MUCIN-1 SPECIFIC BINDING MEMBERS AND METHODS OF USE THEREOF

Abstract: MUC1-specific binding members for cancer-associated MUC1 protein comprise a MUC1 binding domain, or portion thereof, for binding to an epitope of the protein core of MUC1. The MUC1-specific binding members comprise various antibody molecules and fragments thereof, including Fab antibodies; scFv antibodies; double scFv antibodies; diabodies; recombinant, full-length immunoglobulins; and immunocytokine fusion proteins; that are used in methods of diagnosing and treating cancer in various tissues, including breast, ovary, bladder, and lung, and in methods of purifying or isolating MUC1 protein. Polynucleotide molecules encoding MUC1-specific binding members, or portions thereof, are also described.

Description: MUCIN-1 SPECIFIC BINDING MEMBERS AND METHODS OF USE THEREOF FIELD OF THE INVENTION This invention is generally in the field ofthe detection and treatment of cancer. In particular, the invention describes molecules that specifically bind to an epitope ofthe protein core of tumor-associated antigen mucin-1 (MUC-1), which is overexpressed and underglycosylated in human cancers of diverse origins, including breast, ovary, bladder, and lung tissues. BACKGROUND OF THE INVENTION An increasing amount of evidence indicating that cytotoxic T cells, which recognize tumor associated/specific antigens ("TAA"), can selectively kill tumor cells, makes active immunotherapy an attractive option for therapy of cancer (reviewed by Boon et al., Immunol. Today, 18: 267-8 (1997)). The tumor associated glycoprotein mucin-1 ("MUCl", "MUC-1"), also known as polymorphic epithelial mucin ("PEM"), is one ofthe most intensively studied targets because, in contrast with normal tissues, it is abundantly present in a non-polar fashion in adenocarcinoma (Burchell et al., Cancer Res. ,47: 5476-5482 (1987)). The protein core consists of a high and variable number of tandem repeats ("VNTR") of 20 amino acids (Gendler et al., J. Biol. Chem. Sep., 263: 12820-12823 (1988)). The tandem repeats are exposed as new peptide epitopes of MUCl in adenocarcinoma because of their reduced glycosylation compared to MUCl on normal tissues (Burchell et al., Cancer Res., 47: 5476-5482 (1987)). Murine monoclonal antibodies ("MAb") against MUCl have successfully been used to target adenocarcinoma, supporting the potency of MUCl as a tumor target (Granowska et al., Eur J Nucl Med., 20: 483-489 (1993), Perkins et al., Nucl. Med. Commun., 14: 578-586 (1993), Maraveyas et al., Cancer Res., 55: 1060-1069 (1995), Mariani et al., Cancer Res., 55: 5911s- 5915s (1995), Kramer et al., J. Nucl. Med., 34: 1067-74 (1993)). A cellular cytotoxic response towards MUCl has been demonstrated in breast cancer and ovarian cancer patients (Ioannides et al., J. Immunol, 151: 3693-703 (1993), Jerome et al., Cancer Res., 51: 2908-16 (1991), Plunkett et al., Cancer Treat Rev., 24: 55-67 (1998)). This response has been associated with a better protection against breast cancer (Jerome et al., Cancer Immunol Immunother,, 43: 355-60 (1997)). Active immunotherapy related to MUCl (reviewed in Plunkett et al., Cancer Treat Rev., 24: 55-67 (1998) and Miles et al., Pharmacol. Then, 82: 97-106 (1999)) has been studied with variable success in humans and has mainly involved active immunization with non-glycosylated MUCl peptides containing a VNTR as a source of epitopes that become exposed when MUCl is expressed in an underglycosylated form by cancer cells. Immunization in humans with (MUC1) 5 + Bacillus Calmette-Guerin (BCG) (Goydos et al., J. Surg. Res. ,63: 298-304 (1996)) or in animal models with MUCl presenting dendritic cells (e.g., in mice (Gong et al, Proc. Natl. Acad. Sci. USA., 95: 6279-83 (1998)) or in chimpanzees (Pecher et al., Proc. Natl. Acad. Sci. USA, 93: 1699-704 (1996)) showed, respectively, that it is possible to restore T cell function and to increase the cytotoxic T cell precursor frequency to MUCl. In spite of these reports, and in contrast to results obtained in mice, a poor cytotoxic T cell response and high antibody titers were observed by immunization with MUCl-mannan fusion proteins in humans (Karanikas et al., J. Clin. Invest, 100: 2783-92 (1997)). The B cell response is thought to be related to the presence in humans of natural anti-α-galactosyl (l->3) galactose antibodies which cross-react with MUCl (Apostolopoulos et al., Nat. Med., 4: 315-20 (1998)). Moreover, amongst its many biological functions, MUCl inhibits T cell proliferation and it has been postulated that this could be one ofthe reasons for the presence of anergic tumor infiltrating lymphocytes (TIL) in adenocarcinoma patients (Agrawal et al., Nat. Med., 4: 43-9 (1998), Agrawal et al., Mol. Med Today, 4: 397-403 (1998)). This immunosuppressive effect or anergy may be due either to the direct interaction of soluble or surface bound MUCl expressed by tumor cells with multiple T cell-receptor molecules (Plunkett et al., Cancer Treat. Rev., 24: 55-67 (1998), Agrawal et al., Nat. Med., 4: 43-9 (1998)), or by the interaction by other, MUC1- associated components, which are not yet identified (Paul et al., Cancer Immunol. Immunother., 48: 22-8 (1999)). Such anergy can be reversed by IL-2 (Agrawal et al., Nat. Med., 4: 43-9 (1998)), and it has been proposed that active immunization with a MUCl peptide (without any repeats) together with IL-2 administration would be able to stimulate MUCl -specific cytotoxic T lymphocytes (CTLs) (Agrawal et al., Mol. Med. Today, 4: 397-403 (1998)). However, systemic IL-2 administration is known to cause an undesirable nonspecific activation of T cells, and is also associated with dose-dependent toxicity, whose symptoms are known to include malaise, nausea, multi organ failure, shock, and even death (Rosenberg et al., Ann. Surg., 210: 474-84; see, discussion 484-5 (1989)). It has been demonstrated that IL-2 targeting by immunocytokines (i.e., antibody- cytokine fusion proteins) efficiently impairs growth of other tumor cells due to the induction of CD8 + T cell and NK-cell mediated anti-tumor responses (reviewed in Reisfeld et al., J. Clin. Lab. Anal, 10: 160-6 (1996) and Melani et al, Cancer Res., 58: 4146-54 (1998)). In contrast to active therapy using defined TAA-derived molecules, such hybrid fusion proteins may not only stimulate T cells specific for one TAA but also other specific TIL present in the microenvironment ofthe tumor (Becker et al., Proc. Natl. Acad. Sci. USA., 93: 7826-31 (1996)). Moreover, tumor specific anergic T cells, which are often present in the carcinomas, could be rescued with the IL-2 part ofthe molecule (Beverly et al., Int. Immunol, 4: 661-671 (1992)). SUMMARY OF THE INVENTION This invention provides various antibody molecules and derivatives thereof, including immuoglobulin molecules and immunocytokine fusion proteins, which are binding members that specifically bind an epitope ofthe protein core of mucin-1 (MUCl). Such MUCl-specific binding members may be used in the diagnosis and/or treatment of cancer in various tissues, such as adenocarcinomas present in various tissues, especially breast, ovary, bladder, and lung. Variant forms ofthe MUCl-specific binding members are also provided which possess an additional feature or moiety, which enables the member to be especially useful in diagnosis, imaging, or treatment of cancers. Variants include fusion proteins that possess additional properties, such as MUCl-specific immunocytokine molecules, which have a MUCl binding domain and a cytokine domain, which provides an additional therapeutic or prophylactic effect on the development or spread ofa cancer. In one embodiment ofthe invention, MUCl-specific binding members are provided that contain a MUCl antigen binding domain (MUCl binding domain) formed from a Fab antibody light chain variable region (V L ) and from an antibody heavy chain variable region (V ), or portions thereof. For example, a MUCl-specific binding member ofthe invention may comprise a V L amino acid sequence of SEQ ID NO: 1, and/or a V H amino acid sequence of SEQ ID NO:3, or portions thereof, especially those portions encoding complementarity determining regions (CDRs). Thus, the invention also provides isolated CDRs from MUCl-specific binding domains, such as RSSQSLLHSNGYTYLD (amino acids 24 to 39 of SEQ ID NO:l) for a V L CDR1; SGSHRAS (amino acids 55 to 61 of SEQ ID NO:l), for a V L CDR2; MQGLQSPFT (amino acids 94 to 102 of SEQ ID NO:l) for a V L CDR3; SNAMG (amino acids 31 to 35 of SEQ ID NO:3) for a V H CDR1; GISGSGGSTYYADSVKG (amino acids 50 to 66 of SEQ ID NO:3) for a V H CDR2; HTGGGVWDPIDY (amino acids 99 to 110 of SEQ ID NO:3) for a V H CDR3. One or more of these CDRs may be used to form MUCl binding domains in a variety of MUCl - specific binding members of the invention. In another embodiment, the invention provides an isolated MUCl-specific binding member comprising an antigen binding domain, wherein the antigen binding domain comprises an amino acid sequence ofthe formula: X, X 2 His Thr Gly X 3 Gly Val Trp X 4 Pro X 5 X 6 X 7 (SEQ ID NO:28), wherein X, is Ala, Ser, Thr, or Val; X 2 is Lys, He Arg, or Gin; X 3 is Gly, Arg, Val, Glu, Ser, or Ala; X 4 is Asp or Asn; X 5 is He, Leu, Met, Phe, or Val; X 6 is Asp, Gly, Lys, Asn, Ala, His, Arg, Ser, Val, or Tyr; and X 7 is Tyr, His, Lys, Asn, Asp, Ser, Pro. In a preferred embodiment, the invention provides MUCl-specific binding members comprising an antigen binding domain, wherein the antigen binding domain comprises any ofthe amino acid sequences listed in Table 9. In yet another embodiment, the invention provides MUCl-specific binding members comprising a V H region, or CDR thereof, from the DP47 V H germ line and/or a V L region, or CDR thereof, from the DPK15 V L germ line. In another embodiment, the invention provides MUCl-specific binding members formed by inserting one or more ofthe CDRs described herein into the framework regions (FRs) of antigen binding domains from other germ lines or from other antibodies. In still another embodiment, the MUCl-specific binding members ofthe invention have a MUCl-specific binding domain comprising a V L and/or V H region, or portions thereof, as described above, and is an antibody molecule selected from the group consisting of full-length immunoglobulin molecules (such as, IgG, IgM, IgA, IgE), Fab antibodies, F(ab') 2 antibodies, diabodies, single chain antibody (scFv) molecules, Fv molecules, double-scFv molecules, domain antibody (dAb) molecules, and immunocytokines. MUCl-specific, full-length immunoglobulin molecules ofthe invention include recombinant immunoglobulin proteins in which the V L and/or V H region of a MUCl-specific Fab antibody has been genetically engineered into a complete, human immunoglobulin molecule, such as a human antibody of isotype IgGl . The benefits of such a recombinant, full-length, human immunoglobulin with MUCl binding specificity derived from a Fab antibody include the presence of two contiguous MUCl binding sites, a decreased immunogenicity to avoid the classic HAMA response in humans, an enhanced half-life in humans, and a significantly enhanced affinity for MUCl expressed on cancer cells and tissues, particularly ovarian and breast cancer cells and tissues, compared to the single MUCl binding site ofthe corresponding Fab antibody. The MUCl-specific immunoglobulins of the invention include isotypic variants and allotypic variants. Preferred embodiments of MUCl-specific immunoglobulins provided by the invention include immunoglobulin molecules comprising a V L having the amino acid sequence of SEQ ID NO:l and a V H having the amino acid sequence of SEQ ID NO:3. In another preferred embodiment, the invention provides a recombinant, human immunoglobulin, which comprises a light chain (i.e., V L and C L kappa light chain constant region) having the amino acid sequence of SEQ ID NO: 24 and a heavy chain (V H and C H heavy chain constant region for the human gamma-1 isotype) having the amino acid sequence of SEQ ID NO:26. In another preferred embodiment, a MUCl-specific binding member ofthe invention is an immunocytokine, which comprises a MUCl-specific binding domain and a cytokine domain, which confers an immunomodulatory activity on the MUCl-specific binding member. Preferred cytokines for use in such MUCl-specific binding members include IL-2, GM-CSF, and TNF, or portions thereof, though others may be used. More preferably, the immunocytokine is a fusion protein comprising a diabody fused to a cytokine, such as the IL-2 cytokine. Most preferably, the immunocytokine is the bivPHl-IL-2 ofthe invention having the amino acid sequence of SEQ ID NO:5. In another aspect ofthe invention, variant forms of MUCl-specific binding members are provided that are linked, preferably covalently, to other molecules, including, but not limited to other proteins, polypeptides, peptides, such as cytokines or enzymes; anti-cancer drugs; fluorescent labels; radioactive compounds, such as magnetic resonance imaging compounds or anti-cancer radioactive compounds; and heavy metals. Such variants are especially well suited for use in the diagnostic, imaging, purification, or therapeutic methods ofthe invention. The invention also provides MUCl-specific binding members that are proteins, polypeptides, and peptides that comprise an amino acid sequence that is homologous to any of the amino acid sequences described herein. Such homologous proteins, polypeptides, or peptide molecules bind MUCl or form part ofa MUCl-specific binding domain and comprise an amino acid sequence that is about 70% or more, preferably about 80% or more, or more preferably about 90%, 95%, 97%, or even 99% or more homologous to an amino acid sequence described herein. Even more preferably, such a homologous protein, polypeptide, or peptide ofthe invention comprises a V H and/or V L region, or CDR thereof, that is about 70% or more, preferably about 80% or more, and more preferably about 90%, 95%, 97%, or 99% or more homologous to the amino acid sequence of SEQ ID NO:l (for the V L region, and CDRs therein) and/or to the amino acid sequence of SEQ ID NO:3 (for the V H region, and CDRs therein). In another embodiment, the invention provides MUCl-specific binding members and portions thereof, such as a V L or V H region, or CDR, that comprise an amino acid sequence described herein in which one or more ofthe amino acids have been conservatively substituted with another amino acid. The invention also provides methods of diagnosing MUCl -expressing cancer, such as adenocarcinoma, using MUCl-specific binding members and variants thereof. Such diagnostic methods comprise contacting cells, tissues, or a body fluid of an individual with a MUCl- specific binding member and detecting the MUCl-specific binding member bound to MUCl on the cells or tissues or present in the fluid ofthe individual. Preferably, the methods ofthe invention are used to diagnose ovarian, breast, bladder, and lung cancer. Diagnostic methods of the invention include the use of a MUCl binding member described herein in methods of imaging cells, tissues, and/or organs to detect the presence ofa cancer in the cells, tissues, and/or organs. In another embodiment, the MUCl-specific binding members and variants thereof may be used in methods of purifying cancer-associated MUCl, underglycosylated forms of MUCl, or non-glycosylated MUCl molecules in a mixture or extract. In yet another embodiment, MUCl-specific binding members, and variants thereof, may be used in methods for therapeutically or prophylactically treating MUCl -expressing cancer in an individual. The treatment methods ofthe invention may be in vivo or ex vivo methods. The in vivo methods of treating cancer comprise administering to an individual a MUCl-specific binding member, or variant thereof, described herein. The MUCl-specific binding member, or variant thereof, may be administered by any of a variety of routes including parenterally, such as intravenously or intramuscularly; orally; by inhalation; topically; or by direct injection into or close to a tumor or affected site. Various pharmaceutical compositions comprising a MUCl- specific member may be prepared that are particularly suited for a chosen route of administration. Preferably, the MUCl-specific binding member is administered parentally, and more preferably intravenously. In a preferred method of treatment, the MUCl-specific binding member is an immunocytokine or is an immunoglobulin, which may be linked to an anti-tumor compound. More preferably, the method of treatment comprises administering the immunocytokine bivPHl-IL-2 having the amino acid sequence of SEQ ID NO:5 or the immunoglobulinn comprising light chains having the amino acid sequence of SEQ ID NO:24 and heavy chains having the amino acid sequence of SEQ ID NO:26. More preferably, the method of treating a cancer using an immunocytokine described herein comprises administering to an individual an unconjugated (free) form ofa cytokine before, contemporaneously with, or after administering an immunocytokine described herein. A preferred method of treating a cancer according to the invention comprises administering to an individual in need of treatment a MUCl-specific immunoglobulin described herein linked (preferably covalently) to an anti-cancer compound, such as a derivative or variant of doxorubicin or a toxin molecule. In another aspect ofthe invention, ex vivo methods of cancer treatment comprise extracting cells, tissues, or a body fluid from an individual, contacting the extracted cells, tissues, or body fluid with a MUCl-specific binding member, or variant thereof, as described herein; collecting the cells, tissues, or body fluid depleted or purged of cancer-associated MUCl and/or MUCl -expressing cancer cells; and then returning the remaining cells, tissues, or body fluid, which do not express or contain cancer-associated MUCl to the individual. It is another aspect ofthe invention to provide polynucleotide molecules encoding the various MUCl-specific binding members, V L region, V H region, CDRs, and framework (FR) regions described herein. In a preferred embodiment, isolated polynucleotide molecules are provided that encode the V L and/or V H region, or portions thereof, ofthe binding domain ofa MUCl-specific binding member, such as the PHI Fab antibody described herein. In another preferred embodiment, the polynucleotide molecules comprise the nucleotide sequence of SEQ ID NO:2 encoding a V L region having the amino acid sequence of SEQ ID NO: 1 , or portions thereof, and/or the nucleotide sequence of SEQ ID NO:4 encoding a V H region having the amino acid sequence of SEQ ID NO:3, or portions thereof. In another preferred embodiment, the invention provides polynucleotide molecules comprising nucleotide sequences that encode one or more CDRs from an antibody V L or V H region ofthe PHI Fab antibody such as: AGGTCTAGTCAGAGCCTCCTGCATAGTAATGGATACACCTATTTGGAT (nucleotides 70- 117 of SEQ ID NO:2), which encodes a V L CDRl ; TCGGGTTCTCATCGGGCCTCC (nucleotides 163 to 183 of SEQ ID NO:2), which encodes a V L CDR2; ATGCAGGGTCTACAGAGTCCATTCACT (nucleotides 280-306 of SEQ ID NO:2), which encodes a V L CDR3; AGTAACGCCATGGGC (nucleotides 91 to 105 of SEQ ID NO:4), which encodes a V H CDRl; GGTATTAGTGGTAGTGGTGGCAGCACATACTACGCAGACTCCGTGAAGGGC (nucleotides 148-198 of SEQ ID NO:4), which encodes a V H CDR2; CATACCGGGGGGGGCGTTTGGGACCCCATTGACTAC (nucleotides 295 to 330 of SEQ ID NO:4), which encodes a V H CDR3; and combinations thereof. The polynucleotide molecules ofthe invention also include polynucleotide molecules comprising degenerate forms of one or more ofthe previously mentioned nucleotide sequences, which encode the same protein, polypeptide, or peptide. In yet another embodiment ofthe invention, polynucleotide molecules are provided which have a nucleotide sequence that is homologous to any ofthe nucleotide sequences listed herein. A homologous polynucleotide molecule of this invention comprises a nucleotide sequence that is about 60%, more preferably 70%, even more preferably 80%, and most preferably 90%), 95%, 97%, or even 99% or more, homologous to a nucleotide sequence described herein that encodes a MUCl-specific binding member, a MUCl-specific binding domain, or a portion thereof, such as a CDR or a CDR and selected amino acid residues of an adj acent FR of a MUC 1 -specific binding domain. The invention also provides methods of producing MUCl-specific binding members using the polynucleotide molecules described herein. Such polynucleotide molecules may be inserted in any of a variety of prokaryotic or eukaryotic vectors for production ofa MUCl- specific binding member in cultures of appropriate prokaryotic or eukaryotic host cells. Such vectors useful in the methods ofthe invention include plasmids, phage, phagemids, and eukaryotic viral vectors. In another embodiment of the invention, MUCl-specific binding members ofthe invention are expressed and displayed on the surface of cells or phage particles. Preferably, MUCl-specific binding members described herein are expressed and displayed on the surface of cells or phage particles using phage, phagemid, or yeast display vectors. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows diagrams (A-D) ofthe cloning schedule for the construction ofthe bivalent diabody bivPHl and bivPHl-IL-2 immunocytokine. Figure 1A is a diagram ofthe starting PHI Fab gene in the vector plasmid pCESl. Figure IB is a diagram ofthe cloning ofthe PHI V H and restriction sites into the plasmid vector pCantab6. Figure 1C illustrates the insertion ofthe PHI V L to retrieve the bivPHl diabody from the plasmid vector pKaPal. Figure ID diagrams the construction of plasmid pKaPa2 by insertion ofthe IL-2 coding sequence to retrieve the bivalent immunocytokine bivPHl -IL-2. Abbreviations: pLacZ, the LacZ promoter; rbs, ribosome binding site; S, signal sequence; PH1VH, heavy chain variable region of Fab fragment PHI; PH1VL, light chain variable region of Fab fragment PHI; H, tag encoding 6 histidines; tag, myc-tag sequence; *, stop codon; LI, linker 1 nucleotide sequence encoding 5 amino acid LI linker peptide; L2, linker 2 nucleotide sequence encoding 9 amino acid L2 linker peptide. Figure 2 shows the graphs ofthe binding characteristics of different antibody formats on BIAcore. Abbreviations: open triangles, scFv 10A; open circles, Fab PHI; open squares, bivalent diabody bivPHl-IL-2. MUCl 80-mer was coupled to a chip at a density of 90 Response' Units (RU), binding ofthe three MUCl antibodies was measured. Figures 3A and 3B show a comparison ofthe binding of antibodies scFv 10A, PHI Fab, bivPHl diabody, bivPHl-IL-2 immunocytokine to cell lines 3T3, the 3T3 MUCl -transfected cell line ETA, OVCAR-3, T47D and LS174T in flow cytometry. Binding characteristics ofthe antibodies to the different cell lines are given in overlayed histograms. Binding intensities ofthe antibodies to the cells were measured by secondary staining with FITC-labeled antibodies, and fluorescence was measured (FL1-H). Number of stained cells were measured (COUNTS). Unbroken line indicates binding of antibody; alternating broken and dotted line indicates negative control (in the case ofthe 3T3 MUCl -transfected cell line ETA, the negative control was the non-transfected cell line 3T3); and broken line indicates competition for cell binding with MUCl 60-mer. Figure 4 shows the results of induction of CTLL-16 proliferation by rIL-2 (open circles) and bivPHl -IL-2 (open squares) by uptake of radioactive 3 H-thymine measured in counts per minute (cpm). Figure 5 shows the results of stimulation of resting PBL by rIL-2 or bivPHl-IL-2, without or with the addition of MUCl measured by 3 H-thymidine uptake assay. Medium alone (stipled bars); PHA without MUCl (open bars); PHA with MUCl (diagonal bars). Uptake of Η- thymidine was measured in counts per minute (cpm). Figure 6 shows the results ofthe 51 chromium-release assay with antibody coated OVCAR-3 target cells (T) by resting PBL effector cells (E). E:T ratios: 100:1 (stipled bars); 50:1 (white bars); 25:1 (horizontal bars); 12.5:1 (diagonal bars). Percent (%) lysis ofthe OVCAR-3 target cells was calculated by 100 x (cpm test sl Cr released - cpm minimal 51 Cr released/cpm maximal 51 Cr released - cpm minimal 51 Cr released). DETAILED DESCRIPTION The invention provides MUCl-specific binding members that preferentially bind to the protein core of MUCl. The specific binding members of MUCl described herein include those binding members that comprise a MUCl antigen binding domain, which comprises a variable light chain region (V L ) having the amino acid sequence of SEQ ID NO: 1, or portion thereof, such as one or more ofthe complementarity deteremining regions (CDRs) of V L , and/or a variable heavy chain region (V H ) having the amino acid sequence of SEQ ID NO:3, or portion thereof, such as one or more CDRs of V H , as found in or isolated from a human Fab antibody or monoclonal antibody (MAb). As discussed below, MUCl-specific binding members ofthe invention may be fusion or recombinant proteins. Such fusion proteins include those that comprise a MUCl-specific binding portion and an immunomodulatory portion, such as a cytokine, such as IL-2, or active fragment thereof. The recombinant proteins ofthe invention include recombinant, immunoglobulin molecules, in which a MUCl-specific binding domain of a Fab antibody or other binding member has been engineered into an immunoglobulin molecule. Such recombinant immunoglobulins exhibit enhanced affinity and avidity for MUCl over MUCl -binding members that have a single MUCl binding site. The MUCl-specific binding members ofthe invention may be used to diagnose or treat cancer, such as adenocarcinoma, which may be found in a wide variety of tissues including mammary (e.g., breast cancer), ovary, lung, and bladder and which is characterized by overexpression of a glycoform of MUC 1. MUC 1 molecules that are produced by cancer cells and tissues (cancer-associated MUCl) are underglycosylated and, therefore, expose the core protein epitopes that are recognized and bound by the MUCl-specific binding members described herein. In order that the invention may be more fully understood, the following terms are defined: "Specific binding member" or "binding member" as used and understood herein, refers to a member of a pair of molecules, which have binding specificity for one another. The members of such a specific binding pair may be naturally derived or wholly or partially synthetically produced. One member ofthe pair of molecules has an area on its surface, or a cavity, which specifically binds to and is therefore complementary to the three-dimensional