USOO7393534B2
(12) United States Patent (10) Patent No.: US 7,393,534 B2 Aylsworth et al. (45) Date of Patent: Jul. 1, 2008
(54) COMPOSITIONS AND METHODS FOR 5,821,047 A 10, 1998 Garrard et al. MMUNOTHERAPY OF CANCER AND 5,969,108 A 10/1999 McCafferty et al. NFECTIOUS DISEASES 6,001,363 A 12/1999 Tomley et al. (75) Inventors: Charles Aylsworth, Holt, MI (US); 6,008,342 A 12/1999 Binger et al. Siu-Cheong Ho, East Lansing, MI (US); 6,100,241 A 8, 2000 Kok et al. David Juckett, East Lansing, MI (US); 6,331415 B1 1 1/2001 Lind John W. Judge, Holt, MI (US); Igor V. 6,407,213 B1 6/2002 Carter et al. Zlatkin, Lansing, MI (US); Tatiana 6,451,984 B1 9/2002 Lillehoi et al. Zlatkin, Lansing, MI (US) 6,528,485 B1 3/2003 Veronese et al. (73) Assignee: Barros Research Institute, Holt, MI (US) (*) Notice: Subject to any disclaimer, the term of this FOREIGN PATENT DOCUMENTS patent is extended or adjusted under 35 EP 239.400 A2 9, 1987 U.S.C. 154(b) by 0 days. EP 519596 A1 12, 1992 (21) Appl. No.: 10/892,659 EP 592106 A1 4f1994 WO WO 86,05807 A1 6, 1987 (22) Filed: Jul. 15, 2004 WO WO 89.01.036 A1 2, 1989 WO WO90,02809 A1 3, 1990 (65) Prior Publication Data WO WO91f10737 A1 7, 1991 US 2005/O169935A1 Aug. 4, 2005 WO WO91f10741 A1 7, 1991 WO WO92/04461 3, 1992 Related U.S. Application Data WO WO92,06180 A1 4f1992 (60) Provisional application No. 60/487.336, filed on Jul. WO WO 91.01134 A1 10, 1992 15, 2003. (51) Int. Cl. A6 IK 38/00 (2006.01) (Continued) A6 IK 39/00 (2006.01) (52) U.S. Cl...... 424/185.1: 514/2 OTHER PUBLICATIONS (58) Field of Classification Search ...... 435/69.7; Aliberti et al. (2003), "Molecular mimicry of a CCR5 binding 424/185.1: 514/2 clomain in the microbial activation of dendritic cells”. Nature Immu See application file for complete search history. nology 4(5): 485-490. (56) References Cited (Continued) U.S. PATENT DOCUMENTS Primary Examiner Karen Cochrane Carlson 4,444,887 A 4, 1984 Hoffmann Assistant Examiner—Marsha Tsay 4,716,111 A 12/1987 Osband et al. (74) Attorney, Agent, or Firm Wolf, Greenfield & Sacks, 4,816,397 A 3, 1989 BOSS et al. P.C. 4,816,567 A 3/1989 Cabilly et al. 4,980,286 A 12/1990 Morgan et al. (57) ABSTRACT 5,122,464 A 6, 1992 Wilson et al. 5,223,409 A 6, 1993 Ladner et al. 5,225,539 A 7, 1993 Winter 5,403.484 A 4/1995 Ladner et al. The present invention provides compositions and methods for 5,427,908 A 6, 1995 Dower et al. the prevention and treatment of primary and metastatic neo 5,436,146 A 7, 1995 Shenk et al. plastic diseases and infectious diseases, for stimulating an 5,516,637 A 5/1996 Huang et al. immune response in a Subject, and for use as an alternative to 5,530,101 A 6/1996 Queen et al. interleukin-12 (IL-12) treatment. In particular, the present 5,565,332 A 10/1996 Hoogenboom et al. invention provides Apicomplexa-related proteins (ARPs) that 5,571,698 A 11/1996 Ladner et al. have immune stimulatory activity and thus have uses in the 5,580,717 A 12/1996 Dower et al. treatment and prevention of cancer and infectious diseases 5,585,089 A 12/1996 Queen et al. and in immune modulation. Compositions comprising an 5,658,727 A 8, 1997 Barbas et al. ARP are provided. Methods of use of an ARP for the preven 5,698,426 A 12, 1997 Huse tion and/or treatment of cancer and/or infectious diseases, for 5,733,743 A 3, 1998 Johnson et al. use as an alternative to interleukin-12 (IL-12) treatment, and 5,750,753 A 5, 1998 Kimae et al. for eliciting an immune response in a subject, are also pro 5,766,886 A 6, 1998 Studnicka et al. vided. 5,780,225 A 7/1998 Wigler et al. 5,792,644 A 8, 1998 Vermeulen et al. 5,807,715 A 9, 1998 Morrison et al. 13 Claims, 38 Drawing Sheets US 7,393,534 B2 Page 2
FOREIGN PATENT DOCUMENTS nucleoside Reverse Transcriptase Inhibitor of HIV Type I” AIDS Res. Hum. Retroviruses 1997, 13.9 pp. 789-796. WO WO92, 18619 A1 10, 1992 Burton et al., “Human Antibodies from Combinatorial Libraries” WO WO92/2O316 A2 11/1992 1994, Advances in Immunology 57:191-280. WO WO92,22324 A1 12, 1992 Cabilly et al. “Generation of antibody activity from immunoglobulin WO WO92,22635 A1 12, 1992 polypeptide chains produced in Escherichia coli Proc Natl AcadSci WO WO 93.14188 A1 7, 1993 USA. 81(11):3273-7 (1984). WO WO93/15199 A1 8, 1993 Caldas et al., “Design and sythesis of germline-based hemi-human WO WO93, 1520O A1 8, 1993 ized single-chain Fv against the CD18surface antigen” Protein Eng. WO WO93f2O221 A1 10, 1993 13(5):353-60 (2000). WO WO94,08598 A1 4f1994 Campos et al., “Activation of Toll-Like Receptor-2 by WO WO 94,12649 A2 6, 1994 Glycosylphosphatidylinositol Anchors from a Protrozoan Parasite s WO WO93, 17105 A1 12, 1994 The J. Immunol. 2001, 167:416–423. WO WO95/15982 A2 6, 1995 Carpenter, et al., “Antiretroviral Therapy for HIV infection in 1998” WO WO95/2O401 A1 8, 1995 JAMA 280(1): 78-86 (1998). WO WO96,33735 A1 10, 1996 Chomczynsky, P., and N. Sacchi. “Single-Step Method RNA Isola WO WO 96/34096 A1 10, 1996 tion by Acid Guanidinium Thiocyanate-Phenol-Chloroform Extrac WO WO 97.13844 A1 4f1997 tion 1987. Anal. Biochem. 162:156-159. WO WO 98.24893 A2 6, 1998 Chothia et al., 1998, "Structural Determinants in the Sequences of WO WO 98,46645 A2 10, 1998 Immunoglobulin Variable Domain” J. Mol. Biol. 278: 457-479. WO WO 93,11236 A1 3, 1999 Chou, P. and Fasman, G., “Prediction of Protein Conformation” WO WO 98.16654 A1 6, 1999 1974, Biochemistry vol. 13: No. 2 222. WO WO91,09967 A1 7, 1999 Cline, "Perspectives For Gene Therapy. Inserting New Genetic Infor WO WO92fO1047 A1 10, 1999 mation Into Mammalian Cells By Physical Techniques And Viral WO WO98,50433 A2 5, 2001 Vectors' 1985, Pharmac. Ther. 29:69-92. WO WOO1,77137 A1 10, 2001 Clowes et al., “Long-Term Biological Response of Injured Rat WO WO 2004/020630 3, 2004 Carotid Artery Seeded with Smooth Muscle Cells Expressing Retrovirally Introduced Human Genes' 1994, J. Clin. Invest. 93:644 OTHER PUBLICATIONS 651. Cockettet al., “High Level Expression Of TissueIbnhibitor OfMetal Allen & Fetterer, (2002), “Recent Advances in Biology and loproteinases. In Chinese Hamster Ovary Cells Using Glutamine Immunobiology of Eimeria Species and in Diagnosis and Control of Synthetase Gene Amplification”. Jul. 1990, Bio/Technology 8: pp. Infection with These Occidian Parasites of Poultry.” Clin. Microbiol. 662-667. Rev. 15:58-65. Cotten et al., 1993, "Receptor-Mediated Transport of DNA into Almeida and Gazzinelli, "Proinflammatory activity of Eukaryotic Cells” Meth. Enzymol. 217:618-644. glycosylphosphatidylinositol anchors derived from Trypanosoma Colberre-Garapin et al., “A New Dominant Hybrid Selective Marker cruzi: structural and functional analyses' J Leukoc Biol. 70(4):467 for Higher Eukaryotic Cells”, 1981, J. Mol. Biol. 150: 1-14. 77 (2001). Couto et al., Designing Human Consensus Antibodies with Minimal Altschulet al., “Gapped BLAST and PSI-BLAST: a new generation of Positional Templates' Cancer Res. 55 (23 Supp):5973s-5977s (Dec. protein database search programs”Nucleic Acids Res. 25(17):3389 1, 1995). 3402, 1997. Couto et al., “Anti-BA46 Monoclonal Antibody Mc3. Humanization Ames et al., “Conversion of murine Fabs isolated from a Using a Novel Positional Concensus and in Vivo and in Vitro Char combinatoria phage display library to full length immunoglobulins' acterization” Cancer Res. 55(8): 1717-22 (Apr. 15, 1995). 1995, J. Immunol. Methods 184:177-186. Crouse et al., “Expression and Amplification of Engineered Mouse Ausubel et al., (eds.), “Informatics For Molecular Biologists' Cur Dihydrofolate Reductase Minigenes” (Feb. 1983), Mol. Cell. Biol. rent Protocols in Molecular Biology, Ch. 19 John Wiley & Sons, NY vol. 3, No. 2, pp. 257-266. (2002). Davies etal “Isolation of DNA from Plants” (Methods in Molecular Baca et al., “Antibody Humanization Using Monovalent Phage Dis Biology vol. 28: Protocols for nucleic acid analysis by non-radioac play” J. Biol. Chem. 272(16):10678-84 (1997). tive probes, Isaac, P.G. (ed) pp. 9-15, Humana Press Inc., TotowaN.J. Bebbington and Hentschel. The use of vectors based on gene ampli USA (date?)). fication for the expression of cloned genes in mammalian cellsvol. 3. Dracopoli et al. (eds), Chapters 12 and 13, Current Protocols in (Academic Press, New York, 1987) pp. 163-189. Human Genetics, John Wiley & Sons, NY (1994). Better et al., “Escherichia coli Secretion of an Active Chimeric Anti Engstrom, A., “The Arrangement Of The Protein Molecules In body Fragment” 1988, Science 240: 1041-1043. Nuclear-Polyhedrosis Inclusions' 1974, Biochem. Exp. Biol. 11:7- Beverley, Cell, “Hijacking the Cell: Parasites in the Driver's Seat” 13. 1996 87: 787-789. Faber et al., “Eimeria Infections in Cows in the Periparturient Phase, Bitter et al., “Expression and Secretion Vectors For Yeast, 1987. And Their Calves. Oocyst Excretion and Levels of Specific Serum and Methods in Enzymol. 153:516-544. Colostrum Antibodies”. (2002), Vet. Parasitol. 104:1-17. Blackman & Bannister, (2001), 'Apical Organelles of Apicomplexa. Foecking et al., “Powerful and Versatile Enhancer-Promoter Unit for biology and isolation by subcellular fractionation” Mol. Biochem. Mammalian Expression Vectors' 1986, Gene 45: 101-105. Parasitol. 117:11-25. Gillies et al., "High-level Expression of Chimeric Antibodies Using Boesen et al., “Circumvention of chemotherapy-induced myelosup Adapted Cdna Variable Region Cassettes”, 1989, J. Immunol. Meth pression by transfer of the mar I gene' 1994, Biotherapy 6:291-302. ods 125:191-202. Boutet al. “Lung Gene Therapy. In Vivo Adenovirus-Mediated Gene Goldspiel et al., “Human Gene Therapy”, Jul. 1993, Clinical Phar Transfer to Rhesus Monkey Airway Epithelium”(1994, Human Gene macy 12:488-505. Therapy 5:3-10. Greenspan & Bona, "Idiotypes. Structure and immunogenicity Brightbill et al., “Host Defense Mechanisms Triggered by Microbial Mar. 1993, FASEB J. 7(5):437-444. Lipoproteins Through Toll-Like Receptors' Science Grossman and Wilson, “Retroviruses. delivery vehicle to the liver'. 285(5428):732-6 (1999). 1993, Curr. Opin. in Genetics and Devel. 3:110-114. Brinkman et al., “Phage display of disulfide-stabilized Fyfragments' Gulick, R.M. et al., “Treatment With Indinavir, Zidovudine, and 1995, J. Immunol. Methods 182:41-50. Lamivudine in Adults With Human Immunodeficiency Virus Infection Buckheit et al., “Efficacy, Pharmacokinetics, and in Vivo Antiviral and Prior Antiretroviral Therapy” N. Engl. J. Med. Sep. 11, 1997, Activity of UC781, a Highly Potent, Orally Bioavailable Non 337:734-739. US 7,393,534 B2 Page 3
Hammer, S.M. etal. A Controlled Trial of Two NucleosideAnalogues Mastrangeli et al., “Diversity of Airway Epithelial Cell Targets for In plus Indinavir in Persons With Human Immunodeficiency Virus Vivo Recombinant Adenovirus-medicated Gene Transfer”. Jan. 1993, Infectio and CD4 Cell Counts of 200 Per Cubic Millimeter or Less N. J. Clin. Invest. 91:225-234. Engl. J. Med. Sep. 11, 1997, 337:725-733. TIBTECH Biotechnology “Gene Therapy proceeding from labora Hammerling et al., “Production of hybridomas in the rodent system” tory to clinic”, 11(5): 155-215, May 1993. in: Monoclonal Antibodies and T Cell Hybridomas 563 681 Merrifield, “Solid Phase Peptide Synthesis. I. The Synthesis of a (Elsevier, N.Y., 1981). Tetrapeptide'. Journal of The American Chemical Society, vol. 14. Hollingshead et al., “In vivo drug screening applications of HIV Jul. 20, 1964 pp. 2149-2154. infected cells cultivated within hollow fibers in two physiologic Miller et al., “Use of Retroviral Vectors for Gene Transfer and compartments of mice'. Antiviral Res. 1995, 28:265-279. Expression”, 1993, Meth. Enzymol. 217:581-599. Hopp and Woods, "Prediction of protein antigenic determinants from Minet al., Adjuvant effects of IL-1BIL-2, IL-8, IL-15. IFN-O, IFN amino acid sequences”. Jun. 1981, Proc. Natl. Acad. Sci. U.S.A. YTGF-B4 and lymphotactin on DNA vaccination against Elmeria 78:6, 3824-3828. acervulina (2001), Vaccine 20:267-274. Hutchinson, C. et al., “Mutagenesis at a Specific Position in a DNA Morea et al., “Antibody Modeling. Implications for Engineering and Sequence”, Feb. 25, 1978, J. Biol. Chem 253:18, 6551-6560. Design”, Methods 2003):267-79 (2000). Inouye & Inouye, "Up-promoter mutations in the lipp gene of Morgan and Anderson, "Human Gene Therapy” 1993, Ann. Rev. Escherichia coli, 1985, Nucleic Acids Res. 13:9, 3101-3109. Biochem, 62:191-217. Jenkins et al. “Eimeria acervulina. DNA Cloning and Characteriza Morrison, “Transfectomas Provide Novel Chimeric Antibodies”. tion of Recombinant Sporozoite and Merozoite Antigens” (1988), 1985, Science 229:1202-1207. Exp. Parasitol. 66:96-107. Mossman, “Rapid Colorimetric Assay for Cellular Growth and Sur Joliot et al., “Antennapedia homeobox peptide regulates neural vival. Application to Proliferation and Cytotoxicity Assays”. T. J. Immunol. Methods 65:55-63 (1983). morphogenesis', Mar. 1, 1991, Proc. Natl. Acad. Sci. USA 88:1864 Mulligan & Berg, “Selection for animal cells that express the 1868. Escherichia coli gene coding for xanthine-guanine Kettleborough et al., “Isolation of tumor cell-specific single-chain Fy phosphoribosyltransferase' 1981, Proc. Natl. Acad. Sci. USA from immunized mice using phage-antibody libraries and the re T8:2O72-2O76. construction of whole antibodies from these antibody fragments”. Mulligan, “The Basic Science of Gene Therapy”, 1993, Science 1994, Eur, J. Immunol. 24:952-958. 260:926-932. Kiem et al., “Retrovirus-Mediated Gene Transduction Into Canine Mullinax et al., “Expression of a Heterodimeric Fab Antibody Protein Peripheral Blood Repopulating Cells'. Mar. 15, 1994, Blood 83:6. in One Cloning Step', 1992, BioTechniques 12(6):864-869. 1467-1473. Nissinoff, "American Association of Immunologists Presidential Kohler, "Immunoglobulin chain loss in hubridoma lines' Apr. 1980, Address Idotypes. Concepts And Applications' 1991, J. Immunol. Proc. Natl. Acad. Sci. USA 77:42-2199 Apr. 1980. 147(8):2429-2438. Koller and Smithies, "Inactivating the f-microglobulin locus in O'Hare et al., “Transformation of mouse fibroblasts to methotrexate mouse embryonic stem cells by homologus recombination” Nov. resistance by a recombinant plasmid expressing a prokaryotic 1989, Proc. Natl. Acad. Sci. USA 86:8932-8935. dihydrofolate reductase' 1981, Proc. Natl. Acad. Sci. USA78: No. 3, Kozarsky and Wilson, “Gene Therapy. Adenovirus Vectors' 1993, 1527-1531. Current Opinion in Genetics and Development 3:499-503). Oi et al., 1986, BioTechniques vol. 4, No. 3.214-211. Kutmeier et al., “An Acetylated (Nuclease-Free) Bovine Serum Albu Padlan, “A Possible Procedure For Reducing The Immunogenicity Of min in a PCR Buffer Inhibits Amplification', 1994, BioTechniques Antibody Variable Domains While Preserving Their Ligand-Binding 17:2, 242-246. Properties” 1991, Molecular Immunology vol. 28(4/5);489-498. Laemmli, U.K. “Cleavage of Structural Proteins during the Assem Pedersen et al., “Comparison of Surface Accessible Residues in bly of the Head of Bacteriophage T4, (Aug. 15, 1970), Nature 227. Human and Murine Immunoglobulin Fv Domains”. J. Mol. Biol. 680-685. 235(3):959-73 (1994). Laurent et al., “The Immunodominant Eimeria acervulina sporozoite Persic et al., “An integrated vector system for the eukaryotic expres antigen previously described as p160p.240 is a 19-kilodalton antigen sion of anibodies or their fragments afier selection from phage dis present in several Eimeria species” (1994), Mol. Bioch. Parasitol. play libraries”, 1997, Gene 187:9-18. 63:79-86. Pittelkow and Scott, “New Techniques for the In Vitro Culture of Lillehojet al., “A Recombinant Eimeria Protein Inducing Interferon Human Skin Keratinocytes and Perpectives on Their Use for Grafiing y-Production. Comparison of Different Gene Expression Systems and of Patients With Extensive Burns’, Mayo Clinic Proc. 1986, 61:771 Immunization Strategies for Vaccination Against Coccidiosi, 777. Proudfoot, “Transcriptional interference and termination between (2000), Avian Diseases 44:379-389. duplicated O-globin gene constructs suggests a novel mechanism for Loeffler and Behr, "Gene Transfer into Primary and Established gene regulation' 1986, Nature 322:52. Mammalian Cell Lines with Lipopolyamine-Coated DNA” 1993, Quenelle et al., Evaluation of anti-AIDS drugs in conventional mice Meth. Enzymol. 217:599-618. implated with a permeable membrane device containing human T Logan & Shenk, “Andenovirus tripartite leader sequence enhances cells infected with HIV, Antiviral Res. 1997, 35:123-129. translation of mRNAs late afier infection” Jun. 1984, Proc. Natl. Rheinwald, "Serial Cultivation of Normal Human Epidermal Acad. Sci. USA, vol. 81:3655-3659. Keratinocytes” Meth. Cell Bio. 1980, 21A:229-254. Magez et al., “The Glycosyl-Inositol-Phosphate and Riechmann et al., “Reshaping human antibodies for therapy”, 1988, Dimyristoylglycerol Moieties of the Glycosylphosphatidylinositol Nature 332:323. Anchor of the Trypanosome Variant-Specific Surface Glycoprotein Roguska et al., “Humanization of murine monoclonal antibodies Are Distinct Macrophage-Activating Factors'” The Journal of through variable domain resurfacing 1994, PNAS 91:969-973. Immunology. 15:160(4): 1949-56 (1998). Roguska et al., “A comparison of two murine monoclonal antibodies Marchler-Bauer et al., “CDD, a database of conserved domain align humanize by CDR-grafiing and variable domain resurfacing'. Pro ments with links to domain three-dimensional structure' (2002) tein Eng.9(10):895-904 (1996). Nucleic Acids Research vol. 30, No. 1, pp. 281-283. Ropert and Gazzinelli, "Signaling of immune system cells by Marchler-Bauer et al., “CDD: a curated Entrez database of con glycosylphosphatidylinositol (GPI) anchor and related structures served domain alignments” (2003) Nucleic Acids Reserch vol. 31. derived from parasitic protozoa”, Curr Opin Microbiol. 3(4):395-403 No. 1, pp. 383-387. (2000). Martin et al., “Molecular analysis of instability in flower pigmenta Rose, (1987), Eimeria, Isopora, and Cryptosporidium, in: Immune tion of Antirrhinum majus, following isolation of the pallida locus by Responses in Parasitic Infections: Immunology, Immunopathology, transposon tagging” (EMBOJ 4:1625-1630 (1985)). and Immuneoprophylaxis (ed. E.J.L. Soulsby) pp. 275-311. US 7,393,534 B2 Page 4
Rosenfeld et al., “Adenovirus-Mediated Transfer of a Recombinant Stacey et al., “Isolation of DNA from Plants'. Methods in Molecular O. 1-Antitrypsin Gene to the Lung Epithelium in Vivo”, 1991, Science Biology, vol. 28, pp. 9-15. 252:431-434. Ajioka, J. W. et al., 1998, "Gene Discovery by EST Sequencing in Rosenfeld et al., “In Vivo Transfer of the Human Cystic Fibrosis Toxoplana gondii Reveals Sequences Restricted to the Transmembrane Conductance Regulator Gene to the Airway Epithe Apicomplexa, Genome Research. lium” 1992, Cell 68: 143-155. Aliberti, J. & Sher, A., 2002, “Role of G-protein-coupled signaling in Ryley et al., “Methods in coccidiosis research. separation of oocysts the induction and regulation of dendritic cell function by Toxoplasma from faeces, Parasitology, 73:311-362(1976). gondii,” Microbes and Infection 4: 991-997. Salmons and Gunzberg, “Targeting of Retroviral Vectors for Gene Aliprantis A. et al., 1999, "Cell Activation and Apoptosis by Bacterial Therapy” 1993, Human Gene Therapy 4: 129-141. Lipoproteins Through Toll-like Receptor-2.” Science, 285:736-739. Sandhu JS, "A rapid procedure for the humanization of monoclonal Ashley S. & Wells S., 1988, “Tumors of the Small Intestine.” Semi antibodies' Gene 150(2):409-10 (1994). nars in Oncology, 15(2):116-128. Santerre et al., “Expression of prokaryotic genees for hygromycin B Bennett, L. et al., 1951. “Effect of Ultracentrifugal Fractions of Small and G4 18 as dominant-selection markers in mouse L cells', 1984, Intestinal Tissue upon Transplanted Lymphosarcoma.” Proc. Society Gene 30:147-156. of Experimental Biology, 700-701. Sawai et al., “Direct Production of the Fab Fragment Derived From Billiau, A., 1996, “Interferon-Y Biology and Role in Pathogenesis.” the Sperm Immobilizing Antibody. Using Polymerase Chain Reaction Advanced in Immunology, 62: 61-130. and CDNa Expression Vectors”, 1995, AJRI 34:26-34. Scopes, Protein Purification, “Principles of Afinity Chromatogra Brake, D.A., 2002. 'Vaccinology for control of apicomplexan para phy”. Principles and Practice, 3rd Ed., Springer (1994) pp. 187-237. sites: a simplified language of immune programming and its use in Shilo and Weinberg, “DNA sequences homologous to vertebrate vaccine design.” IntJ Parasitol. 23(5): 509-15. oncogenes are conserved in Drosophila melanogaster', 1981, Proc. Cairns, J., 1975, "Mutation selection and the natural history of can Natl. Acad. Sci. U.S.A. vol. 78, No. 11,6789-6792. cer, Nature, 255:197-200. Sigma Chemical MTT “Tissue Culture Media and Reagents”. Calman, K. 1974, “Why are small bowel tumors rare? An experimen (M5655) product application note Biochemicals and Reagents, 2000 tal model. Gut, 15:552-554. 2001. Chan P. et al., 1975, "Small bowel extracts in the inhibition of tumour Song, et al., “A DNA vaccine encoding a conserved Eimeria protein growth.” Gut, 16:50-52. induces protective immunity against live Eimeria acervulina chal Fong, L. & Engleman, E. G., 2000, "Dedritic Cells in Cancer lenge” (2001), Vaccine 19:243-252. Immunotherapy.” Annu Rev Immunol. 18:245-273. Stemple and Anderson, “Isolation of a Stem Cell for Neurons and Gately, M. et al., 1993, 'Administration of recombinant IL-12 to Glia from the Mammalian Neural Crest Cell 1992, 71:973-985. normal mice enhances cytolytic lymphocyte activity and induces Studnicka et al., “Human-engineered monoclonal antibodies retain production of IFN-Y in vivo.” Intl Immuno, 6(1): 157-167. full specific binding activity by preserving non-CDR complementar Hayday A. et al., 2001, “Intraepithelial lymphocytes: exploring the ity-modulating residues”, 1994, Protein Engineering 7(6):805-814. Tan et al., “Superhumanized” Antibodies: Reduction of Immuno Third Way in Immunology.” Nature Immunology, 2(11): 997-1003. genic Potential by Complementarity-Determining Region Grafting Horvath-Arcidiacono J. et al., 1996, “IL-12 administered in vivo to with Human Germline Sequences: Application to an Anti-CD28' J. young and aged mice. Discrepancy between the effects on tumor Immunol. 169:1119-25(2002). growth in vivo and cytotoxic T lymphocyte generation ex vivo: Tijssen, Techniques in Biochemistry and Molecular Biology-Hybrid dependence on IFN-Y.” Intl Immuno. 8(5): 661-673. ization with Nucleic Probes, "Overview of principles of hybridiza Lightdale, C. et al., 1982, "Small Intestine.” Cancer Epidemiology tion and the strategy of nucleic acid assays” (1993). and Prevention, 692-702. Tolstoshev, 1993, “Gene Therapy, Concepts, Current Trials And Lingeman C. et al. 1972, “Comparative Study of Intestinal Future Directions'. Ann. Rev. Pharmacol. Toxicol. 32:573-596. Tomley, “Techniques for Isolation and Characterization of Apical Adenocarcinomas of Animals and Man.” Natl Cancer Inst. 48(2): Organelles from Eimeria tenella Sporozoites'. Methods: A Compan 325-340. ion to Methods in Enzymology 13:171-176 (1997). Maldonado-Lopez, R. & Moser M., 2001, "Dedritic cell subsets and Van Heeke & Schuster, "Expression of Human Asparagine Sythetase the regulation of Th1/Th2 responses.” Immunology, vol. 0: 1-8. in Escherichia coli, 1989, J. Biol. Chem. 24:5503–5509. Moulton, J. (ed.), 1990, "Tumors in Domestic Animals.” Univ Cal. Walsh et al., 1993, “Gene Therapy for Human Hemoglobinopathies” Press, 403, 435. Proc. Soc. Exp. Biol. Med. 204:289-300. Neutra, M. et al., 2001, "Collaboration of epithelial cells with orga Wang et al., 1995, “A packaging cell line for propagation of recom nized mucosal lymphoid tissues.” Nature Immunology, 2(11): 1004 binant adenovirus vectors containing two lethal gene-region dele 1009. tions' Gene Therapy 2:775-783. Wang, “Instability, stabilization, and formulation of liquid protein Okamura H. et al., 1995, “Cloning of a new cytokine that induces pharmaceuticals' International J. of Pharmaceutics, 185:129-188 IFN-y production in T cells.” Nature 378: 88-91. (1999). Pardoll, D. M., 2002, "Spinning Molecular Immunology into Suc Wigler et al., “Transformation of mammalian cells with an amplifi cessful Immunotherapy.” Nature, 2:227-238. able dominate-acting gene' 1980, Natl. Acad. Sci. USA vol. 77. No. PCT International Search Report, PCT/US2004/023113, dated Jul. 1, 6:3567-3570. 2005. Wu and Wu, “Receptor-mediated in Vitro Gene Transformation by a Pollak, Y, et al., 2003, “The EAE-associated behavioral syndrome: II. Soluble DNA Carrier System”, Apr. 5, 1987, J. Biol. Chem. vol. 262, Modulation by anti-inflammatory treatments. J Neuroimmunology, No. 10:4429-4432. Wu and Wu, “Delivery Systems For Gene Therapy”, 1991, 137: 100-108. Biotherapy 3:87-95. Reise Sousa, C. et al., 1997, “In Vivo Microbial Stimulation Induces Zambrano-Villa et al., “How protozoan parasites evade the immune Rapid CD40 Ligand-independent Production of Interleukin 12 by response'. Trends Parasitol. 18(6):272-8 (2002). Dendritic Cells and their Redistribution to T Cell Areas.” J Exp. Med., Zijlstra et al., “Germ-line transmission of a disrupted 186(11): 1819-1829. f3 microglobulin gene produced by homologous recombination in Stern A. et al., 1996, “Interleukin-12 an Integral Cytokine in the embryonic stem cells”, Nov. 23, 1989, Nature 342:435-438. Immune Response.” Life Sciences, 58(8): 639-654. Xu et al., “In Vivo Anti-Hiv Activity of (+)-Calanolide A In The Tatusova, T. & Madden T.L., 1999, “BLAST 2 Sequences, a new tool Hollow Fiber Mouse Model, Bioorg. Med. Chem. Lett. 1999,9:133 for comparing protein and nucleotide sequences.” FEMS Microbiol 138. Wang et al., “A Phosphatidylinositol 3,4,5-Trisphosphate Analogue ogy Letters 174: 247-250. with Low Serum Protein-Binding Afinity”. Biororganic & Van der Auwera, P., 1989, Ex Vivo Study of Serum Bacterial Titers NMedicinal Chemistry 9 (2001) 133-139. and Killing Rates of Daptomycin (LY 146032) Combined or Not Aliprantis et al., “Cell Activation and Apoptosis by Bacterial Combined with Amikacin Compared with Those of Vancomycin. Lipoproteins Through Toll-like Receptor-2, Jul. 30, 1999, Science Yarovinsky, F. et al., 2005. “TLR 11 Activation of Dedritic Cells by Vo. 285, pp. 736-739. a Protozoan Profilin-Like Protein. Science, 308: 1626-1629. U.S. Patent Jul. 1, 2008 Sheet 1 of 38 US 7,393,534 B2
Eineria bovis 52 (cattle) Isopora robini 96 (robin) Eineria nieschulzi 99 (rat)
Eineria falcifornis (mouse) Cyclospora Cayetanensis 1OO (man) Cyclospora colobi (colobus monkey) 1 OO Cyclospora papionis (baboon) 97 Cyclospora cercopitheci 1 (green monkey) 79 1OO Cyclospora cercopitheci 2 (green monkey) Eineria tenella 100 (chicken) Eineria necatrix (chicken) 1 OO Eineria a cervulina (chicken) 95 Eineria mitis 1 OO (chicken) Eineria nivati (chicken) Eimeria praecox 52 (chicken) 58 Eineria brunetti (chicken) Eineria maxina O.01 (chicker) Fig. 1 U.S. Patent
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Ma - CVS r t O CN w O O O O S. 3 s CN CN re (Cl-I/6d) AllAllow U.S. Patent Jul. 1, 2008 Sheet 8 of 38 US 7,393,534 B2 Probable N terminus SSFCFLFOI IFYLVCKMG EADTOAWDTS VREWLVDTGR VFAGGVASIA ARVFIVCSFF VFPYSVKMGE EADTOAWDTS VKEWLVDTGK VYAGGIASIA EWLVDTGK VFAGGVASIA IASIA EtO3: YOIEVP OEDGTSISWD CDEAETLROA 3-LE: YQIEVI, QEDGSSTQED CDEAETLROA bov: M YQIESV QEDNGTVQ OA 50-iner: DGCRMFGAST DSGGDPYAEL VQ--TGYOIEVL OEDGSSTOED CDEAE Et3: VVDGRAPNGV YIGGTKYKLA EVKRDFTFND QNYDVAILGK NKGGGFLIKT 3-1E : IVDGRAPNGV YIGGIKYKLA EVKRDFTYND QNYDVAILGK NKGGGFLIKT bov: IV APDGV YIGGVK GGGFLIKT EtO3: PNENVVIALY DEEKEONKAD ALTTALNFAE YLHOSGF 3-E : PNDNVVIALY DEEKEQNKAD ALTTALAFAE YLYQGGF bov: PNENIAIALY DEEKEQNKAD ALTTALNFAD FLYQ Note: A weakly conserved region is shown in highlight. Gaps in the bovine sequence indicate no data. Hyphens indicate display shifts for alignment purposes. Figure 4 U.S. Patent US 7,393,534 B2 ·|00z·I-·- (eep peZeuJou) (u/6d) Zl U.S. Patent Jul. 1, 2008 Sheet 10 of 38 US 7,393,534 B2 1200 1000 800 600 DC assay of samples of uneven volumes 400 200 1O 15 20 25 0.20 Fraction 0. 1. 5 I 0.10 C8 RP-HPLC Of E. tenella extract O.05 0.00 20 40 60 80 1OO 120 Minutes Figure 5(B) U.S. Patent Jul. 1, 2008 Sheet 11 of 38 US 7,393,534 B2 -000Z -000|| •009 (eep pezieu Jou) (u/6d) at U.S. Patent Jul. 1, 2008 Sheet 12 of 38 US 7,393,534 B2 DNA SEQUENCE OF THE CLONE EtU3 TCCAGTTTTTGCTTTCTTTTCCAAATTATTTTCTATTTAGTTTGCAAAAIG GGAGAAGCAGACACCCAGGCCTGGGACACTTCGGTCCGCGAGTGGCTGGTT GACACCGGCAGGGTCTTCGCCGGCGGCGTTGCTAGCATAGCCGACGGCTGC CGGCTCTTCGGAGCAGCAGTGGAGGGCGAGGGCAACGCCTGGGAAGAACTC GTCAAGACCAACTACCAAATTGAAGTCCCCCAGGAAGACGGAACCTCTATT TCAGTGGATTGCGACGAGGCGGAGACTCTGCGGCAGGCGGTGGTGGACGGC CGCGCGCCCAACGGCGTCTACATCGGCGGCACCAAGTACAAGCTCGCCGAA GTCAAAAGGGACTTCACCTTCA ACGACCAAAACTATGATGTGGCGATTCTG GGAAAAAACAAAGGCGGAGGGTTTTTGATTAAAACTCCAAACGAAAATGTT GTTATAGCTTTGTATGATGAAGAAAAAGAACAAAACAAAGCTGATGCTCTC ACAACAGCTCTTAACTTCGCGGAGTACCTTCACCAGTCCGGCTTCTAA Figure 7(A) U.S. Patent Jul. 1, 2008 Sheet 13 of 38 US 7,393,534 B2 1000 - A - Forward -- Reverse 500 O ------1E-04 1E-03 1.E-02 1E-G1 E--00 Relative Conic Figure 7(B) U.S. Patent Jul. 1, 2008 Sheet 14 of 38 US 7,393,534 B2 S 8 C N (luffd) all O C C O C O O C C CP s (uu Gla) eoueqJosaw U.S. Patent US 7,393,534 B2 (%) emiAuns Figure 8 U.S. Patent Jul. 1, 2008 Sheet 16 of 38 US 7,393,534 B2 Figure 9 0. 6 0. 4. 0. 2 O 20 40 60 80 100 Days O.8 0.6 O 20 40 60 80 100 Days -o-36 -o-3.6 -O-0.36 -- 0.036 0.0036 O 20 40 60 80 100 Days U.S. Patent Jul. 1, 2008 Sheet 17 Of 38 US 7,393,534 B2 N O O OO N CO w cy) CN va O (ulfidu ZI) AAOW OOut Se O) o 9. Vr CC o / o cr) CN w cr) CN ver O vs A30e yN%09 Joy uonput U.S. Patent Jul. 1, 2008 Sheet 18 of 38 US 7,393,534 B2 1st Course 600 500 400 3. O O 2 O O Day 1 Day 2 Day 3 Day 4 Day 5 Day 9 2nd Course 600 500 400 300 200 1 OO Day 1 Day 2 Day 3 Day 4 Day 5 Day 10 Figure 11 U.S. Patent Jul. 1, 2008 Sheet 19 Of 38 US 7,393,534 B2 i (). NOO-) ALIAELow Aliwal o o r d t r c s s o est e. e s e ra i s s s (tuffd)6H U.S. Patent Jul. 1, 2008 Sheet 20 of 38 US 7,393,534 B2 4500 DC Assay of E. tenella Extract 3000 - 1500 - O |- - - - - I Cy CS S.& Y & & 8 Š ess s s X S wn 800 DC Assay of BBX-01c S 3 400 N 0 . - Na g x S S) & x Q S) NM S. & $ 3 3 & Š 8 X& go 3 Š s &Y Y a? is x w sS Figure 13(A) U.S. Patent US 7,393,534 B2 0006 0009 000/ 0009 U.S. Patent Jul. 1, 2008 Sheet 22 of 38 US 7,393,534 B2 BBX-01C1 - GM-CSF 0.5 BBX-01c 2 BBX-01c #1 GM-CSF (hatched) O 50 100 150 days after tumor Figure 14 U.S. Patent Jul. 1, 2008 Sheet 23 of 38 US 7,393,534 B2 200 150 100 5 O Guernsey Holstein Hereford Shorthorn Angus Colored - - Dairy ------Beef ------ Figure 15 US 7,393,534 B2 (V)9,?Infil loo Jed Messe Ou peseee (uffd) all U.S. Patent Jul. 1, 2008 Sheet 25 of 38 US 7,393,534 B2 I II ultnoted a. S ureuS w r CN H C G Aep - Xepui Ioun effee AV U.S. Patent US 7,393,534 B2 ZLeinfil (uffd). U.S. Patent Jul. 1, 2008 Sheet 27 of 38 US 7,393,534 B2 Figure 18 o 2 N i s / y se OC C OC i c) C C s s C s s (uu GZ) eoue CuOSCW U.S. Patent Jul. 1, 2008 Sheet 29 of 38 US 7,393,534 B2 2Ol 223 C parvum NCKKLLCN...... E. acervu in DALTTALAFA . . EYLYOGGF . . . E tenella DALTTALNFA. . . EYLYQGGF .. N caninum NSMTSALAFA . . EYLHQSGY . . . N caninuat2 NSRTSALAFA . . EYLHOSGY . P falciparu NSKIAALTFA. K. ELAESSQ.. . . . P. yoelii NSKNAALAFS K. ELAESTDA GAA S. neurora NSRTCALAFA HH. LNFLGC.. T gondiil NSKIAALTFA. K. ELAESSQ. T gondii.2 NSRTSALAFA . . EYLHQSGY T gondii.3 NSRTSALAFA . . EYLRQSGY IDENTITY g F. . . E ...... SNARTY N A. FA. K. EY. . . S.Y Figure 19 Cont. U.S. Patent Jul. 1, 2008 Sheet 30 of 38 US 7,393,534 B2 DNA sequences of E1 and U3 clones l SO E. ATGGGTGAAG AGGCTGATAC TCAGGCCTGG GACACTTCGG TCCGCGAGTG U3 ATGGGAG AAGCAGACAC COAGGCCTGG GACACTTCGG TCCGCGAGTG S. OO E. GCTGGTTGAC ACCGGCAGGG TCTTCGCCGG CGGCGTTGCT AGCATAGCCG U3 GCTGGTTGAC ACCGGCAGGG TCTTCGCCGG CGGCGTTGCT AGCACAGCCG O1 1SO ACGGCTGCCG GCTCTTCGGA GCAGCAGTGG AGGGCGAGGG CAACGCCTGG ACGGCTGCCG GCTCTTCGGA GCAGCAGTGG AGGGCGAGGG CAACGCCTGG S. 200 E1. GAAGAACTCG TCAAGACCAA CTACCAAATT. GAAGTCCCCC AGGAAGACGG U3 GAAGAACTCG TCAAGACCAA CTACCAAATT GAAGTCCCCC AGGAAGACGG 20. 250 AACCTCTATT TCAGTGGATT GCGACGAGGC GGAGACTCTG CGGCAGGCGG AACCTCTAT TCAGTGGATT GCGACGAGGC GGAGACTCTG CGGCAGGCGG 2S3 3 O O E. TGGTGGACGG CCGCGCGCCC AACGGCGTCT ACATCGGCGG CACCAAGTAC U3 TGGTGGACGG CCGCGCGCCC AACGGCGTCT ACATCGGCGG CACCAAGAC 3 O1 350 E. AAGCTCGCCG AAGTCAAAAG GGACTTCACC TTCAACGACC AAAACTATGA U3 AAGCTCGCCG AAGTCAAAAG GGACTTCACC TCA ACGACC AAAACTATGA 351 4 OO E. TGTGGCGATT CTGGGAAAAA ACAAAGGCGG AGGGTTTTTG ATTAAAACTC U3 TGTGGCGATT CTGGGAAAAA ACAAAGGCGG AGGGTTTTTG ATTAAAACTC 4 O1 450 E. CAAACGAAAA TGTTGITATA GCTTTGTATG ATGAAGAAAA AGAACAAAAC U3 CAAACGAAAA TGTTGITATA GCTTTGTATG ATGAAGAAAA AGAACAAAAC 451. 500 E. AAAGCTGATG CTCTCACAAC AGCTCTTAAC TTCGCTGAGT ACCTGTACCA U3 AAAGCTGATG CTCTCACAAC AGCTCTTAAC TTCGCGGAGT ACCTTCACCA 50. 513 E. GGGCGGCTTC TAA U3 GTCCGGCTTC TAA FG. 20 U.S. Patent Jul. 1, 2008 Sheet 31 of 38 US 7,393,534 B2 Protein Sequences of El and U3 clones l 4 O E. MGEEADTOAW DTSVREWLVD TGRVFAGGVA SIADGCRLFG U3 MGEADTOAW DTSVREWLVD TGRVFAGGVA SIADGCRLFG 41 8.0 E. AAVEGEGNAW EELVKTNYOI EVPOEDGTSI SVDCDEAETL U3 AAVEGEGNAW EELVKTNYOI EVPQEDGTSI SVDCDEAET 80 12O E. ROAVVDGRAP NGVYGGTKY KLAEWKRDFT FNDQNYDVAI U3 ROAVVDGRAP NGVYIGGTKY KAEVKRDFT FNDONYDVAI 21 16 O E. LGKNKGGGFT IKTPNENVVI ALYDEEKEQN KADATTTALN U3 LGKNKGGGFL KTPNENVVI ALYDEEKEQN KADATTALN 16. 17 O E. FAEYLYOGGF arr vergr T ray U3 AAC. L. lurid Qy Figure 20 Cont. U.S. Patent Jul. 1, 2008 Sheet 35. Of 38 US 7,393,534 B2 B s S s s 2 Cl b 9 s V K) CN o O L 4. S. 2 S 3 O C cS c G CD (uugla) 'OO l U.S. Patent Jul. 1, 2008 Sheet 36 of 38 US 7,393,534 B2 06|#ZITIul/OGunuºsosnoW-salduesºcm)(uo?oº?u???? ------+------;}||..—*—||(os)O-H1? 0000Z- E Figure 25 U.S. Patent Jul. 1, 2008 Sheet 37 of 38 US 7,393,534 B2 Figure 26 00’0009 00'O sdo "Aisuelu U.S. Patent Jul. 1, 2008 Sheet 38 of 38 US 7,393,534 B2 Figure 27 800 1e-7 1e-6 1e-5 1e-4 1e-3 Relative Concentraion -O- Oxidized - O - Reduced US 7,393,534 B2 1. 2 COMPOSITIONS AND METHODS FOR of cancers of the small intestine. See Shimizu et al. (1991), J. MMUNOTHERAPY OF CANCER AND Radiat. Res. Supplement, 212-230. NFECTIOUS DISEASES In Familial Polyposis Coli, a genetic disease, it is known that patients have a predisposition to large intestine cancers. RELATED APPLICATIONS However, the potential for cancers of the small intestine is very low even in those individuals where the polyposis This application claims the priority of U.S. application Ser. appears in the entire gastrointestinal tract. See Lightdale et No. 60/487,336 filed on Jul. 15, 2003. al., (1982), Small Intestine, in: Cancer epidemiology and The present invention relates to compositions and methods Prevention (eds, Schottenfield et al.) W.B. Saunders Co. for stimulating an immune response and the prevention and 10 Finally, and most significantly, the spread of metastases treatment of primary and metastatic neoplastic diseases and from cancers arising in other sites to the Small intestine is very infectious diseases. More particularly, the present invention rare. This suggests that whatever mechanism protecting the relates to compositions comprising Apicomplexa-related Small intestine from local cancers also operates to block the proteins (ARPs), a fragment, a derivative, a homolog or an occurrence of most other cancers as well. analog thereof, and their uses in immune stimulation, cancer 15 The very low incidence of cancers in, or of the small and infectious disease prevention and treatment. intestine is a robust fact of nature, and cries for explanation. The facts described above have been known for a long time. They have astonished many scientists and stimulated them to BACKGROUND OF INVENTION speculate on its causes. The low incidence of Small intestine tumors cannot be attributed to the relative size of the organs of Based on the National Cancer Institute's Surveillance, Epi the digestive tract, since in humans the Small intestine con demiology, and End Results (SEER) database and the popu stitutes 90% of the length of the tract, and 98% of the absorp lation data from the Bureau of the Census, in 2000, 1.22 tive surface area (Gabos et al., (1993) International J. of million new cases of invasive cancer (619,700 men, 600,400 Epidemiology 22: 198-206). This is also true for other ani women) were diagnosed and 552,200 people died from can 25 mals as well. The low incidence cannot be attributed to a low cer in the United States. See Braunwald et al., HARRISON'S cell turnover rate, as it is in neural cells and muscle cells, since PRINCIPLES OF INTERNAL MEDICINE, 15 ed., the epithelium of the small intestine crypts have the fastest McGraw-Hill (2001), at pp. 491. Cancer is still the second turnover rates of all organs of the body. Potten and Loeffler leading cause of death behind heart disease in the United (1990, Development 110: 1001-1020) have commented, States. Id. Much resource and effort are invested in seeking 30 “Since there are about 7.5x10 crypts in the ileum of the effective ways to prevent and treat cancer. mouse there must be between3x10' and 1.2x10 stem cells in Small Intestine Tumors the ileum. Thus, there are between 10 and 10' stem cell divisions in the mouse Small intestine in its 3-year life span. As pervasive as cancer is, tumors of the Small intestine are With a spontaneous mutation rate of approximately 1 in 10, a rare event (see, e.g., Juckett & Rosenberg, (1988) Mech. 35 about 10 or 10 spontaneous mutations would be expected, Ageing Dev. 43: 239-257). Over the years, preliminary i.e. about 1-10 per day. It is therefore surprising that the small experiments have reported attempts to determine the cause of intestine rarely develops cancer. It must be very well pro this natural tumor-free environment. However, the results tected by ill-understood mechanisms.” were inconclusive and the work was not pursued vigorously. Ashley and Wells (1988, Seminars in Oncology 15: 116 The Small intestine's profound resistance to cancer can be 40 128) listed various speculations regarding the protected status demonstrated by the low number of small intestine cancer of the small intestine. They include: a) protection from expo deaths compared to other gastro-intestinal cancers. The frac Sure to noxious Substances by the liquidity and rapid transit of tional ratio of small intestine cancers compared to all others is its contents; b) the alkalinity of the interior lumen of the small only 0.0071. Parkinet al.eds (1997) Cancer Incidence in Five intestine; c) the high concentrations of secretory immuno Continents, Vol. V (IARC Scientific Publications No. 143) 45 globulins; d) the low bacterial concentration; and e) the pres Lyon, IARC. This is approximately the same for all the years ence of hydroxylases which may inactivate potential carcino Publications No. 143) Lyon, IARC. This is approximately the gens. Another mechanism was proposed by Cairns (1975, same for all the years examined and for all the various Nature 255: 197-200). This involves the physical movement cohorts. Id. Incidence of small intestine tumors also follows of epithelial cells, which begin as stem cells at the bottom of the same trend as mortality (Parkinet al., eds., (1997), Cancer 50 the crypt, move along the Surface of the crypt and then the Incidence in Five Continents, Vol. VII (IARC Scientific Pub villus, and are finally shed into the lumen. This might prevent lications No. 143) Lyon, IARC). The low incidence rate is the accumulation of variant (abnormal) stem cells which may almost universally true for the human species, for all genetic eventually be further mutated into transformed cells (cancer). pools, and over long periods of time. It also appears to be true None of these speculations have been proven. for almost all other mammalian species (with the possible 55 In a paper by Bennett et al. (1951, Proceedings of Society exception of two varieties of sheep). See Lingeman et al., for Experimental Biology 78: 790-791, referred as “Bennett” (1972), J. of the National Cancer Institute 48: 325-346. herein after), it was suggested that the Small intestine con Additional observations, regarding the unusual nature of tained a chemical inhibitory to tumor growth. Cells of the the Small intestine, come from studies on radiation-induced Small intestine of mice were extracted and separated into 3 illness (primarily from Nagasaki and Hiroshima) and in 60 fractions—the pellet arising from a low speed centrifugation genetic diseases of the intestine. Large doses of high-energy of the raw material (fraction 4), a pellet arising from a high radiation cause death by the destruction of the cells of the speed centrifugation (fraction 90), and its Supernatant (Super bone marrow and the epithelial cells lining the Small intestine, natant). Lymphosarcoma cells were incubated with each frac both of which are easily damaged or destroyed by radiation. tion for a specified time and both were inoculated into mice. In bomb survivors that received a non-lethal dose of radiation, 65 The results were that few animals developed tumors using there is a much higher than normal incidence of cancers of the fraction 90, but a larger number of animals developed a tumor bone marrow at later times, but there is no increased incidence and died using the cells incubated with the Supernatant and US 7,393,534 B2 3 4 fraction 4. Similar extracts from liver, spleen and muscles organelles known as micronemes and rhoptries, polar rings failed to show any tumor inhibition. Bennett concluded that composed of microtubules, and in Some species a conoid the tests confirmed their hypothesis. Bennett also showed in which lies within the polar rings. At some point during their further tests that the Supernatant activity was due to contami life cycle, members of the Apicomplexa either invade or nation with components of fraction 90 (containing attach to host cells. The apical organelles play a role in these microsomes), and could be eliminated by more careful prepa host-parasite interactions. ration. Thus the supernatant was not active by itself. Bennett A few Eimeria species are known to cause severe morbid also stated that they have eliminated proteolytic enzymes as ity and occasionally mortality in certain animals. In particu the probable cause of the activity. lar, E. tenella, E. acervulina, and E. maxima are a problem in In the mid 1970s, another group examined small intestine 10 the chicken industry. The stress and crowded conditions exac extracts (Chan et al., 1975, J. of the British Society of Gas erbate Eimeria infections and cause massive diarrhea and troenterology 16:50-52, referred as “Chan herein after) and death. A similar problem can occur in the cattle industry, came to a different explanation of Bennett's result. Chan where crowding among calves can lead to severe disease. concluded that the in vitro incubation of tumor cells with Usually, adult animals are immune to Eimeria but a low, small intestine extracts resulted in killing the cells prior to 15 asymptomatic infection is endemic in most vertebrates. Upon inoculation into animals; that the extracts kill normal cells as first exposure, a brief humoral immune response has been well as tumor cells; and that the activity was predominantly in documented in animals (Faber et al., (2002), Vet. Parasitol. the microsomal fraction and not in the Supernatant. They 104: 1-17: Allen & Fetterer, (2002), Clin. Microbiol. Rev. 15: Suggested that the enzymatic activity of the epithelium was 58-65; Rose, (1987), Eimeria, Isopora, and Cryptospo responsible for the observations. ridium, in: Immune Responses in Parasitic Infections: Immu In a paper by Calman (1974, Gut 15:552-554), the anti nology, Immunopathology, and Immuneoprophylaxis (ed. E. tumor effect in the small intestine was shown to require the J. L. Soulsby) pp. 275-230), but this is replaced by a localized presence of an intact immune system. Thymectomy and cellular immune response that appears to provide lasting pro radiation was shown to allow tumors to be transplanted to the tection throughout a host’s life. The exact mechanism of this Small intestine. In animals that received the same treatment, 25 protection is not fully known and why low levels of the but with their immune system reconstituted with thymus organism Survive without undergoing the explosive growth of grafts, tumors would again no longer grow in the Small intes its life cycle remains a mystery. tine. In both groups, tumors could be transplanted Success Aliberti et al. (2003, Nature Immunology 4(5): 485-490) fully to the stomach. Thus, the protective effect of the small showed a purified protein, C-18, which is an isoform of Toxo intestine appeared to be related to an enhancement of the local 30 plasma gondii cyclophilin (which does not contain a profilin immune system. However, the search for factor(s) that con domain), can stimulate production of interleukin-12 (IL-12) trolled the local immune system was not performed. from dendritic cells (DCs). However, recombinant T. gondii C-18 showed reduced IL-12 stimulatory activity relative to Protozoa and Immune Responses of their Hosts the crude T. gondii tachyzoite extract (STAg). Parasites have evolved multiple evasion strategies allowing 35 them to survive in their primary host and to complete their life The 19 kD Sporozoite Antigen cycle through a series of stages that usually involve passage Vaccination against Eimeria has been the Subject of Sub through the environment and often through secondary hosts. stantial research in the animal husbandry of poultry, and to a These strategies include antigen shedding, antigen shifting, lesser extent, cattle. Chemical prophylaxes are standard treat living intracellularly for long periods, manipulating host cell 40 ment in the poultry industry, but resistant strains of Eimeria biochemistry, infecting multiple organs, and encystment (see, are beginning to be a problem. The development of vaccines e.g., Beverley, (1996), Cell 87: 787-789; Zambrano-Villa et is considered an alternative approach and has focused on the al., (2002), Trends in Parasitol. 18: 272-278). Such mecha use of surface antigens from the sporozoite stage, which is the nisms have been fine-tuned through evolutionary pressures to stage that invades the epithelial layer of the small intestine help the protozoan avoid the immune response of the host. In 45 (see e.g., Allen & Fetterer, (2002), Clin. Microbiol. Rev. 15: many cases, protozoans are so specialized in these avoidance 58-65; Blackman & Bannister, (2001), Mol. Biochem. Para mechanisms that they can only complete their life cycle in one sitol. 117: 11-25; Minet al., (2001), Vaccine 20: 267-274). host species or genera. Merozoite Surface antigens are also of interest because this Apicomplexa are spore-forming single-celled parasites of protozoan stage is released after the first round of multiplica animals and include species that cause various disease. Such 50 tion and infects neighboring cells. During this process an as malaria, coccidiosis, redwater disease, corridor disease, effective immune response could limit the expansion of the east coast fever and binary fever. Four major classes within infection. the phylum Apicomplexa are: Coccidia (genera include, e.g., One of the surface molecules, eventually examined for use Besnoitia, Caryospora, Cryptosporidium, Eimeria, Frenke as a vaccine, was isolated several times from cDNA expres lia, Hammondia, Hepatozoon, Isospora, Lankesterella, 55 sion libraries of E. acervulina. It was originally identified by Neospora, Sarcocystis, and Toxoplasma); Gregarinia (genera Jenkins et al. (1988, Exp. Parasitol. 66: 96-107) as a low include, e.g., Gregarina, Monocystis, Ophriocystis, and abundance surface protein (clone cSZ-1) detected by rabbit Pseudomonocystis); Haemosporida or haemosporidians antibodies made to acerVulina membranes. Sera from (genera include, e.g., Haemoproteus, Hepatocystis, and Plas infected chickens, however did not react with it. They iden modium); and Piroplasmida or Piroplasmids (genera include, 60 tified two bands in Western blots at 240 and 160 kD and e.g., Babesia, Cytauxzoon, and Theileria). Seven species that showed that the B-galactosidase fusion protein, made in E. infect humans have been identified (Plasmodium, Babesia, coli, could activate T-cell division. Another group (Laurent et Cryptosporidium, Isospora, Cyclospora, Sarcocystis, Toxo al., (1994), Mol. Bioch. Parasitol. 