE INVENTION

The present invention relates to immunoconjugates comprising antibodies, for example monoclonal antibodies or mesothelin-binding fragments thereof, which bind to cytotoxic substances, for example maytansinoids or their derivatives, and / or are used with one or more additional anti-cancer substances. The immunoconjugates of the invention can be used to treat and / or diagnose and / or monitor disorders associated with mesothelin, such as cancer.

The aim of the invention is the creation of immunoconjugates, including antibodies or antigen-binding fragments of antibodies or their variants, highly selective for 40 kDa, the extracellular part of the precursor of the mesothelin polypeptide, and binding of mesothelin in the presence of cancer antigen 125 (CA125; MUC16), and the effector part. The specific characteristics of mesothelin antibodies are described in PCT / EP2008 / 009756, as well as in one aspect of the invention, the combination of their special ability to specific immunoreaction with mesothelin in the presence of CA125 in combination with a cytotoxic substance, for example maytansinoid, the compound promotes the activity of antibodies with blocking functions that compete with CA125 for binding to mesothelin.

In one aspect of the present invention, the antibodies or fragments thereof are IgG antibodies or IgG fragments. Antibodies or fragments may also be IgG1, IgG2a, IgG2b, IgG3, IgM, IgD, IgE, IgA, or IgM antibodies. Fab fragments, F (ab ') 2 fragments, scFv fragments, Fv fragments, double-body antibodies, linear antibodies, single-chain antibodies, biospecific antibodies, multispecific antibodies or chimeric antibodies (for example, including a framework of a human antibody attached to a human or non-human binding antibody site, or framework of a non-human antibody attached to a human or non-human antibody binding site). Chimeric antibodies may include, for example, fragments of a framework of antibodies from non-human sources, such as, for example, cows, mice, llamas, camels or rabbits. Further information on antibody development can be found in the literature, for example, Holliger and Hudson, Nature Biotechnology, (Sep, 2005) 23: 1126-1136, to which reference is made in this document. The above fragments can be obtained from immunoglobulin or in a specific way, for example, using recombinant expression, in fragmentary form.

Antibodies or fragments of the present invention can be made human, characterized in that the CDR sequences or regions (e.g., CDR1, CDR2, CDR3) can be non-human, e.g., murine.

Antibodies or antibody fragments of the present invention or compositions comprising antibodies or fragments may include a cytotoxic substance that is attached to the antibody or fragment thereof. In one aspect, the cytotoxic substance is maytansinoid or a derivative thereof, however, there are also other cytotoxic substances, which may include, for example, aplidine, oristatin, azaribine, anastrozole, azacytidine, bleomycin, bortezomide, bryostatin-1, busulfan, calicheamicin, camptothecin, 10-hydroxycamptothecin, carmustine, celebrex, chlorambucil, cisplatin, irinocetan (CPT-I 1), SN-38, carboplatin, cladribine, cyclophosphamide, cytarabine, dacarbazine, docetaxil, dactiomycin, daunoicidone dauncidone dauncidene dauncidene dauncidene dauncidone n. Any of the cytotoxic substances may also include functional analogs or derivatives thereof.

In another aspect, the present invention provides immunoconjugates in which the cytotoxic substance is not immunogenetic, i.e. does not increase the immunogeneticity of the parent antibody by involving human or mammalian cell epitopes, or T cell epitopes in drug preparation technology. The compositions of the invention can, in addition to antibodies and fragments (with or without the cytotoxic substance combined above) include various anti-cancer substances, which may include, for example, bleomycin, docetaxel (Taxotere), doxorubicin, edatrexate, erlotinib (Tarceva), etoposide, finasteride (Proscar ), flutamide (Eulexin), gemcitabine (Gemzar), gefitinib (Irresa), goserelin acetate (Zoladex), granisetron (Kitril), imatinib (Glivec), irinocetan (Kampto / Kamptosar), ondansetron (Zofels), Pak pegaspargaz (Onkaspar), nail files arpin hydrochloride (Saladzhen), sodium porphymer (Photofrin), interleukin-2 (Proleikin), rituximab (Rituxan), topotetsan (Gikamtin), trastuzumab (Herceptin), Triapine, vincristine, and vinorelbine tartrate (Navelbin or fragments, or an anti-angiogenic substance, such as, for example, angiostatin, bevicizumab (Avastin®), sorafenib (Nexavar®), baculostatin, canstatin, olives, anti-VEGF antibodies or polypeptides, antibodies with anti-placental growth factor or peptides, anti- Flk-l antibodies, anti-Fit-l antibodies or peptides, laminin peptides, fibronectis peptides, plasminogen activator inhibitors, tissue metalloproteinase inhibitors, interferons, interleukin 12, IP-IO, Gro-β, thrombospondin, 2-methoxyestradiol, polypherin proteins, carboxymidotriazole, CM1O1, marimastat, pentosan polysulfonate, 2-pentosan polisulfonate, 2-pentosanophenesulfonate , PNU145156E, 16K prolactin fragment, linomide, thalidomide, pentoxifyline, genistein, TNP-470, endostatin, paclitaxel, nektin, cidofovir, vincristine, bleomycin, AGM-1470, thrombocidal factor 4 or minocycline.

The present invention further provides, in another aspect, a method of treating a mesothelin-related disorder by controlling an effective treatment amount of an immunoconjugate of the invention or compositions of the invention that include an immunoconjugate of the invention. The disorder associated with mesothelin may include, for example, cancer, such as cancer with the formation of a solid tumor. A solid tumor can be in the ovaries, pancreas, respiratory tract, lung, colon, stomach, esophagus, cervix, liver, chest, head and neck.

These and other embodiments of the invention are disclosed and clearly shown in the following Detailed Description.

DESCRIPTION OF FIGURES

Figure 1 shows the antitumor efficacy of the anti-mesothelin immunoconjugate MF-T-SPDB-DM4 in pancreatic tumor cells transfected with mesothelin, both in the xenograft model (A) transfected with mesothelin and in the control non-transfected tumors.

Figure 2 shows the antitumor efficacy of antimesothelin immunoconjugates with persistent and cleavable, polar and non-polar linkers in the HeLaMATU xenograft model with cancer cells expressing mesothelin internally.

Figure 3 shows the antitumor efficacy of anti-mesothelin immunoconjugates with persistent and cleavable, polar and non-polar linkers in both the xenograft model (A) transfected with mesothelin and non-transfected control tumors (B).

Figure 4 shows an example dose-response curve of the toxicity of MF-T-SPDP-DM4 on mesothelin-positive HelaMatu cells.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the discovery of new immunoconjugates that are mesothelin-specific or have strong resemblance to it and may benefit the treatment described in the invention. The immunoconjugates of the invention can be used in various contexts, which are most fully described herein. You must understand the present invention is not limited to the specific details set forth in this document regarding aspects of the invention, including antimesothelin antibodies, immunoconjugates, treatment methods, protocols, cell lines, species or families of animals, constructs and reagents that are subject to change. It should also be understood that the terminology of the invention is used only to describe particular embodiments of the invention and does not limit the purpose of the present invention.

Definitions

Unless otherwise specified, all technical and scientific terms in this document have the meaning generally accepted in the field to which the invention belongs. The following references may, however, suggest the field to which the invention belongs, with the general meaning of a variety of terms, and may be used as long as such definitions correspond to the meaning generally accepted in science. Such references include, but are not limited to. Singleton et ah, Dictionary of Microbiology and Molecular Biology (2d ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); Hale & Marham, The Harper Collins Dictionary of Biology (1991); and Lackie et al., The Dictionary of Cell & Molecular Biology (3d ed. 1999); and Cellular and Molecular Immunology, Eds. Abbas, Lichtman and Pober, 2nd Edition, W. B. Saunders Company. You can also ask for help regarding any additional technical resources available to a scientist that contain definitions of terms used in this document that have a meaning generally accepted in science. For the purposes of the present invention, the following terms are defined below. Additional terms are defined elsewhere in the description. As used herein and in the appended claims, the singular forms are intended to include the plural, unless the context implies otherwise. So, for example, “gene” means one or more genes, as well as their equivalents, known in science.

As used herein, the term “antibody” includes immunoglobulin molecules (for example, any species comprising IgG, IgE, IgM, IgD, IgA and IgY, and / or any class comprising IgG1, IgG2, IgG3, IgG4, IgA1 and Ig A2) isolated from the natural environment or obtained by recombinant means. Antibodies are meant to include antigen-binding antibody fragments such as Fab, F (ab ') 2, scFv (one-valued Fvs), Fv, single-chain antibodies, double bodies, disulfide-bound Fvs (sdFv), and fragments comprising VL or VH domain obtained from the original immunoglobulins or by recombinant means.

Antibodies and / or antigen binding fragments of antibodies of the present invention can be monospecific (e.g., monoclonal), bispecific, trispecific or more. Multispecific antibodies may be specific for different epitopes of an antigen or for epitopes of more than one antigen. See, for example, WO 93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt, et al., 1991, J. Immunol. 147: 60 69; U.S. Pat. Nos. 4,474,893; 4,714,681; 4,925,648; 5,573,920; 5,601,819; Kostelny et al., 1992, J. Immmunol. 148: 1547 1553, each of which is incorporated into this application by reference.