geometry and chemistry ofthe other member ofthe pair of molecules. Thus, the members ofthe binding pair have the property of binding specifically to each other. Examples of types of specific binding pairs are antigen-antibody, biotin-streptavidin or avidin, hormone-hormone receptor, receptor- ligand, enzyme-substrate. It is also understood that one member ofa specific binding pair may also be a member of other specific binding pairs, for example, as is the case with an antigenic protein and different antibodies, where each antibody binds to a different site (epitope) on the same antigen or to the same site, but with a different or same affinity or avidity. This invention is concerned with antigen-antibody type binding members. More specifically, this invention is concerned with specific binding member pairs consisting of a MUCl-specific binding member molecule, such as an antibody molecule as defined below, which has an antigen binding site formed by a variable light (V L ) chain region, or portion thereof, and/or variable heavy (V H ) chain region, or portion thereof, from a human Fab antibody and ofthe other binding member ofthe pair, which is a protein or polypeptide that comprises a MUCl VNTR (variable number of tandem repeats) protein core amino acid sequence. "Antibody" or "antibody molecule", as used and understood herein, refers to a specific binding member that is a protein molecule or portion thereof or any other molecule, whether produced naturally, synthetically, or semi-synthetically, which possesses an antigenic binding domain formed by an immunoglobulin variable light chain region or domain (V L ), or portion thereof, and/or an immunoglobulin variable heavy chain region or domain (V H ), or portion thereof. The term also covers any polypeptide or protein molecule that has an antigen binding domain which is identical, or homologous to, an antibody binding domain of an immunoglobulin. Examples of an antibody molecule, as used and understood herein, include any ofthe well known classes of immunoglobulins (e.g., IgG, IgM, IgA, IgE, IgD) and their isotypes; fragments of immunoglobulins that comprise an antigen binding domain, such as Fab or F(ab') 2 molecules; single chain antibody (scFv) molecules; double scFv molecules; single domain antibody (dAb) molecules; Fd molecules; and diabody molecules. Diabodies are formed by association of two diabody monomers, which form a di er that contains two complete antigen binding domains wherein each binding domain is itself formed by the intermolecular association of a region from each ofthe two monomers (see, e.g., Holliger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993)). It is possible to take an antibody molecule, such as a Fab antibody or monoclonal antibody (MAb) molecule, and use techniques of recombinant DNA technology available in the art to produce other molecules, which retain the specificity ofthe original (parent) antibody or a particular region ofthe original antibody. Such techniques may involve introducing DNA comprising a nucleotide sequence(s), which, for example, encodes the immunoglobulin variable regions ofthe variable light (V L ) and/or variable heavy (V H ) immunoglobulin chains of a Fab or other MUCl-specific antibody, or which encodes portions ofthe V L and/or V H , such as one or more ofthe complementarity determining regions (CDRs), in frame with another DNA sequence, such as a nucleotide sequence encoding an immunoglobulin constant region or constant region and framework (FR) regions of a different immunoglobulin (see, e.g., EP-A-184187, GB 2188638A , EP-A-239400). For example, new, recombinant MUCl-specific immunoglobulins may be produced by cloning nucleotide sequences encoding V and V H regions, or portions thereof, from one (parent) MUCl -binding member, into plasmid expression vectors used for expressing the light and heavy chains of an immunoglobulin molecule, such as an IgG. The recombinant plasmids are then transfected into a compatible host cell for expression of the recombinant immunoglobulin, which has the MUCl -binding specificity ofthe parent molecule. Such recombinant immunoglobulins may also exhibit enhanced avidity for MUCl compared to the parent molecule, owing to the divalent structure (two identical binding sites) for MUCl and/or other features (see, e.g., Example 3). A hybridoma or other cell that produces an antibody molecule may also be subjected to genetic mutation or other changes, which may alter the binding specificity or other property ofthe antibody molecule produced by that cell to form a new MUCl binding member of this invention. As antibodies can be modified in a number of ways, the term "antibody" is understood to cover any specific binding member or substance having a binding domain as described herein with the required specificity for the other member, i.e., MUCl. Thus, "antibody" or "antibody molecule" covers antibody fragments, derivatives, functional equivalents and homologues of antibodies, including any polypeptide comprising an immunoglobulin binding domain, whether natural or wholly or partially synthetic. Fusion or chimeric protein molecules comprising an immunoglobulin binding domain or CDRs thereof, or equivalent, fused to another polypeptide, such as a cytokine, another immunoglobulin, enzyme, or protein toxin, are also included. Cloning and expression of some examples of chimeric antibodies are described in EP-A-0120694 and EP-A-0125023. Various fragments of a whole immunoglobulin molecule are generally known to be capable of performing the function of binding antigens or of being recombined, for example using recombinant DNA methods, to form binding members with the same specificity as a whole immunoglobulin but having a smaller size. For example, classically a Fab fragment is an antibody that can be generated by papain digestion of an immunoglobulin molecule and has a single antigen binding domain (monovalent) consisting ofthe V L , V H , the constant domain ofthe light chain (C L ), and the CHI constant domain ofthe heavy chain. Fab antibodies can also be produced synthetically or in vivo from cells containing recombinant expression vectors, which encode and express a particular Fab antibody. Fab antibodies ofthe invention also include those molecules selected from a phage display library of human Fab molecules for the ability to bind a MUCl epitope (see, e.g., Examples 1 and 2). A F(ab') 2 fragment is an antibody, which classically has been generated by pepsin digestion of an immunoglobulin molecule to yield two linked Fab fragments and, therefore, two complete antigen binding domains (bivalent), which are capable of binding and cross-linking antigen molecules. An Fd fragment or antibody consists of the V H and CHI domains ofthe immunoglobulin heavy chain. Another example of a portion of an immunoglobulin that is capable of binding the same antigen as full-length immunoglobulin is an Fv antibody molecule consists ofthe V L and V H regions of a single immunoglobulin (and absent constant domains). Another antigen-binding portion of a full-length immunoglobulin is a dAb fragment or antibody, which consists of a V H domain (Ward, et al., Nature, 341: 544-546 (1989)). In addition, an isolated CDR region, either alone or together with one or more other CDRs of an immunoglobulin, may form an antigen binding domain. A single chain Fv (scFv) antibody molecule is a monovalent molecule wherein a V H domain and a V L domain are linked by a peptide linker, which allows the two variable domains to associate intramolecularly to form a complete antigen binding site (see, e.g., Bird et al., Science, 242: 423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA, 85: 5879-5883 (1988)). It is also possible to form bispecific scFv dimers, which bind two different epitopes (see, e.g., PCT/US92/09965). Diabodies (discussed in more detail below) may be bivalent or even multivalent or multispecific molecules are also typically constructed by gene fusion in which a DNA molecule encoding one or more V L domains is linked in frame with a DNA molecule encoding one or more V H domains. Diabodies (or diabody antibodies) are multimers (e.g., dimers, tetramers) of polypeptides, wherein each polypeptide comprises a V L region and V H region of an immunoglobulin antigen binding domain that are linked to one another, e.g., by a relatively short peptide linker, such that the two regions are unable to associate with each other intramolecularly to form an antigen binding site. Complete antigen binding domains are only assembled intermolecularly by the association ofthe V domain of one polypeptide (monomer) with the V H domain of another polypeptide (monomer) which occurs when a multimer forms (see, e.g., PCT publication number WO 94/13804; P. Holliger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993)). Where bispecific antibodies, i.e., antibody molecules having binding domains for two different antigens or epitopes, are to be used, these may be conventional bispecific immunoglobulin antibodies, which can be produced by various techniques, including, for example, by chemical modifications, from hybrid hybridomas, or by recombinant immunoglobulin expression vectors transfected into appropriate host cells, or may be any ofthe bispecific antibody fragments mentioned above (see, e.g., Holliger and Winter, Current Opinion Biotechnol, 4: 446-449 (1993)). Alternatively, it may be preferable to use scFv dimers or diabodies, rather than whole antibodies. Diabodies and scFv molecules can be constructed using variable domains without an Fc region in order to reduce potential effects of anti-idiotypic reactions. Other forms of bispecific antibodies include the single chain "Janusins" described in Traunecker et al., EMBO J., 10: 3655-3659 (1991). Bispecific diabodies, as opposed to bispecific whole immunoglobulin molecules, may also be particularly useful because they can be conveniently constructed and expressed in procaryotic cells, such as E. coli. Furthermore, diabodies and many other antibody fragments, as described above, of appropriate binding specificity can be readily selected from libraries using phage display (see, e.g., WO 94/13804 and Examples below). In addition, bispecific diabodies may be constructed by maintaining one domain ofthe diabody having a specificity that is directed against one antigen, while selecting from a library for a different specificity in the other binding domain. "Antigen", as used and understood herein refers to any molecule that can elicit an immune response and/or that can be bound by an antibody. An antigen as used herein is not limited by molecular size and includes any molecule, whether produced naturally, synthetically, or semi-synthetically, which can be bound by an antibody molecule. In addition, it is understood that an antigen molecule has one, several, or many different sites at which an antibody may bind. "Antigenic determinant" or "epitope" are used synonymously and refer to the specific site on an antigen at which an antibody molecule binds. The antigenic determinant or epitope of an antigen is complementary to the antigen binding domain (see, below) of an antibody. An antigen may have only one or, as is usually the case, several or even many epitopes. Epitopes of a given antigen molecule may be present as multiple copies of structurally identical moieties, as in case of repetitive amino acid sequences in a protein, or distinctly different, in which case each epitope could be bound by a different antibody. "Antigen binding domain," as used and understood herein refers to the region of an antibody molecule which specifically binds to and is complementary to a particular site on an antigen, which is a specific binding member or partner to the antibody molecule. An antigen binding domain may be provided by one or more antibody variable regions. The antigen binding domain of an immunoglobulin antibody or fragment thereof, such as a Fab or F(ab') 2 antibodies, comprises an antibody V L region and an antibody V H , which variable regions consists of complementarity determining regions (CDRs) and framework regions (FRs). CDRs are highly variable regions within the V L and V H regions of an antibody and contain the critical amino acid sequences for the specificity and avidity for binding to a particular site (i.e., an epitope) on an antigen (see, e.g., Fundamental Immunology. 4th ed. (Paul, William E., ed.) (Lippincott-Raven, Philadelphia, 1999), pages 58-60). CDRs are located among framework regions (FRs), which provide a structural context to the variable regions necessary for binding to a specific site on an antigen. Using recombinant DNA techniques, it is possible to construct DNA molecules that code for each variable region or domain (V L , V H ), or even portions of a variable region, such as individual CDRs or a CDR and contiguous residues of adjacent FRs, which in turn may be inserted into a gene coding for a different antibody, or other protein to form a recombinant antibody protein that has a new antigen binding domain (see, e.g., Example 3). "Specific," as used and understood herein refers to the preference of one member ofa specific binding pair to bind with the other member. The term is also applicable where an antigen binding domain is specific for a particular epitope which is carried by a number of antigens, in which case the specific binding member carrying the antigen binding domain will be able to bind to the various antigens carrying that epitope. Likewise the term is applicable where an antigen binding domain is specific for a particular epitope of a binding member and the same antigen binding domain is carried by different types of antibody molecules, e.g., scFv or Fab antibodies, in which case the different types of antibody molecules are able to bind to and are, therefore, understood to be, "specific" for the same epitope. "Functionally equivalent variant" or simply "variant", unless noted otherwise, as used and understood herein, refers to a molecule (the variant), which although having structural differences from another molecule (the parent), has retained some significant homology or at least some ofthe biological function ofthe parent molecule, such as the ability to bind a particular antigenic determinant or epitope of MUCl. Variants may be in the form of fragments, such as Fabs or F(ab') 2 antibodies, which are fragments of larger immunoglobulin molecules, or mutant antibody protein molecules in which the amino acid sequence of a parent antibody protein has been altered to yield a variant antibody, which retains the specificity ofthe parent for an epitope, but now has an enhanced (or, for some applications, possibly decreased) avidity for the epitope. For example, a selected antibody can be affinity matured for enhanced affinity for an antigen or epitope according to procedures known to persons skilled in the art and described herein by introducing diversity in a nucleotide sequence of a polynucleotide molecule encoding the parent antibody, or portion thereof, by replacing the V H or V L genes with a repertoire of V H or V L genes or by introducing mutations, and then selecting variants against the desired antigen or epitope by phage display (see, e.g., Example 2, De Haard et al., Adv. Drug Del Rev., 31: 5-31 (1998); Hoogenboom et al., Trends in Biotech., 15: 62-70 (1997)). The variants can then be screened for enhanced affinity. Variant mutant proteins may be produced synthetically or biologically using recombinant DNA techniques in which case the variant is the expressed product (mutant protein) ofa mutated σpnp Δ ■ vαricir.f lir.V-tt-.rr f α-T Λ f r ΛΛτro1n«f1>ιτ -f -. * -.« * *«.«-< ■ "Homologues" ofthe MUCl -binding members described herein may be formed by substitution, addition, or deletion of one or more amino acids employing methods well known in the art and for particular purposes known in the art. Such "homologous" proteins, polypeptides, or peptides will be understood to fall within the scope ofthe present invention so long as the substitution, addition, or deletion of amino acids does not eliminate its ability to bind MUCl or to form part of a MUCl binding domain. The term "homologous", as used herein, refers to the degree of sequence similarity between two polymers (i.e., polypeptide molecules or nucleic acid molecules). When the same nucleotide or amino acid residue occupies a sequence position in the two polymers under comparison, then the polymers are homologous at that position. For example, if the amino acid residues at 60 of 100 amino acid positions in two polypeptide sequences match or "are homologous", then the two sequences are 60% homologous. The homology percentage figures referred to herein reflect the maximal homology possible between the two polymers, i.e., the percent homology when the two polymers are so aligned as to have the greatest number of matched (homologous) positions. Various computer programs are available for aligning two polymers and also for calculating the percent homology between the two polymers. For example, alignment and/or percent homology calculations between two polymers of interest are routinely performed using the BLAST sequence bank computer program (see, e.g., http://www.ncbi.nlm.nih.gov/blast/) or the MCVECTOR ® computer program. For germ line homology studies, Vbase (see, e.g., http://www.mrc-cpe.cam.ac.uk/imt-doc/) performs alignments between new and known germ line sequences in order to determine the source of individual V L or V H regions of an antibody molecule. Protein, polypeptide, and peptide homologues within the scope ofthe present invention will be about 70%, preferably about 80%, and more preferably about 90% or more (including about 95%, about 97%, or even about 99% or more) homologous to a MUCl -binding member, a MUCl binding domain, or portion thereof, including a CDR or a CDR and selected contiguous framework (FR) residues, as disclosed herein. Polynucleotide homologues within the scope ofthe present invention will be about 60%, preferably about 70%, more preferably about 80%, and even more preferably about 90% or more (including about 95%, about 97%, or even about 99% or more) homologous to the nucleotide sequences described herein that encode a MUCl-specific binding member, a MUCl binding domain, or portion thereof (such as V L , V H , CDR), as disclosed herein. The amino acid sequences ofthe proteins, polypeptides, and peptides described herein are recited using either the conventional one letter or three letter abbreviations for amino acids known in the art. Anti-MUCl PHI Fab Antibody The origin ofthe MUCl binding domain of all ofthe MUCl-specific binding members ofthe invention is an anti-MUCl human Fab fragment (Fab antibody), designated PHI, which was obtained by screening a naive (non-immunized) phage display library containing 3.7 X 10 10 different Fab fragments (see, Examples below). The phage displaying the PHI Fab fragment was identified and isolated by selection and screening for the ability to bind a VNTR sequence ofthe MUCl core protein and for binding to MUCl -expressing cells. The genes encoding the V H and V regions of PHI encoded on a phagemid were isolated and sequenced. The PHI V L region is encoded by the nucleotide sequence of SEQ ID NO:2 and has the amino acid sequence of SEQ ID NO: 1. The PHI V H region is encoded by the nucleotide sequence of SEQ ID NO:4 and has the amino acid sequence of SEQ ID NO:3. Each variable region ofthe PHI Fab antibody contains both structural framework (FR) sequences and the highly variable complementarity- determining regions (CDRs), which confer the specificity and avidity ofthe antigen-binding domain for the epitope of MUCl. For the V L region ofthe PHI Fab molecule, CDRl is encoded by the nucleotide sequence and reading frame AGG TCT AGT CAG AGC CTC CTG CAT AGT AAT GGA TAC ACC TAT TTG GAT (nucleotides 70 to 117 of SEQ ID NO:2) and has the amino acid sequence of RSSQSLLHSNGYTYLD (amino acids 24 to 39 of SEQ ID NO:l); CDR2 is encoded by the nucleotide sequence and reading frame TCG GGT TCT CAT CGG GCC TCC (163 to 183 of SEQ ID NO:2) and has the amino acid sequence of SGSHRAS (amino acids 55 to 61 of SEQ ID NO:l); and CDR3 is encoded by the nucleotide sequence and reading frame ATG CAG GGT CTA CAG AGT CCA TTC ACT (nucleotides 280 to 306 of SEQ ID NO:2) and has the amino acid sequence of MQGLQSPFT (amino acids 94 to 102 of SEQ ID NO:l). FR1 ofthe V L region of PHI is encoded by the nucleotide sequence and reading frame GAA ATT GTG CTG ACT CAG TCT CCA CTC TCC CTG CCC GTC ACC CCT GGA GAG CCG GCC TCC ATC TCC TGC (nucleotides 1 to 69 of SEQ ID NO:2) and has the amino acid sequence of EIVLTQSPLSLPVTPGEPASISC (amino acids 1 to 23 of SEQ ID NO:l); FR2 ofthe V L region of PHI is encoded by the nucleotide sequence and reading frame TGG TAC CTG CAG AAG CCA GGG CAG TCT CCA CAG CTC CTG ATC TAT (nucleotides 118 to 162 of SEQ ID NO:2) and has the amino acid sequence of WYLQKPGQSPQLLIY (amino acids 40 to 54 of SEQ ID NO:l); and FR3 ofthe ofthe V L region of PHI is encoded by the nucleotide sequence and reading frame GGG GTC CCT GAC AGG TTC AGT GGC AGT GTA TCA GGC ACA GAT TTT ACA CTG AGA ATC AGC AGA GTG GAG GCT GAG GAT GTT GGA GTT TAT TAC TGC (nucleotides 184 to 279 of SEQ ID NO:2) and has the amino acid sequence GVPDRFSGSVSGTDFTLRISRVEAEDVGVYYC (amino acids 62 to 93 of SEQ ID NO: 1). For the V H region ofthe PHI Fab molecule, CDRl is encoded by the nucleotide sequence and reading frame AGT AAC GCC ATG GGC (nucleotides 91 to 105 of SEQ ID NO:4) and has the amino acid sequence of SNAMG (amino acids 31 to 35 of SEQ ID NO:3); CDR2 is encoded by the nucleotide sequence and reading frame GGT ATT AGT GGT AGT GGT GGC AGC ACA TAC TAC GCA GAC TCC GTG AAG GGC of (nucleotides 148 to 198 of SEQ ID NO:4) and has the amino acid sequence of GISGSGGSTYYADSVKG (amino acids 50 to 66 of SEQ ID NO:3); and CDR3 is encoded by the nucleotide sequence and reading frame CAT ACC GGG GGG GGC GTT TGG GAC CCC ATT GAC TAC (nucleotides 295 to 330 of SEQ ID NO:4) and has the amino acid sequence of HTGGGVWDPIDY (amino acids 99 to 110 of SEQ ID NO:3). FR1 ofthe V H region of PHI is encoded by the nucleotide sequence and reading frame CAG GTC CAG CTG GTG CAG TCT GGG GGA GGC TTG GTA CAG CCT GGG GGG TCC CTG AGA CTC TCC TGT GCA GCC TCT GGA TTC ACG TTT AGA (nucleotides 1 to 90 of SEQ ID NO:4) and has the amino acid sequence of QVQLVQSGGGLVQPGGSLRLSCAASGFTFR (amino acids 1 to 30 of SEQ ID NO:3); FR2 of the V H region of PHI is encoded by the nucleotide sequence and reading frame TGG GTC CGC CAG GCT CCA GGG AAG GGG CTG GAG TGG GTC TCA (nucleotides 106 to 147 of SEQ ID NO:4) and has the amino acid sequence of WVRQAPGKGLEWVS (amino acids 36 to 49 of SEQ ID NO:3); and FR3 ofthe ofthe V H region of PHI is encoded by the nucleotide sequence and reading frame CGG TTC ACC ATC TCC AGA GAC AAT TCC AAG AAC ACG CTG TAT CTG CAA ATG AAC AGC CTG AGA GCC GAG GAC ACG GCC GTA TAT TAT TGT GCG AAA (nucleotides 199 to 294 of SEQ ID NO:4) and has the amino acid sequence RFTISPJ)NSKNTLYLQMNSLRAEDTAVYYCAK (amino acids 67 to 98 of SEQ ID NO:3). By indirect epitope finge rinting (Henderikx et al., Cancer Res., 58: 4324-32 (1998)), the minimal binding epitope in the VNTR ofthe protein core of MUCl for the PHI Fab antibody molecule was determined to have the tripeptide amino acid sequence of Pro Ala Pro. The PHI Fab bound 3T3-MUC1 cells (expressing MUCl). In BIAcore binding studies using an 80-mer MUCl core peptide (i.e., four core protein repeat units of a polypeptide having the 20 amino acid sequence of SEQ ID NO:7) as the antigen binding member, PHI exhibited a slower off-rate (k off = 1 x 10 "3 sec "1 ) than other anti-MUCl scFv antibody molecules, such as scFv-lOA (k off = 1 x 10 "2 sec "1 ), previously retrieved from a scFv phage library (Henderikx et al., Cancer Res., 58: 4324-32 (1998)). Affinity Maturation of PHI Fab Antibody MUCl-Binding Site The PHI Fab antibody was evaluated for affinity for its MUCl epitope by surface plasmon resonance (SPR) using a BIAcore 2000 apparatus (BIAcore AB, Uppsala, Sweden) in which the surface of a biotin chip was coated with a MUCl 60-mer peptide antigen (NH 2 - (VTSAPDTRPAPGSTAPPAHG) 3 -COOH (i.e., containing three copies of SEQ ID NO:8 (von Mensdorff-Pouilly et al., Tumor Biol, 19: 186-195 (1998)). By this analysis, the affinity ofthe PHI Fab antibody was determined as a dissociation constant (Kd) for the MUCl 60-mer peptide antigen to be 1.4 micromolar (μM). According to the invention, the intrinsic affinity of a monovalent Fab antibody, such as the monovalent PHI Fab antibody, for its MUCl epitope can be improved, for example, by using an in vitro affinity maturation procedure involving phage display to select variants (mutants) of a parent Fab antibody (e.g., PHI Fab) that bind MUCl, preferably with higher affinity. Details of an actual example of affinity maturation ofthe PHI Fab binding site are provided in Example 2, below. Using affinity maturation and phage display, variants ofthe PHI Fab antibody were selected. A list of representative variants of PHI Fab antibody obtained in one selection (Example 2), is provided in Table 9 (below), which shows that the listed variants contained mutations in the FR3-CDR3 region ofthe parent PHI Fab antibody. Dissociation constants (Kds) were calculated for the variants by BIAcore analysis of affinity for the MUCl 60-mer peptide antigen. The results indicated that the affinity ofthe selected variants for the MUCl 60- mer peptide antigen ranged from about 400 nanomolar (nM), i.e., a 3.5-fold improvement in the PHI Fab affinity, to about 1.4 μM, i.e., similar to the parent PHI Fab affinity. Other MUCl -Specific Binding Member Molecules In addition to the MUCl-specific Fab antibodies described above, the invention provides other MUCl-specific binding members. The availability of polynucleotide and amino acid molecules encoding specific V H and V regions of one MUCl-specific binding molecule, such as the PHI Fab antibody, along with the knowledge ofthe specific FR and CDR sequences within each variable region of the molecule provide the means for producing any of a variety of other MUCl-specific binding members, or portions thereof, using recombinant DNA procedures or in vitro peptide synthesis protocols. For example, a DNA molecule encoding the antigen binding domain ofthe PHI Fab antibody, or portion thereof (such as V L , V H , or one or more CDRs), can be inserted into vectors for expressing new MUCl-specific binding members with the specificity or binding properties ofthe parent PHI Fab antibody. Such additional MUCl-specific binding members may include, but are not limited to, full-length immunoglobulin molecules (such