63: 79-86) subsequently plasma). reported that a 19 kD surface protein, referred to as IP, was A defining characteristic of the Apicomplexa is a group of 65 conserved across three poultry Eimeria species, shared the organelles found at one end—called the apical end—of the same cDNA sequence as the cSZ-1 clone of Jenkins et al., and organism. This "apical complex includes secretory Suggested that iodination may have given the anomalously US 7,393,534 B2 5 6 high molecular weights in the previous work. The nucleotide ARP is purified to homogeneity as assayed by, e.g., Sodium sequence of their clone was submitted to GenBank under the dodecyl Sulfate polyacrylamide gel electrophoresis, or agar name of 19 kD sporozoite antigen (Accession Z26584). ose gel electrophoresis. Most recently, Lillehojet al. (2000, Avian Diseases 44: 379 The present invention provides a method of treating or 389) isolated a slightly longer protein reading frame from a preventing a disease in an animal, where the method com cDNA clone (3-1E) that reacted with rabbit antiserum to E. prises administering to the animal a pharmaceutically effec acervulina merozoites (Accession Z26584). The translation tive amount of a pharmaceutical composition which provides included 17 additional amino acids at the N terminus, but the a protein to the animal, where the protein has at least 25%, at remainder of the molecule was virtually identical. They dem least 30%, at least 35%, at least 40%, at least 45%, at least onstrated that the 3-1 E protein activated cell mediated immu 10 50%, at least 55%, at least 60%, at least 65%, at least 70%, at nity by showing in vitro IFNY production from spleen cell least 75%, at least 80%, at least 85%, at least 90%, at least isolated from chickens doubly challenged with E. acervulina 95%, at least 98% or at least 99% identity to the sequence of and then exposed to the protein. They also showed that the SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:28 or SEQ ID protein can be used as a modestly effective vaccine to prevent NO:30 as determined by a BLAST 2.0 algorithm set to default coccidiosis from Eimeria. This group continues to explore 15 parameters, and where the protein possesses immunostimu this potential using the nucleotide sequence as a DNA vaccine latory activity. The present invention further provides a phar (Song, et al., (2001), Vaccine 19:234-252: Min, et al., (2001), maceutical composition which provides the protein to an Vaccine 20: 267-274). As the sole agent, the DNA vaccine animal as a protein, a nucleic acid encoding the protein or a generated only partial protection from challenge. Addition of cell transfected with a nucleic acid encoding the protein. plasmids carrying various chicken cytokine genes offered The present invention provides a method of treating or Some improvement, but still did not reduce oocyst production preventing cancer in an animal comprising administering to by more than two-fold. the animal a pharmaceutical composition which provides the Citation or discussion of a reference herein shall not be protein to the animal as: (a) an isolated protein comprising construed as an admission that Such is prior art to the present SEQ ID NO:1; (b) an isolated protein comprising SEQ ID 25 NO:2; (c) an isolated protein comprising SEQID NO:28; (d) invention. an isolated protein comprising SEQID NO:30; (e) an isolated protein comprising SEQIDNO:3, 4, 5, 6 and 7: (f) an isolated SUMMARY OF INVENTION nucleic acid comprising a nucleotide sequence encoding any of (a)-(e), above; or (f) a cell transformed with a nucleic acid As used herein, the term "isolated” means that the refer 30 comprising a nucleotide sequence encoding any of (a)-(e) enced material is removed from its native environment, e.g., a above, the nucleotide sequence being operably linked to a cell. Thus, an isolated biological material can be free of some promoter. or all cellular components, i.e., components of the cells in The present invention provides a method of treating or which the native material is occurs naturally (e.g., cytoplas preventing cancer where a pharmaceutical composition pro mic or membrane component). In the case of nucleic acid 35 vides a protein to an animal as: (a) an isolated protein that is molecules, an isolated nucleic acid includes a PCR product, the product of a process comprising the steps of: (1) produc an isolated mRNA, a cDNA, or a restriction fragment. In ing a cell extract of Eimeria infected tissue or cells; (2) another embodiment, an isolated nucleic acid is preferably centrifuging the cell extract to produce a Supernatant; (3) excised from the chromosome in which it may be found, and fractionating the Supernatant by ammonium sulfate to pre more preferably is no longer joined or proximal to non-cod 40 cipitate proteins to a pellet, and resuspending the pellet into a ing regions (but may be joined to its native regulatory regions Solution; (4) applying the Solution to a HIC column; (5) or portions thereof), or to other genes, located upstream or eluting material bound to the HIC column to produce eluted downstream of the gene contained by the isolated nucleic acid material: (6) dialyzing the eluted material to produce a molecule when found in the chromosome. In yet another sample for loading on a DEAE column; (7) applying the embodiment, the isolated nucleic acid lacks one or more 45 sample to a DEAE column; (8) eluting material bound to the introns. Isolated nucleic acid molecules include sequences DEAE column with buffer to produce an eluted fraction and inserted into plasmids, cosmids, artificial chromosomes, and concentrating the eluted fraction; (9) applying the concen the like, i.e., when it forms part of a chimeric recombinant trated eluted fraction to a size exclusion column: (10) collect nucleic acid construct. Thus, in a specific embodiment, a ing and pooling fractions eluted from the size exclusion col recombinant nucleic acid is an isolated nucleic acid. An iso 50 umn: (11) applying the pooled fractions to a HIC column to lated protein may be associated with other proteins or nucleic remove calcium binding proteins; (12) applying eluted frac acids, or both, with which it associates in the cell, or with tions from the HIC column to a HPLC HIC column and cellular membranes if it is a membrane-associated protein. An collecting active fractions, wherein said active fractions have isolated organelle, cell, or tissue is removed from the ana the ability to stimulate interleukin-12 release from dendritic tomical site in which it is found in an organism. An isolated 55 cells, said dendritic cells having been isolated from a mam material may be, but need not be, purified. mal within 10 hours of assaying said ability; (13) dialyzing The term “purified in reference to a protein or a nucleic the active fractions; and (14) applying the dialyzed active acid, refers to the separation of the desired substance from fractions to a HPLC DEAE column and collecting one or contaminants to a degree sufficient to allow the practioner to more active fractions, wherein said active fractions have the use the purified substance for the desired purpose. Preferably 60 ability to stimulate interleukin-12 release from dendritic this means at least one order of magnitude of purification is cells, said dendritic cells having been isolated from a mam achieved, more preferably two or three orders of magnitude, mal within 10 hours of assaying said ability; (b) a recombi most preferably four or five orders of magnitude of purifica nately expressed protein; (c) an isolated nucleic acid com tion of the starting material or of the natural material. In prising a nucleotide sequence encoding (a) above; or (d) a cell specific embodiments, a purified ARP is at least 60%, at least 65 transformed with a nucleic acid comprising a nucleotide 80%, or at least 90% of total protein or nucleic acid, as the sequence encoding (a) above, said nucleotide sequence being case may be, by weight. In a specific embodiment, a purified operably linked to a promoter. US 7,393,534 B2 7 8 The present invention provides a method of treating or eluting material bound to the HIC column to produce eluted preventing cancer comprising a pharmaceutical composition material: (6) dialyzing the eluted material to produce a which is an isolated protein that is the product of a process sample for loading on a DEAE column; (7) applying the comprising the steps of: (a) producing a homogenate of a sample to a DEAE column; (8) eluting material bound to the purified Apicomplexan organism or a cell infected with an DEAE column with buffer to produce an eluted fraction and Apicomplexan organism; (b) centrifuging the homogenate to concentrating the eluted fraction; (9) applying the concen obtain a pellet; (c) treating the pellet with a solution compris trated eluted fraction to a size exclusion column: (10) collect ing a phospholipase; (d) centrifuging the Solution to obtain a ing and pooling fractions eluted from the size exclusion col Supernatant; (e) applying the Supernatant to a reverse phase umn: (11) applying the pooled fractions to a HIC column to HPLC column; and (f) eluting bound protein from the reverse 10 remove calcium binding proteins; (12) applying eluted frac phase HPLC column, to produce eluted protein, wherein said tions from the HIC column to a HPLC HIC column and protein has the activity to stimulate interleukin-12 release collecting active fractions, wherein said active fractions have from dendritic cells, said dendritic cells having been isolated the ability to stimulate interleukin-12 release from dendritic from a mammal within 10 hours of assaying said activity. cells, said dendritic cells having been isolated from a mam The present invention provides a method of treating or 15 mal within 10 hours of assaying said ability; (13) dialyzing preventing cancer in an animal comprising administering to the active fractions; and (14) applying the dialyzed active the animal a pharmaceutical composition which provides the fractions to a HPLC DEAE column and collecting one or protein to the animal as: (a) an isolated protein comprising more active fractions, wherein said active fractions have the SEQID NO: 3, 4, 6 or 7; (b) an isolated protein comprising a ability to stimulate interleukin-12 release from dendritic variant of any one of SEQID NO:1, SEQID NO:2, SEQID cells, said dendritic cells having been isolated from a mam NO:28 or SEQ ID NO:30, wherein said variant has only mal within 10 hours of assaying said ability; (b) a recombi conservative amino acid substitutions relative to SEQ ID nately expressed protein; (c) an isolated nucleic acid com NO:1, SEQ ID NO:2, SEQ ID NO:28 or SEQ ID NO:30, prising a nucleotide sequence encoding (a) above; or (d) a cell respectively; (c) an isolated protein comprising a variant of transformed with a nucleic acid comprising a nucleotide SEQ ID NO: 3, 4, 6 or 7, wherein said variant has only 25 sequence encoding (a) above, said nucleotide sequence being conservative amino acid substitutions relative to SEQID NO: operably linked to a promoter. 3, 4, 6 or 7, respectively; (d) an isolated protein comprising a The present invention provides a method of treating or PROF (profilin) domain; (e) an isolated Apicomplexan pro preventing infectious disease comprising a pharmaceutical tein comprising an amino acid sequence encoded by a nucleic composition which is an isolated protein that is the product of acid consisting of a nucleotide sequence hybridizable to SEQ 30 a process comprising the steps of: (a) producing a homoge IDNO:15, 16, 17, 18, 19, 20 or 27, or a complement of any of nate of a purified Apicomplexan organism or a cell infected the foregoing SEQID NOs, under conditions of low strin with an Apicomplexan organism; (b) centrifuging the homo gency, (f) an isolated Apicomplexan protein comprising an genate to obtain a pellet; (c) treating the pellet with a solution amino acid sequence encoded by a nucleic acid consisting of comprising a phospholipase; (d) centrifuging the solution to a nucleotide sequence hybridizable to SEQID NO:15, 16, 17, 35 obtain a Supernatant; (e) applying the Supernatant to a reverse 18, 19, 20 or 27, or a complement of any of the foregoing SEQ phase HPLC column; and (f) eluting bound protein from the ID NOS, under conditions of high Stringency, (g) an isolated reverse phase HPLC column, to produce eluted protein, nucleic acid comprising a nucleotide sequence encoding any wherein said protein has the activity to stimulate interleukin of (a)-(f), above: (h) a cell transformed with a nucleic acid 12 release from dendritic cells, said dendritic cells having comprising a nucleotide sequence encoding any of (a)-(f) 40 been isolated from a mammal within 10 hours of assaying above, said nucleotide sequence being operably linked to a said activity. promoter, and wherein said pharmaceutical composition has The present invention provides a method of treating or the ability to stimulate interleukin-12 release from dendritic preventing infectious disease in an animal comprising admin cells, said dendritic cells having been isolated from a mam istering to the animal a pharmaceutical composition which mal within 10 hours of assaying said ability. 45 provides the protein to the animal as: (a) an isolated protein The present invention provides a method of treating or comprising SEQID NO: 3, 4, 6 or 7; (b) an isolated protein preventing infectious disease in an animal comprising admin comprising a variant of any one of SEQ ID NO:1, SEQ ID istering to the animal a pharmaceutical composition which NO:2, SEQID NO:28 or SEQID NO:30, wherein said variant provides the protein to the animal as: (a) an isolated protein has only conservative amino acid Substitutions relative to comprising SEQID NO: 1; (b) an isolated protein comprising 50 SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:28 or SEQ ID SEQ ID NO:2; (c) an isolated protein comprising SEQ ID NO:30, respectively; (c) an isolated protein comprising a NO:28; (d) an isolated protein comprising SEQID NO:30; (e) variant of SEQID NO: 3, 4, 6 or 7, wherein said variant has an isolated protein comprising SEQID NO: 3, 4, 5, 6 and 7: only conservative amino acid substitutions relative to SEQID (f) an isolated nucleic acid comprising a nucleotide sequence NO:3, 4, 6 or 7, respectively; (d) an isolated protein compris encoding any of (a)-(e), above; or (f) a cell transformed with 55 ing a PROF (profilin) domain; (e) an isolated Apicomplexan a nucleic acid comprising a nucleotide sequence encoding protein comprising an amino acid sequence encoded by a any of (a)-(e) above, the nucleotide sequence being operably nucleic acid consisting of a nucleotide sequence hybridizable linked to a promoter. to SEQID NO:15, 16, 17, 18, 19, 20 or 27, or a complement The present invention provides a method of treating or of any of the foregoing SEQID NOs, under conditions of low preventing infectious disease where a pharmaceutical com 60 stringency, (f) an isolated Apicomplexan protein comprising position provides a protein to an animal as: (a) an isolated an amino acid sequence encoded by a nucleic acid consisting protein that is the product of a process comprising the steps of a nucleotide sequence hybridizable to SEQID NO:15, 16, of: (1) producing a cell extract of Eimeria infected tissue or 17, 18, 19, 20 or 27, or a complement of any of the foregoing cells; (2) centrifuging the cell extract to produce a Superna SEQ ID NOs, under conditions of high stringency; (g) an tant; (3) fractionating the Supernatant by ammonium sulfate 65 isolated nucleic acid comprising a nucleotide sequence to precipitate proteins to a pellet, and resuspending the pellet encoding any of (a)-(f), above, (h) a cell transformed with a into a solution; (4) applying the solution to a HIC column; (5) nucleic acid comprising a nucleotide sequence encoding any US 7,393,534 B2 10 of (a)-(f) above, said nucleotide sequence being operably Section 12, infra), (f) an isolated nucleic acid comprising a linked to a promoter; and wherein said pharmaceutical com nucleotide sequence encoding any of (a)-(g), above; and (h) a position has the ability to stimulate interleukin-12 release cell transformed with a nucleic acid comprising a nucleotide from dendritic cells, said dendritic cells having been isolated sequence encoding any of (a)-(g) above, said nucleotide from a mammal within 10 hours of assaying said ability. 5 sequence being operably linked to a promoter. The present invention provides a method of treating or The present invention also provides a method of treating or preventing cancer comprising administering to a Subject in preventing cancer comprising administering to a Subject in whom Such treatment or prevention is desired therapeutically whom such treatment or prevention is desired a therapeuti or prophylactically effective amount of a substance selected cally or prophylactically effective amount of a Substance from the group consisting of (a) an isolated protein compris- 10 selected from the group consisting of: (a) an isolated protein ing SEQID NO:1 (ARP of E. tenella); (b) an isolated protein comprising SEQ ID NO:3, 4, 6 or 7; (b) an isolated protein comprising SEQ ID NO:2 (ARP of E. acervulina); (c) an comprising a variant of SEQIDNO:1, SEQID NO:2, SEQID isolated protein comprising SEQ ID NO: 3, 4, 5, 6 and 7 NO:28 or SEQID NO:30 wherein said variant has only con (partial amino acid sequence of bovine Eimeria spp. ARP; (d) servative amino acid substitutions relative to SEQID NO:1, an isolated protein comprising SEQID NO:28 (ARP of clone 15 SEQ ID NO:2, SEQ ID NO:28 or SEQ ID NO:30 respec E1); (e) an isolated protein comprising SEQID NO:30 (ARP tively; (c) an isolated protein comprising a variant of SEQID of clone E1 drug product). SEQID NOs: 3-7 are not neces NO:3, 4, 6 or 7, wherein said variant has only conservative sarily contiguous, there may be intervening or adjacent amino acid substitutes relative to SEQ ID NO:3, 4, 6 or 7. sequences to each fragment. Preferably, a protein comprises respectively; (d) an isolated protein that has at least 25%, at SEQ ID NOS:3, 4, 5, 6 and 7 in an order of SEQID NO:3 to 20 least 30%, at least 35%, at least 40%, at least 45%, at least SEQID NO:7 from the N terminus to the C terminus): (f) an 50%, at least 55%, at least 60%, at least 65%, at least 70%, at isolated protein that is a product of process comprising the least 75%, or at least 80% identity to the sequence of SEQID steps of: (1) producing a cell extract of Eimeria infected tissue NO:1, SEQ ID NO:2, SEQID NO:28 or SEQID NO:30 as or cells; (2) centrifuging the cell extract to produce a Super determined by a BLAST 2.0 algorithm set to default param natant; (3) fractionating the Supernatant by ammonium Sul- 25 eters; (e) an isolated protein comprising a PROF (profiling) fate to precipitate proteins to a pellet, and resuspending the domain, (f) an isolated Apicomplexan protein comprising an pellet into a solution; (4) applying the solution to a hydropho amino acid sequence encoded by a nucleic acid consisting of bic interaction column (hereinafter “HIC); (5) eluting mate a nucleotide sequence hybridizable to SEQID NO:15, 16, 17, rial bound to the HIC column to produce eluted material: (6) 18, 19, 20 or 27, or a complement of any of the foregoing SEQ dialyzing the eluted material to produce a sample for loading 30 ID NOS, under conditions of low Stringency; (g) an isolated on a diethylaminoethyl (hereinafter "DEAE) column; (7) nucleic acid comprising a nucleotide sequence encoding any applying the sample to a DEAE column; (8) eluting material of (a)-(f) above: (h) a cell transformed with a nucleic acid bound to the DEAE column with buffer to produce an eluted comprising a nucleotide sequence encoding any of (a)-(f) fraction and concentrating the eluted fraction; (9) applying above, said nucleotide sequence being operably linked to a the concentrated eluted fraction to a size exclusion column; 35 promoter, and wherein said Substance has immune stimula (10) collecting and pooling fractions eluted from the size tory ability, e.g., has the ability to stimulate interleukin-12 exclusion column; (11) applying the pooled fractions to a HIC release from dendritic cells, said dendritic cells having been column to remove calcium-binding proteins; (12) applying isolated from a mammal within 10 hours of assaying said eluted fractions from the HIC column to a high performance ability. liquid chromatography (hereinafter “HPLC) HIC column 40 The present invention also provides a method of treating or and collecting active (immunostimulatory) fractions, preventing an infectious disease, wherein said infectious dis wherein said active fractions have the ability to stimulate ease is not caused by infection with a human immunodefi interleukin-12 release from dendritic cells, said dendritic ciency virus (HIV) or an Eimeria organism, comprising cells having been isolated from a mammal within 10 hours of administering to a Subject in whom such treatment or preven assaying said ability (or as assayed by one or more assays 45 tion is desired a therapeutically or prophylactically effective described in Section 5.6 and Section 12, infra); (13) dialyzing amount of a Substance selected from the group consisting of the active fractions; and (14) applying the dialyzed active (a) an isolated protein comprising SEQID NO:1 (ARP of E. fractions to a HPLC DEAE column and collecting one or tenella); (b) an isolated protein comprising SEQ ID NO:2 more active (immunostimulatory) fractions, wherein said (ARP of E. acervulina); (c) an isolated protein comprising active fractions have the ability to stimulate interleukin-12 50 SEQ ID NO:28 (ARP of E1 clone); (d) an isolated protein release from dendritic cells, said dendritic cells having been comprising SEQID NO:30 (ARP of E1 clone drug product): isolated from a mammal within 10 hours of assaying said (e) an isolated protein comprising SEQID NO: 3, 4, 5, 6 and ability (or as assayed by one or more assays described in 7 (partial amino acid sequence of bovine Eimeria spp. ARP): Section 5.6 and Section 12, infra); (g) an isolated protein that (f) an isolated protein that is a product of process comprising is the product of a process comprising the steps of: (1) pro- 55 the steps of: (1) producing a cell extract of Eimeria infected ducing a homogenate of a purified Apicomplexan organism or tissue or cells; (2) centrifuging the cell extract to produce a a cell infected with an Apicomplexan organism; (2) centri Supernatant; (3) fractionating the Supernatant by ammonium fuging the homogenate to obtain a pellet; (3) treating the Sulfate to precipitate proteins to a pellet, and resuspending the pellet with a solution comprising a phospholipase; (4) centri pellet into a solution; (4) applying the Solution to a HIC fuging the Solution to obtain a Supernatant; (5) applying the 60 column; (5) eluting material bound to the HIC column to Supernatant to a reverse phase HPLC column; and (6) eluting produce eluted material: (6) dialyzing the eluted material to bound protein from the reverse phase HPLC column, to pro produce a sample for loading on a DEAE column; (7) apply duce eluted protein, wherein said protein has the ability (im ing the sample to a DEAE column; (8) eluting material bound munostimulatory ability) to stimulate interleukin-12 release to the DEAE column with buffer to produce an eluted fraction from dendritic cells, said dendritic cells having been isolated 65 and concentrating the eluted fraction; (9) applying the con from a mammal within 10 hours of assaying said ability (or as centrated eluted fraction to a size exclusion column: (10) assayed by one or more assays described in Section 5.6 and collecting and pooling fractions eluted from the size exclu US 7,393,534 B2 11 12 sion column; (11) applying the pooled fractions to a HIC tory ability, e.g., has the ability to stimulate interleukin-12 column to remove calcium-binding proteins; (12) applying release from dendritic cells, said dendritic cells having been eluted fractions from the HIC column to a HPLCHIC column isolated from a mammal within 10 hours of assaying said and collecting active (immunostimulatory) fractions, ability. wherein said active fractions have the ability to stimulate 5 The present invention also provides a method of stimulat interleukin-12 release from dendritic cells, said dendritic ing an immune response comprising administering to a Sub cells having been isolated from a mammal within 10 hours of ject, with the proviso that said Subject is not an avian species assaying said ability (or as assayed by one or more assays and is not infected with a human immunodeficiency virus described in Section 5.6 and Section 12, infra); (13) dialyzing (HIV), an effective amount of a substance selected from the the active fractions; and (14) applying the dialyzed active 10 group consisting of: (a) an isolated protein comprising SEQ fractions to a HPLC DEAE column and collecting one or ID NO: 1 (ARP of E. tenella); (b) an isolated protein compris more active (immunostimulatory) fractions, wherein said ing SEQID NO:2 (ARP of E. acervulina); (c) and isolated active fractions have the ability to stimulate interleukin-12 protein comprising SEQID NO:28 (ARP of E1 clone); (d) an release from dendritic cells, said dendritic cells having been isolated protein comprising SEQID NO:30 (ARP of E1 clone isolated from a mammal within 10 hours of assaying said 15 drug product); (e) an isolated protein comprising SEQ ID ability (or as assayed by one or more assays described in NO: 3, 4, 5, 6 and 7 (partial amino acid sequence of bovine Section 5.6 and Section 12, infra); (g) an isolated protein that Eimeria spp. ARP): (f) an isolated protein that is a product of is the product of a process comprising the steps of: (1) pro process comprising the steps of: (1) producing a cell extract of ducing a homogenate of a purified Apicomplexan organism or Eimeria infected tissue or cells; (2) centrifuging the cell a cell infected with an Apicomplexan organism; (2) centri extract to produce a Supernatant; (3) fractionating the Super fuging the homogenate to obtain a pellet; (3) treating the natant by ammonium sulfate to precipitate proteins to a pellet, pellet with a solution comprising phospholipase; (4) centri and resuspending the pellet into a solution; (4) applying the fuging the Solution to obtain a Supernatant; (5) applying the solution to a HIC column; (5) eluting material bound to the Supernatant to a reverse phase HPLC column; and (6) eluting HIC column to produce eluted material: (6) dialyzing the bound protein from the reverse phase HPLC column, to pro 25 eluted material to produce a sample for loading on a DEAE duce eluted protein, wherein said protein has the ability (im column; (7) applying the sample to a DEAE column; (8) munostimulatory ability) to stimulate interleukin-12 release eluting material bound to the DEAE column with buffer to from dendritic cells, said dendritic cells having been isolated produce an eluted fraction and concentrating the eluted frac from a mammal within 10 hours of assaying said ability (or as tion; (9) applying the concentrated eluted fraction to a size assayed by one or more assays described in Section 5.6 and 30 exclusion column; (10) collecting and pooling fractions Section 12, infra): (h)an isolated nucleic acid encoding any of eluted from the size exclusion column; (11) applying the (a)-(g), above; and (i) a cell transformed with a nucleic acid pooled fractions to a HIC column to remove calcium-binding comprising a nucleotide sequence encoding any of (a)-(g) proteins; (12) applying eluted fractions from the HIC column above, said nucleotide sequence being operably linked to a to a HPLCHIC column and collecting active (immunostimu promoter. 35 latory) fractions, wherein said active fractions have the ability The present invention provides a method of treating or to stimulate interleukin-12 release from dendritic cells, said preventing an infectious disease, wherein said infectious dis dendritic cells having been isolated from a mammal within 10 ease is not caused by infection with a human immunodefi hours of assaying said ability (or as assayed by one or more ciency virus (HIV) or an Apicomplexan organism, compris assays described in Section 5.6 and Section 12, infra); (13) ing administering to a Subject in whom Such treatment or 40 dialyzing the active fractions; and (14) applying the dialyzed prevention is desired a therapeutically or prophylactically active (immunostimulatory) fractions to a HPLC DEAE col effective amount of a substance selected from the group con umn and collecting one or more active fractions, wherein said sisting of: (a) an isolated protein comprising SEQID NO:3, 4, active fractions have the ability to stimulate interleukin-12 6 or 7; (b) an isolated protein comprising a variant of SEQID release from dendritic cells, said dendritic cells having been NO:1 or SEQ ID NO:2, wherein said variant has only con 45 isolated from a mammal within 10 hours of assaying said servative amino acid substitutions relative to SEQID NO:1, ability (or as assayed by one or more assays described in SEQ ID NO:2, SEQ ID NO:28 or SEQ ID NO:30 respec Section 5.6 and Section 12, infra); (g) an isolated protein that tively; (c) an isolated protein comprising a variant of SEQID is the product of a process comprising the steps of: (1) pro NO:3, 4, 6 or 7, wherein said variant has only conservative ducing a homogenate of a purified Apicomplexan organism or amino acid substitutes relative to SEQ ID NO:3, 4, 6 or 7. 50 a cell infected with an Apicomplexan organism; (2) centri respectively; (d) an isolated protein that has at least 25%, at fuging the homogenate to obtain a pellet; (3) treating the least 30%, at least 35%, at least 40%, at least 45%, at least pellet with a solution comprising a phospholipase; (4) centri 50%, at least 55%, at least 60%, at least 65%, at least 70%, at fuging the Solution to obtain a Supernatant; (5) applying the least 75%, or at least 80% identity to the sequence of SEQID Supernatant to a reverse phase HPLC column; and (6) eluting NO:1 or SEQID NO:2, SEQID NO:28 or SEQID NO:30 as 55 bound protein from the reverse phase HPLC column, to pro determined by a BLAST 2.0 algorithm set to default param duce eluted protein, wherein said protein has the ability (im eters; (e) an isolated protein comprising a PROF (profilin) munostimulatory ability) to stimulate interleukin-12 release domain, (f) an isolated Apicomplexan protein comprising an from dendritic cells, said dendritic cells having been isolated amino acid sequence encoded by a nucleic acid consisting of from a mammal within 10 hours of assaying said ability (or as a nucleotide sequence hybridizable to SEQID NO:15, 16, 17, 60 assayed by one or more assays described in Section 5.6 and 18, 19, 20 or 27, or a complement of any of the foregoing SEQ Section 12, infra): (h) an isolated nucleic acid comprising a ID NOS, under conditions of low Stringency; (g) an isolated nucleotide sequence encoding any of (a)-(f), above; and (i) a nucleic acid comprising a nucleotide sequence encoding any cell transformed with a nucleic acid comprising a nucleotide of (a)-(f) above: (h) a cell transformed with a nucleic acid sequence encoding any of (a)-(f) above, said nucleotide comprising a nucleotide sequence encoding any of (a)-(f) 65 sequence being operably linked to a promoter. above, said nucleotide sequence being operably linked to a The present invention provides a method of stimulating an promoter, and wherein said Substance has immune stimula immune response comprising administering to a subject, with US 7,393,534 B2 13 14 the proviso that said subject is not infected with a human stitutes relative to SEQID NO:3, 4, 6 or 7, respectively; (d) an immunodeficiency virus (HIV), an effective amount of a sub isolated protein that has at least 25%, at least 30%, at least stance selected from the group consisting of: (a) an isolated 35%, at least 40%, at least 45%, at least 50%, at least 55%, at protein comprising SEQ ID NO: 3, 4, 5, 6 and 7; (b) an least 60%, at least 65%, at least 70%, at least 75%, or at least isolated protein that is a product of a process comprising the 80% identity to the sequence of SEQIDNO:1, SEQID NO:2, steps of: (1) producing a cell extract of Eimeria infected tissue SEQ ID NO:28 or SEQ ID NO:30, as determined by a or cells, wherein said Eimeria do not naturally infect an avian BLAST 2.0 algorithm set to default parameters; (e) an iso species; (2) centrifuging the cell extract to produce a Super lated protein comprising a PROF (profilin) domain; (f) an natant; (3) fractionating the Supernatant by ammonium Sul isolated Apicomplexan protein comprising an amino acid fate to precipitate proteins to a pellet, and resuspending the 10 sequence encoded by a nucleic acid consisting of a nucleotide pellet into a solution; (4) applying the solution to a HIC sequence hybridizable to SEQID NO:15, 16, 17, 18, 19, 20 or column; (5) eluting material bound to the HIC column to 27, or a complement of any of the foregoing SEQID NOS, produce eluted material: (6) dialyzing the eluted material to under conditions of low stringency; (g) an isolated nucleic produce a sample for loading on a DEAF column; (7) apply acid comprising a nucleotide sequence encoding any of (a)- ing the sample to a DEAE column; (8) eluting material bound 15 (f) above: (h) a cell transformed with a nucleic acid compris to the DEAE column with buffer to produce an eluted fraction ing a nucleotide sequence encoding any of (a)-(f) above, said and concentrating the eluted fraction; (9) applying the con nucleotide sequence being operably linked to a promoter, and centrated eluted fraction to a size exclusion column: (10) wherein said Substance has immune stimulatory ability, e.g., collecting and pooling fractions eluted from the size exclu has the ability to stimulate interleukin-12 release from den sion column; (11) applying the pooled fractions to a HIC dritic cells, said dendritic cells having been isolated from a column to remove calcium-binding proteins; (12) applying mammal within 10 hours of assaying said ability. eluted fractions from the HIC column to a HPLCHIC column The present invention provides a method of elevating sys and collecting active (immunostimulatory) fractions, temic level of interleukin-12 (IL-12) in a subject, wherein the wherein said active fractions have the ability to stimulate Subject is not infected with a human immunodeficiency virus interleukin-12 release from dendritic cells, said dendritic 25 (HIV), comprising administering to said subject in whom cells having been isolated from a mammal within 10 hours of such elevation of IL-12 level is desired an effective amount of assaying said ability (or as assayed by one or more assays a Substance selected from the group consisting of: (a) an described in Section 5.6 and Section 12, infra); (13) dialyzing isolated protein comprising SEQ ID NO:1; (b) an isolated the active fractions; and (14) applying the dialyzed active protein comprising SEQ ID NO:2; (c) an isolated protein fractions to a HPLC DEAF column and collecting one or 30 comprising SEQID NO:28; (d) and isolated protein compris more active (immunostimulatory) fractions, wherein said ing SEQID NO:30; (e) an isolated protein comprising SEQ active fractions have the ability to stimulate interleukin-12 ID NO:3, 4, 5, 6 and 7: (f) an isolated protein that is a product release from dendritic cells, said dendritic cells having been of process comprising the steps of: (1) producing a cell extract isolated from a mammal within 10 hours of assaying said of Eimeria infected tissue or cells; (2) centrifuging the cell ability (or as assayed by one or more assays described in 35 extract to produce a Supernatant; (3) fractionating the Super Section 5.6 and Section 12, infra); (c) an isolated protein that natant by ammonium sulfate to precipitate proteins to a pellet, is the product of a process comprising the steps of: (1) pro and resuspending the pellet into a solution; (4) applying the ducing a homogenate of a purified Apicomplexan organism or solution to a HIC column; (5) eluting material bound to the a cell infected with an Apicomplexan organism; (2) centri HIC column to produce eluted material: (6) dialyzing the fuging the homogenate to obtain a pellet; (3) treating the 40 eluted material to produce a sample for loading on a DEAE pellet with a solution comprising a phospholipase; (4) centri column; (7) applying the sample to a DEAE column; (8) fuging the Solution to obtain a Supernatant; (5) applying the eluting material bound to the DEAE column with buffer to Supernatant to a reverse phase HPLC column; and (6) eluting produce an eluted fraction and concentrating the eluted frac bound protein from the reverse phase HPLC column, to pro tion; (9) applying the concentrated eluted fraction to a size duce eluted protein, wherein said protein has the ability (im 45 exclusion column; (10) collecting and pooling fractions munostimulatory ability) to stimulate interleukin-12 release eluted from the size exclusion column; (11) applying the from dendritic cells, said dendritic cells having been isolated pooled fractions to a HIC column to remove calcium-binding from a mammal within 10 hours of assaying said ability (or as proteins; (12) applying eluted fractions from the HIC column assayed by one or more assays described in Section 5.6 and to a HPLCHIC column and collecting active (immunostimu Section 12, infra); (d) an isolated nucleic acid comprising a 50 latory) fractions, wherein said active fractions have the ability nucleotide sequence encoding any of (a)-(c), above; and (e) a to stimulate interleukin-12 release from dendritic cells, said cell transformed with a nucleic acid comprising a nucleotide dendritic cells having been isolated from a mammal within 10 sequence encoding any of (a)-(c) above, said nucleotide hours of assaying said ability (or as assayed by one or more sequence being operably linked to a promoter. assays described in Section 5.6 and Section 12, infra); (13) The present invention also provides a method of stimulat 55 dialyzing the active fractions; and (14) applying the dialyzed ing an immune response comprising administering to a Sub active fractions to a HPLC DEAE column and collecting one ject, with the proviso that said subject is not an avian species or more active (immunostimulatory) fractions, wherein said and is not infected with a human immunodeficiency virus active fractions have the ability to stimulate interleukin-12 (HIV), an effective amount of a substance selected from the release from dendritic cells, said dendritic cells having been group consisting of: (a) an isolated protein comprising SEQ 60 isolated from a mammal within 10 hours of assaying said IDNO:3, 4, 6 or 7; (b) an isolated protein comprising a variant ability (or as assayed by one or more assays described in of SEQID NO:1, SEQID NO:2, SEQID NO:28 or SEQID Section 5.6 and Section 12, infra); (g) an isolated protein that NO:30, wherein said variant has only conservative amino is the product of a process comprising the steps of: (1) pro acid substitutions relative to SEQ ID NO:1, SEQ ID NO:2, ducing a homogenate of a purified Apicomplexan organism or SEQ ID NO:28 or SEQ ID NO:30, respectively; (c) an iso 65 a cell infected with an Apicomplexan organism; (2) centri lated protein comprising a variant of SEQID NO:3, 4, 6 or 7. fuging the homogenate to obtain a pellet; (3) treating the wherein said variant has only conservative amino acid Sub pellet with a solution comprising a phospholipase; (4) centri US 7,393,534 B2 15 16 fuging the Solution to obtain a Supernatant; (5) applying the cells having been isolated from a mammal within 10 hours of Supernatant to a reverse phase HPLC column; and (6) eluting assaying said ability; (k) applying the active fractions eluted bound protein from the reverse phase HPLC column, to pro from the low pressure DEAE column to a size exclusion duce eluted protein, wherein said protein has the ability (im column; and (1) eluting the active fractions from the size munostimulatory ability) to stimulate interleukin-12 release 5 exclusion column, wherein said active fractions have the abil from dendritic cells, said dendritic cells having been isolated ity to stimulate interleukin-12 release from dendritic cells, from a mammal within 10 hours of assaying said ability (or as said dendritic cells having been isolated from a mammal assayed by one or more assays described in Section 5.6 and within 10 hours of assaying said ability. Section 12, infra): (h) an isolated nucleic acid comprising a The present invention also provides a method of treating or nucleotide sequence encoding any of (a)-(g), above; and (i) a 10 preventing cancer comprising administering to a Subject in cell transformed with a nucleic acid comprising a nucleotide whom such treatment or prevention is desired a therapeuti sequence encoding any of (a)-(g) above, said nucleotide cally or prophylactically effective amount of a purified Api sequence being operably linked to a promoter. complexan organism or a protein-containing membrane frac The present invention also provides a method of elevating tion of a purified Apicomplexan organism. systemic level of interleukin-12 (IL-12) in a subject, wherein 15 The present invention also provides a method of treating or the Subject is not infected with a human immunodeficiency preventing an infectious disease, wherein said infectious dis virus (HIV), comprising administering to said subject in ease is not caused by infection with an human immunodefi whom such elevation of IL-12 level is desired an effective ciency virus (HIV) or an Eimeria organism, comprising amount of a Substance selected from the group consisting of administering to a Subject in whom such treatment or preven (a) an isolated protein comprising SEQID NO:3, 4, 6 or 7; (b) tion is desired a therapeutically or prophylactically effective an isolated protein comprising a variant of SEQ ID NO:1, amount of a purified Apicomplexan organism or a protein SEQID NO:2, SEQ ID NO:28 or SEQID NO:30, wherein containing membrane fraction of a purified Apicomplexan said variant has only conservative amino acid Substitutions organism. relative to SEQID NO:1, SEQID NO:2, SEQID NO:28 or The present invention further provides a method of stimu SEQID NO:30, respectively; (c) an isolated protein compris 25 lating an immune response comprising administering to a ing a variant of SEQID NO:3, 4, 6 or 7, wherein said variant subject, with the proviso that said subject is not infected with has only conservative amino acid substitutes relative to SEQ human immunodeficiency virus (HIV) and is not an avian ID NO:3, 4, 6 or 7, respectively; (d) an isolated protein that species, a therapeutically or prophylactically effective has at least 25%, at least 30%, at least 35%, at least 40%, at amount of a purified Apicomplexan organism or a protein least 45%, at least 50%, at least 55%, at least 60%, at least 30 containing membrane fraction of a purified Apicomplexan 65%, at least 70%, at least 75%, or at least 80% identity to the organism. sequence of SEQID NO:1, SEQID NO:2, SEQID NO:28 or The present invention provides a purified polypeptide SEQID NO:30, as determined by a BLAST 2.0 algorithm set selected from the group consisting of: (a) a purified polypep to default parameters; (e) an isolated protein comprising a tide which comprises SEQID NO: 3, 4, 6, or 7; (b) a purified PROF (profilin) domain; (f) an isolated Apicomplexan pro 35 polypeptide which comprises SEQID NOs: 3, 4, 5, 6, and 7: tein comprising an amino acid sequence encoded by a nucleic (c) a purified polypeptide which comprises an amino acid acid consisting of a nucleotide sequence hybridizable to SEQ sequence which has at least 90%, at least 91%, at least 92%, IDNO:15, 16, 17, 18, 19, 20 or 27, or a complement of any of at least 93%, at least 94%, or at least 95% identity to SEQID the foregoing SEQ ID NOs, under conditions of low strin NO: 29 as determined by a BLAST 2.0 algorithm set to gency; (g) an isolated nucleic acid comprising a nucleotide 40 default parameters; and (d) a purified polypeptide which sequence encoding any of (a)-(f) above: (h) a cell transformed comprises a variant of an amino acid sequence comprising with a nucleic acid comprising a nucleotide sequence encod SEQID NO: 3, 4, 5, 6 and 7, wherein said variant has only ing any of (a)-(f) above, said nucleotide sequence being oper conservative amino acid substitutions relative to SEQ ID ably linked to a promoter, and wherein said Substance has NO:3, 4, 5, 6 and 7, respectively. The present invention also immune stimulatory ability, e.g., has the ability to stimulate 45 provides a pharmaceutical composition comprising a thera interleukin-12 release from dendritic cells, said dendritic peutically effective amount of the polypeptide and a pharma cells having been isolated from a mammal within 10 hours of ceutically acceptable carrier. assaying said ability. The present invention also provides a method of isolating a The present invention also provides a method of extracting polypeptide comprising the steps of: (a) producing a cell ARP proteins expressed in recombinant cells comprising (a) 50 extract of Eimeria infected tissue or cells; (b) centrifuging the producing an extract of said host cell; (b) centrifuging the host cell extract to produce a Supernatant; (c) fractionating the cell extract to produce a Supernatant; (c) fractionate the Super Supernatant by ammonium Sulfate to precipitate proteins to a natant by ammonium sulfate to precipitate proteins; (d) cen pellet, and resuspending the pellet into a solution; (d) apply trifuging the precipitate to obtain a pellet; (e) resuspending ing the solution to a HIC column; (e) eluting material bound the pellet into a solution, (f) applying the Solution to a low 55 to the HIC column to produce eluted material: (f) dialyzing pressure hydrophobic interaction column; (g) collecting the eluted material to produce a sample for loading on a active fractions eluted from the low pressure hydrophobic DEAE column; (g) applying the sample to a DEAE column; interaction column, wherein said active fractions have the (h) eluting material bound to the DEAE column with buffer to ability to stimulate interleukin-12 release from dendritic produce an eluted fraction and concentrating the eluted frac cells, said dendritic cells having been isolated from a mam 60 tion: (i) applying the concentrated eluted fraction to a size mal within 10 hours of assaying said ability, (h) dialyzing the exclusion column: (i) collecting and pooling fractions eluted eluted active fractions from the low pressure hydrophobic from the size exclusion column; (k) applying the pooled interaction column: (i) applying the dialyzed active fractions fractions to a HIC column to remove calcium-binding pro to a low pressure DEAE column: 0) collecting one or more teins: (1) applying eluted fractions from the HIC column to a active fractions eluted from the low pressure DEAE column, 65 HPLC HIC column and collecting active (immunostimula wherein said active fractions have the ability to stimulate tory) fractions, wherein said active fractions have the ability interleukin-12 release from dendritic cells, said dendritic to stimulate interleukin-12 release from dendritic cells, said US 7,393,534 B2 17 18 dendritic cells having been isolated from a mammal within 10 acid is a vector. In yet another embodiment, the vector further hours of assaying said ability (or as assayed by one or more comprises a promoter operably linked to said nucleic acid assays described in Section 5.6 and Section 12, infra); (m) sequence encoding said polypeptide. dialyzing the active fractions; (n) applying the dialyzed active The present invention also provides a host cell comprising fractions to a HPLC DEAE column and collecting one or 5 an expression vector, wherein said expression vector com more active (immunostimulatory) fractions, wherein said prises a promoter operably linked to a nucleotide sequence, active fractions have the ability to stimulate interleukin-12 wherein said promoter is non-native with respect to said release from dendritic cells, said dendritic cells having been nucleotide sequence, and wherein said nucleotide sequence isolated from a mammal within 10 hours of assaying said encodes (a) a polypeptide comprising SEQID NO: 3, 4, 6 or ability (or as assayed by one or more assays described in 10 7; (b) a polypeptide comprising SEQID NOs: 3, 4, 5, 6 and 7: Section 5.6 and Section 12, infra). In certain embodiments, or (c) a polypeptide comprising an amino acid sequence the method of producing a polypeptide further comprising which has at least 90%, at least 91%, at least 92%, at least (O) applying the active fractions of step (n) to a reverse phase 93%, at least 94%, or at least 95% identity to SEQID NO:29 HPLC column; and (p) eluting bound protein from the reverse as determined by a BLAST 2.0 algorithm set to default phase HPLC column, to produce eluted protein, wherein said 15 protein has the ability (immunostimulatory ability) to stimu parameters. late interleukin-12 release from dendritic cells, said dendritic In one embodiment, the host cell is a mammalian host cell. cells having been isolated from a mammal within 10 hours of In another embodiment, the host cell is a yeast cell. In another assaying said ability (or as assayed by one or more assays embodiment, the host cell is an insect cell. In yet another described in Section 5.6 and Section 12, infra). The present embodiment, the host cell is a bacterial cell. In a specific invention also provides a purified polypeptide that is the embodiment, the bacterial cell is an E. coli. The present product of this process. The present invention further pro invention further provides a method of producing a polypep vides a pharmaceutical composition comprising a therapeu tide comprising growing said cell. Such that the encoded tically effective amount of the polypeptide and a pharmaceu polypeptide is expressed by the cell, and recovering the tically acceptable carrier. 