Chronic lymphocytic leukemia cell line Number:7,435,412 from the United States Patent and Trademark Office (PTO) owispatent The preparation and characterization of antibodies that bind to antigens on CLL or other cancer cells, especially to antigens upregulated in the cancer cells, and the identification and characterization of antigens present on or upregulated by cancer cells are useful in studying and treating cancer.<H lymphocytic, leukemia, Chronic, lymphoc, 7435412.html, Patent, pto, 7435412

Title: Chronic lymphocytic leukemia cell line

Abstract: The preparation and characterization of antibodies that bind to antigens on CLL or other cancer cells, especially to antigens upregulated in the cancer cells, and the identification and characterization of antigens present on or upregulated by cancer cells are useful in studying and treating cancer.

Patent Number: 7,435,412 Issued on 10/14/2008 to Bowdish, et al.

Inventors: Bowdish; Katherine S. (Del Mar, CA), McWhirter; John (San Diego, CA)
Assignee: Alexion Pharmaceuticals, Inc. (Chesire, CT)

Appl. No.: 10/379,151

Filed: March 4, 2003

Related U.S. Patent Documents


Application Number	Filing Date	Patent Number	Issue Date
PCT/US01/47931	Dec., 2001
60254113	Dec., 2000

Current U.S. Class: 424/133.1 ; 424/135.1; 424/138.1; 424/155.1; 424/156.1

Current International Class: A61K 39/00 (20060101); C07K 16/18 (20060101)

References Cited [Referenced By]

U.S. Patent Documents


3940475	February 1976	Gross
4289747	September 1981	Chu
4376110	March 1983	David et al.
5223409	June 1993	Ladner et al.
5225539	July 1993	Winter
5403484	April 1995	Ladner et al.
5427908	June 1995	Dower et al.
5434131	July 1995	Linsley et al.
5508717	April 1996	Miller
5571698	November 1996	Ladner et al.
5580717	December 1996	Dower et al.
5780279	July 1998	Matthews et al.
5916560	June 1999	Larsen et al.
6011138	January 2000	Reff et al.
6040136	March 2000	Garrard et al.
6338851	January 2002	Gorczynski
6652858	November 2003	Gorczynski et al.
6749854	June 2004	Gorczynski et al.
6955811	October 2005	Gorczynski et al.
6984625	January 2006	Gorczynski
7238352	July 2007	Gorczynski et al.
2002/0031515	March 2002	Caliguira et al.
2002/0168364	November 2002	Gorczynski et al.
2002/0192215	December 2002	Hoek et al.
2003/0017491	January 2003	Shi et al.
2004/0018972	January 2004	Gorczynski et al.
2004/0054145	March 2004	Gorczynski
2004/0175692	September 2004	Bowdish et al.
2004/0198661	October 2004	Bowdish et al.
2005/0048069	March 2005	Gorczynski et al.
2005/0107214	May 2005	Gorczynski et al.
2005/0129690	June 2005	Bowdish et al.
2005/0169870	August 2005	Truitt et al.

Foreign Patent Documents


WO-8403508	Sep., 1984	WO
WO-8503508	Aug., 1985	WO
WO-8806630	Sep., 1988	WO
WO-9215679	Sep., 1992	WO
WO-9428027	Dec., 1994	WO
WO 95/18825	Jul., 1995	WO
WO-9627011	Sep., 1996	WO
WO 96/38557	Dec., 1996	WO
WO-9708320	Mar., 1997	WO
WO-9721450	Jun., 1997	WO
WO-9721450	Jun., 1997	WO
WO-9924565	May., 1999	WO
WO-0211762	Feb., 2002	WO
WO-02/059280	Aug., 2002	WO
WO-02095030	Nov., 2002	WO
WO-03025202	Mar., 2003	WO
WO-2004078937	Sep., 2004	WO
WO-04078938	Oct., 2004	WO