as, IgG, IgM, IgA, IgE), other Fab antibodies, F(ab') 2 antibodies, diabodies, scFv molecules, double- scFv molecules, Fv molecules, domain antibody (dAb) molecules, immunocytokines, and immunotoxins. MUCl-specific immunoglobulins may be produced by cloning polynucleotides encoding the V H and V L regions ofthe PHI Fab antibody into any eukaryotic expression systems available in the art for producing immuoglobulin light and heavy chains, which then assemble into a whole immunoglobulin molecule. An example of such an expression system uses the vectors, VHexpress (encoding the human gamma- 1 heavy constant region) and VKexpress (encoding the human kappa constant domain) (Persic et al., Gene, 187: 9-18 (1997)). Details of a working example of using these expression vectors to produce a completely human, recombinant, MUCl- specific IgGl antibody ("PHl-IgGl") from DNA encoding the V H and V L regions ofthe PHI Fab antibody are provided in Example 3, below. The PHI -IgGl comprises an immunoglobulin kappa light chain (V L and C L light chain constant region) having the amino acid sequence of SEQ ID NO:24, which is encoded by the nucleotide sequence of SEQ ID NO:25, and an immunoglobulin heavy chain (V H and heavy chain constant region) having the amino acid sequence of SEQ ID NO:26, which is encoded by the nucleotide sequence of SEQ ID NO:27. BIAcore analysis using the MUCl 60-mer peptide antigen indicated that the PHl-IgGl molecule exhibited a 100-fold higher apparent Kd (8.7 nM) compared to the Kd ofthe parent PHI Fab (1.4 μM). This improved affinity was due to the presence ofthe two identical MUCl binding sites of PHI. The recombinant, human PHl-IgGl antibody specifically recognizes tumor cells expressing the peptide core epitope of MUCl of breast and ovarian cancer cell lines, but not colon cancer cell lines, which have heavily glycosylated MUCl on their surface. Immunohistochemical analysis of PHl-IgGl indicated that this immunoglobulin intensely stained (i.e., bound) tumor tissue in mammary, ovary, bladder, and lung tissue. In addition, PHl- IgGl was internalized rapidly into vesicles by human ovarian carcinoma cell line OVCAR-3 cells (see, Example 3). The tumor-associated binding characteristics, the internalization behavior in cancer cells, and the completely human nature ofthe recombinant, PHl-IgGl molecule make this molecule, and molecules like PHl-IgGl, particularly well-suited for use immunotherpeutic, immunodiagnostic, and immunoimaging compositions and procedures. For example, various drugs, polypeptides, and detectable labels (such as, toxins or cytokines, radiolabels or other detectable signals, epitope tags, and imaging compounds) may be conjugated to a MUCl-specific immunoglobulin molecule, such as PHl-IgGl, using standard recombinant DNA methods or in vitro conjugation procedures. The resulting variant is a MUCl-specific immuoglobulin linked to an additional moiety that provides an additional function or label. Such variants can be used as MUCl-specific reagents in various procedures directed or targeted at cancer cells and tissue, especially those directed to tumors found in breast, ovarian, bladder, and lung adenocarcinoma. In addition, variants of recombinant immunoglobulins may also be prepared from all or a portion ofthe V H and V L regions from other MUCl binding members, such as Fab antibodies having improved affinities over the parent PHI molecule (see, Table 9 and Example 2). It is also understood that the MUCl-specific immunoglobulins ofthe invention encompass MUCl-specific immunoglobulin variants, which contain variations in the constant heavy chains ofthe immunoglobulin molecule, including isotypic variants, such as gamma- 1, 2, 3, and 4 isotypes or the alpha- 1 and 2 isotypes, and allotypic (intraspecies allelic) variants, such as allotypic variants of gamma- 1 or of another isotype. The V H and V coding sequences have also been reformatted into a plasmid vector to produce an anti-MUCl diabody molecule, designated bivPHl. As with all diabodies, bivPHl is normally (physiological conditions) a dimer of two monomers, each having the motif "V H -L-V L ", where the linker peptide L is a short peptide (for bivPHl, a pentapeptide having the amino acid sequence of G G G A L (amino acids 122 to 126 of SEQ ID NO:5), which restricts intramolecular formation ofthe MUCl binding domain from the V H and V L regions. The presence ofthe linker peptide drives dimer formation resulting in the intermolecular recreation of two MUCl binding domains. Thus, each bivPHl diabody dimer is a bivalent antibody capable of binding to two identical epitopes ofa MUCl core protein VNTR sequence. The anti-MUCl diabodies of this invention may bind at two identical epitopes in a single MUCl protein or at the same epitope on two separate MUCl molecules. Such binding properties are used to advantage in various therapeutic, diagnostic (including imaging), and purification methods described herein. The invention provides proteins, polypeptides, or peptides that bind MUCl or that form all or part of a MUCl binding domain (such as a V L , V H , or one or more CDRs). Such proteins include fusion proteins that are formed by fusing a selected protein of interest to a MUC1- specific binding member, or portion thereof, such as a V L , V H , or CDR(s) from the PHI Fab antibody described herein. The selected protein of interest may provide the fusion protein with an additional domain useful for purification, diagnostic, or therapeutic application. Thus, the protein of interest for use in a fusion protein ofthe invention may be any protein, or portion thereof, that can be fused, for example, by recombinant DNA methods, to a MUCl-specific binding member, or portion thereof, described herein and that retains its useful function, activity, or other property in the fusion protein. An example ofa fusion protein ofthe invention is an . immunotoxin comprising a MUCl-specific binding portion, such as the bivPH-1 diabody, and a toxin portion, which will be toxic to MUCl-expressing tumor cells. Another example of a fusion protein ofthe invention is an immunocytokine comprising a MUCl-specific binding portion, such as the bivPH-1 diabody, and an active cytokine portion, such as IL-2, as described below. In a further construction, IL-2 was fused to bivPHl diabody to form a fusion protein, which is an immunocytokine molecule, designated bivPHl-IL-2. The bivPHl-IL-2 has IL-2 immunostimulatory activity as demonstrated by the ability to stimulate peripheral blood lymphocytes (PBL) to lyse cells ofthe ovarian carcinoma cell line OVCAR-3 in a standard 51 Cr- release assay. In this assay, the bivPHl diabody did not stimulate lysis by PBL, although the addition of rIL-2 produced a significant increase in killing. The bivPHl -IL-2 immunocytokine enhanced lysis ofthe OVCAR-3 target cells by the PBL more than the level seen in mixtures of bivPHl diabody and rIL-2 (see, Figure 5). Surprisingly, complete killing of tumor cells was achieved using the bivPHl -IL-2 immunocytokine in combination with rIL-2 (Figure 5). The bivPHl-IL-2 immunocytokine is a representative of MUCl-specific immunocytokines that comprise a specific MUCl binding portion fused (conjugated) to an immunomodulatory portion comprising an immunomodulatory protein or peptide, such as a cytokine. The amino acid sequence of bivPHl -IL-2 is shown in SEQ ID NO:5 and a nucleotide sequence encoding the bivPHl-IL-2 immunocytokine is shown in SEQ ID NO:6. Thus, other cytokines could be substituted for the IL-2 immunomodulatory moiety in bivPHl-IL-2, including, but not limited to, GM-CSF and TNF. The MUCl-specific immunocytokines ofthe invention provide a safer or more efficient means of employing cytokines in cancer therapy because the immunocytokine molecule is able to specifically target MUCl -expressing cancer cells for delivery ofthe cytokine. The dosage levels used to see an anti-cancer effect with an unconjugated (free) cytokine may also result in a number of undesirable side effects that may even be life-threatening. However, MUCl-specific immunocytokines described herein offer a means for using a cytokine at a relatively low or less toxic dosage level to achieve a therapeutic anti-cancer benefit compared to treatment methods that employ the free cytokine alone. MUCl-specific immunocytokines may be readily produced by using recombinant DNA techniques in which the V H and V L coding sequences for the PHI Fab antibody molecule are cloned into a diabody expression vector that also provides a site for the insertion and fusion of a coding sequence for the cytokine of interest, as was done for IL-2 (see, Examples for details). Such immunocytokine fusion proteins are particularly useful for targeting MUCl -expressing cancer cells for killing by a lymphocyte population. The therapeutic effect of using an immunocytokine, such as bivPHl-IL-2, may be further enhanced by additionally administering an unconjugated form of a cytokine (free cytokine), or other compounds, to counteract an anergic or suppressor effect on T cells that is often seen in the area of cancer cells or to augment the anti- tumor effect. The immunocytokine bivPHl-IL-2 is also an example ofthe various types of antibody molecules, other than the PHI Fab antibody, that are provided by the invention which comprise the V L region and/or V H region ofthe PHI Fab antibody (SEQ ID NOS:l and 3, respectively), or may contain one or more CDRs ofthe PHI Fab antibody described herein. The MUCl binding members ofthe invention also include derivative proteins that contain amino acid changes (deletions, additions or substitutions) that do not significantly diminish or destroy the MUCl binding property as described for the various examples of MUCl binding members provided herein. Such changes in the amino acid sequence of a MUCl binding member include, but are not limited to, what are generally known as conservative amino acid substitutions, such as substituting one or more amino acids of a V H , V L , CDR, FR, and/or bivPHl-IL-2 amino acid sequence (for example, SEQ ID NOS:l, 3, and 5) with another of similar structure, charge, or hydrophobicity. Any addition or substitution to a MUCl-specific binding member amino acid sequence that maintains MUCl binding, but also improves another property, such as stability in vivo or in situ, is also useful in the diagnostic, purification, or therapeutic methods of this invention. An analysis ofthe PHI Fab antibody revealed that the heavy chain variable (V H ) region is a V H region ofthe DP47 human germ line and that the light chain variable (V L ) region is a V region ofthe DPK15 human germ line (see, Example 1, Table 2). Accordingly, the invention also provides MUCl-specific binding members comprising a MUCl-specific binding domain, which binding domain comprises a V H and/or a V L region, or portion thereof (e.g., one or more CDRs), which is encoded on a polynucleotide sequence ofthe DNA from the DP47 and/or the DPK15 human germ lines. Furthermore, one or more ofthe CDRs described herein may be inserted into the FRs from other known germ lines or other cloned antibody domains for cloning and expressing V L and/or V H , or portions thereof, for example using various recombinant DNA methods, to produce additional forms of MUCl-specific antibody molecules. The invention also provides an isolated MUCl-specific binding member comprising an antigen binding domain, wherein the antigen binding domain comprises an amino acid sequence ofthe formula: X ! X 2 His Thr Gly X 3 Gly Val Tip X 4 Pro X 5 X 6 X 7 (SEQ ID NO:28), wherein X ! is Ala, Ser, Thr, or Val; X 2 is Lys, He Arg, or Gin; X 3 is Gly, Arg, Val, Glu, Ser, or Ala; X 4 is Asp or Asn; X 5 is He, Leu, Met, Phe, or Val; X 6 is Asp, Gly, Lys, Asn, Ala, His, Arg, Ser, Val, or Tyr; and X 7 is Tyr, His, Lys, Asn, Asp, Ser, Pro. Preferably, the MUCl-specific binding member comprises the amino acid sequence selected from the group consisting of: Ala Lys His Thr Gly Gly Gly Val Trp Asp Pro He Asp Tyr (amino acids 97-110 of SEQ ID NO:3); Ala Lys His Thr Gly Arg Gly Val Trp Asp Pro He Gly Tyr (SEQ ID NO:29); Ala Lys His Thr Gly Gly Gly Val Trp Asp Pro He Lys His (SEQ ID NO:30); Ala Lys His Thr Gly Gly Gly Val Trp Asp Pro He Gly Tyr (SEQ ID NO:31); and Ala He His Thr Gly Gly Gly Val Trp Asp Pro He Lys Tyr (SEQ ID NO:32). Such MUCl-specific binding members include any antibody ofthe various known antibody formats, including immunoglobulin, scFv, double scFv, Fab, F(ab') 2 , Fv, dAb, and diabody antibody formats. The invention also provides proteins, polypeptides, and peptides comprising amino acid sequences that are not identical, but are homologous, as defined above, to the particular amino acid sequences described herein. In particular, a homologous protein, polypeptide, or peptide useful in the compositions and methods ofthe invention binds MUCl or forms all or part ofa MUCl-specific binding domain and comprises an amino acid sequence that is about 70 %, preferably about 80%, and more preferably about 90% or more (including about 95%, about 97%, or even about 99% or more) homologous to an amino acid sequence for a MUCl-specific binding member, V L , V H , CDR, or portions thereof, described herein. As mentioned above, the invention also provides MUCl-specific binding members that are variant forms of other MUCl-specific binding members linked to additional domains or molecules, which provide a desirable activity or property. Such variant forms may be formed by linking, preferably covalently, a MUCl-specific binding member molecule described herein to a moiety, such as one or more other proteins or molecules including, but not limited to, a cytokine, a receptor protein, a toxin (e.g., doxorubicin and related drugs, diphtheria toxin, anthrax toxin), an epitope tag (such as a hemagglutinin, polyhistidine, or myc epitope tag), a fluorescein dye, streptavidin, biotin, an enzyme (e.g., horseradish peroxidase (HRP), β-galactosidase, or a site- specific protease), or a radioactive compound, such as 125 I or 99ra Tc, and the like. Linkage ofthe moiety to the MUCl-specific binding member may involve the use of "linker molecule or peptide" that connects the binding member to the moiety. Such variants find use in purification, diagnostic, imaging, and therapeutic methods ofthe invention, particularly those directed to adenocarcinoma tumors in mammary, ovary, bladder, and lung tissue. The invention also provides isolated polynucleotide molecules that encode an amino acid sequence for the various proteins, polypeptides, and peptides described herein that bind MUCl or that form all or part of a MUCl binding domain (such as a V L , V H , or a CDR). Such polynucleotide molecules may be DNA or RNA (wherein in RNA contains uracil instead of thymine). Polynucleotide molecules ofthe invention also comprise degenerate sequences, i.e., nucleotide sequences that differ from sequences specifically listed herein in that they contain different codons that code for the same amino acid according to the genetic code, and therefore encode the same protein, polypeptide, or peptide, e.g., MUCl-specific binding member, V L , V H , and/or portions thereof such as CDRs and FRs. Polynucleotide molecules ofthe invention also include polynucleotide molecules that have nucleotide sequences that are homologous, as defined above, to the particular sequences described herein (e.g., SEQ ID NOS:2, 4, 6, 25, and 27). In one embodiment, a homologous polynucleotide molecules ofthe invention may comprise a nucleotide sequence that is about 60%), preferably about 70%, more preferably about 80%, and even more preferably 90%> or more, homologous to a nucleotide sequence described herein and encodes a MUCl-specific binding member, a MUCl -binding domain, or portion thereof (such as a CDR). A homologous polynucleotide molecule ofthe invention may also comprise a degenerate polynucleotide sequence as described above. Isolated nucleic acid molecules, especially DNA molecules, ofthe invention comprise nucleotide sequences that encode all or a portion ofthe MUCl binding domain ofthe PHI Fab antibody, including the V L and/or V H regions of PHI (SEQS ID NOS:2 and 4, respectively), or one or more CDRs and/or FRs ofthe V L or V H regions. The nucleic acid molecules ofthe invention, which comprise a nucleotide sequence encoding a MUCl binding member or MUCl binding domain, or portion thereof, may be in a variety of forms, including but not limited to, plasmids, which include cloning and expression plasmid vectors used in prokaryotes; phage genomes or phagemids, which include lysogenic phages that may integrate into the bacterial chromosome; eukaryotic expression and cloning plasmid or viral vectors; linear nucleic acid molecules, which include linear DNA or RNA molecules, such as mRNA molecules; and synthetically made nucleic acid molecules. The various nucleic acid molecules described above may be used to produce MUCl- specific binding members ofthe invention using recombinant nucleic acid methodologies. For example, nucleic acid molecules comprising nucleotide sequences described herein may be combined or synthesized in vitro using standard cloning methods or chemical synthesis to encode any ofthe MUCl-specific binding members ofthe invention and then inserted into an appropriate expression vector, such as an expression plasmid, phagemid, or other expression viral vector. A nucleic acid molecule having a sequence encoding the MUCl-specific binding member must be operably linked to a promoter in the expression vector. The recombinant expression vector containing the coding sequence for the MUCl-specific binding member is then placed or inserted, e.g., by transformation, transfection, electroporation, into an appropriate host cell that will express the MUCl-specific binding member encoded on the vector. The host cell may be a prokaryotic or eukaryotic cell depending on the type of expression vector used. In addition, a nucleic acid molecule encoding a MUCl-specific binding member may be operably linked in a display vector to an anchor sequence, which encodes all or part ofa surface protein, so that the expressed MUCl-specific binding member is displayed on the surface of a particular genetic package, i.e., a phage or cell, which includes, but is not limited to, M13- derived phage, M13-derived phagemids, and yeast cells (see, e.g., VanAntwerp et al., Biotechnol. Prog, 16: 31-37 (2000); Wittrup, Trends In Biotechnol, 17: 423-424 (1999); Kieke et al., Proc. Natl Acad. Sci. USA, 96: 5651-5656 (1999)). Such display systems are useful for mutagenizing a gene segment encoding a MUCl-specific binding member (e.g., by introducing alternative CDR sequences) to produce a population of genetic packages, each carrying one member of a family of variant genes and displaying that variant MUCl-specific binding member. From the population of displayed variants, individual variants having a superior property, such as an enhanced avidity or affinity for MUCl, can then be selected by methods known in the art. Preferably, enhancing affinity (affinity maturation) of a MUCl -binding member is carried out using a yeast display vector and an appropriate yeast host cell. Any ofthe various polynucleotide molecules ofthe invention described herein also find use as probes for genes encoding MUCl-specific binding proteins or portions thereof, including alleles or mutated gene sequences encoding corresponding allelic or variant forms of a MUCl- specific binding protein or portion thereof. Diagnostic. Purification, and Therapeutic Methods of Use The MUCl-specific binding members ofthe invention may be used in methods for diagnosing and imaging MUCl -expressing cancer cells and tissue; for purifying or isolating non- glycosylated, underglycosylated, or cancer-associated forms of MUCl or MUCl epitope- containing molecules; and/or for therapeutically or prophylactically treating (i.e., antibody-based passive immunotherapy) MUCl -expressing cancer, such as adenocarcinoma. For diagnosing cancer, such as adenocarcinoma, a sample, such as cells, tissues (e.g., biopsy sample), and/or body fluid (e.g., bone marrow, urine, and/or blood) obtained from an individual is contacted with a MUCl-specific binding member described herein. As noted above, the MUCl-specific binding members of this invention comprise a V L and/or V H region, or portion thereof (such as CDRs), which forms a binding domain for an epitope in the VNTR of the MUCl protein core. Thus, the diagnostic methods described herein may be used to test for evidence of cancer in an individual by detecting binding of a MUCl-specific binding member of this invention to MUCl -expressing cells or tissues or to MUCl present in blood or other fluid of an individual. Such diagnostic methods may be performed completely in vitro, as with many standard clinical diagnostic tests. Alternatively, a diagnostic procedure may be performed in vivo and involve the administration of a MUCl-specific binding member to a individual. The binding ofthe administered MUCl-specific binding member to cells or tissues may then be detected either in vivo (e.g., by imaging methods) or in vitro. A variety of detection systems are available to detect antibody bound to an antigen on cells or tissues or in a fluid, and such detection systems may be employed by the skilled practitioner in the diagnostic methods of this invention to detect bound MUCl-specific binding member. The detection of a bound MUCl-specific binding member will usually involve detecting a signal from a label or tag linked or bound either directly to the MUCl-specific binding member or to a separate detection molecule, which in turn will bind to a MUCl-specific binding member. Whether linked directly to the MUCl-specific binding member or to a separate detection molecule, such labels or tags that are useful in the diagnostic methods of this invention include, without limitation, enzymes, fluorescent labels, radioactive labels, heavy metals, and magnetic resonance imaging (MPJ) labels, such as used for diagnostic tumor imaging. If the label is an enzyme, the binding can be detected by using a substrate that produces a detectable signal, such as a colorigenic, bioluminescent, or chemiluminescent substrate. Enzyme label detection systems include those using the biotin-streptavidin (or avidin) pair, for example, in which the MUCl-specific binding member or a detection molecule is conjugated to biotin (or streptavidin) which in turn will bind to streptavidin- (or biotin) conjugated to an enzyme ofthe detection system, such as β-galactosidase, horseradish peroxidase, or luciferase. For example, a detection antibody linked to a label or tag, such as an enzyme or radioactive label, may also be used to detect a MUCl-specific binding member that has bound to MUCl on the cells or tissues or in the blood or fluid of an individual. The label or tag on the detection antibody is then detected to determine the amount of and/or location ofthe bound MUCl-specific binding member. Various methods for detecting such labeled or tagged molecules are well known to those skilled in the art and include, without limitation, enzyme-linked immunosorbent assay (ELISA) or immunoprecipitation protocols. Such methods may employ fully or semi-automated devices to more efficiently read and process multiple test samples. If the label or tag is radioactive, the detection means is anything that is sensitive to the radioactivity, such as, X-ray film, scintillation counters, Geiger counters, or body imagining or scanning devices, such as magnetic resonance imagining (MPJ) machines. The MUCl-specific binding members of this invention may also be used to purify or extract MUCl protein molecules in a mixture or sample. Procedures that use antibodies for isolating or purifying an antigen may be adapted by substituting a particular MUCl-specific binding member ofthe invention for the conventional antibody component. Such procedures include without limitation direct binding to MUCl molecules in solution followed by precipitation, such as in immunoprecipitations, ELISA, and affinity chromatography. For affinity chromatography, resins may be prepared in which a MUCl-specific binding member of this invention is conjugated to resin particles using methods already established for conjugating immunoglobulins and other binding proteins. As with any affinity resin, the ability to bind a cognate partner or ligand on the resin, such as MUCl molecules, will depend on the availability of exposed MUCl epitopes on the resin particles after conjugation ofthe specific binding member to the resin. The MUCl-specific binding members described herein may also be used as therapeutic or prophylactic reagents to treat cancer, such as adenocarcinoma. MUCl-specific binding members provided herein may be used either in an unmodified form, or as a variant in which a MUCl-specific binding member is bound to, conjugated to, or engineered as a fusion protein to possess another moiety having an effector function that would damage or kill cancerous cells or tissues or that would stimulate or promote an anti-rumor immune response. Thus, the invention provides therapeutic and prophylactic methods of treating cancer, especially adenocarcinoma, in an individual. The methods of treating cancer according to the invention include both in vivo and ex vivo methods. One method of treating adenocarcinoma in an individual comprises administering to the individual a completely human, recombinant, MUCl-specific immunoglobulin, such as PHl- IgGl (see, Example 3). Preferably, the immunoglobulin is also linked to another moiety that provides an anti-cancer function, such as an anti-cancer compound or cell toxin, which only is toxic to cells that bind and internalize the MUCl-specific immunoglobulin. In another treatment method, certain cells are delivered to a MUCl -expressing cancer tumor or cancerous tissue using a MUCl-specific binding member ofthe invention. To deliver cells, such as T cells or killer cells to a MUCl -expressing tumor or tissue, a MUCl binding member may be conjugated or fused to another binding domain, such as a receptor, that specifically binds a marker antigen on the surface ofthe particular cells to be delivered, so that the resultant MUCl binding member now binds to both MUCl and the cells to be delivered. A MUCl-specific immunocytokine of this invention, such as the bivPHl -IL-2 immunocytokine, which is a fusion protein containing an active IL-2 domain, may be administered to an individual to target the IL-2 immunostimulatory function to cancer cells in the body in order to promote a T cell-mediated anti-tumor immune response. The anti-tumor immune response may be further enhanced