KR20120084343A - Polypeptides and antibodies derived from chronic lymphocytic leukemia cells and uses thereof - Google Patents

Abstract

The present invention relates to cell line "CLL-AAT" deposited under ATCC Accession No. PTA-3920.

Description

Polypeptides and antibodies derived from chronic lymphocytic leukemia cells and their uses {POLYPEPTIDES AND ANTIBODIES DERIVED FROM CHRONIC LYMPHOCYTIC LEUKEMIA CELLS AND USES THEREOF}

This application was filed on December 15, 2003 by U.S. Application No. It is a continuing application in part of 10/736,188, which was filed on March 4, 2003 by U.S. Application No. It is a continuation application in part of 10/379,151, the application is a continuation application in part of PCT/US01/47931 filed on December 10, 2001, and the international application is a continuation application in part of U.S. Division application No. It claims priority to 60/254,113. The U.S. mentioned above. Applications, international applications, and divisional applications are purely by reference.

Cells derived from chronic lymphocytic leukemia (CLL) cells and their use in the study and treatment of CLL and other diseases are disclosed. In particular, the present application relates to the CLL cell line, “CLL-AAT”, deposited on November 28, 2001 in the American Type Culture Collection (Manassas, Virginia, USA) under the ATCC accession number PTA-3920 according to the Budapest Treaty. All.

Chronic lymphocytic leukemia (CLL) is a leukocyte disease and is the most common form of leukemia in the western hemisphere. CLL represents a diverse group of diseases associated with the growth of malignant lymphocytes that grow slowly but exist for a long time. CLL is classified into various types including classic and hybrid B-cell chronic lymphocytic leukemia (B-CLL), B-cell and T-cell prolymphatic leukemia, hairy cell leukemia, and large granular lymphocytic leukemia. do.

Among the different types of CLL, B-CLL accounts for approximately 30% of all leukemias. Although the disease is more frequent in individuals over 50 years of age, the incidence of the disease is increasing in young people. B-CLL is characterized by the accumulation of morphologically normal but biologically immature B-lymphocytes and consequent loss of function. Normally, lymphocytes function to suppress infection. However, in B-CLL, lymphocytes accumulate in the blood and bone marrow and cause lymph nodes to dilate. Normal bone marrow and blood cell production are reduced, and patients often experience severe anemia, as well as a reduced platelet count. This can lead to the risk of fatal bleeding and the development of serious infections due to a decrease in the white blood cell population.

In order to better understand diseases such as leukemia, it is important to secure appropriate cell lines that can be used as tools for etiology, disease development, and ecological studies. Examples of malignant human B-lymphoid cell lines are pro-B acute lymphocytic leukemia (Reh), diffuse large cell lymphoma (WSU-DLCL2), Waldenstrom's macroglobulinemia (WSU-WM), and the like. Unfortunately, most existing cell lines do not represent the most clinically common types of leukemia and lymphoma.

The use of Epstein Barr Virus (EBV) infection in vitro resulted in some CLL-derived cell lines, especially B-CLL cell lines representing malignant cells. The phenotype of these cell lines differs from that of tumors in vivo, but rather the characteristics of B-CLL cell lines are similar to those of lymphoblastoid cell lines. Attempts to perpetuate B-CLL cells with EBV infection have not been successful. The cause of this is uncertain, but is not known to be due to the absence of EBV receptor expression, binding or absorption. Wells et al. found that B-CLL cells were arrested at the G1/S phase of the cell cycle and that the EBV DNA associated with transformation was not expressed. This suggests that the interaction of EBV and B-CLL cells is different from that of normal B cells. In addition, the EBV-transformed CLL cell line appears to differentiate and has a more similar morphology to the lymphogenic cell line (LCL) perpetuated by EBV.

The EBV-negative CLL cell line, WSU-CLL, has already been established (Mohammed et al., (1996) Leukemia 10(1):130-7). However, no other such cell line is known.

Various mechanisms play a role in the body's response to disease states, including cancer and CLL. For example, CD4 ⁺ T helper cells play an important role in effective immune responses against various malignancies by providing stimulating factors to effector cells. Cytotoxic T cells are considered to be the most effective cells to eliminate cancer cells, and T helper cells trigger cytotoxic T cells by secreting Th1 cytokines such as IL-2 and IFN-γ. In a variety of malignant tumors, T helper cells have been found to possess a modified phenotype compared to cells found in healthy individuals. One of the main features of modification is reduced Th1 cytokine production and conversion to Th2 cytokine production (Kani, 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., Leak 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)). Reversal of cytokine conversion with the Th1 profile has been demonstrated to enhance the anti-tumor effect of T cells (Winter, et al., Immunology 108:409-419 (2003); Inagawa, et al., Anticancer Res 18: 3957-3964 (1998)).

