HK1228261B - Anti-mesothelin immunoconjugates and uses therefor - Google Patents

Description

Anti-mesothelin immunoconjugates and their applications

This patent application is a divisional application of the patent application with the same title and patent application number 201080029126.1 and application date April 16, 2010.

Technical Field

The present invention provides an immunoconjugate comprising an antibody or fragment thereof specific for mesothelin and a therapeutic agent. Such an immunoconjugate composition can be used to treat, prevent, or diagnose mesothelin-related disorders, such as cancer.

Background Art

Cancer is most commonly associated with aging, with 65% of all new cancer cases occurring in patients aged 65 and older. In the United States, cancer is the second leading cause of death, behind heart disease. In fact, the American Cancer Society has estimated that if current mortality rates remain constant, one in four deaths in the United States will be attributable to cancer. In the United States alone, 1,437,180 new cancer cases and 565,650 deaths from cancer are expected in 2008.

Antibody-based therapies have been shown to be very effective in treating a variety of cancers, including solid tumors. For example, Herceptin® has been successfully used to treat breast cancer. The key to developing successful antibody-based therapies is to isolate antibodies that are directed against cell surface proteins that have been found to be preferentially expressed on tumor cells. The mesothelin precursor polypeptide is a glycophosphatidylinositol (GPI)-anchored, glycosylated cell surface protein that is proteolytically cleaved into a 30 kDa N-terminal secreted polypeptide and a 40 kDa C-terminal polypeptide, which exists primarily in a membrane-bound, GPI-anchored form (Chang, K. and I. Pastan, Proc. Natl. Acad. Sci. USA, (1996) 93(1):136), and is herein designated mesothelin. Mesothelin is preferentially expressed by certain tumor cells (particularly mesothelioma cells, pancreatic tumor cells, and ovarian cancer cells), while its expression in normal tissues is restricted, making it an attractive target for the development of tumor therapies (Argani, P. et al. , Clin. Cancer Res. (2001) 7(12): 3862; Hassan, R., et al ., Clin. Cancer Res. (2004) 10(12 Pt 1): 3937). The function of mesothelin is unknown, and no obvious reproductive, hematological, or anatomical abnormalities were observed in mice deficient in the mesothelin gene (Bera, TK and I. Pastan, Mol. Cell. Biol. (2000) 20(8): 2902).

Antibody-based targeted therapy against mesothelin-expressing cancer cells has been proposed for the treatment of lung, ovarian, and pancreatic cancers. Mab K1 was the first antibody to be described that targets the membrane-bound mesothelin polypeptide (Chang, K., et al. , Int. J. Cancer, (1992) 50(3):373). Mab K1 was prepared by immunizing mice. Due to the low affinity and poor internalization rate of this antibody, an immunotoxin consisting of Mab K1 linked to a chemically modified, truncated form of Pseudomonas exotoxin A was considered unsuitable for clinical development (Hassan, R., et al. , J. Immunother. (2000) 23(4):473; Hassan, R., et al. , Clin. Cancer Res. (2004) 10(12 Pt 1):3937). Subsequently, single-chain antibodies with higher affinity were developed, including SS1-(dsFv)-PE38, which showed activity in killing tumor cells in vitro (Hassan, R., et al. , Clin. Cancer Res. (2002) 8(11): 3520) and efficacy in a mouse model of tumors expressing human mesothelin (Fan, D., et al. , Mol. Cancer Ther. (2002) 1(8): 595). These data confirm that mesothelin is an attractive target for developing immunotherapies for the treatment of various cancers. SS1-(dsFv)-PE38 has been shown to have rapid blood clearance, and attempts to increase molecular weight by pegylation of the fusion protein have been reported (Filpula, D., et al. , Bioconjugate Chem. (2007) 18(3): 773).

MS-1, MS-2, and MS-3 are antibodies that bind to mesothelin and elicit immune effector activity at the cell surface due to their human IgG1 isotype and internalization into mesothelin-expressing cells (WO 2006/099141 A2). One of these antibodies, the unconjugated, chimeric (mouse/human) IgG1 anti-mesothelin antibody MORAb 009, is currently being tested in clinical trials for its therapeutic efficacy in the treatment of pancreatic cancer. The postulated mechanism of action of MORAb 009 is to trigger immune effector functions such as ADCC and functional blockade.

New therapies with improved efficacy in combating aggressive cancers, such as ovarian, pancreatic, and lung cancers, are highly desirable and represent an advance in the art. Thus, the present invention discloses novel immunoconjugate compositions that can be used to treat, prevent, and/or diagnose mesothelin-related disorders, such as cancer.

Summary of the Invention

The present invention relates to immunoconjugates comprising an antibody (e.g., a monoclonal antibody) or fragment thereof that binds to mesothelin, conjugated to a cytotoxic agent (e.g., a maytansinoid or a derivative thereof), and/or co-administered or co-formulated with one or more additional anti-cancer agents. The immunoconjugates of the present invention can be used to treat and/or diagnose and/or monitor mesothelin-related disorders, such as cancer.

One object of the present invention is to provide immunoconjugates comprising: an antibody, or antigen-binding antibody fragment or variant thereof, that is highly selective for the 40 kDa C-terminal extracellular portion of the mesothelin precursor polypeptide and does not bind to mesothelin in the presence of cancer antigen 125 (CA125; MUC16), and an effector moiety. The specific properties of mesothelin antibodies have been described in PCT/EP2008/009756, and in one aspect of the present invention, their exceptional ability to specifically immunoreact with mesothelin in the presence of CA125, combined with conjugation to a cytotoxic agent (e.g., a maytansine class), provides enhanced efficacy over function-blocking antibodies that compete with CA125 for mesothelin binding.

In one aspect, antibody of the present invention or its fragment is IgG antibody or IgG fragment.Described antibody or fragment can also be IgG1, IgG2a, IgG2b, IgG3, IgM, IgD, IgE, IgA or IgM antibody, Fab fragment, F (ab ') 2 fragment, scFv fragment, Fv fragment, bispecific antibody, linear antibody, single-chain antibody, biospecific antibody, multispecific antibody or chimeric antibody (for example comprising the human antibody scaffold that is transplanted on people or non-human antibody binding region, or transplanting on people or non-human antibody binding region non-human antibody scaffold).Described chimeric antibody can comprise, for example, from the antibody scaffold district of non-human source (for example, ox, mouse, llama, camel or rabbit).About other information of antibody engineering, can reference, for example, Holliger and Hudson, Nature Biotechnology, (September 2005) 23: 1126-1136, it is incorporated herein by reference. The aforementioned fragments can be obtained from immunoglobulins, or prepared in the form of fragments by appropriate means (eg, recombinant expression).

The antibodies or antibody fragments of the present invention may also be humanized, wherein the CDR sequences or regions (eg, CDR1, CDR2, CDR3) may be non-human, such as murine.

The antibodies or antibody fragments of the present invention, or compositions comprising the same, may include a cytotoxic agent conjugated to the antibody or fragment thereof. In one aspect, the cytotoxic agent is a maytansine or a derivative thereof, however, other cytotoxic agents are also provided and can include, for example, and other cytotoxic agents, for example, aplidin, auristatin, azaribine, anastrozole, azacytidine, bleomycin, bortezomib, bryostatin-1, busulfan, calicheamicin, camptothecin, 10-hydroxycamptothecin, carmustine, celebrex, chlorambucil, cisplatin, irinotecan (CPT-1 1), SN-38, carboplatin, cladribine, cyclophosphamide, cytarabine, dacarbazine, docetaxel, dactinomycin, daunorubicin glucuronide, daunorubicin, dexamethasone, diethylstilbestrol, doxorubicin, doxorubicin glucuronide, epirubicin glucuronide, ethinyl estradiol, estramustine, etoposide, etoposide glucuronide, etoposide phosphate, azauridine (FUdR), 3',5'-O-dioleoyl-FudR (FUdR-dO), fludarabine, flutamide, fluorouracil, fluoxymesterone, gemcitabine, hydroxyprogesterone caproate, hydroxyurea, idarubicin, ifosfamide, L-asparaginase, folinic acid, lomustine, nitrogen mustard, medroxyprogesterone acetate, megestrol acetate, melphalan, mercaptopurine, 6-mercaptopurine, methotrexate, mitoxantrone, mithramycin, mitomycin, mitotane, phenyl butyrate, prednisone, procarbazine, paclitaxel, pentostatin, PSI-341 , semustine, streptozocin, tamoxifen, taxanes, taxol, testosterone propionate, thalidomide, thioguanine, thiotepa, teniposide, topotecan, uramustine, velcade, vinblastine, vinorelbine, vincristine, ricin, abrin, ribomiclease, onconase, rapLRI, DNase I, Staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin, diphtheria toxin, or a combination thereof. Any of the cytotoxic agents may also include functional analogs or derivatives thereof.

In another aspect, the invention provides immunoconjugates wherein the cytotoxic agent is non-immunogenic, ie, does not increase the immunogenicity of the parent antibody by contributing human or mammalian B-cell epitopes or T-cell epitopes to the pharmaceutical preparation.