25 expressed polypeptide. In a specific embodiment, the The present invention further provides a method of isolat expressed polypeptide is recovered by a method comprising ing a polypeptide comprising the steps of: (1) producing a the steps of: (1) producing an extract of said cell; (2) centri homogenate of a purified Apicomplexan organism or a cell fuging the cell extract to produce a Supernatant; (3) fraction infected with an Apicomplexan organism; (2) centrifuging ating the Supernatant by ammonium sulfate to precipitate the homogenate to obtain a pellet; (3) treating the pellet with 30 proteins; (4) centrifuging the precipitate to obtain a pellet; (5) a solution comprising a phospholipase; (4) centrifuging the resuspending the pellet into a solution; (6) applying the solu solution to obtain a supernatant; (5) applying the Supernatant tion to a low pressure column; (7) collecting active (immu to a reverse phase HPLC column; and (6) eluting bound nostimulatory) fractions eluted from the low pressure col protein from the reverse phase HPLC column, to produce umn, wherein said active fractions have the ability to eluted protein, wherein said protein has the ability (immuno 35 stimulate interleukin-12 release from dendritic cells, said stimulatory ability) to stimulate interleukin-12 release from dendritic cells having been isolated from a mammal within 10 dendritic cells, said dendritic cells having been isolated from hours of assaying said ability (or as assayed by one or more a mammal within 10 hours of assaying said ability (or as assays described in Section 5.6 and Section 12, infra); (8) assayed by one or more assays described in Section 5.6 and dialyzing the active fractions; (9) applying the dialyzed active Section 12, infra). The present invention also provides a puri 40 fractions to a HPLC DEAE column: (10) collecting one or fied polypeptide that is the product of this process. The more active (immunostimulatory) fractions eluted from the present invention further provides a pharmaceutical compo HPLC DEAE column, wherein said active fractions have the sition comprising a therapeutically effective amount of the ability to stimulate interleukin-12 release from dendritic polypeptide and a pharmaceutically acceptable carrier. cells, said dendritic cells having been isolated from a mam The present invention provides an antibody which binds a 45 mal within 10 hours of assaying said ability (or as assayed by first protein, the amino acid sequence of which first protein one or more assays described in Section 5.6 and Section 12, consists SEQ ID NO:26, which antibody does not bind to a infra); (11) applying the active fraction to a size exclusion second protein, the amino acid sequence of which second column; and (12) eluting the active (immunostimulatory) protein consists of SEQ ID NO:1 or SEQ ID NO:2. In a fractions from the size exclusion column, wherein said active specific embodiment, the antibody is a monoclonal antibody. 50 fractions have the ability to stimulate interleukin-12 release In another specific embodiment, the present invention pro from dendritic cells, said dendritic cells having been isolated vides a molecule comprising a fragment of said antibody, from a mammal within 10 hours of assaying said ability (or as which fragment is capable of binding said first protein. assayed by one or more assays described in Section 5.6 and The present invention provides an isolated nucleic acid Section 12, infra). selected from the group consisting of: (a) an isolated nucleic 55 acid comprising a nucleotide sequence, which nucleotide sequence encodes a polypeptide comprising SEQID NO: 3, Conventions and Abbreviations 4, 6 or 7; (b) an isolated nucleic acid comprising a nucleotide sequence, which nucleotide sequence encodes a polypeptide ARP Apicomplexa-related protein (the active molecule of the invention) comprising SEQID NOs: 3, 4, 5, 6 and 7; and (c) an isolated 60 SIE Small intestine extract nucleic acid molecule comprising a nucleotide sequence, BEX Bovine Small intestine extract which nucleotide sequence encodes a polypeptide compris GM-CSF Granulocyte macrophage-colony stimulating factor IL2 Interleukin 2 ing an amino acid sequence which has at least 90%, at least IL12 Interleukin 12 91%, at least 92%, at least 93%, at least 94%, or at least 95% IFN-gamma. Interferon-gamma identity to SEQ ID NO:29 as determined by a BLAST 2.0 65 DC Dendritic cell algorithm set to default parameters. In one embodiment, the LGL Large granular lymphocytes nucleic acid is purified. In another embodiment, the nucleic US 7,393,534 B2 19 20 (B) Activity from two cultures of E. coli containing plasmids -continued incorporating either forward or reverse orientations of the cDNA shown in (A). (C) C8HPLC separation of the purified Conventions and Abbreviations minimal media conditioned by E. coli containing the cloning NK or NK cells Natural killer cells plasmid with the cloned ARP for E. tenella, and the corre in NK NK to target cell ratio, where in represents seeding ratio sponding DC assay activity of the fractions. CMC Cell-mediated cytotoxicity spp. Various species of a given genus FIG. 8. Compilation of survival data of tumor-bearing cDNA Complementary DNA Balb/c mice from control and partially purified ARP (stage 1 BEX) treated groups. These survival times were obtained 10 from Screening experiments designed to check the efficacy of the enriched ARP (stage 1 extraction) product. The number of BRIEF DESCRIPTION OF THE FIGURES control is not equal to the number in the treatment group because several batches were often screened simultaneously FIG.1. Phylogenetic tree for small subunit ribosomal RNA against the same control. sequences of Cyclospora and Eimeria species. Quartet puz 15 Zling maximum likelihood results are shown, with Toxo FIG. 9. Anti-tumor activity of E. tenella extracts and plasma gondii as the outgroup. After analysis, the outgroup recombinant E. tenella ARP in the murine tumor model. Three or five dosing groups for each treatment are shown. The branch was removed for clarity. Numbers to the left of the dosing protocol consisted of the indicated dose in ng/mouse? nodes indicate the quartet puzzling Support for each internal day given daily for 5 consecutive days beginning one day branch. The scale bar indicates an evolutionary distance of after tumor injection. The amount of ARP in the extract was 0.01 nucleotides per position in the sequence. Vertical dis determined by C8HPLC chromatography. The control group tances are for clarity only. (This figure is taken from Eberhard received a sham injection of buffer. A) recombinant E. tenella et al., 1999, Emerging Infectious Diseases, 5(5): 651-658.) ARP from the EtU3 clone, B) recombinant E. tenella ARP FIG. 2. (A) Example of a molecular sieving chromatogra from the E1 clone, C) E. tenella ARP extract from sporulated phy step using SuperdexTM 75 and the activity on the NK and 25 DC assays. (B) Typical total recovery from the purification oocysts. scheme outlined in section 6, at the end of the SuperdexTM FIG. 10. Correlation of the in vitro NK and DC assays with size exclusion step. The activity within the crude material anti-tumor response in mice. (A) Each batch of BEX was routinely tested in mice and in the NK assay. A series of (corrected for total volume) is shown on the far left. The total batches, spanning approximately 1 year of extracts, were recovered after SuperdexTM chromatography is shown at the 30 far right. used in this figure. The in vivo anti-tumor activity was repre FIG. 3. (A) HIC gradient chromatography of BBX after sented by a tumor index calculated from increased life span Calcium-binding protein removal step. (B) DEAE gradient and survival, with 0 representing 100% cures and 4 represent ing no Survival advantage over negative controls. The in vitro chromatography of the active fractions from the HIC gradi NK assay activity was represented by the logarithm of the ent. (C) C8 reverse phase gradient of the active fractions from 35 dilution need to reach 50% cell kill. The solid line represents the DEAE gradient. (D) Correlation of absorbance and activ the best linear regression through the points. (B) Experimen ity in C8 active fractions. tal dosages were pre-tested in the DC assay and the log of the FIG. 4. Comparison of protein sequences from three Eime activity observed for the dose was plotted against a tumor ria species. The sequences of E. tenella (EtU3) and E. acer index based on degree of palpable tumors, presence of vulina (3-1E: Accession # AF1 13613) were obtained from 40 cDNA translation. The cDNA for EtU3 was obtained from E. cachexia, and Survival status. This index has the same end tenella mRNA as described in section 10. The segments from points as above. The regression fit parameters are displayed a presumed bovine Eimeria of unknown species was obtained on each plot. by Edman and MS/MS amino acid sequencing of tryptic FIG. 11. Serum IL-12 levels in patient #5-RW, during two peptides. 45 courses of the 5 day low-dose BBX-01c. Serum levels were FIG.5. (A) Dilution profile of unpurified E. tenella extracts measured prior to injections of BBX-01c on day 1 through on the DC assay. Samples were prepared according to first day 5. A final sample was drawn either on day 9 or 10. extraction of section 8. Five replicate dilution curves, span FIG. 12. Correlation of induced NK-CMC with elevated ning two separate DC assays, were averaged. The error bars levels of IFNY in the media of treated LGL cultures. LGL/S indicate the standard deviation of the 5 replicates. (B) C8 50 180 co-cultures were treated with varying concentrations of HPLC separation of the second E. tenella extract and the BEX and analyzed for NK-CMC with the standard 4 day in corresponding DC assay activity of the fractions. vitro NK assay. LGL cultures were treated with the same FIG. 6: Dose/response curves for various sources of ARP. concentrations of BEX and the conditioned media was ana The impure E. tenella extract and the DEAE-purified recom lyzed for IFNY concentration following an overnight culture. binant sample contain ARP that is about 4% of total protein 55 FIG. 13. (A) Response of the DC assay to E. tenella and weight. The pure bovine extract and the recombinant E. Bovine BBX-01c as a function of synergistic additives. In tenella protein are pure but approximately 95% denatured each panel, activity is measured for the antigen alone, the from the reverse-phase chromatographic conditions. These additives alone, the antigen with all the additives (ALL), the estimates were used to calculate protein concentration in antigen plus a single additive (denoted with +), and the anti assays. The highly pure raRP sample was obtained from 60 gen with only one additive missing (denoted with a -). The size-exclusion chromatography from the DEAF active frac two panels represent two separate assays and absolute values tion. Its purity and protein content were evaluated on a small between the two are not comparable. (B) Loss of sensitivity of sample by C8 reverse-phase chromatography and its activity freshly isolated CD11c" dendritic cells to BBX-01c as a was plotted without corrections for concentration. Response function of time in tissue culture conditions. curves were normalized to similar maximal levels. 65 FIG. 14. Synergism of BBX-01c (enriched ARP) and GM FIG. 7. (A) DNA sequence of the cDNA clone obtained CSF in mouse tumor model. Two sub-therapeutic doses of from E. tenella mRNA using primers described in section 10. BBX-01c, with and without GM-CSF, were injected one day US 7,393,534 B2 21 22 after tumor. GM-CSF alone was not significantly different FIG. 27. Dose/response curves for both the oxidized and from sham injection with vehicle. reduced forms of ARP. The curves are adjusted to constant FIG. 15. DC assay activities of various bovine breeds com protein concentration. pared to a benchmark pool. Boxes represent 25" percentile (bottom) to 75" percentile (top). Lines above and below DETAILED DESCRIPTION OF THE INVENTION boxes are the 95" and 5" percentile indicators, respectively. The median in denoted as a line inside the box. The present invention provides compositions and methods FIG. 16. (A) DC activity of extracts from sections of the for the prevention and treatment of primary and metastatic bovine intestinal system with comparison to natural tumor neoplastic diseases and infectious diseases, for stimulating an incidence. (B) Antitumor activity of bovine intestinal extracts 10 immune response in a Subject, and for use as an alternative to in the murine tumor model. The tumor index, as described in interleukin-12 (IL-12) treatment. In particular, the present section 12.1.2, was used to evaluate an experiment in invention provides Apicomlexa-related proteins (ARPs) that progress. Pools of three adjacent fractions along the Small and have immune stimulatory activity and thus have uses in the large intestine were included with the cecum sample. Each treatment and prevention of cancer and infectious diseases, group consisted of a group of 5 mice following the protocol of 15 and in immune modulation. Compositions comprising ARPS section 12.1.1. are provided. Methods of use of ARPs for the prevention and/or treatment of cancer and infectious diseases, for use as FIG. 17. DC activity and murine tumor index data of an an alternative to interleukin-12 (IL-12) treatment, and for Eimeria tenella crude membrane preparation. Dendritic cell eliciting an immune response in a subject, are also provided. IL12 production is shown for various dilutions of the E. While not bound by any theory, the invention is based, in part, tenella crude membrane fragment Suspension, where 1.0 on the Applicants discovery that a molecule purified from indicates no dilution of the Suspension and is the concentra Eimeria infected bovine Small intestine extracts, which is tion administered to mice in the in Vivo anti-tumor assay. highly homologous to a Surface antigen (3-1E. Lellehoetal. Tumor index for the ongoing test of membrane Suspension 2000, Avian Diseases 44: 379-389) of Apicomplexan proto activity is also shown. The lower value for the test treatment 25 Zoan Eimeria acervulina, is a robust activator of dendritic indicates positive anti-tumor activity. cells at pico- and femtomolar levels and has cross species FIG. 18. C8 reverse phase chromatography of ARP immune stimulatory effects both in vitro and in vivo. released from the crude membrane preparation from E. For clarity of disclosure, and not by way of limitation, the tenella oocysts after incubation in 50 mM tris-HCl pH 7.4, 1 detailed description of the invention is divided into the fol mM PMSF at 37° C. 4 hours (section 9). 30 lowing Subsections: FIG. 19. Alignment of various Apicomplexa expressed (i) ARPs sequence tag (EST) translations. Alignments were performed (ii) ARPs of Purified Apicomplexa by visual observation. A threshold for “similarity” at a posi (iii) Derivatives and Analogs of ARPs tion was that no more than two amino acids that would be (iv) Antibodies to ARPs, Derivatives and Analogs considered non-conservative changes relative to the other 35 (v) Structure Prediction and Functional Analysis of ARPs amino acids that were present at that position. If this condition (vi) Characterization and Demonstration of ARP activities is satisfied, the recorded amino acid is the one occurring most (vii) Uses of the Compositions of the Invention commonly in the column. Average length from start Methion (viii) Other Anti-cancer Therapies ine is 191. Among the ESTs compared, 41 positions (41/ (iv) Other Anti-infection Agents 191=21%) have 100% identity, and 106 positions (106/ 40 (X) Dosage Regimens 191=55%) have at least 80% similarity by amino acid property. (xi) Administrations. Formulations and Kits FIG. 20. Alignment of DNA sequences of E. tenella ARP (xii) Article of Manufacture E1 clone and the EtU3 (also referred as U3 in the figure) (xiii) Monitoring of Effects During Treatment clone. 45 ARPS FIG. 21. Chromatogram of the HIC gradient chromatogra In some embodiments, an ARP of the invention is a protein phy separation of the 80% ammonium sulfate fraction of an Apicomplexan organism that has immune stimulatory obtained from the E1 clone extract and the corresponding DC activity, as well as a fragment, a derivative, a variant, a assay activity profile. homolog or an analog thereof. An Apicomplexan organism is FIG.22. Chromatogram of the DEAE gradient chromatog 50 one of those of the phylum Apicomplexa. Non-limiting raphy separation of the active fractions from the HIC chro examples of Apicomplexan organisms includebut not limited matography of the E1 clone extract and the corresponding DC to, those that are listed in Table 1, infra. In specific embodi assay activity profile ments, an ARP of the invention include, but are not limited to, FIG. 23. Chromatogram of the Superdex isocratic chroma SEQID NO:1 (ARP of E. tenella), SEQID NO:2 (ARP of E. 55 acervulina), and a protein that comprises SEQID NOs: 3-7 tography separation of the active fractions from the DEAE (partial amino acid sequence of bovine Eimeria spp. ARP. chromatography of the E1 clone extract and the correspond SEQID NOs: 3-7 are not necessarily contiguous, there may ing DC assay activity profile be intervening or adjacent sequences to each fragment. Pref FIG. 24. Chromatogram of the C8 HPLC separation of a erably, a protein comprises SEQID NOs: 3, 4, 5, 6 and 7 in an small portion of the pooledactive fractions from the Superdex 60 order of SEQID NO:3 to SEQID NO:7 from the N terminus chromatography of the E1 clone extract. This indicates that to the C terminus). the SuperdeX product is highly pure. In a specific embodiment, an ARP of the invention is an FIG. 25. ELISA assessment of IL-12 within the mice that Apicomplexan protein (encoded by genome of an Apicom were treated with an E. tenella ARP (EtU3). plexan organism) that has at least 25%, at least 30%, at least FIG. 26. Deconvoluted electrospray MS spectrum showing 65 35%, at least 40%, at least 45%, at least 50%, at least 55%, at the expected masses of 18,414 and 18.809 for the reaction of least 60%, at least 65%, at least 70%, at least 75%, at least Ellman's reagent with ARP E1. 80%, at least 85% identity to the sequence of SEQID NO:1 or US 7,393,534 B2 23 24 SEQ ID NO:2 as measured by a BLAST algorithm with forth in SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, default parameters using the BLAST 2.0 suite of programs SEQ ID NO:19, or SEQID NO:20, or to its reverse comple (Altschul et al., Nucleic Acids Res, 2: 3389-3402, 1997), ment, or to a nucleic acid encoding an ARP derivative, or to its wherein the Apicomplexan protein has immune stimulatory reverse complement), wherein the Apicomplexan protein has and/or anti-cancer activity. Such activity (immunostimula immune stimulatory and/oranti-cancer activity. Such activity tory) can be measured by, e.g., assays described in Section 5.6 can be measured by, e.g., assays described in Section 5.6 and and Section 12.3, infra. In a specific embodiment, an ARP of Section 12.3, infra. Stringent conditions are sequence-depen the invention is a naturally occurring Apicomplexan protein. dent and circumstance-dependent, for example, longer In some embodiments, the ARPs of the invention exist in a sequences hybridize specifically at higher temperatures. An soluble form. In some embodiments, the ARPs of the inven 10 extensive guide to the hybridization of nucleic acids is found tion exist in a membrane-linked form. The BLAST family of in Tijssen, Techniques in Biochemistry and Molecular Biol programs which can be used for database similarity searches ogy-Hybridization with Nucleic Probes, “Overview of prin includes: BLASTN for nucleotide query sequences against ciples of hybridization and the strategy of nucleic acid nucleotide database sequences; BLASTX for nucleotide assays” (1993). See also Martin etal (EMBO.J. 4: 1625-1630 query sequences against protein database sequences: 15 (1985)) and Davies et al (Methods in Molecular Biology Vol BLASTP for protein query sequences against protein data 28: Protocols for nucleic acid analysis by non-radioactive base sequences: TBLASTN for protein query sequences probes, Isaac, P. G. (ed) pp. 9-15, Humana Press Inc., Totowa against nucleotide database sequences; and TBLASTX for N.J., USA). All of which are incorporated herein by reference nucleotide query sequences against nucleotide database in their entireties. sequences. See, Current Protocols in Molecular Biology, In a specific embodiment, a nucleic acid which is hybrid Chapter 19, Ausubel, et al., Eds. Greene Publishing and izable to an ARP nucleic acid or its reverse complement under Wiley-Interscience, New York, 1995. New versions of the conditions of low stringency is provided. By way of example above programs or new programs altogether will undoubtedly but not limitation, procedures using Such conditions of low become available in the future, and can be used with the stringency areas follows (see also Shilo and Weinberg, 1981, present invention. It is to be understood that default settings of 25 Proc. Natl. Acad. Sci. U.S.A. 78,6789-6792): filters contain the parameters can be readily changed as needed in the future. ing DNA are pretreated for 6 hours at 40°C. in a solution In a specific embodiment, an ARP of the invention is a containing 35% formamide, 5xSSC, 50 mM Tris-HCl protein that contains a conserved PROF (profilin) domain, (pH7.5), 5 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and wherein the protein has immune stimulatory and/or anti-can 500 ug/ml denatured salmon sperm DNA; hybridizations are cer activity. Such activity can be measured by, e.g. assays 30 carried out in the same solution with the following modifica described in Section 5.6 and Section 12.3, infra. Conserved tions: 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 g/ml domains are defined based on recurring sequence patterns or salmon sperm DNA, 10% (wt/vol) dextran sulfate, and 5-20x motifs. In one embodiment, the conserved PROF (profilin) is 106 cpm 32P-labeled probe is used; filters are incubated in determined by using Conserved Domain Database (CDD V. hybridization mixture for 18-20 hours at 40° C., and then 1.60) and an RPS-BLAST (Reverse Position-Specific 35 washed for 1.5 hours at 55° C. in a solution containing BLAST) algorithm set to default parameters. Current CDD 2xSSC, 25 mM Tris-HCl (pH7.4), 5 mM EDTA, and 0.1% database contains domains derived from two popular collec SDS; the wash solution is replaced with fresh solution and tions, Smart and Pfam, plus contributions from NCBI. In incubated an additional 1.5 hours at 60° C.; filters are blotted Conserved Domain Database, the PROF (profilin) domain is dry and exposed for autoradiography. If necessary, filters are also identified as Smart 00392 or cd 00148 domain, or pfam 40 washed for a third time at 65-68°C. and re-exposed to film. In 00235 domain (PSSM Id's 14983, 14824 and 801 corre another example, low Stringency hybridization is carried out spondingly). To identify conserved domains in a protein at 62° C. without formamide. Other conditions of low strin sequence, the RPS-BLAST algorithm can be used. The query gency which may be used are well known in the art (e.g., as sequence is compared to a position-specific score matrix pre employed for cross-species hybridizations). pared from the underlying conserved domain alignment. Hits 45 In another specific embodiment, a nucleic acid which is may be displayed as a pairwise alignment of the query hybridizable to an ARP nucleic acid, or its reverse comple sequence with a representative domain sequence, or as a ment, under conditions of high Stringency is provided. By multiple alignment. See, Marchler-Bauer et al., (2003) way of example but not limitation, procedures using Such Nucleic Acids Research 31:383-387 (2003); Marchler-Bauer conditions of high Stringency areas follows: prehybridization et al., (2002) Nucleic Acids Research 30:281-283, all of 50 of filters containing DNA is carried out for 8 hours to over which are incorporated by reference in their entirety. The night at 65° C. in buffer composed of 6xSSC, 50 mM Tris “PROF domain is represented by profilin, which is ubiqui HCl (pH7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% tous in nature, occurring in organisms from amoeba to mam BSA, and 500 ug/ml denatured salmon sperm DNA; filters mals. Profilin is involved in the regulation of actin polymer are hybridized for 48 h at 65°C. in prehybridization mixture ization and may link the cytoskeleton with major signaling 55 containing 100 g/ml denatured salmon sperm DNA and pathways by interacting with components of the phosphati 5-20x10 cpm of‘P-labeled probe; washing offilters is done dylinositol and Ras pathway. See e.g. Korenbaum et al., Bio at 37° C. for 1 hour in a solution containing 2xSSC, 0.01% chemistry (1998) 37(26): 9274-83: Schluter et al., Biochim PVP 0.01% Ficoll, and 0.01% BSA; this is followed by a Biophys Acta. (1997) 1359(2): 97-109, all of which are incor washin 0.1 xSSC at 50° C. for 45 min before autoradiography. porated by reference in their entireties. In a specific embodi 60 In another example, high Stringency hybridization is carried ment, an ARP of the invention is a plant profilin e.g. SEQID out at 62°C. with 50% formamide. Other conditions of high NO: 11, 12, 13, or 14. stringency which may be used are well known in the art. In a specific embodiment, an ARP of the invention is an In another specific embodiment, a nucleic acid which is Apicomplexan protein whose encoding nucleic acid (Api hybridizable to an ARP nucleic acid, or its reverse comple complexan cDNA or genomic nucleic acid) hybridizes under 65 ment, under conditions of moderate stringency is provided. stringent conditions (high, moderate or low stringent condi Selection of appropriate conditions for Such stringencies is tion) to an ARP nucleic acid (e.g., having a sequence as set well known in the art (see e.g., Sambrook et al., 1989, US 7,393,534 B2 25 26 Molecular Cloning, A Laboratory Manual. 2d Ed., Cold relative to the natural or original state from which the nucleic Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.: acid is derived. The enrichment can be achieved by preferen see also, Ausubel et al., eds., in the Current Protocols in tial reduction in the amount of other nucleic acid present, or Molecular Biology series of laboratory technique manuals, by a preferential increase in the amount of the specific nucleic (C) 1987-1997, Current Protocols, (C) 1994-1997 John Wiley acid of interest, or by a combination of the two. It should be and Sons, Inc.). In one non-limiting example, moderate strin noted that "enriched' does not imply that there are no other gency hybridization can be carried out at 62° C. with 20% nucleic acids present. The term also does not imply that there formamide. are no nucleic acids present from other sources. The other In an embodiment where a bovine ARP (which comprises Source nucleic acids may, for example, comprise nucleic SEQID NOs: 3-7) or its derivatives are used therapeutically 10 acid(s) encoded by a genome of another species, or of a or prophylactically, the ARP comprises a disulfide bond. In cloning vector. The term is meant to cover only those situa one embodiment, a bovine ARP has not been subjected to tions in which man has intervened to elevate the proportion of reducing conditions that would disrupt a disulfide bond. ARP the desired nucleic acid. protein that has been reduced has activity that is 2-5 times In a preferred embodiment, an ARP of the invention is greater than the oxidized form. 15 purified. The term “purified in reference to a protein or a In some embodiments, an ARP of the invention is post nucleic acid means at least one order of magnitude of purifi translationally modified. In some embodiments, an ARP of cation is achieved, preferably two or three orders of magni the invention is not post-translationally modified. In specific tude, most preferably four or five orders of magnitude of embodiments, an ARP of the invention is glycosylated. In purification of the starting material or of the natural material. specific embodiments, an ARP of the invention is unglycosy Thus, the term “purified as used herein does not mean that lated. the material is 100% purified and thus does not mean that a In some embodiments, an ARP of the invention is mem purified protein or a nucleic acid excludes any other material. brane-linked. In some embodiments, an ARP of the invention In specific embodiments, a purified ARP is at least 60%, at is not membrane-linked. In specific embodiments, an ARP of least 80%, or at least 90% of total protein or nucleic acid, as the invention is glysosylphosphatidylinos-itol (GPI)-linked. 25 the case may be, by weight. In a specific embodiment, a In specific embodiments, an ARP of the invention is not purified ARP is purified to homogeneity as assayed by, e.g., GPI-linked. In one embodiment, an ARP of the invention is a Sodium dodecyl Sulfate polyacrylamide gel electrophoresis, lipoprotein. In another embodiment, an ARP of the invention or agarose gel electrophoresis. is not a lipoprotein. In a specific embodiment, an ARP of the invention is In a specific embodiment, an ARP of the invention is a 30 present in the form of purified viable or inactivated Apicom native protein. In a specific embodiment, an ARP of the plexan organisms at any developmental stage or a protein invention is a recombinantly produced protein. In specific fraction thereof (e.g., a protein-containing membrane prepa embodiments, an ARP of the invention has a molecular ration thereof, or a storage granule preparation thereof). In a weight in the range of 18 kD to 25kD, and an isoelectric point specific embodiment, an Apicomplexan organism is an Eime (pl) between 4.0 and 4.7. 35 ria, which is species-specific and usually cannot cause symp Therapeutic ARPs of the invention can be tested in vitro for tomatic infection in a host of a different species from its native the desired activity by any one or more assays known in the art host. In another specific embodiment, an inactivated Apicom or assays as described in Section 5.6. plexan organism is a life cycle defective Apicomplexan An ARP of the invention typically has potent immune organism. stimulatory activity, even at very low concentration (e.g., 40 pico- or femtomolar levels). In specific embodiments, athera Preparation and Purification of ARP peutic or prophylactic composition of the invention for elic In some embodiments, the compositions of the invention iting an immune response and for the prevention and treat that are used in prevention or treatment of cancer and/or ment of cancer and infectious diseases comprises an enriched infectious diseases comprise an enriched, an isolated, or a ARP. As used herein, the term "enriched in reference to a 45 purified ARP. In accordance with the methods described protein (e.g., a peptide, polypeptide or fusion protein) means herein, an ARP employed in a composition of the invention that the protein constitutes a higher fraction of the total can be in the range of 0.001 to 100 percent of the total mg amount of protein present in the composition of interest, protein, or at least 0.001%, at least 0.003%, at least 0.01%, at relative to the natural or original state from which the protein least 0.1%, at least 1%, at least 10%, at least 30%, at least is derived. The enrichment can be achieved by preferential 50 60%, or at least 90% of the total mg protein. In one embodi reduction in the amount of other protein present, or by a ment, an ARP employed in a composition of the invention is preferential increase in the amount of the specific protein of at least 4% of the total protein. In another embodiment, an interest, or by a combination of the two. It should be noted ARP is purified to apparent homogeneity, as assayed, e.g., by that "enriched' does not imply that there are no other proteins Sodium dodecyl Sulfate polyacrylamide gel electrophoresis. present. The term also does not imply that there are no pro 55 The ARPs of the invention can be purified, e.g., from the teins present from other sources. The other source proteins homogenates of infected tissue or a purified Apicomplexan may, for example, comprise protein(s) encoded by a genome organism with the use of either soluble fractions (Superna of another species, or of a cloning vector. The term is meant to tants) or insolubles (pellets), or from intact cells (e.g., mam cover only those situations in which man has intervened to malian cells expressing a recombinant protein or Apicom elevate the proportion of the desired protein. In specific 60 plexa cells) with the use of enzymes acting on cell Surface. embodiments, an ARP is greater than 0.001%, greater than Any techniques known in the art can be used in purifying an 0.003%, greater than 0.01%, greater than 0.05%, greater than ARP, including but are not limited to, separation by precipi 0.1%, greater than 0.5%, greater than 1%, greater than 10%, tation, separation by adsorption (e.g., column chromatogra greater than 20%, greater than 30% of total proteinby weight. phy, membrane adsorbents, radial flow columns, batch The term "enriched in reference to nucleic acid means that 65 adsorption, high-performance liquid chromatography, ion the nucleic acid constitutes a higher fraction of the total exchange chromatography, inorganic adsorbents, hydropho amount of nucleic acids present in the composition of interest, bic adsorbents, immobilized metal affinity chromatography, US 7,393,534 B2 27 28 affinity chromatography), or separation in Solution (e.g., gel the end of the size exclusion chromatography step. In specific filtration, electrophoresis, liquid phase partitioning, detergent embodiments (e.g., when Small intestine extracts are used, partitioning, organic Solvent extraction, and ultrafiltration). bacterial endotoxin is always a contaminant), the endotoxin See Scopes, PROTEIN PURIFICATION, PRINCIPLES levels are monitored with the LAL assay (see Section 5.3. AND PRACTICE, 3' ed., Springer (1994), the entire text is infra) at each step in the purification. Endotoxin levels are incorporated herein by reference. During the purification, the reduced approximately 1000-fold resulting in final levels that immune-stimulatory activity and/or the anti-tumor activity of are well below those that can interfere with the DC assay the ARP should be monitored by one or more in vitro or in (threshold of 1000 U/mL for minimal DC activity. See Sec vivo assays as described in Section 5.6 and Section 12. The tion 5.6 and Section 12 for detailed description of the DC present invention also encompasses any modification to the 10 assay). methods of isolation of ARP that affects ARP activity and/or In some embodiments, to obtain enough material for final aggregation state through regulation of binding of divalent purification, pools are created that contained the extracts from cations. The purity of ARP can be assayed by any methods multiple samples (e.g., animal intestines). Preferably, these known in the art, such as but not limited to, gel electrophore are pre-selected for high activity in the crude Supernatants sis. See Scopes, Supra. 15 (for an example of such pretesting of the activity, see Section 6.2.1). Several batches of intestinal or cell preparations are Purification of Soluble ARPs from Tissue and Cell Extracts combined, concentrated, cleared of calcium binding proteins In one embodiment, an ARP is purified from Apicomplexa (by an HIC column, see Section 6.2.2., infra) and designated infected tissue of a vertebrate animal (the animal may have as a pool. symptomatic infection or non-symptomatic infection), To obtain a purified product, a pool is initially separated on including but not limited to, a cow, a sheep, a pig, a goat, a a high performance liquid chromatography (hereinafter horse, a dog, a cat, a rodent, an avian, a primate (e.g., mon “HPLC) HIC column. The active fractions (wherein said key), a mammal, and a human. In another embodiment, an active fractions have the ability to stimulate interleukin-12 ARP is purified from a cell culture infected with an Apicom release from dendritic cells, said dendritic cells having been plexan organism. In yet anther embodiment, an ARP is puri 25 isolated from a mammal within 10 hours of assaying said fied from a preparation of an Apicomplexan organism. A ability (or as assayed by one or more assays described in procedure that can be used to purify ARPs from infected Section 5.6 and Section 12, infra)) are dialyzed to remove tissues, a cell culture, or a preparation of Apicomplexan ammonium sulfate and are applied to an HPLC DEAE col organism, is presented by way of example but not limitation umn in 0.15M NaCl (see Section 6.2.2., infra). A NaCl gra in Section 6, infra. 30 dient is applied and the activity (immunostimulatory activity, Briefly, by way of example but not limitation, a preparation as assayed by one or more assays described in Section 5.6 and of infected tissue or cells (e.g., a homogenized small intestine Section 12, infra) eluted with a major peak near 0.3M ionic tissue or a preparation of the cells of intestinal mucosa) is strength. Preferably, DEAE runs are used to process the active centrifuged. The resulting liquid Supernatants are brought to material from the HIC. In specific embodiments, the pooled 45% saturation with solid ammonium sulfate, centrifuged 35 active region is applied to a reverse phase HPLC column(e.g., and the pellet is discarded. The Supernatants are brought to C8 reverse phase HPLC column, see Section 6.2.2., infra)and 80% saturation using Solid ammonium sulfate, centrifuged eluted with an aqueous-acetonitile gradient. The activity (im again, and the Supernatant is discarded. The pellets are dis munostimulatory activity, as assayed by one or more assays solved in 1.5 M Ammonium Sulfate/50 mM Sodium Phos described in Section 5.6 and Section 12) eluted near 40% phate (pH 6.8) after resuspending completely within the 40 acetonitrile on a slow, shallow gradient elution protocol. The bottles or tubes. Non-dissolved material is removed by cen question of purity of these active fractions is addressed by trifugation. plotting activity versus absorbance along a finely-sampled In a preferred embodiment, to reduce the macromolecular separation. A reasonably robustlinear regression line and the load prior to size-exclusion chromatography, two bulk pro intersection near Zero indicate the lack of contaminating mol cessing steps are used. The first step uses a hydrophobic 45 ecules. interaction column (hereinafter “HIC column. See Section The reverse phase HPLC column purification step dena 6.2.2., infra). A large amount of inactive material, particularly tures most of the protein. In a preferred embodiment, the polynucleic acid, is removed in the flow-through and wash. eluted protein from a reverse phase HPLC column is rena The active (immunostimulatory) material (wherein said tured by a method known in the art, preferably by the method active fractions have the ability to stimulate interleukin-12 50 described in Section 13. In another embodiment, reverse release from dendritic cells, said dendritic cells having been phase HPLC column is not used to purify an ARP of the isolated from a mammal within 10 hours of assaying said invention. ability (or as assayed by one or more assays described in In some embodiments, a soluble ARP can be isolated by a Section 5.6 and Section 12, infra)) is eluted after a change to process comprising the steps of: (1) producing a solution with a buffer free of ammonium sulfate. The eluted material is 55 enriched ARP, e.g., an extract of cells expressing a recombi prepared for DEAE chromatography by dialysis against 4 L nant ARP protein; (2) centrifuging the Solution to obtain a of 0.2M NaCl/10 mM Sodium Phosphate (pH7.4) at 4° C. Supernatant; (3) fractionating the Supernatant by ammonium with buffer exchanges. The elution is processed in a similar Sulfate to precipitate proteins, centrifuging the precipitate to fashion on DEAE ion exchange chromatography (see Section obtain a pellet, and resuspending the pellet into a solution; (4) 6.2.2., infra) where the flow-through and wash are discarded. 60 applying the Solution to a low pressure HIC column and The elution at higher NaCl concentration is retained, concen collecting active (immunostimulatory) fractions (wherein trated on a membrane pressure cell, and added to a size said active fractions have the ability to stimulate interleukin exclusion column (e.g., a SuperdexTM column). Specific 12 release from dendritic cells, said dendritic cells having activity (with respect to protein content, as assayed by one or been isolated from a mammal within 10 hours of assaying more assays described in Section 5.6 and Section 12) is deter 65 said ability (or as assayed by one or more assays described in mined on the retained fractions from each step in the purifi Section 5.6 and Section 12, infra)); (5) dialyzing the active cation. Approximately 7000-fold purification is reached at fractions; (6) applying the dialyzed active fractions to HPLC US 7,393,534 B2 29 30 DEAE column and collecting one or more active (immuno stimulatory) fractions (wherein said active fractions have the TABLE 1-continued ability to stimulate interleukin-12 release from dendritic cells, said dendritic cells having been isolated from a mam Apicomplexa mal within 10 hours of assaying said ability (or as assayed by Eineria Catronensis one or more assays described in Section 5.6 and Section 12, Eimeria chaetodipi Eimeria choiboiari infra)); and (7) applying the active fractions to a size exclu Eimeria crandais sion column and collect eluted active (immunostimulatory) Eimeria dipodomysis fractions (wherein said active fractions have the ability to Eimeria falciformis stimulate interleukin-12 release from dendritic cells, said 10 Eimeria faurei Eimeria langebarteli dendritic cells having been isolated from a mammal within 10 Eimeria leucopi hours of assaying said ability (or as assayed by one or more Eineria maxima assays described in Section 5.6 and Section 12, infra)) (see Eimeria meleagrimitis Section 10.2, infra). Eineria mitis 15 Eineria nivai In a specific embodiment, a soluble ARP is isolated by a Eineria necatrix process comprising the steps of: (1) producing mineral media Eimeria nieschulzi spent by bacteria expressing a recombinant ARP; (2) apply Eimeria onychomysis ing the media to a DEAE column; (3) eluting material bound Eimeria ovinoidais Eimeria papillata to the DEAE column with buffer to produce an eluted frac Eimeria peromysci tion; (4) applying the eluted fraction to a size exclusion col Eimeria pillarensis umn; and (5) collecting the fractions eluted from the size Eimeria polita exclusion column. Eimeria porci Eimeria praecox In another embodiment, an ARP is purified from an Api Eimeria reedi complexan protozoan, 4 including but is not limited to, those Eimeria rioarribaensis that are listed in Table 1. FIG. 1 also provides phylogenetic 25 Eimeria scabra information on some of the Apicomplexan protozoa. A non Eimeria scholtysecki limiting example of the purification process is given in Sec Eimeria separata Eimeria Sevilleiensis tion 8. Eimeria Stiedae Eimeria teleki TABLE 1. 