GLYCOSYLATED TRANSFERRIN RECEPTOR 1 TUMOR ANTIGEN

Abstract

This invention relates to compositions and methods for treating or diagnosing cancer.

Claims

1. A composition or method comprising a purified human transferrin receptor 1 (TFR1) protein or a purified cell surface glycosylated peptide fragment thereof, for use as a biomarker for detection of malignancy.

2. The composition of claim 1, wherein said TFR1 protein or peptide fragment thereof comprises glycosylation at an aberrant site compared to a wild type TFR1 protein.

3. The compositon of claim 1, wherein said protein or peptide fragment does not comprise a N-linked glycosylation at amino acid position 251, 317, or 727 of SEQ ID NO:1.

4. The composition of claim 1, wherein said protein or peptide fragment comprises an N-linked glycosylation at amino acid position 50 or 55 of SEQ ID NO:1.

5. The composition of claim 1, wherein said protein or peptide fragment comprises an O-linked glycosylation at amino acid position 104 of SEQ ID NO:1.

6. A purified monoclonal antibody that binds to a human transferrin receptor 1 (TFR1) for use in immunotherapy, as a vehicle for delivery of anti-tumor compounds, or as a diagnostic agent, wherein said antibody binds to an aberrant glycosylation site of said TFR1.

7. The monoclonal antibody of claim 6, wherein said TFR1 comprises glycosylation at an aberrant site compared to a wild type TFR1 protein.

8. The monoclonal antibody of claim 6, wherein said antibody comprises AF-20.

9. The monoclonal antibody of claim 6, wherein said antibody does not comprise AF-20.

10. The composition or method of claim 1, wherein said TFR1 or glycosylated peptide thereof, is in the absence of other compounds with which it naturally occurs in a mammal, said compounds being selected from the group consisting of Heat Shock Protein 90 (HSP90) and/or a Na.sup.+/K.sup.+ ATPase or Mg++ ATPase (Transporting ATPase).

11. A method for delivering a cargo molecule to a subject, the method comprising: administering an antibody conjugated to said cargo molecule to the subject, wherein the antibody binds to a glycosylated human transferrin receptor-1 antigen, and wherein said cargo molecule is selected from the group consisting of a therapeutic agent or a detectable label.

12. The method of claim 11, wherein said therapeutic agent comprises a cytotoxic compound.

13. The method of claim 11, wherein said detectable label comprises a fluorescent compound or a radioisotope.

14. The method of claim 11, wherein said subject is characterized as comprising a malignancy.

15. The method of claim 11, wherein said antibody comprises AF-20.

16. The method of claim 11, wherein said antibody does not comprise AF-20.

17. The composition or method of claim 1, wherein said receptor or peptide thereof comprises asparagine (N)-linked glycosylation.

18. A purified glycosylated TFR1 antigen epitope comprising N-linked glycosylation at amino acid position 50 of SEQ ID NO:1 or amino acid position 55 of SEQ ID NO:1.

19. A purified glycosylated TFR1 antigen epitope comprising O-linked glycosylation at amino acid position 104 of SEQ ID NO:1.

20. The antigen epitope of claim 18 or 19, wherein said epitope comprises a length of at least 5 consecutive amino acids of SEQ ID NO:1.

21. A method of diagnosing a malignancy, comprising contacting a bodily tissue or fluid of a subject with an antibody that binds to TFR1, wherein said TFR1 comprises glycosylation at an aberrant site compared to a wild type TFR1 protein.

22. The method of claim 21, further comprising administering to said subject a cancer therapeutic composition.

23. The method of claim 11 or 21, wherein said subject comprises or is at risk of developing a colon cancer, a liver cancer, or a lung cancer.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0034] FIG. 1 is a blot showing the comparison of AF20 antigen levels in different cell lines. Cell lysate containing 200 ?g of proteins was subjected to immunoprecipitation with AF20 antibody followed by Western blot using the same antibody. The 50-kDa protein band in the blot corresponds to the heavy chain of AF20 antibody (IgG). Pro RG: proliferating HepaRG cells.

[0035] FIG. 2A is an elution profile showing the purification of AF20 antigen from Huh7 cells for proteomic analysis. Cell pellet (2 grams) was lyzed by freeze/thaw and diluted with Tris buffer. The sample was loaded onto a DEAE-cellulose column. After flow through (FT), the column was washed with Tris buffer (wash). Proteins were eluted successively with 100 mM, 200 mM and 400 mM of NaCl, and protein concentration was determined by BCA assay using known concentrations of albumin as a standard.

[0036] FIG. 2B is a blot showing that AF20 antigen was imunoprecipitated from the three protein peaks by AF20 mAb using Western blot analysis (minigel format).

[0037] FIG. 2C is an SDS-PAGE gel (large gel format) stained with Coomassie blue showing the pooled protein peaks.

[0038] FIG. 3A is a blot showing Huh7 cells transiently transfected with DDK tagged TFR1 cDNA. Lysate of transfected or non-transfected cells was immunoprecipitated with DDK or AF20 mAb, followed by sequential detection of the blot with DDK and TFR1 antibodies.

[0039] FIG. 3B is a blot showing lysate of nontransfected Huh7 cells incubated with AF20 mAb immobilized on protein G beads, or protein G beads alone to serve as a negative control, followed by Western blot detection of beads-associated proteins by antibodies against AF20, TFR1, HSP and N.sup.+/K.sup.+ ATPase, respectively.

[0040] FIG. 4 are images showing the affinity of AF20 mAb for TFR1 but not N.sup.+(K.sup.+ ATPase or HSP90 by immunofluorescent staining.

[0041] FIG. 5A is an image showing that TFR1 transfected NIH 3T3 cells were fixed and stained with either AF20 mAb.

[0042] FIG. 5B is an image showing that TFR1 transfected NIH 3T3 cells were fixed and stained with TFR1 mAb.

[0043] FIG. 5C is an image showing stacks of X axis for FIG. 5A and 5B, respectively.

[0044] FIG. 6A is a blot showing lysate of LS180 cells subjected to immunoprecipitation with AF20 mAb in the absence (w/o) or presence of 33 ?g/ml of apo or holo transferrin (TF), and retained proteins were detected by Western blot with AF20 mAb.

[0045] FIG. 6B is a blot showing that increasing concentrations (3.3, 16.5, and 66 ?g/ml) of holo transferrin added during immunoprecipitation with AF20 mAb, followed by Western blot with the same antibody.

[0046] FIG. 7 is a blot showing that AF20 mAb failed to recognize deglycosylated TFR1. Huh7 cell lysate was subject to immunoprecipitation with AF20 mAb and retained proteins on protein G beads were treated with PNGase F at 37? C. for lhr. Samples were directly loaded onto 10% SDS-PAGE (minigel) in duplicate followed by Western blot detection using AF20 (left) and TFR1 antibodies (right), respectively. An additional protein band above the 75-kd size marker was detected by TFR1 antibody from PNGase F treated sample, most likely corresponding to deglycosylated form of TFR1 (79kDd).

[0047] FIG. 8 are images of tissue sections showing a correlation of AF20 expression of malignant transformation of colon tissues. A total of 4 pairs of tissue sections were stained with AF20 antibody followed by incubation with HRP-conjugated anti-mouse antibody. Two representative pairs are shown (upper and lower panels). AF20 was clearly detectable in colon polyps (middle panels), and strongly expressed in colon cancer (right panels), but not in normal colon tissue (left panels). Images were taken at 20? magnifications, with the scale bar provided.

[0048] FIG. 9A is a tissue section image of a normal colon sample stained with TFR1 (upper panels, 1:50 dilution) and AF20 mAb (lower panels, 1:500 dilution), respectively. Images were taken at 1000? magnification. Overexpression of both TFR1 and AF20 antigen was not observed in normal colon samples. The expression pattern and localization as revealed by TFR1 and AF20 Ab are indistinguishable.

[0049] FIG. 9B is a tissue section image of a colon cancer sample stained with TFR1 (upper panels, 1:50 dilution) and AF20 mAb (lower panels, 1:500 dilution), respectively. Images were taken at 1000? magnification. Overexpression of both TFR1 and AF20 antigen was observed in colon cancer samples. The expression pattern and localization as revealed by TFR1 and AF20 Ab are indistinguishable.

[0050] FIG. 10 is blot showing Huh7 cells transfected with cDNA encoding DDK-tagged TFR1 or DDK-tagged Na.sup.+/K.sup.+-ATPase. Cells were harvested two days later, and cell lysate in duplicate was immunoprecipitated with DDK mAb followed by blotting with either DDK mAb or AF20 mAb. The results suggest that TFR1 (the AF20 antigen) and ATPase could be co-precipitated. The protein band of 75 kd in DDK blot was due to non-specific binding of the antibody

DETAILED DESCRIPTION OF THE INVENTION

[0051] Since the generation of the AF20 monoclonal antibody more than 20 years ago, the molecular identity of its target antigen has remained a longstanding puzzle.

