HK1201457B - Treatment of cancer with novel anti-il 13 monoclonal antibodies - Google Patents

Description

Treatment of cancer with novel anti-IL 13 monoclonal antibodies

The application is a divisional application of Chinese patent application with the application number of 200480038820.4, the application date of 2004 is 12 months and 23 days, and the invention name is 'treating cancer by using a novel anti-IL 13 monoclonal antibody'.

Background

IL13 is mainly composed of CD4⁺T-helper type 2 cells, as well as NKT cells, basophils and mast cells produce pleiotropic Th2 cytokine (Hershey, GKK, jallergyclin immunol. (2003)111: 677-90). In addition to its etiologic role in asthma, fibrosis, chronic pulmonary obstructive disease and ulcerative colitis (Wynn, TA, AnnuRevImmunol. (2003)21: 425-56; WynnTA., NatRevImmunol. (2004)4: 583-94; HellerFetal., Immunity (2002)17:629-38), IL13 is also known to play an important role in tumor growth (KappUet., JExpmed. (1999)189: 1939-4; Trieuyetal., Cancer Res.2004; 64:3271-5) and regulation of tumor Immunity (Terabel. et., cancer Immunol. 2004; 53: 79-85; Terabel. Metal., Immunol.2000; 1: 515-20). Therefore, IL13 and its receptor are potential therapeutic targets for cancer.

Hodgkin's Lymphoma (HL) is a lymph node malignancy characterized by the aberrant production of multiple cytokines from the malignant population of HL, Reed-Stemberg (RS) cells (see Kapp et al and Trieu et al, supra). IL13 has also been shown to promote HL proliferation by an autocrine mechanism. Neutralizing monoclonal antibodies (MAbs) against IL13 were shown to inhibit HL cell proliferation in vitro (Trieu et al, supra).

Cumulative evidence suggests that the IL13 receptor is expressed on a variety of human malignant cell lines (e.g., glioblastoma, head and neck tumors, squamous cell carcinoma, renal cell carcinoma, AIDS-related Kaposi's carcinoma, prostate cancer, pancreatic cancer, and epithelial cancers such as gastric, colon, and skin adenocarcinomas) (see, e.g., Debinski Weal. JBiolChem. (1995)270: 16775-80; PuriRKet. blood (1996)87: 4333-9; MainAetal. Jurol. (1997)158: 948-53; Debinski Weal. Clin cancer Res. (1995)1: 1253-8; Kormann Martin et. anticancer Res. (1999)19: 125-31; Husain blood. Blod (SRblood) (95: 6-13; Kakawami KeceKe. cancer. (6161) 2001: 6200). A recombinant fusion protein comprising IL13 conjugated to a mutant form of pseudomonas exotoxin was shown to specifically kill these tumor cells in vitro. Thus, these data suggest that the IL13 receptor is an attractive target for mediating selective tumor killing.

The major mediator of anti-tumor immunity is now known to be CD4⁺Th1 cells and CD8⁺Cytotoxic T Lymphocytes (CTL). Since immune excursions toward Th2 suppressed Th1 development, it has been suggested that induction of Th2 responses in cancer patients is a major mechanism to suppress tumor immune surveillance. Terabe et al showed that an inhibitor of IL13 (sIL13R2-Fc) inhibited tumor recurrence in a mouse model. Similar observations were also found in mice knocked out with STAT6 or IL4R, but similar results were not observed in mice knocked out with IL 4. Taken together, these results suggest that IL13 plays an important role in inhibiting anti-tumor immunity in vivo. Therefore, inhibition of IL13 may promote anti-tumor immunity in cancer patients.

Antibody-based therapies have proven to be very effective in treating various cancers. For example,have been successfully used to treat breast cancer and non-hodgkin's lymphoma, respectively. The present invention provides additional methods of treating cancer that overcome the limitations of conventional therapeutic methods and provide additional advantages that will be apparent from the detailed description below.

Summary of The Invention

The present invention relates to the treatment of cancers and/or tumors expressing IL13 with a novel anti-IL 13 monoclonal antibody. The antibodies used in the present invention include novel anti-IL 13 antibodies that specifically and with high affinity bind glycosylated and non-glycosylated human IL13 but do not bind mouse IL13 and neutralize human IL13 activity at a molar ratio of about 1:2(MAb: IL 13). Antibodies comprising antigen binding regions derived from the light and/or heavy chain variable regions of the antibodies are also encompassed by the invention. The antibody of the invention may be monoclonal, and the monoclonal antibody may be a human antibody, a chimeric antibody or a humanized antibody.

Such antibodies are, for example, 228B/C-1, 228A-4, 227-26 and 227-43. Hybridomas producing these antibodies were deposited at 20.11.2003 with the American type culture Collection (American type culture Collection,10801university blvd., Manassas, VA 20110-2209) under accession numbers PTA-5657, PTA-5656, PA-5654 and PTA-5655, respectively. These antibodies can target IL 13-expressing tumor cells in vivo. These antibodies are described in co-pending patent application (WO05062972, filed as 2004, 12/23), which is incorporated herein by reference.

Antibodies useful in the present invention also include the following: an antibody having a VL sequence having at least 95% homology with the sequence shown in SEQ ID NO. 3 and a VH sequence having at least 95% homology with the sequence shown in SEQ ID NO. 4; an antibody having a VL sequence having at least 95% homology with the sequence shown in SEQ ID NO.5 and a VH sequence having at least 95% homology with the sequence shown in SEQ ID NO. 6; an antibody having a VL sequence having at least 95% homology with the sequence shown in SEQ ID NO. 7 and a VH sequence having at least 95% homology with the sequence shown in SEQ ID NO. 8. The invention also includes a recombinant antibody molecule or fragment thereof that binds IL13 comprising at least one antibody heavy chain or fragment thereof that binds IL13 and at least one antibody light chain or fragment thereof that binds IL13 comprising non-human CDRs from a mouse anti-IL 13 antibody at positions 31-35 (CDR1), 50-65 (CDR2) and 95-102(CDR3) (Kabat numbering) and a framework region from a human monoclonal antibody, wherein the at least one antibody heavy chain or fragment thereof that binds IL13 has amino acids Gly26, Phe27, Ser28, Leu29, Asn30(seq id no:18) at positions 27-30 and the at least one antibody light chain IL13 comprises non-human CDRs from a mouse anti-IL 13 antibody at positions 24-34 (CDR1), 50-56 (CDR2) and 89-97(CDR 3).

Antibodies useful in the invention also include human antigen-binding antibody fragments of the antibodies of the invention, including but not limited to Fab, Fab 'and F (ab')₂Fd, single-chain fv (scFv), single-chain antibody, disulfide-linked fv (sdFv). The invention also includes single domain antibodies comprising a VL or VH domain. One example is an scFv having the sequence shown in SEQ ID NO: 152.

The antibodies useful in the present invention also include the humanized sequences of monoclonal antibody 228B/C-1. These humanized recombinant antibody molecules comprise a variable light chain region comprising an amino acid sequence of the formula FRL1-CDRL1-FRL2-CDRL2-FRL3-CDRL3-FRL4, wherein FRL1 consists of any one of the sequences of seq id nos 20-25; CDRL1 consists of any one of SEQ ID NOs 99-103; FRL2 consists of SEQ ID NO: 29; CDRL2 consists of any one of the sequences of SEQ ID NOs 104-114; FRL3 consists of any sequence of SEQ ID NO. 30-56; CDRL3 consists of any one of the sequences of SEQ ID NO. 115-116; FRL4 consists of any sequence of SEQ ID NO. 57-59; the humanized recombinant antibody molecule further comprises a variable heavy chain region comprising an amino acid sequence of the formula FRH1-CDRH1-FRH2-CDRH2-FRH3-CDRH3-FRH4, wherein FRH1 consists of any one of the sequences of seq id nos 60-66; CDRH1 consists of any one of the sequences of SEQ ID NO: 117-122; FRH2 consists of any sequence of SEQ ID NO. 67-75; CDRH2 consists of any one of the sequences of SEQ ID NO: 123-134; FRH3 consists of any sequence of SEQ ID NO. 76-90; CDRH3 consists of any one of the sequences of SEQ ID NO. 135-141; FRH4 consists of any one of SEQ ID NO 91-92. The variable heavy chain region may further comprise at least the CH1 domain of the constant region or the CH1, CH 2and CH3 domains of the constant region. The heavy chain constant region may comprise an IgG antibody, wherein the IgG antibody is an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, or an IgG4 antibody.

In addition, the antibodies encompassed by the present invention comprise recombinant antibody molecules wherein the variable light chain is selected from any one of the sequences of seq id nos. 3,5, 7, 93, 95, 97, 142, 144, and 150 and the variable heavy chain is selected from any one of the sequences of seq id nos. 4,6, 8, 94, 96, 98, 143, 145, 146, 147, 148, and 149. A particular antibody comprises a variable light chain having the sequence shown in SEQ ID NO:142 and a variable heavy chain having the sequence shown in SEQ ID NO: 143.

The binding epitope of MAb228B/C-1 is localized to a unique site on IL13 responsible for interaction with IL4R α, which forms part of the multimeric IL13R complex. This binding site on IL13 is remote from the site responsible for IL13R interaction, therefore, 228B/C-1 can bind to IL13 bound on tumor cells overexpressing IL 13R. The invention also provides antibodies that compete for binding to the same epitope recognized by any of the monoclonal antibodies described above. The invention also includes antibodies that bind to the same epitope as that bound by 228B/C-1. Epitope peptides include peptides that substantially comprise or consist of ESLINVSG (SEQ ID NO:18) or YCAALESLINVS (SEQ ID NO: 19).

In another embodiment, an isolated anti-IL 13 monoclonal antibody is provided that inhibits the growth of IL 13-expressing cancer cells in vivo or that is toxic to such cells and tumors containing such cells in vivo. The present invention provides anti-IL 13 antibodies conjugated to a cytotoxic agent or a growth inhibitory agent. The cytotoxic agent may be a toxin, a cytotoxic small molecule drug, a high-energy radioisotope, a photoactivatable drug, a pro-apoptotic protein or drug, a lytic enzyme or a nucleolytic enzyme.

Antibodies useful in the present invention may comprise a human IgG1 constant region that mediates killing of tumor cells by complement-mediated cell lysis (CMC) and antibody-dependent cell-mediated cytotoxicity (ADCC). Such antibodies may also inhibit IL 13-dependent tumor cell growth.

The anti-IL 13 antibodies in the preceding embodiments include intact (full-length) antibodies as well as antibody fragments. In one embodiment, the anti-IL 13 antibody of any of the preceding embodiments is a chimeric, humanized or human antibody. Antibody fragments of the antibodies of the invention that bind to human antigens include, but are not limited to, Fab 'and F (ab')₂Fd, single-chain fv (scFv), single-chain antibody, disulfide-linked fv (sdFv). The invention also includesA single domain antibody of a VL or VH domain. One example is a polypeptide having the sequence of seq id no: 152.

The invention also encompasses the use of a composition comprising any of the anti-IL 13 antibodies of the embodiments described above and a carrier in the methods of the invention. The carrier is a pharmaceutically acceptable carrier. These compositions may be provided in an article of manufacture or kit for use in the treatment of cancer.

Yet another aspect of the invention is a method of killing a cancer cell that expresses IL13, comprising contacting the cancer cell with an anti-IL 13 antibody of any one of the embodiments described above, thereby killing the cancer cell. Another aspect of the invention is a method of reducing or treating a cancer that expresses IL13 in a mammal, the method comprising administering to the mammal a therapeutically effective amount of an anti-IL 13 antibody of the invention. In embodiments of the foregoing methods, the cancer is renal cell carcinoma, glioma, brain tumor, hodgkin's lymphoma or other tumor or cancer that expresses IL13 receptor on its surface. In a preferred embodiment of these methods, the anti-IL 13 antibody is a human or humanized antibody. In another preferred embodiment, the antibody is conjugated to a cytotoxic agent such as a toxin or radioisotope and a cytostatic agent such as an inhibitor of cyclin-dependent kinase.

Methods of alleviating cancers that express IL13 it is contemplated that anti-IL 13 antibodies are administered in combination with other forms of cancer treatment such as radiation therapy and chemotherapy. In the latter case, the mammal also receives at least one chemotherapeutic agent. In a specific embodiment, the chemotherapeutic agent is selected from the group of drugs such as, but not limited to, doxorubicin, 5-fluorouracil, cytarabine, cyclophosphamide, Thiotepa (Thiotepa), Busulfan (busufan), Cytoxin, Taxol, methotrexate, cisplatin, Melphalan (Melphalan), Vinblastine (Vinblastine), bleomycin, and Carboplatin (Carboplatin). In another specific embodiment, the anti-IL 13 may be used in combination with other anti-tumor antibodies such as, but not limited to, anti-VEGFMAb, anti-Her 2MAb, anti-egfr MAb, anti-EpCamMAb, anti-ganglioside MAb, anti-tissue factor MAb, and anti-integrin MAb.

In another aspect, the invention provides an article of manufacture (articlef manufacturing) comprising a container and a composition contained therein, wherein the composition comprises an anti-IL 13 antibody of the above embodiment, the article of manufacture further comprising a package insert indicating that the composition is useful for alleviating or treating a cancer expressing IL 13.

Another aspect of the invention includes diagnosing a cancer or tumor overexpressing IL13, the diagnosis comprising detecting overexpression of IL13 in a biological sample taken from a patient suspected of having the cancer or tumor using an anti-IL 13 antibody of the invention.

Brief Description of Drawings

FIG. 1 depicts the binding of an anti-IL 13 monoclonal antibody to human IL 13.

FIG. 2 depicts the binding of an anti-IL 13 monoclonal antibody to mutant IL 13-Fc.

FIG. 3 illustrates that mAbJES10-5A2(Pharmingen) does not inhibit the binding of mAb228B/C-1 to human IL 13.

FIG. 4 illustrates the effect of anti-IL 13 monoclonal antibody on cell proliferation of Hodgkin lymphoma L-1236.

FIG. 5 illustrates the effect of anti-IL 13 monoclonal antibodies on IL 13-induced inhibition of CD14 expression in human monocytes.

Figure 6 illustrates the effect of anti-IL 13 monoclonal antibodies on IL 13-induced upregulation of CD23 expression in human monocytes.

FIG. 7 illustrates the effect of anti-IL 13 monoclonal antibodies on IL 13-induced phosphorylation of STAT6 in THP-1 cells.

FIG. 8 depicts the amino acid sequences of the VH and VL regions of monoclonal antibody 228B/C-1.