In addition to the antibodies and fragments (with or without the aforementioned conjugated cytotoxic agents), the compositions of the present invention may comprise various anticancer agents, which may include, for example, bleomycin, docetaxel (Taxotere), doxorubicin, edatrexate, erlotinib (Tasifar), etoposide, finasteride (Proscar), flutamide (Eulexin), gemcitabine (Gemzar), gefitinib (Lrresa), goserelin acetate (Zoralide), granisetron (Kateri), imatinib (Gleevec), irinotecan (Campto/Camptosar), ondansetron (Zofran), paclitaxel (Taxol), pegaspargase (Oncaspar), pilocarpine hydrochloride (Salagen), porfibrin sodium (Photofrin), interleukin-2 (Proleukin), rituximab (Rituxan), topotecan (Hemisil), trastuzumab (Trastuzumab), (Herceptin), Triapine, vincristine and vinorelbine tartrate (Navelbine), or therapeutic antibodies or fragments thereof, or anti-angiogenic agents, e.g., angiostatin, bevacizumab (Avastin®), sorafenib (Nexavar®), baculostatin, canstatin, maspin, anti-VEGF antibody or peptide, anti-placental growth factor antibody or peptide, anti-Flk-1 antibody, anti-Fit-1 antibody or peptide, laminin peptide, fibronectin peptide, plasminogen activator inhibitor, tissue metalloproteinase inhibitor, interferon, interleukin 12, IP-10, Gro-β, thrombospondin, 2-methoxyestradiol, proliferator-activated protein, carboxiamidotriazole, CM101, marimastat, pentosan polysulfate, angiopoietin 2, interferon-α, herbimycin A, PNU145156E, 16K Prolactin fragments, linomide, thalidomide, pentoxifylline, genistein, TNP-470, endostatin, paclitaxel, accutin, cidofovir, vincristine, bleomycin, AGM-1470, platelet factor 4, or minocycline.

In another aspect, the present invention further provides a method for treating a mesothelin-related disorder, wherein a therapeutically effective amount of an immunoconjugate of the invention, or a composition of the invention comprising an immunoconjugate of the invention, is administered. The mesothelin-related disorder may include, for example, cancer, such as a solid tumor cancer. The solid tumor may be in or originate from the ovary, pancreas, respiratory tract, lung, colon, stomach, esophagus, cervix, liver, breast, head, and neck.

These and other embodiments are disclosed or are obvious from and included in the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the antitumor efficacy of the anti-mesothelin-immunoconjugate MF-T-SPDB-DM4 against mesothelin-transfected human pancreatic cancer cells in a mesothelin-transfected xenograft model (A) and in untransfected control tumors (B).

Figure 2 shows the anti-tumor efficacy of anti-mesothelin immunoconjugates with stable and cleavable, as well as polar and non-polar linkers in the HeLaMATU xenograft model, which contains cancer cells that endogenously express mesothelin.

Figure 3 shows the antitumor efficacy of anti-mesothelin immunoconjugates with stable and cleavable, as well as polar and nonpolar linkers in a mesothelin-transfected xenograft model (A) and in untransfected control tumors (B).

FIG4 shows an example of a dose-response curve of the toxicity of MF-T-SPDP-DM4 to mesothelin-positive HelaMatu cells.

DETAILED DESCRIPTION

The present invention is based on the discovery of novel immunoconjugates that are specific for mesothelin, or have a high affinity for mesothelin, and can deliver therapeutic benefits to a subject. The immunoconjugates of the present invention can be used in a number of situations as more fully described herein. It should be understood that the invention described herein is not limited to the specific details set forth herein with respect to any aspect of the invention, including the anti-mesothelin antibodies, immunoconjugates, treatment methods, protocols, cell lines, animal species or genera, constructs, and reagents described, which themselves can vary. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to limit the scope of the invention.

definition

Unless otherwise defined, all technical and scientific terms used in this article have the same meaning as those generally understood by those skilled in the art. However, the following references can provide general definitions of many terms used in the present invention for those skilled in the art, and can be referenced and used as long as such definitions are consistent with the meanings generally understood in the art. Such references include, but are not limited to: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd edition, 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 (3rd edition, 1999); and Cellular and Molecular Immunology, Abbas, Lichtman and Pober ed., 2nd edition, W.B. Saunders Company. Any other technical resources available to those skilled in the art can be consulted as long as the definitions of the terms used herein have the meanings generally understood in the art.

For the purposes of the present invention, the following terms are further defined. Other terms are defined elsewhere in the specification. As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a gene" refers to one or more genes and includes equivalents thereof known to those skilled in the art, and so on.

The term "antibody" as used herein includes immunoglobulin molecules (e.g., any type, including IgG, IgE, IgM, IgD, IgA and IgY, and/or any class, including IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) isolated from nature or prepared by recombinant means. Antibodies are also intended to include antigen-binding antibody fragments such as Fab, F(ab')2, scFv (single-chain Fvs), Fv, single-chain antibodies, bispecific antibodies, disulfide-linked Fv (sdFv) and fragments containing VL or VH domains, which are prepared from intact immunoglobulins or prepared by recombinant means.

The antibodies and/or antigen-binding antibody fragments of the present invention may be monospecific (e.g., monoclonal), bispecific, trispecific, or have greater multispecificity. Multispecific antibodies may be specific for different epitopes of an antigen, or may be specific for epitopes of more than one antigen. See, for example, PCT Publications WO 93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt, et al., 1991, J. Immunol. 147:6069; U.S. Patent Nos. 4,474,893; 4,714,681; 4,925,648; 5,573,920; 5,601,819; Kostelny et al., 1992, J. Immunol 148:1547-1553, each of which is incorporated herein by reference.

Antigen-binding antibody fragments may comprise a variable region alone or in combination with all or a portion of the following regions: hinge region, CH1, CH2, CH3, and CL domains. The present invention also includes antigen-binding antibody fragments further comprising any combination of a variable region and hinge region, CH1, CH2, CH3, and CL domains.

Preferably, the antibody or antigen-binding antibody fragment is human, humanized, murine (e.g., mouse and rat), donkey, sheep, rabbit, goat, guinea pig, camelid, horse or chicken. "Human" antibodies as used herein include antibodies having the amino acid sequence of a human immunoglobulin, and include antibodies isolated from a human immunoglobulin library, from human B cells, or from a transgenic animal of one or more human immunoglobulins, as described below, and as described, for example, by Kucherlapati et al. in U.S. Patent No. 5,939,598. The term antibody also extends to other protein scaffolds that are capable of orienting the antibody CDR insert into the same active binding conformation found in natural antibodies, such that the binding of the target antigen observed with these chimeric proteins is maintained compared to the binding activity of the natural antibody from which the CDRs are derived. The term "humanized" forms of non-human (e.g., murine) antibodies as used herein are chimeric antibodies containing minimal sequence derived from non-human immunoglobulins. Typically, a humanized antibody is a human immunoglobulin (recipient antibody) in which the hypervariable region residues (e.g., complementarity determining regions "CDRs") of the recipient are replaced with hypervariable region residues (CDRs) from a non-human species (such as mouse, rat, rabbit, or non-human primate) (donor antibody) with desired specificity, affinity, and capacity. In some cases, the framework region (FR) residues of the human immunoglobulin may be replaced with corresponding non-human residues. In addition, the humanized antibody may contain residues that are not present in the recipient antibody or in the donor antibody. Such modifications are made to further optimize antibody performance. In general, the humanized antibody may contain substantially all of at least one or typically two variable domains, wherein all or substantially all of the hypervariable regions correspond to the hypervariable regions of a non-human immunoglobulin, and all or substantially all of the FRs are FRs of human immunoglobulin sequences. The humanized antibody may also optionally contain at least a portion of an immunoglobulin constant region (Fc) (typically the Fc of a human immunoglobulin). For review, 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). For preparation of humanized antibodies, see U.S. 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, each of which is incorporated herein in its entirety.

As used herein, the term "single-chain Fv" or "sFv" antibody fragment comprises the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Typically, the Fv polypeptide additionally comprises a polypeptide linker between the VH and VL domains, which enables the sFv to form the structure required for antigen binding. For a review, 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 "bispecific antibody" refers to a small antibody fragment with two antigen-binding sites, which comprises a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker (which is not long enough to allow pairing between the two domains on the same chain), the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Bispecific antibodies are more fully described in, for example, EP404,097; WO 93/11161; and Hollinger et al. (Proc. Natl. Acad. Sci. USA 90:6444-6448, 1993), each of which is incorporated by reference.

The expression "linear antibody" refers to antibodies described in the art, for example, in Zapata et al. (Protein Eng. 8(10): 1057-1062, 1995), which is incorporated herein by reference. Briefly, such antibodies comprise a pair of tandem Fd segments (VH-CH1-VH-CH1) that form a pair of antigen-binding regions. Linear antibodies can be bispecific or monospecific.

