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Patent Application Publication Nov. 27,2003 Sheet 4 of 5 US 2003/0219434 Al
Fig. 5A: Alignment of VL domains of human anti-DAF antibodies §■
LU30 1 QSVLTQPPSASGSPGQSVTISCTG[TSSDVGGYNYV]SWYQQHPGKAPKFMI g
LU13 1 QSVLTQPASVSGSPGQSITVSCTG[TSSDVGGYNYV]SWYQQHPGKAPKLMI
LU20 1 DIQMTQSPSTLSASIGDRVTITCRA[SEGIY HWL]AWYQQKPGKAPKLLI |
;LU30 51 Y[DVS]KRPSGVSNRFSGSKSGNTASLTISGVQAEDEADYYCSS[YTSASTV]I £
;LU13 51 Y[EGS]KRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSS[YTTRSTR]V g
LU20 4 9 Y[KAS]SLASGAPSRFSGSGSGTDFTLTISSLQPDDFATYYCQQ[YS-NYPL]T |
LU30 101 FGGGTKLTVL (SEQ ID N0:1)
LU13 101 FGGGTKLTVL (SEQ ID NO:2) ©
LU20 99 FGGGTKLEIK (SEQ ID NO: 3) 'k>
Fig. 5B:Alignment of VH domains of anti-DAF antibodies
LU30 1 QVKLQESGGGLVQPGGSLKLSCAAS[GFTFSGY]GMSWIRQTPDKRLEWVAT S
LU13 1 QVQLQESGGNLVQPGGSLRLSCAAS[GFTFSSY]AMSWVRQAPGKGLEWVSA £
LU20 1 EVQLVETGGGLVQPGRSLRLSCAAS[GFTFEDY]GMHWVRQAPGKGLEWVSG £
LU30 51 IN[SGGS]YTYYSDSVKGRFTISRDNVKNTLYLQMSSLKSEDTAMYYCARR[N
LU13 51 IS[GSGG]NTYYADSVKGRFTISRDNSKNTLYLQMNTLRAEDTAVYYCARR[A C
LU20 51 IN[WNGG]STGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCARD[A £
LU3 0 101 GTLY-YYLMD]YWGRGTLVTVSS (SEQ ID NO:4) g
LU13 101 SYD]YWGQGTMVTVSS (SEQ ID NO:5) S
LU20 101 PSGSYGYWFD]PWGQGTLVTVSS (SEQ ID NO:6) £
ANTIBODIES FOR CANCER THERAPY AND
 This application is a non-provisional application filed under 37 CFR 1.53(b)(1), claiming priority under 35 USC 119(e) to provisional application No. 60/122,262 filed 3/t/99, the contents of which are incorporated herein by reference.
BACKGROUND OF THE INVENTION  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 (Riethrmiller et al., Lancet, 343: 1177-1183 (1994); Riethrmiller et al., /. Clin. Oncol, 16: 1788-1794 (1998); Maloney et al, Blood, 90: 2188-2195 (1997); McLaughlin et al., /. Clin. Oncol, 16: 2825-2833 (1998); and Baselga et al, /. Clin. Oncol, 14: 697-699 (1996)) antibodies as well as with radiolabeled murine antibodies (Press et al., TV. 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.,/. Clin. Oncol, 14: 1974-1981 (1996)). Indeed a chimeric antiCD20 antibody (Reff et al, Blood, 83: 435-445 (1994)) and a chimeric/humanized anti-HER2 antibody (Carter et al. PNAS (USA) 89:42854289 (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)).
 Naive antibody phage libraries have proved to be a rapid and general method for identifying antibodies binding
to purified antigens (Griffiths et nl,EMBOJ., 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., 11: 1145-1149 (1992); Portolano et al, /. 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 alProc. 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 alProc. 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. /. 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., /. 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. /. 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. /. Path., 149:129-142 (1996)).
 Other publications relating to DAF include Hara et al. Immunology Letters 37:145-152 (1993); NicholsonWeller and Wang/. Lab. Clin. Med. 123(4):485491 (1994); Lublin et al. /. Immunol. 137:1629-1635 (1986); W099/ 43800; W098/39659; 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.