FIG. 9 depicts the amino acid sequences of the VH and VL regions of some humanized antibodies derived from monoclonal antibody 228B/C-1.

Detailed Description

The invention is not limited to the particular methodology, protocols, cell lines, vectors or reagents described herein, as these may vary. Furthermore, the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention. As used herein and in the appended claims, the singular forms "a", "an", and the like include plural referents unless the context clearly dictates otherwise, e.g., "a host cell" includes a plurality of such host cells.

Unless otherwise defined, all technical and scientific terms and any abbreviations used herein have the same meaning as commonly understood by one of ordinary skill in the art. Although any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, the use of equipment and materials is described herein.

All patents and publications mentioned herein are incorporated herein by reference to the extent allowed by law for the purpose of describing and disclosing the proteins, enzymes, vectors, host cells and methodologies reported in the patents and publications that might be used in connection with the invention. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

As used herein, the term "antibody" refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds an antigen. The immunoglobulin molecules of the invention may be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1hIgA 2) or subclass of immunoglobulin molecule. In addition, the term "antibody" (Ab) or "monoclonal antibody" (mAb) is intended to include intact molecules as well as antibody fragments (e.g., Fab and F (Ab')₂Fragments). Fab and F (ab')₂Fc fragment whose fragment lacks an intact antibodyClearance from the circulation of animals or plants is faster and can be lower for non-specific tissue binding than for intact antibodies (Wahletal, J.Nucl. Med.24:316-325 (1983)).

As used herein, the term "human" antibody includes antibodies having the amino acid sequence of a human immunoglobulin, and includes antibodies isolated from a human immunoglobulin library or from an animal transgenic for one or more human immunoglobulins but which do not express endogenous immunoglobulins, as described below and, for example, in U.S. patent No.5,939,598 to Kucherlapati et al.

Antibody "effector function" refers to those biological activities attributable to the Fc region of an antibody (either the native sequence Fc region or the amino acid sequence variant Fc region), and varies with antibody isotype. Antibody effector functions include, for example: c1q binding and complement dependent cytotoxicity; fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down-regulation of cell surface receptors (e.g., B cell receptors) and B cell activation.

"antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted Ig bound Fc γ receptors (Fc γ R) present on certain cytotoxic cells (e.g., Natural Killer (NK) cells, neutrophils, and macrophages) such that these cytotoxic effector cells specifically bind to antigen-bearing target cells, which in turn kill the target cells with cellular enzymes or oxidative radicals. Antibodies "arm" cytotoxic cells and are required for such killing. The main cells mediating ADCC, NK cells, express FC γ RIII only, whereas monocytes express FC γ RI, FC γ RII and FC γ RIII. To assess ADCC activity of a molecule of interest, an in vitro ADCC assay as described below may be performed. Effector cells used in such assays include Peripheral Blood Mononuclear Cells (PBMC) and Natural Killer (NK) cells. Alternatively or additionally, the ADCC activity of the molecule of interest can be assessed in vivo, for example in an animal model such as that disclosed by Clynesetal. PNAS (USA)95: 652-.

Immunogens

Mice were immunized with recombinant IL13 to generate hybridomas producing monoclonal antibodies of the invention. Recombinant IL13 is commercially available from a number of sources (see, e.g., R & DSystems, Minneapolis, MN, pepotech, inc., NJ, and SanofiBio-Industries, inc., Tervose, PA.). Alternatively, the gene or cDNA encoding IL13 may be cloned into a plasmid or other expression vector and expressed in any of a variety of expression systems according to methods well known in the art. Methods for cloning and expressing IL13 and the nucleic acid sequence of IL13 are well known (see, e.g., U.S. patent 5,652,123). Due to the degeneracy of the genetic code, a plurality of nucleotide sequences encoding IL13 polypeptides may be generated. One can alter the nucleotide sequence by selecting combinations based on possible codon usage. These combinations were generated based on the standard triplet genetic code for the nucleotide sequence encoding the naturally occurring IL13 polypeptide, all of which variations were considered. Any of these polypeptides can be used to immunize an animal to produce antibodies that bind IL 13.

When beneficial, the immunogenic IL13 polypeptide may be expressed as a fusion protein with IL13 polypeptide attached to the fusion segment. The fusion segment generally facilitates protein purification, for example, by allowing the fusion protein to be isolated and purified via affinity chromatography. Fusion proteins can be produced by culturing recombinant cells transformed with a fusion nucleic acid sequence encoding a protein that includes a fusion segment attached to the carboxy-terminus and/or the amino-terminus of the protein. Fusion segments can include, but are not limited to, immunoglobulin Fc region, glutathione-S-transferase, beta-galactosidase, polyhistidine segments capable of binding divalent metal ions, and maltose binding protein.

Antibodies of the invention are produced using fusion proteins comprising a mutated form of human IL 13. This mutant form of IL13 contains a single mutation, resulting in an inactive form of the protein (thompson et al, j.biol.chem.274:2994 (1969)). In order to generate neutralizing antibodies with high affinity, the fusion protein comprises a mutant IL13 protein fused to an immunoglobulin Fc, in particular IgG1, and is expressed in a mammalian cell line, whereby the recombinant protein is natively glycosylated. The Fc portion of the fusion protein may provide a conformational structure that exposes critical epitopes. The glycosylation can increase the immunogenicity of the epitope, allowing the production of antibodies against this particular epitope.

IL13 polypeptide expressed in e.coli lacks glycosylation and the commercially available antibodies tested were produced using this protein. We tested these antibodies, such as R & DSystems and Pharmingen, and found that they did not cross-react with the epitope to which the antibody of the invention binds.

Antibody production

The antibodies of the invention may be produced by any suitable method known in the art. The antibody of the present invention may comprise a polyclonal antibody. Methods for making polyclonal Antibodies are known to those skilled in the art (Harlow, et al, Antibodies: aLaboratoriarmoual, (Coldspring Harbor laboratory Press,2nd d. (1988), which is incorporated herein by reference in its entirety).

For example, the above immunogens can be administered to various host animals, including but not limited to rabbits, mice, rats, etc., to induce the production of serum containing polyclonal antibodies specific to the antigen. The immunogen may be administered with one or more injections of the immunizing agent and, if desired, an adjuvant. Depending on the host species, various adjuvants may be used to increase the immunological response, including, but not limited to, Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG (Bacille calcium-Guerin) and Corynebacterium parvum (Corynebacterium parvum). Examples of other adjuvants that may be employed include MPL-TDM adjuvant (monophosphoryl lipid A, synthetic trehalose dicorynomycolate). Immunization protocols are well known in the art and may be carried out by any method that elicits an immune response in a selected host. Adjuvants are also well known to those skilled in the art.

Typically, the immunogen (with or without adjuvant) is injected into the mammal by multiple subcutaneous or intraperitoneal injections, or intramuscularly or by intravenous injection. The immunogen may comprise an IL13 polypeptide, a fusion protein or variant thereof. Depending on the nature of the polypeptide (i.e., percent hydrophobicity, percent hydrophilicity, stability, net charge, isoelectric point, etc.), the immunogen may be conjugated to a protein known to be immunogenic in the animal being immunized. Such conjugation includes chemical conjugation by derivatizing the immunogen and the active chemical functional groups of the immunogenic protein to be conjugated, thereby forming covalent bonds, or conjugation by fusion protein-based methodologies or other methods known to those skilled in the art. Such immunogenic proteins include, but are not limited to, keyhole limpet hemocyanin, ovalbumin, serum albumin, bovine thyroglobulin, soybean trypsin inhibitor, and promiscuous T helper cell peptides. Various adjuvants may be used to increase the immune response as described above.

The antibodies of the invention include monoclonal antibodies. Monoclonal Antibodies can be prepared using hybridoma technology, such as Kohlerand Milstein, Nature,256:495(1975) and U.S. Pat. No.4,376,110, Harlow et al, Antibodies: Arabidopsis, (Coldspring Harbor laboratory Press,2. sup.nd. (1988), Hammerling, et al, monoclonal Antibodies and cell hybrids (Elsevier, N.Y., (1981)), or other methods known to the skilled artisan.

Using typical hybridoma technology, a host such as a mouse, humanized mouse (a mouse with a human immune system), hamster, rabbit, camel, or any other suitable host animal is typically immunized with an immunogen to elicit lymphocytes that produce or are capable of producing antibodies that specifically bind IL 13. Alternatively, lymphocytes may be immunized with an antigen in vitro.

Generally, in the preparation of antibody-producing hybridomas, Peripheral Blood Lymphocytes (PBLs) are used if cells of human origin are desired, and spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent such as polyethylene glycol to form a hybridoma cell (Goding, monoclonal antibodies: Prinipposite, academic Press, (1986), pp. 59-103). Immortalized cell lines are generally transformed mammalian cells, in particular myeloma cells of rodent, bovine or human origin. Typically, rat or mouse myeloma cell lines are used. The hybridoma cells can be cultured in a suitable medium that preferably contains one or more substances that inhibit the growth or survival of unfused immortalized cells. For example, if the parental cells lack hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium of the hybridoma typically includes the substances hypoxanthine, aminopterin, and thymidine (HAT medium) that prevent the growth of HGPRT-deficient cells.

Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. More preferred immortalized cell lines are murine myeloma cell lines, which may be obtained, for example, from the salk institutes cell distribution center, san diego, calif, and american type culture collection, Manassas, Va. Human myeloma and mouse-human hybrid myeloma cell lines may also be used to produce human monoclonal antibodies (Kozbor, J.Immunol.,133:3001 (1984); Brodeutal, monoclonal antibody production techniques and applications, Marcel Dekker, Inc., New York, (1987) pp.51-63).

The culture medium in which the hybridoma cells were cultured was then analyzed for the presence of monoclonal antibodies to IL 13. The binding specificity of monoclonal antibodies produced by hybridoma cells is determined, for example, by immunoprecipitation methods or by in vitro binding assays such as Radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). Such techniques are known in the art and are within the skill of the artisan. The binding affinity of monoclonal antibodies to IL13 can be determined, for example, by Scatchard analysis (Munsonnetal, anal. biochem.,107:220 (1980)).

After identifying the desired hybridoma cells, these clones can be subcloned by limiting dilution procedures and cultured by standard methods (Goding, supra). Suitable media for this purpose include, for example, Dulbecco's Modifiedeagle's medium and RPMI-1640. Monoclonal antibodies secreted by these subclones can be isolated or purified from the culture medium by conventional immunoglobulin purification procedures, such as protein a-sepharose, hydroxyapatite chromatography, gel exclusion chromatography, gel electrophoresis, dialysis, or affinity chromatography.

There are many methods for producing monoclonal antibodies available in the art, and thus the antibodies of the present invention are not limited to production only in hybridomas. For example, monoclonal antibodies can be produced by recombinant DNA methods, such as those described in U.S. patent No.4,816,567. As used herein, the term "monoclonal antibody" refers to an antibody derived from a single eukaryotic cell, phage, or prokaryotic cell clone. DNA encoding the monoclonal antibodies of the invention can be isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of specifically binding genes encoding the heavy and light chains of murine antibodies or those chains from human, humanized or other sources). The hybridoma cells of the invention are a preferred source of such DNA. After isolation, the DNA may be placed into an expression vector, which is then transformed into a host cell such as an NSO cell, simian COS cell, Chinese Hamster Ovary (CHO) cell, or myeloma cell that does not otherwise produce immunoglobulin protein, to synthesize a monoclonal antibody in the recombinant host cell. The DNA may also be modified, for example, by replacing the homologous murine sequences with the coding sequences for the human heavy and light chain constant domains (U.S. Pat. No.4,816,567; Morrisonetal, supra) or by covalently linking the immunoglobulin coding sequence to all or part of the coding sequence for a non-immunoglobulin polypeptide. Such non-immunoglobulin polypeptides may replace the constant domains of an antibody of the invention, or may replace the variable domains of one antigen combining site of an antibody of the invention, to produce a chimeric bivalent antibody.

The antibody may be a monovalent antibody. Methods for making monovalent antibodies are well known in the art. For example, one method involves recombinant expression of an immunoglobulin light chain and a modified heavy chain. The heavy chain is typically truncated at any point in the Fc region to prevent heavy chain cross-linking. Alternatively, the relevant cysteine residue is substituted or deleted with another amino acid residue to prevent cross-linking.

Antibody fragments that recognize specific epitopes can be generated by known techniques. For example, Fab and F (ab')₂Fragments can be generated by using enzymes such as papain (to generate Fab fragments) or pepsin (to generate F (ab')₂Fragments) are produced by proteolytic cleavage of immunoglobulin molecules. F (ab')₂The fragment contains the variable region, the light chain constant region and the CH1 domain of the heavy chain.

For some applications, including the use of antibodies in vivo and in vitro detection assays, it may be preferred to use chimeric, humanized or human antibodies. Chimeric antibodies are molecules in which different portions of the antibody are derived from different animal species, such as antibodies having variable regions derived from murine monoclonal antibodies and human immunoglobulin constant regions. Methods for generating chimeric antibodies are known in the art, see, e.g., Morrison, Science229:1202 (1985); oietal, BioTechniques4:214 (1986); gilliesetal, (1989) J.Immunol.Methods125: 191-202; U.S. Pat. Nos. 5,807,715; 4,816,567 and 4,816,397, which documents and patents are incorporated herein by reference in their entirety.

Humanized antibodies are antibody molecules produced in non-human species that bind to a desired antigen and have one or more Complementarity Determining Regions (CDRs) from the non-human species and a Framework Region (FR) from a human immunoglobulin molecule. Typically, framework residues in the human framework regions are substituted with corresponding residues from the CDR donor antibody to alter, preferably improve, antigen binding. These framework substitutions are identified by methods well known in the art, for example by modeling the interaction of the CDRs with the framework residues to identify framework residues important for antigen binding, and performing sequence alignments to identify unusual framework residues at specific positions (see, e.g., queeneteal, U.S. Pat. No.5,585,089; riechmannetal, Nature332:323(1988), which are incorporated herein by reference in their entirety). Antibodies can be humanized using a variety of techniques known in the art, such as CDR grafting (EP239,400; PCT publication WO 91/09967; U.S. Pat. No.5,225,539; 5,530,101 and 5,585,089), vectoring or resurfacing (EP592,106; EP519,596; Padlan, molecular immunology28(4/5):489-498 (1991); Studnickal, protein engineering7(6):805-814 (1994); Roguska. et. PNAS91:969-973(1994)) and chain shuffling (U.S. Pat. No.5,565,332).