30 Eimeria tenelia Eimeria tropidura Apicomplexa Eimeria weybridgensis Goussia Coccidia Goussiajanae Eimeriida Isospora Cryptosporidiidae 35 Isospora belli Cryptosporidium andersoni Isosporafeis Cryptosporidium baileyi Isospora gryphoni Cryptosporidium canis Isospora insularitis Cryptosporidium felis Isospora Ohioensis Cryptosporidium galli Isospora peromysis Cryptosporidium meieagridis Isospora robini Cryptosporidium nitris 40 Isospora Stiis Cryptosporidium parvin Sarcocystidae Cryptosporidium satirophilum Toxoplasma Cryptosporidium serpentis Eucoccidiorida Cryptosporidium wrairi Adeleorina Cryptosporidium sp. Lankesterellidae unclassified Cryptosporidium 45 Lankestereiia Eimeriidae Colpodellidae Caryospora Colpodeila Caryospora bigenetica Colpodeila pontica Caryospora sp. Colpodella sp. ATCC50594 Choleoeineria Gregarinia Choleoeimeria sp. 50 Eugregarinida Cyclospora Gregarinidae Cyclospora Cayetanensis Lecludinidae Cyclospora Cercopitheci Leidyanidae Cyclospora colobi Monocystidae Cyclospora papionis Ophryocystidae Cyclospora sp. 55 Pseudomonocystis Cyclospora sp. strain Gombe 22 Pseudomonocystis lepidiota Cyclospora sp. strain Gombe 34 Haemosporida (haemosporidians) Cyclospora sp. strain Gombe 40 Haemoproteus Eineria Haemoproteiischelodinae Eimeria acervuina Haemoproteus columbae Eimeria adeneodei Haemoproteus kopiki Eimeria ahsata 60 Haemoproteus majoris Eimeria alabamensis Haemoproteus piyodactyli Eimeria albigulae Haemoproteus Sylvae Eineria anirozoi Haemoproteus sp. Eineria arizonensis unclassified Haemoproteus Eimeria aubernensis Hepatocystis Eimeria bovis 65 Hepatocystis sp. Eimeria brunetti Leucocytozoon US 7,393,534 B2 31 32 In one embodiment, ARP is purified from the class coccid TABLE 1-continued ian. In another embodiment, ARP is purified from the family Eimeriidae. In yet another embodiment, ARP is purified from Apicomplexa the genus Eimeria. Leucocytozoon dutbretti Briefly, by way of example but not limitation, ARP can be Leucocytozoon Simondi purified from E. tenella (see Section 8) as follows: sporulated Piasmodium oocysts are suspended in PBS and centrifuged. The superna Plasmodium agamae Plasmodium atheruri tant is removed and retained. The pellet is suspended in 30 Plasmodium azurophilum mM Hepes and homogenized on ice. This processing will Plasmodium berghei 10 result in most oocysts to be broken, releasing sporocysts that Plasmodium brasianum Plasmodium chaibaudi remain largely intact. The homogenate is transferred to a Plasmodium chiricahuae microcentrifuge tube and centrifuged. Plasmodium cuculus In a second preparation, the homogenate is Sonicated with Plasmodium cynomolgi care taken to keep the homogenate cooled during the Sonica Plasmodium elongatum 15 Plasmodium fairchildi tion. Approximately 80% of the released sporocysts are dis Plasmodium falcipartin (malaria parasite Pfalcipartin) rupted. The Suspensions are pooled and microcentrifuged. Plasmodium fieldi The pellet is retained and stored at 4°C. for future processing. Plasmodium floridense The starting material for the 3rd extract of sporulated Plasmodium fragile Plasmodium gallinaceum oocysts is the residual pellet of the 2nd extract preparation. Plasmodium giganteum The pellet material is suspended in Hepes and transferred to a Plasmodium gonderi beadbeater tube containing glass beads. The tube is then Plasmoditingutanggong completely filled with Hepes and processed in a Mini-Bead Plasmodium heteronucleare Plasmodium hylobati beater with care taken to prevent excessive heating. The Sus Plasmodium inii pension is microcentrifuged briefly and the Supernatant is Plasmodium iuxtanticleare 25 separated from the glass beads. This Supernatant is microcen Plasmodium knowiesi trifuged and is filter-sterilized and used for purification of Plasmodium lophurae Plasmodium maiariae ARP by a reverse phase HPLC chromatography (e.g., C8 Plasmodium cf. maiariae reverse phase HPLC chromatography). The pellet can be used Plasmodium mexicanum for purification of the membrane-linked form of ARP. Plasmodium nucleophilum 30 Plasmodium ovale (malaria parasite P ovale) Purification of Membrane-Linked ARPs Plasmodium reichenowi Plasmodium reicium In a specific embodiment, the therapeutic or prophylactic Plasmodium rouxi compositions of the invention comprise a protein-containing Plasmodium simiovaie membrane preparation of Apicomplexan protozoa (see sec Plasmodium simium 35 tion 9). While not bound by any theory, the ARP is believed to Plasmodium vinckei exist in two forms: a soluble form and a membrane-linked Plasmodium vivax (malaria parasite P vivax) Plasmodium yoeli form. In a specific embodiment, an ARP of the invention is Plasmodium sp. linked to a cell membrane through a glycosylphosphatidyli unclassified Piasmodium nositol (GPI) anchor (for description of protozoan-derived unidentified Haemosporida Piroplasmida (Piroplasmids) 40 GPI and its receptor (Toll-like receptor 2) see, e.g., Almeida et Babesiidae al., J. of Leukocyte Biology, 2001, 70:467477; Campos et al., Babesia the J. of Immunology, 2001, 167: 416423: Ropert et al., Theileriidae Current Opinion in Microbiology, 2000, 3: 395-403). When Cytauxzoon Theileria an Apicomplexa organism infects a host, Such as a cow, Theileria-related sp. 45 soluble ARP may also originate from the membranes of the unclassified Piroplasmida Apicomplexa organism due to cleavage by endogenous phos Piroplasmida gen. sp. BH1 pholipase C or D present in the host’s blood. An ARP can be Piroplasmida gen. sp. BH3 Piroplasmida gen. Sp. CA1 extracted from a membrane by a non-enzymatic or a enzy Piroplasmida gen. Sp. CA3 matic method. In a specific embodiment, an ARP is extracted Piroplasmida gen. Sp. CA4 50 from a membrane by a detergent. In another specific embodi Piroplasmida gen. sp. FD1 ment, an ARP is released from a membrane by an enzymatic Piroplasmida gen. sp. MD1 Piroplasmida gen. sp. WA1 method, e.g., using internal or external phospholipase or a Piroplasmida gen. sp. WA2 protease. Co-sedimenting large ARP aggregates (if any) are unclassified Apicomplexa dissolved simultaneously if the incubation buffer is free of Tridacna hemolymph apicomplexan 55 divalent cations. Apicomplex.asp. 72141 In certain embodiments, a membrane-bound ARP is iso unidentified symbiont Type N lated by a process comprising the steps of: (1) producing a For more information, see e.g., the website at ncbi.nlm.nih.gov Taxonomy homogenate of a purified Apicomplexan organism, cells taxonomy home.htmlindex.cgi, Plasmodium falcipartin, Apicomplexa; the infected with an Apicomplexan organism, or cells expressing website at plasmodb.org; or the website at biology.unm.edu/biology coc 60 a recombinant ARP; (2) centrifuging the homogenate to cidia. obtain a pellet; (3) Suspending the pellet in a solution and A purified Apicomplexan organism can be obtained by any treating the solution to release the ARP by an enzymatic method known in the art. See e.g., Tomley, Methods: A Com method (e.g., using a phospholipase); (4) centrifuging the panion to Methods in Enzymology 13: 171-176 (1997); solution to obtain a supernatant highly enriched in ARP; (5) Ryley et al., Parasitology, 73:311-362 (1976); U.S. Pat. Nos. 65 applying the Supernatant to a reverse phase HPLC column; 6,001,363, 6,100,241, 5,792,644, 6,451,984 and 6,008,342. and (6) eluting bound protein from the reverse phase HPLC For a detailed example, also see Section 8. column. In specific embodiments, the eluted bound protein of US 7,393,534 B2 33 34 step (6) is renatured by any technique known in the art. The An expression construct, as used herein, refers to a nucle immunostimulatory activity of an ARP is monitored by one or otide sequence encoding ARP or a fragment thereof operably more assays described in Section 5.6 and Section 12 during associated with one or more regulatory regions which enable the isolation process. In a specific embodiment, a membrane expression of ARP in an appropriate host cell. “Operably bound ARP is prepared by the following method: associated refers to an association in which the regulatory Briefly, by way of example but not limitation, crude mem regions and the ARP sequence to be expressed are joined and brane preparation from the sporozoites of E. tenella can be positioned in Such a way as to permit transcription, and ulti prepared as follows: a Suspension of oocysts in homogeniza mately, translation. tion buffer (hereinafter “HB) (50 mM Tris pH 7.4, 1 mM The regulatory regions necessary for transcription of the PMSF, 50 ul/ml of protease inhibitors cocktail) is sonicated 10 ARP can be provided by the expression vector. A translation on ice followed by freeze-thaw cycles (liquid nitrogen/room initiation codon (ATG) may also be provided if the ARP gene temperature). This procedure results in almost complete sequence lacking its cognate initiation codon is to be breakage of oocysts, sporocysts and sporozoites. 10 mM expressed. In a compatible host-construct system, cellular ZnCl, can be added to inhibit the release of an ARP from transcriptional factors, such as RNA polymerase, will bind to membranes by internal phospholipase and/or facilitate ARP 15 the regulatory regions on the expression construct to effect aggregation to increase the yield. transcription of the modified ARP sequence in the host organ The cell homogenate is then centrifuged to pellet mem ism. The precise nature of the regulatory regions needed for branes. This centrifugation regime also pellets nuclei, other gene expression may vary from host cell to host cell. Gener organelles, pelletable ARP aggregates and storage granules, if ally, a promoter is required which is capable of binding RNA any. Organelles can be disrupted in further purification cycles polymerase and promoting the transcription of an operably in HB without ZnCl. associated nucleic acid sequence. Such regulatory regions Pellet is suspended in 1 ml of HB and centrifuged again. may include those 5' non-coding sequences involved with Centrifugation/resuspension is repeated once more with and initiation of transcription and translation, Such as the TATA 2-3 times more without ZnCl2 in the suspension buffer. Super box, capping sequence, CAAT sequence, and the like. The natants from centrifugation cycles done without ZnCl2 con 25 non-coding region 3' to the coding sequence may contain tain significant amounts of ARP. transcriptional termination regulatory sequences, such aster The final pellet is suspended in 100 ul of HB with or minators and polyadenylation sites. without protease inhibitors and incubated at 37°C. 1-4 hrs to In order to attach DNA sequences with regulatory func release additional quantities of ARP. Phospholipase C at 16 tions, such as promoters, to the ARP gene sequence or to U/ml facilitates the ARP release. The ARP released into solu 30 insert the ARP gene sequence into the cloning site of a vector, tion can be separated from the insoluble material by centrifu linkers or adapters providing the appropriate compatible gation, followed by two washing cycles. All three superna restriction sites may be ligated to the ends of the cDNAs by tants are combined. They contain ARP with small amount of techniques well known in the art (Wu et al., 1987, Methods in contaminants which can be removed by a single separation by Enzymol. 152: 343-349). Cleavage with a restriction enzyme reverse phase chromatography (e.g., C8 reverse phase chro 35 can be followed by modification to create blunt ends by matography). digesting back or filling in single-stranded DNA termini Antibody-Affinity Purification of ARP before ligation. Alternatively, a desired restriction enzyme In another embodiment, antibody is used to purify an ARP site can be introduced into a fragment of DNA by amplifica or a fragment thereof. Any ARP preparations, e.g., an Api tion of the DNA by use of PCR with primers containing the complexa infected tissue extract, cell culture, or a protein 40 desired restriction enzyme site. containing membrane preparation, can be used in antibody An expression construct comprising an ARP sequence affinity purification of ARP. Antibodies can also be used in operably associated with regulatory regions can be directly purifying a recombinant ARP product or a synthesized ARP. introduced into appropriate host cells for expression and pro Antibodies can be monoclonal or polyclonal, and antibodies duction of ARP without further cloning. See, e.g., U.S. Pat. 45 No. 5,580,859. The expression constructs can also contain can be raised against the ARP or against a fragment of ARP DNA sequences that facilitate integration of the ARP (e.g., comprising a hydrophilic portion). In a specific embodi sequence into the genome of the host cell, e.g., via homolo ment, an antibody that binds to SEQID NO:26 (bovine ARP gous recombination. In this instance, it is not necessary to fragment 1 (SEQID NO:3) fused to bovine ARP fragment 2 employ an expression vector comprising a replication origin (SEQ ID NO:4) fused to bovine ARP fragment 3 (SEQ ID NO:5) fused to bovine ARP fragment 4 (SEQ ID NO:6) and 50 Suitable for appropriate host cells in order to propagate and fused to bovine ARP fragment 5 (SEQID NO:7)) is used to express ARP in the host cells. purify an ARP or a fragment thereof. When ARP is engi A variety of expression vectors may be used including, but neered to express as a fusion protein, antibodies raised against not limited to, plasmids, cosmids, phage, phagemids or modi a portion of the fused protein that is not ARP can be used to fied viruses. Such host-expression systems represent vehicles purify the ARP. Antibodies can be produced by any method 55 by which the coding sequences of interest may be produced known in the art or as described in Section 5.4., infra. and Subsequently purified, but also represent cells which may, Methods of using antibodies to purify a protein are stan when transformed or transfected with the appropriate nucle dard and well known in the art, and are not described in detail otide coding sequences, express ARP in situ. These include, here. See, e.g., Scopes, PROTEIN PURIFICATION, PRIN but are not limited to, microorganisms such as bacteria (e.g., 60 E. coli and B. subtilis) transformed with recombinant bacte CIPLES AND PRACTICE, 3' ed., Springer (1994), at pp riophage DNA, plasmid DNA or cosmid DNA expression 187-236, the entire text is incorporated herein by reference. vectors containing ARP coding sequences; yeast (e.g., Sac Recombinant Expression of ARP charomyces, Pichia) transformed with recombinant yeast Methods known in the art can be utilized to recombinantly expression vectors containing ARP coding sequences; insect produce ARP. A nucleic acid sequence encoding ARP can be 65 cell systems infected with recombinant virus expression vec inserted into an expression vector for propagation and expres tors (e.g., baculovirus) containing ARP coding sequences; sion in host cells. plant cell Systems infected with recombinant virus expression US 7,393,534 B2 35 36 vectors (e.g., cauliflower mosaic virus, CaMV; tobacco e.g., Logan & Shenk, 1984, Proc. Natl. Acad. Sci. USA 81: mosaic virus, TMV) or transformed with recombinant plas 355-359). Specific initiation signals may also be required for mid expression vectors (e.g., Ti plasmid) containing ARP efficient translation of inserted ARP coding sequences. These coding sequences; or mammalian cell systems (e.g., COS, signals include the ATG initiation codon and adjacent CHO, BHK,293, NS0, and 3T3 cells) harboring recombinant sequences. Furthermore, the initiation codon must be in phase expression constructs containing promoters derived from the with the reading frame of the desired coding sequence to genome of mammalian cells (e.g., metallothionein promoter) ensure translation of the entire insert. These exogenous trans or from mammalian viruses (e.g., the adenovirus late pro lational control signals and initiation codons can be of a moter; the vaccinia virus 7.5K promoter). Preferably, bacte variety of origins, both natural and synthetic. The efficiency rial cells Such as Escherichia coli and eukaryotic cells, espe 10 of expression may be enhanced by the inclusion of appropri cially for the expression of whole recombinant ARP ate transcription enhancer elements, transcription termina molecule, are used for the expression of a recombinant ARP tors, etc. (see, e.g., Bittner et al., 1987, Methods in Enzymol. molecule. For example, mammalian cells Such as Chinese 153: 51-544). hamster ovary cells (CHO) can be used with a vector bearing In addition, a host cell strain may be chosen which modu promoter element from major intermediate early gene of 15 lates the expression of the inserted sequences, or modifies and cytomegalocirus for effective expression of ARPs (Foecking processes the gene product in the specific fashion desired. et al., 1986, Gene 45: 101; and Cockett et al., 1990, Bio/ Such modifications (e.g., glycosylation) and processing (e.g., Technology 8: 2). cleavage) of protein products may be important for the func In bacterial systems, a number of expression vectors may tion of the protein. Different host cells have characteristic and be advantageously selected depending upon the use intended specific mechanisms for the post-translational processing and for the ARP molecule being expressed. For example, when a modification of proteins and gene products. Appropriate cell large quantity of such an ARP is to be produced, for the lines or host systems can be chosen to ensure the correct generation of pharmaceutical compositions of an ARP mol modification and processing of the foreign protein expressed. ecule, vectors which direct the expression of high levels of To this end, eukaryotic host cells which possess the cellular fusion protein products that are readily purified may be desir 25 machinery for proper processing of the primary transcript and able. Such vectors include, but are not limited to, the E. coli post-translational modification of the gene product, e.g., gly expression vector pCR2.1 TOPO (Invitrogen), in which the cosylation and phosphorylation of the gene product, may be ARP coding sequence may be directly ligated from PCR used. Such mammalian host cells include, but are not limited reaction and may be placed inframe to the lac Zcoding region to, PC12, CHO, VERY, BHK, Hela, COS, MDCK, 293, 3T3, so that a fusion protein is produced; plN vectors (Inouye & 30 WI38, BT483, Hs578T, HTB2, BT20 and T47D, NS0 (a Inouye, 1985, Nucleic Acids Res. 13:3101-3109; Van Heeke murine myeloma cell line that does not endogenously pro & Schuster, 1989, J. Biol. Chem. 24: 5503–5509) and the like. duce any immunoglobulin chains), CRL7030 and HsS78Bst Series of vectors like pFLAG (Sigma), pMAL (NEB), and cells. Expression in a bacterial or yeast system can be used if pET (Novagen) may also be used to express the foreign post-translational modifications turn to be non-essential for polypeptides as fusion proteins with FLAG peptide, malE-, or 35 the activity of ARP. CBD-protein. These recombinant proteins may be directed For long term, high yield production of properly processed into periplasmic space for correct folding and maturation. ARP, stable expression in cells is preferred. Cell lines that The fused part can be used for affinity purification of the stably express ARP may be engineered by using a vector that expressed protein. Presence of cleavage sites for specific pro contains a selectable marker. By way of example but not tease like enterokinase allows to cleave off the APR. The 40 limitation, following the introduction of the expression con pGEX vectors may also be used to express foreign polypep structs, engineered cells may be allowed to grow for 1-2 days tides as fusion proteins with glutathione 5-transferase (GST). in an enriched media, and then are Switched to a selective In general. Such fusion proteins are soluble and can easily be media. The selectable marker in the expression construct purified from lysed cells by adsorption and binding to matrix confers resistance to the selection and optimally allows cells glutathione agarose beads followed by elution in the presence 45 to stably integrate the expression construct into their chromo offree glutathione. The pGEX vectors are designed to include Somes and to grow in culture and to be expanded into cell thrombin or factor Xa protease cleavage sites so that the lines. Such cells can be cultured for a long period of time cloned target gene product can be released from the GST while ARP is expressed continuously. moiety. A number of selection systems may be used, including but In an insect system, many vectors to express foreign genes 50 not limited to, antibiotic resistance (markers like Neo, which can be used, e.g., Autographa Californica nuclear polyhedro confers resistance to geneticine, or G-418 (Wu and Wu, 1991, sis virus (AcNPV) can be used as a vector to express foreign Biotherapy 3: 87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. genes. The virus grows in cells like Spodoptera frugiperda Toxicol. 32:573-596; Mulligan, 1993, Science 260:926-932: cells. The ARP coding sequence may be cloned individually and Morgan and Anderson, 1993, Ann. Rev. Biochem. 62: into non-essential regions (for example the polyhedrin gene) 55 191-217; May, 1993, TIBTECH 11 (5): 155-2 15); Zeo, for of the virus and placed under control of an AcNPV promoter resistance to Zeocin, Bsd, for resistance to blasticidin, etc.); (for example the polyhedrin promoter). antimetabolite resistance (markers like Dhfr, which confers In mammalian host cells, a number of viral-based expres resistance to methotrexate, Wigler et al., 1980, Natl. Acad. sion systems may be utilized. In cases where an adenovirus is Sci. USA 77: 357: O'Hare et al., 1981, Proc. Natl. Acad. Sci. used as an expression vector, the ARP coding sequence of 60 USA 78: 1527); gpt, which confers resistance to mycophe interest may be ligated to an adenovirus transcription/trans nolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. lation control complex, e.g., the late promoter and tripartite USA 78: 2072); and hygro, which confers resistance to leader sequence. This chimeric gene may then be inserted in hygromycin (Santerre et al., 1984, Gene 30: 147). In addition, the adenovirus genome by in vitro or in Vivo recombination. mutant cell lines including, but not limited to, tha-, hgprt- or Insertion in a non-essential region of the viral genome (e.g., 65 aprt-cells, can be used in combination with vectors bearing region E1 or E3) will result in a recombinant virus that is the corresponding genes for thymidine kinase, hypoxanthine, viable and capable of expressing ARP in infected hosts (see, guanine- or adenine phosphoribosyltransferase. Methods US 7,393,534 B2 37 38 commonly known in the art of recombinant DNA technology pET-Blue2 vector (Novagen)) in the inverse orientation. This may be routinely applied to select the desired recombinant plasmid is transformed into a bacterial host. The bacterial clone, and Such methods are described, for example, in strain harboring the pET-Blue2 plasmid has the ability to Ausubeletal. (eds.), Current Protocols in Molecular Biology, generate single strand (SS) phage DNA after infection with the John Wiley & Sons, NY (1993); Kriegler, Gene Transfer and helper phage R408. The positive strand of the insert will be Expression, A Laboratory Manual, Stockton Press, NY made during the phage formation. The positive strand with (1990); and in Chapters 12 and 13, Dracopoli et al. (eds), the inverse orientation would correspond to the negative Current Protocols in Human Genetics, John Wiley & Sons, strand of the original ARP gene cloned into the vector. This NY (1994); Colberre-Garapin et al., 1981, J. Mol. Biol. 150: ssDNA will be complimentary to a targeted ARP mRNA. The 1. 10 ssDNA can be biotinylated and added to the total Apicom The recombinant cells may be cultured under standard plexan protozoan mRNA preparation to allow hybridization conditions of temperature, incubation time, optical density of the two complimentary Strands. The complex can be iso and media composition. However, conditions for growth of lated by binding to streptavidin magnetic beads. After wash recombinant cells may be different from those for expression ing of the unbound mRNA, the bound targeted Apicomplexan of ARP. Modified culture conditions and media may also be 15 ARP mRNA will be eluted under low salt condition, while the used to enhance production of ARP. Any techniques known in biotinylated ssDNA will remain bound to the magnetic bead the art may be applied to establish the optimal conditions for through strong biotin-streptavidin binding. The resulting producing ARP. mRNA, which is specific for targeted Apicomplexan ARP gene, will be used for synthesizing first strand DNA, second Isolation of ARP Gene strand DNA, and cloned into protein expression vector for The gene encoding an ARP can be isolated by any tech DNA sequencing and protein expression. DNA sequence is nique known in the art. Specific embodiments for the isola confirmed by homology to an ARP gene. The recombinant tion of an ARP gene follow. For example, a protozoa of genus protein is expressed to test DC activation function. In a spe Apicomplexa can be used to isolate its total RNA. Once the cific embodiment, antitumor activity is confirmed by mouse total RNA of an Apicomplexaprotozoan is available, the ARP 25 teSt. gene can be isolated by methods known in the art, including In another embodiment, using the total mRNA isolated but are not limited to, screening of a cDNA library, affinity from an Apicomplexa protozoan, the first strand can be Syn purification of ARP mRNA, and polymerase chain reaction thesized by MMLV reverse transcriptase using oligo dT as (PCR). primer. The resulting sample will be used to perform poly In specific embodiments, an isolated ARP gene comprises 30 merase chain reaction (PCR) by using Taq polymerase and a nucleic acid molecule encoding SEQID NO: 1 (ARP of E. specific primers determined by an ARP gene sequence (e.g., tenella), SEQID NO:2 (ARP of E. acervulina), or SEQID SEQID NOs: 15, 16, 17, 18, 19, 20 or 27) or consensus of NOs: 3-7 (partial sequences of bovine Eimeria spp. ARP. ESTs from GenBank. PCR products can be analyzed by DNA SEQID NOS:3-7 are not necessarily contiguous, there may be agarose gel to determine the molecular weight as compared to intervening or adjacent sequences to each fragment. Prefer 35 molecular weight standard and the expected value from an ably, the nucleic acid molecule encodes a protein comprising ARP gene. PCR products can be introduced into cloning SEQ ID NOS:3, 4, 5, 6 and 7 in an order of SEQID NO:3 to vector (e.g., pCR 2.1 TOPO from Invitrogen) and the plas SEQ ID NO:7 from the N-terminus to the C-terminus). In mids with the correct insert can be selected on a basis of specific embodiments, an isolated ARP gene consists a analysis of their size, and PCR amplification pattern with nucleic acid molecule encoding SEQ ID NO:1, SEQ ID 40 primers specific to the ARP gene. Finally the gene can be NO:2, or SEQ ID NO:26 (SEQ ID NO:3 fused to SEQ ID re-cloned into an expression vector. The cloned gene can be NO:4fused to SEQID NO:5 fused to SEQID NO:6 and fused subjected to DNA sequence analysis for final confirmation of to SEQID NO:7). ARP sequence and precise determination of the ARP gene. In one embodiment, cDNA library is generated by using In another embodiment, polymerase chain reaction (PCR) any method known in the art (e.g., OrientExpress cloNA 45 is used to amplify the desired sequence in a genomic or cDNA Library Construction System (Novagen)). Total mRNA are library, prior to selection. Oligonucleotide primers represent used to synthesize the first strand, second strand, and ligated ing an ARP sequences can be used as primers in PCR. In a into lambda (W) bacteriophage arms. The constructed phage preferred aspect, the oligonucleotide primers represent at DNA can be packaged into bacteriophage by, e.g., Phage least part of conserved segments of strong homology between Maker Kit (Novagen). The bacteriophages containing differ 50 ARP genes of different species. The synthetic oligonucle ent DNA inserts are used to infect the bacterial host to gen otides may be utilized as primers to amplify sequences from erate the lambda bacteriophage cDNA library. Normally, the a source (RNA or DNA), preferably a cDNA library, of poten library will contain 10 to 10° primary clones. The phage tial interest. PCR can be carried out, e.g., by use of a Perkin library are amplified and plated out to e.g., about 10 clones Elmer Cetus thermal cycler and Taq polymerase (e.g., Gene per plate. The library can then be screened by membrane 55 AmpTM). The nucleic acid being amplified can include lifting technique, using an ARP gene probe (e.g., an E. tenella mRNA or cDNA or genomic DNA from any species. One ARP). Alternatively, the cDNA library can be screened by may synthesize degenerate primers for amplifying homologs induction of protein expression by isopropyl-1-thio-B-D-ga from other species in the PCR reactions. lactoside (IPTG). The membranes lifted from the plates con It is also possible to vary the stringency of hybridization tained the expressed proteins are screened by using antibody 60 conditions used in priming the PCR reactions, to allow for generated against an ARP (e.g., SEQ ID NO:1 or 2). The greater or lesser degrees of nucleotide sequence similarity identified clones are analyzed by DNA sequencing. The cor between the ARP nucleotide sequences and a nucleic acid rect DNA insert are subcloned into protein expression vector homolog being isolated. For cross species hybridization, low for protein expression. The recombinant protein can be tested stringency conditions are preferred. For same species hybrid for DC activating property. 65 ization, moderately stringent conditions are preferred. After In another embodiment, an ARP gene (e.g., SEQ ID Successful amplification of a segment of an ARP homolog, NO:15, 16, 17, 18, 19, 20 or 27) is cloned into a vector (e.g., that segment may be cloned and sequenced by standard tech US 7,393,534 B2 39 40 niques, and utilized as a probe to isolate a complete cDNA or istered to a Subject for treatment or prevention of cancer. Any genomic clone. This, in turn, permits the determination of the technique known in the art that is able to cause an Apicom gene's complete nucleotide sequence, the analysis of its plexan organism to have a defective life cycle can be used. expression, and the production of its protein product for func Such techniques include but not limited to, genetic engineer tional analysis, as described below. In this fashion, additional ing, use of chemical toxins, or radiation. Whetheran Apicom genes encoding ARPs and ARP analogs may be identified. plexan organism is having a defective life cycle can be tested Peptide Synthesis in animals by infection followed by biopsy to verify that the An alternative to producing ARP or a fragment thereof by organism has entered cells. An appropriate waiting period recombinant techniques is peptide synthesis. For example, an would indicate if the organism is unable to complete its life entire ARP, or a peptide corresponding to a portion of ARP 10 cycle and create oocysts in the feces. can be synthesized by use of a peptide synthesizer. Conven Apicomplexa have complex life cycles, and there is much tional peptide synthesis or other synthetic protocols well variation among different apicomplexa groups. Both asexual known in the art may be used. and sexual reproduction are involved, although some apicom Peptides having the amino acid sequence of ARP or a portion thereof may be synthesized by solid-phase peptide 15 plexa skip one or the other stage. The basic life cycle may be synthesis using procedures similar to those described by Mer said to start when an infective stage, or sporozoite, enters a rifield, 1963, J. Am. Chem. Soc., 85: 2149. During synthesis, host cell, and then divides repeatedly to form numerous mero N-C-protected amino acids having protected side chains are Zoites. Some of the merozoites transform into sexually repro added stepwise to a growing polypeptide chain linked by its ductive cells, or gamonts. Gamonts join together in pairs and C-terminal and to an insoluble polymeric Support, i.e., poly form a gamontocyst. Within the gamontocyst, the gamonts styrene beads. The peptides are synthesized by linking an divide to form numerous gametes. Pairs of gametes then fuse amino group of an N-C-deprotected amino acid to an O-car to form Zygotes, which give rise by meiosis to new sporozoi boxyl group of an N-C-protected amino acid that has been tes, and the cycle begins again. Apicomplexans are transmit activated by reacting it with a reagent such as dicyclohexyl ted to new hosts in various ways; some, like the malaria carbodiimide. The attachment of a free amino group to the 25 parasite, are transmitted by infected mosquitoes, while others activated carboxyl leads to peptide bond formation. The most may be transmitted in the feces of an infected host, or when a commonly used N-C-protecting groups include Boc which is predator eats infected prey. acid labile and Fmoc which is base labile. Details of appro The class Coccidia tend to infect vertebrates through priate chemistries, resins, protecting groups, protected amino 30 mucosal Surfaces, such as the gut. The family Eimeriidae, acids and reagents are well known in the art and so are not within the order Eimerida, within the Coccidia, is character discussed in detail herein (See, Atherton et al., 1989, Solid ized by a life cycle that requires only one host and a brief Phase Peptide Synthesis: A Practical Approach, IRL Press, and Bodanszky, 1993, Peptide Chemistry, A Practical Text passage through the environment in the oocyst stage. The book, 2nd Ed., Springer-Verlag). genus Eimeria is characterized by sporozoite invasion, Purification of the resulting ARP or a fragment thereof is 35 merogony, gamogony and oocyst formation confined to a accomplished using conventional procedures, such as pre region of the intestine that is specific to each Eimeria species. parative HPLC using gel permeation, partition and/or ion The oocysts sporulate in the environment, are ingested, and exchange chromatography. The choice of appropriate matri the oocyst walls are broken by bile salts and trypsin in the ces and buffers are well known in the art and so are not duodenum. The sporozoites penetrate the epithelial cell walls described in detail herein. 40 where they protect themselves with a specialized membrane. Within this safe haven, they produce as many as 120,000 ARPs of Purified Apicomplexa merozoites as a Schizont. Upon maturation, these burst out In specific embodiments, the present invention provides a and invade other cells. This process is repeated two or three purified Apicomplexan organism that containing an ARP for times and is followed by a gamont stage in which sexual the treatment of cancer and infectious diseases, and for elic 45 iting an immune response. In a preferred embodiment, the reproduction occurs and oocysts are formed. These are purified Apicomplexan organism is also an inactivated Api excreted with the feces to complete the cycle. In accordance with the present invention, an Apicomplexan complexan organism or an Apicomplexan organism that can organism at any developmental stage, e.g., a merozoite, a not cause the disease in an intended host. A purified or an gamont, a gamontocyst, a gamete, a sporozoite, or an oocyst, inactivated Apicomplexan organism containing ARP can be 50 directly administered, preferably orally, to a subject for treat can be used to treat or prevent cancer oran infectious disease, ment or prevention of cancer. or to eliciting an immune response. In a preferred embodi Some genus of Apicomplexa, Such as Eimeria, is highly ment, a sporozoite, a merozoite or an oocyst of an Apicom species-specific. For example, an Eimeria that infect avian plexan organism is used to treat or prevent cancer or an species usually does not cause symptomatic infections in 55 infectious disease, or to elicit an immune response. More non-avian species. In one embodiment of the invention, an preferably, a sporozoite, a merozoite oran oocyst of an Eime Apicomplexan organism containing ARP is adminitered to a ria species is used to treat or prevent cancer or an infectious Subject that is not a natural host of the organism for the disease, or to eliciting an immune response. treatment and prevention of cancer. In another embodiment of the invention, a dead Apicom In a preferred embodiment, Eimeria sporulated oocysts 60 plexan organism containing ARP is administered to a subject containing ARPs are administered to a subject that is not a for treatment or prevention of cancer. An Apicomplexan natural host of the organism for the treatment or prevention of organism can be killed by any method known in the art, CaCC. including but not limited to, chemical toxins and radiations. In another embodiment of the invention, an Apicomplexan In yet another embodiment, a protein fraction, preferably, a organism containing ARP is engineered to have a defective 65 membrane preparation or a storage granule preparation of an life cycle so that it cannot cause a symptomatic infection in a Apicomplexan organism containing ARP is administered to a host, and the engineered Apicomplexan organism is admin Subject for treatment or prevention of cancer. US 7,393,534 B2 41 42 Derivatives and Analogs of ARP or fragments thereof, or whose encoding nucleic acid is The invention further provides derivatives (including but capable of hybridizing to an ARP nucleic acid sequence. not limited to fragments), and analogs of ARPs. The produc The ARP derivatives and analogs of the invention can be tion and use of derivatives and analogs related to ARP are produced by various methods known in the art. The manipu lations which result in their production can occur at the gene within the scope of the present invention. or protein level. For example, the cloned ARP gene sequence In a specific embodiment, the derivative or analog is func can be modified by any of numerous strategies known in the tionally active, i.e., capable of exhibiting one or more func art (Maniatis, T., 1989, Molecular Cloning, A Laboratory tional activities associated with a full-length, wild-type ARP. Manual. 2d ed., Cold Spring Harbor Laboratory, Cold Spring Preferably, the derivative or analog is an activator of dendritic 10 Harbor, N.Y.). The sequence can be cleaved at appropriate cells as assayed by the DC assay (see section 5.6 and section sites with restriction endonuclease(s), followed by further 12.3). Derivatives or analogs of ARP can be tested for the enzymatic modification if desired, isolated, and ligated in desired activity by procedures known in the art, including but vitro. In the production of the gene encoding a derivative or not limited to the assays described in Section 5.5 and Section analog of ARP, care should be taken to ensure that the modi 12, infra. 15 fied gene remains within the same translational reading frame In particular, ARP derivatives can be made by altering ARP as ARP uninterrupted by translational stop signals, in the sequences by Substitutions, insertions or deletions that pro gene region where the desired ARP activity is encoded. vide for functionally equivalent molecules. Preferably, such Additionally, the ARP-encoding nucleic acid sequence can alteration of an ARP sequence is done in a variable region be mutated in vitro or in vivo, to create and/or destroy trans (i.e., that is not highly conserved among different ARP mol lation, initiation, and/or termination sequences, or to create ecules, for example, see FIG. 19), e.g. an alteration is done variations in coding regions and/or form new restriction within the first fifteen amino acids of an ARP. Due to the endonuclease sites or destroy preexisting ones, to facilitate degeneracy of nucleotide coding sequences, other DNA further in vitro modification. Any technique for mutagenesis sequences which encode Substantially the same amino acid known in the art can be used, including but not limited to, in sequence as an ARP gene may be used in the practice of the 25 vitro site-directed mutagenesis (Hutchinson, C. et al., 1978, J. present invention. These include, but are not limited to, nucle Biol. Chem. 253: 6551), use of TABTM linkers (Pharmacia), otide sequences comprising all or portions of ARP genes etc. which are altered by the substitution of different codons that Manipulations of the ARP sequence may also be made at encode a functionally equivalent amino acid residue within the protein level. Included within the scope of the invention the sequence, thus producing a silent change. Likewise, the 30 are ARP fragments or other derivatives or analogs which are ARP derivatives of the invention include, but are not limited differentially modified during or after translation, e.g., by to, those containing, as a primary amino acid sequence, all or glycosylation, acetylation, phosphorylation, amidation, part of the amino acid sequence of an ARP including altered derivatization by known protecting/blocking groups, pro sequences in which functionally equivalent amino acid resi teolytic cleavage, linkage to an antibody molecule or other dues are Substituted for residues within the sequence resulting 35 cellular ligand, etc. Any of numerous chemical modifications in a silent change (i.e., conservative Substitutions). For may be carried out by known techniques, including but not example, one or more amino acid residues within the limited to, reagents useful for protection or modification of sequence can be substituted by another amino acid of a simi free NH2-groups, free COOH-groups, OH-groups, side lar polarity which acts as a functional equivalent, resulting in groups of Trp-, Tyr-Phe-, His-, Arg-, or Lys-, specific chemi a silent alteration. Substitutes for an amino acid within the 40 cal cleavage by cyanogen bromide, hydroxylamine, BNPS sequence may be selected from other members of the class to Skatole, acid, or alkali hydrolysis; enzymatic cleavage by which the amino acid belongs. For example, the nonpolar trypsin, chymotrypsin, papain, V8 protease, NaBH; acetyla (hydrophobic) amino acids include alanine, leucine, isoleu tion, formylation, oxidation, reduction; metabolic synthesis cine, Valine, proline, phenylalanine, tryptophan and methion in the presence of tunicamycin; etc. ine. The polar neutral amino acids include glycine, serine, 45 In addition, analogs and derivatives of ARP can be chemi threonine, cysteine, tyrosine, asparagine, and glutamine. The cally synthesized. For example, a peptide corresponding to a positively charged (basic) amino acids include arginine, portion of an ARP which comprises the desired domain, or lysine and histidine. The negatively charged (acidic) amino which mediates the desired aggregation activity in vitro, or acids include aspartic acid and glutamic acid. ARP deriva binding to a receptor, can be synthesized by use of a peptide tives of the invention also include, but are not limited to, those 50 synthesizer. Furthermore, if desired, nonclassical amino containing, as a primary amino acid sequence, all or part of acids or chemical amino acid analogs can be introduced as a the amino acid sequence of an ARP including altered substitution or addition into the ARP sequence. Non-classical sequences in which amino acid residues are Substituted for amino acids include, but are not limited to, the D-isomers of residues with similar chemical properties (i.e., conservative the common amino acids, C.-amino isobutyric acid, 4-ami Substitutions). In specific embodiments, 1, 2, 3, 4, or 5 amino 55 nobutyric acid, hydroxyproline, sarcosine, citrulline, cysteic acids are substituted. In a specific embodiment, SEQID NO:1 acid, t-butylglycine, t-butylalanine, phenylglycine, cyclo amino acid residue 165 His can be changed to Tyr, and the hexylalanine, B-alanine, designer amino acids such as B-me new protein still has the same activity as SEQID NO: 1. In a thyl amino acids, CC.-methyl amino acids, and NC.-methyl specific embodiment, SEQID NO:1 amino acid residue 167 amino acids. Ser can be changed to Ala, and the new protein still has the 60 In a specific embodiment, the ARP derivative is a chimeric, same activity as SEQ ID NO: 1. In anther specific embodi or fusion, protein comprising an ARP or fragment thereof ment, SEQ ID NO: 1 amino acid residue 165 His can be fused via a peptide bond at its amino- and/or carboxy-termi changed to Tyr, and amino acid residue 167 Ser can be nus to a non-ARP amino acid sequence. In a preferred changed to Ala, the new protein still has the same activity as embodiment, the non-ARP amino acid sequence is fused at SEQID NO:1. 65 the amino-terminus of an ARP or a fragment thereof. In Derivatives or analogs of ARP include, but are not limited another preferred embodiment, the non-ARP amino acid to, those peptides which are substantially homologous to ARP sequence is fused at a variable region of an ARP or a fragment US 7,393,534 B2 43 44 thereof, e.g., the first 15 amino acids of an ARP gene. In one ID NO:3) fused to fragment 2 (SEQID NO:4) fused to frag embodiment, Such a chimeric protein is produced by recom ment 3 (SEQID NO:5) fused to fragment 4 (SEQ ID NO:6) binant expression of a nucleic acid encoding the protein and fused to fragment 5 (SEQ ID NO:7)) is used in immu (comprising an ARP-coding sequence joined in-frame to a noassays to measure the amount of an ARP or in immunoaf non-ARP coding sequence). Such a chimeric product can be finity purification of an ARP. custom made by a variety of companies (e.g., Retrogen, The antibodies that immunospecifically bind to an ARP or Operon, etc.) or made by ligating the appropriate nucleic acid an antigenic fragment thereof can be produced by any method sequences encoding the desired amino acid sequences to each known in the art for the synthesis of antibodies, in particular, other by methods known in the art, in the proper coding by chemical synthesis or preferably, by recombinant expres frame, and expressing the chimeric product by methods com 10 sion techniques. monly known in the art. Alternatively, such a chimeric prod Polyclonal antibodies immunospecific for ARP or an anti uct may be made by protein synthetic techniques, e.g., by use genic fragment thereof can be produced by various proce of a peptide synthesizer. In a specific embodiment, a chimeric dures well-known in the art. For example, an ARP can be nucleic acid encoding a mature ARP with a heterologous administered to various host animals including, but not lim signal sequence is expressed such that the chimeric protein is 15 ited to, rabbits, mice, and rats, to induce the production of sera expressed and processed by the cell to the mature ARP pro containing polyclonal antibodies specific for the ARP. Vari tein. The primary sequence of ARP and non-ARP gene may ous adjuvants may be used to increase the immunological also be used to predict tertiary structure of the molecules response, depending on the host species, including but are not using computer simulation (Hopp and Woods, 1981, Proc. limited to, Freund's (complete and incomplete), mineral gels Natl. Acad. Sci. U.S.A. 78:3824-3828); the chimeric recom Such as aluminum hydroxide, Surface active Substances Such binant genes could be designed in light of correlations as lysolecithin, pluronic polyols, polyanions, peptides, oil between tertiary structure and biological function. Likewise, emulsions, keyhole limpet hemocyanins, dinitrophenol, and chimeric genes comprising an essential portion of ARP mol potentially useful human adjuvants such as BCG (bacille ecule fused to a heterologous (non-ARP) protein-encoding Calmette-Guerin) and corynebacterium parvum. Such adju sequence may be constructed. In a specific embodiment. Such 25 vants are also well known in the art. chimeric construction can be used to enhance one or more Monoclonal antibodies can be prepared using a wide vari desired properties of an ARP, including but not limited to, ety of techniques known in the art including the use of hybri ARP stability, solubility, or resistance to proteases. In another doma, recombinant, and phage display technologies, or a embodiment, chimeric construction can be used to target ARP combination thereof. For example, monoclonal antibodies to a specific site, e.g., a chimeric construction comprising an 30 can be produced using hybridoma techniques including those ARP fused to an antibody to a specific type of cancer allows known in the art and taught, for example, in Harlow et al., ARP to be delivered to the cancer site. In yet another embodi Antibodies: A Laboratory Manual, (Cold Spring Harbor ment, chimeric construction can be used to identify or purify Laboratory Press, 2nd ed. 1988); Hammerling et al., in: an ARP of the invention, Such as a His-tag, a FLAG tag, a Monoclonal Antibodies and T Cell Hybridomas 563 681 green fluorescence protein (GFP), B-galactosidase, a maltose 35 (Elsevier, N.Y., 1981). The term “monoclonal antibody” as binding protein (MalE), a cellulose binding protein (CenA) or used herein is not limited to antibodies produced through a mannose protein, etc. hybridoma technology. The term “monoclonal antibody” In another specific embodiment, the ARP derivative is a refers to an antibody that is derived from a single clone, fragment of ARP of one specie of Apicomplexa comprising a including any eukaryotic, prokaryotic, or phage clone, and region of homology with an ARP of another specie of Api 40 not the method by which it is produced. complexa. As used herein, a region of a first protein shall be Methods for producing and Screening for specific antibod considered “homologous to a second protein when the ies using hybridoma technology are routine and well known amino acid sequence of the region is at least 25%, at least in the art. Briefly, mice can be immunized with a non-murine 30%, at least 45%, at least 55%, at least 65%, at least 75% antigen and once an immune response is detected, e.g., anti either identical or involving conservative changes, when 45 bodies specific for the antigen are detected in the mouse compared to any sequence in the second protein of an equal serum, the mouse spleen is harvested and splenocytes iso number of amino acids as the number contained in the region. lated. The splenocytes are then fused by well known tech In a specific embodiment, the invention provides ARP niques to any Suitable myeloma cells, for example cells from derivatives and analogs, in particular ARP fragments and cell line SP20 available from the ATCC. Hybridomas are derivatives of Such fragments that comprise one or more 50 selected and cloned by limited dilution. The hybridoma domains of the ARP. clones are then assayed by methods known in the art for cells In a specific embodiment, relating to an ARP of a species that secrete antibodies capable of binding a polypeptide of the other than Eimeria, preferably other species in the Apicom invention. Ascites fluid, which generally contains high levels plexa phylum, the fragments comprising specific portions of of antibodies, can be generated by immunizing mice with ARP are those comprising portions in the respective ARP 55 positive hybridoma clones. most homologous to specific fragments of an Eimeria ARP. The present invention provides methods of generating monoclonal antibodies as well as antibodies produced by the Antibodies to ARPs, Derivatives and Analogs method comprising culturing a hybridoma cell secreting an In various embodiments, monoclonal or polyclonal anti antibody of the invention wherein, preferably, the hybridoma bodies specific to ARP, or a domain of ARP can be used in 60 is generated by fusing splenocytes isolated from a mouse immunoassays to measure the amount of ARP or used in immunized with a non-murine antigen with myeloma cells immunoaffinity purification of an ARP. A Hopp & Woods and then screening the hybridomas resulting from the fusion hydrophilic analysis (see Hopp & Woods, Proc. Natl. Acad. for hybridoma clones that secrete an antibody able to bind to Sci. U.S.A. 78: 3824-3828 (1981) can be used to identify the antigen. hydrophilic regions of a protein, and to identify potential 65 Antibody fragments which recognize specific particular epitopes of an ARP. In a specific embodiment, an antibody epitopes may be generated by any technique known to those that binds to SEQID NO:26 (bovine ARP fragment 1 (SEQ of skill in the art. For example, Fab and F(ab')2 fragments of US 7,393,534 B2 45 46 the invention may be produced by proteolytic cleavage of that express full-length antibodies, e.g., IgG, using tech immunoglobulin molecules, using enzymes such as papain niques known to those of skill in the art. (to produce Fab fragments) or pepsin (to produce F(ab')2 For some uses, including in vivo use of antibodies in fragments). F(ab')2 fragments contain the variable region, the humans and in vitro detection assays, it may be preferable to light chain constant region and the CH1 domain of the heavy use humanized antibodies or chimeric antibodies. Human chain. Further, the antibodies of the present invention can also antibodies can be made by a variety of methods known in the be generated using various phage display methods known in art including phage display methods described above using the art. antibody libraries derived from human immunoglobulin sequences. See also U.S. Pat. Nos. 4,444,887 and 4,716,111; In phage display methods, functional antibody domains are 10 and International publication Nos. WO 98/46645, WO displayed on the Surface of phage particles which carry the 98/50433, WO 98/24893, WO98/16654, WO 96/34096, WO polynucleotide sequences encoding them. In particular, DNA 96/33735, and WO 91/10741. sequences encoding V and V, domains are amplified from A chimeric antibody is a molecule in which different por animal clNA libraries (e.g., human or murine cDNA librar tions of the antibody are derived from different immunoglo ies of affected tissues). The DNA encoding the V and V, 15 bulin molecules. Methods for producing chimericantibodies domains are recombined together with an scFv linker by PCR are known in the art. See e.g., Morrison, 1985, Science 229: and cloned into a phagemid vector. The vector is electropo 1202; Oi et al., 1986, BioTechniques 4: 214; Gillies et al., rated in E. coli and the E. coli is infected with helper phage. 