The term "monoclonal antibody" as used herein means an antibody obtained from a group of substantially homologous antibodies, that is, comprising individual antibodies that are identical to a group except for possible naturally occurring mutations (which may exist in smaller amounts). Monoclonal antibodies are highly specific, that is, directed against a single antigenic site. In addition, compared to conventional (polyclonal) antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier "monoclonal" indicates that the antibody is characterized by being obtained from a group of substantially homologous antibodies, and should not be construed as requiring the antibody to be produced by any ad hoc method. For example, the monoclonal antibody to be used according to the present invention can be prepared by the hybridoma method described first by the like (Nature 256:495,1975) of Kohler, or can be prepared by recombinant DNA methods (see, for example, U.S. Patent number 4,816,567). Monoclonal antibodies can also be isolated from phage antibody libraries using, for example, the techniques described by Clackson, et al. (Nature 352:624-628, 1991) and Marks, et al. (J. Mol. Biol. 222:581-597, 1991).

The monoclonal antibodies herein also include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (see, e.g., U.S. 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 term "biological sample" or "patient sample" refers to a sample obtained from an organism or from a component (e.g., a cell) of an organism. The sample may belong to any biological tissue or fluid. The sample may be a "clinical sample," i.e., a sample derived from a patient. Such samples include, but are not limited to, sputum, blood, serum, plasma, blood cells (e.g., leukocytes), tissue samples, biopsy samples, urine, peritoneal and pleural fluid, saliva, semen, breast exudate, cerebrospinal fluid, tears, mucus, lymph, cytosol, ascites, amniotic fluid, bladder washes, and bronchoalveolar lavage fluid or cells derived therefrom, as well as other body fluid samples. The patient sample may be fresh or frozen and may be treated with heparin, citrate, or EDTA. Biological samples may also include tissue sections, such as frozen sections made for histological purposes.

The term "cancer" includes, but is not limited to, solid tumors such as pancreatic cancer, breast cancer, respiratory tract cancer, brain cancer, genital cancer, digestive tract cancer, urinary tract cancer, eye cancer, liver cancer, skin cancer, head and neck cancer, thyroid cancer, parathyroid cancer, and their distant metastases. The term also includes sarcomas, lymphomas, leukemias, and plasma cell myeloma.

Respiratory tract tumors include, but are not limited to, small cell lung cancer, non-small cell lung cancer, bronchial adenomas, and pleuropulmonary blastomas. Breast tumors include, but are not limited to, invasive ductal carcinoma, invasive lobular carcinoma, ductal carcinoma in situ, and lobular carcinoma in situ. Brain tumors include, but are not limited to, brainstem and hypothalamic gliomas, cerebellar astrocytomas, cerebral astrocytomas, medulloblastomas, ependymomas, as well as neuroectodermal and pineal tumors. Male genital tumors include, but are not limited to, prostate and testicular cancer. Female genital tumors include, but are not limited to, endometrial, cervical, ovarian, vaginal, and vulvar cancers, as well as uterine sarcomas. Digestive tract tumors include, but are not limited to, anal, colon, colorectal, esophageal, gallbladder, stomach, pancreatic, rectal, small intestine, and salivary gland cancers. Urinary tract cancers include, but are not limited to, bladder cancer, penile cancer, kidney cancer, renal pelvis cancer, ureter cancer, and urethral cancer. Eye cancers include, but are not limited to, intraocular melanoma and retinoblastoma. Liver cancers include, but are not limited to, hepatocellular carcinoma (hepatocellular carcinoma with or without fibrolamellar variant), cholangiocarcinoma (intrahepatic bile duct carcinoma), and mixed hepatocellular cholangiocarcinoma. Skin cancers include, but are not limited to, squamous cell carcinoma, Kaposi's sarcoma, malignant melanoma, Merkel cell skin cancer, and non-melanoma skin cancer. Head and neck cancers include, but are not limited to, laryngeal/hypopharyngeal/nasopharyngeal/oropharyngeal cancer, as well as lip and oral cancer. Lymphomas include, but are not limited to, AIDS-related lymphoma, non-Hodgkin's lymphoma, cutaneous T-cell lymphoma, Hodgkin's disease, and central nervous system lymphoma. Sarcomas include, but are not limited to, soft tissue sarcoma, osteosarcoma, malignant fibrous histiocytoma, lymphosarcoma, and rhabdomyosarcoma. Leukemias include, but are not limited to, acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, and hairy cell leukemia.

As used herein, the term "epitope" refers to any antigenic determinant on an antigen (e.g., mesothelin) to which an antibody binds via its antigen-binding site. Determinants, or antigenic determinants, typically consist of chemically active surface clusters of molecules (such as amino acids or sugar side chains) and typically possess specific three-dimensional structural characteristics as well as specific charge characteristics.

The term "specifically immunoreactive" refers to the binding reaction between an antibody and a protein, compound, or antigen having an epitope recognized by the antibody's antigen binding site. This binding reaction determines the presence of the protein, antigen, or epitope having the recognized epitope in a heterogeneous population of proteins and other biological contexts. In the context of an immunoassay, a specifically immunoreactive antibody can bind to a protein having the recognized epitope, and if bound, binds to a less detectable degree than other proteins present in the sample that lack the epitope. In the in vivo context, "specifically immunoreactive" can refer to the conditions under which an animal develops an immune response to a vaccine or antigen, such as a humoral response to the antigen (the generation of antibodies under immunoreactive conditions directed against a vaccine, protein, compound, or antigen presented thereto) or a cell-mediated response (also referred to herein as a "cellular immune response," i.e., a response mediated by T lymphocytes directed against a vaccine, protein, compound, or antigen presented thereto). As used herein, the term "immunoreactive conditions" is used in the context of immunoassays or in vitro reactions, wherein the physical conditions of the reaction are provided or adjusted, including, for example, temperature, salt concentration, pH, reagents and their concentrations, and concentrations of antigens and corresponding antibodies specifically immunoreactive with the antigens, to allow the corresponding antibodies to bind to the antigens. Immunoreactive conditions depend on the format of the antibody binding reaction and are typically those used in immunoassay protocols. For a description of immunoassay formats and conditions, see Harlow and Lane (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Publications, New York. As used herein, the term "patient" or "subject" includes mammals (e.g., humans and animals).

As used herein, the term "invariant binding" of a particular antibody to mesothelin refers to its ability to bind to mesothelin on a wide range of mesothelin-expressing cancer cell lines, including those expressing different forms of mesothelin. Invariant binding can be due to, but is not limited to, the antibody, or its antigen-binding antibody fragment, or variant thereof, recognizing an epitope of mesothelin that is not masked by another extracellular antigen that interacts with mesothelin, such as cancer antigen 125 (CA125). For invariant binding antibodies, the EC50 values measured by FACS titration on two different cancer cell lines may differ by no more than 10-fold, or preferably 5-fold, and most preferably 1-3-fold.

As used herein, the term "immunoconjugate" refers to a conjugate molecule comprising at least one antibody or antigen-binding fragment thereof bound (preferably via a suitable linker or precursor thereof) to a cytotoxic agent (eg, a maytansine or derivative thereof).

Immunoconjugates of the present invention

The present invention relates to methods for inhibiting the growth of mesothelin-positive cancer cells and the progression of neoplastic diseases by providing anti-mesothelin immunoconjugates. The antibody portion of the provided immunoconjugate is specifically immunoreactive against the 40 kDa C-terminal domain of the mesothelin precursor polypeptide (SEQ ID NO 36), which is referred to herein as 'mesothelin'.

In one aspect of the present invention, the antibodies, antigen-binding antibody fragments, and variants of the antibodies and fragments of the present invention are described in PCT/EP2008/009756 and comprise a light chain variable region and a heavy chain variable region. Variants of the antibodies or antigen-binding antibody fragments contemplated by the present invention are molecules in which the binding activity of the antibodies or antigen-binding antibody fragments for mesothelin is maintained.

The present invention also relates to immunoconjugates consisting of anti-mesothelin antibodies, antigen-binding antibody fragments, and variants of the antibodies and fragments of the invention, different from those already described in PCT/EP2008/009756, which have been linked to a chemotherapeutic agent (e.g. a maytansine or a derivative thereof).

Maytansines that can be used in the present invention are well known in the art and can be isolated from natural sources according to known methods or prepared synthetically according to known methods.

Examples of suitable maytansinoids include maytansinol and maytansinol analogs.Examples of suitable maytansinol analogs include those with modified aromatic rings and those with modifications at other positions.

Specific examples of suitable maytansinol analogs having modified aromatic rings include:

(1) C-19-dechlorination (U.S. Patent No. 4,256,746) (prepared by LAH reduction of ansamitocin P2);

(2) C-20-hydroxy (or C-20-demethyl) +/- C-19-dechloro (U.S. Patent Nos. 4,361,650 and 4,307,016) (prepared using Streptomyces or Actinomyces for demethylation, or LAH for dechlorination); and

(3) C-20-demethoxy, C-20-acyloxy (-OCOR), +/-dechloro (U.S. Patent No. 4,294,757) (prepared by acylation with an acyl chloride).