Typically, one or more amino acid residues from a non-human source are introduced into the humanized antibody. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain. Humanization is essentially performed according to the methods described by Winter and co-workers (Joneset al, Nature,321:522-525 (1986); Reichmannetal, Nature,332:323-327 (1988); Verhoeyenetal, Science,239:1534-1536 (1988)) by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody.

Fully human antibodies are particularly desirable for treatment of human patients. Human antibodies can be produced by a variety of methods known in the art, including the phage display methods described above using antibody libraries derived from human immunoglobulin sequences. See also, for example, U.S. Pat. Nos. 4,444,887 and 4,716,111, and PCT publications WO98/46645, WO98/50433, WO98/24893, WO98/16664, WO96/34096, WO96/33735, and WO 91/10741; all of which are incorporated by reference in their entirety. The techniques of Cole et al and Boerder et al can also be used to prepare human monoclonal antibodies (Coleatal, monoclonal antibodies and cells therapy, AlanR. Riss, (1985); Boernereal, J.Immunol.,147(1):86-95, (1991)).

Human antibodies can also be produced using transgenic mice that do not express functional endogenous immunoglobulins but express human immunoglobulin genes. For example, human heavy and light chain immunoglobulin gene complexes can be introduced randomly or by homologous recombination into mouse embryonic stem cells. Alternatively, in addition to human heavy and light chain genes, human variable, constant, and diversity regions can be introduced into mouse embryonic stem cells. Introduction of human immunoglobulin loci by homologous recombination can render the mouse heavy and light chain immunoglobulin genes separately or simultaneously non-functional. In particular, homozygous deletion of the JH region prevents endogenous antibody production. The modified embryonic stem cells are expanded and microinjected into blastocysts to generate chimeric mice. The chimeric mice are then bred to produce homozygous progeny expressing human antibodies. The transgenic mice are immunized in a normal manner with a selected antigen, e.g., all or a portion of a polypeptide of the invention. Monoclonal antibodies against the antigen can be obtained from immunized transgenic mice by conventional hybridoma techniques. The human immunoglobulin transgene contained in the transgenic mice rearranges during B cell differentiation, and after that undergoes class switching and somatic mutation (somatometation). Thus, it is possible to produce therapeutically useful IgG, IgA, IgM and IgE antibodies using this technique. For an overview of this technique for producing human antibodies, see Lonberg and Huszar, int.Rev.Immunol.13:65-93 (1995). For a detailed discussion of the techniques for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e.g., PCT publications WO98/24893, WO92/01047, WO96/34096, WO96/33735, European patent No.0598877, U.S. Pat. Nos. 5,413,923, 5,625,126, 5,633,425, 5,569,825, 5,661,016, 5,545,806, 5,814,318, 5,885,793, 5,916,771, and 5,939,598, which are incorporated herein by reference in their entirety. In addition, companies such as Abgenix, inc. (Freemont, Calif.), Genpharm (san jose, Calif.), and Medarex, inc. (Princeton, n.j.) may be entrusted with providing human antibodies to selected antigens using techniques similar to those described above.

Human mabs can also be prepared by immunizing mice transplanted with human peripheral blood leukocytes, splenocytes, or bone marrow (e.g., the triple hybridoma technology (triomantechniques) by XTL). A technique known as "guided selection" can be used to generate fully human antibodies that recognize selected epitopes. In this method, selection of a complete human antibody recognizing the same epitope is guided by a selected non-human monoclonal antibody, such as a mouse antibody (Jesperseal., Bio/technology12:899-903 (1988)).

In addition, antibodies directed against the polypeptides of the invention may then be used to generate anti-idiotypic antibodies that "mimic" the polypeptides of the invention using techniques well known to those skilled in the art (see, e.g., Greenspan & Bona, FASEBJ.7(5): 437-444; (1989) and N.isinoff, J.Immunol.147(8):2429-2438 (1991)). For example, an anti-idiotypic antibody that "mimics" the multimerization and/or binding region of a polypeptide and thus binds and neutralizes the polypeptide and/or its ligand may be generated using an antibody that binds to and competitively inhibits polypeptide multimerization and/or binding of a polypeptide of the invention to a ligand. In a therapeutic regimen, the polypeptide ligand may be neutralized with these neutralizing anti-idiotype antibodies or Fab fragments of these anti-idiotype antibodies. For example, these anti-idiotype antibodies can be used to bind to the polypeptide of the invention and/or to its ligand/receptor and thus block its biological activity.

The antibody of the invention may be a bispecific antibody. Bispecific antibodies are monoclonal antibodies, preferably human or humanized monoclonal antibodies, which have binding specificities for at least two different antigens. In the present invention, one of the binding specificities may be for IL13, the other may be for any other antigen, preferably for a cell surface protein, receptor subunit, tissue specific antigen, virus derived protein, virus encoded envelope protein, bacteria derived protein, or bacteria surface protein, etc.

Methods for making bispecific antibodies are well known. Traditionally, recombinant production of bispecific antibodies has been based on the co-expression of two immunoglobulin heavy/light chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature,305:537-539 (1983)). Due to the random combination of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a possible mixture of ten different antibody molecules, only one of which has the correct bispecific structure. Purification of the correct molecule is often accomplished with an affinity chromatography step. Similar methods are disclosed in WO93/08829 published at 13.5.1993 and in Truneckereral, EMBOJ, 10:3655-3659 (1991).

Antibody variable regions (antibody-antigen binding sites) with the desired binding specificity can be fused to immunoglobulin constant region sequences. The fusion is preferably with an immunoglobulin heavy chain constant region comprising at least a portion of the hinge, CH2, and CH3 regions. It may have a first heavy chain constant region (CH1) containing the site necessary for light chain binding present in at least one of the fusions. The DNA encoding the immunoglobulin heavy chain fusion and, if desired, the immunoglobulin light chain, is inserted into separate expression vectors and co-transformed into a suitable host organism. See, e.g., surereshel, meth. inezyme, 121:210(1986) for a more detailed description of bispecific antibody production.

The invention also includes heteroconjugate antibodies (heteroconjugate antibodies). Heteroconjugate antibodies consist of two covalently linked antibodies. These antibodies have been proposed, for example, to target immune system cells to unwanted cells (U.S. Pat. No.4,676,980). Antibodies can be prepared in vitro using methods known in the art of synthetic protein chemistry, including those involving cross-linking agents. For example, immunotoxins may be constructed using disulfide interchange reactions or by forming thioester bonds. Examples of suitable agents for this purpose include iminothiolate (iminothiolate) and methyl-4-mercaptoimidate, and those disclosed in, for example, U.S. Pat. No.4,676,980.

In addition, single domain antibodies to IL13 may be generated. Examples of this technology regarding antibodies derived from Camelidae (Camelidae) heavy chain Ig have been described in WO9425591, and the isolation of single domain fully human antibodies from phage libraries is described in US 20030130496.

Identification of anti-IL 13 antibodies

The present invention provides antagonistic monoclonal antibodies that inhibit and neutralize the effects of IL 13. In particular, the antibodies of the invention bind to IL13 and inhibit activation of the IL13 receptor complex. The antibodies of the present invention include antibodies designated 228B/C-1, 228A-4, 227-26, and 227-43, and humanized clones of 228B/C-1 are disclosed. The present invention also includes antibodies that bind to the same epitope as monoclonal antibody 228B/C-1.

Candidate anti-IL 13 antibodies were tested using enzyme-linked immunosorbent assay (EILISA), Western immunoblotting, or other immunochemical techniques. Assays for identifying individual antibodies include: (1) inhibit IL 13-autocrine proliferation of Hodgkin's lymphoma (Hodgkin's SLymphoma) cell lines HDLM-2 and L-1236; (2) inhibits IL 13-induced STAT6 phosphorylation in THP-1 cells; (3) inhibition of IL 13-induced inhibition of CD14 expression in primary human monocytes; and (4) inhibition of IL 13-induced upregulation of CD23 expression on primary human monocytes. Details of the experiments are described in the examples.

Antibodies of the invention include, but are not limited to, polyclonal antibodies, monoclonal antibodies, monovalent antibodies, bispecific antibodies, heteroconjugate antibodies, multispecific antibodies, human antibodies, humanized or chimeric antibodies, single chain antibodies, single domain antibodies, Fab fragments, F (ab') fragments, fragments produced by Fab expression libraries, anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies directed against an antibody of the invention), and epitope-binding fragments of any of the above.

The antibody can be an antibody fragment of the invention that binds to a human antigen, including but not limited to Fab, Fab 'and F (ab')₂Fd, single chain fv (scFv), single chain antibodies, disulfide linked fv (sdFv), and single domain antibodies comprising a VL or VH domain. Antigen-binding antibody fragments, including single chain antibodies, may include variable regions alone or in combination with all or a portion of: hinge region, CH1, CH 2and CH3 domain. The invention also includes antigen binding fragments comprising any combination of the variable region and the hinge, CH1, CH2, and CH3 domains. The antibodies of the invention may be from any animal source including birds and mammals. Preferably, the antibody is from a human, non-human primate, rodent (e.g., mouse and rat), donkey, sheep, rabbit, goat, guinea pig, camel, horse or chicken.

The antibodies of the invention may be monospecific, bispecific, trispecific or have a higher multispecific character. The multispecific antibody may be specific for a different epitope of IL13, or may be specific for IL13 and a heterologous epitope, such as a heterologous polypeptide or solid phase support material. See, e.g., PCT documents WO93/17715, WO92/08802, WO91/00360, WO92/05793, Tutt, et al, J.Immunol.147:60-69 (1991); U.S. Pat. nos. 4,474,893, 4,714,681, 4,925,648, 5,573,920, 5,601,819; kostelnyetal, J.Immunol.148:1547-1553 (1992).

The antibodies of the invention may be described or specified in terms of the epitope or portion of IL13 that the antibody recognizes or specifically binds. Epitopes or polypeptide portions can be specified as described herein, for example by N-terminal and C-terminal positions, by the size of contiguous amino acid residues, or as listed in the tables and figures.

The antibodies of the invention may also be described or specified in terms of their cross-reactivity. The invention also includes antibodies that bind to an IL13 polypeptide, which IL13 polypeptide has at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, and at least 50% identity to IL13 (as calculated using methods known in the art and described herein).

In particular embodiments, the antibodies of the invention cross-react with the monkey homolog of human IL13 and its corresponding epitope. In a particular embodiment, the above cross-reactivity is with respect to any single specific antigenic or immunogenic polypeptide, or with respect to a combination of specific antigenic and/or immunogenic polypeptides as described herein.

The invention also includes antibodies that bind to a polypeptide encoded by a polynucleotide that hybridizes under stringent hybridization conditions to a polynucleotide encoding IL 13. The antibodies of the invention may also be described or specified in terms of their binding affinity to the polypeptides of the invention. Preferred binding affinities include those having from 10^-8To 10^-15Equilibrium dissociation constant of M or K_DThe antibody of (1). The invention also provides antibodies that competitively inhibit binding of an antibody to an epitope of the invention as determined according to any method known in the art for determining competitive binding, e.g., the immunoassays described herein. In preferred embodiments, the antibody competitively inhibits at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, or at least 50% binding to the epitope.

The invention further includes antibodies that bind to the same epitope as the anti-IL 13 antibodies of the invention bind. To determine whether an antibody competitively binds to the same epitope as an anti-IL 13 antibody of the invention (including antibodies produced by using the hybridomas deposited with the ATCC), a cross-blocking assay, such as a competitive ELISA assay, can be performed. In one competitive ELISA example, IL13 coated on wells of a microtiter plate is pre-incubated with or without candidate competitor antibodies, followed by addition of a biotin-labeled anti-IL 13 antibody of the invention. The amount of labeled anti-IL 13 antibody bound to IL13 antigen in the well was determined using avidin-peroxidase conjugate and a suitable substrate. The antibody may be labeled with a radioactive or fluorescent label or some other detectable and measurable label. The amount of labeled anti-IL 13 antibody bound to the antigen is indirectly related to the ability of the candidate competing antibody (test antibody) to compete for binding to the same epitope, i.e., the higher the affinity of the test antibody to the same epitope, the less labeled antibody binds to the antigen-coated wells. A candidate competitor antibody is considered to be an antibody that binds the same epitope sufficiently or competes with the anti-IL 13 antibody of the invention for binding to the same epitope if the candidate antibody can block binding of at least 20%, preferably at least 20-50%, more preferably at least 50% of IL13 antibody compared to a control experiment run in parallel without the candidate competitor antibody. It is understood that variations of this analysis may be performed to achieve the same quantitative value.

Vectors and host cells

In another aspect, the invention provides vector constructs comprising a nucleotide sequence encoding an antibody of the invention and host cells comprising such a vector. Cell lines expressing the antibodies of the invention can be prepared using standard techniques for cloning and transformation.

Recombinant expression vectors containing nucleotide sequences encoding the antibodies of the invention can be prepared using well-known techniques. The expression vector includes a nucleotide sequence operably linked to a suitable transcription or translation regulating nucleotide sequence, such as those derived from a mammalian, microbial, viral, or insect gene. Examples of regulatory sequences include transcriptional promoters, operators, enhancers, mRNA ribosome binding sites, and/or other suitable sequences that control transcription and translation initiation and termination. Nucleotide sequences are "operably linked" when the regulatory sequence is functionally related to the nucleotide sequence of a suitable polypeptide. Thus, a promoter nucleotide sequence is operably linked to, for example, an antibody heavy chain sequence if it controls the transcription of the appropriate nucleotide sequence.

In addition, sequences encoding suitable signal peptides not naturally associated with the antibody heavy and/or light chain sequences may be incorporated into the expression vector. For example, the nucleotide sequence of the signal peptide (secretory leader) may be fused in-frame (in-frame) to the polypeptide sequence so that the antibody may be secreted into the periplasmic space or into the culture medium. A signal peptide functional in the desired host cell enhances extracellular secretion of the appropriate antibody. After secretion of the antibody from the cell, the signal peptide may be cleaved from the polypeptide. Examples of such secretion signals are well known and include those described, for example, in US5698435, US5698417, and US 6204023.