1989, J. Immunol. Methods 125:191-202; and U.S. Pat. Nos. Phage used in these methods are typically filamentous phage 5,807,715, 4,816,567, 4,816,397, and 6,311415. including fl and M13 and the V and V, domains are usually A humanized antibody is an antibody or its variant or recombinantly fused to either the phage gene III or gene VIII. fragment thereof which is capable of binding to a predeter Phage expressing an antigen binding domain that binds to a mined antigen and which comprises a framework region hav particular antigen can be selected or identified with antigen, ing Substantially the amino acid sequence of a human immu e.g., using labeled antigen or antigenbound or captured to a noglobulin and a CDR having Substantially the amino acid Solid Surface or bead. Examples of phage display methods 25 sequence of a non-human immuoglobulin. A humanized anti that can be used to make the antibodies of the present inven body comprises Substantially all of at least one, and typically tion include those disclosed in Brinkman et al., 1995, J. two, variable domains (Fab, Fab', F(ab'), Fabc. Fv) in which Immunol. Methods 182: 41-50; Ames et al., 1995, J. Immu all or substantially all of the CDR regions correspond to those nol. Methods 184: 177-186: Kettleborough et al., 1994, Eur.J. of a non-human immunoglobulin (i.e., donor antibody) and Immunol. 24: 952-958: Persic et al., 1997, Gene 187: 9-18: 30 all or substantially all of the framework regions are those of a Burton et al., 1994, Advances in Immunology 57: 191-280; human immunoglobulin consensus sequence. Preferably, a International application No. PCT/GB91/O1 134; Interna humanized antibody also comprises at least a portion of an tional publication Nos. WO 90/02809, WO 91/10737, WO immunoglobulin constant region (Fc), typically that of a 92/01047, WO92/18619, WO 93/11236, WO95/15982, WO human immunoglobulin. Ordinarily, the antibody will con 95/20401, and WO97/13844; and U.S. Pat. Nos. 5,698,426, 35 tain both the light chain as well as at least the variable domain 5,223,409, 5,403.484, 5,580,717, 5,427,908, 5,750,753, of a heavy chain. The antibody also may include the CH1, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, hinge, CH2, CH3, and CH4 regions of the heavy chain. The 5,658,727, 5,733,743 and 5,969,108. humanized antibody can be selected from any class of immu As described in the above references, after phage selection, noglobulins, including IgM, IgG, Ig), IgA and IgE, and any the antibody coding regions from the phage can be isolated 40 isotype, including IgG1, IgG2, IgG3 and IgG4. Usually the and used to generate whole antibodies or any other desired constant domain is a complement fixing constant domain antigen binding fragment, and expressed in any desired host, where it is desired that the humanized antibody exhibit cyto including mammalian cells, insect cells, plant cells, yeast, toxic activity, and the class is typically IgG. Where Such and bacteria, e.g., as described below. Techniques to recom cytotoxic activity is not desirable, the constant domain may binantly produce Fab, Fab' and F(ab')2 fragments can also be 45 be of the IgG class. The humanized antibody may comprise employed using methods known in the art such as those sequences from more than one class or isotype, and selecting disclosed in PCT publication No. WO92/22324; Mullinax et particular constant domains to optimize desired effector func al., 1992, BioTechniques 12(6): 864-869; Sawai et al., 1995, tions is within the ordinary skill in the art. The framework and AJRI 34: 26-34; and Better et al., 1988, Science 240: 1041 CDR regions of a humanized antibody need not correspond 1043. 50 precisely to the parental sequences, e.g., the donor CDR or the To generate whole antibodies, PCR primers including V consensus framework may be mutagenized by Substitution, or V, nucleotide sequences, a restriction site, and a flanking insertion ordeletion of at least one residue so that the CDR or sequence to protect the restriction site can be used to amplify framework residue at that site does not correspond to either the V, or V, sequences in scFv clones. Utilizing cloning the consensus or the import antibody. Such mutations, how techniques known to those of skill in the art, the PCR ampli 55 ever, will not be extensive. Usually, at least 75% of the fied V. domains can be cloned into vectors expressing a V humanized antibody residues will correspond to those of the constant region, e.g., the human gamma 4 constant region, parental framework region (FR) and CDR sequences, more and the PCR amplified V, domains can be cloned into vectors often 90%, and most preferably greater than 95%. Human expressing a V, constant region, e.g., human kappa or lamba ized antibody can be produced using variety of techniques constant regions. Preferably, the vectors for expressing the 60 known in the art, including but not limited to, CDR-grafting V or V, domains comprise an EF-1C promoter, a secretion (European Patent No. EP239,400; International Publication signal, a cloning site for the variable domain, constant No. WO91/09967; and U.S. Pat. Nos. 5,225,539, 5,530,101, domains, and a selection marker such as neomycin. The V, and 5,585,089), veneering or resurfacing (European Patent and V, domains may also cloned into one vector expressing Nos. EP592,106 and EP 519,596; Padlan, 1991, Molecular the necessary constant regions. The heavy chain conversion 65 Immunology 28(4/5): 489-498: Studnicka et al., 1994, Pro vectors and light chain conversion vectors are then co-trans tein Engineering 7(6): 805-814; and Roguska et al., 1994, fected into cell lines to generate stable or transient cell lines PNAS 91: 969-973), chain shuffling (U.S. Pat. No. 5,565, US 7,393,534 B2 47 48 332), and techniques disclosed in, e.g., U.S. Pat. No. 6,407, sequence to identify, e.g., a cDNA clone from a cDNA library 213, U.S. Pat. No. 5,766,886, WO 93.17105, Tan et al., J. that encodes the antibody. Amplified nucleic acids generated Immunol. 169: 1119-25 (2002), Caldas et al., Protein Eng. by PCR may then be cloned into replicable cloning vectors 13(5): 353-60 (2000), Morea et al., Methods 20(3): 267-79 using any method well known in the art. (2000), Baca et al., J. Biol. Chem. 272(16): 10678-84 (1997), Once the nucleotide sequence of the antibody is deter Roguska et al. Protein Eng.9(10): 895-904 (1996), Couto et mined, the nucleotide sequence of the antibody may be al., Cancer Res. 55 (23 Supp): 5973s-5977s (1995), Couto et manipulated using methods well known in the art for the al., Cancer Res. 55 (8): 1717-22 (1995), Sandhu J S. Gene manipulation of nucleotide sequences, e.g., recombinant 150(2): 409-10 (1994), and Pedersen et al., J. Mol. Biol. DNA techniques, site directed mutagenesis, PCR, etc. (see, 235(3): 959-73 (1994). Often, framework residues in the 10 for example, the techniques described in Sambrook et al., framework regions will be substituted with the corresponding 1990, Molecular Cloning, A Laboratory Manual. 2d Ed., Cold residue from the CDR donor antibody to alter, preferably Spring Harbor Laboratory, Cold Spring Harbor, N.Y. and improve, antigenbinding. These framework Substitutions are Ausubel et al., eds., 1998, Current Protocols in Molecular identified by methods well known in the art, e.g., by modeling Biology, John Wiley & Sons, NY, which are both incorpo of the interactions of the CDR and framework residues to 15 rated by reference herein in their entireties), to generate anti identify framework residues important for antigen binding bodies having a differentamino acid sequence, for example to and sequence comparison to identify unusual framework resi create amino acid Substitutions, deletions, and/or insertions. dues at particular positions. (See, e.g., Queen et al., U.S. Pat. In a specific embodiment, one or more of the CDRs is No. 5,585,089; and Riechmannet al., 1988, Nature 332:323.) inserted within framework regions using routine recombinant Further, the antibodies that immunospecifically bind to DNA techniques. The framework regions may be naturally ARP or an antigenic fragment thereof can, in turn, be utilized occurring or consensus framework regions, and preferably to generate anti-idiotype antibodies that “mimic' ARP or an human framework regions (see, e.g., Chothia et al., 1998, J. antigenic peptide thereof using techniques well-known to Mol. Biol. 278: 457-479 for a listing of human framework those skilled in the art. (See, e.g., Greenspan & Bona, 1989, regions). Preferably, the polynucleotide generated by the FASEB J. 7(5): 437-444; and Nissinoff, 1991, J. immunol. 25 combination of the framework regions and CDRs encodes an 147(8): 2429-2438). antibody that specifically binds to a particular antigen. Pref Polynucleotide Sequences Encoding an Antibody erably, as discussed Supra, one or more amino acid substitu tions may be made within the framework regions, and, pref The invention provides polynucleotides comprising a erably, the amino acid substitutions improve binding of the nucleotide sequence encoding an antibody or fragment 30 antibody to its antigen. Additionally, Such methods may be thereofthat immunospecifically binds to ARP or an antigenic used to make amino acid Substitutions or deletions of one or fragment thereof. The invention also encompasses polynucle more variable region cysteine residues participating in an otides that hybridize under high Stringency, intermediate, or intrachain disulfide bond to generate antibody molecules lower stringency hybridization conditions to polynucleotides lacking one or more intrachain disulfide bonds. Other alter that encode an antibody of the invention. 35 ations to the polynucleotide are encompassed by the present The polynucleotides may be obtained, and the nucleotide invention and within the skill of the art. sequence of the polynucleotides determined, by any method known in the art. The nucleotide sequence of antibodies Recombinant Expression of an Antibody immunospecific for a desired antigen can be obtained, e.g., Recombinant expression of an antibody of the invention, from the literature or a database such as GenBank. Once the 40 derivative, analog or fragment thereof (e.g., a heavy or light amino acid sequences of an ARP or an antigenic fragment chain of an antibody of the invention or a portion thereof or a thereof is known, nucleotide sequences encoding this anti single chain antibody of the invention), requires construction body or a fragment thereof (e.g., a CDR) can be determined of an expression vector containing a polynucleotide that using methods well known in the art, i.e., nucleotide codons encodes the antibody. Once a polynucleotide encoding an known to encode particular amino acids are assembled in 45 antibody molecule or a heavy or light chain of an antibody, or Such a way to generate a nucleic acid that encodes the anti portion thereof (preferably, but not necessarily, containing the body. Such a polynucleotide encoding the antibody may be heavy or light chain variable domain), of the invention has assembled from chemically synthesized oligonucleotides been obtained, the vector for the production of the antibody (e.g., as described in Kutmeier et al., 1994, BioTechniques molecule may be produced by recombinant DNA technology 17:242), which, briefly, involves the synthesis of overlapping 50 using techniques well-known in the art. See, e.g., U.S. Pat. oligonucleotides containing portions of the sequence encod No. 6,331,415. Thus, methods for preparing a protein by ing the antibody, annealing and ligating of those oligonucle expressing a polynucleotide containing an antibody encoding otides, and then amplification of the ligated oligonucleotides nucleotide sequence are described herein. Methods which are by PCR. well known to those skilled in the art can be used to construct Alternatively, a polynucleotide encoding an antibody may 55 expression vectors containing antibody coding sequences and be generated from nucleic acid from a suitable source. If a appropriate transcriptional and translational control signals. clone containing a nucleic acid encoding a particular anti These methods include, for example, in vitro recombinant body is not available, but the sequence of the antibody mol DNA techniques, synthetic techniques, and in vivo genetic ecule is known, a nucleic acid encoding the immunoglobulin recombination. The invention, thus, provides replicable vec may be chemically synthesized or obtained from a suitable 60 tors comprising a nucleotide sequence encoding an antibody source (e.g., an antibody clNA library, or a cDNA library molecule of the invention, a heavy or light chain of an anti generated from, or nucleic acid, preferably poly A+ RNA, body, a heavy or light chain variable domain of an antibody or isolated from, any tissue or cells expressing the antibody, a portion thereof, or a heavy or light chain CDR, operably such as hybridoma cells selected to express an antibody of the linked to a promoter. Such vectors may include the nucleotide invention) by PCR amplification using synthetic primers 65 sequence encoding the constant region of the antibody mol hybridizable to the 3' and 5' ends of the sequence or by cloning ecule (see, e.g., International Publication No. WO 86/05807 using an oligonucleotide probe specific for the particular gene and WO 89/01036; and U.S. Pat. No. 5,122,464) and the US 7,393,534 B2 49 50 variable domain of the antibody may be cloned into such a (CDD v. 1.60) as determined by a RPS-BLAST (Reverse vector for expression of the entire heavy, the entire light Position-Specific BLAST) algorithm set to default param chain, or both the entire heavy and light chains. eters. The expression vector is transferred to a host cell by con Once an ARP is identified, it may be isolated and purified ventional techniques and the transfected cells are then cul 5 by standard methods including chromatography (e.g., ion tured by conventional techniques to produce an antibody of exchange, affinity, and sizing column chromatography), cen the invention. Thus, the invention includes host cells contain trifugation, differential solubility, or by any other standard ing a polynucleotide encoding an antibody of the invention or technique for the purification of proteins. The in vitro activi fragments thereof, or a heavy or light chain thereof, orportion ties and in vivo biological functions of the foregoing may be thereof, or a single chain antibody of the invention, operably 10 evaluated using any suitable assay (including immunoassays linked to a heterologous promoter. In preferred embodiments as described infra). for the expression of double-chained antibodies, vectors Alternatively, once an ARP produced by a recombinant encoding both the heavy and light chains may be co-ex host cell is identified, the amino acid sequence of the ARP(s) pressed in the host cell for expression of the entire immuno can be determined by standard techniques for protein globulin molecule, as detailed below. 15 sequencing, e.g., with an automated amino acid sequencer. A variety of host-expression vector systems may be uti The ARP sequence can be characterized by a hydrophilic lized to express the antibody molecules of the invention (see, ity analysis (Hopp, T. and Woods, K., 1981, Proc. Natl. Acad. e.g., U.S. Pat. No. 5,807,715, and those described in Section Sci. U.S.A. 78; 3.824). A hydrophilicity profile can be used to 5.1.2, supra). The expression levels of an antibody molecule identify the hydrophobic and hydrophilic regions of the ARP can be increased by vector amplification (for a review, see and the corresponding regions of the gene sequence which Bebbington and Hentschel, The use of vectors based on gene encode such regions. amplification for the expression of cloned genes in mamma Secondary structural analysis (Chou, P. and Fasman, G., lian cells in DNA cloning, Vol. 3. (Academic Press, New 1974, Biochemistry 13: 222) can also be done, to identify York, 1987)). When a marker in the vector system expressing regions of the ARP that assume specific secondary structures. antibody is amplifiable, increase in the level of inhibitor 25 Other methods of structural analysis can also be employed. present in culture of host cell will increase the number of These include, but are not limited to, X-ray crystallography copies of the marker gene. Since the amplified region is (Engstom, A., 1974, Biochem. Exp. Biol. 11: 7-13) and com associated with the antibody gene, production of the antibody puter modeling (Fletterick, R. and Zoller, M. (eds.), 1986, will also increase (Crouse et al., 1983, Mol. Cell. Biol. 3: Computer Graphics and Molecular Modeling, in Current 257). 30 Communications in Molecular Biology, Cold Spring Harbor The host cell may be co-transfected with two expression Laboratory, Cold Spring Harbor, N.Y.). vectors of the invention, the first vector encoding a heavy The functional activity of an ARP or afragment thereof can chain derived polypeptide and the second vector encoding a be assayed by various methods known in the art. light chain derived polypeptide. The two vectors may contain For example, where one is assaying for the ability of an identical selectable markers which enable equal expression of 35 ARP from one genus to bind or compete with an ARP from heavy and light chain polypeptides. Alternatively, a single another genus for binding to an antibody, various immunoas vector may be used which encodes, and is capable of express says known in the art can be used, including but not limited to, ing, both heavy and light chain polypeptides. In Such situa competitive and non-competitive assay systems using tech tions, the light chain should be placed before the heavy chain niques such as radioimmunoassays, ELISA (enzyme linked 40 immunosorbent assay), 'sandwich’ immunoassays, immu to avoid an excess of toxic free heavy chain (Proudfoot, 1986, noradiometric assays, gel diffusion precipiting reactions, Nature 322:52; and Kohler, 1980, Proc. Natl. Acad. Sci. USA immunodiffusion assays, in situ immunoassays (using colloi 77: 2 197). The coding sequences for the heavy and light dal gold, enzyme or radioisotope labels, for example), west chains may comprise cDNA or genomic DNA. ern blots, precipitation reactions, agglutination assays (e.g., Once an antibody molecule of the invention has been pro 45 gel agglutination assays, hemagglutination assays), comple duced by recombinant expression, it may be purified by any ment fixation assays, immunofluorescence assays, protein A method known in the art for purification of an immunoglobu assays, and immunoelectrophoresis assays, etc. Antibody lin molecule, for example, by chromatography (e.g., ion binding can be detected by detecting a label on the primary exchange, affinity, particularly by affinity for the specific antibody. Alternatively, the primary antibody is detected by antigen after Protein A, and sizing column chromatography), 50 detecting binding of a secondary antibody or reagent to the centrifugation, differential solubility, or by any other standard primary antibody, particularly where the secondary antibody technique for the purification of proteins. Further, the anti is labeled. Many means are known in the art for detecting bodies of the present invention or fragments thereof may be binding in an immunoassay and are within the scope of the fused to heterologous polypeptide sequences described present invention. herein or otherwise known in the art to facilitate purification. 55 The half life of a protein is a measurement of protein Structure Prediction and Functional Analysis of ARPs stability and indicates the time necessary for a one halfreduc The invention provides ARPs which can be from various tion in activity of the protein. The half-life of an ARP can be species of Apicomplexa. Thus, it would be valuable if the determined by any method measuring activity of ARP in structure of an ARP or a fragment thereof may be predicted samples from a Subject over a period of time. Such as but not based on the amino acid sequence. Structure prediction, 60 limited to, DC assay and NK assay (see Section 5.6 and analysis of crystallographic data, sequence alignment, as well Section 12). The normalization to concentration of ARP in the as homology modeling, can be accomplished using computer sample can be done by immunoassays using anti-ARP anti software programs available in the art, such as BLAST, bodies (or antibody to a fused part of a gene wherein an ARP CHARMm release 21.2 for the Convex, and QUANTA v. 3.3, is fused to another moiety) to measure the levels of the ARP (Molecular Simulations, Inc., York, United Kingdom). For 65 molecules in samples taken over a period of time after admin example, some ARPs comprise a conserved PROF (smart istration of the ARP, or detection of radiolabelled ARP mol 00392 or cd 00148) domain in Conserved Domain Database ecules in Samples taken from a subject after administration of US 7,393,534 B2 51 52 the radiolabeled ARP molecules. In specific embodiments, Briefly, by way of example but not limitation, a DC assay can techniques known in the art can be used to prolong the halflife be carried out as follows: freshly isolated dendritic cells of an ARP in vivo. For example, albumin or inert polymer (CD11c splenocytes, e.g., isolated from the spleens of molecules Such as high molecular weight polyethyleneglycol BALB/c mice within ten hours of assaying the activity) are (PEG) can be used. See, e.g., International Publication Nos. suspended at a density of 0.5x10 cells per mL in cytokine WO 93/15199, WO 93/15200, and WO 01/77137; and U.S. supplemented culture media (GM-CSF, 1.0 ng/ml. IL-4, 1.0 Pat. No. 6,528,485. ng/ml. IFNg, 3.0 ng/ml; anti mouse CD40, 0.5 mg/ml). The Other methods will be known to the skilled artisan and are dendritic cells are then added (100 uL/well) to 96-well tissue within the scope of the invention. culture plates containing ARP diluted in 100 uL media per 10 well and cultured overnight at 37° C. in 5% CO, 95% air. Characterization and Demonstration of ARP Activity Mouse IL-12 release from the dendritic cells (CD11c sple The immunostimulatory activity and/or anti-cancer activ nocytes) is then measured using ELISA. ity of the ARPs used in accordance with the present invention Compounds for use in therapy can be tested in Suitable can be determined by any method known in the art. In one animal model systems prior to testing in humans, including embodiment, the immunostimulating activity and/or anti 15 but not limited to in rats, mice, chicken, cows, monkeys, cancer activity of an ARP is determined by using various rabbits, etc. The principle animal models for cancer known in experimental animal models, including but not limited to, the art and widely used include, but not limited to, mice, as cancer animal models such as Scidmouse model or nude mice described in Hann et al., 2001, Curr Opin Cell Biol 2001 with human tumor grafts known in the art and described in December; 13(6): 778-84. Yamanaka, 2001, Microbiol Immunol 2001; 45(7): 507-14, In a preferred embodiment, The S-180 cell line (ATCC which is incorporated herein by reference. CCL 8, batch F4805) is chosen as the tumor model because Various in vitro and in vivo assays that test the immuno the same line is capable of growing both in animals and in stimulatory activities and/or anti-cancer activities of an ARP culture (in both serum-containing and serum-free condi are used in purification processes of an ARP. The protocols tions). Tumors are established in mice (BALB/c) by injection and compositions of the invention are also preferably tested in 25 of cell Suspensions obtained from tissue culture. Approxi vitro, and then in vivo, for the desired therapeutic or prophy mately 1x10° to 3x10° cells are injected intra-peritoneally per lactic activity, prior to use in humans. mouse. The tumor developed as multiple solid nodules at In one embodiment, a NK cell assay is used to assess multiple sites within the peritoneal cavity and cause death in ARP-induced cytotoxic activity of NK-like cells (see section most of the animals within 10 to 15 days. In addition to 12.2). Briefly, by way of example but not limitation, a NK cell 30 monitoring animal Survival, their condition is qualitatively assay can be carried out as follows: large granular lympho assessed as tumor growth progressed and used to generate a cytes (LGLs) are co-cultured with tumor cells at various tumor index as described in the following paragraph. ratios (effector to target ratio) for four days at 37°C., 5% CO. To establish an estimate of drug activity in tumor model The culture medium also contains a low concentration of IL2 experiments, an index can be developed that combines obser plus an ARP to be tested. The culture is terminated at the end 35 Vational examination of the animals as well as their Survival of day 4 and vitally stained using a MTT cell viability quan status. For example, mice are palpated once or twice weekly tification assay (see Mossman, T.J. Immunol. Methods 65:55 for the presence, establishment and terminal progression of (1983); and Sigma Chemical MTT (M5655) product appli the intraperitoneal S180 tumor. Tumor development and pro cation note). Culture media is aspirated and the remaining gression is assessed in these mice according to the following cells are washed and replaced with DMEM/F12 plus FCS, 40 scale: “0” no tumor palpated; “1” initial tumor appears to containing MTT. The cells are further incubated for 4-5 hours be present; Small in size (~ 1 mm); no distended abdomen; at 37° C. Absorbance is measured with the aid of a plate “2' tumor appears to be established; some distension of the reader. Decreased absorbance indicated a decrease in the abdomen; no apparent cachexia; '3' tumor is well estab number of viable cells per well (i.e., cytotoxicity). Absor lished, marked abdominal distension, animal exhibits bance is measured again after the MTT is solubilized by 45 cachexia; and, '4' animal is dead. The index value for a replacement of the medium with 2-propanol containing HCl, treatment group is the average of the individual mouse indices which gives a uniform color throughout the well. NK-induc in the group. ing activity is calculated relative to negative (PBS/BSA) and Further, any assays known to those skilled in the art can be positive (internal standard) controls. used to evaluate the prophylactic and/or therapeutic utility of In another embodiment, the ARP activity is measured by 50 IFNY assay (see section 12.4). By way of example but not the combinatorial therapies disclosed herein for treatment or limitation, the production and release of IFNY by LGLs into prevention of cancer and/or infectious diseases. the culture media is determined as follows: LGLS, enriched Uses of the Compositions of the Invention with NK cells, are isolated and seeded into 96 well plates at The compositions of the invention may be administered to densities of 2.5 or 5.0x10 cells/well, in 200 uL of DMEM/ 55 a subject in need thereof to stimulate an immune response, to F12 supplemented with 10% fetal calf serium, gentamycin prevent or treat a cancer or an infectious disease (including (50 ug/mL), and IL2 (125 U/mL). The ARP to be tested is one or more symptoms thereof), or to use as an alternative to added and the cells are cultured overnight, following which interleukin 12 (IL-12) treatment. The compositions of the the condition media (CM) is removed and then centrifuged to invention may be administered alone or in combination with remove any aspirated cells. Aliquots of the CM are then 60 one or more other therapies useful for immune stimulation to measured for IFNY using an commercially available ELISA a Subject in need thereof to elicit an immune response. The kit. compositions of the invention may administered alone or in In a preferred embodiment, dendritic cell (DC) assay is combination with one or more other therapies to a subject in used to follow interleukin-12 (IL-12) release from freshly need thereof to elevate systemic levels of IL-12 in the subject. isolated dendritic cells as an index of DC activation (see 65 The compositions of the invention may also be administered section 12.3). This activity is highly correlated with both alone or in combination with one or more other therapies NK-CMC in vitro and anti-tumor activity in vivo of an ARP. useful for the prevention or treatment of cancer (including, US 7,393,534 B2 53 54 but not limited to, the prophylactic ortherapeutic agents listed ARP. In specific embodiments, the Subject is an avian species. in Section 5.8 hereinbelow) or infectious diseases (including, In other specific embodiments, the Subject is not an avian but not limited to, the prophylactic ortherapeutic agents listed species. in Section 5.9 hereinbelow) to a subject in need thereof to prevent or treat a cancer or infectious diseases. In yet another embodiment, the present invention provides In one embodiment, the present invention provides a a method of elevating systemic level of interleukin-12 (IL-12) method of treating or preventing cancer or an infectious dis in a Subject, wherein said Subject is not infected with a human ease (including one or more symptoms thereof), wherein said immunodeficiency virus (HIV), comprising administering to infectious disease is not caused by infection with a human the subject in whom such elevation of interleukin-12 level is immunodeficiency virus (HIV), comprising administering to 10 desired an effective amount of an ARP. In another embodi a subject in whom Such treatment or prevention is desired a ment, the present invention provides a method of elevating therapeutically or prophylactically effective amount of an systemic level of interleukin-12 (IL-12) in a subject, wherein ARP. In another embodiment, the present invention provides said Subject is not infected with a human immunodeficiency a method of treating or preventing cancer or an infectious virus(HIV), comprising administering to the Subject in whom disease (including one or more symptoms thereof), wherein 15 said infectious disease is not caused by infection with a such elevation of interleukin-12 level is desired an effective human immunodeficiency virus (HIV), comprising adminis amount of a nucleic acid comprising a nucleotide sequence tering to a Subject in whom such treatment or prevention is encoding one or more ARPs. In another embodiment, the desired atherapeutically or prophylactically effective amount present invention provides a method of elevating systemic of a nucleic acid comprising a nucleotide sequence encoding level of interleukin-12 (IL-12) in a subject, wherein said one or more ARPs. In another embodiment, the present inven Subject is not infected with a human immunodeficiency virus tion provides a method of treating or preventing cancer oran (HIV), comprising administering to the Subject in whom Such infectious disease (including one or more symptoms thereof), elevation of interleukin-12 level is desired an effective wherein said infectious disease is not caused by infection amount of a cell transformed with a nucleic acid comprising with a human immunodeficiency virus (HIV), comprising 25 administering to a subject in whom such treatment or preven a nucleotide sequence encoding an ARP, wherein said nucle tion is desired a therapeutically or prophylactically effective otide sequence is operably linked to a promoter. In yet another amount of a cell transformed with a nucleic acid comprising embodiment, the present invention provides a method of a nucleotide sequence encoding an ARP, wherein said nucle elevating systemic level of interleukin-12 (IL-12) in a subject, otide sequence is operably linked to a promoter. In yet another 30 wherein said subject is not infected with a human immuno embodiment, the present invention provides a method of deficiency virus (HIV), comprising administering to the Sub treating or preventing cancer oran infectious disease (includ ject in whom such elevation of interleukin-12 level is desired ing one or more symptoms thereof), wherein said infectious an effective amount of an ARP and an effective amount of one disease is not caused by infection with a human immunode or more immune stimulatory agents other than ARP. ficiency virus (HIV), comprising administering to a subject in 35 Several adjuvants are used experimentally to enhance the whom such treatment or prevention is desired a therapeuti immune response to administered antigens. Only aluminum cally or prophylactically effective amount of an ARP and a hydroxide (alum), however, is approved by the FDA as an therapeutically or prophylactically effective amount of one or adjuvant for human use. This inert inorganic molecule has more agents other than ARP. In a specific embodiment, the been shown to be safe, but is only a moderately good adju cancer to be treated is an ovarian cancer. In specific embodi 40 vant. Other adjuvants (e.g. Bacille Calmette-Guerin (BCG). ments, the infectious disease is caused by infection with an Freund's adjuvants, keyhole limpet hemocyanin (KLH), dini Apicomplexan organism. In other specific embodiments, the trophenol, QS-21, CAP have been used in animal studies or infectious disease is not caused by infection with an Apicom are currently under investigation for humans. Unfortunately, plexan organism. they are either non-specific (CAP, QS-21, dinitrophenol) or In another embodiment, the present invention provides a 45 method of stimulating an immune response comprising toxic (BCG, KLH, Freund's adjuvant). administering to a Subject, with the proviso that said subject is In a further embodiment of the invention, the very potent not infected with a human immunodeficiency virus (HIV), an dendritic cell activation properties of ARP make it an ideal effective amount of an ARP. In another embodiment, the candidate to provide a Superior adjuvant for vaccine produc present invention provides a method of stimulating an 50 tion. Inorganic adjuvants are considered non-specific activa immune response comprising administering to a subject, with tors of the immune system. In contrast, ARP appears to be a the proviso that said subject is not infected with a human specific activator of innate immunity. Thus, it activates a immunodeficiency virus (HIV), an effective amount of a cascade of genetically preprogrammed events that upregulate nucleic acid comprising a nucleotide sequence encoding one the antigen presenting activity of dendritic cells while simul or more ARPs. In another embodiment, the present invention 55 taneously generating the release of the cytokines associated provides a method of stimulating an immune response com with the activation of immediate cellular responses (e.g. NK prising administering to a subject, with the proviso that said cells) and, more importantly, the activation of adaptive immu Subject is not infected with a human immunodeficiency virus nity (both cellular and humoral). This latter function would (HIV), an effective amount of a cell transformed with a facilitate the generation of memory T-cells (cellular response) nucleic acid comprising a nucleotide sequence encoding an 60 ARP, wherein said nucleotide sequence is operably linked to and circulating antibodies (humoral response). a promoter. In yet another embodiment, the present invention The subject as used herein refers to an animal, preferably a provides a method of stimulating an immune response com mammal. In some embodiments, the Subject is a domestic prising administering to a subject, with the proviso that said animal, including but not limited to, chicken, pig, cow, horse Subject is not infected with a human immunodeficiency virus 65 and goat. In some embodiments, the Subject is a pet, including (HIV), an effective amount of an ARP and an effective but not limited to, cat, dog, and bird. In a preferred embodi amount of one or more immune stimulatory agents other than ment, the Subject is a human. US 7,393,534 B2 55 56 Target Infectious Diseases cancer, said methods comprising administering to said Sub Infectious diseases that can be treated or prevented by the ject atherapeutically effective amount of a composition com methods of the present invention are caused by infectious prising an ARP and a therapeutically effective amount of one agents including, but not limited to, viruses, bacteria, fungi, or more therapeutic agents other than ARP. The invention also protozoa and parasites. provides methods for treating cancer by administering a com Viral diseases that can be treated or prevented by the meth position comprising an ARP in combination with any other ods of the present invention include, but are not limited to, anti-cancer treatment (e.g., radiation therapy, chemotherapy those caused by hepatitis type A, hepatitis type B, hepatitis or Surgery) to a patient who has proven refractory to other type C, influenza, Varicella, adenovirus, herpes simplex type treatments. The invention also provides methods for the treat I (HSV-I), herpes simplex type II (HSV-II), rinderpest, rhi 10 ment of a patient having cancer and immunosuppressed by novirus, echovirus, rotavirus, respiratory syncytial virus, pap reason of having previously undergone one or more other illoma virus, papova virus, cytomegalovirus, echinovirus, cancer therapies. The invention also provides alternative arbovirus, huntavirus, coxsackie virus, mumps virus, measles methods for the treatment of cancer where chemotherapy, virus, rubella virus, and polio virus. In accordance with the radiation therapy, hormonal therapy, and/or biological present invention, the disease that is treated or prevented by 15 therapy/immunotherapy has proven or may prove too toxic, the methods of the present invention is not caused by a human i.e., results in unacceptable or unbearable side effects, for the immunodeficiency virus (human immunodeficiency virus Subject being treated. type I (HIV-I), or human immunodeficiency virus type II In specific embodiments, a composition comprising an (HIV-II); e.g., the disease is not AIDS). ARP of the invention is administered to a subject for the Bacterial diseases that can be treated or prevented by the treatment of cancer, wherein the Subject does not have an methods of the present invention are caused by bacteria infection with an Apicomplexan organism. including, but not limited to, mycobacteria, rickettsia, myco Cancers that can be treated by the methods encompassed plasma, neisseria and legionella. by the invention include, but are not limited to, neoplasms, Fungal diseases that can be treated or prevented by the malignant tumors, metastases, or any disease or disorder methods of the present invention include, but not limited to, 25 characterized by uncontrolled cell growth such that it would aspergilliosis, crytococcosis, sporotrichosis, coccidioidomy be considered cancerous. The cancer may be a primary or cosis, paracoccidioidomycosis, histoplasmosis, blastomyco metastatic cancer. Specific cancers that can be treated accord sis, Zygomycosis, and candidiasis. ing to the present invention include, but are not limited to, Protozoal diseases that can be treated or prevented by the those listed in Table 2 (for a review of such disorders, see methods of the present invention are caused by protozoa 30 Fishman et al., 1985, Medicine, 2d Ed., J.B. Lippincott Co., including, but not limited to, leishmania kokZidioa, and try Philadelphia): panosoma. In specific embodiments, protozoal diseases that can be treated or prevented by the methods of the present TABLE 2 invention are not caused by Apicomplexa protozoa. Parasitic diseases that can be treated or prevented by the 35 MALIGNANCIES AND RELATED DISORDERS methods of the present invention are caused by parasites Leukemia including, but not limited to, chlamydia and amebiasis. acute leukemia acute lymphocytic leukemia acute myelocytic leukemia Target Cancer myeloblastic 40 promyelocytic myelomonocytic Therapeutic Uses monocytic In some embodiments, the present invention provides a erythroleukemia chronic leukemia method of treating a cancer (including ameliorating a symp chronic myelocytic (granulocytic) leukemia tom thereof) comprising administering to a subject in whom 45 chronic lymphocytic leukemia such treatment is desired a therapeutically effective amount Polycythemia vera of a composition comprising an ARP. A composition of the Lymphoma Hodgkin's disease invention may, for example, be used as a first, second, third or non-Hodgkin's disease fourth line cancer treatment. In some embodiments, the Multiple myeloma invention provides methods for treating a cancer (including 50 Waldenstrom's macroglobulinemia ameliorating a symptom thereof) in a subject refractory to one Heavy chain disease Solid tumors or more conventional therapies for Such a cancer, said meth sarcomas and carcinomas ods comprising administering to said Subject a therapeuti fibrosarcoma cally effective amount of a composition comprising an ARP. myxosarcoma liposarcoma A cancer may be determined to be refractory to a therapy 55 chondrosarcoma when at least Some significant portion of the cancer cells are Osteogenic sarcoma not killed or their cell division are not arrested in response to chordoma the therapy. Such a determination can be made either in vivo angiosarcoma or in vitro by any method known in the art for assaying the endotheliosarcoma lymphangiosarcoma effectiveness of treatment on cancer cells, using the art-ac 60 lymphangioendotheliosarcoma cepted meanings of “refractory' in Such a context. In a spe synovioma cific embodiment, a cancer is refractory where the number of mesothelioma cancer cells has not been significantly reduced, or has Ewings tumor increased. leiomyosarcoma rhabdomyosarcoma The invention provides methods for treating a cancer (in 65 colon carcinoma cluding ameliorating one or more symptoms thereof) in a rectal cancer Subject refractory to existing single agent therapies for Such a US 7,393,534 B2 57 58 Alternatively or in addition to the presence of abnormal TABLE 2-continued cell growth characterized as hyperplasia, metaplasia, or dys plasia, the presence of one or more characteristics of a trans MALIGNANCIES AND RELATED DISORDERS formed phenotype, or of a malignant phenotype, displayed in pancreatic cancer Vivo or displayed in vitro by a cell sample from a patient, can breast cancer indicate the desirability of prophylactic/therapeutic adminis ovarian cancer tration of the composition of the invention. Such characteris broState cancer Squamous cell carcinoma tics of a transformed phenotype include morphology basal cell carcinoma changes, looser Substratum attachment, loss of contact inhi adenocarcinoma 10 bition, loss of anchorage dependence, protease release, Sweat gland carcinoma increased Sugar transport, decreased serum requirement, Sebaceous gland carcinoma papillary carcinoma expression of fetal antigens, disappearance of the 250,000 papillary adenocarcinomas dalton cell surface protein, etc. (see also id., at pp. 84-90 for cystadenocarcinoma characteristics associated with a transformed or malignant medullary carcinoma 15 phenotype). bronchogenic carcinoma renal cell carcinoma In a specific embodiment, leukoplakia, a benign-appearing hepatoma hyperplastic or dysplastic lesion of the epithelium, or bile duct carcinoma Bowen's disease, a carcinoma in situ, are pre-neoplastic choriocarcinoma Seminoma lesions indicative of the desirability of prophylactic interven embryonal carcinoma tion. Wilms tumor In another embodiment, fibrocystic disease (cystic hyper curvical cancer plasia, mammary dysplasia, particularly adenosis (benign testicular tumor lung carcinoma epithelial hyperplasia)) is indicative of the desirability of Small cell lung carcinoma prophylactic intervention. bladder carcinoma 25 The prophylactic use of the compositions of the invention epithelial carcinoma glioma is also indicated in some viral infections that may lead to astrocytoma cancer. For example, human papilloma virus can lead to cer medulloblastoma Vical cancer (see, e.g., Hernandez-Avila et al., Archives of craniopharyngioma Medical Research (1997) 28: 265-271), Epstein-Barr virus ependymoma 30 pinealoma (EBV) can lead to lymphoma (see, e.g., Herrmann et al., J hemangioblastoma Pathol (2003) 199(2): 140-5), hepatitis B or C virus can lead acoustic neuroma to liver carcinoma (see, e.g., El-Serag, J. Clin Gastroenterol oligodendroglioma (2002) 35(5 Suppl 2): S72-8), human T cell leukemia virus menangioma (HTLV)-I can lead to T-cell leukemia (see e.g., Mortreux et melanoma 35 neuroblastoma al., Leukemia (2003) 17(1): 26-38), and human herpesvirus-8 retinoblastoma infection can lead to Kaposi's sarcoma (see, e.g., Kadow et al., Curr Opin Investig Drugs (2002)3(11): 1574-9). In other embodiments, a patient which exhibits one or more Prophylactic Uses of the following predisposing factors for malignancy is 40 treated by administration of an effective amount of a compo The compositions of the invention can be administered to sition of the invention: a chromosomal translocation associ prevent progression to a neoplastic or malignant state, includ ated with a malignancy (e.g., the Philadelphia chromosome ing but not limited to those disorders listed in Table 2. Such for chronic myelogenous leukemia, t (14; 18) for follicular prophylactic use is indicated in conditions known or Sus lymphoma, etc.), familial polyposis or Gardner's syndrome pected of preceding progression to neoplasia or cancer, in 45 (possible forerunners of colon cancer), benign monoclonal particular, where non-neoplastic cell growth consisting of gammopathy (a possible forerunner of multiple myeloma), a hyperplasia, metaplasia, or most particularly, dysplasia has first degree kinship with persons having a cancer or precan occurred (for review of such abnormal growth conditions, see cerous disease showing a Mendelian (genetic) inheritance Robbins and Angell, 1976, Basic Pathology, 2d Ed., W.B. pattern (e.g., familial polyposis of the colon, Gardner's Syn Saunders Co., Philadelphia, pp. 68-79.). Hyperplasia is a 50 drome, hereditary exostosis, polyendocrine adenomatosis, form of controlled cell proliferation involving an increase in medullary thyroid carcinoma with amyloid production and cell number in a tissue or organ, without significant alteration pheochromocytoma, Peutz-Jeghers syndrome, neurofibro in structure or function. As but one example, endometrial matosis of Von Recklinghausen, retinoblastoma, carotidbody hyperplasia often precedes endometrial cancer. Metaplasia is tumor, cutaneous melanocarcinoma, intraocular melanocar a form of controlled cell growth in which one type of adult or 55 cinoma, Xeroderma pigmentosum, ataxiatelangiectasia, Che fully differentiated cell substitutes for another type of adult diak-Higashi syndrome, albinism, Fanconi's aplastic anemia, cell. Metaplasia can occur in epithelial or connective tissue and Bloom's syndrome; see Robbins and Angell, 1976, Basic cells. A typical metaplasia involves a somewhat disorderly Pathology, 2d Ed., W.B. Saunders Co., Philadelphia, pp. 112 metaplastic epithelium. Dysplasia is frequently a forerunner 113) etc.), and exposure to carcinogens (e.g., Smoking, and of cancer, and is found mainly in the epithelia; it is the most 60 inhalation of or contacting with certain chemicals). disorderly form of non-neoplastic cell growth, involving a In another specific embodiment, a composition of the loss in individual cell uniformity and in the architectural invention is administered to a human patient to prevent pro orientation of cells. Dysplastic cells often have abnormally gression to breast, colon, ovarian, or cervical cancer. large, deeply stained nuclei, and exhibit pleomorphism. Dys plasia characteristically occurs where there exists chronic 65 Gene Therapy irritation or inflammation, and is often found in the cervix, In one embodiment, one or more nucleic acid molecules respiratory passages, oral cavity, and gallbladder. comprising sequences encoding one or more ARPs are US 7,393,534 B2 59 60 administered to a Subject to stimulate an immune response 20316 dated Nov. 26, 1992 (Findeis et al.); WO93/14188 and/or to prevent or treat cancer or infectious diseases by way dated Jul. 22, 1993 (Clarke et al.), WO 93/20221 dated Oct. of gene therapy. Gene therapy refers to therapy performed by 14, 1993 (Young)). Alternatively, the nucleic acid can be the administration to a Subject of an expressed or expressible introduced intracellularly and incorporated within host cell nucleic acid. 5 DNA for expression, by homologous recombination (Koller Any of the methods for gene therapy available in the art can and Smithies, 1989, Proc. Natl. Acad. Sci. USA 86: 8932 be used according to the present invention. Exemplary meth 8935; Zijlstra et al., 1989, Nature 342:435-438). ods are described below. In one embodiment, viral vectors that contain nucleic acids For general reviews of the methods of gene therapy, see encoding one or more ARPs are used in accordance with the Goldspiel et al., 1993, Clinical Pharmacy 12: 488-505; Wu 10 invention (see Miller et al., 1993, Meth. Enzymol. 