Other References

Kneitz et al (Leukemia, 1999, vol. 13, pp. 98-104). cited by examiner .
Auchincloss, "Strategies to Induce Tolerance," Transplantation Immunology, Bach and Auchincloss, Eds., Wiley-Liss, New York, Chapter 11, pp. 211-218 (1995). cited by other .
Barclay, "Different reticular elements in rat lymphoid tissue identified by localization of Ia, Thy-1 and MRC OX 2 antigens," Immunology, 44:727-736(1981). cited by other .
Barclay and Ward, "Purification and Chemical Characterisation of Membrane Glycoproteins From Rat Thymocytes and Brain, Recognised by Monoclonal Antibody MRC OX2," European J. Biochemistry, 129:447-458(1982). cited by other .
Borriello et al., "Characterization and localization of Mox2, the gene encoding the murine homolog of the rat MRC OX--2 membrane glycoprotein," Mammalian Genome, 9(2):114-118 (1998). cited by other .
Borriello et al., "MRC OX-2 Defines a Novel T Cell Costimulatory Pathway," J. Immunol., 158:4549-4554(1997). cited by other .
Chen et al., "Cloning and characterization of the murine homologue of the rat/human MRC OX--2 gene," Biochemica et Biophysica Acta, 1362(1):6-10(1997). cited by other .
Gorczynski et al., "Increased expression of the novel molecule OX--2 is involved in prolongation of murine renal allograft survival," Transplantation, 65(8):1106-1114(1998). cited by other .
Gorczynski et al., "An Immunoadhesin incorporating the Molecule OX-2 is a Potent Immunosuppressant That Prolongs Allo- and Xenograft Survival," J. Immunol., 163:1654-1660(1999). cited by other .
Preston et al., "The leukocyte/neuron cell surface antigen OX2 binds to ligand on macrophages", European J. of Immunol., 27(8):1911-1918(1997). cited by other .
Bach, "Immunosuppressive therapy of autoimmune diseases," Immunology Today, 14(6)322-326(1993). cited by other .
Bohen, S.P., "Variation in gene expression patterns in follicular lymphoma and the response to rituximab," PNAS, 100(4):1926-1930(2003). cited by other .
Boon, Thierry., "Toward a Genetic Analysis of Tumor Rejection Antigens," Advances in Cancer Res., 58: 177-210(1992). cited by other .
Broderick et al., "Constitutive Retinal CD200 Expression Regulates Resident Microglia and Activin State of Inflammatory Cells During Experimental Autoimmune Uveoretinitis," Am. J. of Pathology, 161(5):1669-1677(2002). cited by other .
Clark, D.A., "Intralipid as Treatment for Recurrent Unexplained Abortion?", Am. J. of Reprod. Immunol., 32:290-293(1994). cited by other .
Clark et al., Amer. Soc. for Reprod. Medicine, 55th Annual Meeting (1999). Abstract Only. cited by other .
Clark et al., "The OX-2 Tolerance Signal Molecule at the Fetomaternal Interface Determines Pregnancy Outcome," Amer. Journal of Reprod Immunol., 43:326(2000). Abstract Only. cited by other .
Chaouat and Clark, FAS/FAS Ligand Interaction at the Placental Interface is not Required for the Success of Allogeneic Pregnancy in Anti-Paternal MHC Preimmunized Mice, Presented at the 6th Congress of the Adria-Alps Soc. of Immunol. of Reprod., (2000) / Amer. J. of Reprod. Immunol., 45:108-115(2001). cited by other .
Clark et al., "Fg12 prothrombinase expression in mouse trophoblast and decidua triggers abortion but may be countered by OX-2," Mol. Human Reprod., 7-185-194(2001). cited by other .
Cohen, P.L., "Systemic Autoimmunity," in Fundamental Immunology, Fourth edition, W.E. Paul, Editor, Lippincott-Raven Publishers, Philadelphia, Ch. 33, p. 1067-1088(1999). cited by other .
Dick et al., "Control of Myeloid Activity During Retinal Inflammation," J. of Leukocyte Bio., 74:161-166(2003). cited by other .
Gorczynski et al., "Does Successful Allopregnancy Mimic Transplantation Tolerance?", Graft, 4(5):338-345(2001). cited by other .
Hoek, et al., "Down-Regulation of the Macrophage Lineage Through Interaction with OX2 (CD200)," Science, 290:1768-1771(2000). cited by other .
Huang, "Structural chemistry and therapeutic intervention of protein-protein interactions in immune response, human immunodeficiency virus entry, and apoptosis," Pharmacol. Therapeutics, 86:201-215(2000). cited by other .
Jain, "The next frontier of molecular medicine: Delivery of therapeutics," Nature Medicine, 4(6):655-657(1998). cited by other .
Keil et al., American Society for Reproductive Immunology XXIst Annual Meeting, Jun. 9-12, 2001, Chicago, IL., p. 343, Abstract Only. cited by other .
Kim et al., "Divergent Effects of 4-1BB Antibodies on Antitumor Immunity and on Tumor-reactive T-Cell Generation," Cancer Res., 61:2031-2037(2001). cited by other .
Kjaergaard et al., "Therapeutic Efficacy of OX-40 Receptor Antibody Depends on Tumor Immunogenicity and Anatomic Site of Tumor Growth," Cancer Res. 60:5514-5521(2000). cited by other .
Pardoll, Drew., "Therapeutic Vaccination for Cancer," Clin. Immunol., 95(1):S44-S62(2000). cited by other .
Ragheb et al., "Preparation and functional properties of monoclonal antibodies to human, mouse and rat OX-2", Immunol. Letters, 68:311-315(1999). cited by other .
Romagnani, Sergio., "Short Analytical Review: TH1 and TH2 in Human Diseases," Clin. Immunol. Immunopath, 80(3):225-235(1996). cited by other .
RosenWald et al., "Relation of Gene Expression Phenotype to Immunoglobulin Mutation Genotype in B Cell Chronic Lymphocytic Leukemia," J. of Exp. Medicine, 194(11):1639-1647(2001). cited by other .
Steinman, Lawrence., "Assessment of Animal Models for MS and Demyelinating Disease in the Design of Rational Therapy," Neuron, 24:511-514(1999). cited by other .
Tangri and Raghupathy, "Expression of Cytokines in Placentas of Mice Undergoing Immunologically Mediated Spontaneous Fetal Resorptions," Biology of Reprod., 49:850-856(1993). cited by other .
Toder et al., "Mouse Model for the Treatment of Immune Pregnancy Loss," Am. J. of Reprod. Immunol., 26:42-46(1991). cited by other .
Mjaaland et al., "Modulation of immune responses with monoclonal antibodies, I. Effects on regional lymph node morphology and on anti-hapten responses to haptenized monoclonal antibodies", Eur. J. Immunol., 20:1457-1461(1990). cited by other .
Barclay et al., "Neuronal/Lymphoid Membrane Glycoprotein MRC OX-2 is a Member of the Immunoglobulin Superfamily with a Light-Chain-Like Structure," Biochem. Soc. Symp., 51:149-157(1985). cited by other .
McCaughan et al., "Characterization of the Human Homolog of the Rat MRC OX-2 Membrane Glycoprotein," Immunogenetics, 25:329-335(1987). cited by other .
Paterson et al., "Antigens of Activated Rat T Lymphocytes Including A Molecule of 50,000 Mr Detected Only on CD4 Positive T Blasts," Molecular Immunology, 24(12):1281-1290(1987). cited by other .
Heaney et al., "Severe asthma treatment: need for characterising patients," Lancet, 365:974-976(2005). cited by other .
Gorczynski, "CD200 and its receptors as targets for immunoregulation," Current Opinion in Investigational Drugs, 6:483-488(2005). cited by other .
Ni et al., "An immunoadhesion incorporating the molecule OX-2 is a potent immunosuppressant which prolongs allograft survival", FASEB Journal 13(5):A983(1999). Abstract Only. cited by other .
Clark et al., "Labile CD200 tolerance signal important in transfusion-related immunomodulation (TRIM) prevention of recurrent miscarriages," Amer. J. Reprod. Immunol., 45:361(2001). Abstract Only. cited by other .
