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Antibodies for cancer therapy and diagnosis

Antibodies for cancer therapy and diagnosis description/claims

This application is a continuation application of co-pending U.S. application Ser. No. 10/447,331, filed on May 28, 2003, which is a divisional application of Ser. No. 09/515,825, filed on Feb. 29, 2000, incorporated herein by reference, and to which priority is claimed under 35 U.S.C. § 120, which co-pending application is a non-provisional application filed under 37 C.F.R. 1.53(b)(1) which claims priority under 35 U.S.C. 119(e) to provisional application No. 60/122,262 filed Mar. 1, 1999, the contents of which are incorporated herein by reference.

1. Field of the Invention

The present invention concerns a method for making antibodies which can, for example, be used for cancer diagnosis or therapy. The invention further provides a method for identifying an antigen which is differentially expressed on the surface of distinct cell populations. The present invention additionally provides human antibodies directed against decay accelerating factor (DAF), as well as therapeutic compositions comprising such antibodies. Moreover, the invention pertains to a method of treating lung cancer with antibodies directed against DAF.

2. Description of Related Art

The demonstration of significant anti-tumor efficacy of antibodies has long been sought-after in the clinic and recently obtained using “naked” chimeric/humanized antibodies (Riethmüller et al., Lancet, 343: 1177-1183 (1994); Riethmüller et al., J. Clin. Oncol., 16: 1788-1794 (1998); Maloney. et al., Blood, 90: 2188-2195 (1997); McLaughlin et al., J. Clin. Oncol., 16: 2825-2833 (1998); and Baselga et al., J. Clin. Oncol., 14: 697-699 (1996)) antibodies as well as with radiolabeled murine antibodies (Press et al., N. Engl. J. Med., 329: 1219-1224 (1993); Press et al., Lancet, 346: 336-340, (1995); Kaminski et al., N. Engl. J. Med., 329: 459-465 (1993); Kaminski et al., J. Clin. Oncol., 14: 1974-1981 (1996)). Indeed a chimeric anti-CD20 antibody (Reff et al., Blood, 83: 435-445 (1994)) and a chimeric/humanized anti-HER2 antibody (Carter et al. PNAS (USA) 89:4285-4289 (1992)) have recently been approved by US Federal Drug Administration for the treatment of non-Hodgkin's lymphoma and metastatic breast cancer, respectively. These successes with anti-tumor antibodies in patients has led to renewed interest in the identification of novel tumor-associated antigens suitable for antibody targeting.

The traditional approach to obtaining tumor-specific antibodies has been to immunize mice with tumor cells and to screen the resultant monoclonal antibodies for their binding specificity. Unfortunately tumor-binding antibodies obtained in this way often cross-react with many normal cells, which may interfere with their clinical utility. Ideally one would like to select rather than screen for antibodies that bind selectively to tumor. The advent of antibody fragment display on phage (McCafferty et al., Nature, 348: 552-554 (1990)) and the development of large (>1010 clone) phage display libraries (Griffiths et al., EMBO J., 13:3245-3260 (1994), Vaughan et al. Nat. Biotechnol. 14: 309-314 (1996)) offers a potential way of making antibodies. With antibody phage screening, unlike hybridoma technology, it is readily possible to obtain antibodies binding antigens that are highly conserved between mouse and man (Nissim et al., EMBO J., 13:692-698 (1994)).

Naïve antibody phage libraries have proved to be a rapid and general method for identifying antibodies binding to purified antigens (Griffiths et al., EMBO J., 13:3245-3260 (1994); Vaughan et al. Nat. Biotechnol. 14: 309-314 (1996); Nissim et al., EMBO J., 13:692-698 (1994)). In contrast, panning cellular targets with antibody phage has proved much more difficult because of the much lower effective antigen concentration, greater antigen complexity and the tendency of phage to bind non-specifically to cells. Nevertheless, antibodies against cell surface antigens have been identified (Marks et al., Bio/Technol., 11145-1149 (1992); Portolano et al., J. Immunol., 151:2839-2851 (1993); de Kruif et al, Proc. Natl. Acad. Sci. USA, 92:3938-3942 (1995); Van Ewijk et al., Proc. Natl. Acad. Sci. USA, 94:3903-3908 (1997); Cai et al, Proc. Natl. Acad. Sci. USA, 92:6537-6541 (1995); Cai et al, Proc. Natl. Acad. Sci. USA, 93:6280-6285 (1996); Cai et al., Proc. Natl. Acad. Sci. USA, 94:9261-9266 (1997)). Melanoma specific antibodies have been identified by selecting for antibody phage that bind to melanoma cells but not melanocytes using antibody phage libraries constructed from human donors immunized with their own tumor cells (Cai et al, Proc. Natl. Acad. Sci. USA, 92:6537-6541 (1995); Cai et al Proc. Natl. Acad. Sci. USA, 93:6280-6285 (1996); Cai et al., Proc. Natl. Acad. Sci. USA, 94:9261-9266 (1997)).