by also administering one or more doses of a nonconjugated form ofthe same or related cytokine, for example, recombinant IL-2, or another more preferred immunostimulatory compound. Such a supplemental or augmentation dose of a nonconjugated cytokine or other compound may be administered prior to, contemporaneously with, or subsequently to administering the MUCl-specific binding member to the individual. A MUCl-specific binding member of this invention may be used alone or as a component in a more complex anti-cancer regimen, which may include one or more other anti- cancer drugs and/or radiation treatments. Also, multiple treatments may be administered to an individual. Preferably, the particular MUCl-specific binding member used for multiple administrations is a protein or polypeptide molecule of human source, such as PHI Fab, bivPHl- IL-2, or PHl-IgGl antibody, so that the individual's immune system does not raise antibodies that would inactivate or rapidly clear the MUCl-specific binding member from the body. Thus, MUCl-specific binding members described herein, may be used to target a wide variety of anti-tumor effector functions to tumors or pre-cancerous cells and tissues including, but not limited to, the immunomodulatory activity of a cytokine, such as IL-2; an anti-cancer drug; a toxin; a radioactive compound; T cells; killer cells; heavy metals; and other anti-cancer molecules. The MUCl-specific binding members ofthe invention may also be used in ex vivo methods for treating cancer, which deplete or purge MUCl and MUCl -expressing cancer cells from cells, tissues, or body fluids, such as bone marrow, blood, or peripheral blood stem cells. For example, in one preferred embodiment, the ex vivo method of cancer treatment comprises extracting a body fluid containing MUCl and/or MUCl -expressing cancer cells from an individual and contacting the extracted body fluid with a MUCl-specific binding member. Preferably, the MUCl-specific binding member is immobilized on a solid support or surface. The body fluid so treated is thereby depleted or purged ofthe MUCl and/or MUCl -expressing cancer cells and returned to the individual. More preferably, the ex vivo methods of treating cancer ofthe invention comprise using an immobilized MUCl-specific binding member comprising an amino acid sequence selected from the group consisting of SEQ ID NO:l; amino acids 24 to 39 of SEQ ID NO:l, amino acids 55 to 61 of SEQ ID NO:l, amino acids 94 to 102 of SEQ ID NO:l, SEQ ID NO:3, amino acids 31 to 35 of SEQ ID NO:3, amino acids 50 to 66 of SEQ ID NO:3, amino acids 99 to 110 of SEQ ID NO:3, conservatively substituted versions of any ofthe preceding sequences, and combinations thereof. A variety of systems are available that may be used to immobilize a MUCl-specific binding member to a surface. Such systems may involve direct or indirect conjugation ofa MUCl -binding member to a solid surface such as plastic, Sepharose, magnetic or paramagnetic beads, or various other resins. The body fluid taken from an individual may be contacted with the immobilized MUCl-specific binding member in a batch protocol or using a column or other surface containing the immobilized MUCl-specific binding member. Immobilization ofthe MUCl-specific binding member may be done before, during or after the addition ofthe cells, tissues, or body fluid taken from an individual. The ex vivo methods ofthe invention may employ automated, semi-automated, or manually operated devices. In addition, body fluid may be contacted with the immobilized MUCl-specific binding member in a non-continuous or continuous flow system. Furthermore, the extracted body fluid must be kept from contamination and may be further treated to prevent or eliminate contamination by undesirable cells, viruses, chemicals, and/or antigens. In another embodiment, one or more anti-cancer agents, antibiotics, or other therapeutic compounds are added to the depleted or purged body fluid prior to its return to the individual. Such anti-cancer agents may include an MUCl-specific binding member described herein. Pharmaceutical Compositions and Modes of Administration A MUCl-specific binding member is preferably administered to an individual (human or other animal) in a "therapeutically effective amount", which is understood to mean an amount that is sufficient to show a benefit to a patient. Such a benefit may be at least an amelioration of at least one symptom of a cancer, such as adenocarcinoma, including but not limited to, death of tumor cells, stasis of tumor growth, decrease in development of tumor size, decrease in or prevention of metastasis, increase in patient strength or vigor, healthy tissue weight gain, prolongation of survival time, and absence of relapse. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity ofthe cancer being treated. Prescription of treatment and selection of dosages to use for a patient are within the knowledge and responsibility ofthe skilled healthcare practitioner. In addition, appropriate doses of immunoglobulin antibody molecules are well known in the art and provide guidance for deciding on a dose or range of doses of MUCl- specific binding members of this invention to be used in a particular therapeutic regimen (see, e.g., Ledermann et al., Int. J. Cancer, 47: 659-664 (1991); Bagshawe et al., Antibody, Immunoconjugates and Radiopharmaceuticals, 4: 915-922 (1991)). Pharmaceutical compositions or medicaments according to the present invention comprise at least one MUCl-specific binding member provided by the invention as an active ingredient and may comprise, in addition to the active ingredient, a pharmaceutically acceptable excipient, carrier, buffer, stabilizer, or other materials that are well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy ofthe active ingredient. The precise nature ofthe carrier or other material will depend on the route of administration, which may be oral, topical, or parenteral, e.g,, by intravenous or intramuscular injection. The pharmaceutical compositions or medicaments provided by the invention may be prepared in any of a variety of forms particularly suited for the intended mode of administration, including solid, semi-solid or liquid dosage forms, for example, tablets, lozenges, pills, capsules, powders, suppositories, liquids, aqueous or oily suspensions, liposomes or polymer microcapsules or microspheres, syrups, elixirs, and aqueous solutions. Preferably, the pharmaceutical composition is in a unit dosage form suitable for single administration of a precise dosage, which may be a fraction or multiple of a dose, which is calculated to produce an effect on adenocarcinoma tumor cells or the T cell-mediated anti-tumor response ofthe patient. The compositions will include, as noted above, a therapeutically effective amount of a selected MUCl-specific binding member in combination with a pharmaceutically acceptable carrier and/or buffer, and, in addition, may include other medicinal and anti-cancer agents or pharmaceutical agents, carriers, diluents, fillers and formulation adjuvants, or combinations thereof, which are nontoxic, inert, and pharmaceutically acceptable. In liquid mixtures or preparations, a pharmaceutically acceptable buffer, such as a phosphate buffered saline may be used. By "pharmaceutically acceptable" is meant a material that is not biologically, chemically, or in any other way, incompatible with body chemistry and metabolism and also does not adversely affect the MUCl-specific binding member or any other component that may be present in the pharmaceutical composition. For solid compositions, conventional nontoxic solid carriers include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium carbonate, and the like. Pharmaceutically acceptable liquid compositions can, for example, be prepared by dissolving or dispersing a MUCl-specific binding member as described herein and optimal pharmaceutical adjuvants in an excipient, such as, water, saline, aqueous dextrose, glycerol, ethanol, and the like, to thereby form a solution or suspension. If desired, the pharmaceutical composition to be administered may also contain minor amounts of nontoxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like, for example, sodium acetate, triethanolamine oleate. For intravenous injection, or direct injection into a tumor or at a site of affliction, the selected MUCl-specific binding member of this invention will preferably be formulated in a parenterally acceptable aqueous solution, which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as sodium chloride injection, Ringer's injection, lactated Ringer's injection. Preservatives, stabilizers, buffers, antioxidants and/or other additives may be included, as required. Formulations comprising a MUCl-specific binding member described herein may also be prepared for injection or infusion into an individual using pumps or slow drip devices. Also within the scope of this invention, a MUCl-specific binding member may alternatively be prepared as a bolus, which may contain a mordant for gradual release from an injection site. One approach for parenteral administration involves use ofa slow release or sustained release system, such that a constant level of dosage is maintained (see, for example, U.S. Patent No. 3,710,795). Additional embodiments and features ofthe invention will be apparent from the teaching and guidance provided by the following non-limiting examples of MUCl-specific binding members. EXAMPLES The following examples ofthe invention describe production and use of MUCl-specific binding members, such as MUCl-specific Fab antibodies, a fully human anti-MUCl immunoglobulin, and an immunocytokine fusion protein. Such MUCl-specific binding members have an unexpected enhanced avidity for the protein core of MUCl . In addition, MUCl-specific binding members that also comprise an immunomodulatory domain, such as the immunocytokine bivPH-l-IL-2, described below, are able to stimulate T cells and, therefore, counteract MUCl -related inhibition of T cell activation, which is necessary for a T cell mediated anti-cancer immune response Example 1: Selection. Characterization, and Use ofthe Cell Binding Fab PHI Antibody to the core protein of MUCl A MUCl negative murine fibroblast cell line 3T3 and a MUCl -transfected 3T3 cell line 3T3-MUC1 (Acres et al., J Immunother., 14: 136-43 (1993)), a biotinylated MUCl 100-mer peptide with the sequence NH 2 -(PAHGVTSAPDTRPAPGSTAP) 5 -COOH (i.e., containing five copies ofthe sequence of SEQ ID NO:7) (Krambovitis et al., J. Biol. Chem., 273: 10874-10879 (1998)) and a MUCl 60-mer peptide NH 2 -(VTSAPDTRPAPGSTAPPAHG) 3 -COOH (i.e., containing three copies ofthe sequence of SEQ ID NO: 8) (von Mensdorff-Pouilly et al., Tumor Biol, 19: 186-195 (1998)) were used during the selection. A large, naive, human Fab library expressed on phage, containing 3.7 x 10 10 different antibodies (de Haard et al., J. Biol. Chem., 274: 18218-18230 (1999)) was used. Cell selections were carried out as described (Hoogenboom et al., Eur. J. Biochem., 260: 714-&4 (1999)) after depletion with a cell line not expressing MUCl. Briefly, adherent, confluent cells were washed twice with PBS (0.15 M NaCl, 8 mM Na 2 HP0 4 , 7.8 mM KH 2 P0 4 , pH 7.2) and subsequently trypsinized (trypsin/EDTA). Cells and human Fab library were preincubated in 2 g skimmed milk per 100 ml PBS (M-PBS). To deplete fibroblast cell binders from MUCl -transfected cell binders, 5 x 10 13 phages were preincubated with 5 x 10 7 3T3 cells for 1 hour at room temperature in 5 ml M-PBS. Cells were centrifuged (3 minutes at 611 x g), and the supernatant liquid containing the depleted phage library was added to 1 x 10 7 3T3-MUC1 cells for 1 hour at room temperature. Cells were washed 10 times with 5 ml M-PBS/10% fetal calf serum (FCS) and 2 times with PBS. After the last wash, the cell pellet was resuspended in 0.6 ml H 2 0 and phages were released from the cells by the addition of 0.6 ml triethylamine (200 mM). The suspension was neutralized with 0.6 ml 1M Tris-HCl (pH 7.4) and spun down for 5 minutes at 21,000 x g. The supernatant contained the selected phages. Two different selection strategies were compared: 4 rounds of selection on cells or two rounds of selection on cells followed by three more selections on the MUCl 60-mer (to avoid remaining cell binders and/or glycosylated MUCl binders) as described before (Henderikx et al., Cancer Res., 58: 4324-32 (1998)). The latter selection strategy (selections on MUC1- expressing cells followed by selections on the MUCl 60-mer) yielded the PHI Fab antibody described herein. Screening and characterization of clones selected from the Fab library Screening and characterization of cell binding clones by whole cell ELISA, fingerprint analysis, flow cytometry, sequencing, indirect epitope fingerprinting and immunohistochemistry was performed according to the methods we described before (Hoogenboom et al., Eur. J. Biochem., 260: 774-84 (1999), Henderikx et al, Cancer Res., 58: 4324-32 (1998)). For screening purposes, individual clones were picked and transferred to 96-well plate and phage was produced as described in (Marks et al., J. Mol. Biol, 222: 581-597 (1991)). Individual clones of rounds 4 and 5 were tested for their specificity by whole cell ELISA (Hoogenboom et al., Eur. J. Biochem., 260: 774-84 (1999)) on a MUCl-negative murine fibroblast cell line 3T3 and a MUCl- transfected 3T3 cell line. Clones were considered positive in whole cell ELISA when the A 450 (horseradish peroxidase staining reaction) ofthe MUCl -transfected 3T3 cell line was at least 3 times higher than the A 50 ofthe MUCl-negative 3T3 cell line. Positive clones were screened for diversity in fingerprint analysis by polymerase chain reaction (PCR), using primer CH1FOR (5'-GTC CTT GAC CAG GCA GCC CAG GGC-3') (SEQ ID NO:9), from the constant CHI region of Fab antibodies, and pUC-reverse (5'-AGC GGA TAA CAA TTT CAC ACA GG-3') (SEQ ID NO:10), followed by BsMl enzyme digestion and analysis ofthe restriction fragments by agarose gel electrophoresis (Marks et al., J. Mol. Biol, 222: 581-597 (1991), Gussow et al, Nucleic Acids Res., 77: 4000 (1989)). Cell binding of unique positive clones was evaluated by flow cytometric analysis of phage binding pattern (Rousch et al, Br. J. Pharmacol, 125: 5-16 (1998)) on the same cell lines as used during the selection. The V-genes of one Fab, clone PHI, were sequenced using a cycle sequencing kit according to the directions ofthe manufacturer (Edge Biosystems, Gaithersburg, MD). Primers were the same as for fingerprinting. Nucleotide sequences and their corresponding deduced amino acid sequences were aligned and compared to the germ line sequences ofthe Sanger Center Sequence database (http://www.sanger.ac.uk/DataSearch/gq_search.shtml) (Table 2). As shown in Table 2, the V H region ofthe PHI Fab antibody is a V H region from the DP47 germ line and the V L region is a V L region from the DPK15 germ line. The selection strategies used here are compared with selections on MUCl that were previously described (see, Table 1; de Haard et al, JBiol Chem., 274: 18218-18230 (1999), Henderikx et al. Cancer Res., 58: 4324-32 (1998)). Likewise, the further characterization ofthe clones and constructs was performed by methods previously described (see, Henderikx et al. Cancer Res., 58: 4324-32 (1998)) and are specified only briefly herein. Table 1 : Selections for specific MUCl antibodies with a very large Fab library on cells compared with previously published selections ScFv library" Fab library b Method of selection Nr Abs Nr Cell k off (s " ') Nr Abs Nr Cell k ff Cs "1 ) binders (range) binders (range) MUCl peptide coated on tubes 3 0 - Biotinylated MUCl 100-mer 5 C IO-'- IO- 14 b 0 ιo- 3 -ιo- < MUCl expressing cells 0 C 0 0 MUCl expressing cells/coating d 0 6 1 10 "2 - 10 "3 a (Vaughan et al, Nat Biotechnol, 14: 309-314 (1996)), (de Haard et al, JBiol Chem., 274: 18218- 18230 (1999)), c (Henderikx et al. Cancer Res., 58: 4324-32 (1998)), d coating + cell selection for scFv library or cell selection + coating for Fab library, e not done Table 2 : Deduced amino acid sequence of MUCl specific antibody PHI compared with genn line sequences" FRl CDRl FR2 CDR2 10 20 30 40 50 60 DP47 ΞVQLLESGGGLVQPGGSLRLSCAASGFTFS SYAMS WVRQAPGKGLEWVS AISGSGGSTYYADSVKG PHl a q vq R -N-G- G FR3 CDR3 FR4 70 80 90 DP47 RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK (SEQ ID NO: 18) PHl a HTGGGVWDPIDY WGQGTLVTVSS (SEQ ID NO: 3) FRl CDRl FR2 CDR2 10 20 30 40 50 60 DPK15 DIVMTQSPLSLPVTPGEPASISC RSSQSLLHSNGYNYLD WYLQKPGQSPQLLIY LGSNRAS PHl a e—1 T S-H FR3 CDR3 FR4 70 80 90 100 DPK15 GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC MQALQTP (SEQ ID NO: 19) PHl a V R FT FGPGTKVDIKR (SEQ ID NO:l) "lower case, primer encoded mutations; upper case, amino acid mutations. FR, framework region; CDR, complementarity determining region. Specificity of MUCl cell binding was tested in flow cytometry on the murine fibroblast cell lines 3T3, the 3T3 MUCl -transfected line ETA, the breast carcinoma line T47D, the ovarian carcinoma line OVCAR-3, and the colon cancer cell line LS174T. The relative amounts of antibodies were compared using dot blots. The same amount of scFv, PHI, and bivPHl, and 3 times less bivPHl -IL-2 was used, as determined in dot blot. MUCl specificity was confirmed by preincubation of the antibodies with 100 μg/ml ofthe synthetic MUCl 60-mer for 1 hour at room temperature. Tumor tissue binding was evaluated by immunohistochemistry on paraffin embedded tissues of breast, ovarian and colon carcinoma and normal tissues. Fine specificity was measured by indirect epitope fingerprinting (Henderikx et al. Cancer Res., 58: 4324-32 (1998)). Generation of a bivalent diabodv-IL-2 fusion protein bivPHl-IL-2. an immunocytokine MUCl- specific binding member The Fab antibody PHI was chosen for the construction ofa dimeric, bivalent antibody fused to IL-2. The cloning schedule for the immunocytokine into a bacterial expression plasmid is shown schematically in Fig. 1. The first cloning step included the insertion into plasmid pCANTAB6 (McGuinness et al. Nature Biotechnol, 14: 1149-54 (1996)), digested with Sfil and EcolU, of two fragments: (1) the heavy chain variable region (V H ) of PHI (as Sfil-BsiEII restriction fragment), and, (2) a region from the diabody vector pDial (as a BsiEΣL-EcoRl fragment), (Roovers et al, 1999, unpublished) providing the unique restriction site Notl and the myc-tag (GAA CAA AAA CTC ATC TCA GAA GAG GAT CTG AAT GGG GCC GCA (SEQ ID NO:21), encoding the myc-tag amino acid sequence EQKLISEEDLNGAA (SEQ ID NO:20)), and a polyhistidine ("hexaHis") tag (i.e., CAT CAC CAT CAT CAC CAT (SEQ ID NO:23), encoding the six-histidine amino acid sequence HHHHHH (SEQ ID NO:22)) to yield plasmid pC6-PHl-VH (Fig. IB). The tags are needed as handles for subsequent detection and purification ofthe diabody. In a second step (Fig. 1C), a two-step PCR was performed with a first amplification ofthe V L -C L ofthe parental Fab PHI with primers V L backward 35: 5'-ACC GCC TCC ACC AGT GCA CTT GAA ATT GTG CTG ACT CAG TCT CC (SEQ ID NO: 11) and V L forward: ACC GCC TCC ACC GGG CGC GCC TTA TTA ACA CTC TCC CCT GTT GAA GCT CTT (SEQ ID NO: 12). A second PCR ofthe V L was performed with primers designed to add a 5 amino acid linker (LI) and restriction sites needed for following cloning steps. A linker of 5 residues favors the folding of scFvs as a diabody (Rousch et al, Br. J. Pharmacol, 125: 5-16 (1998)). The primers were: PHI V L backward: 5' GCCGATCGCTCTGGTCACCGTCTCAAGCGGAGGCGGTGCACTTGAAATT GTGCTGACTCAG (SEQ ID NO: 13) and PHI V L forward: 5' GTCTCGCGAGCGGCCGCCGA TTGGATATCCACTTTGGTCCCAGGGCCGAA) (SEQ ID NO: 14). This PCR product was cloned into the pC6-PHl-VH via BstElVNotl, resulting in plasmid pKaPal . From this vector, the antibody fragment PHI will be expressed as a bivalent MUCl specific diabody bivPHl (Fig. 1C). In a third step, we fused IL-2 to the diabody construct (Fig. ID). The template for the PCR amplification ofthe IL-2 encoding gene was obtained by reverse-transcriptase-PCR (RT-PCR), (kit supplied by Perkin Elmer, Branchburg, N.J.), of total RNA (RNAzol, Campro Scientific, Veenendaal, The Netherlands) derived from PBL stimulated with phytohaemagglutinin (PHA) for 8 h for maximal expression of IL-2 (Fan et al, Clin. Diagn. Lab. Immunol, 5: 335-40 (1998)). The IL-2 gene was inserted in the diabody vector between PH1V L and the tag - encoding fragment (i.e., the myc-tag followed by the six-histidine peptide tag), through NotVEcoΕN, resulting in a phage vector, pKaPa2, encoding a secreted diabody-IL-2 fusion protein (bivPHl-IL-2) (see, Fig. ID). ScFv-IL-2 fusion proteins with linkers between 4 and 13 residues (Melani et al. Cancer Res., 58: 4146-54 (1998), Savage et al, Br, J. Cancer, 67: 304-10 (1993)) have been described. A nine amino acid encoding linker (GGG GGT GGA TCA GGC GGC GGG GCC CTA) (SEQ ID NO: 15) was chosen in order to avoid potential steric hindrance between the two antigen binding sites ofthe diabody and IL-2 and to minimize enzymatic cleavage. This sequence was primer encoded (PH1-IL-2 backward: 5' ACCAAAGTGGATATCAAACGAGGGGGTGGATCAGGCGGCGGGGCCCTAGCACCTAC TTCAAGTTCTACA (SEQ ID NO: 16); PH1-IL-2 forward: 5' GTCCCGCGTGCGGCCGCAGT CAGTGTTGAGATGATGCTTTGACAAAAGG) (SEQ ID NO: 17)). BIAcore analysis of scFv. Fab, and bivalent antibody fragments The selected Fab PHI and other antibody constructs were evaluated by surface plasmon resonance on a BIAcore 2000 apparatus (Pharmacia). A CM-5 chip was coated with the MUCl 80-mer (containing four copies ofthe amino acid sequence of SEQ ID NO:7) at a density of 90 or 800 response units (RU) in 10 mM acetate buffer at pH 4.6. An empty, activated and subsequently deactivated surface was used as a negative control. The Fab fragment PHI, scFv 10A (Henderikx et al. Cancer Res., 58: 4324-32 (1998)), and the engineered diabody fragments were injected in HBS buffer (Pharmacia, Uppsala, Sweden). To minimize rebinding ofthe antigen binding fragments, a flow rate of 20 μl/s was used. Purification of antibody fragments For assays, involving cell culture, antibody fragments were purified by immobilized metal affinity chromatography (Roovers et al, Br. J. Cancer, 78: 1407-16 (1998)). Free IL-2 present in the final product was removed by ultra-filtration against PBS in a centrifugal concentrator (3000 rpm) (M r cut-off 30 000, Centricon, Millipore, Bedford, MA). The volume was reconstituted by the addition of PBS to the maximal capacity ofthe concentrator and the sample was concentrated again by centrifugation. The reconstitution and concentration was repeated twice. Absence of non-conjugated IL-2 was checked by sodium-dodecyl-sulfate polyacrylamide gell electrophoresis (SDS-page) and Western-blot. IL-2 assays IL-2 concentrations ofthe bivPHl -IL-2 construct and the IL-2 control (Boehringer, Mannheim, Germany) were quantitated by means of ELISA for the purpose of later use in in vitro stimulation assays. The ELISA was performed following the directions ofthe supplier (Endogen, Woburn, MA). The activity ofthe bivPHl -IL-2 was measured by stimulation of an IL-2 dependent murine T cell line CTLL-16 (Heeg et al, J. Immunol. Methods., 77: 237-46 (1985), Gillis et al, J. Immunol, 120: 2027-32 (1978)). Cells, cultured in RPMI 1640 (10% FCS, 100 U IL-2 per ml), were washed 3 times. 