Gorczynski, R.M., "Evidence for an Immunoregulatory Role of OX2 with Its Counter Ligand (OX2L) in the Regulation of Transplant Rejection, Fetal Loss, Autoimmunity and Tumor Growth," Arch. Immunol. et Ther. Exp., 49(4):303-309(2001). cited by other .
Nathan and Muller, "Putting the Brakes on innate immunity: a regulatory role for CD200?", Nat Immunol., 2(1):17-19(2001). cited by other .
Clark et al., "Procoagulants in fetus rejection: the role of the OX-2 (CD200) tolerance signal," Seminars in Immunol., 13(4)255-263(2001). cited by other .
Stuart et al., "Monkeying Around with Collagen Autoimmunity and Arthritis," Lab. Invest., 54(1):1-3(1986). cited by other .
Gorczynski and Marsden, "Modulation of CD200 receptors as a novel method of immunosuppression," Expert Opin. Ther. Patents, 13(5):711-715(2003). See also WIPO Patent No. WO02095030 assigned to Transplantation Tech, Inc. cited by other .
Tang et al., Pathogenesis of collagen-induced arthritis: modulation of disease by arthritogenic T-Cell epitope location, Immunology, 113:384-391. cited by other .
Myers et al., "Characterization of a Peptide Analog of a Determinant of Type II Collagen that Suppresses Collagen-Induced Arthritis," J. of Immunology, 161:3589-3595(1998). cited by other .
Gorczynski et al., "Anti-CD200R Ameliorates Collagen-Induced Arthritis in Mice," Clinical Immunol., 104(3):256-264(2002). cited by other .
Chitnis et al., "The Role of CD200 in Immune-Modulation and Neural Protection in EAE," Abstract, 12th International Congress of Immunology and 4th Annual Conference of FOCIS, Montreal, Jul. 21, 2004. Abstract Only. cited by other .
Barclay et al., "CD200 and membrane protein interactions in the control of myeloid cells," Trends in Immunology, 23(6):2002. cited by other .
Gorczynski et al., "Evidence of a role for CD200 in regulation of immune rejection of leukaemic tumour cells in C57BL/6 mice," Clin. Exp. Immunol., 126:220-229(2001). cited by other .
Alizadeh et al., "Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling," Nature, 403:503-511 (2000). cited by other .
Banerjee, D., et al., "Blocking CD200-CD200 receptor axis augments NOS-2 expression and aggravates experimental autoimmune uveoretinitis in Lewis rats," Ocular Immunology and Inflammation, 12(2):115-125 (2004). cited by other .
Blazer, B.R., et al., "CD28/B7 Interactions Are Required for Sustaining the Graft-Versus-Leukemia Effect of Delayed Post-Bone Marrow Transplantation Splenocyte Infusion in Murine Recipients of Myeloid of Lymphoid Leukemia Cells, " J. Immunol., 159:3460-3473 (1997). cited by other .
Bukovsky, A., et al., "Association of lymphoid cell markers with rat ascitic malignant cells," IRCS Med. Sci., 11:866-867 (1983). cited by other .
Bukovsky, A., et al., "Association of some cell surface antigens of lymphoid cells and cell surface differentiation antigens with early rat pregnancy," Immunology, 52:631-640 (1984). cited by other .
Bukovsky, A., et al., "The localozation of Thy-1.1, MRC OX 2 and Ia antigens in the rat ovary and follopian tube," Immunology, 48:587-596 (1983). cited by other .
Bukovsky, A., et al., "The ovarian follicle as a model for the cell-mediated control of tissue growth," Cell Tissue Res., 236:717-724 (1984). cited by other .
Chen, D., et al., "Discrete Monoclonal Antibodies Define Functionally Important Epitopes in the CD200 Molecule Responsible for Immunosuppression Function," Transplantation, 79:282-228 (2005). cited by other .
Chen, D., et al., "Synthetic peptides from the N-terminal regions of CD200 and CD200R1 modulate immunosupressive and anti-inflammatory effects of CD200-CD200R1 interaction," International Immunology, 17(3):289-296 (2005). cited by other .
Cherwinski, H.M., et al., "The CD200 Receptor Is a Novel and Potent Regulator of Murine and Human Mast Cell Function," J. Immunol., 174:1348-1356 (2005). cited by other .
Clark, M.J., et al., "MRC OX-2 antigen: a lymphoid/neuronal membrane glycoprotein with a structure like a single immunoglobulin light chain," EMBO Journal, 4(1):113-118 (1985). cited by other .
Clarke, M.J., "MRC OX-2 lymphoid brain glycoprotein: S1 mapping suggests higher levels of abnormal RNA in the thymus than in the brain," Biochemical Society Transactions, 14:80-81 (1986). cited by other .
Fallorino, F., et al., "Murine Plasmacytoid Dendritic Cells Initiate the Immunosuppressive Pathway of Tryptophan Catabolism in Response to CD200 Receptor Engagement," J. Immunol., 173:3748-3754 (2004). cited by other .
Farber, U., et al., "Loss of heterozygosity on chromosome 3, bands q24>qter, in a diploid meningioma," Cytogenet Cell Genet, 57:157-158 (1991). cited by other .
Gorczynski, L., et al., "Evidence That an OX-2-Positive Cell Can Inhibit the Stimulation of Type 1 Cytokine Production by Bone Marrow-Derived B7-1 (and B7-2)-Positive Dendtritic Cells," J. Immunol., 162:774-781 (1999). cited by other .
Gorczynski, R., et al., "CD200 is a Ligand for All Members of the CD200R Family of Immunoregulatory Molecules," J. Immunol., 172:7744-7749 (2004). cited by other .
Gorczynski, R., et al., "Dendritic Cells Expressing TGFBeta/IL-10, and CHO Cells With OX-2, Increase Graft Survival," Transplantation Proceedings, 33:1565-1566 (2001). cited by other .
Gorczynski, R.M., "Role of Cytokines in Allograft Rejection," Current Pharmaceutical Design, 7:1039-1057 (2001). cited by other .
Gorczynski, R.M., "Synergy in Induction of Increased Renal Allograft Survival after Portal Vein Infusion of Dendtritic Cells Transduced to Express TGFB and IL-10, along with Administration of CHO Cells Expressing the Regulatory Molecule OX-2," Clinical Immunology, 95(3):182-189 (2000). cited by other .
Gorczynski, R.M., "Transplant tolerance modifying antibody to CD200 receptor, but not CD200, alters cytokine production profile from stimulated marcrophages," Eur. J. Immunol., 31:2331-2337 (2001). cited by other .
Gorczynski, R.M., et al., "A CD200FC Immunoadhesin Prolongs Rat Islet Xenograft Survival in Mice," Transplantation, 73(12):1948-1953 (2002). cited by other .
Gorczynski, R.M., et al., "Anti-Rat OX-2 Blocks Increased Small Intestinal Transplant Survival After Portal Vein Immunization," Transplantation Proceedings, 31:577-578 (1999). cited by other .
Gorczynski, R.M., et al., "Augmented Induction of CD4+ CD25+ Treg using Monoclonal Antibodies to CD200R," Transplantation, 79:(4)488-491 (2005). cited by other .
Gorczynski, R.M., et al., "Augmented Induction of CD4+ CD25+ Treg using Monoclonal Antibodies to CD200R," Transplantation, 79(9):1180-1183 (2005). cited by other .
Gorczynski, R.M., et al., "CD200 Immunoadhesin Supresses Collagen-Induced Arthritis in Mice," Clinical Immunology, 101(3):328-334 (2001). cited by other .
Gorczynski, R.M., et al., "Evidence for Persistent Expression of OX2 as a Necessary Component of Prolonged Renal Allograft Survival Following Portal Vein Immunization," Clinical Immunol., 97(1):69-78 (2000). cited by other .
Gorczynski, R.M., et al., "Induction of Tolerance-Inducing Antigen-Presenting Cells in Bone Marrow Cultures In Vitor Using Monoclonal Antibodies to CD200R," Transplantation, 77(8):1138-1144 (2004). cited by other .
Gorczynski, R.M., et al., "Interleukin-13, in Combination with Anti-Interleukin-12, Increases Graft Prolongation After Portal Venous Immunization with Cultured Allogeneic Bone Marrow-Derived Dentritic Cells," Transplantation, 62(11):1592-1600 (1996). cited by other .