Decay Accelerating Factor (DAF), is a GPI-anchored protein that acts together with two other GPI-anchored proteins, CD46 and CD59, in protecting host cells from complement-mediated cell lysis (Nicholson-Weller et al. J. Lab. Clin. Med., 123:485-491 (1994)). DAF is expressed at widely varying levels on tumor cell lines and its overexpression correlates with enhanced resistance to complement-mediated cell lysis in vitro (Cheung et al., J. Clin. Invest., 81:1122-1128 (1988)). DAF overexpression has been observed on a variety of human tumor tissues including 6/9 lung adenocarcinomas and 2/7 lung squamous cell carcinomas (Niehans et al., Am. J. Path., 149:129-142 (1996)). Regarding normal lung tissue, DAF has been detected by immunohistochemistry on the alveolar epithelium, interstitium and endothelium as well as the bronchial epithelium, glands and ducts plus blood vessels (Niehans et al., Am. J. Path., 149:129-142 (1996)).

Other publications relating to DAF include Hara et al. Immunology Letters 37:145-152 (1993); Nicholson-Weller and Wang J. Lab. Clin. Med. 123(4):485-491 (1994); Lublin et al. J. Immunol. 137:1629-1635 (1986); WO99/43800; WO98/39659; U.S. Pat. No. 5,695,945; U.S. Pat. No. 5,763,224; and WO 86/07062.

Vollmers et al. Cancer Research 49: 2471-2476 (1989); and Vollmers et al. Cancer 76(4): 550-558 (1995) describe the human IgM monoclonal antibody “SC-1” which is said inhibit growth of stomach adenocarcinoma cells in vitro and in vivo by inducing apoptosis. Vollmers et al. Oncology Reports 5:549-552 (1998) reports the results of a clinical trial in which patients with poorly differentiated stomach adenocarcinoma were treated with the SC-1 antibody. The later publication, Hensel et al. Cancer Research 59:5299-5306 (1999), identifies DAF as the antigen bound by SC-1.

In the present application, a large naïve antibody phage library was used to search for cancer-associated antigens, thus obviating the need for creating custom libraries from immunized donors. In addition, antibodies were selected using live rather than fixed cells, to obtain antibodies primarily against native rather than denatured antigens. This was done to facilitate subsequent expression cloning of corresponding antigen as well as enhance the therapeutic potential of antibodies obtained. Indeed an antigen corresponding to a scFv fragment identified with significant tumor selectivity was cloned according to the present methods.

Accordingly, the invention provides a method for making an antibody comprising the following steps: (a) binding antibody phage from a naïve antibody phage library to a live cancer cell; (b) selecting an antibody phage or antibody which binds selectively to the live cancer cell; and (c) identifying an antigen to which the antibody phage or antibody binds.

The invention further provides an antibody derived according to the method of the preceding paragraph and optionally including amino acid sequence alterations (e.g. additions, deletions and/or substitutions) compared to the antibody selected in step (b)). Moreover, the invention provides a method for detecting the antigen comprising exposing a sample suspected of containing the antigen to the antibody or altered antibody and determining binding of the antibody or altered antibody to the sample. The invention further provides a method for treating a mammal having a disease or disorder comprising administering the above antibody or altered antibody to the mammal in an amount effective to treat the disease or disorder.

The invention further provides a method for identifying an antigen which is differentially expressed on the surface of two or more distinct cell populations, comprising the following steps: (a) binding antibody phage from a naïve antibody phage library to a first cell population; (b) binding the antibody phage to a second cell population which is distinct from the first cell population; (c) selecting an antibody phage or antibody which binds selectively to the first cell population; and (d) identifying an antigen to which the antibody phage or antibody in (c) binds.

The invention further provides an antagonist, such as an antibody, directed against an antigen, wherein the antigen has been identified according to the method of the previous paragraph.