Host cells useful in the present invention include, but are not limited to, microorganisms such as bacteria (e.g., escherichia coli, bacillus subtilis) transformed with recombinant phage DNA, plasmid DNA, or cosmid DNA expression vectors containing antibody coding sequences; yeast such as Saccharomyces (Saccharomyces), Pichia (Pichia)) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected with recombinant viral expression vectors (e.g., baculovirus) containing antibody coding sequences; plant cell systems infected with recombinant viral expression vectors (e.g., cauliflower mosaic virus (CaMV); Tobacco Mosaic Virus (TMV); or transformed with recombinant plasmid expression vectors containing antibody coding sequences (e.g., Ti plasmid), or mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3 cells) carrying recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., the metallothionein promoter) or promoters derived from mammalian viruses (e.g., the adenovirus late promoter, the vaccinia virus 7.5K promoter).

The vector may be a plasmid vector, a single-or double-stranded phage vector, or a single-or double-stranded RNA or DNA viral vector. These vectors can be introduced into cells as polynucleotides using well-known techniques for introducing DNA and RNA into cells. In the case of phage and viral vectors, the vectors may also be introduced into cells as packaged or encapsulated viruses using well-known techniques of infection and transduction. Viral vectors may be replicable or replication defective. In the latter case, viral propagation will generally occur only in the complementing host cell. Using RNA derived from the present DNA construct, a cell-free translation system may also be used to produce the protein. These vectors may include nucleotide sequences encoding the constant regions of antibody molecules (see, e.g., PCT publication WO86/05807, PCT publication WO89/01036, U.S. Pat. No.5,122,464), and the variable domains of antibodies may be cloned into such vectors for expression of the entire heavy or light chain.

Prokaryotic cells that may be used as host cells in the present invention include gram-negative or gram-positive organisms such as E.coli and Bacillus subtilis. Expression vectors used in prokaryotic host cells typically include one or more phenotypic selectable marker genes. For example, a phenotypic selectable marker gene is a gene that encodes a protein that confers antibiotic resistance or provides autotrophic requirements. Examples of useful expression vectors for prokaryotic host cells include those derived from commercially available plasmids such as pKK223-3(pharmacia Fine Chemicals, Uppsala, Sweden), pGEM1(Promega Biotec, Madison, Wisconsin., USA), and pET (Novagen, Madison, Wisconsin, USA) and pRSET (Invitrogen corporation, Carlsbad, California, USA) series of vectors (Studier, F.W., J.Mol.biol.219:37(1991); Schoepfer, R.Gene124:83 (1993)). Promoter sequences commonly used in recombinant prokaryotic host cell expression vectors include T7(Rosenberg, et al, Gene56,125-135(1987)), beta-lactamase (penicillinase), the lactose promoter system (Change, Nature275:615 (1978); and Goeddiretal, Nature281:544 (1979)), the tryptophan (trp) promoter system (Goeddiretal, Nucl. acid sRs.8: 4057, (1980)), and the tac promoter (Sambrookoketal, 1990, molecular cloning, Arabidopsis Manual,2dEd., Cold spring harbor laboratory, Cold spring harbor, N.Y.).

Yeasts useful in the present invention include those from the genera Saccharomyces, Pichia, Actinomycetes and Kluyveromyces. Yeast vectors often contain an origin of replication sequence from a 2. mu. yeast plasmid, an Autonomously Replicating Sequence (ARS), a promoter region, polyadenylation sequence, transcription termination sequence, and selectable marker gene. Suitable promoter sequences for yeast vectors include, but are not limited to, goldThe promoters of thioproteines, 3-phosphoglycerate kinase (Hitzemanet al, J.biol. chem.255:2073, (1980)) or other glycolytic enzymes (Hollandent, biochem.17:4900, (1978)) such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. Other suitable vectors and promoters for yeast expression are also described in Fleeret al, Gene,107:285-195 (1991). Other suitable promoters and vectors for use in yeast and yeast transformation protocols are well known in the art. Yeast transformation protocols are well known. One such approach is described in hinonental, proc.natl.acad.sci.,75:1929 (1978). Hinnen protocol selects Trp in selection medium⁺A transformant.

Recombinant antibodies can also be expressed using mammalian or insect host cell culture systems, such as baculovirus systems for the production of heterologous proteins. In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV) can be used as a vector for expressing a foreign gene. The virus grows in Spodoptera frugiperda (Spodoptera frugiperda) cells. Antibody coding sequences can be individually cloned into non-essential regions of the virus (e.g., polyhedrin gene) and placed under the control of an AcNPV promoter (e.g., polyhedrin promoter).

NS0 or Chinese Hamster Ovary (CHO) cells may be used to express the antibodies of the invention in mammals. Control sequences for transcription and translation of mammalian host cell expression vectors can be cleaved from the viral genome. Commonly used promoter and enhancer sequences are derived from polyoma virus, adenovirus 2, simian virus 40(SV40), and human Cytomegalovirus (CMV). The DNA sequence derived from the SV40 viral genome may be used to provide additional genetic elements for expression of structural gene sequences in mammalian host cells, such as SV40 origin, early and late promoters, enhancers, splice and polyadenylation sites. The early and late promoters of the virus are particularly useful because both promoters are readily available from the viral genome as fragments which may also contain the viral origin of replication. Exemplary expression vectors for use in mammalian host cells are commercially available.

Polynucleotides encoding antibodies

The invention also provides polynucleotides comprising nucleotide sequences encoding the antibodies and fragments thereof of the invention. The invention also includes polynucleotides that hybridize under stringent or less stringent conditions to a polynucleotide encoding an antibody of the invention.

The polynucleotide can be obtained and the nucleotide sequence of the polynucleotide determined using any method known in the art. For example, if the nucleotide sequence of an antibody is known, a polynucleotide encoding the antibody can be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeieretal, BioTechniques17:242 (1994)). Briefly, the assembly involves the synthesis of overlapping oligonucleotides containing portions of the sequences encoding the antibodies, annealing and ligation of these oligonucleotides, and subsequent amplification of the ligated oligonucleotides by PCR.

Alternatively, polynucleotides encoding the antibody may be produced from nucleic acids from a suitable source. If no clone containing a nucleic acid encoding a particular antibody is available, but the sequence of the antibody molecule is known, the nucleic acid encoding the immunoglobulin can be obtained by chemical synthesis, or from a suitable source (e.g., an antibody cDNA library, or a cDNA library generated from any tissue or cell expressing the antibody, e.g., hybridoma cells selected for expression of the antibody of the invention, or isolated nucleic acid therefrom, preferably poly A, using synthetic primers hybridizable to the 3 'and 5' ends of the sequence⁺RNA) by PCR amplification or by cloning using oligonucleotide probes specific for the particular gene sequence to identify c encoding the antibody, e.g., from a cDNA libraryAnd (5) cloning the DNA. The amplified nucleic acid produced by PCR can then be cloned into a replicable cloning vector using any method well known in the art.

Once the nucleotide sequence of an antibody, as well as the corresponding amino acid sequence, has been determined, the nucleotide sequence of the antibody can be manipulated using methods well known in the art for manipulating nucleotide sequences, such as recombinant DNA techniques, site-directed mutagenesis, PCR, and the like (see, e.g., sambrook et al, 1990, molecular cloning, alabor manual,2d et al, cold spring harbor laboratory, cold spring harbor, n.y. and ausubel, eds.,1998, current protocol analytical biology, john wiley & Sons, NY, incorporated herein by reference in its entirety) to generate antibodies having different amino acid sequences, such as to generate amino acid substitutions, deletions, and/or insertions.

In a particular embodiment, the amino acid sequence of the heavy and/or light chain variable domains can be examined to identify the sequence of the CDRs by well-known methods, for example, by comparison with known amino acid sequences of other heavy and light chain variable regions to determine the regions of high variability of the sequences. Using conventional recombinant DNA techniques, one or more CDRs can be inserted into a framework region, for example into a human framework region to humanize a non-human antibody, as described above. The framework regions may be naturally occurring or common framework regions, and preferably human framework regions (see, e.g., the list of human framework regions in Chothiacetal, J.mol.biol.278: 457-. Preferably, the polynucleotides produced by the combination of framework regions and CDRs encode antibodies that specifically bind to the polypeptides of the invention. Preferably, as discussed above, one or more amino acid substitutions may be made within the framework regions, and preferably the amino acid substitutions improve binding of the antibody to its antigen. In addition, one or more variable region cysteine residues involved in an intrachain disulfide bond may be substituted or deleted by these methods to produce an antibody molecule lacking one or more intrachain disulfide bonds. Other variations on the polynucleotides are encompassed by the present invention and are within the skill of those in the art.

In addition, techniques developed for producing "chimeric antibodies" by splicing together genes from mouse antibody molecules having suitable antigen specificity and genes from human antibody molecules having suitable biological activity (Morrisonetal., Proc. Natl. Acad. Sci.81:851-855(1984); Neubergertal., Nature312:604-608(1984); Takedaet. Nature314:452-454(1985)) can be used. As noted above, a chimeric antibody is a molecule having different portions derived from different animal species, e.g., a chimeric antibody, e.g., a humanized antibody, having a variable region derived from a mouse MAb and a human immunoglobulin constant region.

Alternatively, the techniques described for producing single chain antibodies (U.S. Pat. No.4,946,778; Bird, Science242:423-42(1988); Hustonet al, Proc. Natl. Acad. Sci. USA85: 5879-. Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge to yield a single chain polypeptide. The technique of assembling functional Fv fragments in E.coli can also be used (Skerraet., Science242: 1038-.

Method for producing anti-IL 13 antibody

The antibodies of the invention may be produced by any method known in the art for the synthesis of antibodies, in particular by chemical synthesis or preferably by recombinant expression techniques.

Recombinant expression of an antibody of the invention or a fragment, derivative or analogue thereof (e.g. a heavy or light chain of an antibody of the invention or a single chain antibody of the invention) requires the construction of an expression vector containing a polynucleotide encoding the antibody or antibody fragment. Once the polynucleotide encoding the antibody molecule has been obtained, recombinant DNA techniques can be used to generate vectors for the production of antibodies. Expression vectors containing antibody coding sequences and appropriate transcriptional and translational control signals were constructed. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination.

The expression vector is transferred into a host cell using conventional techniques, and the transfected cells are then cultured using conventional techniques to produce the antibody of the invention. As described in detail below, in one aspect of the invention, vectors encoding the heavy and light chains may be co-expressed in a host cell so that the entire immunoglobulin molecule is expressed.

As described above, various host expression vector systems can be used to express the antibody molecules of the present invention. Such host expression systems represent not only vectors (vehicles) by which the coding sequence of interest can be produced and subsequently purified, but also cells which can express the antibody molecules of the invention in situ when transformed or transfected with the appropriate nucleotide coding sequences. Recombinant antibody molecules, in particular intact recombinant antibody molecules, are typically expressed in bacterial cells such as E.coli and eukaryotic cells. For example, mammalian cells such as Chinese Hamster Ovary (CHO) cells, which are combined with a vector such as the major intermediate early Gene promoter element from human cytomegalovirus, are efficient expression systems for antibodies (Foeckingage, Gene45:101 (1986); Cockettet al, Bio/Technology8:2 (1990)).

In addition, host cell strains may be selected which regulate the expression of the inserted sequences or modify and process the gene product in the particular manner desired. These modifications (e.g., glycosylation) and processing (e.g., cleavage) of the protein product may be important for the function of the protein. Different host cells have characteristic and specific mechanisms for post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems may be selected to ensure proper modification and processing of the foreign protein expressed. To this end, eukaryotic host cells with the cellular machinery for the correct processing of the primary transcription, glycosylation, and phosphorylation of gene products can be used. These mammalian host cells include, but are not limited to, CHO, COS, 293, 3T3, or myeloma cells.

For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines that stably express the antibody molecule can be engineered. Instead of using an expression vector containing the viral origin of replication, the host cell may be transformed with DNA and a selectable marker under the control of suitable expression control elements (e.g., promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc.). After introduction of the exogenous DNA, the engineered cells can be grown in enrichment medium for 1 to 2 days and then switched to selection medium. The selectable marker in the recombinant plasmid confers resistance to the selection, allowing the cell to stably integrate the plasmid into its chromosome and grow to form foci (foci), which can then be cloned and expanded into a cell line. This method can be advantageously used to engineer cell lines expressing antibody molecules. These engineered cell lines can be used in particular for screening and evaluating compounds that interact directly or indirectly with the antibody molecule.

A variety of selection systems may be used, including, but not limited to, the herpes simplex virus thymidine kinase (Wigieretal, Cell11:223(1977)), hypoxanthine guanine phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA48:202(1992)), and adenine phosphoribosyltransferase (Lowyetal, Cell22:817(1980)) genes that may be employed within tk, hgprt, or aprt-cells, respectively. The following genes can also be selected based on antimetabolite resistance: dhfr (Wigleretel, Proc.Natl.Acad.Sci.USA77: 357(1980); O' Hareeal, Proc.Natl.Acad.Sci.USA78:1527(1981)) which produces resistance to methotrexate; gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, proc. Natl.Acad. Sci. USA78:2072 (1981)); neo which confers resistance to the aminoglycoside G-418 (WuandWu, Biotherapy3:87-95 (1991)); and hygro (Santerreeet, Gene30:147(1984)) which develops resistance to hygromycin. The selection of the desired recombinant clone can be routinely accomplished by methods generally known in the art of recombinant DNA technology, for example, as described in Ausubeletal. (eds), Current protocols molecular biology, John Wiley & Sons, NY (1993), Kriegler, Gene transfer expression, Alaborator Manual, Stockton Press, NY (1990), and intacter 12and13, Dracomatic et. (eds), Current protocols human genetics, John Wiley & Sons, NY (1994), Colberre-Garaper., J.mol.biol.150:1(1981), which are incorporated herein by reference in their entirety.

The expression level of antibody molecules can be increased by vector amplification (for a review see Bebbingtonnandschel, "the use of vector based amplification for the expression of expression genes mammalians" (DNAcloning, Vol.3.academic Press, New York, 1987)). When the marker in the vector system expressing the antibody is an amplifiable marker, an increase in the level of inhibitor present in the host cell culture will increase the copy number of the marker gene. Since the amplified region is associated with an antibody gene, antibody production is also increased (Crouseet al, mol. CellBiol.3:257 (1983)).

Host cells may be co-transfected with two expression vectors of the invention, the first vector encoding a heavy chain-derived polypeptide and the second vector encoding a light chain-derived polypeptide. Both vectors may contain the same selectable marker that allows equal expression of the heavy and light chain polypeptides. Alternatively, a single vector encoding and capable of expressing both the heavy and light chain polypeptides may be used. In this case, the light chain should be placed in front of the heavy chain to avoid the overproduction of toxic free heavy chain (Proudfoot, Nature322:52(1986); Kohler, Proc. Natl. Acad. Sci. USA77:2197 (1980)). The coding sequences for the heavy and light chains may comprise cDNA or genomic DNA.