Antigen-binding antibody fragments may contain different regions, either individually or in combination with the entire hinge region, the CH1, CH2, CH3 and CL domain or parts thereof. Antigen binding fragments of antibodies are also included in the invention, also containing any combination of different regions with a hinge region, CH1, CH2, CH3 and CL domain.

Preferably, the antibodies or antigen-binding antibody fragments are humanized, belong to a human, mouse (e.g., mouse or rat), donkey, sheep, rabbit, goat, guinea pig, camel family, horse or chicken. As used herein, “human” antibodies include antibodies having the amino acid sequence of a human immunoglobulin and antibodies isolated from a library of human immunoglobulin, from human B cells or animals transgenic for one or more human immunoglobulins, as described below and, for example, in U.S. Patent No. 5,939,598 to Kucherlapati et al. The term “antibody” also refers to other protein scaffolds that are capable of directing the CDR of an antibody insert into the same active binding structure as natural antibodies, such that the binding of the target antigen with these chimeric proteins is maintained relative to the binding activity of the natural antibody, from which were obtained by CDR. As used herein, the term “humanized” forms of non-human antibodies (eg, mice) means chimeric antibodies that contain a minimal sequence derived from non-human immunoglobulin. Most humanized antibodies are human immunoglobulins (recipient antibodies) in which hypervariable site radicals (for example, complementarity determining CDR regions) of a recipient are replaced by hypervariable site radicals (CDRs) from non-human species (donor antibody), such as a mouse, rat, rabbit, or non-human primates having the desired specificity, similarity, and activity. In some cases, the radicals of the framework region of a human immunoglobulin can be replaced by corresponding non-human radicals. Moreover, humanized antibodies may contain radicals that are not found in the recipient antibody or antibody donor. Such modifications are made to further improve antibody activity. In general, a humanized antibody may comprise almost all of at least one or usually two variable domains in which all or almost all of the hypervariable regions correspond to regions of a non-human immunoglobulin and all or almost all of the framework regions belong to the human immunoglobulin sequence. A humanized antibody may also contain at least a portion of an immunoglobulin constant region inherent in human immunoglobulin. For reference, see Jones, et al., (Nature 321: 522-525, 1986); Reichmann, et al., (Nature 332: 323-329, 1988); and Presta, (Curr. Op. Struct. Biol. 2: 593-596, 1992). The creation of humanized antibodies can be found in US Patent Nos. 7,049,135, 6,828,422, 6,753,136, 6,706,484, 6,696,248, 6,692,935, 6,667,150, 6,653,068, 6,300,064, 6,294,353, and 5,514,548, which are incorporated herein by reference in their entirety.

As used herein, the term “single chain Fv” or “sFv” antibody fragments includes the VH and VL domains of an antibody that are in the same polypeptide chain. Typically, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains, which helps sFv create the desired structure for antigen binding. For reference, see Pluckthun (The Pharmacology of Monoclonal Antibodies. Vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315, 1994), which is incorporated herein by reference in its entirety.

The term "double bodies" refers to small fragments of antibodies with two antigen binding sites, fragments of which contain a variable domain with a heavy chain (VH) connected to a variable domain with a light chain (VL) in the same polypeptide chain (VH-VL ) Using a linker that is too short to link two domains on the same chain, the domains are forced to connect to additional domains on the other chain and create two antigen-binding sites. Binary bodies are described in more detail, for example, in EP 404,097; WO 93/11161; and Hollinger, et al., (Proc. Natl. Acad. Sci. USA 90: 6444-6448, 1993), each of which is incorporated herein by reference in its entirety.

The expression "linear antibodies" refers to antibodies described in science, for example, in Zapata, et al., (Protein Eng. 8 (10): 1057-1062, 1995), which is incorporated by reference. Briefly, such antibodies comprise a pair of linked Fd segments (VH-CHI-VH-CHI) that form a pair of antigen-binding sites. Linear antibodies may be bispecific or monospecific.

The term "monoclonal antibody", as used herein, refers to an antibody derived from a collection of sufficiently homogeneous antibodies, that is, individual antibodies 10 containing an identical combination, with the exception of possible natural mutations that can be observed in minimal amounts. Monoclonal antibodies are highly specific, that is, directed to a single antigenic site. Moreover, unlike traditional (polyclonal) antibody preparations, which usually include different antibodies directed to different determinants (epitopes), each monoclonal antibody is directed to a single determinant on the antigen. The definition of "monoclonal" indicates the nature of the antibody obtained from a sufficiently homogeneous set of antibodies, and does not imply the need for its presence to create antibodies in any way. For example, monoclonal antibodies used in accordance with the present invention can be created using the hydrioma method first described by Kohler, et al. (Nature 256: 495, 1975), or the recombinant DNA method (see, for example, US Patent No. 4,816,567 ) Monoclonal antibodies can also be isolated from phage libraries of antibodies using the technique described, for example, in Clackson, et al., (Nature 352: 624-628, 1991) and Marks, et al., (J. MoI. Biol. 222 : 581-597, 1991).

Monoclonal antibodies also include “chimeric” antibodies in which the proportion of the heavy and / or light chain is identical or corresponds to homologous sequences in antibodies derived from a particular species or belonging to a particular class or subclass of antibodies, while the remainder of the chain (s) is identical or corresponds to homologically corresponding sequences in antibodies obtained from another species or belonging to another class or subclass of antibodies, as well as fragments of such antibodies, according to how they exhibit the described biological activity (see, for example, US Patent No. 4,816,567; and Morrison, et al., Proc. Natl. Acad. Sci. USA 81: 6851-6855, 1984, each of which is incorporated by reference).

As used herein, the terms “biological sample” or “patient sample” as used herein refer to a sample obtained from an organism or components (eg, cells) of an organism. Samples may consist of any biological tissue or fluid. The sample may be "clinical", that is, obtained from the patient. Such samples include, but are not limited to, saliva, blood, serous fluid, plasma, blood cells (e.g. white bodies), tissue samples, biopsies, urine, abdominal and pleural fluid, semen, breast exudate, cerebrospinal fluid, tears secreting mucus, lymph, cytosols, ascites, amniotic fluid, fluid from the bladder, bronchioalveolar fluid, or their cells. The patient sample may be fresh or frozen and treated with gerarim, cetrate or ethylenediaminetetraacetic acid. Biological samples may also include parts of tissues, for example, frozen parts for histological purposes.

The term “cancer” includes, but is not limited to, solid tumors such as cancer of the pancreas, breast, respiratory tract, brain, reproductive organs, digestive tract, urinary tract, eye, liver, skin, head and neck, thyroid, parathyroid glands and their distant metastases. The term also includes sarcoma, lymphoma, leukemia, and plasma cell myeloma.

Respiratory tract tumors include, but are not limited to, small cell and non-small cell lung cancer, as well as bronchial adenoma and pleuropulmonary blastoma. Breast tumors include, but are not limited to, invasive ductal carcinoma, invasive lobular carcinoma, ductal carcinoma, and lobular carcinoma. Brain tumors include, but are not limited to, brain stem glioma and hypophthalmic glioma, cerebellar or cerebral astrocytoma, medulloblastoma, ependinoma, and neuroectodermal and pineal tumors. Tumors of male reproductive organs include, but are not limited to, prostate cancer and testicular cancer. Tumors of female reproductive organs include, but are not limited to, intrauterine, cervical, ovarian, vaginal and female external genital organs cancer, as well as uterine cancer. Digestive tract tumors include, but are not limited to, cancer of the anal, colon, colorectal, esophagus, gall bladder, stomach, rectal, small intestine, and salivary glands. Tumors of the urinary tract include, but are not limited to, cancer of the bladder, penis, kidney, renal pelvis, ureter, and urethral. Eye cancer includes, but is not limited to, intraocular melanoma and retinoblastoma. Liver tumors include, but are not limited to, hepatocellular cancer (liver cancer cells with or without fibrolamellar variant), cholangiocellular (intrahepatic bile duct cancer), and mixed hepatocellular cholangitis. Skin cancer includes, but is not limited to, squamous cell carcinoma, Kaloshi sarcoma, malignant melanoma, Merkel skin cell cancer and non-melanoma skin cancer. Head and neck cancer includes, but is not limited to, laryngeal / ipopharyngeal / oropharyngeal / rhopharyngeal cancer and lip and oral cancer. Lymphomas include, but are not limited to, AIDS-related lymphomas, non-Hodgkin’s lymphoma, cutaneous T-cell lymphoma, Hodgkin’s disease, and central nervous system lymphoma. Sarcoma includes, but is not limited to, soft tissue sarcoma, osteogenic sarcoma, malignant fibrous histiocytoma, lymphosarcoma, and rhabdomyosarcoma. Leukemia includes, but is not limited to, acute myeloid leukoma, acute lymphoblastic, chronic lymphatic, chronic myelogenous and hair cell leukoma.

The term “epitope” as used in the designation means any antigenic determinant on an antigen, for example a mesothelin protein, to which an antibody binds through an antigen-binding site. Determinants or antigenic determinants usually consist of chemically active surface groups of molecules, such as amino acid or sugar side chains, and usually have specific three-dimensional structural characteristics, as well as specific charging characteristics.