Specific examples of suitable maytansinol analogs having modifications at other positions include:

(1) C-9-SH (U.S. Patent No. 4,424,219) (prepared by 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) (U.S. Patent No. 4,450,254) (prepared from Nocardia sp.);

(4) C-15-hydroxy/acyloxy (U.S. Patent No. 4,364,866) (prepared by transformation of maytansinol by Streptomyces);

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

(6) C-18-N-demethyl (U.S. Patent Nos. 4,362,663 and 4,322,348) (prepared by demethylation of maytansinol by Streptomyces); and

(7) 4,5-deoxy (U.S. Patent No. 4,371,533) (prepared by titanium trichloride/LAH reduction of maytansinol).

The synthesis of thiol-containing maytansines useful in the present invention is fully disclosed in US Pat. Nos. 5,208,020, 5,416,064, and 7,276,497.

Maytansines having a thiol moiety at the C-3 position, the C-14 position, the C-15 position, or the C-20 position are all expected to be useful. The C-3 position is preferred, and the C-3 position of maytansinol is particularly preferred. Also preferred are maytansines containing a C-3 thiol moiety of N-methyl-alanine, and maytansines containing a C-3 thiol moiety of N-methyl-cysteine, and analogs of each. Preferred maytansines are those described in U.S. Patents 5,208,020, 5,416,064, 6,333.410, 6,441,163, 6,716,821, RE39,151, and 7,276,497, each of which is incorporated herein by reference in its entirety. In a preferred embodiment, the esterified maytansinol is selected from: N2'-deacetyl-N2'-(3-mercapto-1-oxopropyl)-maytansine (DM1, CAS Reg. No. 139504-50-0), N2'-deacetyl-N2'-(4-mercapto-1-oxopentyl)-maytansine (DM3, CAS Reg. No. 796073-54-6), and N2'-deacetyl-N2'-(4-methyl-4-mercapto-1-oxopentyl)-maytansine (DM4 CAS Reg. No. 796073-69-3).

Throughout this document, reference is made to the following representative antibodies of the invention: "MF-J," "MOR06640," "MF-226," and "MF-T." MF-J represents an antibody having a heavy chain variable region corresponding to SEQ ID NO: 28 (DNA)/SEQ ID NO: 20 (protein) and a light chain variable region corresponding to SEQ ID NO: 32 (DNA)/SEQ ID NO: 24 (protein). MOR06640 represents an antibody having a heavy chain variable region corresponding to SEQ ID NO: 29 (DNA)/SEQ ID NO: 21 (protein) and a light chain variable region corresponding to SEQ ID NO: 33 (DNA)/SEQ ID NO: 25 (protein). MF-226 represents an antibody having a heavy chain variable region corresponding to SEQ ID NO: 30 (DNA)/SEQ ID NO: 22 (protein) and a light chain variable region corresponding to SEQ ID NO: 34 (DNA)/SEQ ID NO: 26 (protein). MF-T represents an antibody having a heavy chain variable region corresponding to SEQ ID NO: 31 (DNA)/SEQ ID NO: 23 (protein) and a light chain variable region corresponding to SEQ ID NO: 35 (DNA)/SEQ ID NO: 27 (protein). The present invention is not limited to these antibodies used as examples herein. Other useful antibodies are disclosed, for example, in PCT/EP2008/009756.

In one aspect, the present invention provides immunoconjugates that are specifically immunoreactive for mesothelin in the presence of cancer antigen 125 (CA125/MUC 16), thereby effectively targeting cancer cells expressing mesothelin and CA125, such as OVCAR-3 cells.

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

In another aspect, the present invention provides immunoconjugates having an antigen-binding region that is specifically immunoreactive or has high affinity for one or more regions of mesothelin, the amino acid sequence of which is set forth in SEQ ID NO: 36. An immunoconjugate is said to have "high affinity" for an antigen if the affinity measurement is at least 100 nM (monovalent affinity of the Fab fragment). The immunoconjugates of the present invention are preferably specifically immunoreactive for mesothelin, with an affinity of less than about 100 nM, more preferably less than about 60 nM, and even more preferably less than about 30 nM. Further preferred are antibodies that bind to mesothelin with an affinity of less than about 10 nM and more preferably less than about 3 nM. For example, the antibodies of the present invention may have an affinity for mesothelin of about 9.1 nM or 0.9 nM (monovalent affinity of the IgG1 format).

How to use

The term "treatment" includes any process, action, administration, therapy, or the like, in which medical assistance is provided to a subject (or patient), including humans, with the purpose of directly or indirectly improving the subject's condition, or slowing the progression of the subject's condition or disorder, or ameliorating at least one symptom of the disease or disorder under treatment.

The term "combination therapy" or "co-therapy" refers to the administration of two or more therapeutic agents to treat a disease, condition, and/or disorder. Such administration includes co-administration of two or more therapeutic agents in a substantially simultaneous manner, such as in a single capsule having a fixed ratio of active ingredients, or in multiple separate capsules for each inhibitor. In addition, such administration includes the use of each type of therapeutic agent in a sequential manner. There is no limitation on the order of administration of two or more sequentially co-administered therapeutic agents. The phrase "therapeutically effective amount" refers to the amount of each agent administered to achieve the following purpose: to improve the severity and/or symptoms of the disease, condition, and/or disorder, while avoiding or minimizing adverse side effects associated with the administered therapeutic treatment.

The term "pharmaceutically acceptable" refers to a subject item that is suitable for use in pharmaceutical products.

It is expected that the immunoconjugates of the present invention are valuable as therapeutic agents. Thus, one embodiment of the present invention includes a method for treating various conditions in a patient (including a mammal), comprising administering to the patient a composition comprising an amount of the immunoconjugate of the present invention effective to treat the target condition.

The immunoconjugates of the present invention can be used to treat or prevent diseases and/or behaviors associated with the mesothelin protein. These diseases and/or behaviors include, for example, cancers such as pancreatic cancer, ovarian cancer, gastric cancer, esophageal cancer, cervical cancer, colon cancer, liver cancer, respiratory tract cancer, and lung cancer. The present invention also relates to methods for ameliorating symptoms of conditions in which mesothelin is elevated or otherwise abnormally expressed. These disorders include, but are not limited to, pancreatic, ovarian, gastric, esophageal, cervical, colon, liver, respiratory, and lung cancers (see, e.g., Liao, Cancer Res. 57:2827-2831, 1997; Turner, Hum. Pathol. 28:740-744, 1997; Liao, et al., Am. J. Pathol. 145:598-609, 1994; Saarnio, et al., Am. J. Pathol. 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 an immunoconjugate of the invention is administered to a patient having a disorder in which mesothelin is elevated.

The immunoconjugates of the present invention can be administered alone or in combination with one or more additional therapeutic agents. Combination therapy includes administration of a single pharmaceutical dosage formulation containing the immunoconjugates of the present invention and one or more additional therapeutic agents, as well as administration of the immunoconjugates of the present invention and each additional therapeutic agent in its own separate pharmaceutical dosage formulation. For example, the immunoconjugates of the present invention and the therapeutic agent can be administered to the patient together in a single oral dosage composition, or each agent can be administered in a separate oral dosage formulation.

In the case of using separate dosage formulations, the immunoconjugate of the present invention and one or more additional therapeutic agents can be administered substantially simultaneously (e.g., concurrently) or at separately staggered times (e.g., sequentially). There is no limitation on the order of administration of the agents.

For example, in one aspect, it is contemplated that the anti-mesothelin immunoconjugates of the invention are co-administered with one or more anti-cancer agents (to enhance the effect of the anti-mesothelin immunoconjugate or the anti-cancer agent or both) for the treatment of mesothelin-related disorders, e.g., cancer. Such combination therapies can also be used to prevent cancer, prevent cancer recurrence, prevent cancer spread or metastasis, or alleviate or ameliorate symptoms associated with cancer.