Once the antibody molecule of the invention has been produced using animal, chemical synthesis or recombinant expression, purification can be carried out using any method known in the art for purifying immunoglobulin molecules, such as by chromatography (e.g., ion exchange chromatography, affinity chromatography, particularly by affinity for a specific antigen following protein a, and size exclusion chromatography), centrifugation, differential solubility, or any other standard technique for purifying proteins. In addition, the antibodies or fragments thereof of the present invention can also be fused to heterologous polypeptide sequences described herein or known in the art to facilitate purification.

The invention includes antibodies recombinantly fused or chemically conjugated (including both covalent and non-covalent conjugation) to polypeptides. The fusion or conjugate antibodies of the invention can be used to make purification easier. See, e.g., Harbor et al (supra) and PCT references WO93/21232, EP439,095, Naramuraet, Immunol.Lett.39:91-99 (1994); U.S. patent nos. 5,474,981; gilliesetal, Proc. Natl. Acad. Sci.89:1428-1432 (1992); felletal, J.Immunol.146:2446-2452(1991), which is incorporated herein by reference in its entirety.

In addition, the antibodies or fragments thereof of the invention may be fused to a marker sequence, such as a peptide, to facilitate purification. In a preferred embodiment, the marker amino acid sequence is a hexa-histidine peptide, such as the tags provided in the pQE vector (QIAGEN, inc.,9259EtonAvenue, Chatsworth, calif.,91311) and the like, many of which are commercially available. Hexahistidine provides for convenient purification of the fusion protein, as described, for example, in Gentzetal, Proc.Natl.Acad.Sci.USA86:821-824 (1989). Other peptide tags useful for purification include, but are not limited to, the "HA" tag, which corresponds to an epitope derived from the hemagglutinin protein of influenza virus (Wilsonet al, Cell37:767(1984)), and the "flag" tag.

Diagnostic uses of anti-IL 13 antibodies

Antibodies of the invention include modified derivatives, i.e., modifications made by covalent attachment of any type of molecule to the antibody that do not interfere with the binding of the antibody to IL 13. For example, antibody derivatives include those that have been modified by, for example, biotin, HRP, or any other detectable moiety, but this is not a limitation.

The antibodies of the invention may be used, for example, but not limited to, detecting IL13 levels in cancer patients, including in vitro and in vivo diagnostic methods. For example, the antibodies may be used in immunoassays for the qualitative or quantitative determination of IL13 levels in biological samples. See, e.g., Harloweal, Antibodies, Arabidopsis Manual, (ColdSpringHarbor laboratory Press,2 nd.1988) (incorporated herein by reference in its entirety).

As discussed in more detail below, the antibodies of the present invention can be used alone or in combination with other compositions. The antibody may also be recombinantly fused at the N-or C-terminus to a heterologous polypeptide, or chemically conjugated (including covalent or non-covalent conjugation) to a polypeptide or other composition. For example, the antibodies of the invention can be recombinantly fused or conjugated to molecules that serve as labels in detection assays.

The invention also includes antibodies or fragments thereof conjugated to diagnostic agents that can be used diagnostically, e.g., to monitor the occurrence or progression of cancer, e.g., to determine the efficacy of a given treatment regimen, as part of a clinical testing procedure.detection can be facilitated by coupling the antibodies to detectable substances, examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals using various positron emission tomography scans, and nonradioactive paramagnetic metal ions.using techniques known in the art, detectable substances can be coupled or conjugated directly to the antibodies (or fragments thereof) or indirectly via an intermediate (e.g., a linker known in the art), see, e.g., U.S. Pat. No.4,741,900 for metal ions that can be conjugated to antibodies, used as diagnostic agents of the invention, examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β -galactosidase, or acetylcholinesterase, examples of suitable prosthetic group complexes include streptavidin and avidin/biotin, examples of suitable luminescent materials including fluorescein¹²⁵I、¹³¹I、¹¹¹In or⁹⁹Tc。

The antibodies may also be attached to a solid support, which is particularly useful for immunoassay or purification of the target antigen. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene.

Labeled antibodies, and derivatives and analogs thereof, that specifically bind to IL13 can be used for diagnostic purposes to detect, diagnose, or monitor diseases, disorders, and/or conditions associated with aberrant expression and/or activity of IL 13. The invention provides a test for the abnormal expression of IL13 comprising (a) testing the expression of IL13 in cells or body fluid of an individual with one or more antibodies of the invention specific for IL13, and (b) comparing the level of gene expression to a standard level of gene expression, whereby a detected increase or decrease in the level of IL13 expression compared to the standard level of expression is indicative of abnormal expression.

The present invention provides a diagnostic test for diagnosing a disorder comprising (a) detecting the expression of IL13 in a cell or body fluid of an individual with one or more antibodies of the invention specific for IL 13; and (b) comparing the level of gene expression with a standard level of gene expression, wherein a detected increase or decrease in the level of gene expression compared with the standard level of expression is indicative of a particular disease.

The antibodies of the invention can be used to detect protein levels in biological samples using classical immunohistological methods known to those skilled in the art (see, e.g., Jalkane, et., J.cell. biol.101: 976-305 (1985); Jalkane, et., J.cell. biol.105:3087-3096 (1987)). Other antibody-based methods for detecting protein gene expression include immunoassays such as enzyme-linked immunosorbent assays (ELISAs) and Radioimmunoassays (RIA). Suitable antibody assay labels are known in the art and include enzyme labels such as glucose oxidase; radioisotopes such as iodine (A), (B), (C), (D), (C), (D), (C¹²⁵I、¹²¹I) Carbon (C)¹⁴C) Sulfur (S), (S)³⁵S), tritium (³H) Indium (I) and (II)¹¹²In), and technetium (⁹⁹Tc); luminescent labels such as luminol; fluorescent labels such as fluorescein and rhodamine; and biotin.

One aspect of the present invention is the detection and diagnosis of diseases or conditions associated with aberrant expression of IL13 in an animal, preferably a mammal, and most preferably a human. In one embodiment, the diagnosing comprises: a) administering (e.g., parenterally, subcutaneously, or intraperitoneally) to a subject an effective amount of a labeled molecule that specifically binds to IL 13; b) waiting a time interval after administration to allow the labeled molecules to preferentially accumulate in the subject at the site of the expressed polypeptide (and allow unbound labeled molecules to be cleared to background levels); c) determining a background level; and d) detecting the labeled molecule in the subject, such that detection of the labeled molecule above a background level indicates that the subject has the particular disease or disorder associated with aberrant expression of IL 13. Background levels can be determined in a variety of ways, including comparing the amount of labeled molecules detected to predetermined standard values for a particular system.

One skilled in the art will appreciate that the size of the subject and the imaging system used will determine the amount of imaging components needed to produce a diagnostic image. With regard to radioisotope compositions, the amount of radioactivity injected typically ranges from about 5 to 20 millicuries for human subjects⁹⁹Tc. The labeled antibody or antibody fragment will then preferentially accumulate at the site of cells containing the particular protein. In s.w. burchiel, "immunopharmaceutical kinetics of radiolabebele dantibody and therapeutics," Chapter13in turmor imaging: in vivo imaging is described in the radiochemical detectenofc cancer, s.w.burchielan db.a.rhodes, eds., masson publishing inc (1982).

Depending on a number of variables including the type of label used and the mode of administration, the time interval after administration for allowing labeled molecules to preferentially accumulate at locations within the subject and for unbound labeled molecules to be cleared to background levels is 6 to 48 hours or 6 to 24 hours or 6 to 12 hours. In another embodiment, the time interval after administration is 5 to 20 days or 5 to 10 days.

In one embodiment, monitoring of the disease or condition is performed by repeating the method for diagnosing the disease or condition, e.g., 1 month after initial diagnosis, 6 months after initial diagnosis, 1 year after initial diagnosis, etc.

The presence of the labeled molecule in the patient may be detected using methods known in the art for in vivo scanning. These methods depend on the type of label used. One skilled in the art will be able to determine suitable methods for detecting a particular marker. Methods and apparatus that may be used in the diagnostic methods of the present invention include, but are not limited to, Computed Tomography (CT), whole body scans such as Positron Emission Tomography (PET), Magnetic Resonance Imaging (MRI), and ultrasound.

In a particular embodiment, the molecules are labeled with a radioisotope and the labeled molecules are detected in the patient using a radio-responsive surgical device (U.S. Pat. No.5,441,050 to Thurston et al). In another embodiment, the molecules are labeled with a fluorescent compound and the labeled molecules are detected in the patient's body with a fluorescent response scanning device. In another embodiment, the molecule is labeled with a positron emitting metal and the labeled molecule is detected in the patient using positron emission tomography. In another embodiment, the molecule is labeled with a paramagnetic label and the labeled molecule is detected in the patient using Magnetic Resonance Imaging (MRI).

In another aspect, the invention provides a method for diagnosing a patient's susceptibility to developing a disease caused by unregulated cytokine expression. An increase in the amount of IL13 in cells, tissues or body fluids of certain patients may indicate that the patient is susceptible to certain diseases. In one embodiment, the method comprises collecting a cell, tissue, or bodily fluid sample from a subject known to have a low or normal level of IL 13; analyzing the tissue for the presence or absence of IL13 in the tissue or body fluid; and predicting the susceptibility of the patient to certain immune diseases based on the expression level of IL13 in the tissue or body fluid. In another embodiment, the method comprises collecting a sample of cells, tissues, or bodily fluids from the patient known to contain a determined level of IL 13; analyzing the amount of IL13 in a tissue or body fluid; and predicting the susceptibility of the patient to certain diseases based on changes in the amount of IL13 when compared to established, defined or measured levels in normal cells, tissues or body fluids. The determined level of IL13 may be a known amount based on literature values or may be determined in advance by measuring the amount in normal cells, tissues or body fluids. In particular, determination of IL13 levels in certain tissues or body fluids allows for specific and early detection of immune diseases in a patient, preferably before the disease occurs. Immune disorders that can be diagnosed using the present methods include, but are not limited to, the immune disorders described herein. In preferred embodiments, the tissue or body fluid is peripheral blood, peripheral blood leukocytes, biopsy tissue such as lung or skin biopsies, and tissue.

Therapeutic uses of anti-IL 13 antibodies

Antibodies administered alone or in combination with cytotoxic or cytostatic factors (with or without therapeutic components conjugated thereto) may be used as a therapeutic agent. The present invention relates to antibody-based therapies comprising administering an antibody of the invention to an animal, mammal, or human to treat a disease, disorder, or condition mediated by IL 13. Antibodies to IL13 are useful for inhibiting tumor or cancer cell proliferation in animals including, but not limited to, cows, pigs, horses, chickens, cats, dogs, non-human primates, etc., and humans. For example, cancer or tumors can be reduced or eliminated in the treated mammal by administering a therapeutically acceptable dose of an antibody of the invention, or a mixture of antibodies of the invention, or other antibodies from a variety of sources in combination.

Therapeutic compounds of the invention include, but are not limited to, antibodies of the invention (including fragments, analogs and derivatives thereof as described herein) and nucleic acids encoding antibodies of the invention (including fragments, analogs and derivatives thereof as described herein and anti-idiotypic antibodies) as described below. Diseases, disorders, or conditions associated with aberrant expression and/or activity of IL13, including but not limited to any one or more of the diseases, disorders, or conditions described herein, may be treated, inhibited or prevented with the antibodies of the present invention. Treatment and/or prevention of diseases, disorders, or conditions associated with aberrant expression and/or activity of IL13 includes, but is not limited to, alleviation of symptoms associated with such diseases, disorders, or conditions. The antibodies of the invention may be provided in pharmaceutically acceptable compositions as known in the art or described herein.

The anti-IL 13 antibodies of the invention may be used therapeutically in a variety of diseases. The present invention provides a method for preventing or treating IL13 mediated diseases in a mammal. The method comprises administering to the mammal a disease preventing or treating amount of an anti-IL 13 antibody. anti-IL 13 antibodies bind to IL13 and modulate the expression of cytokines and cellular receptors, producing cytokine levels that manifest in non-disease states.

The amount of antibody effective to treat, inhibit and prevent a disease or disorder associated with aberrant expression and/or activity of IL13 can be determined using standard clinical techniques. The antibody may be administered in a therapeutic regimen consistent with the disease, for example, in a single or multiple doses over a period of one to several days to alleviate the disease state, or in a periodic dose over an extended period of time to prevent allergy or asthma. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose employed in the formulation will also depend on the route of administration and the severity of the disease or condition, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems.

For antibodies, the dose administered to the patient is typically 0.1mg to 100mg per kg of patient body weight. Preferably, the dose administered to the patient is between 0.1mg and 20mg per kg body weight of the patient, more preferably between 1mg and 10mg per kg body weight of the patient. In general, human antibodies have a longer half-life in humans than antibodies of other species due to the immune response of humans to foreign polypeptides. Thus, lower doses of human antibodies and less frequent dosing are often possible. In addition, the dosage and frequency of administration of the antibodies of the invention can be reduced by modifying, for example, lipidation to enhance uptake and tissue permeability of the antibody (e.g., into the brain).

The antibodies of the invention may be advantageously employed in combination with, for example, other monoclonal or chimeric antibodies, or lymphokines or hematopoietic growth factors (e.g., IL-2, IL-3, IL-7, IFN, GCSF, GMCSF, Flt3, IL21) or unmethylated CpG-containing oligopeptides, which may increase the number or activity of effector cells interacting with the antibody.

The antibodies of the invention may be administered alone or in combination with other types of therapies such as chemotherapy and radiotherapy.

In a preferred aspect, the antibody is substantially purified (e.g., substantially free of materials that limit its effect or produce undesirable side effects).

anti-IL 13 antibodies can be administered to a mammal in any acceptable form. Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, inhalation, and oral routes. The antibody or composition may be administered by any convenient route, for example by infusion or bolus injection, or by absorption through epithelial or cutaneous mucosal linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and may be administered with other biologically active agents. Administration may be systemic or local. In addition, it may be desirable to introduce a therapeutic antibody or composition of the invention into the central nervous system by any suitable route, including intraventricular and intrathecal injection; intraventricular injection may be facilitated by an intraventricular catheter, for example a catheter connected to a reservoir such as an Ommaya balloon.

Pulmonary administration may also be employed, for example, by use of an inhaler or nebulizer, and formulation of the aerosol. Antibodies can also be administered to the lungs of a patient in the form of a dry powder composition (see, e.g., U.S. Pat. No.6,514,496).