SUMMARY OF THE INVENTION
 In the present application, a large naive 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 naive 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 naive 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 10 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 BIACORETM 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 antigenbinding 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 antiVEGF antibody), an anti-mucin antibody, or a second antibody directed against a different epitope on DAE Such therapy with the combination of the antibody and the second cytotoxic agent may result in a synergistic therapeutic effect against lung cancer.
BRIEF DESCRIPTION OF THE DRAWINGS
 FIG. 1 depicts flow cytometric analysis of phage populations from rounds 1,2 and 3 binding to tumor cell line 1264 (dark) used for selection and non-tumor cell line BEAS-2B (light) used for counter-selection. Also shown is a negative control phage population.
 FIGS. 2A-C depict dendrograms for tumor-selective scFv satisfying primary and secondary selection criteria (Table 1). Comparisons were made between scFv amino acid sequences (FIG. 2A) as well as their component VH domains (FIG. 2B), and VL domains (FIG. 2C).
aTumor selective clones by phage ELISA: robust binding to 1264 cells
(^450—^650 g 0.3) and much weaker binding To BEAS-2B cells (SlO-fold lower signal), as judged by phage ELISA.
Clones LU13 and LU34 are predicted from their nucleotide sequences to generate identical fingerprint patterns, whereas clones LU3 and LU77 share closely related fingerprints that were not distinguishable by our electrophoretic analysis.
cCodon 3 in VH is amber (TAG) that will be read through as glutamine in the supE E. coli strain, TGI.
 FIG. 3 shows flow cytometric analysis of scFv fragments with tumor (1264, A549, CALU6 and SKLU1) and non-tumor (BEAS-2B and NHEK) cell lines.
 FIG. 4 shows binding of LU30 scFv (3 ^g/ml) to 1264 cells in the absence and presence of recombinant human DAF (30 /ug/mT).
 FIGS. 5A and 5B depict the amino acid sequences of the variable light (VL) (FIG. 5A; SEQ ID NOS: 1-3, respectively) and variable heavy (VH) (FIG. 5B; SEQ ID NOS:4-6, respectively) domains of human antibodies LU30, LU13 and LU20 identified in Example 1. Complementarity
Determining Region (CDR) residues are those residues in bold and hypervariable loop residues are within brackets
DETAILED DESCRIPTION OF THE
 I. Definitions
 The term "antibody" is used in the broadest sense and specifically covers intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g. bispecific antibodies) formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological activity.
 "Antibody fragments" comprise a portion of an intact antibody, preferably the antigen binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; "linear antibodies" (U.S. Pat. No. 5,641,870); singlechain antibody molecules such as single chain Fv fragments (scFv); and multispecific antibodies formed from antibody fragments.
 An "intact" antibody is one which comprises an antigen-binding variable region as well as a tight chain constant domain (CL) and heavy chain constant domains, CH1, CH2 and CH3. The constant domains may be native sequence constant domains (e.g. human native sequence constant domains) or amino acid sequence variants thereof. Preferably, the intact antibody has one or more effector functions.
 Antibody "effector functions" refer to those biological activities attributable to the Fc region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody. Examples of antibody effector functions include Clq binding; complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g. B cell receptor, BCR), etc.
 Depending on the amino acid sequence of the constant domain of their heavy chains, intact antibodies can be assigned to different "classes". There are five major classes of intact antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into "subclasses" (isotypes), e.g., IgGl (including human A and non-A allotypes), IgG2, IgG3, IgG4, IgA, and IgA2. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.
 "Antibody-dependent cell-mediated cytotoxicity" and "ADCC" refer to a cell-mediated reaction in which nonspecific cytotoxic cells that express Fc receptors (FcRs) (e.g. Natural Killer (NK) cells, neutrophils, and macrophages) recognize bound antibody on a target cell and subsequently cause lysis of the target cell. The primary cells for mediating ADCC, NK cells, express FcgammaRIII only, whereas monocytes express FcgammaRI, FcgammaRII and FcgammaRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To assess ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in U.S. Pat. No. 5,500,362 or 5,821, 337 may be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally,