CN101384280A - Polypeptides and antibodies derived from chronic lymphocytic leukemia cells and applications thereof - Google Patents

Abstract

Methods of using polypeptides, including antibodies, capable of binding to OX-2/CD 200 and/or OX-2/CD 200 receptors are disclosed herein. These polypeptides are useful for treating diseases characterized by elevated levels of OX-2/CD 200, including cancer and Chronic Lymphocytic Leukemia (CLL).

Description

Polypeptides and antibodies derived from chronic lymphocytic leukemia cells and applications thereof

related application

This application is a continuation-in-part of U.S. Application Serial No. 10/736,188, filed December 15, 2003, which is a continuation-in-part of U.S. Application Serial No. 10/379,151, filed March 4, 2003, which in turn was filed in 2001 Continuation in part of PCT/US01/47931, filed December 10, an international application claiming priority to US Provisional Application No. 60/254,133, filed December 8,2000. The entire disclosures of the aforementioned US, International and Provisional Applications are hereby incorporated by reference.

technical field

Cell lines derived from chronic lymphocytic leukemia (CLL) cells and their use in the study and treatment of CLL and other diseases are disclosed. Specifically, the present disclosure relates to a CLL cell line designated "CLL-AAT" deposited with the American Type Culture Collection (Manassas, Virginia, USA), ATCC Accession No. PTA-3920.

Background of the invention

Chronic lymphocytic leukemia (CLL) is a disease of white blood cells that is the most common form of leukemia in the Western Hemisphere. CLL represents a group of different forms of diseases associated with the growth of malignant lymphocytes that grow slowly but have a prolonged lifespan. CLL is divided into different types including, for example, classic and mixed B-cell chronic lymphocytic leukemia (B-CLL), B-cell and T-cell prolymphocytic leukemia, hairy cell leukemia, and large granular lymphocytic leukemia .

Of all the different types of CLL, B-CLL accounts for approximately 30% of all leukemias. Although it occurs more frequently in individuals over the age of 50, it is also increasingly seen in younger populations. B-CLL is characterized by the accumulation of morphologically normal but biologically immature and nonfunctional B lymphocytes. The normal function of lymphocytes is to fight infection. However, in B-CLL, lymphocytes accumulate in the blood and bone marrow, causing the lymph nodes to swell. Production of normal bone marrow and blood cells is reduced, and patients often experience severe anemia and low platelet counts. This can cause life-threatening bleeding, and serious infections can develop due to a decrease in the number of white blood cells.

To further understand diseases like leukemias, it is important to have appropriate cell lines that can be used as tools to study their etiology, pathogenesis and biology. Examples of malignant human B lymphocyte-like cell lines include pre-B acute lymphoblastic leukemia (Reh), diffuse large cell leukemia (WUS-DLCL2) and Waldenstrom's macroglobulinemia (WUS-WM). Unfortunately, most existing cell lines do not represent the most clinically common types of leukemia and lymphoma.

Some CLL-derived cell lines, especially B-CLL cell lines, are representative of malignant cells by in vitro infection with Epstein Barr virus (EBV). The phenotype of these cell lines differs from that of tumors in vivo, and the characteristics of B-CLL strains tend to resemble those of cell lines of the lymphoblastoid class instead. Attempts to immortalize B-CLL cells with the help of EBV infection have rarely been successful. The reason for this is unclear, but it is known not to be due to lack of expression, binding or uptake of the EBV receptor. Wells et al. found that B-CLL cells were arrested in the G1/S phase of the cell cycle and there was no expression of EBV DNA associated with transformation. This suggests that EBV interacts differently with B-CLL cells than it does with normal B cells. EBV-transformed CLL cell lines were more differentiated, with a morphology more similar to lymphoblastoid-like cell lines (LCL) immortalized with EBV.

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

A number of different mechanisms are at play in the body's response to disease states, including cancer and CLL. For example, CD4 ⁺ helper T cells play an important role in efficient immune responses against various malignancies by providing stimulatory factors to effector cells. Cytotoxic T cells are believed to be the most effective cells for eliminating cancer cells, and helper T cells prime cytotoxic T cells by secreting Th1 cytokines such as IL-2 and IFN-γ. In various malignancies, helper T cells have been shown to have altered phenotypes compared to cells found in normal individuals. A markedly altered feature is reduced production of Th1 cytokines, with a shift to production of Th2 cytokines. (See eg Kiani et al., Haematologica 88:754-761 (2003); Maggio et al., Ann Oncol 13 Suppl 1:52-56 (2002); Ito et al., Cancer 85:2359-2367 (1999); Podhorecka et al., Leuk Res 26 :657-660 (2002); Tatsumi et al., J Exp Med 196:619-628 (2002); Agarwal et al., Immunol Invest 32:17-30 (2003); Smyth et al., Ann Surg Oncol 10:455-462 (2003) ; Contasta et al., Cancer Biother Radiopharm 18:549-557 (2003); Lauerova et al., Neoplasma 49:159-166 (2002).) It has been demonstrated that reversing this cytokine shift to Th1 increases T cell resistance Tumor effects (see Winter et al., Immunology 108:409-419 (2003); Inagawa et al., Anticancer Res 18:3957-3964 (1998)).

Mechanisms underlying the ability of tumor cells to drive the expression of cytokines of helper T cells from Th1 to Th2 include the secretion of cytokines such as IL-10 or TGF-β and the expression of surface molecules that interact with cells of the immune system. OX-2/CD200, a molecule expressed on the surface of dendritic cells with high homology to molecules of the immunoglobulin gene family, has been implicated in immunosuppression (Gorczynski et al., Transplantation 65:1106-1114 (1998) ), evidence that cells expressing OX-2/CD200 are able to suppress stimulation of Th1 cytokine production has been provided. Gorczynski et al. confirmed in a mouse model that infusion of OX-2/CD200Fc inhibited tumor cell rejection in an animal model using leukemia tumor cells (Clin Exp Immunol 126:220-229 (2001)).

Improvements in methods of treating patients with cancer or CLL are desirable, in particular to improve the extent to which they enhance T cell activity.

Brief description of the invention

In one embodiment, a CLL cell line of malignant origin not established by immortalization with EBV is provided. This cell line is derived from primary CLL cells and deposited with the ATCC under accession number PTA-3920. In a preferred embodiment, the cell line is CLL-AAT. CLL-AAT is a B-CLL cell line derived from B-CLL primary cells.

In another aspect, the CLL-AAT cell line is used to produce monoclonal antibodies for the diagnosis and/or treatment of CLL. Antibody production can be achieved by using the cells of the present invention as immunogens to elicit an immune response in animals, from which monoclonal antibodies are isolated. The sequences of these antibodies can be determined, and antibodies and variants thereof can be produced by recombinant techniques. "Variants" in this regard include chimeric, CDR-grafted, humanized and fully human antibodies based on monoclonal antibody sequences.

In addition, antibodies derived from recombinant libraries ("phage antibodies") can be screened by isolating antibodies on the basis of target specificity using cells of the invention or polypeptides derived therefrom as baits.

Alternatively, antibodies can be produced by panning antibody libraries using primary CLL cells or antigens derived therefrom, followed by further screening and/or characterization using a CLL cell line such as those described herein. Accordingly, methods are provided for characterizing antibodies specific for CLL comprising evaluating binding of the antibodies to CLL cell lines.

In another aspect, methods are provided for identifying proteins uniquely expressed in CLL cells, using the CLL-AAT cell line, using methods well known to those skilled in the art, such as immunoprecipitation followed by mass spectrometry. These proteins may be uniquely expressed in CLL-AAT cell lines, or uniquely expressed in primary cells from CLL patients.

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

Nucleic acids isolated from CLL-AAT cell lines can be used in subtractive hybridization experiments to identify CLL-specific genes, or in microarray analysis (eg, gene chip experiments). Genes whose transcription is regulated in CLL cells can be identified. Polypeptides or nucleic acid gene products identified in this manner are used as precursors for the development of antibody or small molecule therapies for CLL.

Preferably, the CLL-AAT cell line can be used to identify endogenous antibodies that bind to cell surface components internalized by the cells. Such antibodies are candidates for therapeutic applications. In particular, scFvs that remain stable in the cytoplasm and retain intracellular binding activity can be screened in this way.

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

The invention also contemplates methods wherein a decision is made on whether OX-2/CD200 is upregulated in the patient, and if so, a polypeptide that binds to OX-2/CD200 is administered to the patient. In another embodiment, the polypeptide binds to the OX-2/CD200 receptor.

In another aspect, the methods of the invention are used to treat a disease state in a patient in which OX-2/CD200 is upregulated by administering to the diseased patient a of peptides. In one embodiment, the disease states treated with these methods include cancer, and in other embodiments specifically CLL.

Brief description of the drawings

Figure 1 schematically illustrates typical steps involved in cell surface panning of antibody libraries by the method of magnetic activated cell sorting (MACS).