217:581 and Wu, 1991, Biotherapy 3: 87-95; Tolstoshev, 1993, Ann. 599). A retroviral vector, for example, can be used in gene Rev. Pharmacol. Toxicol. 32: 573-596; Mulligan, 1993, Sci therapy to deliver an ARP to a subject. These retroviral vec ence 260: 926-932; and Morgan and Anderson, 1993, Ann. tors have been modified to delete retroviral sequences that are Rev. Biochem. 62: 191-217; May, 1993, TIBTECH 11(5): not necessary for packaging of the viral genome and integra 155-215). Methods commonly known in the art of recombi 15 tion into host cell DNA. More detail about retroviral vectors nant DNA technology which can be used are described in can be found in Boesen et al., 1994, Biotherapy 6: 291-302: Ausubel et al. (eds.), 1993, Current Protocols in Molecular Clowes et al., 1994, J. Clin. Invest. 93: 644-651; Kiem et al., Biology, John Wiley & Sons, NY; and Kriegler, 1990, Gene 1994, Blood 83: 1467-1473; Salmons and Gunzberg, 1993, Transfer and Expression, A Laboratory Manual, Stockton Human Gene Therapy 4: 129-141; and Grossman and Wilson, Press, NY. 1993, Curr. Opin. in Genetics and Devel. 3: 110-114. In a preferred embodiment, a composition of the invention Adenoviruses are other viral vectors that can be used in comprises nucleotide sequences encoding one or more ARPs, gene therapy. Adenoviruses are especially attractive vehicles said nucleic acid sequences being part of expression vectors for delivering genes to respiratory epithelia. Adenoviruses that express ARPs in a suitable host. In particular, such naturally infect respiratory epithelia where they cause a mild nucleic acids have promoters, preferably heterologous (non 25 disease. Other targets for adenovirus-based delivery systems native) promoters, operably linked to the ARP coding region, are liver, the central nervous system, endothelial cells, and said promoter being inducible or constitutive, and, optionally, muscle. Adenoviruses have the advantage of being capable of tissue-specific. In another particular embodiment, nucleic infecting non-dividing cells. Kozarsky and Wilson (1993, acid molecules are used in which the ARP coding sequences Current Opinion in Genetics and Development 3: 499-503) and any other desired sequences are flanked by regions that 30 presenta review of adenovirus-based gene therapy. Bout etal. promote homologous recombination at a desired site in the (1994, Human Gene Therapy 5: 3-10) demonstrated the use genome, thus providing for intrachromosomal expression of of adenovirus vectors to transfer genes to the respiratory the ARP nucleic acids (Koller and Smithies, 1989, Proc. Natl. epithelia of rhesus monkeys. Other instances of the use of Acad. Sci. USA 86: 8932-8935; Zijlstra et al., 1989, Nature adenoviruses in gene therapy can be found in Rosenfeld et al., 342:435-438). 35 1991, Science 252: 431-434; Rosenfeld et al., 1992, Cell 68: Delivery of the nucleic acids into a patient may be either 143-155; Mastrangeli et al., 1993, J. Clin. Invest. 91: 225 direct, in which case the patient is directly exposed to the 234; PCT Publication WO94/12649; and Wang et al., 1995, nucleic acid or nucleic acid-carrying vectors, or indirect, in Gene Therapy 2: 775-783. In a preferred embodiment, aden which case, cells are first transformed with the nucleic acids ovirus vectors are used in gene therapy to deliver ARPs to a in vitro, then transplanted into the patient. These two 40 Subject to prevent or treat cancer. approaches are known, respectively, as in vivo or ex vivo gene Adeno-associated virus (AAV) has also been proposed for therapy. use in gene therapy (Walsh et al., 1993, Proc. Soc. Exp. Biol. In a specific embodiment, the nucleic acid sequences are Med. 204: 289-300; U.S. Pat. No. 5,436,146). directly administered in vivo, where it is expressed to produce Another approach to gene therapy involves transferring a the encoded product. This can be accomplished by any of 45 gene to cells in tissue culture by Such methods as electropo numerous methods known in the art, e.g., by constructing ration, lipofection, calcium phosphate mediated transfection, them as part of an appropriate nucleic acid expression vector or viral infection. Usually, the method of transfer includes the and administering it so that they become intracellular, e.g., by transfer of a selectable marker to the cells. The cells are then infection using defective or attenuated retrovirals or other placed under selection to isolate those cells that have taken up viral vectors (see U.S. Pat. No. 4,980.286), or by direct injec 50 and are expressing the transferred gene. Those cells are then tion of naked DNA, or by use of microparticle bombardment delivered to a patient. (e.g., a gene gun; Biolistic, Dupont), or coating with lipids or In this embodiment, the nucleic acid is introduced into a cell-surface receptors or transfecting agents, encapsulation in cell prior to administration in vivo of the resulting recombi liposomes, microparticles, or microcapsules, or by adminis nant cell. Such introduction can be carried out by any method tering them in linkage to a peptide which is known to enter the 55 known in the art, including but not limited to transfection, nucleus, by administering it in linkage to a ligand Subject to electroporation, microinjection, infection with a viral or bac receptor-mediated endocytosis (see, e.g., Wu and Wu, 1987, teriophage vector containing the nucleic acid sequences, cell J. Biol. Chem. 262: 4429-4432) (which can be used to target fusion, chromosome-mediated gene transfer, microcell-me cell, types specifically expressing the receptors), etc. In diated gene transfer, spheroplast fusion, etc. Numerous tech another embodiment, nucleic acid-ligand complexes can be 60 niques are known in the art for the introduction of foreign formed in which the ligand comprises a fusogenic viral pep genes into cells (see, e.g., Loeffler and Behr, 1993, Meth. tide to disrupt endosomes, allowing the nucleic acid to avoid Enzymol. 217:599-618: Cohen et al., 1993, Meth. Enzymol. lysosomal degradation. In yet another embodiment, the 217: 618-644; Cline, 1985, Pharmac. Ther. 29: 69-92) and nucleic acid can be targeted in Vivo for cell specific uptake may be used in accordance with the present invention, pro and expression, by targeting a specific receptor (see, e.g., PCT 65 vided that the necessary developmental and physiological Publications WO92/06180 dated Apr. 16, 1992 (Wu et al.); functions of the recipient cells are not disrupted. The tech WO 92/22635 dated Dec. 23, 1992 (Wilson et al.); WO92/ nique should provide for the stable transfer of the nucleic acid US 7,393,534 B2 61 62 to the cell, so that the nucleic acid is expressible by the cell nitroso ureas, such as Carmustine, Lomustine, Streptozocin, and preferably heritable and expressible by its cell progeny. platinum complexes, such as Cisplatin, Carboplatin, biore The resulting recombinant cells can be delivered to a ductive alkylator, such as Mitomycin, and Procarbazine, Dac patient by various methods known in the art. Recombinant arbazine and Altretamine); the DNA strand-breakage agents, blood cells (e.g., hematopoietic stem or progenitor cells) are e.g., Bleomycin; the intercalating topoisomerase II inhibi preferably administered intravenously. The amount of cells tors, e.g., Intercalators, such as Amsacrine, Dactinomycin, envisioned for use depends on the desired effect, patient state, Daunorubicin, Doxorubicin, Idarubicin, Mitoxantrone, and etc., and can be determined by one skilled in the art. nonintercalators, such as Etoposide and Teniposide; the non Cells into which a nucleic acid can be introduced for pur intercalating topoisomerase II inhibitors, e.g., Etoposide and poses of gene therapy encompass any desired, available cell 10 Teniposde; and the DNA minor groove binder, e.g., Plicamy type, and include, but are not limited to, epithelial cells, din; the antimetabolites including, but not limited to, folate endothelial cells, keratinocytes, fibroblasts, muscle cells, antagonists Such as Methotrexate and trimetrexate; pyrimi hepatocytes; blood cells such as T lymphocytes, B lympho dine antagonists, such as Fluorouracil, Fluorodeoxyuridine, cytes, NK cells, dendritic cells, monocytes, macrophages, CB3717, AZacitidine and Floxuridine; purine antagonists neutrophils, eosinophils, megakaryocytes, granulocytes, 15 Such as Mercaptopurine, 6-Thioguanine, Pentostatin: Sugar autologous cancer cells; various stem or progenitor cells, in modified analogs such as Cytarabine and Fludarabine; and particular hematopoietic stem or progenitor cells, e.g. as ribonucleotide reductase inhibitors such as hydroxyurea; obtained from bone marrow, umbilical cord blood, peripheral tubulin Interactive agents including, but not limited to, col blood, fetal liver, etc. cbicine, Vincristine and Vinblastine, both alkaloids and Pacli In a preferred embodiment, the cell used for genetherapy is taxel and cytoxan; hormonal agents including, but note lim autologous to the patient. ited to, estrogens, conjugated estrogens and Ethinyl Estradiol In one embodiment in which recombinant cells are used in and Diethylstilbesterol, Chlortrianisen and Idenestrol; genetherapy, one or more nucleotide sequences encoding one progestins such as Hydroxyprogesterone caproate, MedroX or more ARPs are introduced into the cells such that the yprogesterone, and Megestrol; and androgens such as test nucleotide sequences are expressible by the cells or their 25 osterone, testosterone propionate; fluoxymesterone, methylt progeny, and the recombinant cells are then administered in estosterone; adrenal corticosteroid, e.g., Prednisone, vivo for therapeutic effect. In a specific embodiment, stem or Dexamethasone, Methylprednisolone, and Prednisolone: progenitor cells are used. Any stem and/or progenitor cells leutinizing hormone releasing hormone agents or gonadotro which can be isolated and maintained in vitro can potentially pin-releasing hormone antagonists, e.g., leuprolide acetate be used in accordance with this embodiment of the present 30 and goserelin acetate; antihormonal antigens including, but invention (see e.g. PCT Publication WO94/08598, dated Apr. not limited to, antiestrogenic agents such as Tamoxifen, anti 28, 1994; Stemple and Anderson, 1992, Cell 71: 973-985: androgen agents such as Flutamide; and antiadrenal agents Rheinwald, 1980, Meth. Cell Bio. 21A: 229; and Pittelkow Such as Mitotane and Aminoglutethimide; cytokines includ and Scott, 1986, Mayo Clinic Proc. 61: 771). In another ing, but not limited to, IL-1.alpha., IL-13, IL-2, IL-3, IL-4, specific embodiment, cancer cells are used. 35 IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, In a specific embodiment, the nucleic acid to be introduced IL-18, TGF-B, GM-CSF, M-CSF, G-CSF, TNF-C., TNF-B, for purposes of gene therapy comprises a constitutive, tissue LAF, TCGF, BCGF, TRF, BAF, BDG, MP, LIF, OSM, TMF, specific, or inducible promoter operably linked to the coding PDGF, IFN-C, IFN-BIFN-y, and Uteroglobins (U.S. Pat. No. region. In a preferred embodiment, the nucleic acid to be 5,696,092); anti-angiogenics including, but not limited to, introduced for purposes of gene therapy comprises an induc 40 agents that inhibit VEGF (e.g., other neutralizing antibodies ible promoter operably linked to the coding region, such that (Kim et al., 1992: Presta et al., 1997: Sioussat et al., 1993: expression of the nucleic acid is controllable by controlling Kondo et al., 1993; Asano et al., 1995, U.S. Pat. No. 5,520, the presence or absence of the appropriate inducer of tran 914), soluble receptor constructs (Kendall and Thomas, 1993; Scription. Aiello et al., 1995; Lin et al., 1998; Millauer et al., 1996), 45 tyrosine kinase inhibitors (Siemeister et al., 1998, U.S. Pat. Other Anti-Cancer Therapies Nos. 5,639,757, and 5,792,771), antisense strategies, RNA The present invention provides methods of preventing or aptamers and ribozymes against VEGF or VEGF receptors treating cancer comprising administering to a subject in need (Saleh et al., 1996; Cheng et al., 1996; Keet al., 1998: Parry thereof a composition comprising ARP alone or in combina et al., 1999); variants of VEGF with antagonistic properties as tion with one or more prophylactic ortherapeutic agents other 50 described in WO 98/16551; compounds of other chemical than ARP. Other therapeutic or prophylactic agents include, classes, e.g., Steroids such as the angiostatic 4.9(11)-steroids but are not limited to, peptides, polypeptides, proteins, fusion and C21-oxygenated steroids, as described in U.S. Pat. No. proteins, nucleic acid molecules, Small molecules, mimetic 5,972.922; thalidomide and related compounds, precursors, agents, Synthetic drugs, inorganic molecules, and organic analogs, metabolites and hydrolysis products, as described in molecules. Any agent ortherapy (e.g., chemotherapies, radia 55 U.S. Pat. Nos. 5,712.291 and 5,593.990; Thrombospondin tion therapies, Surgery, hormonal therapies, and/or biological (TSP-1) and platelet factor 4 (PF4); interferons and metallo therapies/immunotherapies) which is known to be useful, or proteinsase inhibitors; tissue inhibitors of metalloproteinases which has been used or is currently being used for the pre (TIMPs); anti-Invasive Factor, retinoic acids and paclitaxel vention or treatment of cancer can be used in combination (U.S. Pat. No. 5,716,981); AGM-1470 (Ingber et al., 1990); with a composition of the invention in accordance with the 60 shark cartilage extract (U.S. Pat. No. 5,618,925); anionic invention described herein. polyamide or polyurea oligomers (U.S. Pat. No. 5,593,664); Examples of Such agents (i.e., anti-cancer agents) include, oxindole derivatives (U.S. Pat. No. 5,576,330); estradiol but are not limited to, DNA-interactive agents including, but derivatives (U.S. Pat. No. 5,504,074); thiazolopyrimidine not limited to, the alkylating agents (e.g., nitrogen mustards, derivatives (U.S. Pat. No. 5,599,813); and LM609 (U.S. Pat. e.g. Chlorambucil, Cyclophosphamide, Isofamide, Mechlo 65 No. 5,753.230); apoptosis-inducing agents including, but not rethamine, Melphalan, Uracil mustard; AZiridine Such as limited to, bcr-abl. bcl-2 (distinct from bcl-1, cyclin D1; Thiotepa; methanesulphonate esters such as BuSulfan; GenBank accession numbers M14745, X06487; U.S. Pat. US 7,393,534 B2 63 64 Nos. 5,650,491; and 5,539,094) and family members includ tine; amidox; amifostine; aminolevulinic acid; amrubicin; ing Bcl-X1, Mcl-1, Bak, A1, A20, and antisense nucleotide amsacrine; anagrelide; anastrozole; andrographolide; angio sequences (U.S. Pat. Nos. 5,650,491; 5,539,094; and 5,583, genesis inhibitors; antagonist D; antagonist G, antarelix; anti 034). Immunotoxins and coaguligands, tumor vaccines, and dorsalizing morphogenetic protein-1, antiandrogen, prostatic antibodies. carcinoma; antiestrogen; antineoplaston; antisense oligo Specific examples of anti-cancer agents which can be used nucleotides; aphidicolin glycinate; apoptosis gene modula in accordance with the methods of the invention include, but tors; apoptosis regulators; apurinic acid; ara-CDP-DL not limited to: acivicin, aclarubicin; acodazole hydrochlo PTBA, arginine deaminase; asulacrine; atamestane; ride; acronine; adoZelesin; aldesleukin; altretamine; ambo atrimustine; axinastatin 1; axinastatin 2; axinastatin 3; aza mycin; ametantrone acetate; aminoglutethimide; amsacrine; 10 setron; azatoxin; azatyrosine; baccatin III derivatives; bal anastroZole; anthramycin; asparaginase; asperlin; azaciti anol; batimastat; BCR/ABL antagonists; benzochlorins; ben dine; azetepa; azotomycin; batimastat; benzodepa; bicaluta Zoylstaurosporine; beta lactam derivatives; beta-alethine; mide; bisantrene hydrochloride; bisnafide dimesylate: bize betaclamycin B; betulinic acid; bFGF inhibitor; bicaluta lesin, bleomycin Sulfate; brequinar sodium; bropirimine; mide; bisantrene; bisaziridinylspermine; bisnafide; bistratene buSulfan, cactinomycin; calusterone; caracemide; carbe 15 A: bizelesin; breflate; bropirimine; budotitane; buthionine timer, carboplatin: carmustine; carubicin hydrochloride; car Sulfoximine; calcipotriol; calphostin C; camptothecin deriva Zelesin; cedefingol; chlorambucil; cirolemycin; cisplatin: tives; canarypox IL-2; capecitabine; carboxamide-amino cladribine; crisinatol mesylate; cyclophosphamide; cytara triazole; carboxyamidotriazole; CaRest M3; CARN 700; car bine; dacarbazine; dactinomycin; daunorubicin hydrochlo tilage derived inhibitor; carZelesin; casein kinase inhibitors ride; decitabine; dexormaplatin; deZaguanine; deZaguanine (ICOS); castanospermine; cecropin B; cetrorelix; chlorlns: mesylate; diaziquone; docetaxel, doxorubicin, doxorubicin chloroquinoxaline Sulfonamide; cicaprost, cis-porphyrin, hydrochloride; droloxifene; droloxifene citrate; dromo cladribine; clomifene analogues; clotrimazole; collismycin stanolone propionate; duaZomycin; ediatrexate; eflomithine A. collismycin B; combretastatin A4, combretastatin ana hydrochloride; elsamitrucin; enloplatin; enpromate; epipro logue; conagenin, crambescidin 816; crisinatol; cryptophycin pidine; epirubicin hydrochloride; erbulozole; esorubicin 25 8, cryptophycin A derivatives; curacin A; cyclopentan hydrochloride; estramustine; estramustine phosphate thraquinones; cycloplatam, cypemycin; cytarabine ocfosfate; Sodium; etanidazole; etoposide; etoposide phosphate: eto cytolytic factor, cytostatin; dacliximab, decitabine; dehy prine; fadrozole hydrochloride; fazarabine; fenretinide; drodidemnin B; deslorelin; dexamethasone; dexifosfamide: floxuridine; fludarabine phosphate; fluorouracil; fluorocitab dexraZoxane; dexVerapamil; diaziquone; didemnin B; didox; ine; fosquidone; fostriecin Sodium; gemcitabine; gemcitab 30 diethylnorspermine; dihydro-5-azacytidine; dihydrotaxol. ine hydrochloride; hydroxyurea; idarubicin hydrochloride: 9-; dioxamycin; diphenyl spiromustine; docetaxel; ifosfamide: ilmofosine; interleukin II (including recombinant docosanol; dolasetron: doxifluridine; droloxifene; dronab interieukin II, or rIL2), interferon alpha-2a: interferon alpha inol; duocarmycin SA; ebselen; ecomustine: edelfosine; 2b: interferon alpha-n1; interferon alpha-n3; interferon beta-I edrecolomab: eflomithine; elemene; emitefur; epirubicin; a; interferon gamma-Ib, iproplatin; irinotecan hydrochloride; 35 epristeride; estramustine analogue; estrogen agonists; estro lanreotide acetate; letrozole; leuprolide acetate; liarozole gen antagonists; etanidazole; etoposide phosphate; exemes hydrochloride; lometrexol Sodium, lomustine; losoxantrone tane; fadrozole; faZarabine; fenretinide; filgrastim; finas hydrochloride; masoprocol; maytansine; mechlorethamine teride; flavopiridol; flezelastine; fluasterone; fludarabine; hydrochloride; megestrol acetate; melengestrol acetate; mel fluorodaunorunicin hydrochloride; forfenimex; formestane: phalan; menogaril; mercaptopurine; methotrexate; methotr 40 fostriecin, fotemustine; gadolinium texaphyrin; gallium exate Sodium; metoprine; meturedepa; mitindomide; mito nitrate; galocitabine; ganirelix; gelatinase inhibitors; gemcit carcin, mitocromin, mitogillin, mitomalcin, mitomycin; abine; glutathione inhibitors; hepsulfam; heregulin; hexam mitosper, mitotane; mitoxantrone hydrochloride; mycophe ethylene bisacetamide; hypericin; ibandronic acid; idarubi nolic acid; nocodazole; nogalamycin; ormaplatin; oxisuran: cin; idoxifene; idramantone, ilmofosine; illomastat; paclitaxel; pegaspargase; peliomycin; pentamustine; peplo 45 imidazoacridones; imiquimod; immunostimulant peptides; mycin Sulfate; perfosfamide; pipobroman; piposulfan, piroX insulin-like growth factor-1 receptor inhibitor; interferon antrone hydrochloride; plicamycin; plomestane; porfimer agonists; interferons; interleukins; iobenguane; iododoxoru Sodium; porfiromycin; prednimustine; procarbazine hydro bicin; ipomeanol, 4-, iroplact; irsogladine; isobengaZole; iso chloride; puromycin; puromycin hydrochloride; pyrazofurin; homohalicondrin B; itasetron; jasplakinolide; kahalalide F: riboprine; rogletimide; Safingol: Safingol hydrochloride; 50 lamellarin-N triacetate; lanreotide; leinamycin; lenograstim; Semustine; simtraZene; sparfosate Sodium; sparsomycin; lentinan Sulfate; leptolstatin; letrozole; leukemia inhibiting spirogermanium hydrochloride; spiromustine; spiroplatin: factor; leukocyte alpha interferon; leuprolide+estrogen streptonigrin: Streptozocin, Sulofenur; talisomycin; tecogalan progesterone; leuprorelin; levamisole; liarozole; linear Sodium, tegafur, teloxantrone hydrochloride; temoporfin; polyamine analogue; lipophilic disaccharide peptide; lipo teniposide; teroxirone; testolactone; thiamiprine; thiogua 55 philic platinum compounds; lissoclinamide 7: lobaplatin: nine; thiotepa; tiazofurin; tirapazamine; toremifene citrate; lombricine; lometrexol; lonidamine; losoxantrone; lovasta trestolone acetate; triciribine phosphate; trimetrexate; trime tin; loxoribine; lurtotecan; lutetium texaphyrin; lysofylline; trexate glucuronate; triptorelin; tubulozole hydrochloride; lytic peptides; maitansine; mannostatin A; marimastat; maso uracil mustard; uredepa; vapreotide; verteporfin; vinblastine procol; maspin; matrilysin inhibitors; matrix metalloprotein sulfate; Vincristine sulfate; Vindesine; vindesine sulfate; vine 60 ase inhibitors; menogaril; merbarone; meterelin; methioni pidine Sulfate; Vinglycinate Sulfate; Vinleurosine Sulfate; nase; metoclopramide: MIF inhibitor; mifepristone; vinorelbine tartrate; Vinrosidine sulfate; Vinzolidine sulfate; miltefosine; mirimostim; mismatched double stranded RNA; Vorozole; Zeniplatin: Zinostatin: Zorubicin hydrochloride. mitoguaZone, mitolactol; mitomycin analogues; mitonafide; Other anti-cancer drugs include, but are not limited to: mitotoxin fibroblast growth factor-Saporin; mitoxantrone; 20-epi-1.25 dihydroxyvitamin D3; 5-ethynyluracil; abirater 65 mofarotene; molgramoStim; monoclonal antibody, human one, aclarubicin; acylfulvene; adecypenol; adoZelesin; chorionic gonadotrophin, monophosphoryl lipid A+myobac aldesleukin; ALL-TK antagonists; altretamine; ambamus terium cell wall sk; mopidamol; multiple drug resistance gene US 7,393,534 B2 65 66 inhibitor, multiple tumor Suppressor 1-based therapy; mus therapy or brachytherapy wherein a radiaoactive source is tard anticancer agent; mycaperoxide B; mycobacterial cell placed inside the body close to cancer cells or a tumor mass. wall extract; myriaporone; N-acetyldinaline; N-substituted In a preferred embodiment, a composition of the invention benzamides; nafarelin; nagreStip; naloxone-pentazocine; is used in combination with an interleukin-18 (IL-18), a napavin; naphterpin, nartograstim; nedaplatin: nemorubicin; CD40 ligand, an interferon-Y (INFY), an interleukin-4 (IL-4), neridronic acid; neutral endopeptidase; nilutamide; nisamy a granulocyte macrophage-colony stimulating factor (GM cin; nitric oxide modulators; nitroxide antioxidant; nitrullyn; CSF), other cytokines, an anti-CD40 antibody and a O6-benzylguanine; octreotide; okicenone; oligonucleotides; co-stimulatory agonist in stimulating an immune reponse onapristone; ondansetron; ondansetron; oracin; oral cytokine and/or in prevention or treatment of cancer. In a specific inducer, ormaplatin: osaterone; Oxaliplatin, oxaunomycin; 10 embodiment, an ARP of the invention may exhibit synergistic paclitaxel; paclitaxel analogues; paclitaxel derivatives; immune-stimulatory effects with these agents. palauamine; palmitoylrhizoxin; pamidronic acid; panax In specific embodiments, a patient with ovarian cancer is ytriol; panomifene; parabactin; paZelliptine; pegaspargase; administered a prophylactically or therapeutically effective peldesinei; pentosan polysulfate sodium; pentostatin; pentro amount of a composition comprising ARP in combination Zole; perflubron; perfosfamide; perillyl alcohol; phenazino 15 with a prophylactically or therapeutically effective amount of mycini; phenylacetate; phosphatase inhibitors; picibanil; one or more other agents useful for ovarian cancer therapy, pilocarpine hydrochloride; pirarubicin; piritrexim; placetin including but not limited to, intraperitoneal radiation therapy, A: placetin B; plasminogen activator inhibitor; platinum such as P’ therapy, total abdominal and pelvic radiation complex; platinum compounds; platinum-triamine complex; therapy, cisplatin, the combination of paclitaxel (Taxol) or porfimer Sodium; porfiromycin; prednisone; propyl bis-acri docetaxel (Taxotere) and cisplatin or carboplatin, the combi done; prostaglandin J2, proteasome inhibitors; protein nation of cyclophosphamide and cisplatin, the combination of A-based immune modulator, protein kinase C inhibitor, pro cyclophosphamide and carboplatin, the combination of 5-FU tein kinase C inhibitors, microalgal; protein tyrosine phos and leucovorin, etoposide, liposomal doxorubicin, gemcitab phatase inhibitors; purine nucleoside phosphorylase inhibi ine or topotecan. In a particular embodiment, a prophylacti tors; purpurins; pyrazoloacridine; pyridoxylated hemoglobin 25 cally or therapeutically effective amount of a composition of polyoxyethylene conjugate, raf antagonists; raltitrexed: the invention is administered in combination with the admin ramosetron; ras farnesyl protein transferase inhibitors; ras istration of Taxol for patients with platinum-refractory dis inhibitors: ras-GAP inhibitor; retelliptine demethylated; rhe ease. A further embodiment is the treatment of patients with nium Re 186 etidronate; rhizoxin: ribozymes: RII retinamide: refractory ovarian cancer including administration of ifosfa rogletimide; rohitukine; romurtide; roquinimex: rubiginone 30 mide in patients with disease that is platinum-refractory, hex B 1: ruboxyl; safingol; saintopin; SarCNU; sarcophytol A: amethylmelamine (HMM) as salvage chemotherapy after sargramostim; Sdi 1 mimetics: semustine; senescence failure of cisplatin-based combination regimens, and tamox derived inhibitor 1; sense oligonucleotides; signal transduc ifen in patients with detectable levels of cytoplasmic estrogen tion inhibitors; signal transduction modulators; single chain receptor on their tumors. antigen binding protein; sizofuran; Sobuzoxane; sodium 35 borocaptate; sodium phenylacetate; solverol; somatomedin Cancer therapies and their dosages, routes of administra binding protein; Sonermin; sparfosic acid; spicamycin D; tion and recommended usage are known in the art and have spiromustine; splenopentin; spongistatin 1; squalamine; stem been described in such literature as the Physician's Desk cell inhibitor; stem-cell division inhibitors; stipiamide: Reference (56' ed., 2002). stromelysin inhibitors; sulfinosine; Superactive vasoactive 40 intestinal peptide antagonist; Suradista; Suramin; Swainso Other Anti-Infection Agents nine; synthetic glycosaminoglycans; tallimustine; 5-fluorou The present invention provides methods of preventing or racil; leucovorin; tamoxifen methiodide; tauromustine; taZ treating an infectious disease, said methods comprising arotene; tecogalan Sodium, tegafur, tellurapyrylium; administering to a subject in need thereof a composition telomerase inhibitors; temoporfin, temozolomide; tenipo 45 comprising ARP alone or in combination with one or more side; tetrachlorodecaoxide; tetrazomine; thaliblastine; thio prophylactic or therapeutic agents other than ARP. Any agent coraline; thrombopoietin; thrombopoietin mimetic; thymal or therapy which is known to be useful, or which has been fasin; thymopoietin receptor agonist; thymotrinan; thyroid used or is currently being used for the prevention or treatment stimulating hormone; tin ethyl etiopurpurin; tirapazamine; of infectious disease can be used in combination with the titanocene bichloride; top sentin; toremifene; totipotent stem 50 composition of the invention inaccordance with the invention cell factor; translation inhibitors; tretinoin; triacetyluridine: described herein. triciribine; trimetrexate; triptorelin; tropisetron; turosteride; Examples of Such agents to treat bacteria infections tyrosine kinase inhibitors; tyrphostins; UBC inhibitors: ube include, but are not limited to, folate antagonists (e.g., nimex: urogenital sinus-derived growth inhibitory factor, mafenide, silver Sulfadiazine, Succinylsulfathiazole, Sulfac urokinase receptor antagonists; vapreotide; variolin B; vector 55 etamide, Sulfadiazine, Sulfamethoxazole, Sulfasalazine, system, erythrocyte gene therapy; thalidomide; velaresol; Sulfisoxazole, pyrimethoamine, trimethoprim, co-trimox Veramine; verdins; verteporfin; vinorelbine; vinxaltine; azole), inhibitors of cell wall synthesis (e.g., penicillins, vitaxin; Vorozole; Zanoterone; Zeniplatin: Zilascorb; and cephalosporins, carbapenems, monobactams, Vacomycin, Zinostatin stimalamer. bacitracin, clavulanic acid, Sulbactam, taZobactam), protein The invention also encompasses administration of a com 60 synthesis inhibitors (e.g., tetracyclines, aminoglycosides, position comprising ARP in combination with radiation macrollides, chloramphenicol, clindamycin), fluoroquinolo therapy comprising the use of X-rays, gamma rays and other nes (e.g., ciproloxacin, enoxacin, lomefloxacin, norfloxacin, sources of radiation to destroy the cancer cells. In preferred ofloxacin), nalidixic acid, methenamine, nitrofurantoin, ami embodiments, the radiation treatment is administered as nosalicylic acid, cycloserine, ethambutol, ethionamide, iso external beam radiation or teletherapy wherein the radiation 65 niazid, pyrazinamide, rifampin, clofazimine, and dapsone. is directed from a remote source. In other preferred embodi Examples of agent that can be used in combination with ments, the radiation treatment is administered as internal ARP to treat fungal infections include, but not limited to, US 7,393,534 B2 67 68 amphotericin B, fluconazole, flucytosine, itraconazole, keto The dosage of ARP for intravenous administration in a conazole, clotrimazole, econazole, griseofulvin, miconazole, human patient provided by the present invention is preferably and nystatin. less than 100 ug/kg body weight, less than 50 g/kg body Examples of agent that can be used in combination with weight, less than 10 ug/kg body weight, less than 5 g/kg ARP to treat protozoal infections include, but not limited to, body weight, less than 1 Jug/kg body weight, less than 0.5 chloroquine, dehydroemetine, diloxanide furoate, emetine, ug/kg body weight, less than 0.1 ug/kg body weight, less than metronidazole, paramomycin, chloroquine, mefloquine, pri 0.05 g/kg body weight, less than 0.01 g/kg body weight, or maquine, pyrimethamine, quinine, quinidine, melarsoprol, less than 0.001 ug/kg body weight. In a preferred embodi nifurtimox, pentamidine, Suramin, Sodium Stibogluconate, ment, ARP is given at a dosage of 0.1 ug perperson per day (or pyrimethamine, and quinacrine. 10 about 0.14 ng per kg body weight). Examples of antihelmintic agents that can be used in com In the case of treating for an infection with influenza virus bination with ARP to treat helminth (worm) infections the dosage for treating a human can be derived from a Suitable include, but not limited to, ivermectin, mebendazole, pyrantel dose in the mouse which has been found to be about 10,000 ng pamoate, thiabendazole, praziquantel, and niclosamide. per mouse. Given the difference in body weight, a comparable 15 dose in a human would be 28 mg. For treatment of influenza Examples of antiviral agents that can be used in combina virus infection in a human patient using the present invention, tion with ARP to treat viral infections include, but not limited the dose is preferably from about 2.8 mg to about 280 mg. to, amantadine, ribavirin, rimantadine, acyclovir, famciclo more preferably from about 5 mg to about 100 mg and most Vir, foScamet, ganciclovir, trifluridine, Vidarabine, didanosine preferably from about 10 mg to about 50 mg. (ddI), stavudine (d4T), zalcitabine (ddC). Zidovudine (AZT), Prior to administering the first full dose, each patient pref lamivudine, abacavir, delavirdine, nevirapine, efavirenz, erably receives a subcutaneous injection of a small amount saquinavir, ritonavir, indinavir, nelfinavir, amprenavir, lopi (e.g., fraction (/100)} to fraction (/10) of the prescribed navir and interferon. dose) of a composition of the invention to detect any acute Dosage Regimens intolerance. The injection site is examined one and two hours Toxicity and efficacy of the prophylactic and/ortherapeutic 25 after the test. If no reaction is detected, then the full dose is protocols of the present invention can be determined by stan administered. dard pharmaceutical procedures in cell cultures or experi The compositions of the invention can also be administered mental animals, e.g., for determining the LDs (the dose orally. In one embodiment, intact sporulated oocysts of an lethal to 50% of the population) and the EDs (the dose Apicomplexan genus (e.g., Eimeria tenella) are given orally therapeutically effective in 50% of the population). The dose 30 with drinking water. The dosage can be, by way of example, ratio between toxic and therapeutic effects is the therapeutic 100 to 10,000 oocysts in a single administration depending on index and it can be expressed as the ratio LDso/EDso. Pro the cross-species infectivity of the protozoan. phylactic and/or therapeutic agents that exhibit large thera The compositions of the invention can be administered in peutic indices are preferred. While prophylactic and/or thera various forms. In one embodiment, a composition of the peutic agents that exhibit toxic side effects may be used, care 35 invention comprises an ARP in a membrane-linked form. In should be taken to design a delivery system that targets Such another embodiment, a composition of the invention com prises live or dead bacteria with recombinant ARP expressed. agents to the site of affected tissue in order to minimize In another embodiment, a composition of the invention com potential damage to uninfected cells and, thereby, reduce side prises live bacteria, which are specific to a particular organ, effects. 40 The data obtained from the cell culture assays and animal expressing recombinant ARP (e.g., Lactobacillus would live studies can be used in formulating a range of dosage of the in intestinal tissue, while some air-born non-pathogenic bac prophylactic and/or therapeutic agents for use in humans. The teria may be specific to lungs, or the nasal cavity, etc). dosage of such agents lies preferably within a range of circu Administrations, Formulations and Kits lating concentrations that include the EDs with little or no 45 The invention provides methods of treatment and/or pro toxicity. The dosage may vary within this range depending phylaxis by administration to a Subject of an effective amount upon the dosage form employed and the route of administra of a composition comprising ARP. In a preferred aspect, the tion utilized. For any agent used in the method of the inven ARP is purified. The subject is preferably an animal, includ tion, the therapeutically effective dose can be estimated ini ing but not limited to animals such as cows, pigs, chickens, tially from cell culture assays (see Section 5.6 and Section 50 etc., and is preferably a mammal. In a preferred embodiment, 12). A dose may be formulated in animal models to achieve a the Subject is a domestic animal, including but not limited to circulating plasma concentration range that includes the ICso chickens, pigs, cows, goats and horse. In another preferred (i.e., the concentration of the test compound that achieves a embodiment, the Subject is a pet, including but not limited to, half-maximal inhibition of symptoms) as determined in cell cats, dogs, and birds. In another preferred embodiment, the culture. Such information can be used to more accurately 55 Subject is a human. determine useful doses in humans. Levels in plasma may be Various delivery systems are known and can be used to measured, for example, by high performance liquid chroma administer a composition of the invention, e.g., encapsulation tography. in liposomes, microparticles, microcapsules, expression by The amount of the composition of the invention which will recombinant cells, receptor-mediated endocytosis (see, e.g., be effective in the treatment of a particular disorder or con 60 Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432), construc dition will depend on the nature of the disorder or condition, tion of an ARP nucleic acid as part of a retroviral or other and can be determined by standard clinical techniques. The vector, etc. Methods of introduction include, but are not lim precise dose to be employed in the formulation will also ited to, intradermal, intramuscular, intraperitoneal, intrave depend on the route of administration, and the seriousness of nous, Subcutaneous, intranasal, and oral routes. The com the disease or disorder, and should be decided according to 65 pounds may be administered by any convenient route, for the judgment of the practitioner and each patient's circum example by infusion or bolus injection, by absorption through Stances. epithelial or mucocutaneous linings (e.g., oral mucosa, rectal US 7,393,534 B2 69 70 and intestinal mucosa, etc.) and may be administered together pharmaceutical grade water or saline. Where the composition with other biologically active agents. Administration can be is administered by injection, an ampoule of sterile water for systemic or local. injection or saline can be provided so that the ingredients may In a specific embodiment, it may be desirable to administer be mixed prior to administration. the pharmaceutical compositions of the invention locally to The compositions of the invention can be formulated as the area in need of treatment; this may be achieved by, for neutral or salt forms. Pharmaceutically acceptable salts example, local infusion during Surgery, topical application, include those formed with free amino groups such as those e.g., in conjunction with a wound dressing after Surgery, by derived from hydrochloric, phosphoric, acetic, oxalic, tartaric injection, by means of a catheter, by means of a Suppository, acids, etc., and those formed with free carboxyl groups such or by means of an implant, said implant being of a porous, 10 as those derived from Sodium, potassium, ammonium, cal non-porous, or gelatinous material, including membranes, cium, ferric hydroxides, isopropylamine, triethylamine, Such as Sialastic membranes, or fibers. In one embodiment, 2-ethylamino ethanol, histidine, procaine, etc. administration can be by direct injection at the site (or former In one embodiment, purified Apicomplexan organisms are site) of a malignant tumor or neoplastic or pre-neoplastic given orally. In a specific embodiment, intact sporulated tissue. 15 oocysts of an Apicomplexa genus (e.g., Eimeria species. Such In another embodiment, a chimeric construction of an ARP as E. tenella) are given orally with drinking water. is used to target a specific area, i.e., even systemic adminis In addition to the formulations described previously, the tration of the composition would direct the composition to the compositions may also be formulated as a depot preparation. organ of choice (e.g., an antibody to a specific type of cancer Such long acting formulations may be administered by fused to an ARP would let the chimeric construct to be deliv implantation (for example, Subcutaneously or intramuscu ered to the surface of the tumor). larly) or by intramuscular injection. Thus, for example, the In a specific embodiment, the ARP nucleic acid can be compositions may be formulated with Suitable polymeric or administered in vivo to promote expression of its encoded hydrophobic materials (for example, as an emulsion in an protein, by constructing it as part of an appropriate nucleic acceptable oil) or ion exchange resins, or as sparingly soluble acid expression vector and administering it so that it becomes 25 derivatives, for example, as a sparingly soluble salt. Lipo intracellular, e.g., by use of a retroviral vector (see U.S. Pat. Somes and emulsions are well known examples of delivery No. 4,980.286), or by direct injection, or by use of micropar vehicles or carriers for hydrophilic drugs. ticle bombardment (e.g., a gene gun; Biolistic, Dupont), or The compositions may, if desired, be presented in a pack or coating with lipids or cell-surface receptors or transfecting dispenser device which may contain one or more unit dosage agents, or by administering it in linkage to a homeobox-like 30 forms containing the active ingredient. The pack may for peptide which is known to enter the nucleus (see e.g., Joliot et example comprise metal or plastic foil. Such as ablisterpack. al., 1991, Proc. Natl. Acad. Sci. USA 88: 1864-1868), etc. The pack or dispenser device may be accompanied by instruc Alternatively, an ARP nucleic acid can be introduced intrac tions for administration. ellularly and incorporated within host cell DNA for expres The invention also provides kits for carrying out the thera Sion, by homologous recombination. 35 peutic regimens of the invention. Such kits comprise in one or The present invention also provides pharmaceutical com more containers therapeutically or prophylactically effective positions. Such compositions comprise a therapeutically amounts of the composition of the invention in pharmaceuti effective amount of a composition of the invention, and a cally acceptable form. The composition in a vial of a kit of the pharmaceutically acceptable carrier or excipient. Such a car invention may be in the form of a pharmaceutically accept rier includes but is not limited to saline, buffered saline, 40 able solution, e.g., incombination with sterile Saline, dextrose dextrose, water, glycerol, ethanol, and combinations thereof. solution, or buffered solution, or other pharmaceutically The carrier and composition can be sterile. The formulation acceptable sterile fluid. Alternatively, the composition may be should suit the mode of administration. lyophilized or desiccated; in this instance, the kit optionally The composition, if desired, can also contain minor further comprises in a container a pharmaceutically accept amounts of wetting or emulsifying agents, or pH buffering 45 able solution (e.g., Saline, dextrose solution, etc.), preferably agents. The composition can be a liquid solution, Suspension, sterile, to reconstitute the composition to form a solution for emulsion, tablet, pill, capsule, Sustained release formulation, injection purposes. or powder. The composition can be formulated as a Supposi In another embodiment, a kit of the invention further com tory, with traditional binders and carriers such as triglycer prises a needle or syringe, preferably packaged in sterile ides. Oral formulation can include standard carriers such as 50 form, for injecting the formulation, and/or a packaged alcohol pharmaceutical grades of mannitol, lactose, starch, magne pad. Instructions are optionally included for administration of sium Stearate, Sodium saccharine, cellulose, magnesium car the formulations of the invention by a clinician or by the bonate, etc. patient. In a preferred embodiment, the composition is formulated In some embodiments, the present invention provides kits in accordance with routine procedures as a pharmaceutical 55 comprising a plurality of containers each comprising a phar composition adapted for intravenous administration to maceutical formulation or composition comprising a dose of human beings. Typically, compositions for intravenous the composition of the invention Sufficient for a single admin administration are solutions insterile isotonic aqueous buffer. istration. Where necessary, the composition may also include a solu In a specific embodiment, a kit comprises a first container bilizing agent and a local anesthetic Such as lignocaine to ease 60 containing a composition comprising ARP, and a second pain at the site of the injection. Generally, the ingredients are container containing a different treatment modality in an Supplied either separately or mixed together in unit dosage amount that, when administered before, concurrently with, or form, for example, as a dry lyophilized powder or water free after the administration of the ARP in the first container, is concentrate in a hermetically sealed container Such as an effective to improve overall treatment effectiveness over the ampule or Sachette indicating the quantity of active agent. 