[0052] The AF20 antibody was developed after immunizing mice with a HCC cell line (FOCUS) using a schedule designed to generate monoclonal antibodies (mAb) against overexpressed cell surface proteins on the tumor cell line. However, great difficulty was encountered in characterizing the AF20 antigen. The hybridoma producing AF20 was identified, since the antibody highly reacted with human tumors and cell lines derived thereof and included liver, lung, and colon tumors. The antibody did not recognize expression of the antigen on most normal human tissues. Previous attempts to clone the AF20 antigen used a FOCUS HCC cell line derived cDNA library, these attempts were unsuccessful likely because this library was missing the 5 ends of the cDNA which may have encoded for the AF20 antigen. The molecular weight of the AF20 antigen was determined by metabolic labeling with 5.sup.32-Methionine or direct labeling with 1.sup.125 followed by immunoprecipitation experiments, which gave a molecular weight of 180 kDa antigen. The early studies demonstrated that it was synthesized as a 90 kDa homodimer and assembled on the cell surface of tumor cells as a 180 kDa protein comprised of two 90 kDa peptides linked by two disulfide bridges. However, numerous attempts to immunoprecipitate the AF20 antigen in tumors and tumor cell lysates were unsuccessful. For nearly 25 years, a number of investigators were unsuccessful in identifying the molecular identity of the AF20 antigen. The invention has provided a solution to this longstanding problem in cancer diagnosis and therapy.

[0053] The present work describes and confirms why it was so difficult to identify the antigen precisely. The AF20 epitope on the 180 kDa protein was a post translational modification of glycosylation and was actually in a complex with two other proteins of the identical size. The AF20 antigen is actually on transferrin-1 and is generated by abnormal glycosylation by the machinery operating in tumor tissues. The technique for identifying the 3 proteins that comprise the complex is described herein. The surprising discovery made while identifying the AF20 antigen was that the AF20 antigen is generated during malignant transformation by abnormal glycosylation of this transferrin receptor protein. This discovery was completely unexpected.

[0054] The elucidation and purification of this tumor antigen has broad implications for human cancer diagnosis and therapy. The purified antigen and antibodies specific for the antigen has great utility in identification of and treatment of malignancies. For example, it is useful as an imaging agent, as well as a carrier of a drug or isotope since once the antibody binds to the antigen complex, is internalized into the cell. It is also useful as a diagnostic marker of cell transformation such as screening for dysplastic cellular changes in long standing ulcerative colitis which has a high risk of developing colon cancer. It is also used to evaluate prognosis with respect to early tumor reoccurrence and overall survival.

[0055] Extensive studies have revealed specific expression of AF20 antigen in human hepatoma and colon cancer cells. The rapid internalization of AF20 antibody in human hepatoma cell lines raised the possibility of specific and highly efficient treatment of liver cancer by conjugating small molecule drugs into the antibody (Mohr L,et al. Gastroenterology 2004;127:S225-231, and Moradpour D et al. Hepatology 1995;22:1527-1537). While immunofluorescent staining of the antigen indicated its cell surface localization, detection by direct Western blot analysis has been unsuccessful. Rather, a combination of immunoprecipitation (IP) and Western blotting was needed.

[0056] In the present study, IPSDS PAGE was combined with ion-exchange chromatography to purify the AF20 antigen. Curiously, the AF20 antigen failed to be eluted from DEAE-cellulose column as a single peak. Rather, it was present in eluents of all the three NaCl concentrations suggesting variable affinities for the negatively charged column (FIG. 2B). Peptide sequencing of the tryptic digests revealed two or three proteins instead of a single protein despite further purification by IPSDS PAGE, which enriched AF20 antigen according to its immunological property as well as molecular size (90-110 kDa).

[0057] Consequently, the three proteins identified: TFR1, HSP90, and Na.sup.+/K.sup.+ ATPase, shared similar molecular weights. Indeed, in the subsequent reconstitution experiments using cDNA transfection, IPWestern blot analysis revealed ability of the AF20 antibody to immunoprecipitate TFR1, HSP90, as well as Na.sup.+/K.sup.+ ATPase. Considering that a monoclonal antibody recognizes an epitope of just 5-7 residues, the AF20 epitope could be present in all the three proteins. The AF20 epitope is present in just one of the three proteins described, and the other two proteins were co-purified with the true AF20 antigen through complex formation.

[0058] Surprisingly, the AF-20 epitope was found to be present in TFR1 Two lines of experimental evidence supported the second interpretation and implicated TFR1 [alternatively known as TFR, p90, and CD71 (Tortorella S et al. J Membr Biol 2014;247:291-307)], as the bona fide AF20 antigen. First, transient transfection with TFR1 but not Na.sup.+/K.sup.+ ATPase or HSP90 cDNA conferred strong cell surface staining by AF20 antibody in NIH 3T3 cells, which express little endogenous AF20 antigen. Second, holo transferrin could compete for the AF20 antigen - antibody interaction during immunoprecipitation. As TFR1 has much greater affinity for the diferric transferrin (Dautry-Varsat A Proc Nall Acad Sci 1983;80:2258-2262), this finding also indicates the overlap between the transferrin binding site and the AF20 epitope. In addition, known features of TFR1 are also consistent with those of the AF20 antigen. TFR2, on the other hand, is a protein of 355 residues (approximately 39 kDa). Moreover, ability of holo transferrin to interfere with the interaction of AF20 antigenantibody is also compatible with TFR1, which has much greater affinity for diferric transferrin than TFR2.

Transferrin Receptor-1

[0059] TFR1 is a type II transmembrane protein. Its 760 residues consist of the short cytoplasmic domain (residues 1-67), a single transmembrane domain (residues 68-88), and a large extracellular domain containing three N-linked glycosylation sites. Tunicamycin treatment not only reduced size of TFR1 from 94 kDa to 79 kDa, but also prevented its dimerization. Deglycosylated TFR1 lost affinity for transferrin (Reckhow CL and Enns CA. J Biol Chem 1988;263:7297-7301). The finding that deglycosylated TFR1 is unable to bind AF20 antibody indicates a role of N-linked glycans in recognition by the AF20 mAb. Alternatively, deglycosylation of TFR1 renders the protein unstable to prevent binding by the AF20 mAb.

[0060] TFR1 is responsible for iron deposition to most cell types. At neutral pH it has higher affinity for holo transferrin than apo transferrin. Following clathrin-mediated endocytosis, the acidic environment in the endosome triggers iron release from transferrin but increases the affinity of apo transferrin for TFR1 (Dautry-Varsat A et al. Proc Nail Acad Sci 1983;80:2258-2262). After fusion of endosomes with plasma membrane, the neutral pH promotes the release of apo transferrin from TFR1, thus completing the cycle of iron transport. A single cycle takes only 10-20 minutes (Bleil JD and Bretscher MS. EMBO J 1982;1:351-355), which is reminiscent of rapid internalization of the AF20 antibody in human hepatoma cells (Moradpour D, Hepatology 1995;22:1527-1537).

[0061] TFR1 is ubiquitously expressed in normal tissues at low level. In the present study, AF20 was undetectable in normal colon but clearly detected in polyps and strongly positive in colon cancer (FIGS. 8 and 9A and 9B).

[0062] Human Transferrin Receptor-lamino acid sequence:

TABLE-US-00001 1mmdqarsafsnlfggeplsytrfslarqvdgdnshvemklavdeeenadnntkanvtkpk 61rcsgsicygtiavivffligfmigylgyckgvepktecerragtespvreepgedfpaar 121rlywddlkrklsekldstdftgtikllnensyvpreagsqkdenlalyvenqfrefklsk 181vwrdqhfvkiqvkdsaqnsviivdkngrlvylvenpggyvayskaatvtgklvhanfgtk 241kdfedlytpvngsivivragkitfaekvanaeslnaigvliymdqtkfpivnaelsffgh 301ahlgtgdpytpgfpsfnhtqfppsrssglpnipvqtisraaaeklfgnmegdcpsdwktd 361stcrmvtsesknvkltvsnvlkeikilnifgvikgfvepdhyvvvgaqrdawgpgaaksg 421vgtalllklaqmfsdmvlkdgfqpsrsiifaswsagdfgsvgatewlegylsslhlkaft 481yinldkavlgtsnfkvsaspllytliektmqnvkhpvtgqflyqdsnwaskvekltldna 541afpflaysgipavsfcfcedtdypylgttmdtykelieripelnkvaraaaevagqfvik 601lthdvelnldyerynsqllsfvrdlnqyradikemglslqwlysargdffratsrlttdf 661gnaektdrfvmkklndrvmrveyhflspyvspkespfrhvfwgsgshtlpallenlklrk 721qnngafnetlfrnqlalatwtiqgaanalsgdvwdidnef
(SEQ ID NO: 1) GenBank Accession NP_001121620 version 1, incorporated herein by reference.

[0063] Exemplary regions or fragments of Transferrin Receptor-linclude residues 1-67, 20-23 (endocytosis signal), 68-88 (transmembrane region), 100-101, 101-760, 201-377, 385-610, 569-760, 642-750, and 646-648 as well as immunogenic fragments thereof for use in immunotherapy. The region may include the cytoplasmic domain (residues 1-67), or the extracellular domain (residues 89-763). Furthermore, the region may include the ligand-binding region (residues 572-763), the endocytosis signal (residues 20-23), the stop-transfer sequence (residues 58-61), the apical domain (residues 324-368), and the cell attachment site residues 649-651. In some examples, the fragment comprises of a cell surface epitope. Exemplary peptides or fragments of Transferrin Receptor 1 include those which comprise an N-linked glycosylation site, e..g, as described in Medzihradszky K. F., Methods Mol Biol. 2008;446:293-316; hereby incorporated by reference. N-glycosylated proteins are modified at Asn residues. There is a consensus sequence for N-glycosylation: AsnXxxSer/Thr/Cys, where Xxx can be any amino acid except proline. For example, wild type TFR-1 includes N-linked glycosylation sites at amino acid positions 251, 317, and 727 of SEQ ID NO:1 (Williams et al., 1993, J. Biol. Chem. 268(17): 12780-12786; Daniels et al., 2012, Biochim Biophys Acta. 1820(3): 291-317; each of which is hereby incorporated by reference. N-linked glycosylation sites are also present at amino acids 50 and 55 (N50 and/or N55) of SEQ ID NO:1. An O-linked glycosylation site is present at amino acid position 104 (T104) of SEQ ID NO:1

[0064] A fragment has a length that is less than the length of the full-length reference peptide. For example, in the case of the transferrin receptor 1 shown above, a fragment is a peptide that containg greater than 1 amino acid and contains less than 760 amino acids, e.g., the fragment contains or contains less than 5, 8, 9, 10, 12, 15, 20, 25, 30, 40, 50, 75, 100, 200, 300, 400, 500, 600, 700, or 750 contiguous amino acids of SEQ ID NO: 1. For example, a fragment comprises a TFR1-sprefic antibody-binding epitope of 5-7 amino acids.