Gorczynski, R.M., et al., "Persistent expression of OX-2 is necessary for renal allograft survival," FASEB Journal, 14(6):A1069 (2000). cited by other .
Gorczynski, R.M., et al., "Receptor Engagement on Cells Expressing a Ligand for the Tolerance-Inducing Molecule OX2 Induces an Immunoregulatory Population That Inhibits Alloreactivity In Vitro and In Vivo," J. Immunol., 165:4854-4860 (2000). cited by other .
Gorczynski, R.M., et al., "Regulation of Gene Expression of Murine MD-1 Regulates Subsequent T Cell Activation and Cytokine Production," J. of Immunology, 165:1925-1932 (2000). cited by other .
Gorczynski, R.M., et al., "Structural and Functional Heterogeneity in the CD200R Family of Immunoregulatory Molecules and their Expression at the Fetomaternal Interface," AJRI, 52:147-163 (2004). cited by other .
Gorczynski, R.M., et al., "The Same Immunoregulatory Molecules Contribute to Successful Pregnancy and Transplantation," AJRI, 48:18-26 (2002). cited by other .
McCaughan, G.M., et al., "Identification of the human homologue of the rat lymphoid/brain antigen MRC OX-2," Australian and New Zealand Journal of Medicine 17: 142 (Abstract) (1987). cited by other .
Hoek, R.M., et al., "Macrophage regulation by the B7.1/2 homologue OX2?", FASEB Journal, 14(6):A1232, Abstract #193.1 (2000). cited by other .
Hutchings, N.J., et al., "Interactions of Cytoplasmic Region of OX2R Are Consistent with an Inhibitory Function," Annual Congress of the British Society for Immunology, 101(Supplement 1): 24, Abstract #10.6 (2000). cited by other .
Jeurissen, S.H.M., et al., "Characteristics and functional aspects of nonlymphoid cells in rat germinal centers, recognized by two monoclonal antibodies ED5 and ED6," Eur. J. Immunol., 16:562-568 (1986). cited by other .
Kroese, F.G.M., et al., "Germinal centre formation and follicular antigen trapping in the spleen of lethally X-irradiated and reconstituted rats," Immunology, 57:99-104 (1986). cited by other .
Kroese, F.G.M., et al., "The ontogeny of germinal centre forming capacity of neonatal rat spleen," Immunology, 60:597-602 (1987). cited by other .
Marsh, M.N., "Functional and Structural Aspects of the Epithelial Lymphocyte, with Implications for Coeliac Disease and Tropical Sprue," Scandinavian Journal of Gastroenterology 114: 55-75 (1985). cited by other .
McCaughan, G.W., et al., "The Gene for MRC OX-2 Membrane Glycoprotein Is Localized on Human Chromosome 3," Immunogenetics, 25:133-135 (1987). cited by other .
McMaster, W.R., et al., "Identification of Ia glycoproteins in rat thymus and purification from rat spleen," Eur. J. Immunol., 9:426-433 (1979). cited by other .
Mjaaland, S., et al., "The Localization of Antigen in Lymph Node Follicles of Congenitally Athymic Nude Rats," Scand. J. Immunol., 26:141-147 (1987). cited by other .
Mohammad, R.M., et al., "Establishment of a human B-CLL xenograft model: utility as a preclinical therapeutic model," Leukemia, 10:130-137 (1996). cited by other .
Morris, R.J., et al., "Sequential Expression of OX2 and Thy-1 Glycoproteins on the Neuronal Surface during Development," Dev. Neurosci., 9:33-44 (1987). cited by other .
Nagelkerken L., et al., "Accessory Cell Function of Thoracic Duct Nonlymphoid Cells, Dentritic Cells, and Splenic Adherent Cells in the Brown-Norway Rat," Cellular Immunology, 93:520-531 (1985). cited by other .
Ragheb, R.F., "Exploration of OX-2 function in tolerance induction and graft acceptance using an anti-mouse OX-2 monoclonal antibody," University of Toronto, Masters Abstracts International, 38(4):971-972 (2000). cited by other .
Richards, S.J., et al., "Reported Sequence Homology Between Alzhemier Amyloid770 and the MCR OX-2 Antigen Does Not Predict Function," Brain Research Bulletin, 38(3):305-306 (1995). cited by other .
Rosenblum, M.D., et al., "CD200 is a novel p53-target gene involved in apoptosis-associated immune tolerance," Blood, 103(7):2691-2698 (2004). cited by other .
Syme, R., et al., "Comparison of CD34 and Monocyte-Derived Dendritic Cells from Mobilized Peripheral Blood from Cancer Patients," Stem Cells, 23:74-81 (2005). cited by other .
Taylor, N., et al., "Enhanced Tolerance to Autoimmune Uveitis in CD200-Deficient Mice Correlates with a Pronounced Th2 Switch in Response to Antigen Challenge," J. Immunol., 174:143-154 (2005). cited by other .
Webb, M., et al., "Localisation of the MRC OX-2 Glycoprotein on the Surfaces of Neurones," J. Neurochemistry, 43:1061-1067 (1984). cited by other .
Wright, G.J., et al., "Lymphoid/Neuronal Cell Surface OX2 Glycoprotein Recognizes a Novel Receptor on Macrophages Implicated in the Control of Their Function," Immunity, 13:233-242 (2000). cited by other .
Wright, G.J., et al., "The lymphoid/neuronal OX-2 glycoprotein interacts with a novel protein expressed by macrophages," Tissue Antigens, 55(Supplement 1): 11 (2000). cited by other .
Wright, G.J., et al., "Viral homologues,of cell surface proteins OX2 and CD47 have potential to regulate macrophage function," Annual Congress of the British Society for Immunology, 101(Supplement 1): 50 (2000). cited by other .
Yang, C., et al., "Functional maturation and recent thymic emigrants in the periphery: development of alloreactivity correlates with the cyclic expression of CD45RC isoforms," Eur. J. Immunol., 22:2261-2269 (1992). cited by other .
Yu, X., et al., "The role of B7-CD28 co-stimulation in tumor rejection," International Immunology, 10(6):791-797 (1998). cited by other .
Zhang, S., et al., "Molecular Mechanisms of CD200 Inhibition of Mast Cell Activation," J. Immunol., 173:6786-6793 (2004). cited by other .
Zheng, P., et al., "B7-CTLA4 interaction enhances both production of antitumor cytotoxic T lymphocytes and resistance to tumor challenge," Proc. Natl. Acad. Sci. USA, 95:6284-6289 (1998). cited by other .
Jansky, L., et al., "Dynamics of Cytokine Production in Human Peripheral Blood Mononuclear Cells Stimulated by LPS or Infected by Borrelia," Physiol. Res., 52:593-598 (2003). cited by other .
Chitnis, T., et al., "Elevated Neuronal Expression of CD200 Protects Wid.sup.s Mice from Inflammation-Mediated Neurodegeneration," American Journal of Pathology, 170(5):1695-1712 (2007). cited by other .
Dennis, C., "Off by a whisker," Nature, 442,:739-741 (2006). cited by other .
Gura, T., "Systems for Identifying New Drugs Are Often Faulty," Science, 278:1041-1042 (1997). cited by other .
Srivastava, P.K., "Immunotherapy of human cancer: lessons from mice," Nature Immunology, 1(5):363-366 (2000). cited by other .
Wilczynski, J.R., "Immunoligical Analogy Between Allograft Rejection, Recurrent Abortion and Pre-Eclampsia--the Same Basic Mechanism?," Human Immunology, 67:492-511 (2006). cited by other .
Zips, D., et al. "New Anticancer Agents: In Vitro and In Vivo Evaluation," in vivo, 19:1-7 (2005). cited by other .
Caldas et al., "Humanization of the anti-CD18 antibody 6.7: an unexpected effect of a framework residue in binding to antigen," Molecular Immunology, 39(15):941-952 (2003). cited by other .
Chien et al., Significant structural and functional change of an antigen-binding site by a distant amino acid substitution: Proposal of a structural mechanism, PNAS, 86(14):5532-5536 (1989). cited by other .
Cochlovius et al., "Cure of Burkitt's Lymphoma in Severe Combined Immunodeficiency Mice by T Cells, Tetravalent CD3.times.CD19 Tandem Diaboty, and CD29 Costimulation," Cancer Research, 60:4336-4341 (2000). cited by other .