The term “specifically immunoreactive” refers to a binding reaction between an antibody and a protein, a compound or antigen with an epitope detected by an antigen-binding region of an antibody. A binding reaction determines the presence of a protein, antigen, or epitope with a detected epitope among the 10 presence of a heterogeneous protein population and other biologies. In the context of immunological analysis, specific immunoreactive antibodies can bind to a protein with a detected epitope and, to a lesser extent, to other proteins without an epitope present in the sample. Under natural conditions, a “specifically immunoreactive” can refer to conditions in which an immune response to a vaccine or antigen is generated in an animal, for example a humoral response to an antigen (generating antigens, a vaccine, a protein, a compound or antigen that is immunologically reactive), or an indirect reaction (also a “cellular immune response”, that is, a T-lymphocyte-mediated reaction to a vaccine, protein, compound or antigen). The term “immunologically reactive conditions” as used herein is used in the context of an immunological assay or in vivo reaction, the physical reaction conditions including, for example, temperature, salt concentration, pH, reagents and their concentrations, antigen and related antibody concentrations, specifically immunoreactive with respect to the antigen, are designed in such a way as to carry out the attachment of a related antibody to the antigen. Immunologically reactive conditions depend on the nature of the antibody-binding reaction and are commonly used in immunological assay protocols. See Harlow and Lane (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Publications, New York, for a description of the nature and conditions of immunological assays. The term “patient” or “object” as used herein includes mammals (eg, humans or animals).

The term "invariant attachment" of a particular antibody to mesothelin, as used herein, refers to its ability to attach to mesothelin on various cancer cell lines expressing various forms of mesothelin. Invariant binding can be caused, but not necessarily, by the fact that antibodies or antigen-binding fragments of antibodies or their variants show an epitope of mesothelin that is not obscured by another extracellular antibody, such as cancer antigen 125 (CA125), which interacts with mesothelin. For invariant binding antibodies, the EC50 value determined by FACS titration on two separate cancer cell lines may differ by no more than 10 coagulations or preferably 5 coagulations, and most preferably from 1 to 3 coagulations.

As used herein, the term “immunoconjugate” refers to a linked molecule containing at least one antibody or antigen binding fragment thereof bound to a cytotoxic substance, for example a maytansinoid or its derivative, preferably with a suitable connecting group or its precursor.

Immunoconjugates

Inventions

The present invention relates to methods that slow down the growth of mesothelin-positive cancer cells and the development of a neoplastic disease using antimesothelin immunoconjugates. A portion of the antibodies of the provided immunoconjugates are specifically immunoreactive with respect to 40 kDa, a C-terminus domain of the mesothelin previous polypeptide (SEQ ID NO 36), which is referred to herein as “mesothelin”.

In one aspect of the invention, antibodies that bind the antigen of antibody fragments and variants of antibodies and fragments of the invention are described in PCT / EP2008 / 009756 and consist of a variable portion of the molded chain and a variable portion of the heavy chain. Variants of antibodies or antigen-binding antibody fragments of the invention are molecules in which the binding activity of an antibody or antigen-binding antibody fragment for mesothelin is maintained.

The present invention also relates to immunoconjugates consisting of antimesothelin antibodies that bind antigen fragments of antibodies and antibody variants and fragments of the invention that are different from those described in (PCT / EP2008 / 009756) and are attached to a chemotherapeutic agent, for example maytansinoids or their derivatives.

Maytansinoids that can be used in the present invention are well known in science and can be obtained from natural sources using known methods or synthetically using known methods.

Examples of suitable maytansinoids include maytansinol and maytansinol analogs. Examples of suitable analogues of maytansinol include analogues with a modified aromatic nucleus and modifications at other positions.

Specific examples of suitable analogues of maytansinol with a modified aromatic core include:

(1) C-19-dechloro (U.S. Patent No. 4,256,746) (obtained by PAH reduction of ansamitocin P2);

(2) C-20-hydroxy (or C-20-dementyl) +/- C-19-dechloro (US Patent Nos. 4,361,650 and 4,361,650) (obtained by demethylation using Streptomycetes or Actinomycetes or dechlorination using PAH); and

(3) C-20-demethoxy, C-20-acyloxy (-OCOR), +/- dechloro (US Patent No. 4,294,757) (obtained by acylation using acyl chloride).

Specific examples of suitable maytansinol analogues with modifications at other positions include:

(1) C-9-SH (U.S. Patent No. 4,424,219) (obtained from the reaction of maytansinol with H2S or P2S5);

(2) C-14-alkoxymethyl (demethoxy / CH2OR) (U.S. Patent No. 4,331,598);

(3) C-14-hydroxymethyl or acyloxymethyl (CH2OH or CH2OAc) (US Patent No. 4,450,254) (obtained from Nocardia);

(4) C-15-hydroxy / acyloxy (U.S. Patent No. 4,364,866) (obtained from the transformation of maytansinol with streptomycetes);

(5) C-15-methoxy (U.S. Patent Nos. 4,313,946 and 4,315,929) (obtained from Trewia nudiflora);

(6) C-18-N-demethyl (US Patent Nos. 4,362,663 and 4,322,348) (obtained by demethylation of maytansinol with streptomycetes); and

(7) 4,5-deoxy (US Patent No. 4,371,533) (obtained from the reduction of maytansinol with titanium trichloride / PAH).

The synthesis of maytansinoids containing thiol used in the present invention is described in detail in US Patent Nos. 5,208,020, 5,416,064, and 7,276,497.

Maytansinoids with a part of thiol at positions C-3, C-14, C-15 or C-20 are supposedly applicable. The C-3 position is preferred and the C-3 maytansinol position is most preferred. Also preferred are maytansinoid N-methyl-alanine-containing with part of C-3 thiol and maytansinoid N-methyl-cysteine-containing with part of C-3 thiol, and their analogs. Preferred maytansinoids are described in US Patents 5,208,020; 5,416,064; 6,333,410; 6,441,163; 6,716,821; RE39.151 and 7,276,497 to which reference is made in this document. In a preferred embodiment, the esterified maytansinol is prepared from N2'-deacetyl-N2 '- (3-mercapto-1-oxoprofil) -maytansin (DM1, CAS Reg. No. 139504-50-0), N2'-deacetyl-N2'- (4-mercapto-1-oxopentyl) -maitansin (DM3, CAS Per. No. 796073-54-6), and N2'-deacetyl-N2 '- (4-methyl-4-mercapto-1-oxopentyl) -maitansin ( DM4 CAS Per. No. 796073-69-3).

Reference is made herein to the following representative antibodies of the invention: "MF-J", "MOR06640", "MF-226", and "MF-T". MF-J is an antibody with a variable heavy region corresponding to SEQ ID NO: 28 (DNA) / SEQ ID NO: 20 (protein) and a variable light region corresponding to SEQ ID NO: 32 (DNA) / SEQ ID NO: 24 (protein). MOR 06640 is an antibody with a variable heavy region corresponding to SEQ ID NO: 29 (DNA) / SEQ ID NO: 21 (protein) and a variable light region corresponding to SEQ ID NO: 33 (DNA) / SEQ ID NO: 25 ( protein). MF-226 is an antibody with a variable heavy region corresponding to SEQ ID NO: 30 (DNA) / SEQ ID NO: 22 (protein) and a variable light region corresponding to SEQ ID NO: 34 (DNA) / SEQ ID NO: 26 ( protein). MF-T is an antibody with a variable heavy region corresponding to SEQ ID NO: 31 (DNA) / SEQ ID NO: 23 (protein,) and a variable light region corresponding to SEQ ID NO: 35 (DNA) / seq ID NO: 27 (protein). The invention is not limited to these antibodies, which are used as examples. Other applicable antibodies are described, for example, in PCT / EP2008 / 009756.

In one aspect, the invention provides immunoconjugates that are particularly immunoreactive to mesothelin in the presence of cancer antigen 125 (CA 125 / MUC 16) and thereby effectively target cancer cells expressing mesothelin and CA125, for example, OVCAR-3 cells.

In other aspects, the invention provides immunoconjugates that are particularly immunoreactive to one or more amino acids of the epitopes of antibodies MOR 06640 or MF-T. In certain aspects, said immunoconjugates are particularly immunoreactive to at least two, three, four, five or six amino acids of the epitopes of the MOR 06640 or MF-T antibody epitopes. In certain aspects, the immunoconjugates of the present invention are particularly immunoreactive to one or more amino acids of the epitope detected by the MOR 06640 antibody. In alternative aspects, the immunoconjugates of the present invention are particularly immunoreactive to one or more amino acids of the epitope detected by the MF-T antibody.

In another aspect, the invention provides immunoconjugates having an antigen-binding region that is particularly immunoreactive or highly similar to one or more regions of mesothelin, the amino acid sequence of which is described by SEQ ID NO: 36. The immunoconjugate is highly similar to the antigen when provided that the similarity is at least 100 nM (monovalent similarity of the Fab fragment). The immunogonjugate of the invention can preferably be especially immunoreactive to mesothelin with similarities of less than about 100 nM, more preferably less than 60 nM, and more preferably less than about 30 nM. Further preferred are antibodies that bind to mesothelin with similarities of approximately less than 10 nM, and more preferably approximately less than 3 nM. For example, the similarity of an antibody of the invention with respect to mesothelin may be about 9.1 nM or 0.9 nM (monovalent similarity of IgG1 nature).