[0065] Human Transferrin Receptor-1 nucleotide sequence.

TABLE-US-00002 1acgcacagcccccctgggggccgggggcggggccaggctataaaccgccggttaggggcc 61gccatcccctcagagcgtcgggatatcgggtggcggctcgggacggaggacgcgctagtg 121ttcttctgtgtggcagttcagaatgatggatcaagctagatcagcattctctaacttgtt 181tggtggagaaccattgtcatatacccggttcagcctggctcggcaagtagatggcgataa 241cagtcatgtggagatgaaacttgctgtagatgaagaagaaaatgctgacaataacacaaa 301ggccaatgtcacaaaaccaaaaaggtgtagtggaagtatctgctatgggactattgctgt 361gatcgtctttttcttgattggatttatgattggctacttgggctattgtaaaggggtaga 421accaaaaactgagtgtgagagactggcaggaaccgagtctccagtgagggaggagccagg 481agaggacttccctgcagcacgtcgcttatattgggatgacctgaagagaaagttgtcgga 541gaaactggacagcacagacttcaccggcaccatcaagctgctgaatgaaaattcatatgt 601ccctcgtgaggctggatctcaaaaagatgaaaatcttgcgttgtatgttgaaaatcaatt 661tcgtgaatttaaactcagcaaagtctggcgtgatcaacattttgttaagattcaggtcaa 721agacagcgctcaaaactcggtgatcatagttgataagaacggtagacttgtttacctggt 781ggagaatcctgggggttatgtggcgtatagtaaggctgcaacagttactggtaaactggt 841ccatgctaattttggtactaaaaaagattttgaggatttatacactcctgtgaatggatc 901tatagtgattgtcagagcagggaaaatcacctttgcagaaaaggttgcaaatgctgaaag 961cttaaatgcaattggtgtgttgatatacatggaccagactaaatttcccattgttaacgc 1021agaactttcattctttggacatgctcatctggggacaggtgacccttacacacctggatt 1081cccttccttcaatcacactcagtttccaccatctcggtcatcaggattgcctaatatacc 1141tgtccagacaatctccagagctgctgcagaaaagctgtttgggaatatggaaggagactg 1201tccctctgactggaaaacagactctacatgtaggatggtaacctcagaaagcaagaatgt 1261gaagctcactgtgagcaatgtgctgaaagagataaaaattcttaacatctttggagttat 1321taaaggctttgtagaaccagatcactatgttgtagttggggcccagagagatgcatgggg 1381ccctggagctgcaaaatccggtgtaggcacagctctcctattgaaacttgcccagatgtt 1441ctcagatatggtcttaaaagatgggtttcagcccagcagaagcattatctttgccagttg 1501gagtgctggagactttggatcggttggtgccactgaatggctagagggatacctttcgtc 1561cctgcatttaaaggctttcacttatattaatctggataaagcggttcttggtaccagcaa 1621cttcaaggtttctgccagcccactgttgtatacgcttattgagaaaacaatgcaaaatgt 1681gaagcatccggttactgggcaatttctatatcaggacagcaactgggccagcaaagttga 1741gaaactcactttagacaatgctgctttccctttccttgcatattctggaatcccagcagt 1801ttctttctgtttttgcgaggacacagattatccttatttgggtaccaccatggacaccta 1861taaggaactgattgagaggattcctgagttgaacaaagtggcacgagcagctgcagaggt 1921cgctggtcagttcgtgattaaactaacccatgatgttgaattgaacctggactatgagag 1981gtacaacagccaactgctttcatttgtgagggatctgaaccaatacagagcagacataaa 2041ggaaatgggcctgagtttacagtggctgtattctgctcgtggagacttcttccgtgctac 2101ttccagactaacaacagatttcgggaatgctgagaaaacagacagatttgtcatgaagaa 2161actcaatgatcgtgtcatgagagtggagtatcacttcctctctccctacgtatctccaaa 2221agagtctcctttccgacatgtcttctggggctccggctctcacacgctgccagctttact 2281ggagaacttgaaactgcgtaaacaaaataacggtgcttttaatgaaacgctgttcagaaa 2341ccagttggctctagctacttggactattcagggagctgcaaatgccctctctggtgacgt 2401ttgggacattgacaatgagttttaaatgtgatacccatagcttccatgagaacagcaggg 2461tagtctggtttctagacttgtgctgatcgtgctaaattttcagtagggctacaaaacctg 2521atgttaaaattccatcccatcatcttggtactactagatgtctttaggcagcagctttta 2581atacagggtagataacctgtacttcaagttaaagtgaataaccacttaaaaaatgtccat 2641gatggaatattcccctatctctagaattttaagtgctttgtaatgggaactgcctctttc 2701ctgttgttgttaatgaaaatgtcagaaaccagttatgtgaatgatctctctgaatcctaa 2761gggctggtctctgctgaaggttgtaagtggtcgcttactttgagtgatcctccaacttca 2821tttgatgctaaataggagataccaggttgaaagaccttctccaaatgagatctaagcctt 2881tccataaggaatgtagctggtttcctcattcctgaaagaaacagttaactttcagaagag 2941atgggcttgttttcttgccaatgaggtctgaaatggaggtccttctgctggataaaatga 3001ggttcaactgttgattgcaggaataaggccttaatatgttaacctcagtgtcatttatga 3061aaagaggggaccagaagccaaagacttagtatattttcttttcctctgtcccttccccca 3121taagcctccatttagttctttgttatttttgtttcttccaaagcacattgaaagagaacc 3181agtttcaggtgtttagttgcagactcagtttgtcagactttaaagaataatatgctgcca 3241aattttggccaaagtgttaatcttaggggagagctttctgtccttttggcactgagatat 3301ttattgtttatttatcagtgacagagttcactataaatggtgtttttttaatagaatata 3361attatcggaagcagtgccttccataattatgacagttatactgtcggttttttttaaata 3421aaagcagcatctgctaataaaacccaacagatactggaagttttgcatttatggtcaaca 3481cttaagggttttagaaaacagccgtcagccaaatgtaattgaataaagttgaagctaaga 3541tttagagatgaattaaatttaattaggggttgctaagaagcgagcactgaccagataaga 3601atgctggttttcctaaatgcagtgaattgtgaccaagttataaatcaatgtcacttaaag 3661gctgtggtagtactcctgcaaaattttatagctcagtttatccaaggtgtaactctaatt 3721cccattttgcaaaatttccagtacctttgtcacaatcctaacacattatcgggagcagtg 3781tcttccataatgtataaagaacaaggtagtttttacctaccacagtgtctgtatcggaga 3841cagtgatctccatatgttacactaagggtgtaagtaattatcgggaacagtgtttcccat 3901aattttcttcatgcaatgacatcttcaaagcttgaagatcgttagtatctaacatgtatc 3961ccaactcctataattccctatcttttagttttagttgcagaaacattttgtggtcattaa 4021gcattgggtgggtaaattcaaccactgtaaaatgaaattactacaaaatttgaaatttag 4081cttgggtttttgttacctttatggtttctccaggtcctctacttaatgagatagtagcat 4141acatttataatgtttgctattgacaagtcattttaactttatcacattatttgcatgtta 4201cctcctataaacttagtgcggacaagttttaatccagaattgaccttttgacttaaagca 4261gagggactttgtatagaaggtttgggggctgtggggaaggagagtcccctgaaggtctga 4321cacgtctgcctacccattcgtggtgatcaattaaatgtaggtatgaataagttcgaagct 4381ccgtgagtgaaccatcattataaacgtgatgatcagctgtttgtcatagggcagttggaa 4441acggcctcctagggaaaagttcatagggtctcttcaggttcttagtgtcacttacctaga 4501tttacagcctcacttgaatgtgtcactactcacagtctctttaatcttcagttttatctt 4561taatctcctcttttatcttggactgacatttagcgtagctaagtgaaaaggtcatagctg 4621agattcctggttcgggtgttacgcacacgtacttaaatgaaagcatgtggcatgttcatc 4681gtataacacaatatgaatacagggcatgcattttgcagcagtgagtctcttcagaaaacc 4741cttttctacagttagggttgagttacttcctatcaagccagtacgtgctaacaggctcaa 4801tattcctgaatgaaatatcagactagtgacaagctcctggtcttgagatgtcttctcgtt 4861aaggagatgggccttttggaggtaaaggataaaatgaatgagttctgtcatgattcacta 4921ttctagaacttgcatgacctttactgtgttagctctttgaatgttcttgaaattttagac 4981tttctttgtaaacaaatgatatgtccttatcattgtataaaagctgttatgtgcaacagt 5041gtggagattccttgtctgatttaataaaatacttaaacactgaaaaaaaaaaa
(SEQ ID NO: 2) GenBank Accession NM_001128148, version 2 incorporated herein by reference.