Figure 2 shows the results of a whole-cell ELISA demonstrating the binding of selected scFv clones to primary B-CLL cells and not to normal human PBMC. The symbols 2°+3° in this and other figures refer to negative control wells stained with mouse anti-HA only and detected with anti-mouse antibody only. The symbol RSC-S library in this and other figures refers to soluble antibodies prepared from the original rabbit scFv unpanned library. The symbol R3/RSC-S library in this figure and other figures refers to the soluble antibody prepared from the complete scFv antibody library obtained from the third round of panning. Anti-CD5 antibody was used as a positive control to confirm that equal numbers of B-CLL and PBMC cells were placed in each well.

Figures 3a and 3b show the results of a whole cell ELISA comparing the 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 confirm that equal numbers of B-CLL and normal B cells were placed in each well. Other controls were the same as described in the legend of Figure 2 .

Figures 4a and 4b show the results of a whole cell ELISA used to determine whether scFv clones bind to patient-specific (ie idiotype) or blood-specific (ie 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 of the patient or blood specific type.

Figures 5a and 5b show the results of a whole-cell ELISA comparing the binding of scFv clones to primary B-CLL cells and three human leukemia cell lines. Ramos is a mature B cell line derived from Burkitt's lymphoma. RL is a mature B cell line derived from non-Hodgkin's lymphoma. TF-1 is an erythroblastoid cell line derived from erythroleukemia.

Figures 6a, 6b and 6c show the results of a whole cell ELISA comparing the binding of scFv clones to primary B-CLL cells and the cell line CLL-AAT derived from a B-CLL patient. TF-1 cells were included as a negative control.

Figure 7 shows the binding specificity of scFv antibodies of the present invention, analyzed by 3-color flow cytometry. In normal peripheral blood mononuclear cells, antigens recognized by scFv-9 are moderately expressed on B lymphocytes and weakly expressed on T lymphocyte subsets. PBMCs from normal donors were analyzed by 3-color flow cytometry using anti-CD5-FITC, anti-CD19-PerCP and scFv-9/anti-HA-biotin/streptavidin-PE.

Figures 8a, 8b and 8c show the expression levels of antigens recognized by the scFv antibodies of the present invention. Antigens recognized by scFv-3 and scFv-9 were overexpressed on primary CLL tumors from which the CLL-AAT cell line was derived. Primary PBMCs from CLL patients used to establish CLL-AAT cell lines or PBMCs from normal donors were stained with scFv antibodies and analyzed by flow cytometry. scFv-3 and scFv-9 stained CLL cells more brightly than normal cells when measured with intermediate fluorescence intensities.

Figures 9a and 9b provide a summary of the CDR sequences and binding specificities of selected scFv antibodies.

Figure 10 is a table showing a summary of the results of a flow cytometry analysis comparing the expression levels of scFv antigens on primary CLL cells and normal PBMCs depicted in Figures 8a-8c.

Figure 11 is a table showing a summary of the results of flow cytometry analysis comparing the expression level of scFv-9 antigen with the percentage of CD38+ cells in peripheral blood mononuclear cells isolated from 10 CLL patients.

Figure 12 shows the identification of scFv antigens by immunoprecipitation and mass spectrometry. CLL-AAT cells were labeled with a solution of 0.5 mg/ml sulfo-NHS-LC-biotin (Pierce) in PBS, pH 8.0 for 30 min. After washing with excess PBS to remove unreacted biotin, cells were disrupted by nitrogen cavitation and the microsomal fraction was isolated by differential centrifugation. Microsomal fractions were resuspended in NP40 lysis buffer and pre-clarified in excess with normal rabbit serum and protein A Sepharose. Antigens were immunoprecipitated using HA-tagged scFv antibodies coupled to rat anti-HA agarose beads (Roche). After immunoprecipitation, antigens were separated by SDS-PAGE and detected by Western blot using streptavidin-alkaline phosphatase (AP) or staining using Coomassie brilliant blue G-250. Antibody scFv-7, which does not bind to CLL-AAT cells, was used as a negative control. Antigen bands were excised from Coomassie brilliant blue-stained gels and identified by mass spectrometry (MS). MALDI-MS was performed at the Proteomics Core Laboratory of the Scripps Research Institute (La Jolla, CA). LC/MS/MS was performed at the Harvard Microchemical Laboratory (Cambridge, MA).

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

Figure 14 shows that the presence of OX-2/CD200 transfected cells results in negative regulation of Th1 cytokines such as IL-2 and IFN-γ. Addition of 30 μg/ml anti-OX-2/CD200 antibody completely restored the Th1 response.

Figure 15 shows that the presence of CLL cells in a mixed lymphocyte reaction results in negative regulation of the Th1 response to IL-2.

Figure 16 shows that the presence of CLL cells in a mixed lymphocyte reaction results in negative regulation of the Th1 response to IFN-γ.

Figures 17A and B show the mean +/- SD of tumor volumes in all groups of NOD/SCID mice injected subcutaneously with ^4X106 RAJI cells in the presence or absence of humans.

Figure 18 shows the results of the statistical analysis using two parametric tests (Student's t-test and Welch's test) and one non-parametric test, the Wilcox test.

Detailed description of the invention

According to the present invention, methods are provided to determine whether OX-2/CD200 is upregulated in a patient, and if so, to administer to the patient a polypeptide that binds to OX-2/CD200. In general, polypeptides for use in the present invention can be constructed using a variety of techniques well known to those skilled in the art. In one embodiment, the polypeptide is obtained by chemical synthesis. In other embodiments, the polypeptide is an antibody, or is constructed from a fragment or fragments of one or more antibodies.

Preferably, the polypeptides used in the methods of the invention are obtained from CLL cell lines. "CLL" as used herein refers to chronic lymphocytic leukemia involving any lymphocyte, including but not limited to B cells and T cells of various developmental stages, including but not limited to B-cell CLL ("B-CLL "). B-CLL as used herein refers to a leukemia whose B cells have the phenotype of CD5 ⁺ , CD23 ⁺ , CD20 ^dim+ and sIg ^dim+ of mature B cells, but terminate in the G0/G1 phase of the cell cycle. In another aspect, CLL cell lines are used to produce polypeptides, including antibodies, for the diagnosis and/or treatment of disease states in which OX-2/CD200 is upregulated, including cancer and CLL.

The term "antibody" as used herein refers to a whole antibody or antibody fragment capable of binding to a selected target. Including Fv, scFv, Fab' and F(ab')2, monoclonal and polyclonal antibodies, engineered antibodies (including chimeric, CDR-grafted and humanized and fully human antibodies, as well as artificially selected antibodies) and using Synthetic or semi-synthetic antibodies produced by phage display or other techniques. Small fragments such as Fv and scFv have advantageous properties in diagnostic and therapeutic applications because of their small size and superior tissue distribution.

The polypeptides and/or antibodies used in the present invention are specifically indicated for diagnostic and therapeutic applications. They can thus be remodeled with effector proteins such as toxins or markers. Particularly preferred are labels that allow imaging of the distribution of the polypeptide or antibody in vivo. Such markers may be radioactive markers or radiopaque markers such as metal particles so as to be readily visible within the patient's body. Additionally, the marker may be a fluorescent marker or other marker that is visible in a tissue sample removed from the patient.

Antibody Production Monoclonal antibodies can be isolated from an immune response elicited in animals using the cells of the invention as immunogens. The sequences of these antibodies can be determined, and antibodies and variants thereof can be produced by recombinant techniques. In this regard, "variants" include chimeric, CDR-grafted, humanized and fully human antibodies based on the sequences of monoclonal antibodies and polypeptides capable of binding OX-2/CD200.

In addition, antibodies derived from recombinant libraries ("phage antibodies") can be screened by isolating antibodies or polypeptides on the basis of target specificity using the cells described herein or polypeptides derived therefrom as baits.

Alternatively, antibodies and polypeptides can be produced by panning antibody libraries using primary CLL cells or antigens derived therefrom, followed by further screening and/or characterization using a CLL cell line such as those described herein. Accordingly, methods are provided for characterizing an antibody or polypeptide specific for CLL comprising assessing binding of the antibody or polypeptide to a CLL cell line.

Preparation of cell lines

Cell lines can be generated according to existing methods well known to those skilled in the art. Generally, cell lines are generated by culturing primary cells from a patient until immortalized cells spontaneously arise in culture. These cells are then isolated and further cultured to generate clonal cell populations or cells that exhibit resistance to apoptosis.

For example, CLL cells can be isolated from peripheral blood drawn from a patient with CLL. These cells can be washed and immunophenotyped appropriately to determine the cell types present. The cells can then be cultured in a medium, eg, medium containing IL-4. Preferably, all or part of the medium is replaced one or more times during the culture. Cell lines can then be isolated and identified by increased growth in culture.

In one embodiment, a CLL cell line of malignant origin not established by EBV immortalization is provided. By "malignant origin" is meant that the cell line is derived from malignant CLL primary cells rather than, for example, non-proliferative cells transformed with EBV. The cell lines used in the present invention may or may not be phenotypically malignant in their origin. The "malignant" phenotype possessed by CLL cells includes independence of cell growth from substrate media during repeated cell growth cycles and resistance to apoptosis. This cell line derived from primary CLL cells is deposited with the ATCC under accession number PTA-3920. In a preferred embodiment, the cell line is CLL-AAT. CLL-AAT is a B-CLL cell line derived from B-CLL primary cells.

In one embodiment, proteins uniquely expressed in CLL cells are identified using the CLL-AAT cell line using methods well known to those skilled in the art, eg, immunoprecipitation followed by mass spectrometry. These proteins may be uniquely expressed in CLL-AAT cell lines, or uniquely expressed in primary cells from CLL patients.

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

Nucleic acids isolated from CLL-AAT cell lines can be used in subtractive hybridization experiments to identify CLL-specific genes, or in microarray analysis (eg, gene chip experiments). Genes whose transcription is regulated in CLL cells can be identified. Polypeptides or nucleic acid gene products identified in this manner are used as precursors for the development of antibody or small molecule therapies for CLL.

In one embodiment, the CLL-AAT cell line can be used to identify endogenous antibodies that bind to cell surface components internalized by the cells. Such antibodies are candidates for therapeutic applications. In particular, scFvs that remain stable in the cytoplasm and retain intracellular binding activity can be screened in this way.

Preparation of monoclonal antibodies

Recombinant DNA techniques can be used to improve the antibodies produced by the present invention. Therefore, chimeric antibodies can be constructed to reduce their immunogenicity in diagnostic or therapeutic applications. In addition, antibodies can also be humanized by CDR grafting and optional framework modification to minimize immunogenicity. See US Patent No. 5,225,539, the contents of which are incorporated herein by reference.

Antibodies can be obtained from animal serum or, in the case of monoclonal antibodies or fragments thereof, produced in cell culture. Recombinant DNA techniques can be used to produce antibodies in bacterial or preferably mammalian cell culture according to established procedures. Preferably the selected cell culture system will secrete the antibody product.

In another embodiment, the process of producing an antibody of the invention comprises culturing a host, such as E. coli or mammalian cells, which has been transformed with a hybrid vector. A vector contains one or more expression elements. These include a promoter operably linked to a first DNA sequence encoding a signal peptide which is linked in proper reading frame to a second DNA sequence encoding an antibody protein. Antibody proteins are then collected and isolated. Alternatively, the expression element may include a promoter operably linked to a polycistronic, such as a bicistronic, in which the DNA sequence encoding the antibody protein is each operably linked to a signal peptide in an appropriate reading frame .

The in vitro propagation of hybridoma cells or mammalian host cells is carried out in an appropriate medium, including conventional standard medium (such as Dulbecco's Modified Eagle Medium (DMEM) or RPMI 1640 medium), which can be supplemented with mammalian serum (such as fetal calf serum) or trace elements and growth maintenance supplements (e.g. feeder cells such as normal mouse peritoneal exudate cells, splenocytes, bone marrow macrophages, 2-aminoethanol, insulin, transferrin, low-density lipoprotein, oil acid, etc.). Propagation of host cells as bacteria or yeast cells is also carried out in an appropriate medium well known in the art. For example suitable media for bacteria include LE, NZCYM, NZM, Terrific Broth, SOB, SOC, 2x YT or M9 minimal media. Suitable media for yeast include YPD, YEPD, Minimal Medium or Complete Minimal Deficiency Medium (Dropout Medium).

In vitro production provides relatively pure antibody preparations and allows for scale-up to produce large quantities of the desired antibody. Bacterial, yeast, plant or mammalian cell culture techniques are well known in the art and include homogeneous suspension culture (e.g. in an airlift reactor or in a continuously stirred reactor) and immobilized or captured cell culture (e.g. in hollow fibers, in microcapsules, on agarose beads or ceramic cartridges).

Large quantities of the desired antibody can also be obtained by propagating mammalian cells in vivo. For this purpose, hybridoma cells producing the desired antibody are injected into a histocompatible mammal, causing the growth of antibody producing tumors. Alternatively, animals may also be primed with hydrocarbons, particularly mineral oils such as squalane (tetramethylpentadecane), prior to injection. Antibodies are isolated from the body fluids of those animals after 1 to 3 weeks. For example, hybridoma cells obtained by fusing appropriate myeloma cells with antibody-producing splenocytes from Balb/c mice or transfected cells derived from the hybridoma cell line Sp2/0 that produce the desired antibody , by intraperitoneal injection into Balb/c mice optionally pretreated with squalane. After 1 to 2 weeks, ascites fluid was obtained from the animals.

These and other techniques are discussed, for example, in Kohler and Milstein (1975) Nature 256:495-497; U.S. Patent No. 4,376,110; Harlow and Lane, A Guide to Antibody Experimentation (1988) Cold Spring Harbor Press, all cited herein for reference. Techniques for preparing recombinant antibody molecules are described in the aforementioned references and for example WO97/08320; U.S. Patent No. 5427908; U.S. Patent No. 5508717; Smith, 1985, Science, Vol.225, pp1315-1317; Parmley and Smith 1988, Gene 73 , pp305-318; De La Cruz et al., 1988, Journal of Biological Chemistry, 263 pp4318-4322; U.S. Patent No.5,403,484, U.S. Patent No.5223409; WO88/06630; WO92/15679; U.S. Patent No.5780279; No.5571698; U.S. Patent No.6040136; Davis et al., Cancer Metastasis Rev., 1999,18(4):421-5; Taylor et al., Nucleic Acids Research 20(1992):6287-6295; Tomizuka et al., Proc.Nat. Described in Academy of Science USA 97(2)(2000):722-727. The contents of all of these references are hereby incorporated by reference.

Screening for desired antibodies is performed in cell culture supernatants, preferably by immunofluorescent staining of CLL cells, by immunoblotting, by enzyme-linked immunoassays such as sandwich assays or dot assays, or by radioimmunoassays.

For isolation of antibodies, immunoglobulins in culture supernatant or ascitic fluid can be concentrated by, for example, precipitation with ammonium sulfate, dialysis against a hygroscopic material such as polyethylene glycol, filtration with a selective membrane, and the like. If necessary and/or desired, antibodies can be purified by conventional chromatographic methods, such as gel filtration, ion exchange chromatography, DEAE cellulose chromatography and/or (immuno)affinity chromatography, e.g. One or more surface peptides derived from CLL cell lines or protein A or G were subjected to affinity chromatography.

Another embodiment provides a method of producing a bacterial cell line that secretes antibodies directed against the cell line, the method being characterized by immunizing a suitable mammal, such as a rabbit, with a collected CLL patient sample. Phage display libraries generated from immunized rabbits are constructed and the desired antibodies are elucidated therefrom using methods well known in the art, such as those published in various references incorporated herein by reference.

Hybridoma cells secreting monoclonal antibodies are also contemplated. Preferred hybridoma cells should be genetically stable, secrete the monoclonal antibodies of the invention with the desired specificity, and can be activated by thawing and recloning from deep-frozen cultures.

In another embodiment, there is provided a method of making a hybridoma cell line that secretes a monoclonal antibody directed against the CLL cell line of the invention. In this method, a suitable mammal, such as a Balb/c small mouse. Antibody-producing cells in the immunized mammal are either cultured briefly or fused with cells of an appropriate myeloma cell line. Hybrid cells obtained in the fusion are cloned and screened for cell clones secreting the desired antibody. For example, the splenocytes of Balb/c mice immunized with the cell line of the present invention are fused with the cells of the myeloma cell line PAI or the myeloma cell line Sp2/0-Ag14, and the obtained hybrid cells are screened for the secretion of the desired antibody, Positive hybridoma cells were cloned.

A preferred method of producing a hybridoma cell line, characterized in that between 10 ⁶ and 10 ⁷ cells of the cell line of the invention are injected subcutaneously and/or intraperitoneally, over a period of several months, for example 2 to 4 Balb/c mice are injected several times eg 4 to 6 times over a period of a month. Splenocytes are obtained from the immunized mice 2 to 4 days after the last injection and fused with cells of the myeloma cell line PAI in the presence of a fusion promoter, preferably polyethylene glycol. Preferably, the myeloma cells are fused with a 3 to 20 fold excess of splenocytes from the immunized mouse in a solution containing about 30% to about 50% polyethylene glycol having a molecular weight of about 4000. After fusion, the cells are plated on an appropriate medium as previously described, and a selection medium such as HAT medium is added at regular intervals in order to prevent normal myeloma cells from outgrowing the desired hybridoma cells.

In another embodiment, recombinant DNA is produced that contains inserts encoding the heavy chain variable region and/or the light chain variable region of an antibody raised against the aforementioned cell line. The term DNA includes coding single-stranded DNAs, double-stranded DNAs comprising the coding DNAs and complementary DNAs thereto, or the complementary (single-stranded) DNAs themselves.