Application methods

The term “treatment” includes any process, action, application, therapy or the like, in which a subject (patient), including a person, receives medical care using an object that improves the condition of the subject, directly or indirectly, or slowing down the development of a disease or disorder of the subject, or an improvement in at least one symptom of the disease or disorder resulting from treatment.

The term "combination treatment" or "co-treatment" means the use of two or more therapeutic agents for the treatment of a disease, condition and / or disorder. Such use includes the simultaneous use of two or more therapeutic agents, for example, in one capsule containing a fixed amount of active ingredients, or in several separate capsules for each inhibitor substance. In addition, this application includes the consistent use of each type of therapeutic agent. The procedure for applying two or more sequentially applied therapeutic agents is not limited. The phrase "therapeutically effective amount" means the amount of each substance used to obtain positive dynamics of the disease, the condition and / or severity of the disorder, and / or their symptoms, while avoiding or minimizing the side effects of this treatment.

The term "pharmaceutically acceptable" means that the preparation in question is suitable for use in a pharmaceutical product.

The immunoconjugates of the present invention are believed to be useful as therapeutic agents. Accordingly, an embodiment of the present invention includes a method of treating a different condition of a patient (including mammals), which involves the use of a composition comprising an amount of immunoconjugates effective to treat this condition.

The immunoconjugates of the present invention can be used in the treatment or prevention of diseases and / or conditions associated with the mesothelin protein. These diseases and / or conditions include, for example, cancer, such as carcinoma of the pancreas, ovaries, stomach, esophagus, cervix, colon, liver, respiratory tract and lungs. The present invention also relates to methods for improving the symptoms of a disorder in which mesothelin is increased or expressed in another atypical manner. Such disorders include, but are not limited to, carcinoma of the pancreas, ovaries, stomach, esophagus, cervix, colon, liver, respiratory tract, and lungs (see, for example, (Liao, Cancer Res. 57: 2827-2831, 1997; Turner, Hum. Pathol. 28: 740-744, 1997; Liao, et al., Am. J. Patol. 145: 598-609, 1994; Saarnio, et al., Am. J. Patol. 153: 279-285, 1998; Vermylen et al., Eur. Respir. J. 14: 806-811, 1999.) In one embodiment of the invention, a therapeutically effective dose of the immunoconjugates of the invention is applied to a patient with a disorder in which mesothelin rises.

The immunoconjugates of the present invention can be used alone or in combination with one or more additional substances. Combination treatment includes the use of a single pharmaceutical dose of a drug that contains the immunoconjugate of the present invention and additional therapeutic agents, as well as the use of the immunoconjugate of the present invention and each additional therapeutic agent with their own pharmaceutical dose of the drug. For example, the immunoconjugate of the present invention and a therapeutic agent can be applied to a patient simultaneously in the form of a single oral dosage composition or individually.

When using separate dosage forms, the immunoconjugate of the present invention, as well as one or more additional therapeutic agents, can be used practically at the same time (for example, in parallel) or separately at different times (for example, sequentially). The use of agents is not limited.

For example, in one case, the use of the antimesothelin immunoconjugate of the present invention together with one or more antitumor agents to enhance the action of the antimesothelin immunoconjugate or antitumor agent (s), or both, is provided in the treatment of diseases associated with mesothelin, such as cancer. Such comprehensive treatment can also be used to prevent cancer, prevent the recurrence of cancer, to prevent the spread or metastasis of cancer, or to relieve or improve symptoms associated with cancer.

One or more antitumor agents may contain any compound known and suitable in the art, such as, for example, chemoagents, other immunotherapeutic agents, antitumor vaccines, antiangiogenic agents, cytokines, hormonal treatment, gene therapy, and radiation therapy. A chemo-agent (or “antitumor agent” or “antitumor agent” or “therapeutic agent for cancer treatment”) refers to any molecule or compound that aids in the treatment of cancer. Examples of chemo-agents contemplated by the present invention include cytosine arabinoside, taxoids (e.g. paclitaxel, docetaxel), antitubulin agents (e.g. paclitaxel, docetaxel, epothilone B or its analogs), macrolides (e.g. rhizoxin), cisplatin, carboplatin, adriam tenoposide, mitosantrone, discodermolide, eleterobin, 2-cladribine, an alkylating agent (e.g. cyclophosphamide, chloromethine, thioepa, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclotosfamide, busulfan, dibromitomolitol C, cis-dichlorodiamine platinum (II) (DDP) cisplatin, thio-tera), antibiotics (e.g., dactinomycin, former actinomycin), bleomycin, mitramycin, anthramycins, antimetabolite (e.g. methotrexate, 6-mercaptopurine, 6-thiothin, 6-thiothin flavopiridol, 5-fluorouracil, fludarabine, gemcitabine, dacarbazine, thermosolamide), asparaginase, bacillus calmette and guerin, diphtheria toxin, hexamethylmelamine, hydroxycarbamide, LYSODREN.RTM, nucleotaxolit, camacotan, plantactoid, plantacotol, for example, plantaxotan, plantocide, plant CAMPTOSAR, CPT-I 1), Vincris in, vinca alkaloids such as vinblastine), podophyllotoxin (including derivatives such as epidophyllotoxin, VP-16 (etoposide), VM-26 (teniposide)), cytochalazine B, colcin, gramicidin D, ethidium bromide, emethine, mitomycin, procarbazine , chlormethine, anthracyclines (e.g., daunorubicin (formerly daunomidin), doxorubicin, liposomal doxorubicin), digidroksiantratsindion, mitoxantrone, mithramycin, actinomycin D, procaine, tetracaine, lidocaine, propranolol, puromycin, anti-mitotic agents, abrin, ricin A, Pseudomonas exotoxin, factor nerve growth, t rhombocyte derived from growth factor, tissue plasminogen activator, aldesleukin, allutamine, anastrozole, bicalutamide, biomycin, busulfan, capecitabine, carboplatin, chlorabusil, cladribine, cilarabin, dactinomycin, estramuzin, phloxuride, gamicidel-itidolitide, gamicidlatin phospholidin, amcidate phospholitin, gamcidate phospholitin levamisole, lomuslin, chloromethine, magestrol, acetate, mercaptopurine, mesna, mitolank, pegaspargase, pentoslatin, picamine, riuximab, campat-1, straplozocin, thioguanine, tretinoin, vinorelbine, or any other fragments, family members or their odnye, including pharmaceutically acceptable salts thereof. Compositions containing one or more chemo agents (e.g., FLAG, CHOP) are also contemplated by the present invention. FLAG contains fludarabine, cytosine arabinoside (cytarabine), and G-CSF. CHOP contains cyclophosphamide, vincristine, doxorubicin and prednisone.

The chemo agent can be an antiangiogenic agent, such as, for example, angiostatin, bevacizumab (avastin®), sorafenib (nexavar®), baculostatin, canstatin, maspine, vascular endothelial growth factor inhibitor antibodies or peptides, anti-placental growth factor antibodies or peptides, anti-antibodies Flk-1, anti-Fit-l antibodies or peptides, laminin peptides, fibronectin peptides, plasminogen activator inhibitors, tissue metalloproteinase inhibitors, interferons, interleukin 12, IP-10, Gro-β, thrombospondin, 2-methoxyestradiol, white-bound proliferin , Karboksiamidotriazol, SM101, marimastat, pentosan polysulphate, angiopoietin-2, interferon-alpha, herbimycin A, PNU145156E, 16K fragment of prolactin linomide, thalidomide, pentoxifylline, genistein, NP-470, endostatin, paclitaxel, akkutin, cidofovir, vincristine, bleomycin , AGM-1470, platelet factor 4 or minocycline.

In one case, said chemo agent is gemcitabine at a dose of 100 to 1000 mg / m ² / cycle. In one embodiment of the invention said himioagent is dacarbazine dose of 200 to 4000 mg / ^m2 / cycle. Otherwise, the specified dose is from 700 to 100 mg / m ² / cycle. There is also an option where said chemo-agent is fludarabine in a dose of 25 to 50 mg / m ² / cycle. Otherwise, the specified chemical agent is a cytosine arabinoside (nitarabine) with a dose of 200 to 2000 mg / m ² / cycle. There is also an option where said chemo-agent is docetaxel with a dose of 1.5 to 7.5 mg / kg / cycle. Otherwise, said chemo agent is paclitaxel with a dose of 5 to 15 mg / kg / cycle. There is also an option where said chemo-agent is cisplatin at a dose of 5 to 20 mg / kg / cycle. Otherwise, said chemo-agent is 5-fluorourapyl in a dose of 5 to 20 mg / kg / cycle. There is also an option where said chemo-agent is doxorubicin at a dose of 2 to 8 mg / kg / cycle. Otherwise, said chemo-agent is an epidophyllotoxin with a dose of 40 to 160 mg / kg / cycle. There is also an option where the chemo-agent is cyclophosphamide at a dose of 50 to 200 mg / kg / cycle. Otherwise, the specified chemoagent is irinotecan dose of from 50 to 150 mg / m ² / cycle. There is also an option where said chemo-agent is vinblastine at a dose of from 3.7 to 18.5 mg / m ² / cycle. Otherwise, the indicated chemoagent is vincristine at a dose of 0.7 to 2 mg / m ² / cycle. There is also an option where said chemo-agent is methotrexate with a dose of 3.3 to 1000 mg / m ² / cycle.