[0066] Exemplary regions or fragments of Transferrin Receptor-1 nucleic acid sequences include residues 197-199, 200-211, 314-325, 344-406, 1847-2422, 2078-2086, 830-943, 944-1042, and 4932-5057.

AF-20

[0067] AF-20 antibody is a monoclonal antibody and is an anti-hepatocellular carcinoma (HCC) monoclonal antibody that binds to a rapidly internalized 180-kDa homodimeric glycoprotein present in high amounts on the surface membrane of human HCC and other human cancer cell lines.

[0068] Immunizing mice with hepatoma cells derived from an HCC cell line (FOCUS), a handful of monoclonal antibodies were isolated with high affinity to cancer cells but not to normal or non-transformed hepatocytes (Wilson B et al. Proc Natl Acad Sci 1988;85:3140-4). One of the antibodies, namely AF20, recognized a protein of apparent molecular size of 90-110 kDa. The AF20 antigen was found abundantly expressed on cell surface of human HCC cell lines, as well as human colon cancer cell lines such as LS180 and HT-29 (Mohr L et al. Gastroenterology 2004;127:5225-231, and Moradpour D et al. Hepatology 1995;22:1527-1537). Binding of AF20 antibody to the cancer cells was followed by its rapid internalization, which raises the hope of specific and highly efficient delivery of therapeutic drugs or small molecules into cancer cells (Moradpour D et al. Hepatology 1995;22:1527-1537, and Wands J R, et al. J Viral Hepat 1997;4 Suppl 2:60-74). In vivo experiments validated AF20's ability to differentiate human HCC from adjacent normal liver tissue (Wilson B, et al. Proc Natl Acad Sci 1988;85:3140-4). AF20 serves as a biomarker for early detection and diagnosis of malignant transformation, and also as a vehicle for delivery of anti-tumor drugs with high affinity and specificity. Further characterization revealed AF20 antigen as a dimer of 90-kDa glycoprotein linked together by disulfide bonds (Moradpour D et al. Hepatology 1995;22:1527-1537). The AF20 antigen is identical to the glycosylated form of human transferrin receptor 1 (TFR1), which can form a protein complex with heat shock protein 90 (HSP90) and/or transporting ATPase

Human AF-20

[0069] AF20 monoclonal antibody (mAb) was produced and characterized as previously described (Wilson B et al. Proc Natl Acad Sci 1988;85:3140-4 , Moradpour D et al. Hepatology 1995;22:1527-1537, and Wands J R, et al. J Viral Hepat 1997;4 Suppl 2:60-74), incorporated herein by reference.

Heat shock protein 90 (Hsp90)

[0070] Hsp90 is a chaperone protein that assists other proteins to fold properly, stabilizes proteins against heat stress, and aids in protein degradation. It also stabilizes a number of proteins required for tumor growth. Heat shock proteins protect cells when stressed by elevated temperatures and when cells are heated, the fraction of heat shock proteins increases to 4-6% of cellular proteins. Heat shock protein 90 (Hsp90) is one of the most common of the heat-related proteins. The 90 comes from the fact that it weighs roughly 90 kDa.

[0071] Heat shock protein 90 (Hsp90) amino acid sequence:

TABLE-US-00003 1mesltdpskldsgkephislipnkqdrtltivdtgigmtkadlinnlgtitksetkvfme 61vlqagadismigqfsvgfysaysvaekvtvitkhnndeqyawesslrgsfteyrefyksl 121tinwedylavkhfsvegqlefraflfvprlapfelletrkkknkiklsarrdlimdncee 181lipeylnfirgvvdsedlplnifretkdqvanstivqrlwkhgleviytiepideycvqq 241lkefegktlvsvtkedlelpedeeekkkqeegkqktkqkknqslrtsakstygwtanmer 301imkaqalrdnsttgymaakkhleinpdhsfidtlrqkaetdkndksvkdlvillyetall 361ssdfglegpqthanriyrmnklglgtdeddptaddtsaavteempplegdddtsrmek

[0072] (SEQ ID NO: 5) GenBank Accession Q58FG1 version 1, incorporated herein by reference. Exemplary regions or fragments of Heat shock protein 90 (Hsp90) include residues 11-101, 2-418, and 112-410.

[0073] Heat shock protein 90 (Hsp90) nucleotide sequence:

TABLE-US-00004 1gagctccggctgccctgcactggttcccagagactccctccttcccaggtccaaatggct 61gcaggagcgaagtgggcggaaaaaaagcgaaccagcttgagaaagggcttgacgtgcctg 121cgtagggagggcgcatgtccccgtgctccgtgtacgtggcggccgcaggggctagagggg 181ggtcccccccgcaggtactccactctcagtctgcaaaagtgtacgcccgcagagccgccc 241caggtgcctgggtgttgtgtgattgacgcggggaaggaggggtcagccgatccctcccca 301accctccatcccatccctgaggattgggctggtacccgcgtctctcggacaggtcagagc 361gggtcgccgggtggggtcgctgcaaaaaccctgccccggccgcagccgagaggcggacgt 421cgcggggagggggcgggaccgccgagacaggcctggaaactgctggaaatgccgcagtgc 481cgccgccgccccttccgccgcatgtcggcaaagagtccccgccagccccggccggcgccc 541tccccctacgctgagctgcccctcagcgcgaaccctccgcccttcctctactcctgcgag 601agtcgggatctggggctacccaaggttgggtcccgaatgccagtccctctgtcgggacgc 661gagatgtgtagggcagatgctaggaagaagattgggtctgggacgggtggtccgcgtggt 721tagctgcctccgctctttttcggtgtcccccccagtcccgcccttgggtgtggggacgcc 781tgccccacaagtgtttagggaggtcagtgggttcctcgcccgtagagacaccgtttatgc 841caaatgagcactcctcatccccgctcttgatggagtcatgtcctagacgtgaaactatgg 901ggctgtgatcacaagcaaatgtgtgggcggatccgttgcttgggttcttccccgccccct 961ctttttttcggaccatgacgtcaaggtgggctggtggcggcaggtgcggggttgacaatc 1021atactcctttaaggcggagggatctacaggagggcggctgtactgtgcttcgccttatat 1081agggcgacttggggcacgcagtagctctctcgagtcactccggcgcagtgttgggactgt 1141ctgggtatcggaaagcaagcctacgttgctcactattacgtataatccttttcttttcaa 1201ggtaaggctgagatctccgctaggcttctttccctttagtgctgtattcgtgttgttttt 1261gtttttttctgtcctttagggagccttagtctagatgtcggggtggcttgtggataacgc 1321tctggatttttatagggtgagggtagtggtgggtgaggttttttgagtcctcctcggttt 1381tctctagtgtgtttggggggtggggctttctctcggcgcctgctggccgtagcgaggtgg 1441gctgtggggttggggcagtgggcggctggcagctgcacgtggtggccgcgcggcccggga 1501cgctgccatttttgcccctccacttccggacgcggctacggggcgtcggagggggaccgc 1561aggtggcgggggtgcccgctcgggtgactcagcacggccttgtgggactggctttgtcac 1621ctctcttatcggacgcgttgttaaagccttcttgggtgctttgtttctgtgagggagggt 1681tgacggtgtgggaagagagctttcggtctccagcacccgatactccctccttccagatct 1741ttcttgcagtcccggtggaggaggggcggggaggggagcaggttctggaagattcatggg 1801ctccttcctccgcccttcctcgagagctgagattgttctggaagcttctggattctggcg 1861ccccgccccagtgcccggatgctgggggcgagggagggtgcactgcggcgccccctcctc 1921gcgtggtcctggccgacgcatgtccggcagtgacgagtgtcggcctggtggctacggcca 1981ccatctttcttgggtttggtcctgttctgtaattttgtgctgtgaaagggtcgtggtgga 2041gcttttggcttaagaattctttgtccggatttaattgctcctccggtgggtatcgtatgg 2101atcccaggttattcctccctgccctatgggcaggagtgtcccgcccttggactggtctta 2161ggaactgacacctcagggggagcagtttaaagttagtgccatttttatcttaaactagtc 2221actttgacctcccccaaataaagaactgtaggtagtgattttcacatttaaatttgtgta 2281aggattacttgggatctctagatacctgggttggaccaacattatgatttttctgccata 2341ctaccagatgatgctgaggctgctggtcaccattctttaagtaggtgggttctgtgacat 2401ttggttgaagaatatttagcttattttctttttccttctgaattttcaggcctcccactt 2461agtgtgtagtctgagatctttaagagaatgcatttttagtcttgggaagggatagtactc 2521cggttaaaccagtctgaactcactgtctaaggtcctaacaaatgatatgacctttaggat 2581ttttaaacatggggccttagtgttcttttgtaattaatgagatttttattttagatgcct 2641gaggaagtgcaccatggagaggaggaggtggagacttttgcctttcaggcagaaattgcc 2701caactcatgtccctcatcatcaataccttctattccaacaaggagattttccttcgggag 2761ttgatctctaatgcttctgatgtaggtgctctggtttccacatttggcatggtttttttt 2821tttgatactctagaaggaggggaaaggagtggtttggcctttgttggggactactattga 2881agggggtaaacttgcagctattccaaaaagatgggttttactctggccatcttgaacttg 2941gaagggactatgtccagaataagtgggctcatggaactaactggttctaaagcctcaaga 3001tagggggcaatcagatttgaggctgagagaggtaaaccaagattttctttgaagatacgg 3061gctttaagaaagcaaaagtggctgagcgtgttggctcacacctgtaatgccaaggcagga 3121ggatcacttgagcctaggatttcgagggcagcctgggcaaccgcgagaccttgtctctgc 3181aaaaaattaaatattagttgggcacggtggcatgtgctgtagtcccagctacttgggaag 3241ctggggttgggaggatggctcgaacctgggaggtcaaggctgcagtgagctgtcatcctt 3301gccactgcactgtagcttgggcaacagagcaagagtctgtcttggaaagagcaaaagtaa 3361gttgctgtttgtatttccaggccttggacaagattcgctatgagagcctgacagaccctt 3421cgaagttggacagtggtaaagagctgaaaattgacatcatccccaaccctcaggaacgta 3481ccctgactttggtagacacaggcattggcatgaccaaagctgatctcataaataatttgg 3541gaaccattgccaagtctggtactaaagcattcatggaggctcttcaggtattgcagttct 3601gtaggcattcatacttatctgtgttctttggttttttgcttctttaaaacttgtgattga 3661ctttaaacttgttggcaggctggtgcagacatctccatgattgggcagtttggtgttggc 3721ttttattctgcctacttggtggcagagaaagtggttgtgatcacaaagcacaacgatgat 3781gaacagtatgcttgggagtcttctgctggaggttccttcactgtgcgtgctgaccatggt 3841aagttagcttttctgttacaaggtagttgggttaggattttctgggctcacaccagtagc 3901agaaattttgggcatcctgtctgtaaagcagttcttcacagcagttctgctgatacttac 3961taattgctggtctcaactgcatatactttttaccctgttacacgcttgtaattgactctt 4021ctaggtgagcccattggcaggggtaccaaagtgatcctccatcttaaagaagatcagaca 4081gagtacctagaagagaggcgggtcaaagaagtagtgaagaagcattctcagttcataggc 4141tatcccatcaccctttatgtgagtatggacttttaaatcttttacacttaacgtgcagga 4201tgtttcctgttctggagaatctcattgtccctggcttttgctttccctggtagtgttttg 4261tactccaaggctaacttctgtttttgttacttagttggagaaggaacgagagaaggaaat 4321tagtgatgatgaggcagaggaagagaaaggtgagaaagaagaggaagataaagatgatga 4381agaaaaacccaagatcgaagatgtgggttcagatgaggaggatgacagcggtaaggataa 4441gaagaagaaaactaagaagatcaaagagaaatacattgatcaggaagaactaaacaagac 4501caagcctatttggaccagaaaccctgatgacatcacccaagaggagtatggagaattcta 4561caagagcctcactaatgactgggaagaccacttggcagtcaaggtgtgagaagcctttgc 4621atgttggctcaacatgcacatatggagaggaatgagttaggtggaagagtgttgggtaat 4681agacacacggaacttgtccaactgataacagaaatgtgatagccatgtgatttcacttac 4741tgattaccctgtcatagtgaagtgccatcatttctaatgacctcactttctcttcttatg 4801gaaatctgggtaatgtctattggcagccttacacatccagggttctgatcagaggggact 4861gttttctacatacagctagtacccatctagatcgtggagggcattaaggctcagttttct 4921caggagctgcttgttgtgtgtgctctatcccttaggcttagggaggatcattgttccact 4981tttagataatttggtgttggggctaaaaggtcctcttttgaaatgtaccacttatttttg 5041gtttctttcagcacttttctgtagaaggtcagttggaattcagggcattgctatttattc 5101ctcgtcgggctccctttgacctttttgagaacaagaagaaaaagaacaacatcaaactct 5161atgtccgccgtgtgttcatcatggacagctgtgatgagttgataccagagtatctcagtg 5221agtatctccttggcctaatttagttgggtgaagtcttgggaggttttaggcattctgcta 5281ggatattctaaggtaacagttttctgcaatacatagtaggtgtaagggttcaggaggcta 5341ttagagccttctgtttgaatctggggaccaggtctggtctagctgtttttactgagcttt 5401ctcaccctggttgatggcagattttatccgtggtgtggttgactctgaggatctgcccct 5461gaacatctcccgagaaatgctccagcagagcaaaatcttgaaagtcattcgcaaaaacat 5521tgttaagaagtgccttgagctcttctctgagctggcagaagacaaggagaattacaagaa 5581attctatgaggcattctctaaaaatctcaaggtaaaaaggcaaataatgcttattccctt 5641taccactttcttagtaataacaataaattattccattcacattgaaagtgaagttattgt 5701agttaagctggattgtttttcctcttcccacccttcaagcttggaatccacgaagactcc 5761actaaccgccgccgcctgtctgagctgctgcgctatcatacctcccagtctggagatgag 5821atgacatctctgtcagagtatgtttctcgcatgaaggagacacagaagtccatctattac 5881atcactggtgcgttgactctgattgaagcctttttggaggagtggggagcacaattaggg 5941cttcctgggaactggcagtatgaggcattttagtcactgagttcatttaattaccctaca 6001ggtgagagcaaagagcaggtggccaactcagcttttgtggagcgagtgcggaaacggggc 6061ttcgaggtggtatatatgaccgagcccattgacgagtactgtgtgcagcagctcaaggaa 6121tttgatgggaagagcctggtctcagttaccaaggagggtctggagctgcctgaggatgag 6181gaggagaagaagaagatggaagagagcaaggcaaagtttgagaacctctgcaagctcatg 6241aaagaaatcttagataagaaggttgagaaggtaagccattctggggctaggatatatttt 6301gtaacatcttcgaggtgggctccctcacaagcatgtttctatacaattagtggtttgagg 6361cagcctatttactgtttcatgccttcttgcctcttgttctcttctctagtcaggtttaag 6421gctattttaataaaatttggcacagattaggcattgcttcagttaacttctgagagtaga 6481taaaataccatcattttcttttttttttctttttttgagatggggtctcgctctgtcacc 6541caggctggagtgcagtggcacgatctctgctcattgcaagctccgcctcctgggttcacg 6601ccattctcctgccttagcctcctgagtagctgccactacaggcgcccgccaccacacccc 6661ggctaattttttgtatttttagtagagatggggtttcatcgcgttagccaggatggtctc 6721catctcctgaccttgtgattcgcccacctcggcctcccaaagtgctgggattaacaggcg 6781caagccaccatgcctggccgattttttttttttttggactggatctcgctcactgcaaac 6841tcaagtctcctgagtggctgggattacagatgtgtgctaccacacccggttaattttttg 6901tagacagggttttgccatgttggccagcatggtctcaaactcaagtggtctgtccacctc 6961ctccccctgctggaattaggcttgacaatgcctgttttctctttcaaagtggtaatgtca 7021atctaaggcttttgtgatcgtccacaggtgacaatctccaatagacttgtgtcttcacct 7081tgctgcattgtgaccagcacctacggctggacagccaatatggagcggatcatgaaagcc 7141caggcacttcgggacaactccaccatgggctatatgatggccaaaaagcacctggagatc 7201aaccctgaccaccccattgtggagacgctgcggcagaaggctgaggccgacaagaatgat 7261aaggcagttaaggacctggtggtgctgctgtttgaaaccgccctgctatcttctggcttt 7321tcccttgaggatccccagacccactccaaccgcatctatcgcatgatcaagctaggtcta 7381ggtaagtagctttggtacttggtgtggcaaggagtttgtgcaactcgtctcctctatgga 7441tttgacttaatgctatttggtcaagtctcacatggcttaattttacttcaggtattgatg 7501aagatgaagtggcagcagaggaacccaatgctgcagttcctgatgagatcccccctctcg 7561agggcgatgaggatgcgtctcgcatggaagaagtcgattaggttaggagttcatagttgg 7621aaaacttgtgcccttgtatagtgtccccatgggctcccactgcagcctcgagtgcccctg 7681tcccacctggctccccctgctggtgtctagtgtttttttccctctcctgtccttgtgttg 7741aaggcagtaaactaagggtgtcaagccccattccctctctactcttgacagcaggattgg 7801atgttgtgtattgtggtttattttattttcttcattttgttctgaaattaaagtatgcaa 7861aataaagaatatgccgtttttatacagttctgctttcccttgtgaagtggatgttatcct 7921tccctagcttcttcatccctccagctcttgctgttttcatgagcacagcaagttgagctg 7981gttttgtagtgaaaataacagaataccagtgagtcttaagagttcacacactgaagctaa 8041aggcagtttggaaaaactaccatataataatgccctttcagtcaaccaaaacacaggacc 8101aagtccactgcagtaatttaatttaataaaataaaattataagagcaaaaagttacattt 8161ctaaagtaccaaaacctgcaacaggctcatggaacagagcctagggatcc
(SEQ ID NO: 6) GenBank Accession J04988 version 1, incorporated herein by reference. Exemplary regions or fragments of Heat shock protein 90 (Hsp90) include bases 453-466, 463-476, 567-580, 1103-7886, 1202-2634, 2434-2450, 5740-5887, and 7382-7491.

[0074] ATPase (Na.sup.+/K.sup.+ or Mg.sup.++)

[0075] ATPases are a class of enzymes that catalyze the decomposition of ATP into ADP and a free phosphate ion. This dephosphorylation reaction releases energy, which the enzyme (in most cases) harnesses to drive other chemical reactions that would not otherwise occur. Some such enzymes are integral membrane proteins (anchored within biological membranes), and move solutes across the membrane, typically against their concentration gradient. These are called transmembrane ATPases. Transmembrane ATPases import many of the metabolites necessary for cell metabolism and export toxins, wastes, and solutes that can hinder cellular processes. An important example is the sodium-potassium exchanger (or Na.sup.+/K.sup.+ ATPase) that maintains the cell membrane potential. And another example is the hydrogen potassium ATPase (H.sup.+/K.sup.+ ATPase or gastric proton pump) that acidifies the contents of the stomach. Besides exchangers, other categories of transmembrane ATPase include co-transporters and pumps (however, some exchangers are also pumps). Some of these, like the Na.sup.+/K.sup.+ ATPase, cause a net flow of charge, but others do not.

Na.sup.+/K.sup.+-ATPase amino acid sequence:

TABLE-US-00005 1mviqkekkscgqvveewkefvwnprthqfmgrtgtswafillfylvfygfptamftltmw 61vmlqtvsdhtpkyqdrlatpglmirpktenldvivnvsdteswdqhvqklnkflepynds 121mqaqkndvcrpgryyeqpdngvlnypklacqfnrtqlgncsgigdsthygystgqpcvfi 181kmnrvinfyaganqsmnvtcagkrdedaenlgnfvmfpangnidlmyfpyygkkfhvnyt 241qplvavkflnvtpnvevnvecrinaaniatdderdkfagrvafklrinkt
(SEQ ID NO: 7) GenBank Accession AAA51805 version 1, incorporated herein by reference. Exemplary regions or fragments of Na?/K+-ATPase include residues 1-290 and 2-289.

[0076] Na.sup.+/K.sup.+-ATPase nucleotide sequence:

TABLE-US-00006 1gaattcatgctaaattgctggaaggctgcgtctctgctgtggtgtcagttccggatgcct 61catcgccaggggcgcgccgcagccacccaccctccggaccgcggcagctgctgacccgcc 121atcgccatggcccgcgggaaagccaaggaggagggcagctggaagaaattcatctggaac 181tcagagaagaaggagtttctgggcaggaccggtggcagttggtttaagatccttctattc 241tacgtaatattttatggctgcctggctggcatcttcatcggaaccatccaagtgatgctg 301ctcaccatcagtgaatttaagcccacatatcaggaccgagtggccccgccaggattaaca 361cagattcctcagatccagaagactgaaatttcctttcgtcctaatgatcccaagagctat 421gaggcatatgtactgaacatagttaggttcctggaaaagtacaaagattcagcccagagg 481gatgacatgatttttgaagattgtggcgatgtgcccagtgaaccgaaagaacgaggagac 541tttaatcatgaacgaggagagcgaaaggtctgcagattcaagcttgaatggctgggaaat 601tgctctggattaaatgatgaaacttatggctacaaagagggcaaaccgtgcattattata 661aagctcaaccgagttctaggcttcaaacctaagcctcccaagaatgagtccttggagact 721tacccagtgatgaagtataacccaaatgtccttcccgttcagtgcactggcaagcgagat 781gaagataaggataaagttggaaatgtggagtattttggactgggcaactcccctggtttt 841cctctgcagtattatccgtactatggcaaactcctgcagcccaaatacctgcagcccctg 901ctggccgtacagttcaccaatcttaccatggacactgaaattcgcatagagtgtaaggcg 961tacggtgagaacattgggtacagtgagaaagaccgttttcagggacgttttgatgtaaaa 1021attgaagttaagagctgatcacaagcacaaatctttcccactagccatttaataagttaa 1081aaaaagatacaaaaacaaaaacctactagtcttgaacaaactgtcatacgtatgggacct 1141acacttaatctatatgctttacactagctttctgcatttaataggttagaatgtaaatta 1201aagtgtagcaatagcaacaaaatatttattctactgtaaatgacaaaagaaaaagaaaaa 1261ttgagccttgggacgtgcccatttttactgtaaattatgattccgtaactgaccttgtag 1321taagcagtgtttctggcccctaagtattgctgccttgtgtattttatttagtgtacagta 1381ctacaggtgcatactctggtcatttttcaagccatgttttattgtatctgttttctactt 1441tatgtgagcaaggtttgctgtccaaggtgtaaatattcaacgggaataaaactggcatgg 1501taattttttttttttgtttgttttttgttttttggctctttcaaaggtaatggcccatcg 1561atgagcatttttaacatactccatagtcttttcctgtggtgttaggtctttatttttatt 1621tttttcctgggggctggggtgggggtttgtcatgggggaactgccctttaaattttaagt 1681gacactacagaaaaacacaaaaaggtgatgggttgtgttatgcttgtattgaatgctgtc 1741ttgacatctcttgccttgtcctccggtatgttctaaagctgtgtctgagatctggatctg 1801cccatcactttggcctagggacagggctaattaatttgctttatacattttcttttactt 1861tccttttttcctttctggaggcatcacatgctggtgctgtgtctttatgaatgttttaac 1921cattttcatggtggaagaattttatatttatgcagttgtacaattttatttttttctgca 1981agaaaaagtgtaatgtatgaaataaaccaaagtcacttgtttgaaaataaatctttattt 2041tgaactttataaaagcaatgcagtaccccatagactggtgttaaatgttgtctacagtgc 2101aaaatccatgttctaacatatgtaataattgccaggagtacagtgctcttgttgatcttg 2161tattcagtcaggttaaaacaacggacaataaaagaatgaaccgaattc
(SEQ ID NO: 8) GenBank Accession X03747 version 1, incorporated herein by reference. Exemplary regions or fragments of Na.sup.+/K.sup.+-ATPase include bases 127-1038, 598-600, 919-921, 1485-1490, 2001-2006, 2026-2031, and 2187-2193.

Binding Ligands

[0077] As used herein, the term antibody refers to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen binding site that specifically binds (immunoreacts with) an antigen. Such antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, F.sub.ab, F.sub.ab and F.sub.(ab)2 fragments, and an F.sub.ab expression library. By specifically bind or immunoreacts with is meant that the antibody reacts with one or more antigenic determinants of the desired antigen and does not react (i.e., bind) with other polypeptides or binds at much lower affinity (K.sub.d>10.sup.?6) with other polypeptides.

[0078] The basic antibody structural unit is known to comprise a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one light (about 25 kDa) and one heavy chain (about 50-70 kDa). The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function. Human light chains are classified as kappa and lambda light chains. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgA, and IgE, respectively. Within light and heavy chains, the variable and constant regions are joined by a J region of about 12 or more amino acids, with the heavy chain also including a D region of about 10 more amino acids. See generally, Fundamental Immunology Ch. 7 (Paul, W., ea., 2nd ed. Raven Press, N.Y. (1989)). The variable regions of each light/heavy chain pair form the antibody binding site.

[0079] The term monoclonal antibody (MAb) or monoclonal antibody composition, as used herein, refers to a population of antibody molecules that contain only one molecular species of antibody molecule consisting of a unique light chain gene product and a unique heavy chain gene product. In particular, the complementarity determining regions (CDRs) of the monoclonal antibody are identical in all the molecules of the population. MAbs contain an antigen binding site capable of immunoreacting with a particular epitope of the antigen characterized by a unique binding affinity for it.

[0080] In general, antibody molecules obtained from humans relate to any of the classes IgG, IgM, IgA, IgE and IgD, which differ from one another by the nature of the heavy chain present in the molecule. Certain classes have subclasses as well, such as IgG.sub.1, IgG.sub.2, and others. Furthermore, in humans, the light chain may be a kappa chain or a lambda chain.

[0081] The term antigen-binding site or binding portion refers to the part of the immunoglobulin molecule that participates in antigen binding. The antigen binding site is formed by amino acid residues of the N-terminal variable (V) regions of the heavy (H) and light (L) chains. Three highly divergent stretches within the V regions of the heavy and light chains, referred to as hypervariable regions, are interposed between more conserved flanking stretches known as framework regions, or FRs. Thus, the term FR refers to amino acid sequences which are naturally found between, and adjacent to, hypervariable regions in immunoglobulins. In an antibody molecule, the three hypervariable regions of a light chain and the three hypervariable regions of a heavy chain are disposed relative to each other in three dimensional space to form an antigen-binding surface. The antigen-binding surface is complementary to the three-dimensional surface of a bound antigen, and the three hypervariable regions of each of the heavy and light chains are referred to as complementarity-determining regions, or CDRs. The assignment of amino acids to each domain is in accordance with the definitions of Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk J. Mol. Biol. 196:901-917 (1987), Chothia et al. Nature 342:878-883 (1989).

[0082] As used herein, the term epitope includes any protein determinant capable of specific binding to an immunoglobulin, an scFv, or a T-cell receptor. The term epitope includes any protein determinant capable of specific binding to an immunoglobulin or T-cell receptor. Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics. An antibody is said to specifically bind an antigen when the dissociation constant is ?1 ?M; preferably ?100 nM and most preferably ?10 nM.

[0083] Antibodies can be produced according to any method known in the art. Methods of preparing monoclonal antibodies are known in the art. For example, monoclonal antibodies may be prepared using hybridoma methods, such as those described by Kohler and Milstein (1975) Nature 256:495. In a hybridoma method, a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes may be immunized in vitro. The immunizing agent will typically include a full length protein or a fragment thereof. Generally, either peripheral blood lymphocytes (PBLs) are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (see pp. 59-103 in Goding (1986) Monoclonal Antibodies: Principles and Practice Academic Press) Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are employed. The hybridoma cells may be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells. For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells.