In addition, the DNA encoding the heavy chain variable region and/or the light chain variable region of the antibody against the cell line of the present invention may be enzymatically or chemically synthesized with the heavy chain variable region and/or the light chain variable region. The DNA of the actual DNA sequence encoding the variable region may also be a mutant thereof. A mutant of the actual DNA is the DNA encoding the heavy chain variable region and/or the light chain variable region of the above-mentioned antibodies, in which one or more amino acids have been deleted or replaced by one or more other amino acids. Preferably, for humanization and expression optimization applications, the modification is located outside the CDRs of the heavy chain variable region and/or the light chain variable region of the antibody. The term mutant DNA also includes silent mutations in which one or more nucleotides are replaced by other nucleotides, resulting in new codons encoding the same amino acid. The term mutant sequence also includes degenerate sequences. A degenerate sequence refers to degeneracy in the sense of the genetic code, in which an unlimited number of nucleotides are replaced by other nucleotides without resulting in a change in the originally encoded amino acid sequence. Such degenerate sequences may be useful because they have different restriction sites and/or specific codon frequencies preferred by specific hosts, especially E. coli, in order to obtain heavy chain murine variable regions and/or Optimal expression of light chain mouse variable regions.

The term mutant includes DNA mutants obtained by in vitro mutation of real DNA according to methods well known in the art.

For the assembly of complete tetrameric immunoglobulin molecules and the expression of chimeric antibodies, recombinant DNA inserts encoding the heavy and light chain variable regions are fused to the corresponding DNA encoding the heavy and light chain constant regions, then in For example, after being incorporated into a hybrid vector, it is transferred to an appropriate host cell.

Also provided is a recombinant DNA comprising an insert encoding the heavy chain murine variable region of an antibody directed against the cell line of the invention and a human constant region g, such as gamma1, gamma2, gamma3 and gamma4, preferably gamma1 or gamma4 fusion. Also provided are recombinant DNAs comprising the fusion of an insert encoding the light chain murine variable region of an antibody directed against the cell line of the invention to the human constant region kappa or lambda, preferably kappa.

Another embodiment relates to recombinant DNAs encoding recombinant polypeptides in which the heavy and light chain variable regions are linked by a spacer, may also contain a signal sequence to facilitate processing of the antibody in a host cell, and/or contain DNA encoding peptides to facilitate purification of antibodies, and/or hydrolysis sites and/or peptide spacers and/or effector molecules.

DNA encoding an effector molecule refers to DNA encoding an effector molecule useful for diagnostic or therapeutic applications. It is therefore specified that the effector molecule may be a toxin or an enzyme, in particular an enzyme capable of catalyzing the activation of a prodrug. DNA encoding such effector molecules has DNA encoding sequences of naturally occurring enzymes or toxins or mutants thereof, and can be prepared by methods well known in the art.

The antibodies and antibody fragments of the invention are useful in diagnosis and therapy. Accordingly, there is provided a therapeutic or diagnostic composition comprising an antibody of the invention.

In a diagnostic composition, the antibody is preferably provided together with a method of detecting the antibody, which may be enzymatic, fluorescent, radioisotopic or other means. In diagnostic kits for diagnostic purposes, antibodies and detection methods may be provided for simultaneous, separate or sequential use.

Although the above disclosure is directed to antibodies, in certain embodiments polypeptides derived from these antibodies may also be used in the present invention.

Use of CLL cell lines

The development of CLL cell lines has many advantages as it provides an important tool for the development of diagnosis and treatment of CLL, cancer and other disease states characterized by upregulated levels of OX-2/CD200 such as melanoma.

The cell lines of the invention can be used in in vitro studies of the etiology, pathogenesis and biology of CLL and other disease states characterized by upregulated levels of OX-2/CD200. This aids in the identification of appropriate agents useful in the treatment of these diseases.

As noted above, cell lines can also be used to produce polypeptides and/or monoclonal antibodies for in vitro and in vivo diagnosis of CLL, cancer, and other disease states characterized by upregulated levels of OX-2/CD200, such as melanoma , and for screening and/or characterization of antibodies produced by other methods, for example by panning antibody libraries with primary cells and/or antigens from CLL patients.

Cell lines may be used as such, or antigens may be derived therefrom. Advantageously such antigens are cell surface antigens specific for CLL. They can be isolated directly from the cell lines of the invention. In addition, the cDNA expression library prepared from the cell lines described in the present invention can also be used to express CLL-specific antigens for the screening and characterization of anti-CLL antibodies and the identification of new CLL-specific antigens.

Treatment of CLL with monoclonal antibody therapy has been proposed in the art. More recently, Hainsworth (Oncologist 5(5) (2000) 376-384) has described current therapeutic approaches derived from monoclonal antibodies. Lymphocytic leukemia in particular is considered an ideal candidate for this therapy due to the presence of multiple lymphocyte-specific antigens on lymphocytic tumors.

Existing antibody therapies such as Rituximab™, which targets the CD20 antigen expressed on the surface of B lymphocytes, have been successfully used to treat certain lymphocytic disorders. However, in CLL a lower density of the CD20 antigen is expressed on the surface of B lymphocytes (Almasri et al., Am. J. Hematol., 40(4) (1992) 259-263).

The CLL cell lines of the present invention thus allow the development of new anti-CLL antibodies and polypeptides specific for one or more epitopes of the CLL cell lines that can be used in CLL, cancer and other OX-2/CD200-specific Levels are upregulated for the treatment and diagnosis of disease states that are characteristic.

The antibody or polypeptide can bind to the receptor that normally interacts with OX-2/CD200, thereby preventing or inhibiting the binding of OX-2/CD200 to the receptor. Alternatively, the antibody can bind to an antigen that regulates the expression of OX-2/CD200, thereby preventing or inhibiting the normal or increased expression of OX-2/CD200. Since the presence of OX-2/CD200 is associated with reduced immune responses, it is worth considering interfering with the metabolic pathways of OX-2/CD200 so that the patient's immune system can more effectively defend against disease states such as cancer or CLL.

In one particularly useful embodiment, the polypeptide binds to OX-2/CD200. In one embodiment, the polypeptide may be an antibody that binds to OX-2/CD200 and prevents or inhibits the interaction of OX-2/CD200 with other molecules or receptors. Because CLL cells and other cells overexpressing OX-2/CD200 greatly reduce Th1 cytokine production, patients with upregulated OX-2/CD200 levels are given anti-CD200 antibodies or peptides that bind OX-2/CD200 The Th1 cytokine profile was restored. Accordingly, these polypeptides and/or antibodies may be useful therapeutic agents in the treatment of CLL and other cancers or diseases that overexpress OX-2/CD200.

Thus, in another embodiment, the methods of the invention comprise the steps of screening patients for the presence of OX-2/CD200 and using a polypeptide that binds OX-2/CD200. In one particularly useful embodiment, CLL patients are screened for overexpression of OX-2/CD200, and antibodies that bind OX-2/CD200 are administered to the patients. As described in detail below, one such antibody is scFv-9 that binds OX-2/CD200 (see Figure 9B).

To enable those skilled in the art to better practice the compositions and methods described herein, the following examples are presented for purposes of illustration.

Example 1

Isolation of CLL-AAT cell lines

Cell Line Establishment

Peripheral blood was obtained from patients diagnosed with CLL. The white blood cell count was 1.6 x 10 ⁸ /ml. Monocytes were isolated by Histopaque-1077 density gradient centrifugation (Sigma Diagnostics, St. Louis, MO). Cells were washed twice with Iscove's Modified Dulbecco's Medium (IMDM) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and resuspended in 5 ml of ice-cold IMDM/10% FBS. Viable cells were counted by trypan blue staining. Cells were mixed with an equal volume of 85% FBS/15% DMSO, frozen in 1 ml aliquots and stored in liquid nitrogen.

Immunophenotypic analysis showed that more than 90% of the CD45+ lymphocyte population expressed IgD, κ light chain, CD5, CD19 and CD23. This group also expressed low levels of IgM and CD20. Approximately 50% of cells express high levels of CD38. Cells are lambda light chain, CD10 and CD138 negative.

An aliquot of cells was thawed, washed, and resuspended at a density of 10 ⁷ /mL in supplemented 20% heat-inactivated FBS, 2 mM L-glutamine, 100 units/ml penicillin, 100 μg/ml streptavidin In IMDM, 50 μM 2-mercaptoethanol and 5 ng/ml recombinant human IL-4 (R&D Systems, Minneapolis, MN). Cells were incubated at 37°C in a humidified atmosphere containing 5% _CO2 . A portion of the medium was changed every 4 days until stable growth was observed. After 5 weeks, the number of cells in culture began to double approximately every 4 days. This cell line was named CLL-AAT.

The nature of the cell line

Immunophenotypic analysis of the cell lines by flow cytometry revealed high expression of IgM, kappa light chain, CD23, CD38, and CD138, moderate expression of CD19 and CD20, and weak expression of IgD and CD5. Cell lines are lambda light chain, CD4, CD8 and CD10 negative.

Immunophenotyping of cell lines was also performed by whole cell ELISA using a panel of rabbit scFv antibodies that had been screened for specific binding to primary B-CLL cells. All of these CLL-specific scFv antibodies also recognized the CLL-AAT cell line. In contrast, most scFvs did not bind to two cell lines derived from B-cell lymphomas, Ramos, a Burkitt's lymphoma cell line and RL, a non-Hodgkin's lymphoma cell line.