There is an option in which the anti-mesothelin immunoconjugates of the present invention are used in combination with one or more immunotherapeutic agents, such as antibodies or immunomodulators, which include Herceptin®, Retuxan®, OvaRex, Panorex, BEC2, IMC-C225, Vitaxin, Kampath I / H, Smart MI95, LymphoCide, Smart ID 10, as well as Oncolim, rituxan, rituximab, gemtuzumab, or trastuzumab.

The invention also provides for the use of the antimesothelin immunoconjugates of the present invention with one or more antiangiogenic agents, which include angiostatin, thalidomide, double loop domain 5, endostatin, serpin (serine protease inhibitor) antithrombin, 29 kDa N-terminal and 40 kDa C- terminal fibronectin cleavage proteins, 16 kDa prolactin cleavage protein, 7.8 kDa platelet factor 4 cleavage protein, β-amino platelet factor 4 protein (Maione et al., 1990, Cancer Res. 51: 2077), 14 amino acid peptide corresponding to the first fragment of collagen I (Tolma et al., 1993, J. Cell Biol. 122: 497), 19 amino acid peptide corresponding to the fragment of thrombospondin I (Tolsma et al., 1993, J. Cell Biol. 122: 497), 20 peptide amino acids corresponding to the SPARC fragment (Sage et al., 1995, J. Cell. Biochem. 57: 1329-), or any fragment, family member or its derivatives, including pharmaceutically acceptable salts. Other peptides that inhibit angiogenesis and correspond to fragments of laminin, fibronectin, procollagen, and EGF have also been described (See Review of Cao, 1998, Prog. MoI. Subcell. Biol. 20: 161). Monoclonal antibodies and cyclic pentapeptides that block certain integrins connecting RGD proteins (e.g., have the Arg-Gly-Asp peptide motif) have been shown to have antivascularization activity (Brooks et al., 1994, Science 264: 569; Hammes et al ., 1996, Nature Medicine 2: 529). Moreover, inhibition of the urokinase plasminogen activator receptor by antagonists inhibits angiogenesis, tumor growth, and metastases (Min et al., 1996, Cancer Res. 56: 2428-33; Crowley et aL, 1993, Proc Natl Acad. Sci. USA 90: 5021) . The use of such antiangiogenic agents is also provided by the present invention.

In another case, the anti-mesothelin immunoconjugates of the present invention are used in combination with a radiation treatment regimen.

The antimesothelin immunoconjugates of the present invention can also be used in combination with one or more cytokines, which include lymphokines, tumor necrosis factor, tumor necrosis factor cytokines, lymphotoxin-α, lymphotoxin-β, interferon-β, macrophage inflammation protein, colony-stimulating monocyte granulocyte factor, interleukins (including interleukin-1, interleukin-2, interleukin-6, nterleukin-12, interleukin-15, interleukin-18), 0X40, CD27, CD30, CD40 or CD 137 ligands, Fas-Pas ligand, 4-IBBL, endothelial monocyte activating protein or any fragments, family members or their derivatives, including pharmaceutically acceptable salts.

The anti-mesothelin immunoconjugates of the present invention can also be used in combination with an antitumor vaccine, for example, autologous cells or tissues, non-autologous cells and tissues, carcinoembryonic antigen, alpha-fetoprotein, human chorionic gonadotropin, live BCG vaccine, melanocytic M cell line proteins, e.g. 1 / MelanA, TRP-I (gp75), tyrosinase, commonly distributed, associated with the tumor, including tumor-specific antigens (e.g. BAGE, GAGE-I, GAGE-2, MAGE-I, MAGE-3, N-acetylglucosaminyltransferase-V , p15), t tumor-associated antigens (β-catenin, MUM-I, CDK4), nonmelanoma antigens (e.g. HER-2 / neutrophils (breast and ovarian carcinoma), human papillomavirus 5-E6, E7 (cervical carcinoma), MUC-1 (carcinoma of the breast, ovary and pancreas.) Human tumor antigens recognized by the T-cell, see usually Robbins and Kawakami, 1996, Curr. Opin. Immunol. 8: 628. Antitumor vaccines may or may not be purified.

In another embodiment of the invention, the antimesothelin immunoconjugates of the present invention are used together with hormonal treatment. Hormonal therapeutic treatments include hormonal agonists, hormonal antagonists (e.g. flutamide, tamoxifen, leuprorelin acetate (LUPRON), LH-RH antagonists), hormone biosynthesis and treatment inhibitors, and steroids (e.g. dexamethasone, retinoids, cortisone, cortizone, prednisone, dehydrotestosterone, glucocorticoid, mineralocorticoid, estrogen, testosterone, progestin), antigestagens (e.g. mifepristone, onapristone), as well as antiandrogens (e.g. cyproterone acetate).

The anti-mesothelin immunoconjugates of the present invention can be used in combination with anti-MDR (multidrug resistance) phenotypic agent. Many types of human cancer actually express or spontaneously develop resistance to several classes of anticancer drugs at the same time, despite the fact that each class of drugs has different structures and mechanisms of action. This phenomenon, which can be mimicked in cultured mammalian cells, is commonly called multidrug resistance (MDR) or the multidrug resistance phenotype. The MDR phenotype represents a significant barrier to the effective chemotherapeutic treatment of cancer in human patients. Resistance of malignant tumors to numerous chemotherapeutic agents is the main cause of unsuccessful treatment (Wittes et al., Cancer Treat. Rep. 70: 105 (1986); Bradley, G. et al., Biochim. Biophys. Acta 948: 87 (1988); Griswald, DP et al., Cancer Treat Rep. 65 (S2): 51 (1981); Osteen, RT (ed.). Cancer Manual, (1990)). Initially, tumors that are sensitive to cytotoxic agents often recur or develop resistance to various chemotherapeutic drugs (Riordan et al., Pharmacol. Ther. 28:51 (1985); Gottesman et al., Trends Pharmacol. Sci. Sci. 9:54 (1988); Moscow et al., J. Natl. Cancer Inst. 80:14 (1988); Croop, JM et al., J. Clin. Invest. 81: 1303 (1988)). Cells or tissues derived from tumors and grown with selective cytotoxic agents can lead to cross-resistance of other drugs of this class, as well as other classes of drugs, including, but not limited to, anthracyclines, vinca alkaloids, and epidophyllotoxins (Riordan et al. Pharmacol. Ther. 28:51 (1985); Gottesman et al., J. Biol. Chem. 263: 12163 (1988)). Therefore, the acquired resistance to the results of one drug with simultaneous resistance to various groups of drugs, structurally and functionally unrelated. Such resistance can be a problem for both solid and liquid tumors (e.g., cancer of the blood or lymph). One major mechanism of multidrug resistance in mammalian cells involves increased expression of the cell membrane glycoprotein pump system (Juranka et al., FASEB J 3: 2583 (1989); Bradley, G. et al., Blochem. Biophys. Acta 948: 87 (1988) )). The gene encoding this pumping system, sometimes called the drug transporter, has been cloned from cultured human cells and is usually called mdrl. This gene is present in several types of normal tissues, however, physiological substrates transferred for the mdrl gene product in these tissues have not been determined. The MDR1 product is a member of the ABC transport superfamily of a transport protein, a group of proteins that perform the function of volatile exports. The protein product of the mdrl gene, commonly known as P-glycoprotein (P-170, P-gp), is a 170 kDa membrane cell protein containing the aforementioned volatile pump. The expression of P-gp on the cell surface is sufficient to represent cells resistant to numerous cytotoxic agents, including many antitumor agents. P-gp-mediated MDR is an important clinical component of tumor resistance in tumors of various types, and mdrl gene expression corresponds to chemotherapy resistance of various types of cancer. The mdrl gene nucleotide sequence (Gros, P. et al., Cell 47: 371 (1986); Chen, C. et al., Cell 47: 381 (1986)) indicates that it encodes a polypeptide similar or identical to P -glycoprotein, as well as the fact that these are members of a highly conservative class of membrane proteins that are similar to bacterial carriers and participate in normal physiological transfer processes. Sequence analysis of the mdrl gene indicates that Pgp contains 1,280 amino acids distributed between two homologous (43% identity) halves. Each half of the molecule has six hydrophobic transmembrane domains, as well as an ATP binding site inside large cytoplasmic loops. Only about 8% of the molecules are extracellular, and the carbohydrate portion (approximately 30 kDa) is limited to areas of this region.