In a particular embodiment, it is desirable to administer a therapeutic antibody or composition of the invention locally to the site where treatment is desired; this may be achieved, for example, by (but not limited to) local infusion, local application, by injection, by means of a catheter, by means of a suppository, or by means of an implant that is a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers. Preferably, when administering the antibodies of the invention, care must be taken to use materials to which the protein does not adsorb.

In another embodiment, the antibody may be delivered in a vector, particularly a liposome (see, Langer, Science249:1527-1533 (1990); Treatetal, Liposomessitine, therapeutics of Infectious Disaseand Anancer, Lopez-Beresteinand Fidler (eds.), Liss, NewYork, pp.353-365 (1989); LopezBerestein, ibid., pp.317-327; segeneralyibi.).

In another embodiment, the antibody may be delivered using a controlled release system. In one embodiment, a pump may be used (see, Langer, supra; Sefton, CRCCrit. Ref. biomed. Eng.14:201 (1987); Buchwaldet., Surgery88:507 (1980); Saudeketal., N.Engl. J. Med.321:574 (1989)). In another embodiment, polymeric materials may be used (see, medical application of controlled Release, LangerandWise (eds.), CRCPRes, bocaaton, Fla (1974); controlledDrugBioavailabiliy, drug product design development Performance, SmolenandBoll (eds.), Wiley, NewYork (1984); Rangerand Peppas, J., Macromol. Sci. Rev. Macromol. chem.23:61 (1983); also see, Levyetal, Science228:190 (1985); Duringer. Ann. Neurol.25:351 (1989); Howardal, J. Neurosurg.71:105 (1989)). In another embodiment, the controlled release system can be placed in the vicinity of the therapeutic target.

The invention also provides pharmaceutical compositions. These compositions comprise a therapeutically effective amount of the antibody, and a physiologically acceptable carrier. In a particular embodiment, the term "physiologically acceptable" means certified by an approval agency of the federal or a state government or listed in the U.S. pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or carrier with which a therapeutic agent is administered. These physiological carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions may also be employed as liquid carriers, particularly as injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, if desired. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations and the like. The compositions may be formulated as suppositories with conventional binders and carriers such as triglycerides. Oral formulations may include standard carriers such as pharmaceutical pure mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. Examples of suitable carriers are described in "Remington's pharmaceutical sciences" of e.w. martin. These compositions contain an effective amount of the antibody (preferably in purified form) and a suitable amount of a carrier so as to provide a form that can be suitably administered to a patient. The dosage form should be adapted to the mode of administration.

In one embodiment, the composition is formulated according to conventional methods into a pharmaceutical composition suitable for intravenous administration to a human. Compositions for intravenous administration are typically sterile isotonic aqueous buffer solutions. If desired, the composition may also contain a solubilizing agent and a local anesthetic such as lidocaine to reduce pain at the site of injection. Generally, the components may be provided separately or mixed together in a unit dosage form, for example, as a lyophilized powder or anhydrous concentrate in a hermetically sealed container such as an ampoule or sealed bag (sachette) indicating the amount of active agent. If the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile, pharmaceutical grade water or saline. If the composition is to be administered by injection, an ampoule of sterile water or saline for injection may be provided so that the components can be mixed prior to administration.

The invention also provides a pharmaceutical package or kit comprising one or more containers containing one or more components of the pharmaceutical composition of the invention. Optionally accompanying these containers may be instructions in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which instructions reflect certification by the agency regulating the manufacture, use or sale of pharmaceuticals for human administration.

In addition, the antibodies of the invention may be conjugated to various effector molecules such as heterologous polypeptides, drugs, radionuclides, or toxins. See, for example, PCT documents WO92/08495, WO91/14438, WO89/12624, U.S. Pat. No.5,314,995, and EP396,387. The antibody or fragment thereof may be conjugated to a therapeutic component, for example a cytotoxin such as a cytostatic or cytocidal agent, a therapeutic agent or a radioactive metal ion such as an alpha-particle emitting agent, for example 213 Bi. Cytotoxic or cytotoxic agents include any substance that is harmful to cells. Examples include paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emidine (emetine), mitomycin (mitomycin), etoposide (etoposide), etoposide (tenposide), vincristine (vincristine), vinblastine (vinblastine), colchicine (colchicin), doxobicin (doxobicin), daunorubicin (daunorubicin), dihydroyanthronide, mitoxantrone (mitoxantrone), mithramycin (mithramycin), dactinomycin D (dactinomycin), 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-azathioprine, 6-thioguanine, cytarabine, 5-fluorouracil aminoenamine), alkylating agents (e.g., mechlorethamine, chlorambucil (thioacetochlor), melphalan (melphalan), carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, cisplatin), anthracyclines (e.g., daunorubicin (formerly known as daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly dactinomycin), bleomycin, mithramycin and Anthracycline (AMC)), and antimitotics (e.g., vincristine and vinblastine).

Techniques for conjugating these therapeutic components to antibodies are well known, see, e.g., arnonetal, "monoclonal antibodies against immune targeting of drugs in cancer therapy," in monoclonal antibodies against cancer therapy, "reisfeldet al, pp.243-56 (ananr. liss inc.1985); hellstrometal, 'AntibodiesForDrugDelivery', incontrolledDrugDelivery (2)^ndEd.), Robinonenet (eds.), pp.623-53(MarcelDekker, Inc.1987); thorpe, "antibodyCarrierOfCytoxicAgentsInCancer therapy: AReview", InMonoclonaLinedics' 84: biologicals and clinical applications, Pincheraeal, (eds.), pp.475-506 (1985); "Analysis, Results, AndfutureProspecentOfTherapeuticUseOfRadiolabedAnthodifInCanceThe InmonlononalAnmontiodes CanceIdethelationAndrograph, Baldwith et al, pp.303-16(academic Press1985), and thorpe et al," the prepartionodcytotoxoprotectiOfficin antibodies-ToxinConjuugates ", Immunol.Rev.62:119-58 (1982). Alternatively, the antibody may be conjugated to a second antibody to form an antibody heteroconjugate. (see, e.g., U.S. Pat. No.4,676,980 to Segal).

The conjugates of the invention may be used to modify a given biological response and the therapeutic agent or pharmaceutical composition should not be construed as limited to classical chemotherapeutic agents. For example, the pharmaceutical component may be a protein or polypeptide having a desired biological activity. These proteins may include, for example, toxins such as abrin, ricin a, pseudomonas exotoxin, or diphtheria toxin; proteins such as tumor necrosis factor, interferon-alpha, interferon-beta, nerve growth factor, platelet derived growth factor, tissue fibrinolytic enzyme activator; apoptotic agents such as TNF α, TNF β, AIMI (see International publication No. WO97/33899), AIMII (see International publication No. WO97/34911), Fas ligand (Takahashietal, int. immunol.,6: 1567-S1574 (1994)), VEGI (see International publication No. WO99/23105); a thrombotic or anti-angiogenic agent such as angiostatin (angiostatin) or endostatin (endostatin); or biological response modifiers such as lymphokines, interleukin-1 ("IL-1"), interleukin-2 ("IL-2"), interleukin-6 ("IL-6"), granulocyte macrophage colony stimulating factor ("GM-CSF"), granulocyte colony stimulating factor ("G-CSF"), or other growth factors.

Examples

Example 1

Preparation of IL13 immunogen, a mutated, inactivated human IL13/Fc (MT-IL13/Fc)

Cloning and construction of expression plasmid for MT-IL13/Fc

Human IL13 with a mutation at amino acid residue 13 (glutamate mutation to lysine) was reported to bind IL13R α 1 with equal or higher affinity, but it had lost the ability to activate cells with IL13R α 1 (thompson et al, j.biol.chem.,274:29944 (1999)). The mutant, inactivated IL13 (designated MT-IL13) was expressed in human embryonic kidney cells 293-T. Purified recombinant protein was used as immunogen in the present invention to generate anti-IL 13 monoclonal antibody. 2 oligonucleotide primers corresponding to the sequence of MT-IL13 were synthesized

5'AAGCTTTCCCCAGGCCCTGTGCCTCCCTCTACAGCCCTCAGGAAGCTCAT3' (SEQ ID NO9) and

5'CTCGAGGTTGAACCGTCCCTCGCGAAAAAG3' (SEQ ID NO10) and was used as a template in a Polymerase Chain Reaction (PCR) to clone the IL13 gene from a human testis cDNA library (BDbiosciences Clontech, Palo alto, Calif.). The PCR fragment (342 base pairs) lacking the expected signal peptide sequence of IL13 was ligated into the pSecTag/FRT vector (Invitrogen, Carlsbad, CA) containing the secretory signal peptide sequence at its 5 'end and the human Fc γ 1 (hinge and constant regions CH 2and CH3) sequence at the 3' end. The composition of the construct was confirmed by sequencing.

B. Production of MT-IL13/Fc from transfected 293T cells

For transient expression of MT-IL13/Fc, purified plasmid DNA was transfected into 293T cells using Lipofectamine2000(Invitrogen) according to the manufacturer's instructions. Culture supernatants of transfected cells were collected for purification 72 hours after transfection. For stable expression of MT-IL13/Fc, a cell line was established with the Flp-In293T cell line (Invitrogen). To confirm expression, the culture supernatants were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The isolated protein was transferred onto nitrocellulose membrane and detected by reaction with a mouse anti-human IgG (fc) monoclonal antibody (Sigma, st. louis, MO) conjugated to horseradish peroxidase (HRP) or a polyclonal goat anti-IL 13 antibody (R & DSystems, Minneapolis, MN), which was then detected with HRP-donkey anti-goat IgG (jackson immunoresearch laboratories, WestGrove, PA). Immunoreactive proteins were identified on membranes using enhanced chemiluminescence detection (supersignal WestPicoChemilns Substrate, Pierce, Rockford, IL).

Purification of MT-IL13/Fc

MT-IL13/Fc was purified using a hyper-D protein A affinity column (Invitrogen) equilibrated in Phosphate Buffer (PBS). After addition of the cell culture supernatant to the column, the resin was washed with more than 20 column volumes of PBS. Then, the resin was washed with SCC buffer (0.05M sodium citrate, 0.5M sodium chloride, pH6.0) to remove unbound protein. The IL13 fusion protein (0.05M sodium citrate, 0.15M sodium chloride, pH3.0) was then eluted and dialyzed against PBS.

Fractions from the affinity column containing MT-IL13/Fc were analyzed by SDS-PAGE. Protein purity was analyzed by coomassie blue staining and protein identification was performed as described above using Western immunoblotting using goat anti-human igg (fc) antibody (Sigma) and goat anti-human IL13 antibody (R & DSystems).

Example 2

Production of anti-IL 13 monoclonal antibodies

Male A/J mice (Harlan, Indian npoli, IN) 8-12 weeks old were injected subcutaneously with 200. mu.l PBS (pH7.4) containing 20. mu. gMT-IL13/Fc and Freund's complete adjuvant (Difco laboratories, Detroit, MI). Mice were injected subcutaneously twice with 20 μ gMT-IL13/Fc in Freund's incomplete adjuvant at two week intervals. Mice were then re-injected intraperitoneally with PBS containing 20 μ g of the same immunogen, two weeks later and 3 days prior to sacrifice. Splenocytes isolated from mice immunized with one or more antigens are used for fusion. Similar immunization and fusion procedures were also used for human IL13(R & DSystems) expressed in E.coli as immunogen.

Combination of 26.4X10 from two immunized mice in a fusion resulting in anti-IL 13MAb228B/C-1 production⁶Spleen cells and 58.8X10⁶And (4) spleen cells. For each fusion, a single cell suspension was prepared from the spleen of the immunized mice and used for fusion with Sp2/0 myeloma cells. Sp2/0 and splenocytes were fused in a ratio of 1:1 in medium containing 50% polyethylene glycol (M.W.1450) (Kodak, Rochester, N.Y.) and 5% dimethyl sulfoxide (Sigma). The cells were then adjusted to a concentration of 1.5X10 per 250. mu.L suspension in DMEM medium (Invitrogen, CA) supplemented with 10% fetal bovine serum, 100 units/mL penicillin, 100. mu.g/mL streptomycin, 0.1mM hypoxanthine, 0.4. mu.M aminopterin, and 16. mu.M thymidine⁵And (4) spleen cells. 250 microliters of cell suspension is added to each well of about 50 96-well microplates. After about 10 days, culture supernatants were removed for screening for reactivity with MT-IL13/Fc in ELISA.

Wells of Immulon2(Dynatech laboratories, Chantilly, Va.) microtiter plates were coated overnight at room temperature by adding purified MT-IL13/Fc (0.1. mu.g/mL). After removing the coating solution by flicking the plate, 200. mu.L of blocking/dilution buffer (containing 2% bovine serum albumin and 0.05%)PBS) for 1 hour to block non-specificityA site. After 1 hour, PBST buffer (0.05% content)PBS) wash wells. 50 microliters of culture supernatant was collected from each fusion well and mixed with 50 μ L of blocking/dilution buffer before being added to a single well of the microassay plate. After 1 hour incubation, wells were washed with PBST. Bound murine antibodies were then detected by reaction with HRP conjugated goat anti-mouse IgG (Fc specific) (jacksonnorresearch lab, WestGrove, PA) and diluted 1:2,000 with blocking/dilution buffer. Peroxidase substrate solution containing 0.1%3,3,5,5 tetramethylbenzidine (Sigma, St. Louis, Mo.) and 0.003% hydrogen peroxide (Sigma) was added to the wells for development for 30 minutes. Add 50. mu.L of 2MH per well₂SO₄The reaction was terminated. OD of the reaction mixture was determined with a BioTek ELISA reader (BioTek instruments, Winooski, VM)₄₅₀The value is obtained.

Culture supernatants from MT-IL13/Fc screening positive wells were then assayed for negative binding to unrelated Fy 1 fusion proteins. The final positive wells of single cell clones were then selected by limiting dilution. Culture supernatants from the monoclonal antibodies were re-assayed by ELISA to confirm their activity. Selected hybridomas were grown in erlenmeyer flasks and the spent culture supernatant was collected for antibody purification by protein a affinity chromatography.

Purified antibodies were tested with four tests: i) cross-reactivity with MT-IL13/Fc expressed from 293T cells and mouse IL13 expressed from E.coli; ii) inhibits IL13 autocrine proliferation of HDLM-2 and L-1236 cells; iii) inhibition of IL 13-induced STAT6 phosphorylation in THP-1 cells; and iv) inhibiting the expression of CD14 and CD23 of IL 13-regulated human monocytes.