It includes the secretion of cytokines such as TGF-β, 11-10 of the underlying mechanism of tumor cell ability to convert the cytokine expression of T helper cells from Th1 to Th2, as well as 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 a significant level of homology with molecules of the immunoglobulin gene lineage, is involved in immune suppression (Gorczynski et al., Transplantation 65:1 106- 1114 (1998)), provided evidence that OX 2/CD200-expressing cells can inhibit the stimulation of Th1 cytokine production. Gorczinski et al. demonstrated that in a mouse model, OX-2/CD200 Fc inhibits the rejection of tumor cells in an animal model using leukemia tumor cells (Clin Exp Immunol 126:220-229 (2001)).

There is a need for improved methods of treating cancer or CLL patients, especially to the extent that they can enhance the activity of T cells.

summary

In one embodiment, a CLL cell line of malignant origin that has not been established by perpetuation with EBV is presented. The cell line is derived from primary CLL cells and deposited under ATCC 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 generate monoclonal antibodies effective for diagnosis and/or treatment of CLL. Antibodies are generated by using the cells disclosed herein as immunogens and inducing an immune response in animals from which monoclonal antibodies are isolated. The sequence of the antibodies is determined, and these antibodies or variants thereof are produced by recombinant techniques. In this respect, “variants” encompass chimeric antibodies, CDR-fused antibodies, humanized antibodies, and fully human antibodies based on the sequence of a monoclonal antibody.

Furthermore, antibodies derived from a recombinant library (“phage antibodies”) can be selected using the cells described herein or a polypeptide derived therefrom as a bait for separating antibodies based on target specificity.

In another aspect, antibodies are produced by panning an antibody library using primary CLL cells or antigens derived therefrom, and further screened or characterized using a CLL cell line, e.g., the CLL cell line described herein. Let it. Thus, we present a method of characterizing an antibody specific for CLL, the method comprising assessing the binding of the antibody to a CLL cell line.

In another aspect, a method known in the art using a CLL-AAT cell line, for example, immunoprecipitation and subsequent mass spectrometry, to identify a protein uniquely expressed in CLL cells is presented. These proteins are uniquely expressed in the CLL-AAT cell line, or in primary cells derived from CLL patients.

Small molecule libraries (mostly commercially available) can be selected using the CLL-AAT cell line in cell-based tests to identify agents that can modulate the growth characteristics of cells. For example, an agent that modulates apoptosis or inhibits its growth and/or proliferation in a CLL-AAT cell line can be identified. These agents are candidates for the development of therapeutic compounds.

Nucleic acids isolated from CLL-AAT cell lines can be used in negative hybridization experiments to identify CLL-specific genes, or microarray analysis (eg, gene chip experiments). It is possible to identify genes whose transcription is regulated in CLL cells. The polypeptide or nucleic acid gene product identified in this way is effective as a leading material for the development of antibodies for CLL or small molecule therapeutics.

In a preferred aspect, the CLL-AAT cell line is used to identify an internalizing antibody that binds to a cell surface component that is internalized by the cell. These antibodies are candidates for therapeutic use. In particular, single-chain antibodies that are stably present in the cytoplasm and retain intracellular binding activity can be selected in this way.

In another aspect, a method of treatment is provided in which the presence of a polypeptide that is upregulated by malignant tumor cells is investigated in a patient, and an antibody that interferes with the metabolic pathway of such upregulated polypeptide is administered to the patient.

In addition, the present invention relates to a method of determining whether OX-2/CD200 is upregulated in a subject, and in this case administering to the subject a polypeptide that binds to OX-2/CD200. In another embodiment, the polypeptide binds to the OX-2/CD200 receptor.

In another aspect, the method according to the present invention is to treat a disease state in which OX-2/CD200 is upregulated in the subject by administering a polypeptide that binds to the OX-2/CD200 or OX-2/CD200 receptor to the diseased subject. Is used. In one embodiment, the disease condition treated with these methods is cancer, particularly CLL.

In the present invention, it is determined whether OX-2/CD200 is upregulated in an individual, and in this case, a method of administering a polypeptide that binds to OX-2/CD200 to the individual is provided. In general, the polypeptides used in the present invention can be constructed using different techniques 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 several fragments of one or more antibodies.

Suitably, the polypeptide used in the method of the present invention is obtained from a CLL cell line. As used herein, “CLL” is a chronic lymphocytic leukemia involving lymphocytes of various stages of development of B cells and T cells, including, but not limited to, B cell CLL (“B-CLL”). In the present specification, B-CLL is CD5 ⁺ , CD23 ⁺ , CD20 ^{dim +} , sIg ^{dim +} and represents a leukemia with a mature B cell phenotype that is arrested at G0/G1 of the cell cycle. In another aspect, the CLL cell line is used to produce a polypeptide, preferably an antibody, effective for diagnosis and/or treatment of disease states in which OX-2/CD200 is upregulated, preferably cancer and CLL.