Example 2

Screening for B-CLL-specific cell surfaces using antibody phage display and cell surface panning scFv antibody to antigen

Immunization and scFv antibody library construction

Peripheral blood mononuclear cells (PBMC) were isolated from blood drawn from CLL patients at Scripps Clinical Hospital (La Jolla, CA). Two rabbits were immunized with 2 x ¹⁰⁷ PBMCs collected from 10 different CLL-bearing donors. Three immunizations were given at 3 week intervals, two by subcutaneous injection and one by intravenous injection. The titer of the serum was checked by measuring the binding of serum IgG to primary CLL cells using flow cytometry. Five days after the last immunization, spleen, bone marrow and PBMC were collected from the animals. Total RNA was isolated from these tissues using Tri-Reagent (Molecular Research Center, Inc). Single chain Fv (scFv) antibody phage display libraries were constructed as previously described (Barbas et al. (2001) Phage Display Experimental Guide, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York). For cell panning, phagemid particles generated from reamplified libraries were precipitated with polyethylene glycol (PEG), resuspended in phosphate buffered saline (PBS) containing 1% bovine serum albumin (BSA), Dialyzed overnight against PBS.

Antibody selection by cell surface panning

A library of CLL cell surface specific antibodies was enriched by positive-negative screening using a magnetic activated cell sorter (MACS) as described by Siegel et al. (1997, J. Immunol. Methods 206:73-85). Briefly, phagemid particles from the scFv antibody library were pre-incubated in MPBS (2% nonfat dry milk and 0.02% sodium azide in PBS, pH 7.4) at 25°C for 1 hour to block non-specific binding sites . Approximately ¹⁰⁷ primary CLL cells were labeled with mouse anti-CD5 IgG and mouse anti-CD19 IgG linked to paramagnetic microbeads (Miltenyi Biotec, Sunnyvale, CA). Unbound beads were removed by washing. Labeled CLL cells ("target cells") were mixed with an excess of "antigen-negative adsorbed cells", pelleted, and resuspended in 50 μl (10 ¹⁰ -10 ¹¹ cfu) of phage particles. Adsorbent cells are used to take up phage that adhere nonspecifically to the cell surface and that are specific for "common" antigens present on both target and adsorber cells. Adsorbed cells used were TF-1 cells (human erythroleukemia cell line) or normal human B cells isolated from peripheral blood by immunomagnetic negative selection (StemSep System, StemCell Technologies, Vancouver, Canada). The ratio of adsorbed cells to target cells was approximately 10-fold by volume. After incubation at 25°C for 30 minutes, the cell/phage mixture was transferred to a MiniMACS MS ⁺ separation column. The column was washed twice with 0.5 ml MPBS and once with 0.5 ml PBS to remove unbound phage and adsorbed cells. Target cells were eluted from the column with 1 ml PBS and pelleted by centrifugation at maximum speed for 15 seconds in a microcentrifuge. Captured phage particles were eluted by resuspending target cells in 200 μl acidic elution buffer (0.1 N HCl, pH adjusted to 2.2 with glycine, 1 μg/ml BSA added). After incubation at 25°C for 10 minutes, the buffer was neutralized with 12 μl of 2M Tris base at pH 10.5, and the eluted phages were amplified in E. coli for the next round of elutriation. For each round of panning, the titer of incoming and outgoing phage was determined. The incoming titer is the number of reamplified phage particles added to the target/adsorbed cell mixture, and the outgoing titer is the number of captured phage eluted from the target cells. The enrichment factor (E) was calculated using the formula E=(Rn out/Rn in)/(R ₁ out/R ₁ in). In most cases, the enrichment factor should reach 10 ² -10 ³ fold by the third or fourth round.

Analysis of the enriched antibody repertoire after panning

After 3-5 rounds of panning, analyze the captured phage pool for binding to CLL cells by flow cytometry and/or whole-cell ELISA:

1. To generate a complete library of HA-tagged soluble antibodies, infect 2 ml of E. coli strain without suppressor gene (eg TOP10F') with 1 μl (10 ⁹ -10 ¹⁰ cfu) of phagemid particles. The original unpanned library served as a negative control. Carbenicillin was added to a final concentration of 10 μM, and the culture solution was incubated at 37° C. for 1 hour, shaking at 250 rpm. Add 8 ml of SB medium containing 50 μg/ml carbenicillin and grow the culture to an OD600 of approximately 0.8. IPTG was added to a final concentration of 1 mM to induce scFv expression from the Lac promoter, and shaking was continued for 4 hours at 37°C. The culture was centrifuged at 3000xg for 15 minutes. The supernatant containing soluble antibody was filtered and stored in 1 ml aliquots at -20°C.

2. The binding of scFv antibody library to target cells and adsorbed cells was determined by flow cytometry, using high-affinity rat anti-HA (clone 3F10, Roche Molecular Biochemicals) as the secondary antibody, and PE-conjugated donkey anti-HA Mouse antibody was used as the third antibody.

3. The binding of the antibody library to target cells and adsorbed cells was also determined by the following whole cell ELISA.

Screening of individual scFv clones after panning

To screen individual scFv clones after panning, TOP10F' was infected with phage pools as described above, plated on LB plates containing carbenicillin and tetracycline, and incubated overnight at 37°C. Single clones are seeded into deep 96-well plates containing 0.6-1.0 ml of SB-carbenicillin medium per well. Cultures were grown on a HiGro shaker (GeneMachines, San Carlos, CA) at 37°C and 520 rpm for 6-8 hours. At this point, 90 μl aliquots were transferred from each well to a deep 96-well plate containing 10 μl DMSO. The replicated plate was stored at -80°C. Add IPTG to the original plate to a final concentration of 1 mM and continue shaking for 3 hours. Plates were centrifuged at 3000xg for 15 minutes. Supernatants containing soluble scFv antibodies were transferred to another deep 96-well plate and stored at -20°C.

A sensitive whole-cell ELISA method for screening HA-tagged scFv antibodies was developed:

1. ELISA plates were coated with concanavalin A (10 mg/ml, dissolved in 0.1M NaHCO ₃ , pH 8.6, 0.1M CaCl ₂ ).

2. After washing the plate with PBS, add 0.5-1 x 10 ⁵ target cells or 50 μl PBS of adsorbed cells to each well, and centrifuge the plate at 250xg for 10 minutes.

3. Add 50 μl of 0.02% glutaraldehyde dissolved in PBS, and fix the cells overnight at 4°C.

4. After washing with PBS, the non-specific binding sites were blocked with PBS containing 4% skimmed milk powder at room temperature for 3 hours.

5. Cells were incubated with 50 μl of soluble HA-labeled scFv or Fab antibody (TOP10F' supernatant) for 2 hours at room temperature, and then washed 6 times with PBS.

6. Bound antibody was detected using a mouse anti-HA secondary antibody (clone 12CA5) and an alkaline phosphatase (AP)-conjugated anti-mouse IgG tertiary antibody. An approximately 10-fold amplified signal was obtained using AMDEX AP-conjugated sheep anti-mouse IgG as a third antibody (Amersham Pharmacia Biotech). The AMDEX antibody is linked to multiple AP molecules through a dextran backbone. The color produced using PNPP, the substrate for alkaline phosphatase, was measured at 405 nm using a plate reader.

Primary screening of scFv clones was performed by ELISA on primary CLL cells and normal human PBMC. Clones positive for CLL cells and negative for normal PBMCs were re-screened by ELISA against normal human B cells, human B cell line TF-1 cells and CLL-AAT cell lines. Clones were again screened by ELISA on CLL cells isolated from three different patients to eliminate clones recognizing patient-specific or blood group-specific antigens. Results obtained from representative ELISAs are shown in Figures 2-6 and summarized in Figure 9.

The number of unique scFv antibody clones obtained was determined by DNA fingerprinting and sequencing. The scFv DNA insert was PCR amplified from the plasmid and digested with the restriction enzyme BstNI. The resulting fragments were separated on a 4% agarose gel and stained with ethidium bromide. Clones with different restriction fragment types must have different amino acid sequences. Clones of the same type may have similar or identical sequences. Clones with unique BstNI fingerprints were further analyzed by DNA sequencing. 25 distinct sequences were found, which could be divided into 16 groups of antibodies with closely related CDRs (Fig. 9).

Characterization of scFv antibodies by flow cytometry

The binding specificity of several scFv antibodies was analyzed by three-color flow cytometry (Figure 7). PBMCs isolated from normal donors were stained with FITC-conjugated anti-CD5 and PerCP-conjugated anti-CD19 antibodies. Staining for scFv antibodies was performed using biotin-linked anti-HA as a secondary antibody with PE-linked streptavidin. Three antibodies, scFv-2, scFv-3 and scFv-6, were found to specifically recognize the CD19 ⁺ B lymphocyte population (data not shown). The fourth antibody, scFv-9, recognized two distinct cell populations: CD19 ⁺ B lymphocytes and a subset of CD5 ⁺ T lymphocytes (Figure 7). Further characterization of the T cell subset revealed it to be a subset of CD4 ⁺ CD8 ^- T _H cells (data not shown).