10 4 cells per well were incubated with increasing concentrations ranging from 0.2-4 pg/ml of rIL-2 or bivPHl-IL-2 in round bottomed microtiter plates (Corning Costar, Kennebunk, Maine). After 24 h of incubation in a humidified incubator at 37C, 5% C0 2 stimulation of human PBL was tested by the addition of 0.5 μCi/well [3] H- thymidine to the culture media. Cells were harvested after overnight incubation and incorporation of radioactivity was measured. To study the MUCl related inhibition on PHA stimulated PBL (Agrawal et al, Nat. Med., 4: 43-9 (1998)), PHA (10 μl/100 μl) was added to 100,000 freshly prepared PBL from healthy donors/100 μl RPMI, 10% FCS/ well in round bottomed microtiter plates. To test inhibition of T cell stimulation by MUCl, 25 μg/ml MUCl-lOOmer peptide was added. To test the reversal by IL-2 of this inhibition, 60 U/ml IL-2 or bivPHl-IL-2 was added. The MUCl- specific MAb 1G5 was used as a positive control. Cells were incubated for 6 days at 37°C, 5% C0 2 in a humidified incubator followed by 3 H-thymidine labeling, harvesting and counting ofthe cells as described above. Cytotoxicity assay The cytotoxic activity of PBL as effector cells towards the MUCl expressing target population, ovarian carcinoma cell line OVCAR-3, was measured by 51 Cr-release assay. Target cells were preincubated in PBS alone or in PBS with 5 μg/ml bivPHl or bivPHl-IL-2 30 minutes prior to the 60 minute incubation with 1 mCi/ml/10 6 cells 5I Cr at 37C. Incubation volumes were 100 μl. Target cells were washed 3 times and resuspended in RPMI, 10%FCS at 5000 cells/50 μl and seeded into a flat bottom microtiter plate. PBL (50 μl) were added at a target (5000 cells/50 μl/well) to effector ratio (T/E) of 1:100, 1:50, 1:25 and 1:12.5. Maximum release was determined by the addition of Tween-20 to the target cells. For measurement of minimal release, no PBL were added to the target cells. To measure the influence of IL-2, 100 U/ml IL-2 was added to the appropriate wells. After overnight incubation, cells were harvested with a supernatant harvesting system and the released 51 Cr was counted in a γ scintillation counter. Percent (%) of lysis was measured as 100 x (cpm test 51 Cr released - cpm minimal 51 Cr released/cpm maximal 51 Cr released - cpm minimal 51 Cr released). Tests were performed in triplicates and repeated at least three times. IL-2 activity retained in bivPHl -IL-2 immunocytokine The gene cassette encoding the bivalent antibody was fused to the human IL-2 gene. The fusion protein (bivPHl-IL-2) had retained the binding characteristics in BIAcore as bivPHl and flow cytometry (Figs. 3A and 3B) and showed the same binding pattern in immunohistochemistry. In flow cytometry, bivPHl-IL-2 was not competed off with the MUCl 60-mer peptide although a lower concentration of bivPHl -IL-2 was used than for the other antibodies (Figs. 3A and 3B). Comparison of bivPHl-IL-2 to rIL-2 showed that the immunocytokine has the same specific activity as commercially available rIL-2 (Fig. 4), the diabody bivPHl did not stimulate this IL-2 dependent cell line (data not shown). This is in accordance to the results observed by others studying similar immunocytokines (Melani et al. Cancer Res., 58: 4146-54 (1998), Gillies et al, Proc. Natl Acad. Sci. USA, 89: 1428-32 (1992)). The bivPHl -IL-2 stimulated human PBL proliferation to the same extend as native rIL-2 (Fig. 5). In an attempt to reverse MUCl -related inhibition of stimulated PBL by IL-2 as described (Agrawal et al, Nat. Med., 4: 43-9 (1998)), we added the MUCl 100-mer together with PHA and IL-2 to PBL. No inhibition ofthe stimulated lymphocytes by MUCl was detected. It was possible to kill tumor cells by resting PBL when target cells were coated with bivPHl -IL-2 (Fig. 6). Moreover, upon the addition of IL-2 to the cultures, bivPHl -IL-2 as well as bivPHl coated target cells were affected. It has previously been shown that the principal cause of antibody-IL-2 fusion protein (IgG-IL-2) mediated killing by resting PBL in vitro is due to the induction of NK activity by interaction of FcγRIII on NK cells with the constant region ofthe antibodies (Naramura et al, Immunol Lett, 39: 91-9 (1994), Gillies et al. Cancer Res., 59: 2159-66 (1999)). However, this cannot be the explanation ofthe enhanced killing observed in these experiments since no Fc region is present on neither bivPHl nor bivPHl-IL-2. The data suggest that the killing ability is influenced by several modes of action. First, the immunocytokine brings T cells in close proximity to tumor cells through interaction ofthe immunocytokine with both the IL-2 receptor and MUCl. Secondly, the MUCl antibody covers potential inhibiting epitopes on the cellular MUCl and thereby prevents inhibition of T cells. And thirdly, the IL-2 part ofthe immunocytokine rescues T cells from anergy. This direct killing of tumor cells mediated by •resting PBL is influenced by antibody binding to the cells, which is obviously not caused by antibody dependent cell-mediated cytotoxicity (ADCC) through the Fc receptor on NK cells. Selection and characterization of human anti-MUCl antibodies (MUCl-specific binding members) from large non-immunized scFv and Fab phage libraries As a starting point, a fully human anti-MUCl antibody was selected from a large non- immunized human Fab library using phage display technology (de Haard et al, J. Biol. Chem., 274: 18218-18230 (1999)). Since the efficiency of immunocytokines improves when repetitive injections are administered (Melani et al. Cancer Res., 58: 4146-54 (1998)), it is important to use components with a minimal immunogenicity for the immunocytokine. The use of human antibody phage libraries allows the retrieval of human anti-MUCl antibodies (Henderikx et al. Cancer Res., 58: 4324-32 (1998), Griffiths et al, EMBOJ., 12: 725-734 (1993)), and permits design and engineering ofthe antibody format (size, affinity or avidity, multivalency, clearance, etc.) and effector functions for the chosen application (de Haard et al. Adv. Drug Del. Rev., 31: 5-31 (1998), Hoogenboom, Trends in Biotechnol, 15: 62-70 (1997)). To obtain an adenocarcinoma specific, high affinity/avidity antibody binding to MUCl present on cells, a very large, non-immunized (naive) Fab library was used, containing 3.7xl0 10 different antibodies, on a MUCl -transfected cell line (3T3-MUC1). These cell selections were compared with previously published selections on biotinylated synthetic MUCl peptide with the same library (de Haard et al, J. Biol. Chem., 274: 18218-18230 (1999)) and with a large scFv library with 6xl0 9 different scFv (Henderikx et al. Cancer Res., 58: 4324-32 (1998), Vaughan et al. Nature Biotechnol, 14: 309-314 (1996)) (Table 1). When selections were run using an ELISA system with coated MUCl 100-mer peptide, antibodies were only recovered from the scFv library. In contrast, selections were successful with both the scFv and Fab libraries when a biotinylated antigen was used and selection was carried out in solution. The antibodies that were isolated from the scFv library have been described previously (Henderikx et al. Cancer Res., 58: 4324-32 (1998)): briefly, 5 different antibodies were found, with scFv 10A and 10B exhibiting the highest ELISA signal, and binding specifically to adenocarcinoma tissue; both have a relative quick off-rate (best k off : 10 "2 s "1 ) in BIAcore. In terms of number of different antibodies selected and the best off-rate, the Fab library was superior: 14 different antibodies were found, with the best off-rate in the 10 "4 s "1 range. Nevertheless, none ofthe obtained Fabs bound to cells in flow cytometry. Most likely, the flexible peptide displays selection-dominant epitopes (Hoogenboom et al, Eur. J. Biochem., 260: 774-84 (1999)) that drive the selection away from less abundant, possibly conformational epitopes on MUCl, which are present on the cell surface. When MUCl expressing cells were used for selections, even after depletion with MUCl negative cells, no MUCl -peptide specific Fab antibodies were found. When using similar conditions with the scFv library, no MUCl specific antibodies were detected. Furthermore, using a combination of selections on, first, coated MUCl 100-mer, followed by panning on the MUCl-expressing cell line T47D with the scFv library, no new MUCl specific antibodies were obtained, nor were the scFv cell binding antibodies 10A and 10B, which are nevertheless known to be present in the library, obtained. Therefore, the selection strategy was reversed: the first two rounds were carried out on MUC1- transfected 3T3 cells, after an initial depletion step on non-transfected 3T3 cells, and rounds 3 to 5 were performed using coated MUCl 60-mer. After the 4 th selection round with the Fab library, 6 different antibodies, based on the BstNI fingerprint pattern, were identified with one pattern dominating the population (58%, represented by clone PHI). In the 5 th round of selection, 92% ofthe ELISA positive clones had the PHI -clone pattern. In flow cytometry ofthe representative clones of each ofthe six Fab-DNA fingerprints, only Fab PHI bound to ETA MUCl-expressing cells. By BIAcore analysis, human Fab antibody PHI was shown to have a slower off-rate than any ofthe antibodies retrieved from the scFv library (k off : 10 "3 s "1 ) and was, therefore, further characterized. By indirect epitope fingerprinting (Henderikx et al. Cancer Res., 58: 4324-32 (1998)), the minimal binding epitope was determined to be the tripeptide Pro Ala Pro ofthe MUCl protein core (data not shown). By DNA sequence analysis, the V H ofthe PHI human Fab antibody was found to be derived from the germ line segment DP47, and the V L was found to be derived from the germ line sequence DPK15, both with a small number of amino acid mutations (see, Table 2). The nucleotide and corresponding amino acid sequences for the V H region from PHI are shown in SEQ ID NOS:4 and 3, respectively. The nucleotide and corresponding amino acid sequences for the V L region of PHI are shown in SEQ ID NOS:2 and 1, respectively. The sequence data revealed the framework (FR) and CDR sequences ofthe PHI V H and V L regions (see, e.g. Table 2). In addition, these sequences are not related to the sequences of other anti- MUCl antibodies cloned by this laboratory (de Haard et al, J. Biol. Chem., 274: 18218-18230 (1999), Henderikx et al. Cancer Res., 58: Ai' l -i' l (1998)) or by others (Griffiths et al, EMBO J., 12: 725-734 (1993)). Construction, expression and biochemical analysis of bivalent anti-MUCl diabody and immunocytokine molecules With the selected PHI Fab antibody V genes, a fully human immunocytokine ofthe general formula (V H -L-V L )-IL-2 was constructed, in which the PHI V H and V L regions are covalently linked to one another via a linker peptide L, and the V H -L-V L moiety is covalently linked at its carboxy terminal amino acid to the amino terminal amino acid residue of an IL-2 protein. The desired anti-MUCl immunocytokine molecule was designed to have several particularly advantageous properties: (1) to be larger than the 45 kD scFv-IL-2 molecular weight, (i.e., above the renal filtration threshold) for obtaining a longer circulation half-life, (2) to possess an avidity advantage in its binding to MUCl, by having two distinct binding sites on the same molecule, which, unlike the monovalent PHI Fab antibody, fully exploits the multimeric nature ofthe MUCl antigen, and (3) to not have an Fc receptor binding domain (i.e., CH2 and CH3 domains of IgG not present), which was recently shown to interfere negatively with the efficacy of antibody-IL-2 fusion products (Gillies et al. Cancer Res., 59: 2159-66 (1999)). Such properties were attained by constructing a bivalent diabody-IL-2 fusion of 90 kD molecular weight (see, Fig. 1). The V genes were reformatted in the diabody V H -lmker-V L format (Holliger et al, Proc. Natl. Acad. Sci. USA., 90: 6444-8 (1993)). The short, 5 amino acid residue linker (LI) drives the preferential formation of diabodies, i.e., two single-chain Fv molecules that are paired non-covalently to form a dimer with two functional binding sites. The bivalent diabody gene cassette was subsequently fused to the human IL-2 gene. The bivPHl diabody and the bivPHl-IL-2 diabody immunocytokine fusion proteins were both expressed in E. coli, and both fusion proteins were purified from the periplasmic extract using immobilized metal affinity chromatography (IMAC). The binding characteristics ofthe Fab PHI and scFv 10A antibodies were compared with the two diabody constructs, i.e., the bivalent bivPHl diabody and the bivalent bivPHl-IL-2 immunocytokine fusion in BIAcore (Fig. 2). The bivalent diabodies bound with off-rates at least 10 times stronger as compared to the Fab binding (k off : 10 "3 s "1 ). These binding characteristics were measured on synthetic MUCl 80-mer peptide chips (with 90 RU immobilized antigen). The relative off-rate ofthe bivalent diabody molecules measured under these optimal conditions was below 10 "4 s "1 . This relative off-rate was dependent on the conditions of measurement, such as antigen-density on the chip. The 20 amino acid peptide of MUCl was repeated 30 to 100 times on cells, in a variable number of tandem repeats (Swallow et al. Nature, 328: 82-4 (1987)). The avidity effect ofthe bivalent bivPHl antibody on cells was expected to be at the least ofthe same magnitude due to binding and rebinding effects on the same molecule. Hence, the binding effect ofthe monovalent versus bivalent antibodies was measured on cells in flow cytometry (Figs. 3A and 3B). The bivPHl diabody bound considerably better to the MUCl -transfected 3T3 cell line, the ovarian carcinoma cell line OVCAR-3, and the breast cancer cell line T47D, than the scFv 10A and the PHI Fab antibodies, although the same amounts of scFv, PHI and bivPHl were used. This binding was one log higher when bivPHl was compared to scFv 10 A and about 0.5 log better when compared to Fab PHI. This stronger binding to cells was confirmed by preincubation ofthe antibodies with the MUCl 60-mer where the inhibition of antibody cell binding by the MUCl 60-mer was complete in the case ofthe scFv 10A antibody, almost complete in the case ofthe PHI Fab antibody, and partial in the case ofthe bivPHl diabody. This partial inhibition was not due to non-specific binding since none ofthe antibodies bound to the non-transfected murine fibroblast cell line 3T3 nor to the highly glycosylated colon cell line LS147T. Inhibition by the MUCl 60-mer peptide was less pronounced in the case ofthe T47D cell line than in the case ofthe OVCAR-3 cell line. Effector function ofthe bivalent human immunocytokine bivPHl -IL-2 Because ofthe relative short distance between the two MUCl binding regions and the IL-2, it was important to test whether this fusion format would impair the IL-2 activity. Therefore, an IL-2 dependent murine T cell line (CTLL-16) was stimulated with increasing amounts of bivPHl -IL-2 and the stimulatory efficiency was compared with that of commercial available recombinant IL-2 (rIL-2). As shown in Fig. 4, both rIL-2 and bivPHl-IL-2, stimulated the murine T cell line with an equal activity, while bivPHl did not stimulate (data not shown); similarly, rIL-2 and bivPHl -IL-2 stimulated PBL equally well (Fig. 5). In an attempt to verify whether IL-2 and bivPHl -IL-2, were able to reverse the MUCl -related inhibition of T cells, PBL were incubated with PHA and MUCl for 6 days and tried to reverse the inhibition. However, no inhibition of T cell activation by MUCl was observed so that reversal of inhibition could not be studied using this protocol. To prove the functionality of both sites ofthe immunocytokine, a 51 Cr-release assay was performed (Fig. 6). The MUCl expressing target cells OVCAR-3 were preincubated with bivPHl or bivPHl -IL-2 and washed. Resting PBL did not mediate lysis ofthe target cells and the addition of 100 U/ml rIL-2 was not efficient in improving the lysis. The bivPHl diabody did not significantly increase the level of lysis, though with the addition of rIL-2, lysis rose considerably above the background level (p < 0.05). The bivPHl-IL-2 immunocytokine fusion protein enhanced the lysis of target cells by resting PBL more than the non-fusion combination bivPHland rIL-2 (p < 0.03), proving that the MUCl binding site as well as the effector site is functional (see, Fig. 6). Moreover, with the addition of rIL-2 to the immunocytokine coated target cells, complete killing was achieved (p < 0.001). No killing was observed when the colon cell line LS174T, not binding PHI in flow cytometry (Fig. 3B), was used as a target in a similar assay (data not shown). Half-life of dissociation of bivPH-l-IL-2 immunocytokine The PHI Fab antibody was chosen as the source of V H and V regions to construct an immunocytokine because ofthe PHI cell binding properties in flow cytometry, adenocarcinoma associated immunohistological staining pattern, and the slowest off-rate of all the clones tested. For antibody-mediated immunotherapy, increasing evidence has accumulated that high affinity ofthe antibody is important for antibody-mediated killing (Velders et al, Br. J. Cancer, 78: 478- 83 (1998)); similarly, increased binding due to avidity can benefit tumor uptake of recombinant antibody fragments (Adams et al. Cancer Res., 53: 4026-34 (1993)). The off-rate ofthe monovalent PHI Fab on coated 80-mer in BIAcore was 10 "3 s-1, which indicates that a similarly monovalent effector molecule would have a half-life of dissociation from the antigen of 11 minutes. Therefore, an improvement of binding strength was desirable. Since MUCl has a variable number of tandem repeats, the goals were: (1) to improve the avidity by making a bivalent form ofthe PHI Fab (bivPHl) and (2) to obtain the dissociation effect as described for multivalent receptors (Goldstein et al, Immunol. Today, 17: 77-80 (1996)). Indeed, in BIAcore, the bivPHl diabody antibody molecule had a more than 10 times slower off-rate: the half-life of binding improves on this antigen surface from about 11 minutes to 2 hours (see, Fig. 2). The bivalency effect ofthe bivPHl diabody antibody molecule described herein was similarly dramatic on cells that express a VNTR of MUCl when measured by flow cytometry (see, Fig.3). Binding intensity increased by approximately 1 log compared with the scFv 10A and 0.5 log compared with the PHI Fab antibody molecule. Moreover, this binding was not easily competed off by 100 μg/ml ofthe MUCl 60-mer peptide, confirming the importance ofthe number of repeats in the MUCl molecule for the retention binding. The kinetics of dissociation of antibodies from multivalent receptors expressed on the cell surface such as MUCl, has been studied extensively. If no rebinding occurs, the half-life of dissociation ofthe complex, described by the formula t 1/2 * l/k off (InN - ln(ln2) + In2/2N), increases with the valency ofthe antigen (N) (Goldstein et al, Immunol. Today, 17: 77-80 (1996)). The t 1/2 (half-life of dissociation) for bivPHl-IL-2 immunocytokine on cellular MUC lean be calculated using this formula and the value of k off measured on BIAcore. Presuming the MUCl glycoprotein has 100 tandem repeats, this would result in an estimated half-life for dissociation of 14 hours. Furthermore, the rebinding ofthe antibodies is additionally affected by the density ofthe antigen (MUCl) on the cell surface (Goldstein et al, Biophys. J., 56: 955-66 (1989)), which is overexpressed in a variety of adenocarcinomas (Burchell et al. Cancer Res. ,47: 5476-5482 (1987)). Accordingly, the tumor dissociation half-life ofthe bivPHl-IL-2 immunocytokine on cells will be substantially higher than 2 hour. In conclusion, the bivPHl -IL-2 not only directs IL-2 to the tumor site and activates T cells, but also covers potentially inhibitory epitopes, which are desired properties for improving tumor cell killing and further preventing anergy of stimulated T cells in cancers, such as adenocarcinoma. Example 2: Affinity Maturation of Human MUCl -Specific Monovalent PHI Fab Antibody This example demonstrates the use of phage display methodology to carry out an in vitro selection (i.e., affinity maturation) for Fab antibodies containing monovalent binding sites with an enhanced affinity for MUCl from libraries of mutated heavy chain molecules from the PHI Fab antibody described above. Mutagenesis was directed toward residues in the heavy chain CDRl and CDR2 regions that are frequently mutated in vivo (known as "hot spots" of in vivo mutagenesis), and toward the complete heavy chain CDR3 region. Escherichia coli (E. coli) TGI: K12, O(lac-pro), supE, thi, hsdD5/F' traD36,proA*B + , lacF, lacZDM15 was used as the host in the phage display affinity selection procedure. Preparation of V libraries (a) CDR3 libraries The V L C L of PHI was cloned as an ApaLl-Ascl fragment in the phagemid pCESl vector (de Haard et al, 1999), to yield pCES-PHl-VL. The V H of PHI was amplified using primers #206 and one ofthe mutagenic CDR3 primers, as indicated below (see, Table 3). The PCR products were cloned as an Sβl-BstEU fragment in pCES-PHl-VL. (b) Hotspot library In a first PCR, the CDRl and the CDR2 libraries, were prepared with the PH1-VH as template using the primer pair #701 and #87 and primer pair #206 and #702, respectively (see Table 3). The DNA encoding these libraries were combined by a PCR assembly reaction using primers #206 and #87 and the resulting VH-genes cloned as a Sfil-BstEH fragment in pCES- PH1-VL for phage display and selection. Table 3: Ohgonucleotides Used in Affinity Maturation of MUCl Binding Domain of PHI (a) Primers used for introduction of mutations #701 Hotspot CDRl oligo 5' -GGA TTC ACG TTT AGA A*G*T* AAC GCC ATG GGC TGG-3' (SEQ ID NO: 33) #702 Hotspot CDR2 oligo 5' -CAC GGA GTC TGC GTA G*T*A* TGT G*C*T* GCC ACC ACT ACC ACT-3' (SEQ ID NO: 34) CDR3 spiked oligo 5'-CTA TGA GAC GGT GAC CAG GGT TCC CTG GCC CCA G*T*A* G*T*C* A *A*T* G*G*G* G*T*C* C*C*A* A*A*C* G*C*C* C*C*C* C*C*C* G*G*T* A * T *G* τ*T*T* C*G*C* ACA ATA ATA TAC GGC-3' (SEQ ID NO: 35) CDR3 random oligo 1 5'-CTA TGA GAC GGT GAC CAG GGT TCC CTG GCC CCA GTA GTC AAT GGG GTC CCA AAC MNN MNN NN MNN MNN TTT CGC ACA ATA ATA TAC GGC-3' (SEQ ID NO:36) CDR3 random oligo 2 5'-CTA TGA GAC GGT GAC CAG GGT TCC CTG GCC CCA GTA GTC MNN MNN MNN MNN MNN GCC CCC CCC GGT ATG TTT CGC ACA ATA ATA TAC GGC-3' (SEQ ID NO: 37) Asterisked nucleotides indicate the following mixtures : A*=90%A + 2.5%A + 2.5%C + 2.5%G + 2.5%T C*=90%C + 2.5%A + 2.5%C + 2.5%G 4- 2.5%T G*=90%G + 2.5%A + 2.5%C + 2.5%G + 2.5%T T*=90%T + 2.5%A + 2.5%C + 2.5%G 4- 2.5%T (b) Primers used for amplification of V H of PHI Fab antibody #87 , HuJH4-5 -FOR 5' -TGA GGA GAC GGT GAC CAG GGT TCC-3 ' (SEQ ID NO: 38) #206, VHlc back Sfi 5 ' -GTC CTC GCA ACT GCG GCC CAG CCG GCC ATG GCC SAG GTC CAG CTG GTR CAG TCT GG-3' (SEQ ID NO: 39) Nucleotide ambiguity codes: M = A or C; R = A or G; S = C or G; N = A, C, G or T underlined sequences indicate encoded restriction sites Affinity selection (a) Selection on biotinylated MUCl Selections were perfonned on biotinylated MUCl 60-mer as described by Hawkins et al. (J. Mol. Biol, 226: 889-96 (1992)) with some modifications: phage were incubated on a rotator wheel for 1 hour in 2% M-PBST (PBS supplied with 2% skimmed milk powder and 0.1% Tween-20). Meanwhile, 100 μl of streptavidin-conjugated paramagnetic beads (Dynal, Oslo, Norway) were incubated on a rotator wheel for 2 hour in 2% M-PBST. Biotinylated antigen was added to the pre-incubated phage and the mixture was incubated on a rotator wheel for 30 minutes. Next, the streptavidin-beads were added and the mixture was left on the rotator wheel for 15 minutes. After 14 washes with 2% M-PBST and one wash with PBS, phage particles were eluted with 950 μl 0.1 M triethylamine for 5 minutes. The eluate was neutralized by the addition of 0.5 ml Tris-HCl (pH 7.5) and was used for infection of log-phase E. coli TGI cells. The TGI cells were infected for 30 minutes at 37° C and were plated on 2xTY (16 g Bacto-trypton, 10 g yeast extract and 5 g NaCl per liter) agar plates, containing 2% glucose and 100 μg/ml ampicillin. After overnight incubation at 30° C, the colonies were scraped from the plates and used for phage rescue as described (Marks et al, J. Mol Biol. 222, 581-597 (1991)). (b) Selection on MUCl-expressing cells Alternating selections were performed on the T47D breast cancer cell line (Hanisch et al, 1996) and on the OVCAR-3 ovarian carcinoma cell line, both are known to express tumor- associated glycoforms of MUCl. Briefly, 10 12 phage and cells (10 7 T47D, 5 x 10 6 OVCAR, 2 x 10 6 T47D and 2 x 10 6 OVCAR for rounds 1, 2, 3 and 4, respectively) were preincubated with 2% M-PBS (PBS supplied with 2% skimmed milk powder) for 30 minutes; then phages were added to the cells. After 1 hour of incubation, cells were washed 10 times with M-PBS + 10% FCS. Specific phage were eluted and used for infection of exponentially growing TGI cells as described earlier. ELISA and kinetic measurement using surface plasmon resonance in BIAcore Soluble Fabs were produced as described (Roovers et al, Br. J. Cancer, 78: 1407-16 (1998)). ELISAs were performed as described by Henderikx et al (Cancer Res., 58: 4324-4332 (1998)), except the biotinylated MUCl 60-mer was used. The selected PHI and the affinity- matured antibodies were evaluated for affinity by surface plasmon resonance (SPR) on a BIAcore 2000 apparatus (BIAcore AB, Uppsala, Sweden). Channels of a biotin chip were coated with a MUCl 15-mer, containing the minimal PHI epitope, PAP, (Ac-PDTRPAPGSTAPPAL- NH 2 , (SEQ ID NO:40) 50 RU or 320 RU) or a 60-mer (NH 2 -(VTSAPDTRPAPGSTAPPAHG) 3 - COOH (i.e., containing three copies of SEQ ID NO:8 (von Mensdorff-Pouilly et al. Tumor Biol, 19: 186-195 (1998), 50 RU) in HBS-EP buffer (Pharmacia). One surface was blocked with biotin (15 RU) and used as a negative control. The antibodies were injected in HBS-EP buffer. To minimize rebinding ofthe antigen binding fragments, the flow speed was 30 μl/sec. Affinity calculation was performed with the BIA-evaluation software provided by the BIAcore. The affinities ofthe Fabs were calculated according to a 1 :1 stoichiometry at steady state. DNA sequencing The nucleotide sequences ofthe selected Fabs were determined using dideoxy sequencing. Products ofthe sequencing reaction were analyzed on a semi-automated sequencer (Alf Express; Pharmacia). The oligonucleotide used for VH sequencing was CH1FOR: 5'-GTC CTT GAC CAG GCA GCC CAG GGC-3' (SEQ ID NO:9). FACS analysis Specific binding ofthe Fabs was measured by FACScalibur analysis (Becton Dickinson, Oxnard, CA) as described by Henderikx et al. (Cancer Res., 58: 4324-4332 (1998)). For affinity studies on cells with recombinant Fabs, the following flow cytometry experiment was carried out. Fab fragments were purified from the periplasmic fraction by IMAC and gel filtration as described in (Roovers et al, Br. J. Cancer, 78: 1407-1416 (1998)). Protein concentrations were measured with the bicinchoninic acid method (Sigma, St. Louis, MO, USA). Two-fold serial dilutions of these Fabs were made and incubated, for each dilution point, with 2 x lOVlOO μl ETA cells (transfected 3T3 cells (Acres et al, J. Immunol, 14: 136-1443 (1993)). After trypsinisation, cells were washed one time in RPMI 10% FCS, 0.1% NaN 3 (incubation buffer). Then, cells were incubated with appropriate dilution for 1 hour at room temperature (RT), on a rotator. As negative controls, 2 x 10 5 3T3 mouse fibroblast cells were also incubated with the highest concentrations of antibodies. As a secondary negative control, ETA cells without primary antibody were used. Cells were spun down by centrifugation for 3 minutes at 611 x g. Between incubations, cells were washed with incubation buffer. In a second incubation, anti- Myc antibody (6 μg/ml 9E10), directed against the Myc-tagged Fabs, was added for 30 minutes in incubation buffer at RT. After washing once, rabbit anti-mouse-FITC was used at RT for 30 minutes (Dako). Detection of bound antibodies was performed by means of flow cytometry on a FACSCalibur (Becton Dickinson, Oxnard), and results analyzed with the CELLQuest program (Becton Dickinson). Mean intensity was plotted against the concentration ofthe antibodies. Results and Analysis The affinity maturation selection procedure employed in this study involved mutagenesis to the variable region ofthe heavy chain ofthe PHI Fab antibody, and within this VH to two types of residues: (1) the residues which frequently confer a higher affinity to the antibody- antigen interaction in vivo ("hot spots"): residue 31 in VH-CDRl and residues 56 and 5, in the VH-CDR2; and (2) the CDR3 regions, which sits at the heart ofthe antigen combining site, and mutagenesis of which frequently results in higher affinity antibodies (Hoogenboom, Trends Biotechnol, 15: 62-10 (1997) ). Specifically, four different libraries were assembled: one CDRl -2 hot spot library (HSPOT), with mutations at amino acid positions 31, 57, 59 of SEQ ID NO:3; and three libraries for the heavy chain CDR3 (H-CDR3). The HSPOT library was made by assembly-PCR of two DNA fragments, one with the CDRl region harboring a spiked residue 31, the other with a CDR2 region with residues 57 and 59 spiked and a wild-type CDR3, and cloning this VH gene for expression with the PHI light chain as Fab fragments displayed on phage (see, HSPOT CDRl and HSPOT CDR2 ohgonucleotides in Table 3). Since the H-CDR3 has a length of 12 amino acid residues, the theoretical diversity in this region is 20 12 . Two different RANI and RAN2 libraries were made, with only 5 amino acid residues in each library completely randomized (see, CDR3 random ohgonucleotides 1 and 2 in Table 3). The theoretical diversity of these individual libraries would therefore be 3.3 xlO 6 , represented by 3.3 x 10 7 variants in a library with 32 possibilities per codon. To access additional diversity in the neighboring residues ofthe CDR3, in the H3 region at amino acid residues 97 and 98 of SEQ ID NO:3, as well as in the last two