[0084] In some examples the antibodies to an epitope for an interested protein as described herein or a fragment thereof are humanized antibodies. Humanized forms of non-human (e.g., murine) antibodies are chimeric molecules of immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab, F(ab)2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework (FR) regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al. 1986. Nature 321:522-525; Riechmann et al. 1988. Nature 332:323-329; Presta. 1992. Curr. Op. Struct. Biol. 2:593-596). Humanization can be essentially performed following methods of Winter and co-workers (see, e.g., Jones et al. 1986. Nature 321:522-525; Riechmann et al. 1988. Nature 332:323-327; and Verhoeyen et al. 1988. Science 239:1534-1536), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such humanized antibodies are chimeric antibodies (e.g., U.S. Pat. No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species.

[0085] In another example the antibodies to an epitope of an interested protein as described herein or a fragment thereof are human antibodies. Human antibodies can also be produced using various techniques known in the art, including phage display libraries (Hoogenboom and Winter. 1991. J. Mol. Biol. 227:381-388; Marks et al. 1991. J. Mol. Biol. 222:581-597) or the preparation of human monoclonal antibodies (e.g., Cole et al. 1985. Monoclonal Antibodies and Cancer Therapy Liss; Boerner et al. 1991. J. Immunol. 147(1):86-95). Similarly, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in most respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, e.g., in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the following scientific publications: Marks et al. 1992. Bio/Technology 10:779-783; Lonberg et al. 1994. Nature 368:856-859; Morrison. 1994. Nature 368:812-13; Fishwild et al. 1996. Nature Biotechnology 14:845-51; Neuberger. 1996. Nature Biotechnology 14:826; Lonberg and Huszar. 1995. Intern. Rev. Immunol. 13:65-93. U.S. Pat. No. 6,719,971 also provides guidance to methods of generating humanized antibodies.

[0086] Exemplary human AF-20 antibodies include, but are not limited to, antibodies commercially available. AF20 monoclonal antibody (mAb) was produced and characterized as previously described (Wilson B et al. Proc Natl Acad Sci 1988;85:3140-4 , Moradpour D et al. Hepatology 1995;22:1527-1537 , and Wands JR, et al. J Viral Hepat 1997;4 Suppl 2:60-74). The following antibodies were obtained commercially: anti-TFR1 (CD71) (Santa Cruz Biotechnology, Inc.; sc-32272), anti-HSP90 (EMD Millipore; 05-594), anti-N.sup.+/K.sup.+ ATPase (abcam, ab7671) and anti-DDK (Origene; TA100011).

EXAMPLES

Example 1

Comparison of AF20 Protein Levels Among Cell Lines of Diverse Origins

[0087] A robust AF20 expression in hepatoma and colon cancer cell lines was previously demonstrated (Wilson B, et al., Proc Natl Acad Sci 1988;85:3140-4 and Moradpour D et al., Hepatology 1995;22:1527-1537). IF staining indicated that AF20 antigen is localized on cell surface of hepatoma cells. Quantitative analysis revealed highest levels of AF20 antigen in hepatoma cell lines such as FOCUS and Huh7, as well as LS180, a colon cancer cell line. In contrast, NIH 3T3 and COS-1 cells showed no or little AF20 expression (Moradpour D et al., Hepatology 1995;22:1527-1537). To compare expression level of AF20 in cancer cells of diverse origins, quantitative IP-Western blot analysis was performed. AF20 expression was high in LS180, Huh7, HepG2, and proliferating HepaRG cells, a liver stem cell line, but low in Bosc, Cos-1 (human and monkey kidney), and NIH 3T3 (mouse embryonic fibroblast) cells (FIG. 1). These findings are highly consistent with the previous reports.

[0088] Plasmids, cell culture and expression methods: Expression constructs for TFR1 (Myc-DDK-tagged, RC200980, NM_003234.1), HSP90 (untagged, SC108085, NM_007355.2), and Na.sup.+/K.sup.+ ATPase (Myc-DDK-tagged, RC201009, NM_000701) were purchased from Origene. Human hepatoma cell line Huh7 (stock), human kidney cell line BOSC (ATCC), and monkey kidney cell line COS-1 (ATCC) were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS). HepG2 (human hepatoma, ATCC), LS180 (human colon cancer, ATCC), and NIH 3T3 (mouse fibroblast, ATCC) cells were cultured in EMEM supplemented with 10% FBS and 1% non-essential amino acids. HepaRG (hepatic cell line, from Dr. Christian Trepo, Lyon, France) was cultured in William's E medium supplemented with 10% FBS, bovine insulin (5 ?g/ml) and 7?10.sup.?5 M hydrocortisone. Cells grown in 6-well or 24-well plates with cover slips were transfected with 1 ?g or 0.25 ?g of TFR1 or other cDNA expression constructs using polyamine as a carrier (Mirus), and harvested 2 days later for Western blot analysis or fixed for immunofluorescent (IF) staining.

[0089] IP-Western blot analysis methods: Cell lysate was incubated at 4? C. overnight with AF20 antibody immobilized on protein G beads. After washing with PBS, bound proteins were separated in 8% or 10% SDS-PAGE, transferred to polyvinylidene fluoride (PVDF) membrane, blocked with 3% milk in PBS supplemented with 0.05% Tween 20 (PBST), and incubated with AF20 antibody overnight in the same solution. After washing with PBS, the membrane was incubated at room temperature (RT) for 1 hr with anti-mouse antibody conjugated with horseradish peroxidase (HRP). Signals were revealed by enhanced chemiluminescence (ECL) followed by exposure to X-ray film.

[0090] IF staining methods: Cells grown on cover slips were fixed at ?20? C. with acid ethanol (5% acetic acid glacial, 95% ethanol) for at least 2 hrs. IF staining was performed using Immunostaining kit (Active Motif, 15251). Briefly, after washing with PBS and Maxwash, fixed cells were incubated successively, at 4? C. for lhr, with Maxblock, primary antibody in Maxbind, and secondary antibody in Maxbind. After washing with Maxwash, the cover slips were transferred onto glass slide using mounting solution containing DAPI, and signals were examined under UV microscope.

[0091] Immunohistochemistry methods: Formalin fixed, paraffin-embedded sections were deparaffinized by xylene followed by submerged in preheated Antigen Unmasking Solution H-3300 (Vector Laboratories, California) in a pressure cooker (Nordic Ware, Minnesota) for 2 minutes. Sections were cooled to room temperature in a water bath and then submerged in an endogenous peroxidase blocking solution containing 3% H.sub.2O.sub.2 in methanol for 30 minutes. The rest of the staining process, including blocking, primary and secondary antibody incubation, and peroxidase-labeling, was performed using the Elite ABC Kit PK-6102 (Vector Laboratories, California) according to manufacturer's instruction. Primary antibody was diluted 1:50-1:500 and incubated at 4? C. overnight. Sections were washed in PBST between each incubation step. Color development was done using DAB Peroxidase Substrate SK-4103 (Vector Laboratories, California). Developed sections were counterstained by hematoxylin. Finally, sections were dehydrated using a reversed ethanol gradient followed by xylene.

Example 2

AF20 mAb Immunoprecipitated TFR1 and Other Host Proteins

[0092] A combination of ion-exchange chromatography and affinity chromatography was used to purify AF20 protein from Huh7 cells. The cell lysate was loaded onto DEAE-cellulose column, and bound proteins were eluted sequentially with 100, 200, and 400 mM NaCl solution (FIG. 2A). Surprisingly, IP-Western blot analysis revealed presence of AF20 antigen in protein peaks from all the three NaCl concentrations (FIG. 2B). Protein peaks of the three NaCl concentrations were pooled for concentration by Centricon-30. After separation in SDS-PAGE, the large gel was stained with Coomassie blue. Three protein bands in the range of 90 to 110 kDd were visible (FIG. 2C). They were cut out and subject to mass spectrometry as detailed in Materials and Methods. The results, as shown in Table 1 (below), revealed presence of three proteins of similar molecular weights from Huh7 cells: transferrin receptor 1 (TFR1), heat shock protein 90 (HSP90), and Na.sup.+/K.sup.+ ATPase or Mg++ ATPase. Using each of the three bands for proteomic analysis still revealed presence of all the three proteins Similar results were obtained using cell lysate from FOCUS, LS180, and HepG2 cells, although ATPase was not always present (Table 1). Proteins from a single NaCl concentration were not used for proteomic analysis.

TABLE-US-00007 TABLE 1 Protein sequencing results Cell line Protein identity ID MW *FOCUS/LS180 180 Kda: TFR1 gi37433 84848 90-100 Kda: Na.sup.+/K.sup.+ ATPase gi21361181 112842 HSP gp96 precursor gi15010550 90138 TFR1 gi37433 84848 HSP 90-? gi20149594 83212 HSP 90-?2 gi61656603 98052 Huh7 Mg.sup.++ ATPase (Pseudomonas) gi395497517 99809 HSP 90 gi306891 83242 HSP 90-? gi12082136 83046 LS180 TFR1 P02786 84818 HSP 90-? P07900 84607 HepG2 TFR1 P02786 84818 HSP 90-? P08238 83212 Na.sup.+/K.sup.+ ATPase subunit ?1 P05023 112824 HSP 90-? P07900 84607 FOCUS TFR1 P02786 84818 HSP 90-? P08238 83212 *Mixed lysate from FOCUS and LS180 cells

[0093] Purification and identification methods: Two grams of cell pellet was re-suspended in water supplemented with a protease cocktail, followed by four cycles of freezing/thawing in dry ice/methanol bath and 37? C. water bath, respectively. Ten times concentrated phosphate buffered saline (PBS) was added to a final concentration of lx. After centrifugation at 14,000 rpm for 10 min, cell lysate was mixed with 1M Tris-HCL, pH 8.0 to a final concentration of 50 mM, and loaded onto a column containing 20 ml DEAE-cellulose. After flow-through, the column was washed three times with 20 ml each of 50 mM Tris pH 8.0. Proteins bound to DEAE cellulose column were eluted successively with 100 mM, 200 mM, and 400 mM NaCl prepared in 50 mM Tris, pH 8.0. Each fraction was collected and BCA assay was performed to measure protein concentration. Protein peaks were collected and subject to immunoprecipitation (IP) using AF20 antibody followed by Western blot with the same antibody. Fractions containing AF20 antigen were pooled for large scale IP followed by separation in 10% SDS-polyacrylamide gel (PAGE). Protein bands(s) corresponding to the size of AF20 (90-110 kDa) were excised and subject to trypsin digestion, followed by peptide separation and mass spectrometry (MS) analysis on a LTQ Orbitrap mass spectrometer at Keck Biotechnology Resource Laboratory, Yale University.