To determine whether the antigens recognized by scFv antibodies were overexpressed on primary CLL cells, PBMCs from 5 CLL patients and 5 normal donors were stained with scFv and compared by flow cytometry (Fig. 8 and Table 2). By comparing the average fluorescence intensity of positive cell populations, the relative expression levels of antigens on CLL cells and normal cells can be determined. One antibody, scFv-2, always stained CLL cells less intensely than normal PBMCs, while scFv-3 and scFv-6 always stained CLL cells brighter than normal PBMCs. The fourth antibody, scFV-9, stained two of the five CLL samples more strongly than normal PBMC, but only slightly brighter for the other three CLL samples (Figure 8 and Table 2). This indicates that the antigens of scFv-3 and scFv-6 are approximately 2-fold overexpressed on most if not all CLL tumors, whereas scFv-9 is overexpressed 3-6-fold in a subset of CLL tumors.

CLL patients can be divided into approximately equal two groups: those with a poor prognosis (average survival time 8 years) and those with a favorable prognosis (average survival time 26 years). Several poor prognostic signals have been identified for CLL, most notably the presence of VH genes lacking somatic mutations and a high percentage of CD38 ⁺ B cells. Because scFv-9 recognizes an antigen that is overexpressed in only a subset of CLL patients, we sought to determine whether overexpression of scFv-9 antigen correlated with the percentage of CD38 ⁺ cells in blood samples from 10 CLL patients (Fig. 11). The results indicated that the overexpression of scFv-9 antigen and the percentage of CD38 ⁺ cells were completely independent of each other.

Identification of antigens recognized by scFv antibodies by immunoprecipitation (IP) and mass spectrometry (MS)

To identify the antigens of these antibodies, scFvs were used to immunoprecipitate antigens from lysates prepared from microsomal fractions of cell surface biotinylated CLL-AAT cells (Figure 12). Immunoprecipitated antigens were purified by SDS-PAGE and identified by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) or capillary inversion HPLC nanospray tandem mass spectrometry (μLC/MS/MS) (data not shown). scFv-2 immunoprecipitated the 110 kd antigen from both RL and CLL-AAT cells (Figure 12). The antigen was identified by MALDI-MS as the B cell-specific marker CD19. Both ScFv-3 and scFv-6 immunoprecipitated the 45kd antigen from CLL-AAT cells (data not shown). This antigen was identified by MALDI-MS as CD23, a known marker of CLL and activated B cells. ScFv-9 immunoprecipitated a 50 kd antigen from CLL-AAT cells (Figure 12). The antigen was identified by μLC/MS/MS as OX-2/CD200, a known marker of B cells, activated CD4 ⁺ cells and thymocytes. OX-2/CD200 is also expressed on some non-lymphoid cells such as neurons and endothelial cells.

Example 3

The ability of cells overexpressing OX-2/CD200 to shift the cytokine response from a Th1 response (IL-2, IFN-γ) to a Th2 response (IL-4, IL-10) was assessed in mixed lymphocyte responses was performed using monocyte-derived macrophage/dendritic cells from one donor and blood-derived T cells from a different donor. EBNA cells transfected with OX-2/CD200 as described below or samples from CLL patients were used as a source of OX-2/CD200 expressing cells.

Transfection of 293-EBNA cells

2.5 x ¹⁰⁶ 293-EBNA cells (Invitrogen) were seeded in each 100 mm dish. After 24 hours cells were transiently transfected with Polyfect reagent (QIAGEN) following the manufacturer's instructions. Cells were co-transfected with 7.2 μg of OX-2/CD200 cDNA ligated in pCEP4 vector (Invitrogen) and 0.8 μg of pAdVAntage vector (Promega). Cells co-transfected with empty pCEP4 vector plus pAdVAntage vector served as negative control. 48 hours after transfection, approximately 90% of cells expressed OX-2/CD200 on their surface as determined by flow cytometry using scFv-9 antibody.

Maturation of dendritic cells/macrophages from blood monocytes

Buffy coats were obtained from the San Diego Blood Bank and primary blood lymphocytes (PBL) were separated using Ficoll. Cells were adsorbed in Eagles minimal essential medium (EMEM) containing 2% human serum for 1 hour and then rinsed extensively with PBS. Cells were cultured for 5 days in the presence of GM-CSF, IL-4 and IFN-γ or M-CSF, with or without the addition of lipopolysaccharide (LPS) after 3 days. Mature cells were collected and irradiated at 2000 RAD using a γ-irradiator (Shepherd Mark I Model 30 irradiator (Cs ¹³⁷ )).

mixed lymphocyte reaction

Mixed lymphocyte reactions were set up in 24-well plates using 500,000 dendritic cells/macrophages and ^1x106 reaction cells. Responder cells were T cell enriched lymphocytes purified from peripheral blood using Ficoll. Enrichment of T cells was performed by incubating the cells in tissue culture flasks for 1 hour, followed by removal of the nonadsorbed cell fraction. 500,000 OX-2/CD200 transfected EBNA cells or CLL cells in the presence or absence of 30 μg/ml anti-CD200 antibody (scFv-9 converted to intact IgG) 2-4 hours before addition of lymphocytes Added to macrophages/dendritic cells. Supernatants were collected after 48 and 68 hours and analyzed for the presence of cytokines.

Conversion of ScFv-9 to intact IgG

The light and heavy chain variable regions of scFv-9 were amplified by overlap PCR using primers linking the variable regions of each gene to the human lambda light chain constant region gene and the human IgG1 heavy chain constant region CH1 gene, respectively. The variable regions of the light chain gene and heavy chain gene of ScFv-9 were amplified using specific primers, and the human λ light chain constant region gene and the human IgG1 heavy chain constant region CH1 gene were respectively amplified using specific primers. The primers used were as follows :

R9VL-F1QP: 5'GGC CTC TAG ACA GCC TGT GCT GAC TCAGTC GCC CTC3' (SEQ ID NO 26);

R9VL/hCL2-rev: 5'CGA GGG GGC AGC CTT GGG CTG ACCTGT GAC GGT CAG CTG GGT C3' (SEQ ID NO 27);

R9VL/hCL2-F: 5'GAC CCA GCT GAC CGT CAC AGG TCA GCCCAA GGC TGC CCC CTC G3' (SEQ ID NO 28);

R9VH-F1: 5' TCT AAT CTC GAG CAG CAG CAG CTG ATG GAGTCC G3' (SEQ ID NO 29);

R9VH/hCG-rev: 5'GAC CGA TGG GCC CTT GGT GGA GGCTGA GGA GAC GGT GAC CAG GGT GC 3' (SEQ ID NO 30);

R9VH/hCG-F: 5'GCA CCC TGG TCA CCG TCT CCT CAG CCTCCA CCA AGG GCC CAT CGG TC 3' (SEQ ID NO31);

hCL2-rev: 5'CCA CTG TCA GAG CTC CCG GGT AGA AGT C3' (SEQ ID NO32);

hCG-rev: 5'GTC ACC GGT TCG GGG AAG TAG TC 3' (SEQ ID NO 33).

Amplified products were purified and subjected to overlap PCR.

The final product was digested with Xba I/Sac I (light chain) and Xho I/Pin AI (heavy chain) and cloned in the human Fab expression vector PAX243hGL. DNA cloning was performed by DNA sequencing to analyze PCR errors. The hCMV IE promoter gene was inserted into the Not I/Xho I site (before the heavy chain). The vector was digested with Xba I/Pin AI/EcoR I/Nhe I, and the 3472bp fragment containing the light chain plus hCMVIE promoter and heavy chain gene was transferred to the XbaI/Pin AI site of the IgG1 expression vector.

Cytokine analysis

The effect of conversion of scFv-9 to intact IgG on cytokine profiles in mixed lymphocyte reactions was determined.

IL-2, IFN-γ, IL-4, IL-10 and IL-6 found in tissue culture supernatants were quantified using ELISA. Matched capture and detection antibody pairs for each cytokine were obtained from R+D Systems, Inc. (Minneapolis, MN), and standard curves were prepared for each cytokine using recombinant human cytokines. Anti-cytokine capture antibody was coated on the plate at an optimal concentration in PBS. After overnight incubation, the plates were washed and blocked for 1 hour with PBS containing 1% BSA and 5% sucrose. After washing three times with PBS containing 0.05% Tween, the supernatant diluted 2-fold or 10-fold with PBS containing 1% BSA was added. Captured cytokines were detected with appropriate biotinylated anti-cytokine antibodies followed by alkaline phosphatase-conjugated streptavidin and SigmaS substrate. Color development was assessed with an ELISA plate reader (Molecular Devices).

As shown in Figure 14, the presence of OX-2/CD200 transfected cells resulted in downregulation of Th1 cytokines such as IL-2 and IFN-γ, but not in untransfected cells. Addition of anti-CD200 antibody at a concentration of 30 μg/ml completely restored the Th1 response, indicating that the antibody blocks the interaction of OX-2/CD200 with its receptor.