Ebert et al., Selective Immunosuppressive Action of a Factor Produced by Colon Cancer Cells, Cancer Research, 50:6158-6161 (1990). cited by other .
Faisal et al., "Cell-surface Associated p43/Endothelial-monocyte-activating-polypeptide-II in Hepatocellular Carcinoma Cells Induces Apoptosis in T-lymphocytes," Asian Journal of Surgery, 30(1):13-22 (2007). cited by other .
Ginaldi et al., "Levels of Expression of CD52 in Normal and Leukemic B and T Cells: Correlation with In Vivo Therapeutic Response to Campath-1H," Leukemia Research, 22(2):185-191 (1998). cited by other .
Giusti et al., "Somatic diversification of S107 from an antiphosphocholine to an anti-DNA autoantibody is due to a single base change in its heavy chain variable region," PNAS, 84(9):2926-2930 (1987). cited by other .
Gussow et al., "Humanization f Antibodies," Methods in Enzymology, 203:99-121 (1991). cited by other .
Hardy et al., "A lymphocyte-activating monoclonal antibody induces regression of human tumors in severe combined immunodeficient mode," PNAS, 94:5756-5760 (1997). cited by other .
Iwanuma et al., "Antitumor Immune Response of Human Peripheral Blood Lymphocytes Coengrafted with Tumor into Severe Combined Immunodeficient Mice," Cancer Research, 57:2937-2942(1997). cited by other .
Kretz-Rommel et al., "CD200 Expression of Tumor Cells Suppresses Antitumor Immunity: New Approaches to Cancer Immunotherapy," The Journal of Immunology, 178:5595-5605 (2007). cited by other .
Liu et al., "Effect of combined T- and B-cell depletion of allogeneic HLA-mismatched bone marrow graft on the magnitude of kinetics of Epstein-Barr virus load in the peripheral blood of bone marrow transplant recipients," Clinical Transplantation, 18:518-524 (2004). cited by other .
Mariuzza et al., "The Structural Basis of Antigen-Antibody Recognition," Ann. Rev. Biophys. Chem., 16:139-159 (1987). cited by other .
Mori et al., "Establishment of a new anti-cancer drugs-resistant cell line derived from B-chronic lymphocyctic leukemia," Proceedings, Fifty-Ninth Annual Meeting of the Japanese Cancer Association, p. 583, #3788 (Sep. 1, 2000). (abstract only). cited by other .
Riley, "Melanoma and the Problem Malignancy," J. Exp. Med., 204:1-9 (2004). cited by other .
Rudikoff et al., "Single amino acid substitution altering antigen-binding specificity," PNAS, 79:1979-1983 (1982). cited by other .
Schultes et al., "Immunotherapy of Human Ovarian Carcinoma With Ovarex.TM. Mab-B43.13 in a Human-PBL-SCID/BG Mouse Model," Hybridoma, 18(1):47-55 (1999). cited by other .
Snyder et al., "Enhanced Targeting and Killing of Tumor Cells Expressing the CXC Chemokine Receptor 4 by Transducible Anticancer Peptides," Cancer Research, 65(23):10646-10650 (2005). cited by other .
Tanaka et al., "The Anti-Human Tumor Effect and Generation of Human Cytotoxic T Cells in SCID Mice Given Human Peripheral Blood Lymphocytes by the in Vivo transfer of the Interleukin-6 Gene Using Adenovirus Vector," Cancer Research, 57:1335-1343 (1997). cited by other .
Thomsen et al., "Reconstitution of a human immune system in immunodeficient mice: models of human alloreaction in vivo," Tissue Antigens, 66:73-82 (2005). cited by other .
Wright et al., "The unusual distribution of the neuronal/lymphoid cell surface CD200 (OX2) glycoprotein is conserved in humans," Immunology, 102:173-179 (2001). cited by other .
DeNardo et al., "Increased Survival Associated with Radiolabeles Lym-1 Therapy for Non-Hodgkin's Lymphoma and Chronic Lymphocyctic Leukemia." Cancer Supplement (1997) vol. 80, No. 12, pp. 2706-2711. cited by other .
Faguet et al., "Blood Kinectics, Tissue Distribution, and Radioimaging of Anti-Common Chronic Lymphatic Leukemia Antigen (cCLLA) Monoclonal Antibody CLL.sub.2 in Mice Transplanted With cCLLa- Bearing Human Leukemia Cells." Blood. vol. 75, No. 9 (1990) pp. 1853-1861. cited by other .
Funakoshi et al., "Antitumor Effects of Nonconjugated Murine Lym-2 and Human-Mouse Chimeric CLL-1 Monoclonal Antibodies Against Various Human Lumphoma Cell Lines In Vitro and In Vivo." Blood vol. 90, No. 8 (1997) pp. 3160-3166. cited by other .
Zhu et al., "Radioimmunotherapy of Human B-Cell Chronic Lymphocytic Leukemia in Nude Mice." Cancer Research. 54, 5111-5117 (1994). cited by other .
Gorczynski, "Evidence for an Immunoregulartory Role of OX2 with its Counter Ligand (OX2L) in the Regulation of Transplant Rejection, Fetal Loss, Autoimmunity and Tumor Growth," Archibum Immunologiae et Therapiae Experimentalis, 2001, 49, 303-309. cited by other .
Sahin et al., "New monoclonal antibody specific for a 6.5 kDa glycoprotein which presents mainly on a B cell of chronic lymphocytic leukemia (CLL)" Immunology Letters, 2001, 76, 1-6. cited by other .
Zou et al. Human Glioma-Induced Immunosuppression Involves Soluble Factor(s) That Alters Monocyte Cytokine Profile and Surface Markers. Apr. 15, 1999, vol. 162, pp. 4882-4892. cited by other .
Brody et al. "Human Cancer Detection and Immunotherapy with Conjugated and Non-Conjugated Monoclonal Antibodies" Anticancer Research 16:661-674 (1996). cited by other .
Kretz-Rommel, A., et al., "CD200 Expression on Tumor Cells Suppresses Anti-Tumor Immunity: New Approaches to Cancer Immunotherapy," J. Immunother., 29(6):666 (2006). cited by other .
Kretz-Rommel, A., et al., "Immune Evasion by CD200: New Approaches to Targeted Therapies for Chronic Lymphocytic Leukemia," J. Immunother., 28(6):650 (2005). cited by other .
Kretz-Rommel, A., et al., "The Immuno-Regulatory Protein CD200 Is Overexpressed in a Subset of B-Cell Chronic Lymphocytic Leukemias and Plays a Role in Down-Regulating the TH1 Immune Response," J. Immunother., 27(6):S46 (2004). cited by other .
McWhirter, J.R., et al., "Antibodies selected from combinatorial libraries block a tumor antigen that plays a key role in immunomodulation," PNAS, 103(4):1041-1046 (2006). cited by other .
Elgert, K. D. "Immunology : Understanding the Immune System," The Genetic Basis of Antibody Diversity, 123 (1996). cited by other .
Schlom, Jeffrey :Monoclonal Antibodies They're More and Less Than You Think, In: Molecular Foundations of Oncology, Broader, ed., pp. 95-134, 1991. cited by other .
Sehgal, et al., "Generation of the Primary Antibody Repertoire in Rabbits: Expression of a Diverse Set of Igk-V Genes May Compensate for Limited Combinatorial Diversity at the Heavy Chain Locus," Immunogenetics 50:31-42 (1999). cited by other .
Matutes et al. Morphological and Immuniphenotypic Features of Chronic Lymphocytic Leukemia. Rev. Clin. Exp. Hematol. vol. 4.1, Mar. 2000 pp. 22-47. cited by other .
Sebestyen et al., Syndecan-1 (CD138) expression in human non-Hodgkin lymphomas. British Jounal of Hematology. vol. 104, 1999, pp. 412-419. cited by other .
Bauvois et al., Constitutive expression of CD26/dipeptidylpepidase IV on peripheral blood B lymphocytes of patients with B chronic lymphocytic leukaemia. British Journal of Cancer 1999., vol 79. pp. 1042-1048. cited by other .
Database Medline, abstract No. NLM16160950, Rioux P. 1999. cited by other .
Feuerstein et al., 1999, Induction of Autoimmunity in a Transgenic Model of B Cell Receptor Peripheral Tolerance: Changes in Coreceptors and B Cell Receptor-Induced Tyrosine-Phosphoproteins, J. Immunol. 163: 5287-5297. cited by other.