Example 3

Validation of TFR1 as an AF20 Antigen

[0094] The AF20 antigen corresponded to a single host protein. The simultaneous purification of TFR1, HSP90, and ATPase by the AF20 mAb, as well as the presence of AF20 antigen in three concentrations of NaCl, was most likely a consequence of protein complex formation. To establish which one was the bona fide AF20 antigen, in vitro reconstitution experiments were performed using expression constructs for TFR1 (DDK tagged), Na.sup.+/K.sup.+ ATPase (DDK tagged) and HSP90 (untagged). DDK-tagged TFR1 expressed in Huh7 cells through transient transfection could be detected by IPWestern blot analysis using the DDK antibody as expected (FIG. 3A, upper left panel). Interestingly, substituting the DDK antibody with AF20 antibody for IP did not compromise detection of the exogenous TFR1 by the DDK antibody in Western blot (FIG. 3A upper right panel).

[0095] Reprobing the same blot with anti-TFR1 antibody revealed that endogenous TFR1 could be pulled down by the AF20 but not by the DDK antibody from non-transfected cells (FIG. 3A, lower panel). In the non-transfected Huh? cells, TFR1, HSP90, and ATPase could all be pulled down by the AF20 antibody, but not by the empty protein A/G beads (FIG. 3B).

[0096] In a different approach, cDNAs were transfected encoding DDK tagged TFR1 and Na.sup.+/K.sup.+ ATPase, or untagged HSP90 into NIH 3T3 cells, a cell line with little endogenous AF20 expression (FIG. 1). Strong IF staining by AF20 antibody was documented in TFR1 but not Na.sup.+/K.sup.+ ATPase or HSP90 transfected NIH 3T3 cells (FIG. 4), despite the fact that these two proteins were readily stained by DDK mAb or HSP90 antibody (FIG. 4). Moreover, the AF20 antibody and TFR1 antibody revealed similar staining pattern in TFR1 transfected cells (FIG. 4 left and FIGS. 5A-5C).

[0097] These findings strongly implicated TFR1, rather than Na.sup.+/K.sup.+ ATPase or HSP90, as the AF20 antigen. Another interesting observation was that TFR1 transfected to NIH 3T3 cells exhibited not only cell surface localization, but also perinuclear staining as revealed by confocal microscopy (FIG. 4 and FIGS. 5A-5C). Moreover, from 2D or 3D view the staining by AF20 mAb and TFR1 mAb in TFR1 transfected cells was indistinguishable (FIGS. 5A-5C). Overexpression of the three proteins by transient transfection caused toxicity as indicated by morphological changes including cell shrinkage and elongation when compared with non-transfected cells (FIG. 4).

Example 4

Holo but not Apo Transferrin Could Disrupt AF20 Antibody Binding to TFR1

[0098] Diferric (holo) but not iron free (apo) transferrin is the ligand of TFR1 at neutral pH [5], two forms of transferrin were compared for their impact on AF20 antigenantibody interaction. Cell lysate was immunoprecipitated with AF20 antibody in the presence or absence of transferrin, followed by Western blot with AF20 antibody. Holo but not apo transferrin inhibited TFR1 precipitation by the AF20 antibody (FIG. 6A). Increasing the concentration of holo transferrin from 3.3 to 66 ?g/ml caused a dose-dependent inhibition of the antibodyantigen interaction (FIG. 6B). Further increase in the concentration of holo transferrin (?66 ?g/ml) during immunoprecipitation did not further reduce the antigen-antibody reaction.

Example 5

Deglycosylation of the AF20 Antigen Abolished its Affinity for AF20 Antibody

[0099] TFR1 contains three sites for N-linked glycosylation (Reckhow CL and Enns CA J Biol Chem 1988;263:7297-7301; hereby incorporated by reference). To determine whether N-linked glycosylation is essential for TFR1 recognition by the AF20 antibody, proteins pulled down by the AF20 antibody were treated with PNGase F. This enzyme cleaves between the innermost GLcNAc and asparagine residues of high mannose, hybrid, and complex oligosaccharides. Deglycosylated AF20 antigen could be no longer be detected by the AF20 antibody in the Western blot (FIG. 7, left panel), even after prolonged exposure. On the other hand, TFR1 antibody detected both glycosylated and deglycosylated forms of TFR1, although the signal intensity was much reduced for the deglycosylated form (FIG. 7, right panel). Alternatively, it is possible that deglycosylation makes TFR1 unstable with the intact protein dropped below the detection limit.

[0100] Protein deglycosylation methods: Huh7 cell lysate was subject to IP with AF20 antibody as described above. After washing with PBS, bound proteins were incubated with PNGase F at 37? C. for at least 1 hr under conditions recommended by the Manufacturer. Samples without addition of the enzyme were incubated in parallel for controls. Next, samples were loaded to 10% SDS-PAGE for Western blot analysis using AF20 and TFR1 mAbs, respectively. PNGase F was purchased from New England Biolabs (P0704S). Apo transferrin and holo transferrin were purchased from EMB Millipore (616395 and 616397).

Example 6

Evaluation of TFR1 as a Biomarker for Early Detection of Malignant Transformation

[0101] AF20 antigen has been detected in majority of hepatoma and colon cancer cell lines (Wilson B, et al., Proc Natl Acad Sci 1988;85:3140-4). To correlate AF20 expression with the stage of cancer development, immunostaining was performed in four paired samples including normal colon tissue, colon polyps, and colon cancer. While AF20 was undetectable in normal colon tissues, it was clearly detectable in polyps and strongly detected in colon cancer from all four pairs of samples (FIG. 8 for two representative pairs). In addition, relationship between AF20 and TFR1 was sought by their localization in colon cancer tissues. Three pairs of consecutive tissue sections from normal colon and colon cancer were stained with AF20 and TFR1 mAb, respectively. Both AF20 and TFR1 antibodies revealed strong signals in colon cancer with similar localization (FIG. 9B). In contrast, no signal was detected from normal colon tissues by either antibody (FIG. 9A). Together with immunostaining of TFR1 cDNA transfected cells by AF20 mAb (FIG. 4), this finding strongly implicates TFR1 as the AF20 antigen.

Example 7

Therapeutic Advantages

[0102] Therapeutic advantages include a monoclonal antibody that identifies malignant cells. The antibody can be used for clinical diagnosis, delivery of an anti-tumor agent and imaging of a tumor in the body. Furthermore, the invention can aid in the diagnosis and treatment of cancerit is useful for physicians and other healthcare personnel that treat cancer patients.

[0103] The compounds and methods described have broad implications for human cancer diagnosis and therapy and are used as an imaging agent, as a carrier of a drug or isotope, (e.g., U.S. Pat. No. 5,703,213, col. 21-22, herein incorporated by reference) since once the antibody binds to the antigen complex, is internalized into the cell and are used as a diagnostic marker of cell transformation such as screening for dysplastic cellular changes in long standing ulcerative colitis which has a high risk of developing colon cancer. It might also be used to evaluate prognosis with respect to early tumor reoccurrence and overall survival.

[0104] Identification of the extensively characterized AF20 antigen as glycosylated TFR1 explains features of AF20, including its homodimeric structure, cell surface localization, rapid endocytosis of antigen-antibody complex, overexpression in liver and colon cancers, and potential for cancer therapy. It also led to novel findings such as blocking of AF20 antigen-antibody interaction by diferric transferrin. In addition, the current study also revealed ability of TFR1 to form a complex with HSP90 and/or Na.sup.+/K.sup.+ ATPase or Mg++ ATPase. In this regard, the presence of AF20 antigen (TFR1) at eluents from 100 mM, 200 mM, and 400 mM NaCl (FIG. 2A) might reflect its different molecular forms (free form, HSP90 bound, ATPase bound, or tertiary complex). As shown in FIG. 5C, overexpression of TFR1 alone led to its perinuclear staining in addition to cell surface localization. The binding site for AF-20 is a post translation modification of the TFR1 protein. Antibodies that bind to a TFR1 antigen with an aberrant post translational modification, e.g., glycosylation (compared to the normal, wild type TFR1) are useful for cancer diagnosis and monitoring response to anti-tumor therapy.

Other Embodiments

[0105] While the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the disclosure, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

[0106] The patent and scientific literature referred to herein establishes the knowledge that is available to those with skill in the art. All United States patents and published or unpublished United States patent applications cited herein are incorporated by reference. All published foreign patents and patent applications cited herein are hereby incorporated by reference. Genbank, NCBI, UniProt, or other submissions indicated by accession number cited herein are hereby incorporated by reference. All other published references, documents, manuscripts and scientific literature cited herein are hereby incorporated by reference.

[0107] While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the disclosure encompassed by the appended claims.