As shown in Figures 15 and 16, the presence of CLL cells in mixed lymphocyte reactions resulted in downregulation of Th1 cytokines. (Figure 15 shows the results for IL-2; Figure 16 shows the results for IFN-γ.) This is not only in the case of cells overexpressing OX-2/CD200 (IB, EM, HS, BH), And also in the case of cells that do not overexpress OX-2/CD200 (JR, JG and GB) (expression levels of these cells are shown in Figure 11). However, anti-CD200 antibodies could only restore Th1 responses in cells overexpressing OX-2/CD200, suggesting that abolishing OX-2/CD200 and its association with macrophages in patients with OX-2/CD200 overexpression Receptor interaction is sufficient to restore the Th1 response. In patients who do not overexpress OX-2/CD200, other mechanisms appear to be involved in the downregulation of Th1 responses.

Animal model for testing the effect of anti-CD200 antibody on tumor rejection

A model was established in which the growth of RAJI lymphoma tumors was arrested by co-injected PBL's. NOD/SCID mice were injected subcutaneously with 4 x 10 ⁶ RAJI cells in the presence or absence of 1 x 10 ⁶ , 5 x 10 ⁶ or 10 x 10 ⁶ human PBL's from different donors. The length and width of tumors and body weight were measured 3 times a week. Mean +/- SD of tumor volumes in all groups are shown in Figure 17A and B. Statistical analysis was performed using two parametric tests (Student's st-test and Welch's test) and one non-parametric test, Wilcox test. The results of the statistical analysis are shown in FIG. 18 . PAJI cells formed tumors subcutaneously with acceptable deviations. Rejection depends on the specific donor and the number of PBL cells. 1 x 10 ⁶ PBL's are not enough to stop tumor growth. 5 x 10 ⁶ PBL's of donor 2 from 22 to 43 days and 5 x 10 ⁶ or 1 x 10 ⁷ PBL's of donor 3 significantly slowed tumor growth from day 36 onwards. Donor 4 was very close to significant results after 48 days.

To test the effect of anti-CD200 antibody, RAJI cells were stably transfected with CD200. Animals were injected as described in the previous paragraph. In the presence of CD200-transfected cells, tumors could grow even in the presence of human PBL's. Anti-CD200 antibodies were used in this model to assess tumor rejection.

In addition, a liquid tumor model was established. RAJI cells were injected intraperitoneally into NOD/SCID mice. The cells spread to the bone marrow, spleen, lymph nodes and other organs causing paralysis. Simultaneous injection of human PBL's prevented or slowed tumor growth. Tumor growth was monitored by assessing mice for motor impairment and paralysis. Once these were found, mice were sacrificed and the number of tumor cells in different organs including bone marrow, spleen, lymph nodes and blood was assessed by FACS analysis and PCR.

As in the subcutaneous injection model, CD200-transfected cells were injected intraperitoneally. They grow even in the presence of human PBL's. Treatment with anti-CD200 antibodies results in tumor rejection or slowing of tumor growth.

references

The following references are hereby incorporated by reference in order to more fully describe the state of the art to which this invention pertains. Any inconsistencies between the following works or the works previously incorporated by reference and the teachings of the present invention should be resolved in favor of the present invention.

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16) Tatsumi et al., (2002).Disease-associated bias in T helper type 1(Th1)/Th2 CD4(+)T cell responses against MAGE-6 in HLA-DRB1040(+)patients with renal cell carcinoma or melanoma.( An excursion in the ratio of T helper type 1 (Th1) versus Th2CD4(+) T cell responses to MAGE-6 in HLA-DRB1040(+) patients with renal cell carcinoma or melanoma is disease-associated) J Exp Med 196:619-628.

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18) Winter et al., (2003).Tumour-induced polarization of tumorvaccine-draining lymph node T cells to a type 1 cytokine profile predicts inherent strong immunogenicity of the tumor and correlates with therapeutic efficacy in adoptive studies. Tumor-induced shift toward type 1 cytokine profile predicts strong tumor-intrinsic immunogenicity and correlates with therapeutic efficacy in adoptive transfer studies) Immunology 108:409-419.

It will be understood that various modifications may be made to the embodiments described herein. For example, those skilled in the art will recognize that the specific sequences described herein may be slightly altered without necessarily seriously affecting the functionality of the polypeptide, antibody or antibody fragment used to bind OX-2/CD200. For example, one or more amino acid substitutions in the antibody sequence can generally be made without destroying the functionality of the antibody or fragment. Therefore, it should be understood that polypeptides or antibodies having more than 70% homology with the specific antibodies of the present invention are included within the scope of the present invention. In particularly useful embodiments, antibodies having greater than 80% homology to a particular antibody of the invention are contemplated by the invention. In other useful embodiments, antibodies having greater than 90% homology to a particular antibody of the invention are contemplated by the invention. Accordingly, the above description is not to be construed as a limitation, but merely an exemplification of preferred embodiments. Those skilled in the art will envision other modifications within the scope and spirit of the invention.

Claims (51)

1. method comprises:

Determine whether OX-2/CD200 is raised in the patient; And

To the patient use can with the polypeptide of OX-2/CD200 or OX-2/CD200 receptors bind.

2. the method in the claim 1, the step of wherein using polypeptide comprises to the patient uses the bonded antibody with OX-2/CD200.

3. the method in the claim 1, the step of wherein using polypeptide comprises to the patient uses the bonded monoclonal antibody with OX-2/CD200.

4. the method in the claim 1, the step of wherein using polypeptide comprise to the patient uses antibody with the OX-2/CD200 receptors bind.

5. the method in the claim 1, the step of wherein using polypeptide comprise to the patient uses monoclonal antibody with the OX-2/CD200 receptors bind.

6. the method in the claim 1, the step of wherein using polypeptide comprise using to the patient and comprise the polypeptide that one or more are selected from following amino acid sequences: SEQ.ID.NO:1, SEQ.ID.NO:2, SEQ.ID.NO:3, SEQ.ID.NO:4, SEQ.ID.NO:5, SEQ.ID.NO:6, SEQ.ID.NO:7, SEQ.ID.NO:8, SEQ.ID.NO:9, SEQ.ID.NO:10, SEQ.ID.NO:11, SEQ.ID.NO:12, SEQ.ID.NO:13, SEQ.ID.NO:14, SEQ.ID.NO:15, SEQ.ID.NO:16, SEQ.ID.NO:17, SEQ.ID.NO:18, SEQ.ID.NO:19, SEQ.ID.NO:20, SEQ.ID.NO:21, SEQ.ID.NO:22, SEQ.ID.NO:23, SEQ.ID.NO:24 and SEQ.ID.NO:25.

7. method for the treatment of the morbid state that OX-2/CD200 wherein raised, comprise to suffer from this wherein the patient of the disease that raised of OX-2/CD200 use can with the polypeptide of OX-2/CD200 or OX-2/CD200 receptors bind.

8. the method in the claim 7, the step of wherein using polypeptide comprises to the patient uses the bonded antibody with OX-2/CD200.

9. the method in the claim 7, the step of wherein using polypeptide comprises to the patient uses the bonded monoclonal antibody with OX-2/CD200.

10. the method in the claim 7, the step of wherein using polypeptide comprise to the patient uses antibody with the OX-2/CD200 receptors bind.

11. comprising to the patient, the method in the claim 7, the step of wherein using polypeptide use monoclonal antibody with the OX-2/CD200 receptors bind.

12. comprising using to the patient, the method in the claim 7, the step of wherein using polypeptide comprise the polypeptide that one or more are selected from following amino acid sequences: SEQ.ID.NO:1, SEQ.ID.NO:2, SEQ.ID.NO:3, SEQ.ID.NO:4, SEQ.ID.NO:5, SEQ.ID.NO:6, SEQ.ID.NO:7, SEQ.ID.NO:8, SEQ.ID.NO:9, SEQ.ID.NO:10, SEQ.ID.NO:11, SEQ.ID.NO:12, SEQ.ID.NO:13, SEQ.ID.NO:14, SEQ.ID.NO:15, SEQ.ID.NO:16, SEQ.ID.NO:17, SEQ.ID.NO:18, SEQ.ID.NO:19, SEQ.ID.NO:20, SEQ.ID.NO:21, SEQ.ID.NO:22, SEQ.ID.NO:23, SEQ.ID.NO:24 and SEQ.ID.NO:25.

13. a treatment method for cancer comprises:

Determine whether OX-2/CD200 is raised in suffering from the patient of cancer; And

To the patient use can with the polypeptide of OX-2/CD200 or OX-2/CD200 receptors bind.

14. the method in the claim 13, the step of wherein using polypeptide comprises to the patient uses the bonded antibody with OX-2/CD200.

15. the method in the claim 13, the step of wherein using polypeptide comprises to the patient uses the bonded monoclonal antibody with OX-2/CD200.

16. comprising to the patient, the method in the claim 13, the step of wherein using polypeptide use antibody with the OX-2/CD200 receptors bind.

17. comprising to the patient, the method in the claim 13, the step of wherein using polypeptide use monoclonal antibody with the OX-2/CD200 receptors bind.