Primary Examiner: Canella; Karen A
Attorney, Agent or Firm: Ropes & Gray LLP

Parent Case Text

RELATED APPLICATIONS

This application is a continuation in part of PCT/US01/47931 filed on Dec. 10, 2001 which is an international application that claims priority to U.S. Provisional Application No. 60/254,113 filed Dec. 8, 2000. The entire disclosures of both the aforementioned international and provisional applications are incorporated herein by reference.

Claims

We claim:

1. A method of treating chronic lymphocytic leukemia (CLL) comprising administering to a patient suffering from CLL an antibody or antigen-binding fragment thereof that specifically binds to OX-2/CD200, wherein said antibody or antigen-binding fragment thereof comprises a light chain CDR1 having the sequence set forth in SEQ ID NO: 5; a light chain CDR2 having the sequence set forth in SEQ ID NO: 21; a light chain CDR3 having the sequence set forth in SEQ ID NO: 29; a heavy chain CDR1 having the sequence set forth in SEQ ID NO: 50; a heavy chain CDR2 having the sequence set forth in SEQ ID NO: 69; and a heavy chain CDR3 having the sequence set forth in SEQ ID NO: 88.

2. The method of claim 1, wherein said antibody or antigen-binding fragment thereof is selected from the group consisting of a monoclonal antibody, a human antibody, a humanized antibody, a chimeric antibody, Fv, scFv, Fab' and F(ab').sub.2.

3. The method of claim 2, wherein said humanized antibody or antigen-binding fragment thereof comprises a framework modification.

4. The method of claim 1, wherein OX-2/CD200 is overexpressed by CLL cells.

5. The method of claim 4, wherein said CLL is B-cell chronic lymphocytic leukemia (B-CLL).

6. A method of treating chronic lymphocytic leukemia (CLL) comprising administering to a patient suffering from CLL an antibody or antigen-binding fragment thereof that specifically binds to OX-2/CD200, wherein said antibody or antigen-binding fragment thereof comprises a light chain CDR1 having the sequence set forth in SEQ ID NO: 13; a light chain CDR2 having the sequence set forth in SEQ ID NO: 23; a light chain CDR3 having the sequence set forth in SEQ ID NO: 38; a heavy chain CDR1 having the sequence set forth in SEQ ID NO: 56; a heavy chain CDR2 having the sequence set forth in SEQ ID NO: 75; and a heavy chain CDR3 having the sequence set forth in SEQ ID NO: 94.

7. The method of claim 6, wherein said antibody or antigen-binding fragment thereof is selected from the group consisting of a monoclonal antibody, a human antibody, a humanized antibody, a chimeric antibody, Fv, scFv, Fab' and F(ab').sub.2.

8. The method of claim 7, wherein said humanized antibody or antigen-binding fragment thereof comprises a framework modification.

9. The method of claim 6, wherein OX-2/CD200 is overexpressed by CLL cells.

10. The method of claim 9, wherein said CLL is B-cell chronic lymphocytic leukemia (B-CLL).

11. A method of treating chronic lymphocytic leukemia (CLL) comprising administering to a patient suffering from CLL an antibody or antigen-binding fragment thereof that specifically binds to OX-2/CD200, wherein said antibody or antigen-binding fragment thereof comprises a light chain CDR1 having the sequence set forth in SEQ ID NO: 12; a light chain CDR2 having the sequence set forth in SEQ ID NO: 23; a light chain CDR3 having the sequence set forth in SEQ ID NO: 37; a heavy chain CDR1 having the sequence set forth in SEQ ID NO: 55; a heavy chain CDR2 having the sequence set forth in SEQ ID NO: 74; and a heavy chain CDR3 having the sequence set forth in SEQ ID NO: 93.

12. The method of claim 11, wherein said antibody or antigen-binding fragment thereof is selected from the group consisting of a monoclonal antibody, a human antibody, a humanized antibody, a chimeric antibody, Fv, scFv, Fab' and F(ab').sub.2.

13. The method of claim 12, wherein said humanized antibody or antigen-binding fragment thereof comprises a framework modification.

14. The method of claim 11, wherein OX-2/CD200 is overexpressed by CLL cells.

15. The method of claim 14, wherein said CLL is B-cell chronic lymphocytic leukemia (B-CLL).

Description

TECHNICAL FIELD

Cell lines derived from chronic lymphocytic leukemia (CLL) cells and the uses thereof in the study and treatment of CLL disease are disclosed. In particular, this disclosure relates to a CLL cell line designated "CLL-AAT", deposited on December 11, 2001 with the American Type Culture Collection (Manassas, Va., USA) in accordance with the terms of the Budapest Treaty under ATCC accession no. PTA-3920.