joining-region encoded residues ofthe CDR3, amino acid residues 109 and 110 of SEQ ID NO:3, a library called SPIKE was made in which ohgonucleotides (spiked at a level of 7.5% of mutant nucleotides with 92.5%) wild-type) were used to mutagenize a region of 14 residues. The CDR3 - libraries were made by PCR with mutant ohgonucleotides (see, CDR3 spiked oligo in Table 3) of the VH of PHI Fab antibody and cloning ofthe resulting DNA as an Sβl-BstEU. fragment into pCESl-PHl-VL. All actual library sizes were over 10 8 clones (see, Table 4). Table 4. Libraries Library V H Region FR 3 CDR 3 FR4 Size NT pattern/mut. freq. % positive clones CAK HTGGGVWDPIDY G per mutant clone in phage ELISA RANI ***** 1.8 x 10 s NN(T/G) 1/20 RAN2 ***** 2.0 x lO 8 NN(T/G) 3/20 SPIKE ** ************ 2.1 x 10 s 4/42 8/20 HSPOT (wt) 3.1 x 10 s 2/9 17/20 * = mutagenized, (wt) = wild type (PHI) sequence Clones from the unselected libraries were analyzed by sequencing to confirm the mutagenesis pattern, and by ELISA to test for binding to the MUCl antigen. Not surprisingly, a high frequency ofthe clones ofthe HSPOT library were positive as phage antibody for MUCl binding: most are indeed wild type in sequence (data not shown), and this library has only 8000 variants spread over three residues. Similarly the SPIKE library yields a high frequency of antigen binding variants of PHI, here though with 2-3 amino acid alterations per clone (see, Table 5). It was more striking to find many ELISA positives (detectable signal at OD 450 ) in the unselected RANI and RAN2 libraries, where a complete stretch ofthe CDR3 is altered (Table 5). It should be kept in mind that the use of phage particles which can display multiple antibodies per particle, promotes avid binding in this ELISA, and affinity differences between clones are readily masked. Table 5. Sequences of unselected clones V H Domains --FR3-- CDR3 --FR4-- Clone PHI A K H T G G G V D P I D Y G (97-112 of SEQ ID NO: 3) gcg aaa cat ace ggg ggg ggc gtt tgg gac ccc att gac tac tgg ggc (289-336 of SEQ ID NO: 4) ELISA OD 450 =0.692 RANI library 2 A K H N T S K V W D P I D Y W G (SEQ ID NO: 41) gcg aaa cat aat acg tct aag gtt tgg gac ccc att gac tac tgg ggc (SEQ ID NO: 42) _>D 450 =0.115 A K S S T T T V W D P I D Y W G (SEQ ID NO: 43) gcg aaa tct agt act acg acg gtt tgg gac ccc att gac tac tgg ggc (SEQ ID NO: 44) DD 450 =0.138 A K & P M A N V W D P I D Y W G (SEQ ID NO: 45) gcg aaa tag cct atg gcg aat gtt tgg gac ccc att gac tac tgg ggc (SEQ ID NO: 46) ->D 450 =0.361 A K & H T K T V W D P I D Y W G (SEQ ID NO: 47) gcg aaa tag cat acg aag acg gtt tgg gac ccc att gac tac tgg ggc (SEQ ID NO: 48) K I T V S R V W D P I D Y W G (SEQ ID NO: 49) gcg aaa att act gtt agt cgt gtt tgg gac ccc att gac tac tgg ggc (SEQ ID NO: 50) B9 A K R Y L Y D V W D P I D Y W G (SEQ ID NO: 51) gcg aaa cgt tat ctg tat gat gtt tgg gac ccc att gac tac tgg ggc (SEQ ID NO: 52) RAN2 library 10 A K H T G G G T L Q R L D Y W G (SEQ ID NO: 53) gcg aaa cat ace ggg ggg ggc act ttg cag egg ctg gac tac tgg ggc (SEQ ID NO: 54) OD 450 =0.103 11 A K H T G G G T Q T P C D Y W G (SEQ ID NO: 55) gcg aaa cat ace ggg ggg ggc act cag act ccg tgt gac tac tgg ggc (SEQ ID NO: 56) OD 450 =0.100 13 A K H T G G G R R I C H D Y W G (SEQ ID NO: 57) gcg aaa cat ace ggg ggg ggc cgt cgt att tgt cat gac tac tgg ggc (SEQ ID NO: 58) OD 450 =0.4 1 15 A K H T G G G & R & & R D Y W G (SEQ ID NO: 59) gcg aaa cat ace ggg ggg ggc tag egg tag tag egg gac tac tgg ggc (SEQ ID NO: 60) OD 4S0 =0.175 D6 A K H T G G G Q K L Q L D Y W G (SEQ ID NO: 61) gcg aaa cat ace ggg ggg ggc cag aag ctg cag ctg gac tac tgg ggc (SEQ ID NO: 62) SPIKE library 20 A &/S H T G G R G W D P I D Y W G (SEQ ID NO: 63) gcg tsa cat acg ggg ggg cgc ggt tgg gac ccc att gac tac tgg ggc (SEQ ID NO: 64) OD 450 =0.109 21 A N Q T G G G V W D P I D Y W G (SEQ ID NO:65) gcg aac cag act ggg ggg ggc gtt tgg gac ccc att gac tac tgg ggc (SEQ ID NO: 66) OD 450 =0.108 22 A R H T G G G V W D P I Y Y W G (SEQ ID NO: 67) gcg aga cat ace ggt ggg ggc gtk tgg gat ccc ata tac tac tgg ggc (SEQ ID NO: 68) OD 450 =0.663 23 A K P T G G G A W D P I D Y W G (SEQ ID NO: 69) gcg aaa cct ace ggg ggg ggc get tgg gac ccc att gac tac tgg ggc (SEQ ID NO: 70) 25 A K H T G V G V W H P I Y Y W G (SEQ ID NO: 71) gcg aaa cat ace ggg gtg ggc gtt tgg cac ccc ate tac tac tgg ggc (SEQ ID NO: 72) OD 4S0 =0.315 mutated residues indicated in bold; nucleotide "k" indicates residue may be guanine or thymine; "&" indicates end of amino acid sequence because mutation in nucleotide sequence forms a translational stop codon (tga, taa, tag) The three CDR3 libraries did contain a low frequency of clones with the wild-type sequence ofthe PH1-VH (4/21 clones with the mix ofthe 3 libraries; see, Table 6), most likely due to pass-through ofthe original pCESl-PHl-Fab used as PCR-template; provided higher affinity variants of PHI are present in the libraries, these wild-type phage should not cause any problems in the affinity maturation process. The bacterial stocks containing the PHl-based libraries were rescued with helper phage and phage subjected to various selection regimens. To sample these libraries as fully as possible as well as probe cellular MUCl, three different selection conditions were followed, including (a) selections on decreasing amounts ofthe MUCl peptide, (b) selections using the antibody PHI as a competitor, and (c) selections on whole cells. Selection on MUCl peptide First phage from the rescued RANI, RAN2, and SPIKE libraries were mixed and selected on biotinylated MUC-1 60-mer peptide, which contains three times the 20-mer repeat sequence ofthe MUCl-1 sequence. Three rounds of selection with decreasing amounts of antigen (60-mer) were performed; the data on this approach are depicted in Table 6. Table 6. Selections ofthe CDR3 -libraries (mix) on biotinylated MUCl 60-mer Round input [60-mer] I/O % pos. Clones % WT representative (nM) (input/output) (Fab ELISA) seq. clones clones Unselected 6/30 4/21 I 6.3 x 10 ut 10 1.2 x lO 5 18/30 6/12 3B10 II 1.2 x lO 12 10 4.3 x 10 3 26/30 8/18 1 4.3 x 10 4 9/20 3/7 3D6 III 2.9*10 12 * 0.1 1.5 x lO 6 12/30 2/10 0.01 5.2 x lO 6 8/30 1/5 t of each library; * of 1 nM selection; WT = wild type An important issue was how to determine the concentration of antigen for selection. The Fab PHI has an affinity of 1.4 micromolar (μM) for the 60-mer peptide antigen with a very fast off-rate, yet it was selected from a naive phage antibody library. Most likely avidity caused by display of multiple Fabs on the surface ofthe phage particles contributed to its selection. Since the affinity constant for Fab binding to a 15-mer MUC-1 peptide with just once the epitope ofthe antibody, is identical to that of binding to the 60-mer (data not shown), the multivalent nature of the antigen appears to have no significant role. Prior work indicated that antigen concentrations can be 100 to 1000-fold lower than the Kd ofthe antibody, and selection is still possible (Schier et al, J. Mol Biol, 263: 551-567 (1996)). Thus, the first round of selection was carried out using the 60-mer peptide at an initial concentration of 10 nM, and, thereafter, decreasing this number 10-fold in subsequent steps as indicated in Table 6. Since we do not known what the spread of affinities of clones in the library, the correct concentration can only be determined empirically. In the first and second selection, a sharp decrease in the ratio Input/Output (I/O) ofthe phage titers was noted, but further selection in round 3 with less antigen showed an increase again. Similarly, the frequency of positive clones in Fab ELISA increased first to nearly 90% in round 2 when 10 nM antigen was used, and 45% when only InM was used; the frequency decreased again in the third round of selection with 100 and 10 picomolar (pM). This indicated that under these conditions, many lower affinity clones failed to be selected. It was possible that under those conditions the highest affinity clones should become enriched preferentially. This was confirmed by the initial selection and later decrease ofthe frequency of clones with a wild- type sequence. Competitive Selection In a second approach we attempted to select the higher affinity variants of PHI using competition with the wild-type PHI Fab fragment. Libraries were now separately selected on the biotinylated MUC-1 60-mer in the presence of 0.2 or lμM ofthe PHI Fab fragment. After 6 hours of co-incubation of phage, antigen and soluble competitor, the phage that remained bound to the biotinylated antigen were retrieved using streptavidin-coated beads. Phage titers and selection data are summarized in Table 7. Table 7. Selections on biotinylated MUCl in the presence of soluble PHI Fab Round [60-mer] Library I [PHI] I/O % pos. Clones %WT representative (nM) (μM) (Fab ELISA) clone I 10 RANI 6.4 x lO 10 1 7.2 x 10" 1/20 1/1 0.2 9.6 x 10" 0/20 RAN2 5.2 x lO 10 1 2.9 10" 1/20 0.2 4.0 x 10" 0/20 SPIKE 1.6 x 10" 1 1.2 x lO 5 1/20 0/1 0.2 7.6 x 10" 1/20 0/1 5C8 HSPOT 6.4 x lO 10 1 5.8 x 10" 6/20 3/3 0.2 4.6 x 10" 6/20 5/5 πt 1 RANI 2.6 x lO 12 1 2.3 x 10 6 0/25 RAN2 3.7 x lO 12 1 6.2 x 10 6 1/25 0/1 SPIKE 2.6 x lO 12 1 2.5 x 10 6 6/25 1/6 7D1/7F3, 7F9 HSPOT 3.8 x lO 12 1 1.2 x lO 6 10/25 7/9 I = input; O = output; f = outputs from the 1 μM PHI competition selection were used As expected, the I/O ratio decreased when compared to the selection without competition (Table 6), but more strikingly, the frequency of MUCl -positive clones dropped dramatically (from 60%, 18/30 in Table 6) for the mix to 5% (3/60, Table 7) for the individual libraries), indicating that the selection with competition worked. Similarly, the frequency of MUC-1 positives selected from the HSPOT library decreased after 1 selection round (compare frequencies in Tables 4 and 7), but these clones are still wild-type sequence, which of course dominate the unselected library. In the second selection round with only 1 nM MUCl antigen also from the HSPOT library, clones appeared that were not wild type. Selections on cells The two other procedures led to the isolation of variants with up to a 3.5-fold increase of the Kd for the MUCl peptide (see below). However, there was a possibility that there were variants in the libraries that would more strongly recognize the cellular MUCl, but show only a minor improvement ofthe affinity towards the peptidic MUCl antigen. Therefore, as an alternative to the selections on MUCl peptide, cells expressing the (partially glycosylated) form ofthe MUCl antigen were used in a selection. To prevent the unlikely yet theoretically possible selection of clones for antigens other than MUCl, the selections were alternated between two cell lines, the T47D breast carcinoma and OVCAR ovarian carcinoma cell lines. The selection data are depicted in Table 8. Table 8. Alternating selections on T47D and OVCAR cells Library Round Cell line Input (I) Output (O) I / O % positive clones % wild type (Fab ELISA) RANI I T47D 3.4 x lO 12 1.4 x lO 7 2.4 x lO 5 4/20 1/1 II OVCAR 2.2 x lO 12 3.5 x 10 6 6.3 x 10 5 0/20 III T47D 2.3 x 10 12 2.6 x 10 7 8.8 x 10" 15/20 0/5 IV OVCAR 2.5 x lO 12 5.2 x lO 7 4.8 x 10" 2/20 0/1 RAN2 I T47D 3.0 x lO 12 2.3 x 10 7 1.3 x 10 5 3/20 II OVCAR 2.4 x lO 12 3.2 x lO 6 7.5 x 10 5 1/20 III T47D 2.5 x 10 12 1.2 x 10 s 2.1 10" 19/20 0/5 IV OVCAR 2.1 x lO 12 1.0 x lO 8 2.0 x 10" 6/20 0/2 SPIKE I T47D 3.2 x lO 12 1.4 x 10 s 2.3 x 10" 3/20 0/1 II OVCAR 1.0 x lO 12 1.6 x lO 7 6.3 x 10" 5/20 III T47D 1.4 x lO 12 1.8 x lO 9 7.8 x 10 2 20/20 0/7 IV OVCAR 1.7 x lO 12 3.3 x 10 8 5.2 x lO 3 20/20 0/5 HSPOT I T47D 3.0 x lO 12 2.2 x lO 8 1.4 x 10" 0/20 II OVCAR 1.2 x lO 12 5.2 x lO 6 2.3 x 10 5 1/20 III T47D 1.5 x lO 12 1.9 x 10 s 7.9 x lO 3 18/20 1/4 IV OVCAR 1.2 x lO 12 2.7 lO 7 4.4 x 10" 15/20 2/6 Despite some variability, the input-output (I/O) ratio ofthe phage titers did not really increase over the course of four cell selections. Yet an increase was seen in the frequency of clones binding to the MUCl peptide, as well as the appearance of non-wild type clones in all selected libraries (Table 8). Upon sequencing it was revealed that all ofthe clones from the RAN libraries were derived from the SPIKE library, most likely due to cross-contamination between libraries. This suggests that in the RAN libraries, there are not many high affinity variants of PHI present. Analysis of representative clones for sequence and affinity in BIAcore and FACS Clones from the many different selection rounds were screened initially in BIAcore for improvement of binding towards the MUCl peptide. From a large screening effort, in which a few hundred clones were screened from the various selection approaches, the best clones were identified for further characterization. An overview ofthe characterized PHI CDR3 variants from all rounds of selection is given in Table 9. Table 9. Characterization ofa large panel of PHI variants relative Clone ELISA FR3-CDR3 region Freq. in sel. SEQ ID NUMBER signal WT-PHl + (+ AK HTGGGVWDPIDY 97-110 of SEQ ID NO: 3 5C8 +++ -- ---R G- SEQ ID NO: 29 7D1 +++ -- KH SEQ ID NO: 30 7F9 +++ -- G- SEQ ID NO: 31 7F 3 +++ -I K- SEQ ID NO: 32 10C10 +++ -- ---V K- SEQ ID NO: 73 11C1 +++ -- ---E K- SEQ ID NO: 74 3 A7 +++ -- K SEQ ID NO: 75 6B6 +++ -- G- SEQ ID NO: 76 10B 3 +++ -- G- 3x SEQ ID NO: 76 11G9 +++ -- G- SEQ ID NO: 76 10A8 +++ -R G- SEQ ID NO: 77 6F4 +++ S- G- SEQ ID NO: 78 6B 3 +++ __ GH SEQ ID NO: 79 10F9 +++ ._ N--GH SEQ ID NO: 80 3 B9 +++ -- LG- SEQ ID NO: 81 3B10 +++ -- -N SEQ ID NO: 82 3D8 +++ -- N- 2x SEQ ID NO: 83 6B9 +++ -- N- SEQ ID NO: 83 3D10 +++ -- N- 2x SEQ ID NO: 83 6F3 +++ -- N- SEQ ID NO: 83 7D8 +++ -- N- SEQ ID NO: 83 10B6 +++ -- N- 8x SEQ ID NO: 83 11E3 +++ -- N- 3x SEQ ID NO: 83 11B9 +++ -R N- SEQ ID NO: 84 6A9 +++ S N- SEQ ID NO: 85 6C8 +++ -- ND SEQ ID NO: 86 11F7 +++ -_ v MN- SEQ ID NO: 87 11F9 T- N- SEQ ID NO: 88 3E2 +++ -- A- SEQ ID NO: 89 6B5 +++ -- A- SEQ ID NO: 89 7B5 +++ -- A- SEQ ID NO 89 8F5 -- A- SEQ ID NO 89 10E1 +++ -- A- SEQ ID NO 89 3F4 +++ -- AN SEQ ID NO 90 3H1 +++ -- FA- SEQ ID NO 91 11D4 +++ -- MAS SEQ ID NO: 92 3H2 +++ -- M— SEQ ID NO: 93 6C10 +++ -- H- SEQ ID NO: 94 11F2 +++ -I ---A R- SEQ ID NO: 95 11F4 +++ -- SS SEQ ID NO: 96 3G1 -- D SEQ ID NO: 97 6C5 ++ V- V- SEQ ID NO: 98 6E4 + (+) -- v— SEQ ID NO: 99 10F3 + (+) -- VP SEQ ID NO: 100 10C3 + (+) V- A- SEQ ID NO: 101 10A9 + (+ ) -- HN SEQ ID NO: 102 10F8 + (+) -- MH- SEQ ID NO: 103 10E10 + (+) SEQ ID NO: 104 5A6 + V- SEQ ID NO: 105 3B8 + SEQ ID NO -.106 3D7 + SEQ ID NO: 106 3D1 + -Q G- SEQ ID NO: 107 3F3 + SEQ ID NO: 108 3F7 + -- Y- SEQ ID NO: 109 The first number ofthe clone name in Table 9 indicates its origin: 3-4, directly selected on MUCl antigen; 5-6-7-8, selected with PHI competition; 10-11, cell selected. The clones were ranked according to their relative ELISA signal (as soluble Fab fragments). Sequencing of the clones revealed that most ofthe observed variability in the clones with the strongest signals targeted a few residues in the CDR3 only, and were nearly exclusively found as derived from the SPIKE library. Indeed, the residues most frequently mutated in these clones, were not targeted in the RAN libraries. Within these clones, there is a strong conservation visible of most ofthe core region ofthe CDR3, the regions randomized in the RAN libraries, with a lot of mutations visible in the FR3 region and the J-encoded region ofthe CDR3. In many clones residues K98 (in SEQ ID NO:3) and/or D109 (in SEQ ID NO:3) are frequently mutated, thereby most likely disrupting the putative salt bridge between these charged amino acids. Not all substitutions are allowed; for example mutations to valine or alanine may disrupt this salt bridge, but do not confer a higher affinity. There was some variability at position 1 ofthe VH, caused by use of oligonucleotide #206, which allows either glutamate (E) or glutamine (Q) to be incorporated; often both variants were found carrying the same mutations in the FR3-CDR3 region, but this never affected the affinity (data not shown). There was little bias in the diversity ofthe clones selected with the three different procedures (direct selection, selection using competition with a MUCl peptide antigen, or selection on cells). Indeed, variants with a single substitution at position 109, to glycine (G) or to asparagine (N) are frequently selected in all selection procedures (Table 9). From the HSPOT library two variants were tested: clone 7G8 (from the competition selection) and clone 10G9 (from the cell selection). Both had a mutation at position 31 in the CDRl ofthe VH, more specifically S31N and S31R for 7G8 and 10G9, respectively. The ELISA signal for these clones did not reach the signals seen for most CDR3 variants, and the clones were not further analyzed. The CDR3 variants were extensively tested in BIAcore for the kinetics ofthe MUCl peptide interaction. The off-rate ofthe wild-type clone could not be determined because it was too fast for analysis; however, based on its Kd (1.4 micromolar), an improvement ofthe off-rate of over 10-fold should result in a detectable change in off-rate in this assay. Using this off-rate screening with Fabs in the periplasmic extract of is. coli cultures, the clones in Table 9 as well as many more were screened for improvement in off-rate. The best clone, 5C8, was derived from the competition selection (Table 7), and showed a clear increase in off-rate. To get accurate measurements, a Kd assay on the BIAcore was used with the MUCl 15-mer (there was no difference in the kinetics of interaction when the MUCl 60-mer was used, data not shown). Clone 5C8 showed a 3.5-fold increase ofthe Kd over wild type PHI Fab antibody (see, Table 10). Some other candidate clones, including 3D6, from the direct selection, and three other clones from a more stringent competition selection clone (i.e., 7D1, 7F3 and 7F9) were extensively investigated using BIAcore and/or flow cytometry analysis. The BIAcore data in Table 10 highlight the data of both the Kd values for peptide binding in BIAcore as well as the sequence differences between these clones; of all variants, clone 5C8 appears to have the best affinity. A single mutation D109G, as in clone 7F9, yields less than a 2-fold improvement, but an additional G102R mutation, as in clone 5C8, brings the affinity gain to 3.5-fold. A flow cytometry experiment was carried out to determine the relative affinities of these Fabs versus wild type on cellular MUCl, although the data are not directly comparable (data not shown). The relative ranking ofthe three clones from highest to lowest affinity (i.e., 7D1 >7F3>7F9) appeared to have stayed the same, but the positioning of both the wild type clone PHI and best BIAcore mutant 5C8 appearef to be different from what was expected on the basis ofthe BIAcore affinity. This apparent discrepancy between the binding affinity for the MUCl peptide and for the cell surface MUCl is most likely caused by the effect of partial glycosylation ofthe antibody epitopes of MUCl glycoprotein on cells, which may effect binding in a different manner depending on the antibody fine specificity and interaction with the MUCl antigen. Although it appeared preferable to select and screen antibody affinity variants on cellular MUCl, rather than on a peptide source ofthe antigen, the selection ofthe PHI -based antibody libraries on cells did not yield any higher affinity variants than 5C8. Most ofthe MUCl peptide binding selected variants, as well as selected clones without detectable peptide binding, harbored sequence variations that were found in clones selected on MUCl peptide (Table 9 and data not shown). Example 3. Production and Characterization of a Recombinant. Human MUCl -Specific Immunoglobulin Molecule PHI -IgG As described above in Example 1, the MUCl-specific PHI Fab antibody was selected from a very large phage library displaying 3.7 x 10 10 Fab antibody molecules. The PHI Fab antibody has a Kd of 1.4 micromolar (μM) in BIAcore analysis using the MUCl 60-mer peptide antigen. This example demonstrates a method to increase the apparent affinity of a Fab antibody ofthe invention for cellular MUCl expressed on cancer cells and tissues by changing the format from the single (monovalent) antigen binding site ofthe Fab antibody to the two (divalent) binding site format of an immunoglobulin molecule, such as IgG. As described below, a completely human, recombinant PHl-IgGl antibody molecule was made by cloning the V H and V L genes of PHI into a mammalian expression vector system (Persic et al. Gene, 187: 9-18 (1997)). The recombinant expression vectors were then cotransfected into mammalian CHO-K1 cells for expression. Cloning the V H and V τ of PHI Fab antibody into a human IgG molecule The heavy and the light chains (i.e., V H and V L ) ofthe PHI human Fab antibody were recloned into the mammalian VHexpress and VKexpress expression vectors, respectively, for producing a fully human gamma-1/kappa IgGl antibody (Persic et al. Gene, 187: 9-18 (1997)). The V H fragment of PHI was amplified by PCR using specific ohgonucleotides VHIC Back eukaryotic (5'-GGA CTA GTC CTG GAG TGC GCG CAC TCC CAG GTC CAG CTG GTG CAG TCT GGG GGA GGC TTG GTA CAG-3' (SEQ ID NO:l 10)) and M13 commercial sequencing primer (Amersham Pharmacia, Upsala, Sweden), and introduced into the VHexpress vector as BssΗIUBstΕll fragment. An ApaLl/Pacl fragment of PHI V L was generated by PCR using specific oligonucleotides VKexpress-MUC-for (5 '-GCG CTC GCA TTT GCC TGT TAA TTA AGT TAG ATC TAT TCT ACT CAC GTT TGA TAT CCA CTT TGG TCC CAG GGC C-3' (SEQ ID NO:l 11)) and MUCl-VL-Back-APA (5'-CCA GTG CAC TCC GAA ATT GTG CTG ACT CAG TCT CC-3' (SEQ ID NO:112)), and inserted into VKexpress. Transfections of CHO-K1 (ATCC, Manassas, VA) cells were carried out using a non-liposomal transfection reagent FuGene 6 (Roche, Brussels, Belgium) according to manufacturer's instructions. Screening of cell culture supernatants in ELISA Supernatants of clones growing on medium containing selection markers were tested in ELISA for antibody binding to MUCl and to determine V H V L production levels. For MUCl binding tests, the method of Henderickx et al. (Henderickx et al. Cancer Res., 58: 4324-4332 (1998)) was adapted for use in this study. Incubation volumes were 100 μl. MUCl peptide antigen (i.e., 0.5 μg/ml biotinylated MUCl 60-mer) was immobilized indirectly on a flexible microtiter plate via streptavidin bound to biotinylated BSA, which was coated on the wells ofthe microtiter plate. Immobilizing MUCl 60-mer was done overnight at 4° C. After three washes with PBS, plates were blocked by incubating 30 minutes at room temperature (RT) with 2% (w/v) skimmed milk powder (Marvel) in PBS. Plates were washed two times with PBS-0.1% Tween 20 and once with PBS, and supernatants were then incubated for 1.5 hours at RT while shaking (diluted 1 :4 in 2% (w/v) Marvel/PBS). Subsequently, plates were washed five times with PBS-0.1% Tween 20 and once with PBS. Bound IgG was detected with rabbit anti-human HRP-conjugated IgG (1 :6000 diluted in 2% Marvel/PBS). Following the last incubation, staining was performed with tetramethylbenzidine (TMB) and H 2 0 2 as substrate and stopped by adding 0.5 volume of 2N H 2 S0 4 . The optical density was measured at 450 nanometers (nm). Production of human IgG To determine the amount of human PHl-IgGl produced, a plate was coated for 1 hour at 37° C with 0.25 μg/ml rabbit anti-human VK immunoglobulin in PBS. After three washes with PBS, plates were blocked during 30 minutes at RT with 2% (w/v) semi-skim milk powder (Marvel) in PBS. Plates were washed two times with PBS-0.1% Tween 20 and once with PBS. Supernatants were then incubated for 1.5 hour at RT while shaking (diluted 1:4 in 2% (w/v) Marvel/PBS). A 2-fold dilution series of human IgG (hulgG) was used as a standard, starting with a concentration of 500 ng/ml. Subsequently, plates were washed five times with PBS-0.1 % Tween 20 and once with PBS. Bound IgG was detected with rabbit anti-human IgG HRP (1 μg/ml in 2% Marvel/PBS). Following the last incubation, staining was performed with tetramethylbenzidine and H 2 0 2 as substrate and stopped by adding 0.5 volume of 2N H 2 S0 4 ; the optical density was measured at 450 nm. Production and purification ofthe PHl-IgGl from culture media of CHO-K1 clone 7F cells Approximately 3 x 10 8 transfected CHO-K1 cells (clone 7F) were cultured in T175 triple-layer flasks in a humidified incubator at 37° C for 3 weeks. The culture medium contained 0.5% fetal calf serum (FCS) and was exchanged once each week. From each harvest, about 1 liter of culture supernatant was obtained. Anti-MUCl antibody was purified with Protein A. Briefly, 1 liter of culture supernatant was loaded onto a 5 ml HiTrap Protein A column (Amersham/Pharmacia) at a flow rate of 5 ml/minute. The column was extensively washed with PBS. Bound MUCl antibody was eluted with 12.5 mM citric acid and neutralized with 0.5 M HEPES (pH 9). Protein containing fractions were combined, dialyzed against PBS (16 hours, 4° C) and sterile filtered. Purified anti-MUCl antibody was analyzed by SDS-PAGE and silver staining, human IgG specific ELISA, and a BCA micro protein assay (Pierce). PHI -IgG (100- 200 ng), purified with Protein A, was separated on a 10% SDS-PAGE gel (Laemmli et al, J. Mol. Biol, 47: 69-85 (1970)) under reducing conditions, and protein bands were visualized by silver staining. For Western blots, purified PHl-IgG was separated on a 10%> SDS-PAGE gel under reducing conditions and transferred onto nitrocellulose. PHl-IgG heavy chain and light chain were simultaneously detected with a HRP-conjugated polyclonal antibody against human IgG and an HRP-conjugated monoclonal antibody against human kappa chain, respectively. Production amount was measured in a human IgG ELISA described above. Surface plasmon resonance The selected PHl-IgGl and the Fab PHI antibodies were evaluated for their binding characteristics by surface plasmon resonance on a BIAcore 2000 apparatus (BIAcore AB, Uppsala, Sweden). A biotin chip was coated with a MUCl 15-mer, containing the minimal PHI epitope, PAP (Ac-PDTRPAPGSTAPPAL-NH 2 (SEQ ID NO:40) (see Example 2, above), 50 RU and 320 RU) and 60-mer (NH 2 -(VTSAPDTRPAPGSTAPPAHG) 3 -COOH (SEQ ID NO:8) (von Mensdorff-Pouilly et al. Tumor Biol., 19: 186-195 (1998), 50 RU) in HBS-EP buffer (Pharmacia) a surface, blocked with biotin (15 RU), was used as a negative control. The Fab PHI and PHl-IgGl were injected in HBS-EP buffer. To minimize rebinding ofthe antigen binding molecules, a speed of 30 μl/sec was used. Affinity calculation was performed with computer programs provided by BIAcore (BIAEvaluation-version3, BIACore AB). Fitting was accepted when Chi 2 was lowest, on the two channels with a non-saturated amount (50 RU) of MUCl peptide bound. The affinity for the PHI Fab antibody was calculated according to a 1 : 1 Langmuir stoichiometry at steady state (Chi 2 : 50.6). Because ofthe two binding places on the PHl-IgGl, the avidity was calculated as an apparent avidity constant using 1 :1 Langmuir determination with mass transfer limitation (Chi 2 : 42). Flow cytometric analysis Cellular MUCl binding was tested in flow cytometry, with PHl-IgG purified as before and with the murine HMFGl antibody (Autogen Bioclear, Wilthshire, UK). About 500,000 cells were used in each experiment: after trypsinisation, cells were washed one time in RPMI 10% FCS, 0.01% NaN 3 (incubation buffer). To confirm the specificity, the same amount (100 μg/ml) of specific antibodies or non-binding human antibody and with or without 100 μg/ml MUCl 60- mer for 1 hour at room temperature were used. Then the samples were added to the cells and left for 1 hour at room temperature. Cells were spun down by centrifugation for 3 minutes at 611 x g. Between incubations, cells were washed twice with incubation buffer. Anti-human IgGl antibody was added to the cells and incubated for 1 hour at room temperature. Then rabbit anti- mouse-FITC was added to all tubes, and the tubes were incubated for 30 minutes. Detection of bound antibodies was performed by means of flow cytometry on a FACSCalibur (Becton Dickinson, Oxnard), and results analyzed with the CELLQuest program (Becton Dickinson, Oxnard). Cell lines used in the study were the mouse fibroblast cell line 3T3, the MUCl transfected cell line 3T3-MUC1 (ETA) (Acres et al, J. Immunother., 14: 136-143 (1993)), the breast carcinoma lines T47D and MCF-7, the ovarian carcinoma line OVCAR-3, the colon cancer cell line LS 174T, the colon cell line CaCo2, and the T cell line Jurkat (non-transfected cell lines were provided by ATCC). Biotinylation and FITC-labeling of PHl-IgG PHl-IgGl in 50 mM NaHC0 3 , pH 8.5, at a concentration of 250 μg/ml was treated with sulfo-NHS-LC-biotin (Pierce, New York, NY) for 1 hour at RT under gentle agitation. 