18. comprising using to the patient, the method in the claim 13, the step of wherein using polypeptide comprise the polypeptide that one or more are selected from following amino acid sequences: SEQ.ID.NO:1, SEQ.ID.NO:2, SEQ.ID.NO:3, SEQ.ID.NO:4, SEQ.ID.NO:5, SEQ.ID.NO:6, SEQ.ID.NO:7, SEQ.ID.NO:8, SEQ.ID.NO:9, SEQ.ID.NO:10, SEQ.ID.NO:11, SEQ.ID.NO:12, SEQ.ID.NO:13, SEQ.ID.NO:14, SEQ.ID.NO:15, SEQ.ID.NO:16, SEQ.ID.NO:17, SEQ.ID.NO:18, SEQ.ID.NO:19, SEQ.ID.NO:20, SEQ.ID.NO:21, SEQ.ID.NO:22, SEQ.ID.NO:23, SEQ.ID.NO:24 and SEQ.ID.NO:25.

19. a method for the treatment of CLL comprises:

Determine whether OX-2/CD200 is raised in suffering from the patient of CLL; And

To the patient use can with the polypeptide of OX-2/CD200 or OX-2/CD200 receptors bind.

20. the method in the claim 19, the step of wherein using polypeptide comprises to the patient uses the bonded antibody with OX-2/CD200.

21. the method in the claim 19, the step of wherein using polypeptide comprises to the patient uses the bonded monoclonal antibody with OX-2/CD200.

22. comprising to the patient, the method in the claim 19, the step of wherein using polypeptide use antibody with the OX-2/CD200 receptors bind.

23. comprising to the patient, the method in the claim 19, the step of wherein using polypeptide use monoclonal antibody with the OX-2/CD200 receptors bind.

24. comprising using to the patient, the method in the claim 19, the step of wherein using polypeptide comprise the polypeptide that one or more are selected from following amino acid sequences: SEQ.ID.NO:1, SEQ.ID.NO:2, SEQ.ID.NO:3, SEQ.ID.NO:4, SEQ.ID.NO:5, SEQ.ID.NO:6, SEQ.ID.NO:7, SEQ.ID.NO:8, SEQ.ID.NO:9, SEQ.ID.NO:10, SEQ.ID.NO:11, SEQ.ID.NO:12, SEQ.ID.NO:13, SEQ.ID.NO:14, SEQ.ID.NO:15, SEQ.ID.NO:16, SEQ.ID.NO:17, SEQ.ID.NO:18, SEQ.ID.NO:19, SEQ.ID.NO:20, SEQ.ID.NO:21, SEQ.ID.NO:22, SEQ.ID.NO:23, SEQ.ID.NO:24 and SEQ.ID.NO:25.

25. identify the bonded antigenic method of antibody, comprising for one kind:

Use is from the library of the deutero-antibody of tissue preparation of patient's CLL collection;

Is that the lysate of the cell line CLL-AAT of PTA-3920 contacts with one or more members in library with being deposited in the ATCC registration number; And

The bonded antigen of at least one member with the library is characterized.

26. the method in the claim 25, the step that wherein prepares the library comprises with CLL cell or organism of its partial immunity.

27. the method in the claim 25, the step that wherein prepares the library comprises with CLL cell or cell line eluriates synthetic antibody library.

28. the method in the claim 27, screen to separate the member in library by phage display in library wherein.

29. the method in the claim 27, library are wherein used from the isolating elementary CLL cell of the patient of one or more CLL of suffering from and are eluriated.

30. the method in the claim 25, also comprise identify in the antibody library with the CLL cell in the bonded member's of antigen that raised step.

31. the method in the claim 30, wherein the member of antibody library combines with OX-2/CD200.

32. bonded antibody of antigen that is raised with the CLL cell.

33. the antibody in the claim 32, antigen wherein is OX-2/CD200.

34. the antibody in the claim 32, the aminoacid sequence that contains be selected from following sequence and have at least 70% homology: SEQ.ID.NO:1, SEQ.ID.NO:2, SEQ.ID.NO:3, SEQ.ID.NO:4, SEQ.ID.NO:5, SEQ.ID.NO:6, SEQ.ID.NO:7, SEQ.ID.NO:8, SEQ.ID.NO:9, SEQ.ID.NO:10, SEQ.ID.NO:11, SEQ.ID.NO:12, SEQ.ID.NO:13, SEQ.ID.NO:14, SEQ.ID.NO:15, SEQ.ID.NO:16, SEQ.ID.NO:17, SEQ.ID.NO:18, SEQ.ID.NO:19, SEQ.ID.NO:20, SEQ.ID.NO:21, SEQ.ID.NO:22, SEQ.ID.NO:23, SEQ.ID.NO:24 and SEQ.ID.NO:25.

35. the antigenic method of evaluation and antibodies comprises:

Use from being deposited in the ATCC registration number and be the deutero-antibody library of the cell preparation the cell line of PTA-3920;

The lysate of the cell one or more members in library and the tissue of collecting from patient CLL is contacted; And

The bonded antigen of at least one member with the library is characterized.

36. the antigenic method of evaluation and antibodies comprises:

At least a portion immunity organism with the CLL cell the tissue of collecting from patient CLL;

Based on being produced antibody library by the immunoreation of the animal of immunity;

One or more members in library are contacted with the CLL cell; And

The bonded antigen of at least one member with the library is characterized.

37. the method in the claim 36 wherein comprises one or more members in library and the contacted step of CLL cell that the lysate with the cell one or more members in library and the tissue of collecting from patient CLL contacts.

38. identify the bonded antigenic method of antibody, comprising for one kind:

At least a portion immunity organism with the CLL cell;

Based on being produced antibody library by the immunoreation of the animal of immunity;

Is that the lysate of the cell line CLL-AAT of PTA-3920 contacts with one or more members in library with being deposited in the ATCC registration number; And

The bonded antigen of at least one member with the library is characterized.

39. a treatment method for cancer comprises the antibody of using the metabolic pathway that can disturb the polypeptide that is raised by pernicious cancerous cell.

40. the method in the claim 39, antibody wherein combines with the polypeptide that is raised.

41. the method in the claim 39, antibody wherein combines with the polypeptide interaction receptor that is raised with quilt.

42. the method in the claim 39, antibody wherein combines with the antigen of regulating expression of polypeptides.

43. the method in the claim 39, polypeptide wherein is OX-2/CD200.

44. the method in the claim 39, the aminoacid sequence that antibody wherein contains be selected from following sequence and have at least 70% homology: SEQ.ID.NO:1, SEQ.ID.NO:2, SEQ.ID.NO:3, SEQ.ID.NO:4, SEQ.ID.NO:5, SEQ.ID.NO:6, SEQ.ID.NO:7, SEQ.ID.NO:8, SEQ.ID.NO:9, SEQ.ID.NO:10, SEQ.ID.NO:11, SEQ.ID.NO:12, SEQ.ID.NO:13, SEQ.ID.NO:14, SEQ.ID.NO:15, SEQ.ID.NO:16, SEQ.ID.NO:17, SEQ.ID.NO:18, SEQ.ID.NO:19, SEQ.ID.NO:20, SEQ.ID.NO:21, SEQ.ID.NO:22, SEQ.ID.NO:23, SEQ.ID.NO:24 and SEQ.ID.NO:25.

45. a method comprises:

The screening cancer patient is to identify those wherein patients of being raised by pernicious cancerous cell of polypeptide expression; And

Use the antibody of the metabolic pathway that can disturb the polypeptide that is raised to those patients that are found rise.

46. the method in the claim 45, the step of wherein screening patient comprises screening patient CLL.

47. the method in the claim 45, the step of wherein screening patient detects the rise of OX-2/CD200.

48. the method in the claim 45, wherein the step of administration of antibodies comprises and uses the bonded antibody with OX-2/CD200.

49. the method in the claim 45, wherein the step of administration of antibodies comprise use with the bonded antibody of OX-2/CD200 interaction receptor.

50. the method in the claim 45, wherein the step of administration of antibodies comprises the bonded antibody of antigen of the expression of using and regulate OX-2/CD200.

51. the method in the claim 45, wherein the step of administration of antibodies comprises aminoacid sequence that the antibody used contains and is selected from following sequence and has at least 70% homology: SEQ.ID.NO:1, SEQ.ID.NO:2, SEQ.ID.NO:3, SEQ.ID.NO:4, SEQ.ID.NO:5, SEQ.ID.NO:6, SEQ.ID.NO:7, SEQ.ID.NO:8, SEQ.ID.NO:9, SEQ.ID.NO:10, SEQ.ID.NO:11, SEQ.ID.NO:12, SEQ.ID.NO:13, SEQ.ID.NO:14, SEQ.ID.NO:15, SEQ.ID.NO:16, SEQ.ID.NO:17, SEQ.ID.NO:18, SEQ.ID.NO:19, SEQ.ID.NO:20, SEQ.ID.NO:21, SEQ.ID.NO:22, SEQ.ID.NO:23, SEQ.ID.NO:24 and SEQ.ID.NO:25.

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