The one or more anticancer agents can include any known and suitable compound in the art, for example, chemotherapeutic agents, other immunotherapeutic agents, cancer vaccines, anti-angiogenic agents, cytokines, hormone therapy, gene therapy, and radiation therapy. Chemotherapeutic agents (or "anticancer agents" or "antitumor agents" or "cancer therapeutic agents") refer to any molecule or compound that assists in the treatment of cancer. Examples of chemotherapeutic agents contemplated by the present invention include, but are not limited to, cytosine arabinoside, taxanes (e.g., paclitaxel, docetaxel), anti-tubulin agents (e.g., paclitaxel, docetaxel, epothilone B, or analogs thereof), macrolides (e.g., rhizoctonia soln), cisplatin, carboplatin, doxorubicin, tenoposide, mitozantron, discodermolide, eleutherobine, 2-chlorodeoxyadenosine, alkylating agents (e.g., cyclophosphamide, mechlorethamine, thioepa, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) - cisplatin, thiotepa), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, anthramycin), antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, flavopiridol, 5-fluorouracil, fludarabine, gemcitabine, dacarbazine, temozolomide), asparaginase, BCG, diphtheria toxin, hexamethylmelamine, hydroxyurea, LYSODREN.RTM., nucleoside analogs, plant alkaloids (e.g., taxol, paclitaxel, camptothecin, topotecan, irinotecan (CAMPTOSAR, CPT-11), vincristine, vinca alkaloids such as vinblastine), podophyllotoxins (including derivatives such as epipodophyllotoxins, VP-16 (etoposide), VM-26 (teniposide), cytochalasin B, colchicine, gramicidin D, ethidium bromide, emetine, mitomycin, procarbazine, nitrogen mustard, anthracyclines (e.g., daunorubicin (formerly daunomycin), doxorubicin, liposomal doxorubicin), dihydroxyanthracindione, mitoxantrone, mithramycin, actinomycin D, procaine, tetracaine, lidocaine, propranolol, puromycin, antimitotic agents, abrin, ricin A, Pseudomonas exotoxin, nerve growth factor, platelet-derived growth factor, tissue plasminogen activator, aldesleukin, allutamine, anastrozole, bicalutamide, biaomycin, busulfan, capecitabine, carboplatin, chlorabusil, cladribine, cylarabine, daclinomycin, estramustine, floxuridine, gemcitabine, gosereine, idarubicin, ifosfamide, leuprolide acetate The present invention also contemplates compositions comprising one or more chemotherapeutic agents (e.g., FLAG, CHOP). FLAG includes fludarabine, cytosine arabinoside (Ara-C), and G-CSF. CHOP includes cyclophosphamide, vincristine, doxorubicin, and prednisone.

The chemotherapeutic agent can be an anti-angiogenic agent, for example, angiostatin, bevacizumab (Avastin®), sorafenib (Nexavar®), baculostatin, canstatin, maspin, anti-VEGF antibody or peptide, anti-placental growth factor antibody or peptide, anti-Flk-1 antibody, anti-Fit-1 antibody or peptide, laminin peptide, fibronectin peptide, plasminogen activator inhibitor, tissue metalloproteinase inhibitor, interferon, interleukin 12, IP-10, Gro-β, thrombospondin, 2-methoxyestradiol, proliferator-activated protein, carboxyamidotriazole, CM101, marimastat, polysulfate pentosan, angiopoietin 2, interferon-α, herbimycin A, PNU145156E, 16K Prolactin fragments, linomide, thalidomide, pentoxifylline, genistein, TNP-470, endostatin, paclitaxel, accutin, cidofovir, vincristine, bleomycin, AGM-1470, platelet factor 4, or minocycline.

In one aspect, the chemotherapeutic agent is gemcitabine in a dosage range of 100-1000 mg/m ² / cycle. In one embodiment, the chemotherapeutic agent is dacarbazine in a dosage range of 200-4000 mg/m ² / cycle. In another aspect, the chemotherapeutic agent is fludarabine in a dosage range of 25-50 mg/m ² / cycle. In another aspect, the chemotherapeutic agent is cytosine arabinoside (Ara-C) in a dosage range of 200-2000 mg/ ^{m 2} / cycle. In another aspect, the chemotherapeutic agent is docetaxel in a dosage range of 1.5-7.5 mg/kg/ cycle. In another aspect, the chemotherapeutic agent is paclitaxel in a dosage range of 5-15 mg/kg/ cycle. In another aspect, the chemotherapeutic agent is cisplatin in a dosage range of 5-20 mg/kg/ cycle. In other aspects, the chemotherapeutic agent is 5-fluorouracil in a dosage range of 5-20 mg/kg/cycle. In another aspect, the chemotherapeutic agent is doxorubicin in a dosage range of 2-8 mg/kg/cycle. In other aspects, the chemotherapeutic agent is epipodophyllotoxin in a dosage range of 40-160 mg/kg/cycle. In another aspect, the chemotherapeutic agent is cyclophosphamide in a dosage range of 50-200 mg/kg/cycle. In another aspect, the chemotherapeutic agent is irinotecan in a dosage range of 50-150 mg/ ^m2 /cycle. In other aspects, the chemotherapeutic agent is vinblastine in a dosage range of 3.7-18.5 mg/ ^m2 /cycle. In another aspect, the chemotherapeutic agent is vincristine in a dosage range of 0.7-2 mg/ ^m2 /cycle. In one aspect, the chemotherapeutic agent is methotrexate in a dosage range of 3.3-1000 mg/m2/cycle.

In another aspect, the anti-mesothelin immunoconjugates of the invention are administered in combination with one or more immunotherapeutic agents, such as antibodies or immunomodulators, including, but not limited to, Herceptin®, Retuxan®, OvaRex, Panorex, BEC2, IMC-C225, Vitaxin, Campath I/H, Smart MI95, LymphoCide, SmartID 10, and Oncolym, rituxan, rituximab, gemtuzumab, or trastuzumab.

The present invention also contemplates the administration of the anti-mesothelin immunoconjugates of the present invention with one or more anti-angiogenic agents, including, but not limited to, angiostatin, thalidomide, kringle 5, endostatin, serpin (serine protease inhibitor) antithrombin, a 29 kDa N-terminal proteolytic fragment and a 40 kDa C-terminal proteolytic fragment of fibronectin, a 16 kDa proteolytic fragment of prolactin, a 7.8 kDa proteolytic fragment of platelet factor-4, a β-amino acid peptide corresponding to a fragment of platelet factor-4 (Maione et al., 1990, Cancer Res. 51: 2077), a 14-amino acid peptide corresponding to a collagen I fragment (Tolma et al., 1993, J. Cell Biol. 122: 497), a 19 amino acid peptide corresponding to a thrombospondin I fragment (Tolsma et al., 1993, J. Cell Biol. 122: 497), a 20 amino acid peptide corresponding to a SPARC fragment (Sage et al., 1995, J. Cell. Biochem. 57:1329), or any fragment, family member, or derivative thereof, including pharmaceutically acceptable salts thereof. Other peptides that inhibit angiogenesis and correspond to fragments of laminin, fibronectin, procollagen, and EGF have also been described (for review, see Cao, 1998, Prog. Mol. Subcell. Biol. 20:161). Monoclonal antibodies and cyclic pentapeptides that block binding of certain integrins to RGD proteins (i.e., having the peptide motif Arg-Gly-Asp) have been shown to have antiangiogenic activity (Brooks et al., 1994, Science 264:569; Hammes et al., 1996, Nature Medicine 2:529). In addition, inhibition of urokinase plasminogen activator receptor by antagonists inhibits angiogenesis, tumor growth, and metastasis (Min et al., 1996, Cancer Res. 56: 2428-33; Crowley et al., 1993, Proc. Natl. Acad. Sci. USA 90: 5021). The present invention also contemplates the use of such anti-angiogenic agents.

In another aspect, the anti-mesothelin immunoconjugates of the invention are administered in combination with radiation therapy.

The anti-mesothelin immunoconjugates of the present invention can also be administered in combination with one or more cytokines, including but not limited to lymphokines, tumor necrosis factor, tumor necrosis factor-like cytokines, lymphotoxin-α, lymphotoxin-β, interferon-β, macrophage inflammatory protein, granulocyte monocyte colony stimulating factor, interleukins (including but not limited to interleukin-1, interleukin-2, interleukin-6, interleukin-12, interleukin-15, interleukin-18), OX40, CD27, CD30, CD40 or CD137 ligand, Fas-Pas ligand, 4-IBBL, endothelial monocyte activation protein, or any fragment, family member or derivative thereof, including pharmaceutically acceptable salts thereof.

The anti-mesothelin immunoconjugates of the present invention can also be administered in combination with cancer vaccines, examples of which include, but are not limited to, autologous cells or tissues, non-autologous cells or tissues, carcinoembryonic antigen, alpha-fetoprotein, human chorionic gonadotropin, live BCG vaccine, melanocyte lineage proteins (e.g., gap100, MART-1/MelanA, TRP-1 (gp75), tyrosinase, widely shared tumor-associated antigens including tumor-specific antigens (e.g., BAGE, GAGE-1, GAGE-2, MAGE-1, MAGE-3, N-acetylglucosaminyltransferase-V, p15), tumor-associated mutant antigens (β-catenin, MUM-1, CDK4), non-melanoma antigens (e.g., HER-2/neu (breast and ovarian cancer), human papillomavirus-E6, E7 (cervical cancer), MUC-1 (breast, ovarian and pancreatic cancer). For human tumor antigens recognized by T-cells, see generally: Robbins and Kawakami, 1996, Curr. Opin. Immunol. 8: 628. Cancer vaccines may or may not be purified preparations.

In another embodiment, the anti-mesothelin immunoconjugates of the invention are used in combination with hormone therapy. Hormonal therapeutic treatments include hormone agonists, hormone antagonists (e.g., flutamide, tamoxifen, leuprorelin acetate (LUPRON), LH-RH antagonists), inhibitors of hormone biosynthesis and processing, and steroids (e.g., dexamethasone, retinoids, betamethasone, cortisol, cortisone, prednisone, dehydrotestosterone, glucocorticoids, mineralocorticoids, estrogens, testosterone, progesterone), antiprogestins (e.g., mifepristone, onapristone), and antiandrogens (e.g., cyproterone acetate).