65 effectiveness of the administration of each component alone, Where the composition is to be administered by infusion, it or is effective to decrease side effects of the treatment when can be dispensed with an infusion bottle containing sterile each component is used alone. In a preferred specific embodi US 7,393,534 B2 71 72 ment, the invention provides a kit comprising in a first con dosage form of each pharmaceutical agent contained within tainer, a composition of the invention; and in a second con said packaging material. The invention further provides an tainer, a composition comprising a purified cytokine. article of manufacture comprising a needle or Syringe, pref The appropriate and recommended dosages, formulation erably packaged in Sterile form, for injection of the formula and routes of administration for treatment modalities such as tion, and/or a packaged alcohol pad. chemotherapeutic agents, radiation therapy and biological/ In a specific embodiment, an article of manufacture com immunotherapeutic agents such as cytokines are known in the prises packaging material and a pharmaceutical agent and art and described in such literature as the Physicians Desk instructions contained within said packaging material, Reference (56th ed., 2002). wherein said pharmaceutical agent is an ARP or a derivative, 10 fragment, homolog, analog thereof and a pharmaceutically Article of Manufacture acceptable carrier, and said instructions indicate a dosing The present invention also encompasses a finished pack regimen for preventing, treating or managing a subject with aged and labeled pharmaceutical product. This article of cancer. In another embodiment, an article of manufacture manufacture includes the appropriate unit dosage form in an comprises packaging material and a pharmaceutical agent appropriate vessel or container Such as a glass vial or other 15 and instructions contained within said packaging material, container that is hermetically sealed. In the case of dosage wherein said pharmaceutical agent is an ARP or a derivative, forms suitable for parenteral administration the active ingre fragment, homolog, analog thereof, a prophylactic or thera dient is sterile and Suitable for administration as a particulate peutic agent other than an ARP or a derivative, fragment, free solution. In other words, the invention encompasses both homolog, analog thereof, and a pharmaceutically acceptable parenteral solutions and lyophilized powders, each being carrier, and said instructions indicate a dosing regimen for sterile, and the latter being suitable for reconstitution prior to preventing, treating or managing a Subject with a cancer. In injection. Alternatively, the unit dosage form may be a solid another embodiment, an article of manufacture comprises Suitable for oral, transdermal, topical or mucosal delivery. packaging material and two pharmaceutical agents and In a preferred embodiment, the unit dosage form is suitable instructions contained within said packaging material, for intravenous, intramuscular or Subcutaneous delivery. 25 wherein said first pharmaceutical agent is an ARP or a deriva Thus, the invention encompasses solutions, preferably sterile, tive, fragment, homolog, analog thereof and a pharmaceuti suitable for each delivery route. cally acceptable carrier, and said second pharmaceutical In another preferred embodiment, compositions of the agent is a prophylactic ortherapeutic agent other than an ARP invention are stored in containers with biocompatible deter or a derivative, fragment, homolog, analog thereof, and said gents, including but not limited to, lecithin, taurocholic acid, 30 instructions indicate a dosing regimen for preventing, treating and cholesterol; or with other proteins, including but not or managing a subject with a cancer. limited to, gamma globulins and serum albumins. More pref erably, compositions of the invention are stored with human Monitoring of Effects During Treatment serum albumins for human uses, and stored with bovine The effect of immunotherapy with an ARP compositions of serum albumins for veterinary uses. While not bound by any 35 the invention on development and progression of neoplastic theory, a low concentration of ARP tends to be denaturing on diseases can be monitored by any methods known to one the Surface of plastic or glass containers. The handling and skilled in the art, including but not limited to measuring: a) storage as described herein would minimize the loss of ARP delayed hyperSensitivity as an assessment of cellular immu on Surfaces of containers and keep an ARP in its active form. nity; b) activity of cytolytic T-lymphocytes in vitro; c) levels Other methods known in the art can also be used. See Wang, 40 of tumor specific antigens, e.g., carcinoembryonic (CEA) International J. of Pharmaceutics, 185: 129-188 (1999). antigens; d) changes in the morphology of tumors using tech As with any pharmaceutical product, the packaging mate niques such as a computed tomographic (CT) scan; e) rial and container are designed to protect the stability of the changes in levels of putative biomarkers of risk for a particu product during storage and shipment. Further, the products of lar cancer in Subjects at high risk, and f) changes in the the invention include instructions for use or other informa 45 morphology of tumors using a sonogram. tional material that advise the physician, technician or patient Delayed Hypersensitivity Skin Test on how to appropriately prevent or treat the disease or disor Delayed hypersensitivity skin tests are of great value in the der in question. In other words, the article of manufacture overall immunocompetence and cellular immunity to an anti includes instruction means indicating or Suggesting a dosing gen. Inability to react to a battery of common skin antigens is regimen including, but not limited to, actual doses, monitor 50 termed anergy (Sato, T., et al., 1995, Clin. Immunol. Pathol., ing procedures (such as methods for monitoring mean abso 74:35-43). lute lymphocyte counts, tumor cell counts, and tumor size) Proper technique of skin testing requires that the antigens and other monitoring information. be stored sterile at 4C, protected from light and reconstituted More specifically, the invention provides an article of shorted before use. A 25- or 27-gauge needle ensures intrad manufacture comprising packaging material. Such as a box, 55 ermal, rather than Subcutaneous, administration of antigen. bottle, tube, Vial, container, sprayer, insufflator, intravenous Twenty-four and 48 hours after intradermal administration of (i.v.) bag, envelope and the like; and at least one unit dosage the antigen, the largest dimensions of both erythema and form of a pharmaceutical agent contained within said pack induration are measured with a ruler. Hypoactivity to any aging material. The invention also provides an article of given antigen or group of antigens is confirmed by testing manufacture comprising packaging material. Such as a box, 60 with higher concentrations of antigen or, in ambiguous cir bottle, tube, Vial, container, sprayer, insufflator, intravenous cumstances, by a repeat test with an intermediate test. (i.v.) bag, envelope and the like; and at least one unit dosage form of each pharmaceutical agent contained within said In Vitro Activation of Cytotoxic T Cells packaging material. The invention further provides an article 8x10° peripheral blood derived T lymphocytes isolated by of manufacture comprising packaging material. Such as a 65 the Ficoll-Hypaque centrifugation gradient technique, are box, bottle, tube, Vial, container, sprayer, insufflator, intrave restimulated with 4x10' mitomycin C treated tumor cells in 3 nous (i.v.) bag, envelope and the like; and at least one unit ml RPMI medium containing 10% fetal calf serum. In some US 7,393,534 B2 73 74 experiments, 33% secondary mixed lymphocyte culture Monitoring Adverse Effects During Treatment Supernatant or IL-2, is included in the culture medium as a Any adverse effects during the use of an ARP alone or in source of T cell growth factors. combination with another therapy (including another thera In order to measure the primary response of cytolytic peutic or prophylactic agent) are preferably also monitored. T-lymphocytes after immunization, T cells are cultured with Examples of adverse effects of chemotherapy during a cancer treatment or treatment of an infectious disease include, but out the stimulator tumor cells. In other experiments, T cells are not limited to, gastrointestinal toxicity Such as, but not are restimulated with antigenically distinct cells. After six limited to, early and late-forming diarrhea and flatulence; days, the cultures are tested for cytotoxicity in a 4 hour 51 nausea; Vomiting; anorexia; leukopenia; anemia; neutrope Cr-release assay. The spontaneous 51 Cr-release of the targets 10 nia; asthenia; abdominal cramping; fever, pain; loss of body should reach a level less than 20%. For the anti-MHC class I weight; dehydration; alopecia; dyspnea; insomnia; dizziness, blocking activity, a tenfold concentrated Supernatant of mucositis, Xerostomia, and kidney failure, as well as consti W6/32 hybridoma is added to the test at a final concentration pation, nerve and muscle effects, temporary or permanent of 12.5% (Heike M., et al., J. Immunotherapy, 15: 165-174). damage to kidneys and bladder, flu-like symptoms, fluid 15 retention, and temporary or permanent infertility. Adverse Levels of Tumor Specific Antigens effects from radiation therapy include, but are not limited to, Although it may not be possible to detect unique tumor fatigue, dry mouth, and loss of appetite. Other adverse effects antigens on all tumors, many tumors display antigens that include gastrointestinal toxicity Such as, but not limited to, distinguish them from normal cells. The monoclonal anti early and late-forming diarrhea and flatulence, nausea; Vom body reagents have permitted the isolation and biochemical iting; anorexia; leukopenia; anemia; neutropenia; asthenia; characterization of the antigens and have been invaluable abdominal cramping; fever, pain; loss of body weight, dehy diagnostically for distinction of transformed from nontrans dration; alopecia; dyspnea; insomnia; dizziness, mucositis, formed cells and for definition of the cell lineage of trans xerostomia, and kidney failure. Adverse effects from biologi formed cells. The best-characterized human tumor-associ cal therapies/immunotherapies include, but are not limited to, ated antigens are the oncofetal antigens. These antigens are 25 rashes or Swellings at the site of administration, flu-like expressed during embryogenesis, but are absent or very dif symptoms such as fever, chills and fatigue, digestive tract ficult to detectin normal adult tissue. The prototype antigen is problems and allergic reactions. Adverse effects from hor carcinoembryonic antigen (CEA), a glycoprotein found on monal therapies includebut are not limited to nausea, fertility fetal gut and human colon cancer cells, but not on normal problems, depression, loss of appetite, eye problems, head adult colon cells. Since CEA is shed from colon carcinoma 30 ache, and weight fluctuation. Additional undesired effects cells and found in the serum, it was originally thought that the typically experienced by patients are numerous and known in presence of this antigen in the serum could be used to screen the art. Many are described in the Physicians 'Desk Reference patients for colon cancer. However, patients with other (56 ed., 2002). tumors, such as pancreatic and breast cancer, also have elevated serum levels of CEA. Therefore, monitoring the fall 35 EXAMPLE 1. PURIFICATION OF ARP FROM and rise of CEA levels in cancer patients undergoing therapy BOVINE SMALL INTESTINE EXTRACTS has proven useful for predicting tumor progression and responses to treatment. Material Several other oncofetal antigens have been useful for diag nosing and monitoring human tumors, e.g., alpha-fetopro 40 Source of intestines used in the examples: large animal tein, an alpha-globulin normally secreted by fetal liver and intestines were obtained from freshly slaughtered cows. yolk sac cells, is found in the serum of patients with liver and These were routinely obtained from Bellingar Packing, Ash germinal cell tumors and can be used as a marker of disease ley, Mich. Ratand mouse intestines were obtained from Spra Status. gue Dawley rats or BALB-c mice, purchased from Harlan 45 Industries, Indianapolis, Ind. Pig intestines were obtained Computed Tomographic (CT) Scan from Bellingar Packing. Some pig and cow intestines were CT remains the choice of techniques for the accurate stag also obtained from Bain’s Packing & Refrigeration, Howell, ing of cancers. CT has proved more sensitive and specific than Mich. any other imaging techniques for the detection of metastases. Methods 50 Measurement of Putative Biomarkers Enriched or Isolated ARP The levels of a putative biomarker for risk of a specific Extraction and Centrifugation cancer are measured to monitor the effect of the molecular Bovine small intestine ileal sections (25 feet or 40 feet) complex of the invention. For example, in Subjects at proximal to the cecum were excised from animals immedi enhanced risk for prostate cancer, serum prostate-specific 55 ately after slaughter. Each section was slit down its length and antigen (PSA) is measured by the procedure described by the contents flushed free. Washing was initiated at the slaugh Brawer, M. K., et. al., 1992, J. Urol., 147: 841-845, and terhouse in phosphate buffered saline (PBS) plus ciprofloxa Catalona, W. J., et al., 1993, JAMA, 270: 948-958; or in cin (10 mg/L) and was completed in the laboratory. The tissue Subjects at risk for colorectal cancer, CEA is measured as was transported on ice to the laboratory where it was cut into described above in Section 5.10.3; and in subjects at 60 1 foot long Strips and the external fatty deposits removed. enhanced risk for breast cancer, 16-hydroxylation of estradiol Each section was washed in a solution of PBS containing is measured by the procedure described by Schneider, J. et al., ciprofloxacin. This was followed by serial washes through 6 1982, Proc. Natl. Acad. Sci. USA, 79: 3047-3051. beakers (on ice) containing PBS without ciprofloxicin. Each section was blotted free to remove excess fluid and then the Sonogram 65 Soft tissue was scraped from the serosal casing and the serosa A Sonogram remains an alternative choice of technique for was discarded. The Soft tissue was minced in a kitchen-vari the accurate staging of cancers. ety motorized mini food processor in 50 mLaliquots using 10 US 7,393,534 B2 75 76 start and stop cycles of the processor. An equal volume of cold cells on 10 kD disc membranes. The resulting material, 30 mM Hepes was added and the mixture was homogenized denoted Stage 1 BEX (Bovine Extract), was sterile-filtered in a 55 mL glass homogenizer tube with a Teflon pestle. After with Serum AcrodiscTM 0.22 um (Gelman Catil 4525) filters, five down-up cycles in the homogenizer, the material was flash frozen, and stored at -80° C. awaiting Stage 2 BEX centrifuged at 18,000 rpm (25,000 G) in a refrigerated SS-34 purification. Sorvol rotor. The resulting liquid supernatants were prefil tered through a double layer of Millipore pre filters SuperdexTM Size Exclusion (#API507500) housed in a sterilizing stainless 90 mm filter Gel filtration chromatography was used to further purify holder to remove fatty particulates in suspension. The filtrate Stage 1 into Stage 2. A 1200 ml column (Amicon Catil was flash frozen in liquid nitrogen and stored frozen at -80° 10 9644100) was packed with Superdex-75 prep grade (Pharma C. cia Catil 17-1044-01) and equilibrated in 15 mM Sodium Phosphate, 120 mM Sodium Chloride pH 7.0 (PBS). PBS Pretesting of Prefiltered BEX buffer was used for isocratic elution of fractions. Prior to Preparation: loading, Stage 1 BEX was concentrated in CentriPrep-10TM (a) Prepare 2.0 L of 1xPBS buffer from 10xPBS and accli 15 (Amicon Cati 4304) to 35-40 mg/ml, filtered using Serum mate to 4°C. AcrodiscTM 0.22 um (Gelman Catil 4525). Glycerol was (b) Soak an appropriate number (one greater than the num added to a final concentration of 5%, and the sample was ber of intestines to be pre-tested) of 100 mm long, 10 loaded onto the column at 0.1-1% of column volume (1.2-12 mm wide regenerated cellulose dialysis membranes, ml). Chromatographic parameters used: 280 nm, 1.0 Absor MWCO 6-8000, in rodeionized water until pliable. bance full scale, 4.0 ml/min (18-26 psi) flow rate, 5 cm/hr (c) Defrost the 1.2 ml/intestine aliquots of prefiltered BBX chart speed and 2.0 minutes/8.0 mL fractions size. and the intestinal mix. Active fractions were pooled and concentrated to ~40 Test for Activity: mg/ml using Amicon Stir CellTM Model #8200 (Amicon Cath (a) Dialyze 1.0 ml of the individual intestinal extracts and 5123) with a YM10TM disc membrane (Amicon Catil 13632). intestinal mix against 1 L of PBS for 3 hours at 4°C. 25 Stage 2 was sterile filtered and stored at -80°C. for the next (b) Change buffer and dialyze overnight. purification step. (c) Transfer dialysates to sterile 1.2 ml cryotubes. (d) Prepare individual samples for the dendritic cell activ DEAE ity test by diluting the dialyzed stock BBX 1:10 (1 part A DEAE-Sepharose column (1.5x6 cm, DEAE-Sepharose BBX plus 9 parts sterile 0.1% BSA in PBS). From each 30 Fast Flow, Pharmacia Biotech Inc. Piscataway, N.J.) was stock serially dilute 1:2 (501 ul stock plus 50 ul 0.1% equilibrated with 30 mM Hepes and 0.1 MNaCl, pH 7.4. The BSA/PBS) 4x. sample obtained from Superdex 75TM (Stage 2) in PBS was (e) Graphically compare pre-test samples with an appro applied to the column. The column was then washed with 100 priate pre-test control with predetermined activity. ml of 0.1 MNaCl in 30 mM Hepes and then 200 ml of 0.2 M (f) Flash freeze and store pre-test leftovers in the -80° C. 35 NaCl in the same buffer. The column was eluted isocratically freezer. with approximately 120 ml of 0.3 M NaCl in Hepes buffer. Process Control: Fractions were collected every 4 min at 2.5 ml/fraction. The Discard extracts from intestines generating dendritic cell column was further eluted with 0.5 MNaCl in 30 mMHepes. activity less than 20% of the pre-test control. Fractions were analyzed by the NK-mediated cytotoxicity assay and SDS-polyacrylamide gel electrophoresis. ProcipitateTM and CleanasciteTM 40 The initial reductions of non-active macromolecules were facilitated with two commercial bulk-processing products Purification of ARP: Modified Method from Ligochem, Inc. (CleanasciteTM and ProcipitateTM). Three parts extract was treated with one part CleanasciteTM, Extraction and Centrifugation which is a non-ionic adsorbent which selectively removes 45 See the same step in section 6.2.1. lipids, floating cells, cell debris and mucinous impurities. After centrifugation, the resulting Supernatant was treated Pretesting of Prefiltered BEX with the synthetic, water insoluble, anionic polyelectrolyte See the same step in Section 6.2.1. ProcipitateTM at a ratio of four parts supernatant to one part ProcipitateTM to remove selective proteins. This was spun at 50 Ammonium Sulfate Fractionating low speeds according the manufacturers recommendation The extract Supernatant was brought to 45% ammonium and the supernatant retained for further purification. sulfate at 4°C. using solid chemical and stirred for 1 hour. This was centrifuged in 250 mL centrifuge bottles at 13,000 Ultra-Filtration RPM (17,300 G) for 30 minutes and in 40 mL centrifuge A small sample of each week's batch was purified for 55 tubes at 15,000 RPM (17,500 G) for 20 min. The supernatant testing using an Amiconstirred cell (series 800) initially fitted was decanted into a 4 L. glass beaker and the pellets were with a 300 kD disc membrane to remove impurities greater discarded. The supernatants were brought to 80% saturation than 300 kD in size followed by a 10 kD disc membrane to using Solid ammonium sulfate, centrifuged as above, and the remove impurities less than 10 kD as well as to concentrate supernatant discarded. The pellets were dissolved in 1.5M the sample. During this procedure phosphate buffered saline 60 Ammonium Sulfate/50 mM Na Phosphate (pH 6.8) after was used to wash the >300 kD retentate. The resulting 10 to resuspending completely within the bottles or tubes. Non 300 kD sample was tested on the NK assay and/or in mice. dissolved material was removed by centrifugation in 250 mL If the sample was active, the whole batch was processed in centrifuge bottles at 10,000 RPM (10,200 G) for 30 minutes. a large-scale procedure. The solution was filtered through 300 The supernatants were pre-filtered through a double layer of kD filter plates in a Millipore PeliconTM ultrafiltration system 65 Millipore pre-filters (AP1507500) housed in a sterilizing and the <300 kD flow-through material together with its 30 stainless 90 mm filter holder. Samples were taken after each mM HEPES wash(es) were concentrated in AmiconTM stirred step for quality control monitoring. US 7,393,534 B2 77 78 Hydrophobic Interaction Column (Hereinafter “HIC) Step the bound material was eluted with 1 mM EGTA (ethylene Elution Chromatography glycol-bis(B-aminoethyl ether)-N.N.N',N'-tetraacetic acid), A 5 cmx20 cm chromatography column, packed with 300 in 10 mM Tris, pH 7.0. The flow through fraction, which mL phenyl Sepharose 6 fast flow (high Substitution)—Amer contained all DC activity, was used for the next step. sham Pharmacia Biotech 17-0973-05, was equilibrated with 1.5Mammonium sulfate/50 mMSodium Phosphate buffer at HIC Gradient Chromatography pH 6.8. At room temperature the pre-filtered ammonium sul All high performance liquid chromatography (hereinafter fate treated BBX was pumped onto the columnata rate of ~18 “HPLC) was performed with a Waters Associates (Milford, mL/min. Flow was collected from the column until no longer Mass.) 626 LC system and a Linear Instruments (Reno, Nev.) cloudy (~1200 mL). The column was washed with 900 mL of 10 1.5M Ammonium Sulfate/50 mM Sodium Phosphate. The 200 Uvis absorbance detector at either 280 nm (HIC) or 215 active material was eluted with 700 ml of 0.5 M Sodium nm (DEAE and C-8 separations). All chemicals are Ultrapure Phosphate buffer. Bioreagent grade from J. T. Baker (Phillipsburg, N.J.) unless The eluted material was prepared for DEAE chromatogra otherwise noted. phy by dialysis against 4 L of 0.2M NaCl/10 mM Na Phos 15 Sample Volume was measured and mixed with an equal phate (pH7.4) at 4°C. with buffer changes after 3 and 6 hours volume of 3.0 M ammonium sulfate (ACS grade, Mallinck followed by a final dialysis overnight. rodt, Paris Ky.) solution in 50 mM phosphate buffer, pH 7.2. The sample was loaded via the solvent inlet manifold on the DEAE Step-Elution Chromatography HPLC. Solvents are: Buffer A, 1.5 Mammonium sulfate in 50 After the sample was dialyzed against 0.2 MNaCl, 10 mM mM phosphate buffer, pH 7.2 and Buffer B, 50 mM phosphate Sodium phosphate, pH 7.4, the sample was filtered through a buffer, pH 7.2. Samples were applied to 21.5x150 mm Phe 0.45 um membrane to remove particulates, and then applied nyl-5PW column, (TosoHaas, Montgomeryville, Pa.) with 13 to the DEAE-Sepharose column (0.5x8 cm). The column was um particle size, equilibrated in Buffer A at a flow rate of 4 washed with about 800 mL of the same buffer (0.2 M NaCl, mL/min at room temperature. At this time a linear gradient 10 mM Na Phosphate, pH 7.4). When the absorbance at 280 25 nm was downto baseline, the bound material was eluted with was performed from 0-100% Buffer B over 90 minutes at 4 about 400 mL of the eluted buffer (0.5 M NaCl, 10 mM Na mL/min. Fractions were collected at 2.5 minutes using a Phosphate, pH 7.4). The eluted fraction was concentrated to fraction collector (LKB-Frac-100, Pharmacia, Piscataway about 20 mL by Amicon stir cell with YM 10 membrane. The N.J.). Fractions were diluted with phosphate buffered saline sample was divided into two halves and flash frozen in liquid 30 (PBS) and assayed on the DC assay. Active fractions were nitrogen and then stored at -80° C. pooled and dialyzed in Spectra/Por 1 Membrane dialysis tubing, 6-8000 MWCO (Fisher, Pittsburgh Pa.) against two SuperdexTM Size Exclusion changes of PBS. Gel filtration chromatography was used to further purify the material from the DEAE step-elution. A 1200 mL column DEAE Gradient Chromatography 35 (Amicon Catil 9644100) was packed with Superdex-75 prep Samples were loaded via the solvent inlet manifold on the grade (Pharmacia Catil 17-1044-01) and equilibrated in 15 HPLC. Solvents are: Buffer C, PBS, pH 7.2 and Buffer D, 0.5 mM Sodium Phosphate, 120 mM Sodium Chloride pH 7.0 M. NaCl in 15 mM phosphate buffer, pH 7.2. Samples were (PBS). PBS buffer was used for isocratic elution of fractions. Prior to loading, the samples was filtered using Serum Acro applied to DEAE-5PW column, 10um bead, 8x75 mm (Toso discTM 0.22 Lum (Gelman Catil 4525) and loaded onto the 40 Haas) equilibrated in Buffer C at 1 mL/min at room tempera column at 0.1-1% of column volume (1.2-12 ml). Chromato ture. The column was flushed with Buffer C until a stable graphic parameters used: 280 nm, 1.0 Absorbance full scale, baseline was established. At this time a linear gradient was 4.0 ml/min (18-26 psi) flow rate, 5 cm/hr chart speed and 2.0 performed from 0-100% Buffer D over 90 minutes at 1 minutes/8.0 ml fractions size. The sample was flash frozen in mL/min. Fractions were collected by hand to capture either liquid nitrogen and stored at -80° C. 45 individual peaks or at five-minute intervals using the fraction collector. Fractions were diluted with PBS and assayed on the Concentration of SuperdexTM Samples and Calmodulin DC assay. Removal The active regions from 7 batches were thawed and pooled, C8 Reverse-Phase Chromatography which typically generated approximately 1,200 mL. The 50 Samples were loaded either via the solvent inlet manifold sample was centrifuged at 10,000 rpm (10,200 G) for 30 min for large volumes (>5 mL) or via the injector loop for smaller with GSA rotor to remove any precipitate. The sample was samples. Solvents are: Buffer E, 5% Acetonitrile (HPLC then filtered with 0.45 um syringe filter and applied to the grade, Burdick and Jackson, Muskegon Mich.)/95% water DEAE-Sepharose column (0.3x5.5 cm). The column was (HPLC grade, Burdick and Jackson)/0.1% Trifluoroacetic washed with 0.2 M NaCl, 10 mM. Na Phosphate, pH 7.4 55 (about 200 mL). The bound material was eluted with 1 M acid (TFA); and Buffer F, 90% HPLC Acetonitrile/10% NaCl, 0.1 mM CaCl, 10 mM Tris, pH 7.0. The protein peak HPLC water/0.08%TFA. Samples were applied to C8, 5um was collected in approximately 50-60 mL and was dialyzed bead, 300 A, 4.6x150 mm column (Vydac 208TP5415, The overnight against 1 liter of 1 MNaCl, 0.1 mM CaCl2, 10 mM Separations Group, Hesperia Calif.) equilibrated with Buffer E at 1 mL/min at 55° C. The column was flushed with Buffer Tris, pH 7.0. 60 To remove calmodulin and other calcium-binding proteins, E until a stable baseline was established. At this time, a linear an HIC column (1.5x6 cm) was equilibrated with 1 MNaCl, gradient was performed from 30%-55% Buffer F in 150 min 0.1 mM CaCl, 10 mM Tris, pH 7.0 before the sample was utes at 1 mL/min and 55° C. Fractions were collected either at loaded onto the column. After applying the sample, the col five-minute intervals or for individual peaks. Fractions were umn was washed with the same buffer. The flow through 65 diluted with PBS and assayed for activity on the DC assay. fraction was collected and constituted about 75 to 80 ml. The The bulk of the fractions were flash frozen in liquid nitrogen column was washed with the same buffer (about 100 mL) then and stored at -80° C. US 7,393,534 B2 79 80 Results To reduce the macromolecular load prior to SuperdexTM size-exclusion chromatography, two bulk processing steps Enriched or Isolated ARP: were incorporated. The first step used an HIC column, which Small intestine tissues were obtained approximately once is compatible with the re-dissolved pellet of the ammonium per week, typically from six freshly slaughtered cattle. The Sulfate fractionating process. A large amount of inactive extraction & centrifugation, ProcipitateTM & CleanasciteTM, material, particularly nucleic acid, was removed in the flow and ultra-filtration steps produced a soluble mixture of mac through and wash. The active material was eluted after a romolecules with the approximate size range of 10-300 kD. change to a buffer free of ammonium sulfate. The elution was This material was non-toxic, could cure S-180 tumors in processed in a similar fashion on DEAE ion exchange chro mice, induce NK cytotoxicity in vitro, induce IFNY in NK 10 matography where the flow-through and wash were dis cells in vitro, and induce IL-12 release from dendritic cells in carded. The elution at higher NaCl concentration was vitro. The solution was sampled for mycoplasma and bacte retained, concentrated on a membrane pressure cell, and rial testing (MSU Animal Health Diagnostic Laboratory) and added to the SuperdexTM column and the activity was eluted measured for LAL reactivity. It was prepared for size exclu in a region of low absorbance (FIG. 2(A)). The total recovery sion (Stage 2) purification by concentration on ultrafiltration 15 of activity, for a typical batch (I week) is shown in FIG. 2(B). membranes. Separation on the SuperdexTM molecular sieving Specific activity (with respect to protein content) was deter column yielded activity confined to a single region, which is mined on the retained fractions from each step in the purifi centered around 20-30 kD. cation (Table 3). Approximately 7000-fold purification was To enhance purity of Stage 2 (size exclusion purification) reached at the end of the Superdex step. material and reduce any endotoxin burden, the concentrated active fractions of the 1st SuperdexTM separation were TABLE 3 applied to the column again. The central portion of the peak in Purification steps that were performed on each batch the re-run was retained and denoted BBX-01 and used in the 1st leg of the phase 1 clinical trial approved by the FDA. This Activity Activity Total Sepcific material was sterile filtered and concentrated on ultrafiltration 25 Purification Step Location Recovery Protein Activity membranes prior to sterile filling in dosage forms at the GMP Extract Supernatant 1OO 2SSO4 1.O formulating facility at the University of Iowa. 45% Ammonium Sulfate Supernatant 102 11684 2.2 cut DEAE ion exchange chromatography was used to purify 80% Ammonium Sulfate Pellet 8O 11035 1.8 Stage 2 material into Stage 3 material. The active portions of 30 cut the re-run material from the SuperdexTM separations were Hydrophobic Interaction PBS Elution 59 6683 2.2 applied to a freshly cleaned DEAE SepharoseTM column. Due Step Elution DEAE Step Elution Salt Elution 65 460 36.2 to the high ionic strength (0.15M) of the Stage 2 material, the Size Exclusion Selected 62 2.3 682O.O majority of the protein passed through the column unbound. (Superdex TM) Fraction This is denoted Fraction A. The bound material was eluted in Chromatography two steps of increasing ionic strength (0.2M and 0.5M). The 35 first elution is denoted Fraction Band the second, Fraction C. Two types of activity have been detected in Fractions A and C, Since bacterial endotoxin was always a contaminant from respectively, but Fraction B has routinely exhibited weak small intestine extracts, the endotoxin levels were monitored activity. The activity of Fraction A is associated with induc with the LAL assay (see section 13) at each step in the puri tion of high levels of IFNY, which was proved to be IL18. 40 fication. This is shown in FIG. 2(B) for the steps shown in Fraction C contains the majority of the NK-inducing activity FIG.2(A). Endotoxin levels are reduced approximately 1000 and antitumor activity and is the only fraction that activates fold resulting in final levels that are well below those that can dendritic cells. The C fraction contains only 5% of the protein interfere with the DC assay (see section 12.3, threshold of and is denoted BBX-01c. This material was used for the 2nd 1000 U/mL for minimal DC activity). 45 To obtain enough material for final purification, pools were leg of the human phase 1 clinical trial. created that contained the extracts from 21 cattle intestines. Purification of ARP: Modified Method These were pre-selected for high activity in the crude homo The modified purification procedures were developed to genate Supernatants and processed three-at-a-time. Seven enhance recovery and specific activity. The modified proce batches of 3 intestinal preparations were combined, concen dure increased the yield of ARP at the final step possibly by as 50 trated, cleared of calcium binding proteins and designated as much as 10°-10 fold. The first part of the procedure used an a pool. Four such pools were created. The first pool was used implementation of a long-standing method to process crude for method development and the second pool was used to material utilizing ammonium Sulfate fractionation. It was obtain the first sequenceable product. done in two steps. The first step removed approximately 50% To obtain a purified product, a pool was initially separated of protein contaminants together with some other material, 55 on an HPLCHIC column, one-half of the pool at a time. The and inhibited proteolysis of ARP by internal proteases, while activity was located near the end of the chromatogram (see the second step precipitated ARP allowing to reduce the vol FIG.3(A)), which is indicative of a moderately hydrophobic ume and speed up further purification steps, also minimizing molecule. Some earlier activity was occasionally detected, proteolysis. Prevention of proteolysis was most probably but was weak and sporadic. The active fractions were dia responsible for the yield increase and for the increase in the 60 lyzed to remove ammonium Sulfate and were applied to an average length of sequenced ARP peptides, which allowed HPLC DEAE column in 0.15M NaCl. A NaCl gradient was identification of the Eimeria surface antigen by database applied and the activity eluted with a major peak near 0.3 M searches. Experiments to find the best conditions yielded 45% ionic strength. The chromatogram and activity are shown in saturation ammonium sulfate for the first precipitation, fol FIG. 3(B). Three DEAE runs were required to process the lowed by 80% saturation for the second precipitation. The 65 active material from the HIC. The pooled active region was activity was recoverable in the final precipitation pellet, applied to the C8 reverse phase HPLC column and eluted with which was resuspended for further purification. an aqueous-acetonitile gradient (FIG. 3(C)). The activity US 7,393,534 B2 81 82 eluted near 40% acetonitrile on a slow, shallow gradient elu consists of 90% HPLC grade Acetonitrile, 10% HPLC Grade tion protocol. Fourteen separations were performed to pro water, and 0.985% TFA. Absorbance was monitored by a cess the material from the DEAE separation. The activity was Kratos Analytical Spectroflow 783 detector at 214 um. N-ter associated with two major neighboring peaks and two or three minal sequencing of selected peptide fragments was per minor peaks or shoulders. This appeared to represent minor 5 formed on a PerkinElmer/Applied Biosystems 494 cIC Pro variations in the molecule, but these differences have not been cise Protein Sequencer which utilizes Edman Degradation quantified. The question of purity of these active fractions chemistry. was addressed by plotting activity versus absorbance along a finely-sampled separation. This is shown in FIG. 3(D). The Samples for MS-MS analysis were isolated and digested as linear regression line is reasonably robust and the intersection 10 described above. Two samples were sent to M-Scan Inc. near Zero indicates the lack of contaminating molecules. (West Chester, Pa.); a Trypsin autodigest and digested The first major peak, denoted alpha, was obtained from sample. Each sample was applied to a C18 ZipTip and approximately one-half of the C8 separations and was con desalted by washing with 0.1% aqueous formic acid. Peptides centrated by roto-evaporation, digested with trypsin, and Sub were eluted using a solution of 60% acetonitrile, 40% water mitted for peptide separation and Edman sequencing at the 15 containing 0.1% formic acid. The peptide mixture obtained MSU Macromolecular Structure Facility. Considerable from each sample was analyzed initially by positive ion ESI amount of the protein was apparently lost during the concen MS using a Sciex Q-Star/Pulsar mass spectrometer. The trating step. Four peaks generated sequence information, one sample was introduced using a nanospray needle and data was of them containing two sequences from different regions of collected in the MCA mode. The same instrument and sample the bovine ARP. The information was still sufficient to begin introduction method were used for the MS/MS sequencing. searching database by using PattinProt program (PattinProt The amino acid sequences from the tryptic peptides, gen searching can be done, e.g., at the website philibcp.fr (select erated by the Edman and MS/MS procedures, are shown in NPS(a), and then PattinProt)) and led to identification of ARP Table 4. Several robust reads were obtained from various as one of the proteins in the mixture. Since the concentrating peaks using the Edman procedure, although some reads were step involved the multiple transfer of collected fractions into 25 the convolution of two or more separate sequences and most a single tube, we considered that the loss of material must were contaminated, resulting in multiple reads in almost have resulted from protein sticking to the plastic conical every position of every peptide. Most commonly used pro tubes. Experiments demonstrated that to move the molecule grams, like BLAST, could not perform a search with such between tubes required the addition of acetonitrile. Incorpo complicated data. Therefore, the sequences were assigned by rating that methodology change separately on the 2" half of 30 usage of PATTINPROT program (Network Protein Sequence the alpha peak and the 1 half of the second major peak (beta), Analysis (NPS(a): TIBS 2000, 25: 147-150). Multiple amino generated much higher tryptic peptide quantities. These pep acid reads at each position were searched by the program at tides provided sufficient sequences to extend searching the the desired level of similarity (usually higher than 70%), and available public databases (e.g., BLAST, PattinProt) for a list of proteins matching those parameters was generated. sequence matches. 35 Since the list normally contained several dozen of different entries, it was searched again with sequence of a second EXAMPLE 2 peptide. This procedure usually produced a list containing only the entries related to sporozoite antigen. In case any Identification of Sporozoite Antigen Protein identifiable contaminants were on the first list (trypsin, kera 40 tin, albumin), their sequences were Subtracted from the amino Protein sequencing services were provided by the Macro acid reads and the search repeated with the remainder reads. molecular Structure Facility at MSU (Edman procedure) and Once the Eimeria sporozoite antigen was considered as a M-Scan Inc., West Chester, Pa. (MS-MS procedure). likely candidate, some of the multiple reads were deconvo Tryptic peptides for Edman sequencing were prepared luted by finding a match for one possible sequence and then using the following procedure. Active fractions, typically 1 45 determined if the remaining sequence also matched a differ mL, from several C8 separations were placed in microcentri ent part of the protein. The strategy was successful in multiple fuge tubes and evaporated to near dryness under vacuum cases, and resulted in generating several most probably pep (Centrivap Concentrator, Labconco, Kansas City Mo.) at 35° tide sequences for bovine ARP. C. All sample tubes were washed three times with 200 uL of 60% HPLC acetonitrile/40% HPLC water/0.1% TFA, each 50 TABLE 4 time reducing the volume to near dryness. After the third wash, all fractions were combined into a single microcentri Probable sequences generated from Edman fuge tube and were washed twice with 500 uL of HPLC water tryptic peptides or MS/MS analysis again reducing to near dryness after each wash. The combined Fragment source Generated sequence sample, reduced to approximately 15 LL, was diluted to 125 55 c. 18, C-218/318' EWLVDTGKVF uL in the digestion buffer (100 mM Tris (reagent grade, SEQID NO:31 Sigma-Aldrich, St. Louis Mo.) pH8.4.2 mM CaCl). Trypsin MS, MS LVDTGK (sequence grade, Sigma-Aldrich) was added to 2% (W/w) and SEQID NO:32 the sample incubated at 37°C. for four hours. The pH of the C225 VFAGGVASIADG SEQID NO:33 sample was checked and an additional 50 uL of digestion 60 Cl225 AGYQIESVQEDNGTVQ buffer was added. Digestion was halted by flash freezing the SEQID NO:34 sample in liquid nitrogen after 18-20 hours of digestion. C235, C.232/330, C128,332 *MFGASTDSGGDPNAELVQYN SEQID NO:35 Purification of tryptic peptides was performed at the MSU MS/MS2 3MFGASTDS Macromolecular Structure Facility on a microbore reversed SEQID NO:36 phase HPLC using a Vydac C18 column, 5um,300 A, ID 800 65 C-213/B14, MS/MS APDGVYIGGVK um, using a linear gradient from 5% H to 70% H in 160 SEQID NO:37 minutes. G buffer consists of 0.1% TFA in water. H buffer US 7,393,534 B2 83 84 standable contaminants, keratin, trypsin, and bovine albumin, TABLE 4-continued though a small number of the generated sequences remained non-assigned. Probable sequences generated from Edman tryptic peptides or MS/MS analysis EXAMPLE 3 Fragment source Generated sequence Extraction of Activity from e. tenella Oocvsts C18, C218,118 GGGFLIK SEQID NO:38 C250,346, C249,345 TPNENIAIALYDEEKEQNKA Oocysts can be obtained from intentionally infected ani SEQID NO:39 10 mals, from barnyard soils, or from feces of infected animal. MS, MS TPNXXXXIALYDEEKEQNK Methods of isolating oocysts are known in the art. See, e.g., SEQID NO:40 Tomley, Methods: A companion to Methods in Enzymology C253,149, C.140, C-37 ADALTTALNFADFLYQ 13: 171-176 (1997), which is incorporated herein in its SEQID NO:41 entirety by reference. For example, one protocol that can be 'Reads of combined peptides are marked with “?, e.g., c.18/B18. 15 used to obtain sporulated oocysts is as follows: infect 6- to (MS/MS method cannot distinguish between I and L, and often between Q 8-week old Light Sussex chickens by oral dosing with and K, so all the positions with one of those amino acids are not certain from between 10 and 6x10 sporulated oocysts and recover MSMS reads. Since tryptic peptides were used, the position preceding the first readable oocysts from the ceca 7 days later using either enzymatic or amino acid is either R, or K, but it cannot be conclusively determined which chemical treatment (e.g., remove and cut ceca, add phos one of these two is the correct amino acid. phate-buffered distilled water, pH 8.0, and homogenize to a fine pulp with a commercial blender; add trypsin (Dificon Possible errors in amino acid assignments cannot be elimi 1:250 powder) to a final concentration of 1.5% w/v; incubate nated, however, due to the quality of some of the data. Table at 41° C. for 30 min, strain through two thicknesses of muslin, 5 lists possible errors (where plain text represents highly and centrifuge at 1000 g for 10 min; discard the supernatant, confident data, i.e., multiple read and multiple samples: 25 replace with a similar volume of saturated sodium chloride underlined text represents lower confidence; and bold text Solution, mix well, and centrifuge again; collect the oocysts represents lowest confidence): from the top of the Salt using a syringe fitted with a cannula or a long needle; and wash them repeatedly by centrifugation TABLE 5 and resuspension in buffered water) and then sporulate the 30 oocysts (e.g., Suspend oocysts in 2% (w/v) potassium dichro Possible errors in SEQID NO:3, 4, 5, 6 and 7 mate and incubate at 28-30° C. for 48 hours with forced SEQID NO:3 Glu Trp Leu Val Asp Thr Gly Lys Val Phe Ala Gly Gly aeration; remove the potassium dichromate by repeated cen Val Ala Ser Ile Ala Asp Gly trifugation and dilution in water, add sodium hypochlorite to SEQID NO:4 Arg Met Phe Gly Ala Ser Thr Asp Ser Gly Gly/Asn Asp a final concentration of 10% (v/v) and incubate on an ice bath Pro ASn Ala Glu Leu Val Lys Ala Val Gly/Phe Tyr/Ala 35 for 5-10 min: centrifuge and mix the pellet with saturated Gln/Gly Ile? Gly Glu/Val Ser/Ala Val/Ser Gln/Ile Glu/Ala Asp Asn/Gly? sodium chloride solution; overlay the tube contents with dis SEQID NO:5 Gin Ala Ile Val tilled water and centrifuge for 5 min; collect clean, bacteria SEQID NO 6 Ala Pro Asp Gly Val Tyr Ile Gly Gly Val Lys' free oocysts from the interface of the salt and water into sterile SEQID NO.7 Gly/Glu Gly/Trp Gly/Leu Phe, Val Leu/Asp Ile/Thr distilled water and wash repeatedly by centrifugation into Lys? Gly Thr Pro ASn Glu ASn Ile Ala Ile Ala Leu Tyr Asp Glu Glu Lys Glu Glin ASn Lys Ala Asp Ala Leu Thr 40 water). Thr Ala Leu Asn Phe Ala Asp PheLeuTyr Gln Approximately 3x10 sporulated oocysts (a gift of Dr. Ray Fetterer, USDA) was suspended in 9.0 mL of PBS and cen "Lower confidence: two reads convoluted in a single sample. The read with trifuged for 10 minutes at 200xG. The supernatant (“wash’) identity to an Eimeria acervulina conserved region is assigned here (see SEQ ID NO:4 for other read). was removed and retained. The pellet (-0.5 mL packed vol “Lowest confidence: two reads in one sample. The second choice in each 45 ume) was suspended in 0.5 mL 30 mM Hepes and homog case was assigned to SEQID NO:3, due to match in Eimeria. The first enized on ice with a glass homogenizer for a total of approxi choice is assigned here, due to partial match to Eimeria. Lowest confidence: Low S/N (signal to noise ratio) and derived from only mately 15 min (prep 1). Most of the oocysts were broken, one sample. releasing sporocysts that remained largely intact. A few less “Lower confidence: slightly lower S/N, but no other amino acid reads rigid, Smaller structures (perhaps sporozoites) were observ present for these positions. Lower confidence: obtained from only one sample, but S/N is good. Lowest 50 able. Continued homogenization did not appear to apprecia confidence: deconvoluted data from two samples containing two sequences. bly increase the number of these structures. The homogenate The second choice in each case was assigned to fragment 1, due to match was transferred to a microcentrifuge tube and centrifuged for with Eimeria. The corresponding region in fragment 1 was also corroborated by MS/MS data making it high confidence. The first choice from each 20 minutes at 6000xG. The supernatant (-200 ug/mL protein) double read is assigned here, due to match to Eimeria. This makes the was sterile filtered (0.22 Lum) and designated the soluble pro sequence fairely reliable, but the three Gly have a low S.N. 55 tein extract of E. tenella sporulated oocysts (Eth-1). In a second preparation, the homogenate was divided into The second half of the beta peak was sent to M-Scan Inc. 3 microcentrifuge tubes each with a total volume of approxi for MS/MS sequence analysis. Several peptides were identi mately 500 uL. Each suspension was sonicated with 301-sec fied, and the match to the Eimeria antigen was verified. A ond bursts using a Fisher Scientific Ultrasonic Dismembra rather long variable region was also detected, which still 60 torTM (Model 100), with care taken to keep the homogenate matched the known ARP sequences with low similarity and cooled during the sonication. Approximately 80% of the released sporocysts were disrupted into non-light refractory was therefore assigned to a portion of molecule. “disrupted' sporozoites. The suspensions were pooled and When all peptide sequences were evaluated, evidence for a microcentrifuged for 20 minutes at 6000xG. The resulting homologue to the sporozoite antigen was overwhelming. The 65 Supernatant was retained, sterile filtered, and designated Eth alignment to the Eimeria 19 kD antigen3-1E is shown in FIG. 2. The pellet was retained and stored at 4° C. for future 4. Most of other sequences could be assigned to the under processing (see 3' extract). US 7,393,534 B2 85 86 The starting material for the 3rd extract of Eimeria tenella resulted in almost complete oocyst breakage into sporocysts. sporulated oocysts was the residual pellet of the 2nd extract Sonication resulted in almost complete breakage of all struc preparation. This pellet was stored at +4°C. for 18 days. The tures present except sporocyst walls. pellet material was suspended in 500 uL 30 mM Hepes and (2) Membrane Sedimentation and Washing. transferred to a 2 mL beadbeater tube containing approxi The cell homogenate was spun at 15000 g 15min, 4°C. to mately 1.3 mL of 0.5 mm glass beads (BioSpec Products pellet membranes. This centrifugation regime is sufficient to #11079105). The tube was then completely filled with 30 mM pellet also nuclei, other organelles, pelletable ARP aggregates Hepes and processed in a Mini-Beadbeater (BioSpec Prod and storage granules, ifany. Organelles should be disrupted in ucts Model #3110BX) for 2 cycles of 3 minutes each at 4800 further purification cycles in HB without ZnCl Pellet was cpm. The tube was placed on ice for 15 minutes in between 10 Suspended in 1 ml of HB and centrifuged again. Centrifuga the 3 minute cycles to prevent excessive heating. The Suspen tion/resuspension was repeated once more with and 2-3 times sion was microcentrifuged briefly and the Supernatant was more without 10 mM ZnCl in the suspension buffer. The separated from the glass beads. This Supernatant was micro pellet obtained by this procedure with two washing cycles in centrifuged for 20 minutes at 8000xG and was filter-sterilized the presence of 10 mM ZnCl was used as a crude membrane and tested in the DC/mL12 release assay for activity. 