4 μg of biotin ester was used for 100 μg ofthe antibody. The reaction was stopped by treatment with Tris/HCl, pH 7.5, at a final concentration of 50 mM, for 30 minutes. To separate the biotinylated antibody from free biotin, the reaction mixture was dialyzed against PBS. Biotinylation of PHl- IgG was verified by flow cytometry analysis by binding ofthe antibody to the MUCl positive OVCAR3 cells and ETA cells compared to the MUCl negative 3T3 cells. FITC-labeling was performed according to the manufacturer with 200 μg PH-IgGl in 200 μl reaction mixture using a FITC protein labeling kit (Molecular Probes, Leiden, Netherlands). Labeling was checked on MUCl positive and negative cell lines in flow cytometry analysis (ETA, OVCAR-3, 3T3). Immunohistochemistry A variety of formalin-fixed normal and tumor tissues were tested for the binding pattern ofthe PHl-IgGl. Tissues were chosen with a preference for diagnosed adenocarcinoma. HMFG-1 was used as a control for a limited number of tumor tissues. The biotinylated PH1- IgGl antibody was used. Slices (5 μm) of paraffin-embedded tissues were de-paraffinized, rehydrated, hydrogen peroxide treated (0.3 % H 2 0 2 in PBS), and preincubated with PBS, 15 % FCS, 5% human serum (HS) for 20 minutes. Antibodies were diluted to a concentration of 17 μg/ml in PBS, 10 % HS and incubated for 1 hour at room temperature. For PHl-IgGl, slides were then incubated with an avidin-biotin-complex (ABC, Dako, Glostrup, Denmark) for 30 minutes. For HMFGl, slides were first incubated with biotinylated sheep-anti-mouse (RAMPO, Dako) in PBS, 0.1% Tween 20, 1% BSA for 30 minutes and then with the avidin-biotin- complex. For each tissue, a negative control with non-binding human IgGl was used. Between antibody incubation, slides were washed three times for 5 minutes in PBS. Staining was carried out using diaminobenzidin (DAB) and H 2 0 2 . The peroxidase reaction was stopped with water, and slides were counter-stained with hematoxylin. The epithelial tissues were evaluated for their binding reactivity (sporadic: < 10%, focal: 10%<f<80%, diffuse: > 80%) and their localization in the cell (a: apical, polar, c: cytoplasmic, depolarized, m: abundantly expression on the cell membrane). To study glycosylation sensitivity, a normal breast tissue section was pre-treated with periodic acid in acetate buffer 0.05 M, pH 5 for 30 min at room temperature in the dark as described by (Cao et al. Tumour Biol, 19 Suppl. 1: 88-99 (1998)). Evaluation of intemalization using a confocal microscope Antibody was FITC labeled according to the manufacturer's instructions (see above). The FITC-labeled antibody bound in flow cytometry to the ETA and OVCAR-3 cells and not to the MUCl negative 3T3 cell line (data not shown). For intemalization studies, the human tumor cell line OVCAR-3 and the MUCl transfected mouse fibroblast 3T3 cell line, ETA, were used. As negative control, the colon cell line CaCo2 was used. FITC-labeled antibody was added to the cells (10 μg/10 6 cells at a concentration of 100 μg/ml) for an incubation period of 1 hour on ice. The cells were washed and put on ice to check whether the antibody stayed bound to the membrane or placed at 37° C to study intemalization. At each time point (1, 3, 6 hours and overnight), cells were checked on a confocal microscope for membrane binding and intemalization. Fc binding was checked by competition with human IgGl . Staining patterns (membranous or intracellular) were evaluated with a confocal microscope (Asciophat, Zeiss, Atto Instrument, Rockville, MD). Cloning of PHl-IgGl into a mammalian expression vector and selection of transfectants In this study, the human PHI Fab antibody (Example 1) directed to MUCl was recloned as a fully human gamma- 1 /kappa immunoglobulin antibody into the mammalian VHexpress and VKexpress expression vectors. DNA containing a sequence encoding the PH1-V H was cloned into VHexpress, and DNA containing a sequence encoding the PH1-V L fragment was inserted into the expression cassette of VKexpress. Co-transfection of VHexpress and VKexpress recombinant vectors into CHO-K1 cells was carried out using the non-liposomal transfection reagent FuGene 6. At 48 hour after transfection, limiting dilutions were performed into medium containing 700 μg/ml G418. Cells were plated in 96-well plates at 10, 100 and 1000 cells per well. On the 100 cell/well plate, 36 out of 96 wells showed cell growth after 5 days in culture. Supernatants of grown, positive wells were assayed for presence of human gamma- immunoglobulins and binding to MUCl-peptide in ELISA. Of these, 13 were positive for binding to MUCl, with a range of detected human IgG between 5 and 77 ng/ml. Clone 7F (75 ng/ml) was chosen for further study. To guarantee clonality, an additional round of subcloning was carried out (data not shown). Production and purification ofthe PHl-IgGl The MUCl-specific PHl-IgGl antibody was purified from 0.5% FCS containing culture media as described above. Under these conditions, no co-purification of bovine IgG appeared, and more than 90% pure PHl-IgGl protein was obtained as evidenced on silver stained SDS- PAGE. The results of a human IgGl specific ELISA and a BCA total protein detection assay were in good agreement (data not shown). From 1 liter of culture media, about 0.5 mg PHl-IgG were purified, approximately corresponding to an expression level of 0.3 pg per cell, derived from approximately 3 x 10 s cells within 1 week. BIAcore analysis The affinity ofthe antibody was determined using BIAcore. Affinities ofthe Fab PHI were calculated to be an average of 1.4 μM for binding to the 15-mer and 60-mer MUCl peptide antigen coated surfaces. Mean avidity of PHl-IgGl (8.7 nM)' was calculated with the BIACore software from binding curves on low density surfaces being 8.3 nM (15-mer) and 9.06 nM (60- mer). The binding affinity ofthe PHl-IgGl antibody was found to be over 100 times stronger than with the parent Fab PHI antibody molecule. Comparative flow cytometric analysis Since differences in the fine-specificity of MUCl antibodies can lead to differences in the panel of tissues and tumors recognized, the PHl-IgGl antibody was compared with a frequently used murine antibody, HMFGl. PHl-IgGl recognizes the PAP epitope as determined by epitope fingerprinting ofthe PHI Fab (Example 1, above; Henderickx et al. Cancer Res., 58: 43224-4332 (1998)), while HMFGl recognizes the PDTR (amino acids 9-12 of SEQ ID NO:7) epitope. The two antibodies were tested on different tumor cell lines in flow cytometry. Both antibodies bound with the same binding pattern to most ofthe cell lines, except for the ovarian carcinoma cell line OVCAR-3, which apparently exposes more ofthe PHl-IgGl epitope than the HMFGl epitope. Both antibodies bind a small subpopulation ofthe LS174T colon tumor cell line and ofthe T cell line Jurkat, which can be inhibited by MUCl 60-mer. No binding to the CaCo2 colon cell line was observed. Binding of MUCl to cells could be competed off with MUCl peptide, although the competition appeared not to be quantitative. This study indicated that there is a difference in the spread and/or density ofthe various MUCl epitopes or a differential accessibility of these epitopes due to residual glycosylation. To understand the abundance ofthe PHl-IgGl MUCl -epitope, it was necessary to carry out immunohistochemical analysis on a large set of tissues and tumors (see, below). Immunohistochemical analysis of PHl-IgG An immunohistochemical analysis was carried out on a large set of tissues and tumors (see, Table 10 below). The general degree of MUCl localization ("staining") in tumor cells was (from most to least staining) depolarized cytoplasmic (c) > abundant membranous staining ofthe whole cell (m) > polarized apical (a), while in normal tissues the localization pattern was a > c >m (see, Table 10). In addition, staining reactivity was higher in tumor tissues than in normal tissues (data not shown). Table 10: Immunohistological staining of normal and tumor epithelial tissues with PHl-IgGl. Normal tissues Tumor tissues* Tissue Reactivity Localization Reactivity Localization Freq. Remarks Bladder s 1/4 Transitional f 1/4 Urothelial f 1/4 d c, m 1/4 Colon 3/3 1/2 Squamous a, c 1/2 Mucinous Endometrium f 2/6 f 1/6 d, f a, c 2/6 a, c, m 1/1 Epididymis f 3/3 Kidney 5/5 - glomeruli - - prox. tub. - - dist. tub. f - coll. ducts d Liver - 3/3 - bile duct s 1/1 Hepatocellular Lung 6/6 c, m 2/5 1 squamous a, m 1/5 1 squamous a,c,m 1/5 a 1/5 Mamma 4/5 1/5 f a, c, m 3/7 d a, c, m 2/7 d a, m 1/7 f a 1/7 Papiloma Ovarian 2/2 d c, m 2/8 f a, c, m 1/8 d a, c, m 1/8 f c 2/8 d c 1/8 f a 1/8 Sereus Pancreas • acini d a 5/5 ■ exocrine gl. d a (c) - isl. Langerhans f a, c 1/2 d a, m, c 1/2 Parathyroid 3/3 a, c 1/2 c 1/2 Prostate 5/6 1/6 - 1/3 a, c 1/3 c, m 1/3 Salivary gland - ducti d-f a-c 2/2 - acini ~f Skin - sebaceous gl. d m - sweat gland f - hair follicle - Testes _ 3/3 1/1 Tuba f a 2/2 Thyroid - - 2/2 1/1 Vas deferens f a 1/1 Tumors are adenocarcinoma, except when stated differently. Abbreviations: s: sporadic staining (< 10%), f: focal staining (10<s<80%), d: diffuse staining (> 80%); a: polarized apical, c: depolarized cytoplasmic, m: abundantly present on whole cell membrane A summary ofthe study of localization of MUCl using the PHl-IgG antibody in various tissues follows. Normal bladder was negative in cases tested. Tumor tissues ofthe bladder had different staining patterns in which both adenocarcinoma tissues had a depolarized staining pattern. Colon cancer, normal tissues, and squamous carcinoma were negative. A mucinous tumor tested in this study had depolarized cytoplasmic staining. In endometrium, some normal tissues showed a depolarized localization. In normal kidney, the staining pattern was always the same with no staining in glomeruli and proximal tubes, focal apical staining in distal tubes and diffuse, apical staining in collecting ducts. In contrast, with lung tissues, normal lung (negative), and adenocarcinoma ofthe lung was intensively MUCl positive in a depolarized fashion. In most tumors, an extensive staining of whole cell membranes was found. Not all tumor cells, per tissue, reacted with the antibody (i.e., focal staining observed). In breast and ovarian adenocarcinoma tissues, there was a differential staining between normal and adenocarcinoma, being polarized in normal and cytoplasmic with membranous staining in adenocarcinoma (6/6 for breast, 4/7 for ovarian adenocarcinoma). Intensity of staining was less in normal tissue than in tumor tissue. The reactivity was diffuse to focal in tumor tissues and focal in normal tissues. Pancreas adenocarcinoma had a cytoplasmic staining pattern. Normal acini expressed MUCl apically, and exocrine glands showed a polar staining or cytoplasmic staining. In normal tissues ofthe endometrium and sebaceous gland ofthe skin, a depolarized staining pattern for MUCl was observed. Periodate-treated nornial breast epithelium was stained slightly more intensively than the non-treated tissue, indicating that, as expected, de-glycosylation exposes the epitope of PHI. Taken together, the above study showed that a differential expression of MUCl was found between normal tissue and tumor in bladder, lung, breast, ovary, pancreas, parathyroid, and prostate tissue. Apical staining was found in normal tissues as well as in tumor tissues, depolarized cellular (cytoplasmic) staining was most frequently detected in tumors, and aberrant staining ofthe whole cell membrane was only found in tumors with the exception ofthe sebaceous glands ofthe skin. A comparison with the murine HMFGl antibody for a limited amount of tissues is shown in Table 11. Normal tissues were stained mainly focally apical, except for an endometrium tissue that showed cytoplasmic staining with PHl-IgGl. In tumors, small differences in immunoreactivity were seen which can be confirmed with a larger panel of tissues. Table 11. Comparison in immunohistochemistry between human PHl-IgGl and the mouse HMFGl antibodies HMFGl PHI Freq. Distribution Localization Distribution Localization Bladder (N)* - Breast (N) f a f a Breast (T) d a f a,c Breast papiloma - f a Breast (T) f m d a, c,m Breast (T)* d m,c f a Liver - Paratyroid (T) f a d a, m Tuba (N)* f a f a Endometrium (N) f a f c Ovarium (T) f c,m f c,m Ovarian (T) d a f a,c,m Ovarian (N) - Ovarian (T) d a f a,c Ovarian (T) f a f a Ovarian (T) d c,m d c,m *: T: Tumor tissue, N: Normal tissue Abbreviations: a: polarized apical, c: depolarized cytoplasmic, m: abundantly present on whole cell membrane Evaluation of intemalization of PHl-IgGl. using confocal microscope To analyze the extent with which PHl-IgGl after binding would be internalized, an intemalization study using FITC-labeled antibody was carried out. The FITC-labeled antibody bound in FACS analysis to the OVCAR-3 and ETA cell lines, and not to the negative 3T3 cell line (data not shown). After 1 hour of incubation on ice with the human antibody PHl-IgGl, membranous binding was observed on the MUCl expressing OVCAR-3 and ETA cell lines. As in flow cytometry, the intensity of staining was more pronounced for the ETA cell line as compared with the OVCAR-3 cell line. No auto-fluorescence was observed, and no fluorescence was visible on the CaCo2 negative control cell line. At 37° C, the intemalization ofthe PH1- IgGl-FITC became visible for both the ETA cells and the OVCAR-3 cells. After 1 hour, more than 50% of OVCAR-3 cells had internalized the antibody in vesicles, while the ETA cells had mainly membrane bound antibody. After 3 hours of incubation, more than 80 % ofthe FITC- labeled antibody was internalized by the OVCAR-3 cells: vesicles were visible but also cells with a low level of intracellular fluorescence were visible. After 6 hours, all OVCAR-3 cells had internalized the antibody, and most cells had lost the vesicle intemalization pattern and exhibited a low cytoplasmic fluorescence only. At either 3 or 6 hours, OVCAR-3 cells kept on ice had the antibody still bound to the membrane only. The ETA cells had internalized less than 3 % ofthe antibody after 3 hours, but after overnight incubation, the surviving cells had internalized the antibody and no membrane bound antibody was left. In contrast, cells kept overnight on ice showed membranous staining. Analysis This study characterized a recombinant, anti-MUCl antibody formed by recloning the V H amd V L regions ofthe MUCl-specific Fab antibody PHI into a two-vector, mammalian cell expression system to produce a new, fully human, whole IgGl, which has significantly enhanced affinity for MUCl compared to the PHI Fab parent molecule. The somewhat low yield, when compared to the production of other antibodies in CHO-K1 cells (for a review, see Trill et al, Curr. Opin. Biotechnol, 6: 553-560 (1995)), is probably caused by differential expression ofthe light and the heavy chain and the yet not undertaken optimization of culture conditions. The amount produced was, nevertheless, sufficient for the small-scale production ofthe antibody for the various laboratory tests described above. For immunotherapy, such characterization is important in order to determine whether a particular antibody will fit a particular therapy or vice versa, especially since all MUCl antibodies do not behave the same (Cao et al. Tumour Biol, 19 Suppl. 1: 88-99 (1998); Pietersz et al. Cancer Immunol. Immuother., 44: 323-328 (1997)). First, the affinity ofthe antibody is a major determining factor in establishing how fast it will bind to a tumor cell and how quickly it will release itself from the antigen-bearing tumor cell. In this study, the avidity ofthe newly generated antibody was compared with the affinity of the original Fab in BIAcore. Avidities for the PHI Fab and PHl-IgG were 1.4 μM and 8.7 nM respectively, indicating a 100-fold increase for the whole human antibody (PHl-IgGl). This avidity change is solely due to the change from one to two binding sites, since binding on the 60- mer and 15-mer channel are comparable. Comparison between diabodies obtained from single chain antibodies (scFvs) to ErB2 with different affinities showed that the magnitude ofthe decrease in the apparent dissociation rate constant (Kd) for the bivalent molecule was inversely proportional to the affinities ofthe scFvs (Nielsen et al. Cancer Res., 60: 6434-6440 (2000)). The PHI Fab antibody has a relatively low affinity, and the increase of apparent affinity for the corresponding PHl-IgG molecule is very high, confirming the above observation from Nielsen. In flow cytometric analysis, PHl-IgGl was compared with HMFGl, which is reported to recognize a different, glycosylation sensitive, MUCl epitope (Cao et al, 1998; Burchell et al. Epithelial Cell Biol, 2; 155-162 (1993)). The binding pattern on tumor cell lines did not differ significantly between both antibodies, except for the OVCAR-3 cell line, which was stained less by HMFGl, probably due to the different epitope recognition. On colon cancer cell lines, both antibodies hardly showed any binding. Colon cancer cells can be highly glycosylated, and glycosylation sensitive antibodies rarely stain this glycosylated colon mucin (Sikut et al. Tumour Biol, 19 Suppl 1: 122-126 (1998); Blockzjil et al. Tumour Biol, 19 Suppl. 1: 46-56 (1998)). This suggests that the antibody PHl-IgGl recognizes MUCl in an underglycosylated form, which is expected to be tumor-associated. The antibody C595, binding the RPAP epitope, reacts in FACS analysis to OVCAR-3 and MCF-7 cells with the same pattern as HMFGl (Reddish et al. Tumour Biol, 19 Suppl. 1: 57-66 (1998)) and consequently also with PHl-IgGl. The antibodies did bind well to the T47D breast cancer cell line known to express different glycofoπns of MUCl (Hanisch et al, Eur. J. Biochem., 236: 318-327 (1996)). The usage of periodate on a normal breast tissue intensified the apical staining confirming the glycosylation sensitivity of this antibody as for many antibodies recognizing an epitope on the protein core of MUCl (Cao et al, 1998). Immunohistochemical staining revealed a differential staining between tumor tissues and normal tissues, being apical or absent in normal tissues and depolarized in tumor tissues as described for glycosylation sensitive antibodies (Zotter et al. Cancer Rev., 11-12: 56-101 (1988); Cao et al, 1998). In normal tissues ofthe ovary and breast, staining was often heterogeneous (f) and not as intense as in tumor. In breast and ovarian tumors, staining was diffuse or heterogeneous, and intense membrane staining was found in 6/6 breast and 4/7 ofthe ovarian adenocarcinoma. Thus, MUCl is ubiquitously present on cell membranes. In bladder and lung, differences between tumor and normal tissues are highest. In normal tissues, tested the PHl- IgGl epitope is not present. This is in contrast with the findings of weakly to focally positive reactivity with monoclonal antibodies recognizing the PDTR (amino acids 9-12 of SEQ ID NO:7) region of MUCl core protein in normal lung and bladder tissues (Zotter et al, 1988; Walsh et al, Br. J. Urol, 73: 256-262 (1994)). In tumor tissues, heterogeneous staining was observed with mostly focal reactivity in both lung and bladder. In all adenocarcinomas tissues, the PHl-IgGl epitope is expressed in a non-polar fashion. Although the staining pattern ofthe PHI epitope is different with staining patterns of other glycosylation sensitive antibodies (Zotter et al, 1988), in some cases the PHl-IgGl meets or even exceeds expectations. The immunohistochemical staining patterns support, as in flow cytometry, that the antibody PHl-IgGl indeed binds to the underglycosylated tumor-associated MUCl that is abundantly expressed in a depolarized fashion in adenocarcinoma. Such antibodies, recognizing an epitope ofthe MUCl tandem repeat, are described for murine (derived) antibodies and are successfully used in targeting studies in humans (von Hof et al, Cancer Res., 565: 5179-5185 (1996); Biassoni et al, Br. J. Cancer, 77: 131-138 (1998); Kramer et al, Clin. Cancer Res., 4: 1679-1688 (1998)). Although the peptide epitope is PAP (SEQ ID NO: ), PHl-IgGl binds specifically and preferentially to underglycosylated MUCl. Spencer et al. (Cancer Lett., 100: 11-15 (1996)) investigated the influence of glycosylation on antibody binding with their antibody recognizing the minimal epitope RPAP (amino acids 12-15 of SEQ ID NO:7) and concluded that this antibody in positively influence by glycosylation. This in contrast with an antibody recognizing the PDTR (amino acids 9-12 of SEQ ID NO: 7) motif. This could explain the different fine- specificity ofthe PHl-IgG. The Fab antibody PHI was selected by phage display technology, by two rounds of selection on ETA cells and 3 rounds of selection on a MUCl 60-mer (see, Example 1). Possibly, by the way the antibody was selected, it favors binding to an underglycosylated epitope PAP ofthe tandem repeat. The data indicate that the PHl-IgG antibody would be particularly useful as a targeting tool in bladder, lung, mammary, and ovarian cancer where the PHl-IgGl epitope is, in most cases, present on the tumor cells in a depolarized fashion (c, m in Table 11). Because ofthe possible heterogeneous (focal) expression, the PHl-IgG antibody could be used in an immunotherapy that has a bystander effect on surrounding tumor cells, e.g, radio- immunotherapy, a combination of radio-immunotherapy and immunotoxins (see, e.g, Wei et al, Clin. Cancer Res., 6: 631-642 (2000)), or in the use of fusion proteins that stimulate tumor infiltrating lymphocytes (see, e.g. Lode et al, Pharmacol. Therap., 80: 277-292 (1998)). The abundance of expression ofthe PHl-IgGl epitope on the membranes of tumor cells is heterogeneously spread. Because ofthe high amount of MUCl on the their membranes, these cells provide excellent targets for PHl-IgG. Again, supporting the use of PHl-IgG in a therapy with bystander effects. Intemalization studies demonstrated that the FITC-labeled antibody is internalized by both OVCAR-3 and ETA cells, although with a different rapidity. First, the intemalization pattern was almost exclusively in vesicles. Later, the vesicle structure was less abundant and faint staining was found in the cytoplasm. This could be due to the breakdown ofthe antibody, leaving free FITC in the cytoplasm, or due to a modification ofthe FITC, itself, and loss of its fluorescence. After 1 hour at 37° C, more than half of the OVCAR-3 cells exhibited fluorescent vesicles, meaning that the antibody rapidly internalized into vesicles. It has been described that the MUCl antigen recycles 0.9 % of surface fraction/minute (Litvino et al, J. Biol Chem., 268: 221364-21371 (1993)). This study confirms the observation (data not shown) that at 1 hour more than 50% ofthe cells have internalized the antibody. Intemalization of MUCl antibodies is not always the same and may depend on the epitope. Pietersz et al. (1997) compared two antibodies for their intemalization rate, the antibody specific for MUCl epitope RPAP (amino acids 12-15 of SEQ ID NO:7) (CTMOl) internalized much better than the antibody specific for the PDTR (amino acids 9-12 of SEQ ID NO:7) epitope. The PHl-IgG antibody, when assayed with the peptide epitope PAP, appears to have a similar intemalization rate. The MUCl transfected 3T3 cell line, ETA, internalized the FITC- labeled antibody much slower. At first sight this could be due to the fact that mouse cells normally do not express human MUCl and that the intemalization machinery is not effective for this xenogenic protein. Some transfected cell lines may internalize better than others (see, e.g, Pietersz et al, 1997). Because ofthe intemalization, the PHl-IgG antibody can be used in a variety of therapies and combination, such as for immunotherapy with pro-drugs, drags, for gene therapy (for a review of such various therapies, see Syrigos et al, Hybridoma, 18: 219-224 (1999)), and for radio-immunotherapy, where it may not always be necessary that the radiolabel is internalized. In conclusion, the human antibody PHl-IgGl was shown to recognize tumor-associated MUCl on adenocarcinoma. Its affinity is high enough to bind to tumor cells and because the FITC-labeled antibody can be internalized by recycled MUCl, it is a candidate molecule for therapeutic and diagnostic tumor targeting applications, especially in lung, bladder, ovarian, and breast adenocarcinoma. All documents and publications cited above are incorporated herein by reference. Other variations and embodiments ofthe invention described herein will now be apparent to those of ordinary skill in the art without departing from the scope ofthe invention or the spirit ofthe claims below.