73 anti-IL 13 MAbs were obtained from fusions in mice immunized with MT-IL13/Fc and IL 13. 39 mabs were purified to characterize them by ELISA and cell-based assays. Of these 39 mabs, 13 inhibited autocrine IL 13-induced proliferation of HDLM-2 and L-1236 cells (see test description and results in example 5). 4 MAbs were found to react strongly with human IL13 in ELISA and they neutralized human IL13 in a functional cell-based assay. These monoclonal antibodies are designated 228B/C-1, 228A-4, 227-26, and 227-43. All these antibodies were generated using glycosylated MT-IL13/Fc as immunogen.

Example 3

Reactivity of anti-IL 13 monoclonal antibody with human and mouse IL13 measured by ELISA

The reactivity of various anti-IL 13 monoclonal antibodies was tested by ELISA. Different wells of a 96-well microtiter plate were coated with either non-glycosylated human IL13 expressed in E.coli (R & DSystems), glycosylated MT-IL13/Fc expressed in293T cells, or mouse IL13 expressed in E.coli (R & DSystems) by the addition of 100. mu.L of PBS solution containing 0.1. mu.g/mLIL 13 protein. After overnight incubation at room temperature, wells were treated with PBSTB (PBST containing 2% BSA) to saturate the remaining binding sites. The wells were then washed with PBST.

Mu.l of 2-fold serial dilutions of anti-IL 13mAb (from 0.5. mu.g/mL (3.33nM) to 0.05ng/mL (0.00033nM)) were added to the wells for 1 hour at room temperature. anti-IL 13mAbJES-5A2 from (BDbiosciences-Pharmingen, san Diego, Calif.) was also assayed and served as a positive control. This antibody was produced by using human IL13 expressed in e.coli as an immunogen. Isotype-matched mouse anti-HIV-1 gp120MAb was used as an unrelated negative control. The wells were then washed with PBST. Bound antibodies were detected by incubation with diluted HRP-goat anti-mouse igg (fc) (jackson immunoresearch) for 1 hour at room temperature. The peroxidase substrate solution was then added to develop the color as described above. Determination of OD with ELISA reader₄₅₀。

FIG. 1 shows the dose-dependent binding of anti-IL 13mAb228B/C-1, 228A-4, 227-26, 227-43 and negative controls as determined by ELISA. Of these mAbs, 228B/C-1 showed the strongest reactivity. FIG. 2 shows the dose-dependent binding of anti-IL 13mAb to MT-IL13/Fc as determined by ELISA. 228B/C-1 and 228A-4 showed the strongest reactivity with MT-IL13/Fc, while 227-26 and 227-43 showed moderate reactivity.

FIGS. 1 and 2 show that 228B/C-1 has the highest affinity for glycosylated and non-glycosylated human IL13 in all of the anti-IL 13 mAbs tested. All these anti-IL 13 mabs did not cross-react with mouse IL13 in the ELISA assay (data not shown).

Example 4

JES10-5A2 lacks competition for 228B/C-1-Hrp binding to human IL13

To investigate whether JES10-5A 2and 228B/C-1 bound to the same epitope on human IL13, the effect of JES10-5A2 on the binding of 228B/C-1-HRP to human IL13 expressed in E.coli was examined by competitive ELISA. Each well in a 96-well microtiter plate was incubated with 100. mu.L of PBS solution containing 0.1. mu.g/mLIL 13 protein. After overnight incubation at room temperature, wells were treated with PBSTB (PBS containing 2% BSA) to saturate the remaining binding sites. The wells were then washed with PBST. 50 microliters of 228B/C-1 and JES10-5A2 (final concentration from 20. mu.g/mL to 9.76ng/mL) serially diluted 2 fold were mixed with 50. mu.L of pre-titrated 228B/C-1-HRP (1:6,400 dilution). The mixture was then added to the wells and incubated at room temperature for 1 hour. The catalase substrate solution was then added as described above for color development. Determination of OD with ELISA reader₄₅₀。

FIG. 3 demonstrates that JES10-5A2 does not compete for binding of 228B/C-1-HRP to human IL13, indicating that 228B/C-1 and JES10-5A2 bind to different sites on human IL 13.

Example 5

Screening of neutralizing monoclonal antibodies against IL13 by IL-13 autocrine dependent proliferation assay using L-1236 and HDLM-2 cells

L-1236 and HDLM-2 are from Collectino of MicroorganismsandCellCulturs, Germany (DSMZ, Braunschweig, Germany). These cell lines produce IL13, whereas IL13 in turn activates their cell proliferation in an autocrine manner (kappa uetal, j.exp. med.189:1939 (1999)). Cells were incubated at 5% CO with or without different anti-IL 13 mAbs (0.2, 0.02 and 0.002. mu.g/mL)₂Cultured (25,000 cells/well) at 37 ℃ for 3 to 5 days. Then tested (at OD) by using the tetrazolium salt compound MTS (Promega, Madison, Wis.)₄₉₀Read) or by incorporation³H-Thymidine (Amersham biosciences, Piscataway, NJ) measures cell proliferation.

It is expected that addition of an anti-IL 13 neutralizing mAb to the cultures of these cell lines will inhibit cell proliferation by binding to and inactivating IL13 produced by these cells. The results shown in FIG. 4 show the effect of the anti-IL 13mAb of the invention on L-1235 cell proliferation. Of the neutralizing antibodies tested, mAb228B/C-1 showed the highest potency to inhibit L-1236 cell proliferation in a dose-dependent manner. TA1-37 (anti-IL 13mAb generated using E.coli-expressed human IL13 as an immunogen) did not have any inhibitory activity even at doses as high as 0.2. mu.g/mL. Similar results were obtained with HDLM-2 cells.

Example 6

Test for IL 13-regulated CD14 and CD23 expression on primary human monocytes

IL13 induces inhibition of CD14 expression and upregulation of CD23 expression on human monocytes (deWaal Mallefytetal, J.Immunol.,151:6370 (1993); Chomaratetetal., int.Rev.Immunol.,17:1 (1998)). Peripheral Blood Leukocytes (PBLs) were isolated from freshly collected heparinized whole blood of healthy human donors by density gradient centrifugation in Histopaque-1077 (Sigma). To each well of a 96-well tissue culture plate containing recombinant IL13 (final concentration 10ng/mL =0.813nM) and anti-IL 13 monoclonal or irrelevant antibody (three-fold serial dilution, starting from a final concentration of 12 μ g/mL =80nM), PBLs (1.5x 10) suspended in RPMI-1640(Invitrogen) supplemented with 5% calf serum were added⁶). By subjecting to incubationAddition of 0.813nM human IL13 to the medium inhibited or upregulated CD14 expression or CD23 expression, respectively, on monocytes. Control medium contained RPMI-1640/FBS medium but no recombinant IL 13.

Cells were incubated at 5% CO₂Incubated at 37 ℃ for 2 days. Cells were harvested and stained with anti-CD 14-FITC or anti-CD 23-PE (BDbiosciences-Pharmingen). The expression levels of CD14 and CD23 on monocyte populations were determined by flow cytometry and expressed as Median Fluorescence Intensity (MFI).

Figure 5 depicts the effect of anti-IL 13mAb on CD14 expression on human monocytes inhibited by IL 13. Of all the anti-IL 13 mAbs tested, 228B/C-1 had the highest potency at inhibiting the effect of IL13 on CD14 expression. At a concentration of 0.33nM, complete inhibition of the effect of IL13 was achieved. The inhibitory activity of mAbs 227-26 and 228A-4 was moderate, whereas JES10-5A2 had weak activity. JES10-5A2 failed to completely inhibit the effects of IL13 even at 80nM concentrations.

FIG. 6 depicts the effect of anti-IL 13MAb on IL 13-induced upregulation of CD23 expression on human monocytes. Similar to the results in CD14 expression (FIG. 5), 228B/C-1 most potently inhibited the effect of IL13 on CD23 expression in all anti-IL 13 MAbs tested. Complete inhibition was achieved at 228B/C-1 at a concentration of 0.33 nM. JES10-5A2 had weak inhibitory potency.

According to the results shown in FIGS. 5 and 6, complete inhibition of IL13 was achieved by 228B/C-1 at a molar stoichiometric ratio of 1:2(mAb: IL13), and thus 228B/C-1 was a neutralizing MAb against human IL13 with very high affinity.

Example 7

Test for IL 13-induced STAT6 phosphorylation in THP-1 cells

IL13 activates the myeloid cell line THP-1(ATCC, Manassas, Va.) to induce phosphorylation of STAT6, a key step in the signaling pathway of IL13 (MurataTetal, int. Immunol.10:1103-1110 (1998)). In this test, the inhibitory effect of anti-IL 13MAb on IL13 was tested.

THP-1 cells were maintained in Darber Modified Eagle Medium (DMEM) (Invitrogen) supplemented with 5% fetal bovine serum. On the day of the assay, cells were washed and placed in serum-free DMEM at 37 ℃ and 5% CO₂The mixture was incubated for 2 hours. Then 80. mu.L of a reagent containing 0.3X10 was added to each well of a 96-well round bottom plate⁶Serum-free medium of individual cells, and 120 μ L contained human IL13 (final concentration 10ng/mL =0.813nM) and anti-IL 13MAb (5-fold serial dilutions, starting at a final concentration of 0.5 μ g/mL =3.333 nM). Negative control wells contained no IL13 or IL13 and isotype matched unrelated mabs.

Mixing the mixture in 5% CO₂Incubated at 37 ℃ for 10 minutes. The plates were then centrifuged at 300Xg for 3 minutes at 4 ℃. After decanting the supernatant, the cell pellet was resuspended in 100 μ LLaemmli non-reducing sample buffer (SDS-PAGE loading buffer, BioRad, CA) and then transferred into a microcentrifuge tube. The tubes were heated at 95 ℃ for 5 minutes and then centrifuged at 10,000Xg for 10 minutes at room temperature. Supernatants were collected and analyzed by 4-20% gradient SDS-PAGE. The isolated protein was transferred to a PVDF membrane, which was then incubated with a diluted mouse anti-human Stat6(Y641, phosphate-specific) mab (bdbiosciences pharmingen).

Bound antibodies were detected with HRP conjugated goat anti-mouse igg (fc) antibody (jackson immunoresearch laboratories). Immunoreactive proteins on the membrane were identified using an enhanced chemiluminescence assay (supersignal WestPicoChemicals Substrate, Pierce). FIG. 7 depicts the results of the effect of anti-IL 13MAb on IL 13-induced Stat6 phosphorylation in THP-1 cells. Stat6 was phosphorylated in THP-1 cells treated with 0.813nM human IL 13. Dose-dependent inhibition of Stat6 phosphorylation was found when cells were treated with MAb228B/C-1, 228A-4, 227-26, 227-43, and JES10-5A 2. Of the anti-IL 13 mAbs tested, MAb228B/C-1 was the most potent neutralizing antibody. Complete inhibition was achieved with 228B/C-1 at concentrations between 0.667nM and 0.133 nM. The approximate molar stoichiometric ratio between 228B/C-1 and IL13 at full inhibition was 1: 2. This is consistent with the data shown in figures 5 and 6.

Example 8

Molecular cloning of heavy and light chain genes encoding anti-IL 13 monoclonal antibodies

Total RNA was isolated from hybridoma cells using QIAGEN kit (Valencia, CA). The reverse transcription (first strand cDNA) reaction was performed as follows: 1-1.5mg of total RNA was mixed with 1mL of 10mM dNTPs, 50ng of random hexamer, and RNase-free water to a final volume of 12 mL.

The reaction mixture was incubated at 65 ℃ for 5 minutes and immediately placed on ice for 1 minute. After a short centrifugation, the following reagents were added: 4mL of 5X first strand buffer (250mM Tris-HCl, pH8.3, 375mM KCl, 15mM MgCl)₂) 2mL0.1mMDTT, and1 mLRNaseOUTRRNase inhibitor (40U/mL). After mixing, the reaction was incubated at room temperature for 2 minutes. Then, 1mLSuperscriptIIRT (50U/mL) was added to the mixture, and incubated at 25 ℃ for 10 minutes, followed by incubation at 42 ℃ for 50 minutes. After a brief centrifugation, the reaction was incubated at 70 ℃ for 15 minutes to inactivate the reverse transcriptase. Then 1 μ LRNaseH (2U/mL) was added and the reaction was incubated at 37 ℃ for 20 minutes to destroy the RNA.

For the amplification of the variable regions of the heavy and light chains, the methods described by O 'Brien and Jones (O' Brien S.andJones T., "humanizing antibodies by CDRdrafting", antibody engineering, Springer Labmanual, eds. Kontermann and Duble, S (2001)) were used. Briefly, 5 'primers (11 sets for the light chain and 12 sets for the heavy chain degenerate primers) were selected from the signal peptide region and 3' primers were selected from within the constant region of either the light or heavy chain. The 5 'and 3' primers (1.5mL10mM) were mixed with 5mL10XPCR buffer (250mM Tris-HCI, pH8.8, 20mM MgSO)₄，100mMKCl，100mM(NH4)₂SO₄1% Triton X-100, 1mg/mL nuclease-free BSA), 1mL cDNA previously prepared, 1mL LTurbipfu (Stratagene), and adjusted to a final volume of 50mL with water. PCR was performed as follows: 1 cycle at 94 ℃ for 4 minutes; for 25 cycles30 seconds at 94 ℃,30 seconds at 53 ℃, and 45 seconds at 72 ℃; 1 cycle at 72 ℃ for 7 minutes. The reaction mixture was analyzed by electrophoresis on a 1% agarose gel.

The amplified DNA fragment was purified and cloned into pcDNA3.1 vector. Cloning was performed using the invitrogentotopo cloning kit according to the manufacturer's recommended protocol. 15 to 20 clones of transformed E.coli were used for plasmid purification. The plasmid was sequenced with the T7 primer. The dominant sequences of the heavy and light chains (dominant sequences) were cloned into the M13Fab expression vector by hybrid mutagenesis techniques (glaser s. et al. antibody engineering (oxford university press, new york (1995)), NearRI, BioTechniques12:88 (1992)). The binding properties of the expressed Fab were confirmed by ELISA. FIG. 8 depicts the amino acid sequences of the VH and VL chains of 228B/C.