BACKGROUND

Chronic Lymphocytic Leukemia (CLL) is a disease of the white blood cells and is the most common form of leukemia in the Western Hemisphere. CLL represents a diverse group of diseases relating to the growth of malignant lymphocytes that grow slowly but have an extended life span. CLL is classified in various categories that include, for example, B-cell chronic lymphocytic leukemia (B-CLL) of classical and mixed types, B-cell and T-cell prolymphocyic leukemia, hairy cell leukemia, and large granular lymphocytic leukemia.

Of all the different types of CLL, B-CLL accounts for approximately 30 percent of all leukemias. Although it occurs more frequently in individuals over 50 years of age it is increasingly seen in younger people. B-CLL is characterized by accumulation of B-lymphocytes that are morphologically normal but biologically immature, leading to a loss of function. Lymphocytes normally function to fight infection. In B-CLL, however, lymphocytes accumulate in the blood and bone marrow and cause swelling of the lymph nodes. The production of normal bone marrow and blood cells is reduced and patients often experience severe anemia as well as low platelet counts. This can pose the risk of life-threatening bleeding and the development of serious infections because of reduced numbers of white blood cells.

To further understand diseases such as leukemia it is important to have suitable cell lines that can be used as tools for research on their etiology, pathogenesis and biology. Examples of malignant human B-lymphoid cell lines include pre-B acute lymphoblasticleukemia (Reh), diffuse large cell lymphoma (WSU-DLCL2), and Waldenstrom's macroglobulinemia (WSU-WM). Unfortunately, many of the existing cell lines do not represent the clinically most common types of leukemia and lymphoma.

The use of Epstein Barr Virus (EBV) infection in vitro has resulted in some CLL derived cell lines, in particular B-CLL cells lines, that are representative of the malignant cells. The phenotype of these cell lines is different than that of the in vivo tumors and instead the features of B-CLL lines tend to be similar to those of Lymphoblastoid cell lines. Attempts to immortalize B-CLL cells with the aid of EBV infection have had little success. The reasons for this are unclear but it is known that it is not due a lack of EBV receptor expression, binding or uptake. Wells et al. found that B-CLL cells were arrested in the G 1/S phase of the cell cycle and that transformation associated EBV DNA was not expressed. This suggests that the interaction of EBV with B-CLL cells is different from that with normal B cells. EBV-transformed CLL cell lines moreover appear to differentiate, possessing a morphology more similar to lymphoblastoid cell lines (LCL) immortalized by EBV.

An EBV-negative CLL cell line, WSU-CLL, has been established previously (Mohammad et al., (1996) Leukemia 10(1):130-7). However, no other such cell lines are known.

There remains a need in the art, therefore, for a CLL cell line which has not been established by transformation with EBV, and which expresses surface markers characteristic of primary CLL cells.

SUMMARY

In one embodiment an CLL cell line of malignant origin is provided that is not established by immortalisation with EBV. The cell line, which was derived from primary CLL cells, and is deposited under ATCC accession no. PTA-3920. In a preferred embodiment, the cell line is CLL-AAT. CLL-MT is B-CLL cell line, derived from a B-CLL primary cell.

In a further aspect, the CLL-AAT cell line is used to generate monoclonal antibodies useful in the diagnosis and/or treatment of CLL. Antibodies may be generated by using the cells as disclosed herein as immunogens, thus raising an immune response in animals from which monoclonal antibodies may be isolated. The sequence of such antibodies may be determined and the antibodies or variants thereof produced by recombinant techniques. In this aspect, "variants" includes chimeric, CDR-grafted, humanized and fully human antibodies based on the sequence of the monoclonal antibodies.

Moreover, antibodies derived from recombinant libraries ("phage antibodies") may be selected using the cells described herein, or polypeptides derived therefrom, as bait to isolate the antibodies on the basis of target specificity.

In a still further aspect, antibodies may be generated by panning antibody libraries using primary CLL cells, or antigens derived therefrom, and further screened and/or characterized using a CLL cell line, such as, for example, the CLL cell line described herein. Accordingly, a method for characterizing an antibody specific for CLL is provided, which includes assessing the binding of the antibody to a CLL cell line.

In a further aspect, there is provided a method for identifying proteins uniquely expressed in CLL cells employing the CLL-AAT cell line, by methods well known to those, skilled with art, such as by immunoprecipitation followed by mass spectroscopy analyses. Such proteins may be uniquely expressed in the CLL-AAT cell line, or in primary cells derived from CLL patients.

Small molecule libraries (many available commercially) may be screened using the CLL-AAT cell line in a cell-based assay to identify agents capable of modulating the growth characteristics of the cells. For example, the agents may be identified which modulate apoptosis in the CLL-AAT cell line, or which inhibit growth and/or proliferation thereof. Such agents are candidates for the development of therapeutic compounds.

Nucleic acids isolated from CLL-AAT cell lines may be used in subtractive hybridization experiments to identify CLL-specific genes or in micro array analyses (e.g., gene chip experiments). Genes whose transcription is modulated in CLL cells may be identified. Polypeptide or nucleic acid gene products identified in this manner are useful as leads for the development of antibody or small molecule therapies for CLL.

In a preferred aspect, the CLL-AAT cell line may be used to identify internalizing antibodies, which bind to cell surface components which are internalized by the cell. Such antibodies are candidates for therapeutic use. In particular, single-chain antibodies, which remain stable in the cytoplasm and which retain intracellular binding activity, may be screened in this manner.

In yet another aspect, a therapeutic treatment is described in which a patient is screened for the presence of a polypeptide that is upregulated by a malignant cancer cell and an antibody that interferes with the metabolic pathway of the upregulated polypeptide is administered to the patient.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. schematically illustrates typical steps involved in cell surface panning of antibody libraries by magnetically-activated cell sorting (MACS).

FIG. 2. is a graph showing the results of whole cell ELI SA demonstrating binding of selected scFv clones to primary B-CLL cells and absence of binding to normal human PBMC. The designation 2.degree.+3.degree. in this and other figures refers to negative control wells stained with Mouse Anti-HA and detecting antimouse antibodies alone. The designation RSC-S Library in this and other figures refers to soluble antibodies prepared from original rabbit scFv unpanned library. The designation R3/RSC-S Pool in this and other figures refers to soluble antibodies prepared from entire pool of scFv antibodies from round 3 of panning. Anti-CD5 antibody was used as a positive control to verify that equal numbers of B-CLL and PBMC cells were plated in each well.

FIGS. 3a and 3b show the results of whole cell ELISA comparing binding of selected scFv antibodies to primary B-CLL cells and normal primary human B cells. Anti-CD19 antibody was used as a positive control to verify that equal numbers of B-CLL and normal B cells were plated in each well. Other controls were as described in the legend to FIG. 2.

FIGS. 4a and 4b show the results of whole cell ELISA used to determine if scFv clones bind to patient-specific (i.e. idiotype) or blood type-specific (i.e. HLA) antigens. Each clone was tested for binding to PBMC isolated from 3 different B-CLL patients. Clones that bound to <1 patient sample were considered to be patient or blood type-specific.