The anti-mesothelin immunoconjugates of the present invention can be used in combination with anti-MDR (multidrug resistance) phenotype agents, e.g., co-administered. Many human cancers inherently express or spontaneously develop resistance to several classes of anticancer drugs simultaneously, despite each drug class having a different structure and mechanism of action. This phenomenon, which can be mimicked in cultured mammalian cells, is generally referred to as multidrug resistance ("MDR") or a multidrug resistance phenotype. The MDR phenotype represents a significant obstacle to the successful chemotherapeutic treatment of cancer in human patients.

Resistance of malignant tumors to multiple chemotherapeutic agents is a major cause of treatment failure (Wittes et al., Cancer Treat. Rep. 70:105 (1986); Bradley, G. et al., Biochim. Biophys. Acta 948:87 (1988); Griswald, D. P. et al., Cancer Treat. Rep. 65(S2):51 (1981); Osteen, R. T. (ed.), Cancer Manual, (1990)). Tumors that were initially sensitive to cytotoxic agents often relapse or become resistant to multiple chemotherapeutic drugs (Riordan et al., Pharmacol. Ther. 28:51 (1985); Gottesman et al., Trends Pharmacol. Sci. 9:54 (1988); Moscow et al., J. Natl. Cancer Inst. 80:14 (1988); Croop, J. M. et al., J. Clin. Invest. 81:1303 (1988)). Cells or tissues derived from tumors and cultured in the presence of a selective cytotoxic drug can develop cross-resistance to other drugs in this class, as well as to other classes of drugs, including, but not limited to, anthracyclines, vinca alkaloids, and epipodophyllotoxins (Riordan et al., Pharmacol. Ther. 28:51 (1985); Gottesman et al., J. Biol. Chem. 263:12163 (1988)). Thus, acquired resistance to a single drug can simultaneously generate resistance to a diverse set of drugs that are structurally and functionally unrelated.Such resistance can be a problem for both solid and liquid forms of tumors (e.g., blood or lymphoid-based cancers).

A major mechanism of multidrug resistance in mammalian cells involves increased expression of a 170 kDa plasma membrane glycoprotein pump system (Juranka et al., FASEB J 3:2583 (1989); Bradley, G. et al., Biochem. Biophys. Acta 948:87 (1988)). The gene encoding this pump system (sometimes referred to as multidrug transporter) has been cloned from cultured human cells and is commonly referred to as mdrl. This gene is expressed in several types of normal tissues, but the physiological substrates transported by the mdrl gene product in these tissues have not yet been identified. The MDR1 product is a member of the ABC transporter protein superfamily (a group of proteins with energy-dependent export functions). The protein product of the mdrl gene, commonly referred to as P-glycoprotein ("P-170," "P-gp"), is a 170 kDa trans-plasma membrane protein that constitutes the aforementioned energy-dependent efflux pump. The expression of P-gp on the cell surface is enough to make cells tolerate multiple cytotoxic drugs, including many anticancer agents. P-gp-mediated MDR seems to be an important clinical component of tumor resistance in different types of tumors, and mdrl gene expression is relevant to the resistance of different types of cancer to chemotherapy. The nucleotide sequence of the mdrl gene (Gros, P. et al., Cell 47:371 (1986); Chen, C. et al., Cell 47:381 (1986)) shows that it encodes a polypeptide similar or identical to P-glycoprotein, and they are members of a highly conserved membrane protein class similar to bacterial transport proteins, and participate in normal physiological transport processes. Sequence analysis of the mdrl gene shows that Pgp is composed of 1280 amino acids distributed between 2 homologous (43% identity) halves. Each half of this molecule has 6 hydrophobic membrane spaning domains, and each has an ATP binding site in a large cytoplasmic ring. Only about 8% of the molecule is extracellular, and the carbohydrate moiety (approximately 30 kDa) is bound to the site in this region.

Thus, it will be understood that mammalian cells having a "multidrug resistance" or "multidrug-resistant" phenotype are characterized by the ability to sequester, export, or exclude multiple cytotoxic substances (e.g., chemotherapeutic drugs) from the intracellular environment. Cells may acquire this phenotype as a result of selective pressure exerted by exposure to a single chemotherapeutic drug (selective toxin). Alternatively, cells may exhibit this phenotype prior to toxin exposure, as the export of cytotoxic substances may involve mechanisms common to the normal export of cellular secretory products, metabolites, etc. Multidrug resistance differs from simply acquiring resistance to a selected toxin in that cells acquire the ability to export additional cytotoxins (other chemotherapeutic drugs) to which the cells were not previously exposed. For example, Mirski et al. (1987), 47 Cancer Res. 2594-2598, describe the isolation of a multidrug-resistant cell population in which an H69 cell line derived from human small cell lung cancer was cultured in the presence of doxorubicin (doxorubicin) as a selective toxin. The cells that found to survive can tolerate the cytotoxic effects of the following drugs: anthracycline analogs (for example, daunomycin, epirubicin, menolide and mitoxantrone), acivicin, etoposide, gramicidin D, colchicine and vinca rosea-derived alkaloids (vincristine and vinblastine) and doxorubicin. Similar selective culture techniques can be applied to prepare cell colonies of additional multidrug resistance. Therefore, the pharmaceutical composition of the present invention can additionally include compounds that suppress the MDR phenotype and/or the condition relevant to the MDR phenotype. Such compounds can include any known MDR inhibitor compounds in this area, for example, antibodies to MDR component specificity (for example, anti-MDR carrier antibodies) or the small molecule inhibitors of MDR transporters, specifically including tamoxifen, verapamil and cyclosporin A, which are known agents that can reverse or suppress multidrug resistance (Lavie et al. J. Biol. Chem. 271: 19530-10536, 1996, incorporated herein by reference). Such compounds can be found in U.S. Patent Nos. 5,773,280, 6,225,325, and 5,403,574, each of which is incorporated herein by reference. Such MDR inhibitor compounds can be co-administered with the anti-mesothelin immunoconjugates of the present invention for various purposes, including reversing the MDR phenotype after detection thereof, to assist or enhance chemotherapy treatment. MDR inhibitors, such as tamoxifen, verapamil, or cyclosporine A, can be used in combination with the compounds of the present invention to assist in detecting the MDR phenotype. According to this aspect, MDR inhibitors can enhance the uptake and accumulation of the compounds of the present invention in MDR cancer cells because the presence of the MDR inhibitor reduces the ability of the MDR transport system to transport or "pump out" the imaging compound (relative to the substrate domain).

In another embodiment, the anti-mesothelin immunoconjugates of the present invention are used in combination with gene therapy regimens for the treatment of cancer. Gene therapy using recombinant cells that secrete interleukin-2 can be administered in combination with the immunoconjugates of the present invention to prevent or treat cancer, particularly breast cancer (see, e.g., Deshmukh et al., 2001, J. Neurosurg. 94:287).

To assess the ability of a particular immunoconjugate to be therapeutically useful for treating cancer, as an example, the immunoconjugate can be tested in vivo in a mouse xenograft tumor model. Examples of therapeutic models are detailed in Examples 1 and 2. Antibody activity can also be tested using the antibody-dependent cell-mediated cytotoxicity assay described in Example 3.

Pharmaceutical composition and dosage

The immunoconjugates described herein can be provided in pharmaceutical compositions comprising a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier can be pyrogen-free. The compositions can be administered alone or in combination with at least one other agent (such as a stabilizing compound) in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water. A variety of aqueous carriers can be used, including, but not limited to, saline, glycine, and the like. These solutions are sterile and generally free of particulate matter. These solutions can be sterilized by conventional, well-known sterilization techniques (e.g., filtration).

Generally, the phrase "pharmaceutically acceptable carrier" is well known in the art and includes pharmaceutically acceptable substances, compositions, or vehicles that are suitable for administering the compounds of the present invention to mammals. Such carriers include liquid or solid fillers, diluents, excipients, solvents, or encapsulating materials involved in carrying or transporting the subject agent from one organ or part of the body to another organ or part of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the patient. Some examples of substances that can be used 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 carboxymethylcellulose, ethylcellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; 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, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol; phosphate buffered saline; and other nontoxic substances suitable for use in pharmaceutical formulations. Wetting agents, emulsifiers and lubricants (such as sodium lauryl sulfate and magnesium stearate), as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants may also be present in the immunoconjugate compositions of the invention.

Examples of pharmaceutically acceptable antioxidants include: water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, etc.; and metal chelators such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

The compositions may contain pharmaceutically acceptable auxiliary substances as needed to approximate physiological conditions, such as pH adjusters and buffers, etc. The concentration of the immunoconjugates of the invention in such pharmaceutical formulations can vary widely and can be selected based primarily on fluid volume, viscosity, etc., depending on the particular mode of administration chosen. If desired, more than one type of antibody or immunoconjugate (e.g., antibodies with different Ka for mesothelin binding) can be included in the pharmaceutical composition.

The compositions can be administered to a patient alone or in combination with other agents, drugs, or hormones. In addition to the active ingredient, these pharmaceutical compositions can contain suitable pharmaceutically acceptable carriers, including excipients and adjuvants that facilitate processing of the active compound into pharmaceutically acceptable products. The pharmaceutical compositions of the present invention can be administered by any number of routes, including, but not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, parenteral, topical, sublingual, or rectal.