15 preparation in immune-stimulatory activity test, described in The Supernatants from the partially homogenized and Section 12.5.1. washed mixture of broken oocysts, sporocysts, broken spo (3) Release of ARP from the Membranes. rocysts, and sporozoites contained a large amount of DC For purification of the membrane-linked ARP, the final activity in a small amount of protein. As shown FIG.5(A), the pellet was suspended in 100 ul of HB without ZnCl2 and first extract supernatant yielded dilution curves that were incubated at 37° C. 1–4 hours with or without protease inhibi active to low picogram levels of total protein. For a 20 kD tors and phospholipase C from Bacillus cereus, 16 U/ml. protein, this is approximately 50 fM. This assumes that all the (4) Membrane Sedimentation and Washing. protein in the extract is active, which is unrealistic. Therefore, ARP released into solution was separated from the the active molecule is probably active below this level. An insoluble material by centrifugation as described in step 2. unusual second region of activity appeared in all five repli 25 followed by two washing cycles. All three supernatants were cates on two different assay days, which may due to possibil combined. ity that the active molecule forms inactive complexes (dimer, trimer, multimer) with itself or another molecule, such that its (5) C8 Reverse Phase Chromatography. active site is sequestered. Upon Sufficient dilution, this com C8 chromatography was performed as described in Section plex dissociates and activity reappears. 30 6 with linear gradient from 100% buffer E to 100% buffer F in A second extract utilized sonication to facilitate the break 90 minutes. An aliquot from each fraction was assayed for DC age of oocysts and sporocysts. This did substantially change activity after renaturing (FIG. 18). the yield of active material and there was a repeat of the reappearance of activity at high dilution. A third extract, CONCLUSIONS utilizing the Bead BeaterTM to break the cysts with micro 35 beads, generated an anomalously high level of activity in the It was determined that in control samples (without PLC) first DC assay, but Subsequent assays were very similar to significant amount of DC-factor is released into Solution FIG. 6. The amount of dilution required to reach Zero activity spontaneously. Upon addition of external PLC the amount of is truly remarkable. This extended dose/response may be released activity increased in all four experiments, the related to the low dilution “hump' described above. If differ 40 increase ranging from 10-fold to 25%, the data suggesting ent size complexes, with different dissociation constants are that the DC-factor is indeed GPI linked to the outer cell releasing activity, then this long, flat response may result. membrane. Samples of the extracts were separated on C8HPLC using Material released into Solution spontaneously as well as the same protocol as for the bovine extract. The chromato after PLC digestion was pure enough to be analyzed by gram and activity are shown in FIG. 5(B). The position of the 45 reverse phase HPLC chromatography. The region where the activity is shifted to higher retention time compared to the DC-activity eluted (41-55% buffer F) was quite clean, con highly pure bovine molecule. This appears to indicate a taining 2-3 peaks with UV absorbance at 215 nm below 0.01. higher hydrophobicity to the Eimeria molecule, when The position of DC-activity on the gradient was the same for extracted directly from the protozoan. all samples analyzed: Supernatants generated by membrane 50 washing in step 2 and Supernatants generated by membrane EXAMPLE 4 washing in step 4 after incubation at 37° C. for 4 hrs with or without PLC. Crude Membrane Preparation and Purification of the The presence of 10 mM ZnCl2 in the oocyst suspension Membrane-Linked Form of ARP from the during Sonication and membrane washing in step 2 was Sporozoites of e. tenella 55 shown to significantly inhibit the ARP loss in the first two washing steps, the effect, probably, being due to inhibition of the internal phospholipase and to a smaller extent—to aggre Protocols gation of ARP. Less than 1% ARP loss was obtained in the (1) Disruption of Oocysts. presence of ZnCl2 as opposed to ca. 50% loss in the absence 1 ml of suspension of oocysts at 3x10"/ml in homogeniza 60 of ZnCl2. This allows to wash away the intracellular material tion buffer (HB): 50 mM Tris pH 7.4, 1 mM PMSF, 50 ul/ml generated by cell breakage thus obtaining a clean membrane protease inhibitors cocktail and 10 mM ZnCl2 (in some preparation without any loss of DC-factor (ARP) (presence of experiments) was homogenized for 20 minutes in glass ARP aggregates in this preparation is possible). Removal of homogenizer, followed by 15 freeze-thaw cycles (liquid ZnCl, by resuspending the membrane pellet in ZnCl-free nitrogen/37° C.) or by sonication (Fisher Scientific Ultra 65 buffer restores the spontaneous activity release. By washing sonic Dismembrator, model 100) 10 secondsx36-45 bursts on the pellet in ZnCl-free buffer combined with incubation at ice with three freeze-thaw cycles. The homogenization 37° C. for 1 hr about 35-50% of DC-activity of the pellet can US 7,393,534 B2 87 88 be removed into Solution, thus producing a clean preparation with distilled water, and viewed on Transilluminator Model of DC-factor (ARP). The whole procedure takes one day. M-20E (VWR). One major band was spotted, cut from the gel, and cleaned with Wizard PCRTM kit (Promega) using the EXAMPLE 5 manufacturer's procedure. The fragment was eluted from the resin into 50 uL of sterile DDW. Gene Cloning and Expression of emeria tenella The cDNA was inserted in the pCR2.1 TOPO cloning cDNA vector (Invitrogen) in accordance with manufacturers rec ommendations. The individual fragment (4 LL in water) was Cloning and Expression of E. Tenella ARP mixed with 1 uL of vector, incubated for 5 min at room 10 temperature, and placed on ice. For transformation proce Total RNA was obtained from E. tenella by treating ~107 dure, 40 uL of E. coli of either NovaBlueTM competent cells oocysts to Proteinase KTM digestion (Sigma) at a final con (Novagen), or TransforMax EC 100 ElectrocompetentTM centration of 1 mg/mL, in 200LL of buffer containing 50 mM cells (Epicentre) were mixed with 1-2 ul of the above mixture, Tris-HCl pH 7.5, 10 mM EDTA, 1 M guanidine thiocyanate and either electroporated by 1500V. 5 msec in 0.1 cm cuvette (Sigma), and 0.5% of N-lauroylsarcosine (Sigma). The mix 15 (BTX) in Model ECM399 electroporator (BTX) (for EC 100 was shaken for 16 hr at 37° C. in a sealed Eppendorf TM tube cells), or transformed by heat-shock procedure (30 sec, at 43° followed by addition of 120 mg of guanidine thiocyanate, and C., for NovaBlue cells). The transformed mixtures were 1 ml of TRITM reagent (MRC, Inc.). Isolation of total RNA diluted by 500 uL of SOC medium, and incubated for 1 hr at was performed according to the manufacturer recommenda 37° C. The bacterial suspension was then plated at 100 tions based on Chomczynsky and Sachi (1987). The final uL/plate onto pre-heated (37° C.) plates with LB medium pellet of total RNA was washed twice with 75% ethanol, air containing amplicillin (100 g/mL). IPTG and X-gal were dried, and dissolved in 20 uL of sterile distilled, deionized spread over the plate immediately before plating. White colo water (DDW) pre-treated with 0.4% diethyl pyrocarbonate nies (with an insert) were selected and re-plated onto LB (DEP) to inhibit RNases. plates with 40 ug/mL, kanamycinto obtain single colonies. A The first strand cDNA was obtained with SuperScript 25 single colony from each clone was used for analysis of the RTTM (Life Technologies) in accordance with manufacturer insert size by PCR, with primers (M13 forward (-40), and recommendations. About 1 g (6 LL) of total RNA, and 1 Jug M13 reverse) specific for the vector. The conditions for the of oligo(dTo) (Genemed) were mixed together in total Vol PCR were the same as described above, with the exceptions ume 11 u (adjusted with DEP-treated sterile DDW), heated that magnesium concentration was 3 mM and final Volume at 90° C. for 2 min, and placed on ice. To the mixture, 4 uL of 30 was 10 ul, per tube. The fragments were visualized with 5x first strand buffer, 2 uL of 0.1M DTT, 1 uL of a mixture of ethidium bromide, as above. The clones with insert size 0.5- all four dNTPs (at 25 mMeach, Life Technologies) and 30U 0.6 kB were analyzed by PCR with primers (synthesized by of anti RNase (Ambion) were added. This was incubated for Retrogen Inc.) designed to be specific to the E. tenella ARP 2 min at 37° C. followed by addition of 200 U of SuperScript on the basis of comparative analysis of available ESTs: for RTTM and a brief low-speed centrifugation. The reaction pro 35 ward primer AAATGGGAGAAGCAGACACC (SEQ ID ceeded for 100 min at 37° C. followed by 15 min at 70° C. to NO:44), and reverse primer TCCGCGAAGTTAAGAGCT inactivate the enzyme. RNase H(4U, Life Technologies) was GTTG (SEQID NO:45). The PCR program was the same as added for 30 min at 37° C. to digest the RNA from the above with the exception that the annealing temperature was RNA:DNA duplex formed at the previous step. 56° C. at the second stage, and 58° C. at the third stage. The Preparation of double-stranded cDNA was performed by 40 clones that showed a single band in the agarose gel were using PCR with the first strand cDNA, obtained above, and chosen for further investigation. the following primers (Retrogen Inc): forward primer, spe Samples for DC/IL12 assay were prepared by growing cific to the sequences of ARP gene derived from E. tenella selected clones aerobically (shaking for 20 hr at 37°C.) in 3 ESTs, CCAGTTTTTGCTTTCTTTTCC (SEQ ID NO:42, mL of liquid LB medium supplemented with 40 ug/mL of and reverse primer, designed to be complementary to the gene 45 kanamycin within Sterile 15-mL polypropylene centrifuge from several genera related to Eimeria, TARAASCCRSM tubes (Fisher). The bacterial cells were collected into 1.7 mL CTGGTRMAGGTACTC (SEQ ID NO:43), where R=A or SafeSeal MulTITM microcentrifuge tubes (Sorenson Bio G: S-C or G; and M=A or C. A degenerated second primer Science, Inc.) by centrifugation at 10,000xg for 5 min, +4°C., was chosen because of the lack of sequence information for microcentrifuge HSC10KTM (Savant). The supernatant was the C-terminal region of ARP from E. tenella at the time. 50 removed and filter sterilized in Spin-X centrifuge tube with Taq-polymeraseTM (Life Technologies) was used with 2 mM 0.22 Lum cellulose acetate filter (Costar) (5 min at 3,000xg). of MgCl2 and 0.2% BSA. The program performed in PCR The bacterial cells were washed with 0.5 mL of filter steril ExpressTM (Hybaid) was the following: a) one cycle consist ized PBS, re-pelleted at the same conditions as above, and ing of 2 min at 94° C., 45 sec at 50° C., 1 min at 72° C.; b) 20 re-suspended in 0.2 mL of sterile PBS. Bacterial cells were cycles consisting of 30 sec at 92 C., 30 sec at 53° C. (with 55 disrupted by ultrasound in the Sonic Dismembrator Model ramp 1 C./sec), 60 sec at 72° C. (with time increasing 5 100 (Fisher), 3 pulses of 10 sec each at 30 sec intervals; with sec/cycle); c) 15 cycles consisting of 30 sec at 92°C., 45 sec all procedures performed on ice. The samples were clarified at 55° C., 100 sec at 72° C. (with time increase 5 sec/cycle); by centrifugation (10,000xg, 10 min, +4°C.) in HSC10K, and and, d) one cycle consisting of 10 min at 72°C. followed by the Supernatants were filter sterilized in Spin-X tubes as hold step at 4°C. First strand cDNA (0.2 uL) in 19.8 uL of the 60 above. For DC assay, 10 uL from each spent medium, and polymerase mixture was reacted in 0.2 ml MulTI UltraTM Supernatants of disrupted bacteria, was used per each well of tubes (Sorenson BioScience, Inc.). After the completion of the assay. the program, the whole volume was separated at 50 V in 0.8% The recombinant form of ARP generated in E. coli from agarose gel made on 0.5xTAE (Tris-acetate: Acetic Acid, pH the E. tenella cDNA, have approximately the same specific 8.5) buffer using RunOneTM cell and power supply unit (Em 65 activity as purified bovine molecule and ARP extracted from biTec). The gel was visualized by staining for 10 min in E. tenella oocyts. See FIG. 5.50% activity occurs at approxi freshly prepared 0.05% ethidium bromide, briefly washed mately 50 femtomolar active protein concentration. The US 7,393,534 B2 89 90 recombinant material was obtained from the cloning vector, did not result in enhancement in DC activity, arguing that the pCR2.1 TOPO. The vector allows for expression from cDNA foreign part of the recombinant molecule may not impact the placed inframe to Lacz gene. The whole construction as ARP activity. protein fused to B-galactosidase was obtained from the Super For stable expression of ARP in mammalian cells, the E. natant of a Liter of minimal growth media. The Supernatant tenella ARP gene was re-cloned into two vectors: pTracer was purified using DEAE chromatography, with the method CMV2 (Invitrogen) or p3xFLAG-CMV9 (Sigma). Uni-direc described in section 6, and was separated by C8HPLC, using tional cloning into vectorpTracer-CMV2 was performed into the method of section 6. One of two active peaks, containing KpnI and Xbal sites. The fragment for cloning was created by approximately 200 ng of protein, was assayed on the DC PCR amplification of the gene with primers bearing newly assay to yield the response in FIG. 6. 10 created sites Kipn I (in front of start codon) and Xba I (after the stop codon). The ARP gene missing the start codon and cut The cDNA for E. tenella ARP was inserted in the pCR2.1 out of the construction in pMal-p2e vector flanked by HindIII TOPO cloning vector (Invitrogen). Appropriate clones were sites was used for cloning into a Hind III site of the vector selected by their insert size, sequence, and orientation. While p3xFLAG-CMV9. Orientation and in-frame position of the not an optimized expression vector, Some protein was pro 15 gene relative to secretion signal peptide as well as 3xFLAG duced from the insert in a form offusion protein and exhibited peptide were confirmed by complete sequencing of the insert. DC activity. The DNA sequence of the insert, denoted EtU3, Both plasmids were used to transform CHO-KI and S-180 is given in FIG. 7(A). These inserts are being transferred to cell lines with Lipofectamine 2000 (Invitrogen) according to expression vectors for E. coli and eukaryotic cells. The manufacturer recommendations. Individual clones of trans expressed protein will contain a removable tag sequence to formed cells were obtained after selection in growth media facilitate identification and purification. Activities of the containing antibiotic (Zeocine for pTracer-CMV2, and products from two E. coli cultures containing the cloning G-418 for p3xFLAG-CMV9), followed by re-seeding at low vector, with the insert introduced in either direct or reverse cell density into 96-well plate. In both cases the spent media orientation to Lacz promoter, are shown in FIG. 7(B). Only showed high activity on DC-assay (up to 103 dilution) imply the correctly oriented ARP gene exhibited DC activity. This 25 ing that ARP is secreted out of the cells. Analysis of the media demonstrates proof that the activity is associated with this by C8 RP-chromatography revealed the presence of a single molecule because everything else between the two cultures is peak of DC-activity eluting in the position close to that of identical. bacterially expressed ARP. Preliminary calculations showed C8 chromatographic purification of the protein produced that specific activity of ARP expressed in mammalian cells is by the cloning vector in E. coli, growing in minimal mineral 30 comparable to or higher than that of bacterially expressed media, is shown in FIG. 7 (C), with DC activity included in protein. the inserted graph. The activity is weak but is located at the The genes of numerous cytokines, chemokines and growth similar retention time as the activity extracted from the sporo factors which influence the survival and growth of cancer Zoites. The E. coli media was first purified by DEAE chro have recently been incorporated into the gene therapy of matography followed by SuperdexTM chromatography (see 35 cancer. In particular, the gene for interleukin 12 (IL12), has Section 6 for detailed description of the procedures), yielding been introduced into various experimental cancer cells, a highly pure product. The purity is shown as two active peaks resulting in a marked reduction in the tumorgenicity of the on the analytical C8 chromatography column (FIG. 7 (C)). cells and/or the growth of the tumor. Presumably, the IL12 The data Suggest that N-terminal region can be extended released by the cancer cells stimulates a local innate immune significantly without any loss in DC-activity. The ARP mol 40 response which either retards the growth of the tumor or ecule, fused at the N-terminus, to either a portion of B-Galac eliminates the tumor all together. tosidase, maltose binding protein (MalE), FLAG peptide, or The successful isolation and expression of the ARP gene cellulose binding protein (CenA), retained significant activ now makes it possible to propose a similar use in cancer ity. Point mutations at two positions nearby the C-terminal immunotherapy. The use of the ARP gene would likely be end (Y to H, and G to either A, or S) apparently did not change 45 more effective than the use of genes of specific factors (e.g. the DC-activity of the mutant as well. To reveal the impor IL12), since ARP likely induced numerous pathways tance of the C-terminus for activity of the ARP molecule, a involved in DC maturation and activation in addition to IL12 truncated ARP molecule was created by introduction of a release. Therefore, a more “complete' immune response is termination codon 14 amino acids prior to C-terminus by triggered. PCR (primer: CTATGTGAGAGCATCAGCTTTG, SEQ ID 50 An example of the use of the ARP gene in the immuno NO:46). It showed significantly (several orders of magnitude) therapy of cancer is Summarized as follows. The tumor is lower DC-activity compared to full-length control. Both were excised from the cancer patient and put into short term cul expressed from T7 promoter with Shine-Dalgarno ribosomal ture. The cultured cancer cells are transfected with the ARP binding site (RBS) introduced in front of the ATG start codon gene in an exportable expression vector, other potentially by PCR (primer: GAAGGAGTTTGCAAAATGGGAA, 55 useful genes such as cytokines and chemokines may be trans SEQ ID NO:47). Therefore, the missing sequence (TALN fected as well. Lethally irradiated transfected cancer cells are FAEYLHQSGF, SEQ ID NO:48) is important for either re-introduced into the patient. The transfected cancer cells structure or activity of ARP. would release ARP locally in situ which would activate Transferring the cDNA to an expression vector (pMAL nearby DCs to release IL12 and possible other immune pZE, New England Biolabs) have successfully generated 60 modulators. This response could stimulate local immune clones with DC activity. This vector contains the malE gene responses against the transfected cancer cells. Cancer cells (for fusion to maltose binding protein at N-terminus) adjacent are killed and release tumor associated antigens which are to enterokinase cleavage site, which allows rapid separation taken up, processed and presented by DCs, generating a spe and purification of the expressed protein. The fused ARP cific adaptive (memory) immune response against other protein was expressed by IPTG induction, and appeared to be 65 tumor cells existing in the body (primary tumor, metastases, active as checked by DC assay. Release of the ARP protein recurrences). The patient now possesses a permanent immu from the fusion construction by cleavage with enterokinase nity to the cancer. US 7,393,534 B2 91 92 Isolation of E. Tenella ARP from E1 Clone MNaCl, 10 mMNaphosphate, pH 7.0). After the sample was The E. tenella gene coding for ARP was cloned by PCR applied, the column was washed with the same buffer to using forward primer (ATGGGTGAAGAGGCTGATACT remove unbound material as monitored by Abs280. When the CAGG, SEQ ID NO:49) and reverse primer (TTAGAAGC absorbance came to the baseline, a linear gradient (0.2 M-0.6 CGCCCTGGTACAGGTACTCAGCGA, SEQ ID NO:50). 5 M. NaCl in 10 mM. Na phosphate, pH 7.0, 200 ml for each The PCR fragment was ligated to the pET-Blue-1 acceptor buffer) was applied. Fractions of 5 ml were collected. The vector (Novagen) with clonableRT (Novagen) overnight at eluted fractions were analyzed by DC assay, and SDS-PAGE. 16°C. The resulting plasmid was transformed into E. coli host SuperdexTM Size Exclusion Chromatography Nova Blue with carbenicillin/tetracycline and white/blue Final purification and elimination of endotoxin was clone selection. Positive clones were identified by colony 10 PCR using the same forward primer and plT Blue Down accomplished by SuperdexTM 75 chromatography using the primer #70603-3 (Novagen) to assure DNA insert of correct method described in Section 6. Active samples were stored at orientation. The DNA sequence of the insert was confirmed 4°C. instead of freezing. by sequencing. The plasmid of the positive clone was isolated C8 Chromatography for Quality Control and transformed into host strain TurnerTM (DE3) placI 15 Reverse phase HPLC (as described in Section 9) was per (Novagen) for protein expression. One stable clone was des formed on a sample of the pooled active material from the ignated as E1. (See FIG. 20.) Clone E1 was deposited with the Superdex chromatography. American Type Culture Collection on Jul. 15, 2004 and assigned patent deposit number PTA-6120. EXAMPLE 6 Single clone of E1 from agar culture was inoculated in 10 ml LB medium supplemented with 1% glucose in the pres Physical Properties of ARP ence of 50 g/ml carbenicillin and 34ug/ml chloramphenicol. The culture was maintained overnight at 37°C. with shaking During the many years of dealing with an impure prepara at 250 rpm. The culture was then added to 1 liter of the same tion of ARP, it was difficult to obtain information on the medium and allowed to grow for 2.5 hours to absorbance of 25 physical characteristics of the molecule. Initially, for about 0.6-0.8 at 600 nm. Protein expression was induced by example, it was not obvious if it was a protein or another type addition of isopropyl-1-thio-B-D-galactospyranoside (IPTG) of macromolecule. In retrospect, it is clear that the extremely to 0.5 mM final concentration. After 4 hours, the bacteria potent nature of the protein meant that it was always a very were harvested by centrifugation at 8,000 rpm (6,500xg), for minor component in any partially pure preparation. Some 20 min. The Supernatant was removed and saved and the 30 findings related to the nature of ARP molecule are contained pellet was frozen until used. in the following Subsections. The bacterial pellet was resuspended in 30 ml PBS and passed through a French press three times at 1,600 psi to Molecular Weight disrupt the cells. The press cell was rinsed with PBS to Several experiments have indicated that the molecular recover residual cell sample. The sample was centrifuged at 35 weight range of ARP is centered near 20 kD. A large amount 15,000 rpm (17,500xg) with SS34 rotor for 30 minto remove of indirect evidence for this molecular weight has come from cell debris. The supernatant and the pellet were separately gel banding in SDS-PAGE for known active fractions. Simi saved. Total volume of the supernatant totaled to about 38 ml. larly, activity has routinely eluted from sieving chromatogra Ammonium sulfate was then added to the supernatant to 30% phy columns in this range. Direct evidence has been obtained saturation. After 1 hour at 4°C. with stirring, the precipitate 40 from large SDS-PAGE slab gels. Active material has been was removed by centrifugation. The ammonium sulfate was applied to this system, with a slight modification in sample further added to 45% saturation. After 1 hour, the precipitate preparation to avoid boiling in SDS, and after electrophoresis, was removed. Ammonium sulfate was added to 80% satura the gel was sliced and the proteins were eluted, renatured and tion. The sample was stirred overnight at 4°C. The precipitate tested. The only recoverable activity was located in the 20kD was recovered by centrifugation. The pellet was resuspended 45 (18.+-3 kD) range. in 1.5 Mammonium sulfate, 50 mM sodium phosphate, pH This has been validated with final purification and sequenc 7.0. The sample was then centrifuged to remove undisolved ing. The homologous protein from avian Eimeria is reported material. to have an 18.5 kD predicted mass and antibodies to a peptide Hydrophobic Chromatography on Phenyl-SepharoseTM Col segment react with a 20 kD band from E. tenella and E. 50 acervulina (Lillihoi et al., 2000). l The soluble fraction was subjected to hydrophobic chro pI Determination matography on a phenyl-Sepharose column (Fast Flow, (low Several experiments on extracting and re-folding proteins sub), by Pharmacia 3x3.3 cm). After the sample was applied from an acrylamide gel after isoelectric focusing revealed a pl to the column, it was washed with the same buffer until the being in range of 4.0-4.7 for protein active in NKassay, while baseline was obtained. A reverse linear gradient of 1.5M to 0 55 the predicted pl for E. acervulina, and E. tenella ARP pro Mammonium sulfate, 50 mM Naphophate, pH 7.0 (200 ml teins is 4.3-4.5. These results are consistent with acidic pro for each buffer chamber) was applied at room temperature at tein containing a significant number of carboxyl groups. a flow rate of 2 ml/min. 5 ml fractions were collected. Isoelectric focusing was performed with the use of a pre Selected fractions were analyzed by DC assay and SDS cast pH 3-7 IEF gel (Novex) according to company instruc PAGE. The active fractions were pooled and dialyzed over 60 tions. Broad p1 calibration kit (Pharmacia) was used as a night at 4° C. against 0.2 M NaCl, 10 mM Na phosphate, standard for p calculation. pH7.0 to be ready for the DEAE-SepharoseTM chromatogra pH Dependence of ARP Activity phy. Several experiments revealed significant dependence of DEAE-Sepharose Chromatography: 65 ARP activity on pH. In crude materials, NK activity was The DEAE-SepharoseTM column (Fast Flow, by Pharma found to decline significantly at both high pH>0, and low cia; 1.5x5.5 cm) was equilibrated with the initial buffer (0.2 pH-4. In some experiments, minimal activity was observed US 7,393,534 B2 93 94 below pH4.0, while one experiment showed minimal activity agarose gel was affected by the treatment with the enzymes, in the range 3.4 Brain 98% 10 Distribution of Activity Across the Intestinal Wall Small Intestine 200% (5/6) 1.6 70-90% of NK-activity, 80-100% of IFNY-inducing activ Small Intestine 66 ity and 80-100% of DC-activity could be extracted from the Large Intestine 166% (1/6) 5.6 intestinal tissue with the use of 30 mM EDTA. Large Intestine 66 Bovine Small intestinalileal sections were prepared and washed as 15 described in Section 6. The washed pieces approximately 3 ft leum mucosa 254% (2/6) 4f6 total length were cut into 2-3 cm pieces and incubated in PBS leum Sub-mucosa 66 with 0.5-1 mMDTT 5-10 minto remove mucus. The solution leum Smooth musc. 66 Distal ileum (all layers) 200% (1/5) 4f S was decanted and tissue pieces were stirred in 3-4x60 ml Proximal ileum (all layers) 214% (2/5) 3.5 changes of PBS, 30 mM EDTA, 1 mM PMSF for 30-60 min Duodenum 83% (5/5) at 40°C. The procedure removed cells from the mucosal layer eiunum 74% (5/5) containing most of the NK-, DC- and IFNY-inducing activity 'ILS = 100% indicates survival identical to controls. as verified by analysis of the amount of activity in the residual °Tumor-free survival beyond 50 days. tissue. Quantitative extraction of all activity types into the Number of animals in this category, versus total in group. mucosal cell fraction is consistent with the known confine “Representative sample of many experiments. 25 ment of Eimeria infections to the intestinal mucosa. The identification of an Eimeria protein in cattle validated the notion that an environmental agent was critical to activity. EXAMPLE 11 The lack of activity in wild mice, however, Suggested that Eimeria from mice may not work in our assay because the DC Domain Studies assay is based on murine splenic dendritic cells. It further 30 Suggests that only cross-species presentation of the Eimeria The importance of the disulfide bridge has been verified in protein is effective when using the non-intestinal dendritic semi-pure samples of both bovine and E. tenella ARPs. cells of the spleen. Further work to clarify this is being Cleavage of bovine ARP at Asn-Gly site drastically reduced planned. both NK-, and DC-activities. Since the bovine ARP has at 35 least one such site in the middle of the molecule (FIG. 4), it Cattle Breeds shows that the resulting fragments of the ARP molecule con We obtained hundreds of intestinal segments from cattle tain no domains responsible for either NK-, or DC-activity. and routinely observed the wide variation in activity between A synthetic peptide (50-mer) was constructed from a com animals and pools of animal extracts. Some of this variation bination of the variable region of the bovine molecule and the was attributed to different breeds, as shown in FIG. 15, but the 40 flanking regions of the 3-1E protein (FIG. 4). This peptide intra-breed variation was also very high. With beef cattle was synthesized by Alpha Diagnostic International, Inc. and routinely containing higher levels of activity, these were was received as a dry powder. It was tested in both the freshly selected more often from the slaughterhouse. The higher reconstituted state and in a renatured form. Neither form yields allowed for more quickly reaching quantities sufficient exhibited any activity when tested over concentrations span for purification to homogeneity (see Section 6). 45 ning 7 orders-of-magnitude starting at approximately 0.1 Tissue Distribution mg/mL. This synthetic peptide encompasses the domain between the disulfide bonds and its lack of activity indicates Various Organs that either a different domain is important to dendritic cell A limited set of organs and tissues have been examined binding or more of the molecule is needed for correct folding over the years to test the hypothesis that the observed activity of this domain. is confined to the intestinal tract. Brain and spleen from 50 rodents examined showed no anti-tumor activity. Recent tis Experiments on recombinant ARP from E. tenella showed Sue Samples from the spleen, liver, and lung of cattle have that the N-terminal end of the molecule can tolerate fusion of shown no activity in the DC and NK assays. However, due to large non-related proteins and peptides without big changes the limited sample size and the sensitivity of the test, the in DC-activity (Section 10). The truncated molecule with an 55 ATG start codon in 6th position (lacking GEADT, SEQ ID absence of ARP in other organs is not conclusive. NO:51) was at least as active as the full protein, but activity Distribution of Activity Along the Intestine was lost when the start codon was moved to the 21" position Initial studies in activity along the intestinal tract in cattle (forming a truncated molecule lacking GEADTOAWDTS revealed that the ileum of small intestine contained the high VREWLVDT, SEQID NO:52). This suggests that the distal est anti-tumor activity in mice (Table 9, Supra). This was 60 part of N-terminus is probably not involved in formation of examined again when ARP-specific activity could be identi any domains important for manifestation of at least DC fied with the DC assay. The average of three experiments is activity. Two C-terminal mutants of the E. tenella ARP (one shown in FIG.16(A). Preliminary murine anti-tumor results with double exchange Tyr to His and Gly to Ser, the other one for pooled samples along the intestinal tract is shown in FIG. with exchange Gly to Ala; Section 10) have been obtained and 16(B). These results validate the previous findings and give a 65 appeared to be equally active on DC-assay. Another mutant finer detail to the distribution of activity along the intestinal lacking three amino acids from C-terminus (SGF) retained tract. This distribution is consistent with the locations of significant activity while the mutants lacking 7, 10 or 14 US 7,393,534 B2 111 112 amino acids from the C-terminus showed significantly lower Aliprantis et al., Science 285(5428): 736-9 (1999)). Prelimi (if any) DC-activity (Section 10), it suggests that the C-ter nary data on testing the membrane preparation from sporo minal part of the molecule is part of Some domain determin Zoites of E. tenella for anti-tumor activity in vivo imply, as ing DC-activity. This part of the molecule contains no regions one of the possible interpretations, that a membrane-linked highly conserved in the known Apicomplexan ARPs. A com 5 form of ARP can be an effective therapeutic agent, giving it mon motif in the region is: T/S-A/I-L-A/N-F-A-E-(Y)-L, enhanced Stability and/or slow-release characteristics. which, as judged by comparison of ESTs, is conserved within Eimeria, Neurospora, and the most often found ARP EXAMPLE 1.4 sequences from Toxoplasma. 10 Oral Administration of E. tenella Oocysts EXAMPLE 12 Murine tumor model is used to determine if oral adminis Antibodies to Variable and Constant Regions tration of a cross-species Eimeria can generate the anti-tumor response seen by administration of E. tenella extracts. Tumor Polyclonal antibodies to two 20-mer epitopes are being bearing mice, fed with oocysts, are examined for increased generated in rabbits by Alpha Diagnostics International, Inc. lifespan. Mice without tumor, receiving oocysts, are exam One epitope is from the non-conserved region of the bovine ined for DC activity in their spleens and small intestines sequence and spans CRMFGASTDSGGDPYAELVQ (SEQ approximately 2-3 weeks after oocysts ingestion. This will ID NO:53). The other is from the E. acervulina sequence and indicate whether ARP can be elevated in the SI tissue and spans CALYDEEKEQNKADALTTAL (SEQID NO:54). In whether this can extend to other tissues. Oocysts are delivered the first case, the cysteine is part of the molecule, while in the to mice in their drinking water after overnight water restric second case it was added at the beginning to allow conjuga tion. Each mouse drink approximately 0.5 mL, containing tion to keyhole limpet haemocyanin, which is used to improve approximately 100,000 oocysts. its antigenicity. 25 EXAMPLE 1.5 EXAMPLE 13 Evidence for Anti-Viral Activity of ARP in Humans Evidence for Hydrophobic Membrane Anchors During the Phase I human trial of BBX-01, a terminal lung In some embodiments, ARP is an outer Surface antigen of 30 cancer patient reported the complete disappearance of long Eimeria parasites. We investigated the mechanism of its link term warts, likely to be of papillomavirus origin, from two age to the cell membranes (GPI-linkage vs. integral mem body regions. The first along the left and central region of the brane protein) by studying the possibility of ARP release by back (along the line of the spine), and the second along the phospholipase C (PLC), the enzyme specific for GPI-linked upper region of the left arm. The report stated that the warts proteins. The results presented in Example 4 Suggest that 35 suddenly dried up and disappeared. This occurred after the ARP is GPI-linked to the outer membrane of E. tenella sporo patient received three progressively larger single doses of Zoites. BBX-01 spaced at approximately two-week intervals, but Spontaneous release of ARP from the membranes was before receiving a multiple-dose course. The patient was observed in these experiments, which is described in litera under no other therapy during this period. ture for other parasites as “shedding of surface proteins 40 (Zambrano-Villa et al., Trends Parasitol. 18(6): 272-8 EXAMPLE 16 (2002)). The process was stimulated by elevated temperature, 10 mM EGTA, and was inhibited in the presence of 10 mM Identification of Proteins ZnCl2, an inhibitor of endogenous protozoan PLC from Try panosoma. These observations are consistent with the possi 45 bility of enzymatic nature of ARP release, probably due to Homologous to the Eimeria 19kd Sporozoite Antizen endogenous PLC. However, non-enzymatic mechanisms are Sequence homology searches were performed, using the E. not excluded. acervulina ARP (SEQ ID NO:2) and the variable region of Many surface proteins of protozoa are anchored by GPI the Bovine Eimeria protein sequence of the invention in order moieties (Ropert and Gazzinelli, Curr Opin Microbiol. 3(4): 50 to identify other proteins with similar sequence and/or struc 395-403 (2000); Almeida and Gazzinelli, J. Leukoc Biol. ture to that found for the isolated peptide of the invention. The 70(4): 467-77 (2001)) and this moiety alone has previously Basic Local Alignment Research Tool (BLAST)(R), available demonstrated macrophage activation capacity (MageZ. et al., through the website managed by the National Library or J. Immunol. 15: 160(4): 1949-56 (1998)) and is known to bind Medicine (www.ncbi.nlm.nih.gov/BLAST), was used to to the toll-like receptor-2 (Campos, 2001, J. of Immunology 55 identify homologues of the bovine Eimeria protein (Table 167: 416–423), probably through its lipid moiety. Our experi 10). Alternatively, databases containing Plasmodium falci ments suggest that the release of ARP is regulated by proto parum genome sequences were used for comparisons Zoan metabolism through control of the GPI linkage and that between the Eimeria 19kD sporozoite antigen and sequences the membrane linkage, disrupted by PLC is not required for of the Plasmodium falciparum genome, using the BLAST activation of DC cells. Further evidence was generated by 60 algorithm available at the following website: plasmodb.org/ comparing the location of activity on C8 RP-HPLC separa plasmodb/serylet/sv?page=blast (Table 10). tions of highly active E. tenella extracts with and without In brief, the Eimeria 19 kD sporozoite antigen peptide phospholipase treatments. In both cases, the activity sequence was used in a BLAST query of 1) the database of remained unchanged and appeared in the same fractions. This Homo sapiens protein sequences with a total of 39,196 distinguishes ARP from the DC activators that depend on 65 sequences, 2) the database of GenBank+EMBL-DDBJ-- lipid components, such as GPI itself, LPS, and bacterial lipo PDB (but no EST, STS, GSS, or phase 0, 1 or 2 HTGS) proteins (Brightbill et al., Science 285(5428): 732-6 (1999); sequences with a total of 1,687.930, and 3) the database of US 7,393,534 B2 113 114 GenBank+EMBL--DDBJ sequences from EST divisions Zoite antigen and the known EST sequences on deposit in this with a total of 15,724,693 sequences. All of these databases database. Interestingly, only members of Apicomplexa were available for public searching through the website of the resulted from this search. National Center for Biotechnology Information (NCBI) at the The variable region sequence of the Bovine Eimeria pro National Library of Medicine (NLM) and could be found at 5 tein of the invention was used in a BLAST query of the the aforementioned website. database of GenBank+EMBL--DDBJ--PDB (but no EST, STS, GSS, or phase 0, 1 or 2 HTGS sequences) sequences. Pairwise BLAST comparisons, within the database of Pairwise BLAST comparisons within the database of Gen Homo sapiens protein sequences were made using the Bank+EMBL+DDBJ--PDB (but no EST, STS, GSS, or phase “blastp' program option, which is Suitable for protein pro 10 0, 1 or 2 HTGS sequences) sequences were made using the tein comparisons. No significant homology between the “tblastin' program option, which compares the protein Eimeria 19 kD sporozoite antigen and the sequences on “Sequence 1 against the nucleotide “Sequence 2' which has deposit in the Homo sapiens protein database were found. been translated in all six reading frames. No reasonable Pairwise BLAST comparisons within the database of Gen homology was found between the variable region of the Bank+EMBL--DDBJ--PDB (but no EST, STS, GSS, or phase is Bovine Eimeria protein of the invention and any known pro 0, 1 or 2 HTGS) sequences were made using the “tblastin' tein on deposit in the bovine database. program option, which compares the protein “Sequence 1 Identified ARPs have also been analyzed for conserved against the nucleotide “Sequence 2' which has been trans domains using Conserved Domain Database (CDD v. 1.60) lated in all six reading frames. Significant sequence and struc and a Reverse Position-Specific BLAST(R) algorithm set to tural homology was found between the Eimeria 19 kD sporo- 20 default parameters, which are available at the website ncbi.n- Zoite antigen and the known protein sequences on deposit in lm.nih.gov/Structure/cdd/wrpsb.cgi. The analysis showed this database. that the identified ARPs have a conserved PROF (profilin) Pairwise BLAST comparisons within the database of Gen domain, which is represented by profilin. Data showed that a Bank--EMBL--DDBJ sequences from EST divisions were preparation containing birch pollen (e.g., SEQ ID NO:13) made using the “tblastin' program option, which compares the 25 was active in DC-assay. Based on the alignment on protein protein “Sequence I against the nucleotide “Sequence 2 level, birch pollen profilin has 28% identity and 54% similar which has been translated in all six reading frames. Signifi ity to the E. tenella ARP; 31% identity and 57% similarity to cant homology was found between the Eimeria 19 kD sporo the E. acervulina ARP. TABLE 10 Representative BLAST & Results SEQID Bit Percentage of Percentage of nput Sequence Accession # NO. Description Score Identity Positives' EA19 kD 11561.738 8 Plasmodium (chr).phat 327) 116 39 56 porozoite antigen EA19 kD 11S62004 9 Plasmodium (chr).gen 161) 116 38 58 sporozoite antigen EA19 kD 11562590 10 Plasmodium (chr).glm 372) 100 40 58 sporozoite antigen EA19 kD 115136O1 11 Latex profilin Hewb8 chain A 35 26 42 sporozoite rom Hevea Brasiliensis antigen EA19 kD 2781152 12 Profilin I from Arabidopsis 31 sporozoite Thaiiana antigen EA19 kD 1942360 13 Birch pollen profilin from 31 31 57 sporozoite Betulia Pendula antigen EA19 kD 15224838 14 Profilin I from Arabidopsis 31 sporozoite Thaiiana antigen EA19 kD BG235691 21 EtESTed 11 fo8.yl Eimeria 285 83 91 sporozoite tenella S5-2 cDNA Neg antigen Selected Eimeria teneia cDNA 5' similar to TR: Q24777 Q24777 19 KDA SPOROZOITE ANTIGEN. (2) TR: Q03777:, MRNA sequence EA19 kD CBO70888 22 TgESTZye83c06.yl TgME49 169 51 72 sporozoite invivo Bradyzoite cDNA size antigen Selected Toxoplasma gondii cDNA clone TgESTZye83c06.yl 5' similar to TR: Q24777 Q24777 19 KDA SPOROZOITE ANTIGEN. (2) TR: Q03777:, MRNA sequence US 7,393,534 B2 115 116 TABLE 10-continued Representative BLAST & Results SEQID Bit Percentage of Percentage of Input Sequence Accession # NO. Description Score Identity Positives' EA19 kD BM189297 23 TgESTZyb37c04yl TgRH 169 51 72 sporozoite Tachyzoite Subtracted cDNA antigen Library Toxoplasma gondii cDNA clone TgESTZyb37c04yl 5' similar to TR: Q24777 Q24777 19 KDA SPOROZOITE ANTIGEN. (2) TR: Q03777:, MRNA sequence EA19 kD AA167907 24 CpEST,071 129 46 64 sporozoite uniZAPCpIOWASporoLib1 antigen Cryptosporidium parvum cDNA 5' similar to Eimeria acervuina 19 kDa sporozoite antigen, MRNA sequence EA19 kD CA856473 25 PfESToac08g05.yl 122 38 57 sporozoite Plasmodium falciparum 3D7 antigen gametocyte cDNA library Plasmodium falciparum 3D7 cDNA 5' similar to TR: Q24777 Q24777 19 KDA SPOROZOITE ANTIGEN. (2) TR: Q03777:, MRNA sequence Percentage of positives: the extent of similarity between two sequences, which can be based on percent sequence iden tity and/or conservation. EXAMPLE 17 EXAMPLE 1.8 30 Evidence for Anti-Viral Activity of ARP in Mice Use of ARP as an Immunosuppressant by Initiation of Dendritic Cell Paralysis Specific pathogen-free female BALB/c mice were infected While the primary activity of ARP is to induce IL-12 pro intranasally with an LD90 dose of influenza virus A/NWS/33 35 duction in dendritic cells it has been noted that at high doses (HINI). The mice were then treated with ARP E1 by one of (~1000 times stimulatory dose) ARP can suppress the pro two treatment protocols. In the first protocol mice received duction of IL-12. Based on this observation it may be possible 100 ng of ARP E1 given intraperitoneally 48 hours before to use ARP as an immunosuppressant in the treatment of viral exposure, 4 hours after viral exposure (day 0) and on diseases that are caused by an overactive or inappropriate days 3 and 6 after viral exposure. In the second protocol mice 40 immune response. This hyposthesis was tested in the experi received 100, 1,000, or 10,000, ng of ARP E1 intraperito mental autoimmune encephalomyelitis (EAE) model in SJL neally 4 hours after viral exposure (day 0) and on days 3 and mice. EAE is induced in mice by immunization with spinal 6 after viral exposure. Placebo treated mice received bovine cord derived myelin basic protein which induces a disease in the mice which is very similar to multiple schlerosis (MS) in serum albumin in phosphate-buffered saline. Mice were 45 observed daily for death. The survival of the mice is shown in humans. Table 11. The SJL mice were immunized with the myelin basic pro tein on day 0 and on day 10 they received daily treatments of TABLE 11 0.1 Lig, 1.0 ug or 10 ug ARP E1 given i.p. or with 5 mg Linomide given s.c. or with vehicle alone. Treatments were Survival of Mice Exposed to Influenza 50 given daily for 5 days. Linomide is a drug that is known to inhibit EAE and is used as a positive control. EAE exhibits Treatment Mean Day to itselfin mice as progressive paralysis beginning in the tail and Compound Dose (ng/day) Schedule Survive/Total Death + SD progressing forward until death occurs. Severity of EAE is ARPE1 1OO –2, 0, 3, 6 Of 10 12.3 1.2* measured by means of the EAE score which starts at for ARPE1 1OO 0, 3, 6 2.10 12.1 + 1.6 55 normal and increases to a score of 5 when death occurs. For ARPE1 1,000 0, 3, 6 3.10 12.72.7 ARPE1 10,000 0, 3, 6 5/10* 12.0 - 1.4* ethical reasons mice are sacrificed when a score of 4 is Placebo O3 6 2,2O 11.3 1.2 reached. The experiment was terminated on day 15 and the mean EAE scores are shown in Table 12. Mean day to death of mice dying before day 21 *P & O.05 60 TABLE 12 There was a significant increase in the number of Survivors Effect of ARP on EAE in SJL Mice and time to death in the mice treated with 10,000 ng of ARP E1. Arterial oxygen Saturation was also measured in these Treatment Day 15 EAE Score + SD mice on days 3-11. There was a statistically significant reduc 65 Vehicle 18 1.9 tion of the decline in oxygen Saturation in the mice treated Linomide O.6 1.3 with 1,000 ng and 10,000 ng of ARP E1.