Therefore, it will be appreciated that mammalian cells having “multidrug resistance” or the “multidrug resistance” phenotype are characterized by the ability to separate, export, or eliminate the multiplicity of cytotoxic substances (eg, chemotherapeutic drugs) from the intracellular medium. Cells can acquire this phenotype as a result of pressure expressed by exposure to a separate chemotherapeutic drug (selective toxin). Otherwise, the cells may exhibit a phenotype before exposure to the toxin, since the transfer of cytotoxic substances includes a mechanism that acts together with the normal transfer of cell secretion products, metabolites, and the like. Multiple drug resistance differs from simple acquired resistance to selective toxin in that the cell is able to transfer additional cytotoxins (other chemotherapeutic agents) to which the cell has not previously been exposed. For example, Mirski et al. (1987), 47 Cancer Res. 2594-2598, describes the isolation of a multidrug-resistant cell population by growing an H69 cell line derived from a human small cell lung cancer in the presence of adriamycin (doxorubicin) as a selective toxin. The surviving cells have been found to interfere with the cytotoxic effect of the anthracycline analogue (e.g., daunomycin, epirubicin, menogaril, as well as mitoxantrone), acivicin, etoposide, gramicidin D, colchicine and the alkaloids derived from vincicum (vincristine and vinblastine), as well. Similar selective cultivation techniques can be used to produce additional multi-drug resistant cell populations. Therefore, the pharmaceutical compositions of this invention may further include compounds that act to inhibit the MDR phenotype and / or conditions associated with the MDR phenotype. Such compounds may include any MDR inhibitor compounds known in the art, such as antibodies specific for MDR components (e.g. anti-MDR transporter antibodies) or small molecular inhibitors of MDR transporters, including in particular tamoxifen, verapamil, as well as cyclosporin A, which are agents that alter or inhibit multidrug resistance. (Lavie et al. J. Biol. Chem. 271: 19530-10536, 1996, which is incorporated herein by reference). Similar compounds can be found in US Patent Nos. 5,773,280, 6,225,325, and 5,403,574, each of which is referred to herein. Such MDR inhibitory compounds can be used in conjunction with antimesoline immunoconjugates for a variety of purposes, including altering the MDR phenotype and then determining the MDR phenotype for assaying or enhancing chemotherapeutic treatment. An MDR inhibitor, such as, for example, tamoxifen, verapamil or cyclosporin A can be used together with the compounds of this invention to help determine the MDR phenotype. According to this aspect, an MDR inhibitor can enhance the uptake and accumulation of a compound of this invention in an MDR cancer cell, since the ability of the MDR transport system to transfer or “pump” the present compound compared to the substrate domain is impaired by the presence of an MDR inhibitor.

In another embodiment, the antimesothelin immunoconjugates of the present invention are used in conjunction with a gene therapy program in the treatment of cancer. Gene therapy with recombinant cells secreting interleukin-2 can be used in combination with inventive immunoconjugates for the prevention or treatment of cancer, especially breast cancer (see, for example, Deshmukh et ah, 2001, J. Neurosurg. 94: 287).

To evaluate the therapeutic efficacy of a particular immunoconjugate, for example in the treatment of cancer, it must be tested in the body in a xenograft mouse tumor model. Examples of therapeutic models are shown in Examples 1 and 2. Antibody activity can also be tested using antibody-dependent cell-mediated cytotoxicity, as described in Example 3.

Pharmaceutical Compositions and Dosages

The immunoconjugates described herein may be presented in a pharmaceutical composition comprising a pharmaceutically acceptable carrier. A pharmaceutically acceptable carrier may be pyrogen-free. These compositions may be used alone or in conjunction with at least one other agent, such as a stabilizing compound, which can be used in any sterile biocompatible pharmaceutical carrier, including salt, buffered saline, dextrose and water. A number of aqueous vehicles may be used, including salt, glycine, and the like. These solutions are sterile and usually do not contain particulate matter. These solutions can be sterilized by conventional well-known sterilization methods (e.g. filtration).

Typically, the term “pharmaceutically acceptable carrier” is recognized in the art and includes a pharmaceutically acceptable material, composition or vehicle suitable for the use of the compounds of the present invention for mammals. Carriers include a liquid or solid carrier, diluent, filler, solvent or sealant involved in transferring or transporting the necessary agent from an organ, or part of the body to another organ, or part of the body. Each carrier must be “suitable,” that is, be compatible with the other ingredients of the composition, and not harm the patient. Some examples of materials that can serve as pharmaceutically acceptable carriers include: sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powder tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository wax; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerol, sorbitol, mannitol, as well as polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic solution; ringer's solution; ethanol; phosphate buffered saline; as well as other non-toxic compatible substances used in pharmaceutical compositions. Moisturizing agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweeteners, flavoring and flavoring agents, preservatives and antioxidants, may also be present in the immunoconjugate compositions of the present invention.

Examples of pharmaceutically acceptable antioxidants include: water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium hydrogen sulfate, sodium metabisulfite, sodium sulfite and the like; oil soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol and the like; metal chelating agents such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid and the like.

The compositions may contain pharmaceutically acceptable excipients, as required to approximate physiological conditions, such as pH adaptable and buffering agents and the like. The concentration of the immunoconjugate of the present invention in such pharmaceutical compositions can vary significantly, and can also be selected, primarily, depending on the volume of liquid, viscosity, etc., in accordance with a particular selected method of application. If desired, more than one type of antibody or immunoconjugate can be included in the pharmaceutical composition (for example, an antibody with a different Ka for the mesothelin compound).

The compositions may be administered to the patient separately or together with other agents, drugs or hormones. In addition to the active ingredients, these pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers containing adjuvants and additional substances that facilitate the transition of the active substances into preparations that can be used in pharmaceuticals. The pharmaceutical compositions of the invention can be used in various ways, including, but not limited to, the oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, parenteral, local, sublingual or rectal methods.

The compositions of the invention further suggest suitable immunocarriers, such as proteins, polypeptides or peptides, such as albumin, hemocyanin, thyroglobulin and their derivatives, in particular bovine serum albumin (BSA), as well as fissurella hemocyanin (KLH), polysaccharides, carbohydrates, polymers, solid phase. Other protein and non-protein substances are well known to those skilled in the art.

In the case of vaccines, for example, antitumor vaccines, together with the antibodies of the invention, the compositions of the invention can be used with or without an adjuvant. Their use can be carried out in the absence of adjuvant, in order to avoid the toxicity caused by it. A person skilled in the art to which this invention relates, for example, an oncologist, will evaluate and understand how to determine whether an adjuvant should be used, which may depend on the object’s medical history, family examination data, toxicity data, allergy test results, etc. . In embodiments where an adjuvant is used, it is desirable that the adjuvant contribute to the formation of protective antibodies, such as protective antibodies of immunoglobulin. Any suitable adjuvant known to a person skilled in the art is contemplated by the present invention and can be easily adapted to it. Suitable adjuvants used to vaccinate animals may include, but are not limited to, aluminum hydroxide, saponin and its purified Quit A components, Freund's complete adjuvant (CFA), and Freund's incomplete adjuvant (IFA). Dextran sulfate has been shown to be a potent stimulant of the immunoglobulin 2 antibody compared to staphylococcal cell surface agents, and they are also suitable as an adjuvant. Those skilled in the art will appreciate that some adjuvants may be more preferable for use in veterinary medicine, while other adjuvants are more suitable for humans, as well as that adjuvant toxicity is evaluated by those skilled in the art before using the compound for humans.

Compositions suitable for parenteral, subcutaneous, intravenous, intramuscular and similar routes of administration; suitable pharmaceutical carriers; the preparation of compositions and their use can be carried out by any methods known to the skilled person (see, for example, Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 20 th edition, 2000). Liquid dosage forms for oral administration of a compound of the invention include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, liquid dosage forms may contain an inert diluent commonly used in the art, such as, for example, water or others, a solution such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1, 3-butylene glycol, oils (especially cottonseed, peanut, corn, seedling oil, olive, castor and sesame oil), glycerin, tetrahydrofuryl alcohol, polyethylene glycols and sorbitan fatty acid esters , as well as mixtures of them.

The terms “parenteral administration” and “apply parenterally” as used herein mean methods of administration other than enteral and local administration, usually by injection, and include, without limitation, intravenous, intramuscular, intraarterial, subdural, intracapsular, intraocular, intracardial, transtracheal, subcutaneous, intradermal, intraperitoneal, subcapsular, subarachnoid, intraspinal, as well as epigastric injection and infusion.

One of skill in the art can determine the therapeutically effective dose. A therapeutically effective dose refers to the amount of immunoconjugate that can be used to effectively treat a disease (eg, cancer), compared with the efficacy apparent in the absence of a therapeutically effective dose.

Initially, a therapeutically effective dose may be evaluated in animal models (e.g., rats, mice, rabbits, dogs, or pigs). Animal models can also be used to determine the level of concentration and route of administration. Such information can then be used to determine effective doses and routes of administration for humans. Therapeutic efficacy and toxicity (e.g., ED50 is the therapeutically effective dose for 50% of the population, and LD50 is the lethal dose for 50% of the population) of the immunoconjugate can be determined using standard pharmaceutical procedures in cell cultures or in experimental animals. The dose to therapeutic effect ratio is the therapeutic index, which can be expressed by the ratio of LD50 / ED50. Data from animal studies can be used in determining dosage limits for humans. The dosage contained in such compositions may be within a common concentration that includes an ED50 with little or no toxicity. Dosage varies within these limits depending on the dosage used, patient sensitivity, route of administration.

The exact dose may be determined by the physician due to factors related to the patient requiring treatment. Dosage and administration may vary to provide a sufficient level of immunoconjugate or to maintain the desired effect. Factors that can be taken into account include the severity of the disease, the general health of the object, age, height, gender, diet, time and frequency of use, the combination (s) of the drug, the sensitivity of the reaction, and the resistance / response to treatment. Polynucleotides encoding the immunoconjugates of the invention can be constructed and introduced into a cell either externally or into a living organism using generally recognized techniques, including, but not limited to, transferrin-polycation-mediated DNA transfer, transfection with a depleted or coated nucleic acid , liposome-mediated cell fusion, intracellular transfer of DNA-coated latex granules, protoplast fusion, viral infection, electroporation, "gene gun", as well as DEAE- or calcium phosphate-mediated transfection.

The efficacy in a living organism of the toxophore components of the immunoconjugate is in the range of about 5 mg to about 500 mg per 1 kg of patient weight. The method of using the immunoconjugate containing the pharmaceutical compositions of the present invention may be any suitable methods in which the antibody is delivered to the host. For example, the pharmaceutical compositions of the invention may be effective for parenteral administration (e.g., subcutaneously, intramuscularly, intravenously or intranasally). All patents and patent applications referred to in this disclosure are expressly incorporated herein by reference. The above disclosure generally describes the present invention. A more complete understanding can be obtained with reference to the following specific examples, presented only for the purpose of illustration and not designed to limit the scope of the invention.

EXAMPLES

EXAMPLE 1: Efficacy of an immunoconjugate in mesothelin expressing a xenographic mouse model of human pancreatic cancer

To analyze whether antimesothelin immunoconjugates were able to reduce tumor development in a mesothelin-dependent manner, whether human pancreatic cancer cells (MiaPaCa-2) were successfully transferred with mesothelin and used to form a subcutaneous tumor in a mouse model. The HT29 human colon carcinoma cell line was used to establish mesothelin-negative control tumors as part of the study's effectiveness. MiaPaCa cells were maintained as adhesive cultures in DMEM supplemented with 10% (v / v) FCS, 2.5% (v / v) horse serum, 1.5 g / l sodium biocarbonate, 4.5 g / l glucose, 4 mmol of glutamine and 0.4% (volume to volume) hygromycin. NT29 cells were grown in McCoy 5a medium with 1.5 mmol of glutamine, 2.2 g / L sodium biocarbonate and 10% (volume to volume) FCS. The expression of mesothelin of MiaPaCa-2 cells, as well as the absence of mesothelin in HT29 cells was confirmed by FACS (not shown). To evaluate the growth of tumor cells in a living organism, female NMRI nude mice were grafted subcutaneously into the right flank of 3 × 10 ⁶ degree MiaPaCa-2 cells or 1 × 10 ⁶ degree HT29 cells reconstituted in 50% Matrigel ™ or 50% medium. The antimesoline immunoconjugates MF-J-SPDB-DM4, MF-T-SPDB-DM4, MF226-SPDB-DM4 and MOR6640-SPDB-DM4 were tested at therapeutic doses of 0.01 mg / kg, 0.03 mg / kg, 0 , 05 mg / kg and 0.2 mg / kg (associated with the amount of toxophore). MF-J-SPDB-DM4, MF-T-SPDB-DM4, MF226-SPDB-DM4 and MOR6640-SPDB-DM4 were formed using the following procedure: antimesothelin antibodies were modified with 4- [2-pyridyldithio] butanoic acid N- hydroxycynimidine ester (SPDB) for the introduction of dithiopyridyl groups. At 8 mg / ml, a ~ 6-fold molar excess of SPDB (~ 20 mmol of stock solution in ethanol or ethanol) was used to modify the antibodies. Modified antibodies reacted with a 1.7-fold molar excess of the free form of the matansinoid thiol compared to thiopyridyl. The reaction was carried out with 2.5 mg / ml antibody in the presence of 3% dimethylacetamide (3% volume to volume) for 20 hours at room temperature. The conjugation reaction mixture was purified from unreacted preparation and reaction by-products using a Sephadex G25 demineralizing column. The number of matansinoid molecules in the antibody was calculated by measuring absorbance at 252 nanometers and 280 nanometers, using an absorption coefficient of 224,000 M ^-1 · cm ^-1 for the antibody and 5180 M ^-1 · cm ^-1 for DM4 at 280 nanometers. The absorption ratio of 252 nanometers / 280 nanometers is 0.37 for antibodies and 5.05 for DM4.

The treatment was started after the tumor was established on the 5th day after the introduction of the tumor cell, then two stages of treatment followed on the 8th and 12th day after the introduction of the tumor cell. Control mice were treated with either 0.2 mg / kg of the non-targeted immunoconjugate (antilysocim-SPDB-DM4) or equal volumes of the carrier itself (10 mmol of histidine, 130 mmol of glycine, 5% (weight / volume) sucrose, pH 5.5). The treatment was carried out with a dosage of 100 ml / 10 g of body weight by intravenous administration. In each group there were 6 animals. The health status of the mice was checked every day. The length and width of subcutaneous tumors were measured by the electromagnetic method twice a week. The tumor site was measured by the formula: tumor site [mm ² ] = length [mm] × width [mm]. All data obtained during the experiment were documented. An example of the antitumor efficacy of the anti-mesothelin immunoconjugate MF-T-SPDB-DM4 in mesothelin-transfected cells of human pancreatic cancer at various therapeutic doses is shown in Figure 1. Female × NMR female mice were inoculated with 3 × 10 ⁶ mesothelin-positive cancer cells MiaPaCa-2 of the human pancreas or 1 × 10 ⁶ mesothelin-negative HT29 colon cancer cells (B), re-dissolved in 50% Matrigel ™ / 50% medium in their right flank. On the 5th, 8th and 12th day after the introduction of the tumor cell, the mice received 0.01, 0.03, 0.05, 0.2 mg / kg MF-T-SPDB-DM4 (all concentrations relate to the amount of toxophore), or the carrier itself. The length and width of the tumors were measured twice a week, and the tumor area was calculated by multiplying the width by length. The average values and standard deviation of each group, as well as the measured moment of time, are indicated on the graph. All n = 6. Footnotes indicate P-values <0 05.

Treatment of tumor mice showed that all the tested antimesothelin immunoconjugates were able to inhibit the growth of mesothelin-positive MiaPaCa-2 tumors in a living organism at doses of 0.03 mg / kg, 0.05 mg / kg and 0.2 mg / kg. At doses of 0.05 mg / kg and 0.2 mg / kg MF-T-SPDB-DM4, the tumor was completely destroyed without resuming its growth until the end of the observation period, lasting 132 days. 0.05 mg / kg of the non-targeted control of antilysocyme-SPDB-DM4 had no effect on the growth of the mesothelin-positive MiaPaCa tumor (Table 1). Compared to untreated and carrier-treated tumors, the growth of mesothelin-positive HT29 tumors was not significantly stopped by the largest dose of 0.2 mg / kg MF-T-SPDB-DM4. This suggests that significant tumor inhibitory efficacy, MF-T-SPDB-DM4, depends on the expression of mesothelin in the tumor.

Table 1: Tumor inhibitory efficacy of antimesothelin immunoconjugates in a mesothelin-positive xenograft tumor model MF-J-DM4 MOPR-6640-DM4 MF-T-DM4 MF-226-DM4 α Lysozyme-DM4 (control) DM4 / Ab 2,8 3.6 3.6 3.3 4.0-4.1 0.01 mg / kg - - - - but. 0.03 mg / kg but. + + + but. 0.05 mg / kg + + ++ a) + - 9.2 mg / kg + ++ b) ++ a) ++ + b) ++ radical tumor removal a) complete removal in more than 132 days + decrease / regression b) the resumption of tumor growth in all animals (n = 6) for 132 days - lack of value effect, the estimated duration of 20-30 days. but. undefined

EXAMPLE 2: Efficacy in tumors endogenously expressing mesothelin, as well as a comparison of different linkers