The compositions of the present invention further include suitable immunogenic carriers, for example, proteins, polypeptides or peptides such as albumin, hemocyanin, thyroglobulin and its derivatives, in particular bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH), polysaccharides, carbohydrates, polymers and solid phases. Other protein-derived or non-protein-derived substances are known to those skilled in the art.

In aspects involving vaccines (e.g., cancer vaccines with antibodies of the present invention), the compositions of the present invention can be administered with or without an adjuvant. Administration can be performed without an adjuvant to avoid any adjuvant-induced toxicity. Those skilled in the art (e.g., physicians specializing in cancer) will understand and appreciate how to determine whether to use an adjuvant, and this determination may depend on the subject's medical history, family history, toxicity data, allergy-related laboratory results, etc. In embodiments where an adjuvant is used, the adjuvant advantageously promotes the formation of protective antibodies (such as protective IgG antibodies). Any suitable adjuvant known to those skilled in the art is contemplated and readily applicable to the present invention. Adjuvants suitable for animal vaccination include, but are not limited to, aluminum hydroxide, saponin and its purified fraction, Quit A, complete Freund's adjuvant (CFA), and incomplete Freund's adjuvant (IFA). Dextran sulfate has been shown to be an effective stimulator of IgG2 antibodies against Staphylococcal cell surface antigens and is also suitable as an adjuvant. The skilled artisan will appreciate that some adjuvants may be more preferred for veterinary applications while other adjuvants are preferred for use in humans, and that the skilled artisan should consider adjuvant toxicity before administering a compound to humans.

Preparations suitable for parenteral, subcutaneous, intravenous, intramuscular administration, suitable pharmaceutical carriers, and formulation and administration techniques can be prepared by any method well known in the art (see, for example, Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 20th edition, 2000). Liquid dosage forms for oral administration of the compounds of the present invention include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the active ingredient, the liquid dosage form can contain an inert diluent commonly used in the art, for example, water or other solvents, solubilizers, and emulsifiers, such as ethanol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (specifically, cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofuran alcohol, polyethylene glycol, and fatty acid esters of sorbitan, and mixtures thereof.

As used herein, the phrases "parenteral administration" and "parenterally administered" refer to modes of administration other than enteral and topical administration, usually by injection, including, but not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, and intrasternal injection and infusion.

Determination of a therapeutically effective dose is within the capabilities of those skilled in the art. A therapeutically effective dose represents the amount of the immunoconjugate that can be used to effectively treat a disease (eg, cancer) compared to the efficacy observed in the absence of the therapeutically effective dose.

A preliminary estimate of the therapeutically effective dose can be made in animal models (e.g., rats, mice, rabbits, dogs, or pigs). The animal models can also be used to determine appropriate concentration ranges and routes of administration. This information can then be used to determine dosages and routes useful in humans. The therapeutic efficacy and toxicity of the immunoconjugate can be determined using standard pharmaceutical procedures in cell cultures or experimental animals (e.g., ED50—the dose that is therapeutically effective in 50% of the population, and LD50—the dose that is lethal to 50% of the population). The dose ratio of the toxic effect to the therapeutic effect is the therapeutic index, which can be expressed as the ratio of LD50/ED50. Data obtained from animal studies can be used to formulate dosage ranges for humans. The dose contained in such a composition can be within a circulating concentration range that includes the ED50 and has little or no toxicity. The dose varies within this range depending on the dosage form used, the patient's sensitivity, and the route of administration.

The exact dosage can be determined by the practitioner after considering factors relevant to the patient in need of treatment. Dosage and administration can be adjusted to provide adequate levels of the immunoconjugate or to maintain the desired effect. Factors that may be considered include: the severity of the disease state, the general health of the subject, the subject's age, weight and sex, diet, time and frequency of administration, drug combination, sensitivity to treatment and tolerance/response. Polynucleotides encoding the immunoconjugates of the present invention can be constructed and introduced into cells in vitro or in vivo using established techniques, including, but not limited to: transferrin-polycation-mediated DNA transfer, transfection using naked or encapsulated nucleic acids, liposome-mediated cell fusion, intracellular transport of DNA-coated latex beads, protoplast fusion, viral infection, electroporation, "gene gun" and DEAE- or calcium phosphate-mediated transfection.

The effective in vivo dose of the toxin cluster component of the immunoconjugate is in the range of about 5 μg to about 500 μg/kg patient body weight. The mode of administration of the pharmaceutical composition containing the immunoconjugate of the present invention can be any suitable route that delivers the antibody to the host. As an example, the pharmaceutical composition of the present invention can be used for parenteral administration (e.g., subcutaneous, intramuscular, intravenous or intranasal administration). All patents and patent applications cited in this disclosure are expressly incorporated herein by reference. The above disclosure generally describes the present invention. A more complete understanding can be obtained by reference to the following specific examples, which are provided for illustrative purposes only and are not intended to limit the scope of the invention.

Example

Example 1: Efficacy of Immunoconjugates in a Mesothelin-Expressing Human Pancreatic Cancer Xenograft Mouse Model

To analyze whether anti-mesothelin immunoconjugates can reduce tumor growth in a mesothelin-dependent manner, human pancreatic cancer cells (MiaPaCa-2) were stably transfected with mesothelin and used to establish a subcutaneously grown tumor mouse model. For efficacy studies, mesothelin-negative control tumors were established using the human colon cancer cell line HT29. MiaPaCa cells were maintained in adherent culture in DMEM supplemented with 10% (v/v) FCS, 2.5% (v/v) horse serum, 1.5 g/l sodium bicarbonate, 4.5 g/l glucose, 4 mM glutamine, and 0.4% (v/v) hygromycin. HT29 cells were cultured in McCoy's 5a medium supplemented with 1.5 mM glutamine, 2.2 g/l sodium bicarbonate, and 10% (v/v) FCS. Mesothelin expression in MiaPaCa-2 cells and mesothelin deficiency in HT29 cells were confirmed by FACS (not shown). To evaluate the in vivo growth of tumor cells, female NMRI nude mice were subcutaneously inoculated with 3 x 10 ⁶ MiaPaCa-2 cells or 1 x 10 ⁶ HT29 cells resuspended in 50% Matrigel ^™ and 50% culture medium on the right flank. MF-J-SPDB-DM4, MF-T-SPDB-DM4, MF226-SPDB-DM4, and MOR6640-SPDB-DM4, anti-mesothelin immunoconjugates, were tested at therapeutic doses of 0.01 mg/kg, 0.03 mg/kg, 0.05 mg/kg, and 0.2 mg/kg (depending on the amount of toxic cluster). MF-J-SPDB-DM4, MF-T-SPDB-DM4, MF226-SPDB-DM4, and MOR6640-SPDB-DM4 were prepared by modifying anti-mesothelin antibodies with 4-[2-pyridyldithio]butyric acid N-hydroxysuccinimide ester (SPDB) to introduce dithiopyridyl groups. The antibodies were modified using 8 mg/mL of antibody (approximately a 6-fold molar excess of SPDB (~20 mM stock solution in EtOH)). The modified antibodies were reacted with a 1.7-fold molar excess of the free thiol form of the maytansine relative to the thiopyridyl groups. The reaction was carried out with 2.5 mg/mL of antibody in the presence of 3% dimethylacetamide (3% v/v) at room temperature for 20 hours. The conjugation reaction mixture was purified from unreacted drug and reaction byproducts using a desalting Sephadex G25 column. The number of maytansine molecules per antibody was calculated by measuring absorbance at 252 nm and 280 nm, using extinction coefficients of 224,000 ^{M⁻¹ cm⁻¹} for the antibody and 5,180 ^{M⁻¹ cm⁻¹} for DM4 at 280 nm. The 252 nm/280 nm absorbance ratios were 0.37 for the antibody and 5.05 for DM4.

Treatment began on day 5 after tumor inoculation, after tumors had established, and was followed by two additional treatments on days 8 and 12 after tumor inoculation. Control mice were treated with 0.2 mg/kg of the non-targeted immunoconjugate (anti-lysozyme-SPDB-DM4) or an equal volume of vehicle alone (10 mM histidine, 130 mM glycine, 5% (w/v) sucrose, pH 5.5). Treatment was administered intravenously at a dose volume of 100 µl/10 g body weight. Each group consisted of six animals. The health of the mice was examined daily. The length and width of subcutaneous tumors were measured twice weekly using electronic calipers. Tumor area was calculated using the following formula: Tumor area [ ^mm² ] = length [mm] x width [mm]. All data obtained during the experiment were recorded. An example of the antitumor efficacy of different treatment doses of the anti-mesothelin immunoconjugate MF-T-SPDB-DM4 against mesothelin-transfected human pancreatic cancer cells is shown in Figure 1. Female NMRI nude mice were inoculated in the right flank with 3 x 10 ⁶ mesothelin-positive MiaPaCa-2 human pancreatic cancer cells (A) or 1 x 10 ⁶ mesothelin-negative HT29 human colon cancer cells (B), resuspended in 50% Matrigel ^™ /50% culture medium. 5, 8, and 12 days after tumor cell inoculation, mice received 0.01, 0.03, 0.05, 0.2 mg/kg MF-T-SPDB-DM4 (all concentrations were related to the amount of toxic clusters) or vehicle alone. The length and width of the tumors were measured twice a week, and the tumor area was calculated by multiplying the width by the length. The mean and standard deviation for each group and each measurement time point are plotted. All n = 6. Asterisks indicate P-values < 0.05.