Claims: CLAIMS: 1. An isolated MUCl-specific binding member comprising an antigen binding domain, wherein the antigen binding domain comprises a region comprising the amino acid sequence of the formula: X ! X 2 His Thr Gly X 3 Gly Val Trp X 4 Pro X 3 X 6 X 7 (SEQ ID NO:28), wherein X, is Ala, Ser, Thr, or Val; X 2 is Lys, He Arg, or Gin; X 3 is Gly, Arg, Val, Glu, Ser, or Ala; X 4 is Asp or Asn; X 5 is He, Leu, Met, Phe, or Val; X 6 is Asp, Gly, Lys, Asn, Ala, His, Arg, Ser, Val, or Tyr; and X 7 is Tyr, His, Lys, Asn, Asp, Ser, Pro. 2. The MUCl-specific binding member according to Claim 1, wherein the variable region comprises the amino acid sequence selected from the group consisting of: Ala Lys His Thr Gly Gly Gly Val Trp Asp Pro He Asp Tyr (amino acids 97-110 of SEQ ID NO:3); Ala Lys His Thr Gly Arg Gly Val Trp Asp Pro lie Gly Tyr (SEQ ID NO:29); Ala Lys His Thr Gly Gly Gly Val Trp Asp Pro lie Lys His (SEQ ID NO:30); Ala Lys His Thr Gly Gly Gly Val Trp Asp Pro He Gly Tyr (SEQ ID NO:31); and Ala He His Thr Gly Gly Gly Val Trp Asp Pro He Lys Tyr (SEQ ID NO:32). 3. An isolated MUCl-specific binding member comprising an antigen binding domain comprising an antibody V L region comprising the amino acid sequence of SEQ ID NO:l, or portion thereof, and an antibody V H region comprising the amino acid sequence of SEQ ID NO:3, or portion thereof. 4. A MUCl-specific binding member comprising an antigen binding domain, wherein the antigen binding domain comprises a CDR of an antibody V L or V H region, wherein said CDR has an amino acid sequence selected from the group consisting of amino acids 24 to 39 of SEQ ID NO:l, amino acids 55 to 61 of SEQ ID NO:l, amino acids 94 to 102 of SEQ ID NO:l, amino acids 31 to 35 of SEQ ID NO:3, amino acids 50 to 66 of SEQ ID NO:3, amino acids 99 to 110 of SEQ ID NO:3, conservatively substituted sequences of any ofthe preceding sequences, and combinations thereof. 5. The MUCl-specific binding member according to any of Claims 1, 2, 3, or 4, wherein said MUCl-specific binding member is a fusion protein. 6. The MUCl-specific binding member according to any one of Claims 1, 2, 3, or 4, further comprising a detectable label or tag. 7. The MUCl-specific binding member according to Claim 6, wherein the detectable label or tag is selected from the group consisting of epitope tags, fluorescent labels, radioactive labels, heavy metals, anti-cancer drugs, toxins, and magnetic resonance imaging labels. 8. The MUCl-specific binding member according to any one of Claims 1, 2, 3, or 4, wherein the MUCl-specific binding member is an antibody molecule selected from the group consisting of immunoglobulins, Fab antibodies, F(ab') 2 antibodies, diabodies, scFv antibodies, double scFv, Fv molecules, dAb, immunocytokine molecules, and immunotoxin molecules. 9. The MUCl-specific immunocytokine according to Claim 8, comprising the amino acid sequence of SEQ ID NO:5. 10. The MUCl-specific immunocytokine according to Claim 9, further comprising a detectable label or tag. 11. The MUCl-specific binding member according to Claim 10, wherein the detectable label or tag is selected from the group consisting of, epitope tags, fluorescent labels, radioactive labels, and magnetic resonance imaging labels. 12. The MUCl-specific immunoglobulin according to Claim 8, comprising a light chain polypeptide comprising the amino acid sequence of SEQ ID NO: 24 and a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO:26. 13. The MUCl-specific immunoglobulin according to Claim 12, further comprising a detectable label or tag. 14. The MUCl-specific binding member according to Claim 13, wherein the detectable label or tag is selected from the group consisting of enzymes, epitope tags, fluorescent labels, radioactive labels, heavy metals, anti-cancer drags, toxins, and magnetic resonance imaging labels. 15. A MUCl-specific binding member comprising an antibody antigen binding domain comprising a heavy chain variable region, or CDR thereof, from the DP47 germ line. 16. A MUCl-specific binding member comprising an antibody antigen binding domain comprising a light chain variable region, or a CDR thereof, from the DPK15 germ line. 17. A MUCl-specific binding member comprising an antibody antigen binding domain comprising a heavy chain variable region, or CDR thereof, from the DP47 germ line and a light chain variable region, or CDR thereof, from the DPK15 germ line. 18. A MUCl-specific binding member comprising an amino acid sequence that is about 70% or more homologous to any ofthe amino acid sequences of Claims 1, 2, 3, or 4. 19. A MUCl-specific binding member comprising an amino acid sequence that is about 80% or more homologous to any ofthe amino acid sequences of Claims 1, 2, 3, or 4. 20. A MUCl-specific binding member comprising an amino acid sequence that is about 90% or more homologous to any ofthe amino acid sequences of Claims 1, 2, 3, or 4. 21. A MUCl-specific binding member comprising an amino acid sequence that is about 95% or more homologous to any ofthe amino acid sequences of Claims 1, 2, 3, or 4. 22. A MUCl-specific binding member comprising an amino acid sequence that is about 97% or more homologous to any ofthe amino acid sequences of Claims 1, 2, 3, or 4. 23. A MUCl-specific binding member comprising an amino acid sequence that is about 99% or more homologous to any ofthe amino acid sequences of Claims 1, 2, 3, or 4. 24. A polypeptide molecule comprising an amino acid sequence that is about 70% or more homologous to an amino acid sequence selected from the group consisting of SEQ ID NO:l, amino acids 24 to 39 of SEQ ID NO:l, amino acids 55 to 61 of SEQ ID NO:l, amino acids 94 to 102 of SEQ ID NO:l, SEQ ID NO:3, amino acids 31 to 35 of SEQ ID NO:3, amino acids 50 to 66 of SEQ ID NO:3, amino acids 99 to 110 of SEQ ID NO:3, and SEQ ID NO:5. 25. A polypeptide molecule comprising an amino acid sequence that is about 80% or more homologous to an amino acid sequence selected from the group consisting of SEQ ID NO:l, amino acids 24 to 39 of SEQ ID NO:l, amino acids 55 to 61 of SEQ ID NO: l, amino acids 94 to 102 of SEQ ID NO:l, SEQ ID NO:3, amino acids 31 to 35 of SEQ ID NO:3, amino acids 50 to 66 of SEQ ID NO:3, amino acids 99 to 110 of SEQ ID NO:3, and SEQ ID NO:5. 26. A polypeptide molecule comprising an amino acid sequence that is about 90% or more homologous to an amino acid sequence selected from the group consisting of SEQ ID NO:l, amino acids 24 to 39 of SEQ ID NO:l, amino acids 55 to 61 of SEQ ID NO:l, amino acids 94 to 102 of SEQ ID NO:l, SEQ ID NO:3, amino acids 31 to 35 of SEQ ID NO:3, amino acids 50 to 66 of SEQ ID NO:3, amino acids 99 to 110 of SEQ ID NO:3, and SEQ ID NO:5. 27. A polypeptide molecule comprising an amino acid sequence that is about 95% or more homologous to an amino acid sequence selected from the group consisting of SEQ ID NO:l, amino acids 24 to 39 of SEQ ID NO:l, amino acids 55 to 61 of SEQ ID NO: l, amino acids 94 to 102 of SEQ ID NO:l, SEQ ID NO:3, amino acids 31 to 35 of SEQ ID NO:3, amino acids 50 to 66 of SEQ ID NO:3, amino acids 99 to 110 of SEQ ID NO:3, and SEQ ID NO:5. 28. A polypeptide molecule comprising an amino acid sequence that is about 97% or more homologous to an amino acid sequence selected from the group consisting of SEQ ID NO:l, amino acids 24 to 39 of SEQ ID NO:l, amino acids 55 to 61 of SEQ ID NO:l, amino acids 94 to 102 of SEQ ID NO:l, SEQ ID NO:3, amino acids 31 to 35 of SEQ ID NO:3, amino acids 50 to 66 of SEQ ID NO:3, amino acids 99 to 110 of SEQ ID NO:3, and SEQ ID NO:5. 29. A polypeptide molecule comprising an amino acid sequence that is about 99% or more homologous to an amino acid sequence selected from the group consisting of SEQ ID NO:l, amino acids 24 to 39 of SEQ ID NO: l, amino acids 55 to 61 of SEQ ID NO: l, amino acids 94 to 102 of SEQ ID NO:l, SEQ ID NO:3, amino acids 31 to 35 of SEQ ID NO:3, amino acids 50 to 66 of SEQ ID NO:3, amino acids 99 to 110 of SEQ ID NO:3, and SEQ ID NO:5. 30. An isolated polynucleotide molecule comprising a nucleotide sequence encoding an amino acid sequence selected from the group consisting of SEQ ID NO:l, amino acids 24 to 39 of SEQ ID NO:l, amino acids 55 to 61 of SEQ ID NO:l, amino acids 94 to 102 of SEQ ID NO:l, SEQ ID NO:3, amino acids 31 to 35 of SEQ ID NO:3, amino acids 50 to 66 of SEQ ID NO:3, amino acids 99 to 110 of SEQ ID NO:3, SEQ ID NO:5, and combinations thereof. 31. An isolated polynucleotide molecule comprising a nucleotide sequence encoding an amino acid sequence selected from the group consisting of SEQ ID NO:24 and SEQ ID NO:26. 32. An isolated polynucleotide molecule encoding a V L region comprising a nucleotide sequence of SEQ ID NO:2 or degenerate sequences thereof. 33. An isolated polynucleotide molecule encoding a V L region comprising a nucleotide sequence which is about 70% or more homologous to the sequence of SEQ ID NO:2. 34. An isolated polynucleotide molecule encoding a V L region comprising a nucleotide sequence which is about 80%ι or more homologous to the sequence of SEQ ID NO:2. 35. An isolated polynucleotide molecule encoding a V L region comprising a nucleotide sequence which is about 90% or more homologous to the sequence of SEQ ID NO:2. 36. An isolated polynucleotide molecule encoding a V L region comprising a nucleotide which is about 95% or more homologous to the sequence of SEQ ID NO:2. 37. An isolated polynucleotide molecule encoding a V L region comprising a nucleotide sequence which is about 97% or more homologous to the sequence of SEQ ID NO:2. 38. An isolated polynucleotide molecule encoding a V L region comprising a nucleotide sequence which is about 99% or more homologous to the sequence of SEQ ID NO:2. 39. An isolated polynucleotide molecule encoding a V H region comprising a nucleotide sequence of SEQ ID NO:4, or degenerate sequences thereof. 40. An isolated polynucleotide molecule encoding a V H region comprising a nucleotide which is about 70% homologous to the sequence of SEQ ID NO:4. 41. An isolated polynucleotide molecule encoding a V H region comprising a nucleotide which is about 80% homologous to the sequence of SEQ ID NO:4. 42. An isolated polynucleotide molecule encoding a V H region comprising a nucleotide which is about 90%> homologous to the sequence of SEQ ID NO:4. 43. An isolated polynucleotide molecule encoding a V H region comprising a nucleotide which is about 95% homologous to the sequence of SEQ ID NO:4. 44. An isolated polynucleotide molecule encoding a V H region comprising a nucleotide which is about 97% homologous to the sequence of SEQ ID NO:4. 45. An isolated polynucleotide molecule encoding a V H region comprising a nucleotide which is about 99%> homologous to the sequence of SEQ ID NO:4. 46. An isolated polynucleotide molecule encoding a CDR of an antibody variable region comprising a nucleotide sequence selected from the group consisting of nucleotides 70 to 117 of SEQ ID NO:2, nucleotides 163 to 183 of SEQ ID NO:2, nucleotides 280 to 306 of SEQ ID NO:2, nucleotides 91 to 105 of SEQ ID NO:4, nucleotides 148 to 198 of SEQ ID NO:4, nucleotides 295 to 330 of SEQ ID NO:4, degenerate sequences of any ofthe preceding CDR coding sequences, and combinations thereof. 47. An isolated polynucleotide molecule comprising a nucleotide sequence that is about 60% or more homologous to a nucleotide sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, nucleotides 70 to 117 of SEQ ID NO:2, nucleotides 163 to 183 of SEQ ID NO:2, nucleotides 280 to 306 of SEQ ID NO:2, nucleotides 91 to 105 of SEQ ID NO:4, nucleotides 148 to 198 of SEQ ID NO:4, and nucleotides 295 to 330 of SEQ ID NO:4. 48. An isolated polynucleotide molecule comprising a nucleotide sequence that is about 70% or more homologous to a nucleotide sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, nucleotides 70 to 117 of SEQ ID NO:2, nucleotides 163 to 183 of SEQ ID NO:2, nucleotides 280 to 306 of SEQ ID NO:2, nucleotides 91 to 105 of SEQ ID NO:4, nucleotides 148 to 198 of SEQ ID NO:4, and nucleotides 295 to 330 of SEQ ID NO:4. 49. An isolated polynucleotide molecule comprising a nucleotide sequence that is about 80% or more homologous to a nucleotide sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, nucleotides 70 to 117 of SEQ ID NO:2, nucleotides 163 to 183 of SEQ ID NO:2, nucleotides 280 to 306 of SEQ ID NO:2, nucleotides 91 to 105 of SEQ ID NO:4, nucleotides 148 to 198 of SEQ ID NO:4, and nucleotides 295 to 330 of SEQ ID NO:4. 50. An isolated polynucleotide molecule comprising a nucleotide sequence that is about 90% or more homologous to a nucleotide sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, nucleotides 70 to 117 of SEQ ID NO:2, nucleotides 163 to 183 of SEQ ID NO:2, nucleotides 280 to 306 of SEQ ID NO:2, nucleotides 91 to 105 of SEQ ID NO:4, nucleotides 148 to 198 of SEQ ID NO:4, and nucleotides 295 to 330 of SEQ ID NO:4. 51. An isolated polynucleotide molecule comprising a nucleotide sequence that is about 95% or more homologous to a nucleotide sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, nucleotides 70 to 117 of SEQ ID NO:2, nucleotides 163 to 183 of SEQ ID NO:2, nucleotides 280 to 306 of SEQ ID NO:2, nucleotides 91 to 105 of SEQ ID NO:4, nucleotides 148 to 198 of SEQ ID NO:4, and nucleotides 295 to 330 of SEQ ID NO:4. 52. An isolated polynucleotide molecule comprising a nucleotide sequence that is about 97% or more homologous to a nucleotide sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, nucleotides 70 to 117 of SEQ ID NO:2, nucleotides 163 to 183 of SEQ ID NO:2, nucleotides 280 to 306 of SEQ ID NO:2, nucleotides 91 to 105 of SEQ ID NO:4, nucleotides 148 to 198 of SEQ ID NO:4, and nucleotides 295 to 330 of SEQ ID NO:4. 53. An isolated polynucleotide molecule encoding a MUCl-specific binding member comprising the nucleotide sequence of SEQ ID NO:6. 54. The isolated polynucleotide molecule according to any one of Claims 30-53, wherein the polynucleotide molecule is a molecule selected from the group consisting of linear polynucleotide molecules, plasmids, phagemids, bacteriophage vectors, yeast display vectors, and eukaryotic viral vectors. 55. A method of diagnosing cancer in an individual comprising: providing a biological sample from the individual; contacting the biological sample from the individual with a MUCl-specific binding member according to any one of Claims 1-23, conservatively substituted versions of any ofthe preceding sequences, and combinations thereof; and detecting binding of said MUCl-specific binding member to MUCl in the biological sample of the individual. 56. The method of diagnosing cancer in an individual according to Claim 55, wherein the cancer is adenocarcinoma. 57. The method of diagnosing cancer in an individual according to Claim 55, wherein the biological sample from the individual is selected from the group consisting of cells, blood, lymph, urine, mammary tissue, ovary tissue, lung tissue, bladder tissue, and combinations thereof. 58. The method of diagnosing cancer in an individual according to Claim 55, wherein the binding of said MUCl-specific binding member to MUCl is detected by a detection means selected from the group consisting of enzyme-linked immunosorbent assay, magnetic resonance imaging, scintillation counting, and X-ray film. 59. A method of treating cancer in an individual comprising: administering to the individual in need of treatment thereof a MUCl-specific binding member according to any one of Claims 1-23, conservatively substituted versions of any ofthe preceding sequences, and combinations thereof. 60. The method of treating cancer in an individual according to Claim 59, wherein the cancer is adenocarcinoma. 61. The method of treating cancer in an individual according to Claim 59, further comprising administering a cytokine to the individual before, contemporaneously with, or after administering the MUCl-specific binding member. 62. The method of treating cancer in an individual according to Claim 59, wherein the cancer is present in tissue ofthe breast, ovary, lung, or bladder ofthe individual 63. An ex vz ' vo method of treating cancer in an individual comprising: obtaining a body fluid containing MUCl and/or MUCl-expressing cancer cells from an individual; contacting the body fluid with an immobilized MUCl-specific binding member according to any one of Claims 1-23, conservatively substituted versions of any ofthe preceding sequences, and combinations thereof; collecting the body fluid not bound to the immobilized MUCl-specific binding member; and returning the collected body fluid not bound to the immobilized MUCl-specific binding member to the individual. 64. The ex vivo method of treating cancer according to Claim 63, further comprising the step of adding one or more therapeutic agents to the body fluid prior to returning the fluid to the individual. 65. The ex vivo method of treating cancer according to Claim 63, wherein the body fluid is selected from the group consisting of bone marrow, blood, and peripheral blood stem cells. 66. The ex vivo method of treating cancer according to Claim 63, wherein the cancer is adenocarcinoma. 67. The ex vivo method of treating cancer according to Claim 63, wherein the anti-cancer reagent is a MUCl-specific binding member. 68. A method of making a MUCl-specific binding member comprising: preparing an expression vector comprising a polynucleotide sequence according to any of Claims 30-54, conservatively substituted versions of any ofthe preceding sequences, and combinations thereof; inserting said expression vector into a host cell; and culturing said host cell under conditions in which the MUCl-specific binding member is expressed from the expression vector. 69. The method of making a MUCl-specific binding member according to Claim 68, wherein the MUCl-specific binding member is selected from the group consisting of an immunoglobulin, a Fab antibody, F(ab') 2 antibody, a diabody, a scFv, a double scFv, a dAb, a Fv, an immunotoxin, and an immunocytokine.

Inventors: HENDERIKX MARIA P G, HOOGENBOOM HENDRICUS R J M, HENDERIKX, MARIA, P., G., HOOGENBOOM, HENDRICUS, R., J., M.
Applicants: DYAX CORP, DYAX CORP.
Application year: 2001
Main classification: C12N15
IPC codes: A61K35, A61K38, A61K39, A61K47, A61K49, A61K51, A61P35, A61P37, C07K14, C07K16, C07K19, C12N15, C12P21, C12Q1