In order to find out whether antimesothelin immunoconjugates are able to stop the development of endogenous mesothelin expressing tumor cells in the body, a xenograft model with subcutaneous growing cervical cancer cells (HeLaMATU) was used. HeLaMATU cells were maintained as adhesive cultures in DMEM / HAMS12 medium supplemented with 10% (v / v) FCS, 2.5% (v / v) horse serum, 1% sodium pyruvate, 1% (v / v) glutamine. Mesothelin expression was confirmed by FACS analysis in vitro. Female NMRI nude mice were subcutaneously inoculated into the right flank with 1.5 × 10 ⁶ HeLaMATU cells reconstituted in 50% Matrigel ™ / 50% medium. Additionally, it was examined whether the exchange of a cleavable SPDB linker causes a polar (-sulfo-SPDB), stable linker (-SMCC), or a polar and stable linker (- (PEG) 4-mal) to alter the anti-cancer effectiveness of the MOR6640-based immunoconjugate in the body . Tumor mice HeLaMATU received intravenously 0.2 mg / kg of either MOR6640-SPDB-DM4, MOR6640-SMCC-DM1, MOR6640-sulfo-SPDB-DM4, or MOR6640- (PEG) 4-mal-DMl (associated with the amount of toxophore) on the 5th, 8th and 12th day after the introduction of the tumor cell. Control mice were treated with either 0.2 mg / kg of the non-targeted immunoconjugate (antilysocim-SPDB-DM4) or with equal volumes of the carrier itself. In each group there were 6 animals. The health status of the mice was checked every day. The length and width of subcutaneous tumors were measured by the electromagnetic method twice a week. The tumor site was measured by the formula: tumor site [mm ² ] = length [mm] × width [mm]. The data obtained are presented in Figure 2. Treatment of mice with tumors showed that a) antimesothelin immunoconjugates effectively stopped the development of tumors expressing mesothelin endogenously in the body, and b) that conjugates with cleavable linkers (MOR6640-SPDB-DM4 and MOR6640) showed a higher antitumor efficiency compared to conjugates with stable linkers (MOR6640-SMCC-DM1 and MOR6640- (PEG) 4-mal-DMl). In particular, MOR6640-SPDB-DM4 and MOR6640-sulfo-SPDB-DM4 led to the destruction of tumors in all treated animals eleven days after treatment, while the treatment of MOR6640-SMCC-DM1 and MOR6640- (PEG) 4-mal -DMl only halted tumor development. However, eleven days after the last treatment, the areas of the tumors treated with MOR6640-SMCC-DM1 and MOR6640- (PEG) 4-mal-DMl, respectively, became significantly smaller compared to the tumors treated with the carrier or antilysocyme-SPDB-DM4 .

The non-targeted control conjugate did not affect tumor growth. In order to compare the antitumor efficacy of various linkers in the second xenograft model, we used a model with subcutaneously growing vector or mesothelin transfected (# 37) MiaPaCa-2 cells (cells of human pancreatic cancer). MiaPaCa-2 vector and MiaPaCa-2 # 37 cells were maintained as adhesive cultures in DMEM / HAMS12 medium supplemented with 10% (v / v) FCS, 1% (w / v) glutamine and 0.1 mmol of non-basic amino acids. Mesothelin expression was confirmed by FACS analysis, as well as immunohistochemical analysis of subcutaneous tumors outside the body. Female NMRI nude mice were subcutaneously inoculated into the right flank with 3 × 10 ⁶ MiaPaCa-2 vector and MiaPaCa-2 # 37 cells reconstituted in 50% Matrigel ™ / 50% medium, respectively. Tumor mice received 0.05 mg / kg of either MOR6640-SPDB-DM4, MOR6640-SMCC-DM1, MOR6640-sylfo-SPDB-DM4, or MOR6640- (PEG) 4-mal-DMl (associated with the amount of toxophore) by 5 , 8 and 12 days after the introduction of the tumor cell. Control mice were treated with either 0.05 mg / kg of the non-targeted immunoconjugate (antilysocim-SPDB-DM4) or with equal volumes of the carrier itself. In each group there were 6 animals. The health status of the mice was checked every day. The length and width of subcutaneous tumors were measured by the electromagnetic method twice a week. The tumor site was measured by the formula: tumor site [mm ² ] = length [mm] × width [mm]. Tumor growth data are shown in Figure 3. In mice with mesothelin-expressed tumors, treatment with MOR6640-SPDB-DM4 and MOR6640-sulfo-SPDB-DM4 led to the destruction of tumors in all treated animals twelve days after treatment. However, the resumption of growth of these tumors occurred ten days later (Figure 3A). Treatment with MOR6640-SMCC-DM1, MOR6640- (PEG) 4-mal-DMl, as well as antilysocyme-SPDB-DM4, did not significantly affect tumor development. Unlike mesothelin-expressed tumors, none of the treatment methods resulted in a change in the growth of the vector of transfected MiaPaCa-2 cells in the body (Figure 3B).

EXAMPLE 3: cytotoxicity of antimesothelin immunoconjugates in artificial conditions

To assess the cytotoxicity of antimesothelin immunoconjugates, 80-90% were grown, trypsinized and counted various mesothelin-expressed cell lines. The cells were then seeded in 384 flat dishes in 800 with 25 μl of volume per cell in their growth medium for all cell lines. Cells with the medium were installed for blank subtraction. 24 hours after seeding, MF-J-SPDB-DM4, MF-226-SPDB-DM4, MOR6640-SPDP-DM4, MF226-SPDP-DM4, as well as antilysocyme-SPDP-DM4 were divided into doses ranging from 0.01 up to 3.00 nmol. For each dilution, triplicates were established. Final measurements were performed after 96 hours. Cell viability was evaluated by measuring samples of WST-1 (Roche Cat # 1644807). IC ₅₀ values are shown in Table 2. A dose response curve indicating, in vitro, the cytotoxicity of MF-T-SPDP-DM4 of HelaMatu cells is shown in Figure 4.

Table 2: nM IC ₅₀ values of antimesothelin immunoconjugates on expressing mesothelin cell lines IC50 (nM) MF-J-SPDP DM4 MOR06640-SPDP- DM4 MF-T-SPDP-DM4 MF-226-SPDP-DM4 α Lysozyme -SPDP-DM4 (control) MiaPaCa-2 (Mesothelin +) 1.4 0.36 1.3 21 > 100 MiaPaCa-2 (Mesothelin-) > 100 > 100 > 100 > 100 > 100 NT29 C2 (Mesothelin +) 0.3 0.21 1,5 8 eighteen HT29V (Mesothelin-) > 50 > 50 > 50 > 50 > 50 CHOA9 * (Mesothelin +) 0.35 1,2 1,0 1.3 > 50 СНО К1 * (Mesothelin-) > 50 > 50 > 50 > 50 > 50 DU145 5.2 5.1 9 10 - HCT116 9.0 20.7 17,2 26.5 33.1 SW480 > 50 > 50 > 50 > 50 > 50 MDAMB231 > 50 > 50 > 50 > 50 > 50 Mcf10a > 50 > 50 > 50 > 50 > 50 Helamatu 0.2 0.6 0.45 0.9 0.4 OVCAR-3 0.9 1,07 2,8 14.8 > 100 KD Fab (nM) 9.2 0.19 16.3 58.3

Table 3: Antibody Sequences Antibody Hcdr1 Hcdr2 Hcdr3 LCDR1 LCDR2 LCDR3 VH protein VL protein VH nucleotide VL nucleotide SEQID SEQID SEQID SEQID SEQID SEQID SEQID SEQID SEQID SEQID Mf-j one four 7 10 13 16 twenty 24 28 32 MOR6640 one four 7 10 13 17 21 25 29th 33 Mf-226 2 5 8 eleven fourteen eighteen 22 26 thirty 34 Mf-t 3 6 9 12 fifteen 19 23 27 31 35

Claims (5)

1. An immunoconjugate intended for the treatment of tumor disorders associated with mesothelin, containing a cytotoxic agent representing N2′-deacetyl-N2 ′ - (4-methyl-4-mercapto-1-oxopentyl) -maytansin (DM4), and human or a humanized antibody or its functional fragment containing an antigen binding site that is mesothelin-specific (SEQ ID NO: 36), wherein said antibody or its functional fragment exhibits invariant mesothelin binding, where the antigen binding site contains:
HCDR1 according to SEQ ID NO: 3,
HCDR2 according to SEQ ID NO: 6,
HCDR3 according to SEQ ID NO: 9,
LCDR1 according to SEQ ID NO: 12,
LCDR2 according to SEQ ID NO: 15,
LCDR3 according to SEQ ID NO: 19,
moreover, the cytotoxic agent is associated with the antibody via an SPDB linker, which is an ester of 4- [2-pyridyldithio] butanoic acid and N-hydroxysuccinimide. 2. The immunoconjugate as defined in paragraph 1, intended for the treatment of cancer associated with mesothelin. 3. A pharmaceutical composition for treating mesothelin-related tumor disorders, comprising the immunoconjugate of claim 1 or 2 in a therapeutically effective amount, and a pharmaceutically acceptable carrier or excipient thereof. 4. A method for producing an immunoconjugate according to claim 1 or 2, comprising the step of attaching a cytotoxic agent, which is DM4, through an SPDB linker with a human or humanized antibody or a functional fragment thereof containing an antigen binding site that is mesothelin-specific (SEQ ID NO: 36), wherein said antibody or a functional fragment thereof exhibits an invariant binding of mesothelin, where the antigen binding site contains:
HCDR1 according to SEQ ID NO: 3,
HCDR2 according to SEQ ID NO: 6,
HCDR3 according to SEQ ID NO: 9,
LCDR1 according to SEQ ID NO: 12,
LCDR2 according to SEQ ID NO: 15,
LCDR3 according to SEQ ID NO: 19. 5. A method of treating a tumor disorder or condition associated with the undesirable presence of mesothelin, comprising administering to a patient in need of such treatment an effective amount of a pharmaceutical composition according to claim 3.

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