Treatment of tumor-bearing mice revealed that all anti-mesothelin immunoconjugates tested at doses of 0.03 mg/kg, 0.05 mg/kg, and 0.2 mg/kg were able to inhibit the growth of mesothelin-positive MiaPaCa-2 tumors in vivo. MF-T-SPDB-DM4 at doses of 0.05 mg/kg and 0.2 mg/kg completely eradicated tumors, with no tumor regrowth until the end of the 132-day observation period. A non-targeting control, anti-lysozyme-SPDB-DM4, at 0.05 mg/kg had no effect on the growth of mesothelin-positive MiaPaCa tumors (Table 1). The highest dose of MF-T-SPDB-DM4, at 0.2 mg/kg, did not significantly reduce the growth of mesothelin-negative HT29 tumors compared to untreated and vehicle-treated tumors. This demonstrates that the potent tumor-suppressive potency of MF-T-SPDB-DM4 is dependent on the expression of mesothelin within the tumor.

Table 1: Tumor inhibitory efficacy of anti-mesothelin immunoconjugates in the mesothelin-positive MiaPaCa xenograft tumor model

.

Example 2: Efficacy in tumors endogenously expressing mesothelin and comparison of different linkers

To test whether anti-mesothelin immunoconjugates can inhibit the in vivo growth of tumor cells endogenously expressing mesothelin, a xenograft model with subcutaneously grown human cervical cancer cells (HeLaMATU) was used. HeLaMATU cells were maintained in adherent culture in DMEM/HAMS12 medium supplemented with 10% (v/v) FCS, 2.5% (v/v) horse serum, 1% sodium pyruvate, and 1% (w/v) glutamine. Mesothelin expression was confirmed by in vitro FACS analysis. Female NMRI nude mice were subcutaneously inoculated with 1.5 x 10 ⁶ HeLaMATU cells, which were resuspended in 50% Matrigel ^™ /50% culture medium. In addition, we investigated whether replacing the cleavable SPDB-linker with a polar (-sulfo-SPDB), a stable linker (-SMCC), or a polar and stable linker (-(PEG)4-mal) would result in altered in vivo antitumor efficacy of MOR6640-based immunoconjugates. Mice bearing HeLaMATU tumors were treated intravenously with 0.2 mg/kg of MOR6640-SPDB-DM4, MOR6640-SMCC-DM1, MOR6640-sulfo-SPDB-DM4, or MOR6640-(PEG)4-mal-DM1 (depending on the amount of toxic cluster) on days 5, 8, and 12 after tumor cell inoculation. Control mice were treated with 0.2 mg/kg of a non-targeted immunoconjugate (anti-lysozyme-SPDB-DM4) or an equal volume of vehicle alone. Each group consisted of 6 animals. The health of the mice was examined daily. The length and width of subcutaneous tumors were measured twice weekly using electronic calipers. Tumor area was calculated by the following formula: Tumor area [mm ² ] = length [mm] x width [mm]. The resulting data are shown in Figure 2. Treatment of tumor-bearing mice revealed that: a) anti-mesothelin immunoconjugates effectively inhibited the in vivo growth of tumors endogenously expressing mesothelin, and b) conjugates containing cleavable linkers (MOR6640-SPDB-DM4 and MOR6640) exhibited greater anti-tumor efficacy than conjugates containing stable linkers (MOR6640-SMCC-DM1 and MOR6640-(PEG)4-mal-DM1). Specifically, 11 days after the last treatment, MOR6640-SPDB-DM4 and MOR6640-sulfo-SPDB-DM4 resulted in eradication of tumors in all treated animals, while MOR6640-SMCC-DM1 and MOR6640-(PEG)4-mal-DM1 treatment only resulted in a delay in tumor growth. However, 11 days after the last treatment, tumors treated with MOR6640-SMCC-DM1 and MOR6640-(PEG)4-mal-DM1, respectively, were significantly smaller in size compared to tumors treated with vehicle or anti-lysozyme-SPDB-DM4. A non-targeting control conjugate had no effect on tumor growth. To compare the antitumor efficacy of different conjugates in a second xenograft model, we employed a model with subcutaneously grown vector- or mesothelin-transfected (#37) MiaPaCa-2 cells (human pancreatic cancer cells). MiaPaCa-2-vector and MiaPaCa-2#37 cells were maintained in adherent culture in DMEM/HAMS12 medium supplemented with 10% (v/v) FCS, 1% (w/v) glutamine, and 0.1 mM non-essential amino acids. Mesothelin expression was confirmed by FACS analysis and by ex vivo immunohistochemical analysis of subcutaneous tumors. Female NMRI nude mice were subcutaneously inoculated with 3 x 10 ⁶ MiaPaCa-2-vector and MiaPaCa-2#37 cells, respectively, resuspended in 50% Matrigel ^™ /50% culture medium, into the right flank. Tumor-bearing mice were treated with 0.05 mg/kg of MOR6640-SPDB-DM4, MOR6640-SMCC-DM1, MOR6640-sulfo-SPDB-DM4, or MOR6640-(PEG)4-mal-DM1 (depending on the amount of toxic cluster) on days 5, 8, and 12 after tumor cell inoculation. Control mice were treated with 0.05 mg/kg of a non-targeted immunoconjugate (anti-lysozyme-SPDB-DM4) or an equal volume of vehicle alone. Each group consisted of 6 animals. The health of the mice was examined daily. The length and width of the subcutaneous tumors were measured twice weekly using an electronic caliper. Tumor area was calculated by the following formula: Tumor area [mm ² ] = length [mm] x width [mm]. Tumor growth data are shown in FIG3 . In mice bearing mesothelin-expressing tumors, MOR6640-SPDB-DM4 and MOR6640-sulfo-SPDB-DM4 treatment resulted in eradication of tumors in all treated animals 12 days after the last treatment, however, these tumors regenerated after 10 days ( FIG3A ). MOR6640-SMCC-DM1, MOR6640-(PEG)4-mal-DM1, and anti-lysozyme-SPDB-DM4 treatment did not significantly affect tumor growth. None of the treatments resulted in altered in vivo growth of vector-transfected MiaPaCa-2 cells compared to mesothelin-expressing tumors ( FIG3B ).

Example 3: In vitro cytotoxicity of anti-mesothelin immunoconjugates

To evaluate the cytotoxicity of anti-mesothelin immunoconjugates, different mesothelin-expressing cell lines were cultured to 80-90% confluence, trypsinized, and counted. Cells were then seeded at 800 μl per well in their growth medium in 384-well flat-bottom plates for all cell lines. Wells containing only medium were set up for blank subtraction. MF-J-SPDB-DM4, MF-226-SPDB-DM4MOR6640-SPDP-DM4, MF226-SPDP-DM4, and anti-lysozyme-SPDP-DM4 were administered 24 hours after seeding at concentrations ranging from 0.01 to 300 nM. Triplicate dilutions were performed for each dilution. Endpoint measurements were taken at 96 hours. Cell viability was assessed using the WST-1 assay (Roche Cat# 1644807). IC ₅₀ values are shown in Table 2. The dose-response curve showing the in vitro cytotoxicity of MF-T-SPDP-DM4 on HelaMatu cells is shown in FIG4 .

Table 2: nM _IC50 values of anti-mesothelin immunoconjugates against mesothelin-expressing cell lines

Claims (5)

1. An immunoconjugate comprising a cytotoxic agent and a human or humanized antibody, wherein the antibody comprises: i) The variable heavy chain sequence of SEQ ID NO: 23 and the variable light chain sequence of SEQ ID NO: 27, or ii) The variable heavy chain sequence of SEQ ID NO: 20 and the variable light chain sequence of SEQ ID NO: 24, or iii) The variable heavy chain sequence of SEQ ID NO: 21 and the variable light chain sequence of SEQ ID NO: 25, wherein the cytotoxic agent is a metansine, wherein the metansine is DM4, and wherein the antibody and the cytotoxic agent are linked by a cleavable SPDB linker. 2. A pharmaceutical composition comprising the immunoconjugate according to claim 1 and its pharmaceutically acceptable carrier. 3. The immunoconjugate according to claim 1 or the pharmaceutical composition according to claim 2, used as a medicine. 4. The immunoconjugate according to claim 1 or the pharmaceutical composition according to claim 2, for the treatment of cancer. 5. The immunoconjugate according to claim 1 or the pharmaceutical composition according to claim 2, for the treatment of pancreatic cancer, ovarian cancer, lung cancer, or mesothelioma.