The invention additionally relates to an isolated human antibody which is directed against, or specifically binds to, human decay accelerating factor (DAF), obtainable by the methods herein. The invention further provides a human antibody which has better binding affinity for DAF than the human IgM SC-1 antibody has for DAF, e.g. about 10 nM or better binding affinity for human DAF (for instance, in the range from about 1 nM to about 1 pM). An example of an antibody with such strong binding affinity for DAF is the LU30 antibody herein which has a binding affinity (Kd) for DAF of about 13 nM as determined using a BIACORE™ instrument. The antibody optionally binds an epitope on DAF bound by the LU30, LU13 or LU20 antibodies herein disclosed. The human antibody may, for instance, comprise antigen-binding amino acid residues of the LU30, LU13 or LU20 antibodies. The application additionally provides the human antibodies designated LU30, LU13 and LU20 herein as well as variants of any one of those antibodies. Preferred amino acid sequence variants comprise VH and VL domains which together share about 90-100%, and preferably about 95-100%, and most preferably 98-100%, amino acid sequence identity with the VH and VL amino acid sequences of the LU30, LU13 or LU20 antibodies as depicted in FIGS. 5A and 5B herein. One preferred amino acid sequence variant is an affinity matured variant, which comprises one or more amino acid sequence modifications (e.g. about 1-20, and most preferably about 3-10 amino acid substitutions) in one or more hypervariable regions of the LU30, LU13 or LU20 VH and/or VL amino acid sequences disclosed herein. Another type of variant is a glycosylation variant which has altered glycosylation compared to a parent antibody and thus may have altered effector function(s). While Fv fragment forms (e.g. single chain Fv fragments, scFv) of the LU30, LU13 or LU20 antibodies may be used, the variable regions of these antibodies are optionally fused to heterologous polypeptide(s) such as (1) a toxin polypeptide(s) to generate an immunotoxin or (2) antibody constant region sequences to make larger antibody molecules, such as Fab fragments, F(ab′)2 fragments or intact antibodies. Such intact antibodies generally have human heavy and light chain constant regions and, therefore, have antibody effector functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC).

In another embodiment, the invention pertains to a pharmaceutical composition comprising a human antibody directed against DAF and a pharmaceutically acceptable carrier. In addition, the invention provides an article of manufacture comprising the pharmaceutical composition and a package insert instructing the user of the composition to treat a patient having, or predisposed to, lung cancer with the composition. The lung cancer to be treated includes small-cell lung cancer, non-small cell lung cancer, large cell lung carcinoma, lung adenocarcinoma, and squamous cell lung carcinoma.

In yet a further embodiment, the invention relates to method of treating lung cancer comprising administering a therapeutically effective amount of an antibody directed against decay accelerating factor (DAF) to a human patient. Candidates for treatment with the anti-DAF antibody are optionally screened to determine DAF expression by tumor cells. For instance, DAF overexpression, and/or expression of a DAF glycoform, by the tumor may be assessed using diagnostic procedures available in the art, such as immunohistochemistry (IHC) or a DNA-based assay (e.g. fluorescent in situ hybridization, FISH). This way, a subpopulation of cancer patients (e.g. DAF-overexpressing patients or patients expressing a cancer-related variant of DAF) may be identified and those patients can be treated as described herein. The antibody may be administered in the neoadjuvant, adjuvant or metastatic settings. Moreover, the antibody used for such therapy may be conjugated with a cytotoxic agent (examples of which are provided below) in order to generate an immunotoxin. Preferably, the antibody is a human antibody (e.g. one which has a binding affinity for DAF of about 10 nM or better). The antibody for such therapy optionally binds an epitope on DAF bound by any one of the LU30, LU13, LU20, 791T36 or SC-1 antibodies. The antibody for therapy may, therefore, comprise antigen-binding amino acid residues of the LU30, LU13, LU20, 791T36 or SC-1 antibodies. The patient may optionally be treated with a second different cytotoxic agent, wherein the second cytotoxic agent is therapeutically effective against lung cancer. Examples of such second cytotoxic agents include, but are not limited to, navelbine, gemcitabine, a taxoid, carboplatin, cisplatin, etoposide, cyclosphosphamide, mitomycin, vinblastine, an anti-ErbB2 antibody (e.g. HERCEPTIN®, sold by Genentech, Inc., South San Francisco), an anti-angiogenic factor antibody (e.g. an anti-VEGF antibody), an anti-mucin antibody, or a second antibody directed against a different epitope on DAF. Such therapy with the combination of the antibody and the second cytotoxic agent may result in a synergistic therapeutic effect against lung cancer.

• Provisional Patent
• Utility Patent

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