Example 9

Epitope mapping

anti-IL-13 MAb228B/C-1 binds to one conformational epitope and binds to cynomologous monkey IL13 with the same high affinity as human IL 13. However, 228B/C did not bind to murine IL 13. Thus, the strategy designed for epitope mapping was to interchange a small portion of monkey IL13 with the corresponding mouse IL13 sequence. Overlapping oligonucleotides were synthesized. Two rounds of PCR were performed to assemble an IL13 hybridization construct, so that a portion of the sequence of monkey IL13 was replaced with the corresponding sequence from mouse IL 13. The final PCR amplified IL13 coding region was cloned in-frame into the pcDNA3.1 vector with the V5 tag using the TOPO cloning kit (Invitrogen). All PCR amplified regions were confirmed by sequencing to contain only the desired domain exchange mutation (domainswappangmutation) and no other undesired mutations in the expression vector.

The anti-IL 13MAb binding epitope was identified as the 8-peptide ESLINVSG (seq id no18) from amino acids #49 to 56. This epitope is located in the B-helix and BC loop of human IL 13. When epitope peptides derived from cyno-IL13 were used to exchange the corresponding sequences in murine IL13, the hybrid IL13 molecule formed was able to bind 228B/C with a binding affinity similar to that of the original cynoIL13, further confirming that 228B/CMAb binds to cyno or human IL13 on the peptide between residues # 49-56. Sequence comparisons of human, cyno, and murine IL13 show that only three residues Ile52, Val54, Gly56 in human IL13 are not conserved, suggesting that the combination of one or more of these three residues determines the key residues for IL13 and anti-IL 13MAb interaction through this 8 peptide.

The epitope was further confirmed by peptide dot analysis (peptidespetalysis). The entire human IL13 peptide was scanned with a series of overlapping 12 peptides synthesized on cellulose membrane via SPOT. The only anti-IL 13 MAb-reactive peptide was identified as the 12 peptide of amino acids #44-56, YCAALESLINVS (seq id no19), which overlaps with the region identified by the domain swapping assay.

Example 10: ADCC assay for anti-IL-13 MAb

PBMC were isolated from fresh heparinized blood samples (50ml of light yellow surface layer provided-300 × 10) using Ficoll-paque by standard centrifugation techniques⁶PBMC). PBMC were treated with IL-2(10U/ml) in RPMI1640/10% FCS at 37 deg.C, 5% CO₂Under the conditions of (1) (20 × 10) for 24 hours (⁶PBMC)。

After 24 hours, the target cells (2 × 10) of HDLM-2 or L-1236 (Hodgkin lymphoma cell line)⁶Individual cells) passed through 400uCi⁵¹Cr (sodium chromate) was labeled by incubation at 37 ℃ overnight. Will be provided with⁵¹Cr-labeled cells were washed 4 times and resuspended in RPM11640/5% FCS. The labeled cells were then aliquoted into 96-well U-bottom plates (in duplicate wells). IL-2 stimulated PBMC are then added at different E: T ratios (e.g., 80:1, 20: 1).

The anti-IL 13MAb tested was serially diluted and added in equal portions to the wells so that the final MAb concentration was between e.g. 0, 0.5, 5, 50. mu.g/ml. After incubation, the plates were centrifuged at 900rpm for 3 minutes. Supernatants were collected from each well and the amount of radioactivity was counted. The percentage of cell lysis was calculated according to the following equation:

% cell lysis = (Cpm)_test-Cpm_spont)/(Cpm_max-Cpm_spont)×100%

Additional ADCC information see, e.g., l.m. weinerett, cancer res, 48: 2568-; herseyetal, cancer Res.,46: 6083-; hansiketal, Proc.Natl.Acad.Sci.,83:7893-97 (1986).

Example 11: analysis of complement-mediated cytotoxicity (CMC)

Tumor cell (5 × 10)⁴In 50. mu.L DMEM medium), normal human serum (1: 1 dilution in 50. mu.L medium) and various concentrations of humanized anti-IL 13MAb (IgG 1) (in 100. mu.L medium) can be plated in 96-well flat-bottom plates at 37 ℃ with 5% CO₂Incubation for 2 hours at low temperature an irrelevant isotype matched antibody can be used as a negative control.cell proliferation reagent WST-1 (15. mu.L, Roche diagnostics, Basel, Switzerland) was added and incubated at 37 ℃ for 5 hours the optical density of the chromogenic reaction was read at 450nm using an ELISA plate reader, the percentage of inhibition of CMC by anti-IL 13MAb was 100 × (OD 13_s/a－OD_s)/(OD_ns-OD_s)，OD_s/aIs the OD, OD of the wells treated with serum and antibody_sIs the OD of the wells treated with serum_nsIs the OD of wells treated without serum and antibody.

Preservation of

The following cultures have been deposited at the American type culture Collection (American type culture Collection,10801university boulevard, ManassasVa.20110-2209USA (ATCC)):

hybridoma cell	ATCC accession number	Date of storage
			anti-IL 13228B/C-1	PTA-5657	11/20/2003
anti-IL 13228A-4	PTA-5656	11/20/2003
			anti-IL 13227-26	PTA-5654	11/20/2003
anti-IL 13227-43	PTA-5655	11/20/2003

This deposit is made in accordance with the relevant provisions of the International recognition of the Budapest treaty on the preservation of microorganisms for patent procedures and the rules thereof (Budapest treaty). This ensures that the viable culture is maintained for 30 years from the date of preservation. The organism is available through the ATCC under the Budapest protocol, which ensures that the public can permanently, without restriction, obtain progeny of the culture after grant of the relevant U.S. patent.

The assignee of the present application has agreed that if a preserved culture cultured under appropriate conditions dies or is lost or damaged, it will be promptly replaced with a live sample of the same culture upon notification. The deposited strains are not obtained as a license to practice the invention in contravention of the rights granted under the authority of any government authority in accordance with its patent laws.

The description written above is believed to be sufficient to enable one skilled in the art to practice the invention. The invention is not to be limited in scope by the culture deposited since the deposited embodiments are intended as illustrations of one aspect of the invention and any culture that is functionally equivalent is within the scope of the invention. The deposit of material herein does not constitute an admission that the written description contained herein is not sufficient to enable the practice of any aspect of the invention, including the best mode thereof, nor is it intended to be used to limit the scope of the claims to the particular exemplifications presented herein. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and are intended to be within the scope of the appended claims.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments described herein. The following claims include such equivalents.

Claims (31)

1. A pharmaceutical composition for treating cancer comprising a therapeutically effective amount of an antagonistic anti-human IL-13 antibody or antigen binding fragment thereof that specifically binds human IL-13, wherein said antagonistic antibody or antigen binding fragment comprises a variable light chain region comprising a heavy chain variable region consisting of the amino acid sequence of seq id no:99, consisting of the amino acid sequence of seq id no:104 and a CDR2 consisting of the amino acid sequence of seq id no:115 CDR 3; the variable heavy chain region comprises a heavy chain variable region consisting of the amino acid sequence of seq id no:117 and a CDR1 consisting of the amino acid sequence of seq id no:123 and a CDR2 consisting of the amino acid sequence of seq id no:135, and CDR 3.

2. A pharmaceutical composition for treating cancer, comprising a therapeutically effective amount of an antagonistic anti-human IL-13 antibody or antigen-binding fragment thereof that specifically binds human IL-13, wherein said antagonistic antibody or antigen-binding fragment comprises the 6 Complementarity Determining Regions (CDRs) of the antibody produced by hybridoma 228B/C-1 with ATCC accession No. PTA-5657, and a physiologically acceptable carrier.

3. The pharmaceutical composition of claim 1, wherein the antagonistic antibody or antigen binding fragment comprises a humanized sequence of an antibody produced by hybridoma 228B/C-1 with ATCC accession number PTA-5657 or an antigen binding region from the light chain variable region and the heavy chain variable region of an antibody produced by hybridoma 228B/C-1 with ATCC accession number PTA-5657.

4. The pharmaceutical composition of claim 2, wherein the antagonistic antibody or antigen binding fragment comprises a humanized sequence of an antibody produced by hybridoma 228B/C-1 with ATCC accession number PTA-5657 or an antigen binding region from the variable light chain region and the variable heavy chain region of an antibody produced by hybridoma 228B/C-1 with ATCC accession number PTA-5657.

5. The pharmaceutical composition of claim 1, wherein the antagonistic antibody or antigen binding fragment comprises a variable light chain region comprising the amino acid sequence of seq id no: 142. 144 or 150, and the antagonistic antibody or antigen binding fragment comprises a variable heavy chain region comprising the amino acid sequence of seq id no: 143. 145, 146, 147, 148 or 149.

6. The pharmaceutical composition of claim 2, wherein the antagonistic antibody or antigen binding fragment comprises a variable light chain region comprising the amino acid sequence of seq id no: 142. 144 or 150, and the antagonistic antibody or antigen binding fragment comprises a variable heavy chain region comprising the amino acid sequence of seq id no: 143. 145, 146, 147, 148 or 149.

7. The pharmaceutical composition of claim 1, wherein the antagonistic antibody or antigen binding fragment comprises a variable light chain region comprising the amino acid sequence of seq id no:3, and the variable heavy chain region comprises the sequence shown in SEQ ID NO: 4.

8. The pharmaceutical composition of claim 2, wherein the antagonistic antibody or antigen binding fragment comprises a variable light chain region comprising the amino acid sequence of seq id no:3, and the variable heavy chain region comprises the sequence shown in SEQ ID NO: 4.

9. The pharmaceutical composition of claim 1, wherein the antagonistic antibody or antigen binding fragment comprises a variable light chain region and a variable heavy chain region, said variable light chain region comprising the amino acid sequence of seq id no: 142; the variable heavy chain region comprises the amino acid sequence of seq id no: 143.

10. the pharmaceutical composition of claim 2, wherein the antagonistic antibody or antigen binding fragment comprises a variable light chain region and a variable heavy chain region, said variable light chain region comprising the amino acid sequence of seq id no: 142; the variable heavy chain region comprises the amino acid sequence of seq id no: 143.

11. the pharmaceutical composition of any one of claims 1-10, wherein the antagonistic antibody or antigen binding fragment is a monoclonal antibody.

12. The pharmaceutical composition of any one of claims 1-10, wherein the antagonistic antibody or antigen binding fragment is an IgG antibody.

13. The pharmaceutical composition of any one of claims 1-10, wherein the antagonistic antibody or antigen binding fragment is IgG1, IgG2, IgG3, or IgG 4.

14. The pharmaceutical composition of any one of claims 1-10, wherein the antagonistic antibody or antigen binding fragment is a human, chimeric, or humanized antibody or fragment thereof.

15. The pharmaceutical composition of any one of claims 1-10, wherein the antagonistic antibody or antigen binding fragment is a single chain antibody, a monovalent antibody, or a multispecific antibody.

16. The pharmaceutical composition of any one of claims 1-10, wherein said antagonist antibody mediates tumor cell killing by antibody-dependent cell-mediated cytotoxicity and/or complement-mediated cytotoxicity and said antibody comprises the constant region of human IgG 1.

17. The pharmaceutical composition of claim 15, wherein the multispecific antibody is a bispecific antibody.

18. The pharmaceutical composition of any one of claims 1-10, wherein the physiologically acceptable carrier is a diluent, adjuvant, excipient, or vehicle.

19. The pharmaceutical composition of claim 14, wherein the physiologically acceptable carrier is a diluent, adjuvant, excipient, or vehicle.

20. The pharmaceutical composition of any one of claims 1-10, wherein the pharmaceutical composition is for administration by inhalation, bolus injection, or infusion.

21. The pharmaceutical composition of claim 14, wherein the pharmaceutical composition is for administration by inhalation, bolus injection, or infusion.

22. The pharmaceutical composition of any one of claims 1-10, wherein the cancer is hodgkin's lymphoma, skin cancer, gastric cancer, colon cancer, breast cancer, pancreatic cancer, liver cancer, prostate cancer, lung cancer, head and neck cancer, renal cell carcinoma, squamous cell carcinoma, AIDS-related Kaposi's carcinoma, or brain cancer.

23. The pharmaceutical composition of claim 14, wherein the cancer is hodgkin's lymphoma, skin cancer, gastric cancer, colon cancer, breast cancer, pancreatic cancer, liver cancer, prostate cancer, lung cancer, head and neck cancer, renal cell carcinoma, squamous cell carcinoma, AIDS-related Kaposi's cancer, or brain cancer.

24. The pharmaceutical composition of any one of claims 1-10, wherein the antibody or antigen-binding fragment is conjugated to a cytotoxic agent.

25. The pharmaceutical composition of claim 24, wherein the cytotoxic agent is a toxin, a radioisotope, or a cytostatic agent.

26. A composition for diagnosing a cancer or tumor in a biological sample, wherein the cancer or tumor overexpresses IL-13, the composition comprising an antagonistic anti-human IL-13 antibody that specifically binds human IL-13, or an antigen-binding fragment thereof, wherein the antagonistic antibody or antigen-binding fragment comprises a variable light chain region comprising a heavy chain variable region consisting of the amino acid sequence of seq id no:99, consisting of the amino acid sequence of seq id no:104 and a CDR2 consisting of the amino acid sequence of seq id no:115 CDR 3; the variable heavy chain region comprises a heavy chain variable region consisting of the amino acid sequence of seq id no:117 and a CDR1 consisting of the amino acid sequence of seq id no:123 and a CDR2 consisting of the amino acid sequence of seq id no:135, and CDR 3.

27. The composition of claim 26, wherein the antagonistic antibody or antigen binding fragment comprises a humanized sequence of an antibody produced by hybridoma 228B/C-1 with ATCC accession number PTA-5657 or an antigen binding region from the light chain variable region and the heavy chain variable region of an antibody produced by hybridoma 228B/C-1 with ATCC accession number PTA-5657.

28. The composition of claim 26, wherein said antagonistic antibody or antigen binding fragment comprises a heavy chain variable region consisting of the amino acid sequence of seq id no: 142. 144 or 150, and the antibody comprises a VL chain consisting of the amino acid sequence of seq id no: 143. 145, 146, 147, 148 or 149.

29. The composition of claim 26, wherein said antagonistic antibody or antigen binding fragment comprises a VL chain comprising the amino acid sequence of seq id no:3, and the VH chain comprises a sequence shown in SEQ ID NO: 4.

30. The pharmaceutical composition of claim 26, wherein the antagonistic antibody or antigen binding fragment comprises a variable light chain comprising the amino acid sequence of seq id no: 142; the variable heavy chain comprises seq id no: 143.

31. The pharmaceutical composition of claim 22, wherein the cancer is hodgkin's lymphoma.