FIGS. 5a and 5b show the results of whole cell ELISA comparing binding of scFv clones to primary B-CLL cells and three human leukemic cell lines. Ramos is a mature B cell line derived from a Burkitt's lymphoma. RL is a mature B cell line derived from a non-Hodgkin's lymphoma. TF-I is an erythroblastoid cell line derived from a erythroleukemia.

FIGS. 6a, 6b and 6c show the results of whole cell ELISA comparing binding of scFv clones to primary B-CLL cells and CLL-MT, a cell line derived from a B-CLL patient. TF-I cells were included as a negative control.

FIG. 7 shows the binding specificity of scFv antibodies in accordance with this disclosure as analyzed by 3-color flow cytometry. In normal peripheral blood mononuclear cells, the antigen recognized by scFv-9 is moderately expressed on B lymphocytes and weakly expressed on a subpopulation of T lymphocytes. PBMC from a normal donor were analyzed by 3-color flow cytometry using anti-CD5-FITC, anti-CD19-PerCP, and scFv-9/Anti-HA-biotin/streptavidin-PE.

FIGS. 8a, 8b and 8c show the expression levels of antigens recognized by scFv antibodies in accordance with this disclosure. The antigens recognized by scFv-3 and scFv-9 are overexpressed on the primary CLL tumor from which the CLL-MT cell line was derived. Primary PBMC from the CLL patient used to establish the CLL-MT cell line or PBMC from a normal donor were stained with scFv antibody and analyzed by flow cytometry. ScFv-3 and scFv-9 stain the CLL cells more brightly than the normal PBMC as measured by the mean fluorescent intensities.

FIGS. 9A-9C provide a summary of CDR sequences and binding specificities of selected scFv antibodies. As shown in FIG. 9B, the clone numbers listed in the left column are also referred to herein by scFv numbers as follows: A2c (scFv-1), G12.1c (scFv-2), B4.2a (scFv-17), E1c (scFv-3), F2d (scFv-18), E5e (scFv-4), H6.2b (scFv-5), G10.1 (scFv-19), D11.1c (scFv-6), A5.2c (scFv-20), F1d (scFv-7), F1e (scFv-8), E4.2 (scFv-21), E2c (scFv-9), A9c scFv-9), E11e (scFv-10), A1.1 (scFv-11). F5.2 (scFv-12), F10b (scFv-22), F7a (scFv-23), F6b (scFv-13), C12b (scFv-24), D2.1b (scFv-14), D1.1 (scFv-25), D2.2a (scFv-15), and D2.2b (scFv-16).

FIG. 10. is Table 2 which shows a summary of flow cytometry results comparing expression levels of scFv antigens on primary CLL cells vs. normal PBMC as described in FIGS. 8a-8c.

FIG. 11. is a Table showing a summary of flow cytometry results comparing expression levels of scFv-9 antigen with the percentage of CD38+cells in peripheral blood mononuclear cells isolated from ten CLL patients.

FIG. 12. shows the identification of scFv antigens by immunoprecipitation and mass spectrometry. CLL-MT cells were labeled with a solution of 0.5 mg/ml sulfo-NHS-LC-biotin (Pierce) in PBS, pH8.0 for 30'. After extensive washing with PBS to remove unreacted biotin, the cells were disrupted by nitrogen cavitation and the microsomal fraction was isolated by differential centrifugation. The microsomal fraction was resuspended in NP40 Lysis Buffer and extensively precleared with normal rabbit serum and protein A sepharose. Antigens were immunoprecipitated with HA-tagged scFv antibodies coupled to Rat Anti-HA agarose beads (Roche). Following immunoprecipitation, antigens were separated by SDS-PAGE and detected by Western blot using streptavidin-alkaline phosphatase(AP) or by Coomassie G-250 staining. ScFv-7, an antibody which doesn't bind to CLL-AAT cells, was used as a negative control. Antigen bands were excised from the Coomassie-stained gel and identified by mass spectrometry (MS). MALDI-MS was performed at the Proteomics Core Facility of The Scripps Research Institute (La Jolla, Calif.). .mu.LC/MS/MS was performed at the Harvard Microchemistry Facility (Cambridge, Mass.).

FIG. 13. shows that three scFv antibodies bind specifically to 293-EBNA cells transiently transfected with a human OX-2/CD200 cDNA clone. A CD200 cDNA was cloned from CLL cells by RT-PCR and inserted into the mammalian expression vector pCEP4 (Invitrogen). PCEP4-CD200 plasmid or the corresponding empty vector pCEP4 was transfected into 293-EBNA cells using Polyfect reagent (QIAGEN). Two days after transfection, the cells were analyzed for binding to scFv antibodies by flow cytometry.

DETAILED DESCRIPTION

Definitions

"CLL", as used herein, refers to chronic lymphocytic leukemia involving any lymphocyte, including but not limited to various developmental stages of B cells and T cells, including but not limited to B cell CLL. B-CLL, as used herein, refers to leukemia with a mature B cell phenotype which is CD5.sup.+, CD23.sup.+, CD20.sup.dim+, sIg.sup.dim+ and arrested in G0/G1 of the cell cycle.

"Malignant origin" refers to the derivation of the cell line from malignant CLL primary cells, as opposed to non-proliferating cells which are transformed, for example, with EBV. Cell lines according to this disclosure may be themselves malignant in phenotype, or not. A CLL cell having a "malignant" phenotype encompasses cell growth unattached from substrate media characterized by repeated cycles of cell growth and exhibits resistance to apoptosis.

Preparation of Cell Lines

Cell lines may be produced according to established methodologies known to those skilled in the art. In general, cell lines are produced by culturing primary cells derived from a patient until immortalized cells are spontaneously generated in culture. These cells are then isolated and further cultured, to produce clonal cell populations or cells exhibiting resistance to apoptosis.

For example, CLL cells may be isolated from peripheral blood drawn from a patient suffering from CLL. The cells may be washed, and optionally immunotyped in order to determine the type(s) of cells present. Subsequently, the cells may be cultured in a medium, such as a medium containing IL-4. Advantageously, all or part of the medium is replaced one or more times during the culture process. Cell lines may be isolated thereby, and will be identified by increased growth in culture.

Preparation of Monoclonal Antibodies

Antibodies, as used herein, refers to complete antibodies or antibody fragments capable of binding to a selected target. Included are Fv, ScFv, Fab' and F(ab')2, monoclonal and polyclonal antibodies, engineered antibodies (including chimeric, CDR-grafted and humanized, fully human antibodies, and artificially selected antibodies), and synthetic or semi synthetic antibodies produced using phage display or alternative techniques. Small fragments, such Fv and ScFv, possess advantageous properties for diagnostic and therapeutic applications on account of their small size and consequent superior tissue distribution.

The antibodies are especially indicated for diagnostic and therapeutic applic