As used herein, “antibody” refers to a complete antibody or antibody fragment capable of binding to a selected target. These include Fv, scFv, Fab' and F(ab')2, monoclonal and polyclonal antibodies, engineered antibodies (including chimeric antibodies, CDR-fused antibodies, humanized antibodies, fully human antibodies, artificially selected antibodies), and phage. Synthetic or semi-synthetic antibodies produced using exhibition or alternative techniques are included. Small fragments, such as Fv and scFv, possess advantageous properties for diagnostic and therapeutic applications due to their small size and thus good tissue dispersion.

The polypeptides and/or antibodies of the invention are particularly suitable for diagnostic and therapeutic applications. Thus, they can be transformed into effector proteins, such as toxins or labels. In particular, labels that enable imaging of polypeptide or antibody dispersion in vivo are preferred. Such a label may be a radioactive label or a radioplaque label that is readily visible in the patient's body, for example a metallic particle. In addition, the label may be a fluorescent label or other label that is visualized in a tissue sample that is removed from the patient.

Antibodies are generated by using the cells disclosed herein as immunogens and inducing an immune response in animals from which monoclonal antibodies are isolated. The sequence of the antibodies is determined, and these antibodies or variants thereof are produced by recombinant techniques. In this respect, “variants” encompass chimeric antibodies, CDR-fused antibodies, humanized antibodies, fully human antibodies based on the sequence of a monoclonal antibody and a polypeptide capable of binding to OX-2/CD200.

Furthermore, antibodies derived from recombinant libraries (“phage antibodies”) can be selected using the cells described herein or polypeptides derived therefrom as bait for separating antibodies or polypeptides based on target specificity.

In another aspect, antibodies or polypeptides are generated by panning antibody libraries using primary CLL cells or antigens derived therefrom, and further screened or characterized using CLL cell lines, e.g., CLL cell lines described herein. . Accordingly, a method of characterizing an antibody or polypeptide specific for CLL is provided, the method comprising assessing binding of the antibody or polypeptide to a CLL cell line.

Preparation of cell line

Cell lines can be produced according to established methods known to those skilled in the art. In general, cell lines are produced by culturing primary cells derived from patients until perpetuated cells are spontaneously produced in culture. Thereafter, these cells are isolated and further cultured to produce clonal cell populations or cells that are resistant to apoptosis.

For example, CLL cells are isolated from peripheral blood collected from CLL patients. These cells are washed and optionally immunotyping to determine the type of cells present. Then, these cells are cultured in a medium, for example, a medium containing IL-4. Suitably, all or part of the medium is replaced one or more times during the cultivation process. Thereafter, the cell line is isolated and confirmed by increased growth during incubation.

In one embodiment, a CLL cell line of malignant origin that has not been established by perpetuation with EBV is presented. “Malignant origin” refers to the bias of a cell line from malignant CLL primary cells as opposed to non-proliferative cells transformed with EBV, for example. Cell lines useful in the present invention are not malignant or malignant in their phenotype. CLL cells carrying a “malignant” phenotype achieve cell growth and resist apoptosis independent of the matrix medium characterized by repeated cycles of cell growth. The cell line derived from primary CLL cells is deposited under ATCC 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 that are uniquely expressed in CLL cells are identified by methods known in the art using a CLL-AAT cell line, such as immunoprecipitation and subsequent mass spectrometry. These proteins are uniquely expressed in the CLL-AAT cell line, or in primary cells derived from CLL patients.

Small molecule libraries (mostly commercially available) can be selected using the CLL-AAT cell line in cell-based tests to identify agents that can modulate the growth characteristics of cells. For example, an agent that modulates apoptosis or inhibits its growth and/or proliferation in a CLL-AAT cell line can be identified. These agents are candidates for the development of therapeutic compounds.

Nucleic acids isolated from CLL-AAT cell lines can be used in negative hybridization experiments to identify CLL-specific genes, or microarray analysis (eg, gene chip experiments). It is possible to identify genes whose transcription is regulated in CLL cells. The polypeptide or nucleic acid gene product identified in this way is useful as a leading material for the development of antibodies for CLL or small molecule therapeutics.

In one embodiment, the CLL-AAT cell line is used to identify an internalizing antibody that binds to a cell surface component and is then internalized by the cell. These antibodies are candidates for therapeutic use. In particular, single-chain antibodies that are stably present in the cytoplasm and retain intracellular binding activity can be selected in this way.

Monoclonal Preparation of antibodies

Recombinant DNA technology can be used to improve antibodies produced according to the present invention. Thus, it is possible to construct chimeric antibodies with reduced immunogenicity in diagnostic or therapeutic applications. Furthermore, immunogenicity can be minimized by humanizing these antibodies by CDR fusion and, optionally, structural modifications (see U.S. Patent No. 5,225,539).

Antibodies can be obtained from animal serum or, in the case of monoclonal antibodies or fragments thereof, produced in cell culture. Recombinant DNA technology can be used to produce antibodies according to established procedures in bacterial, preferably mammalian cell culture. Suitably, the cell culture system of choice secretes the antibody product.

In one embodiment, the process for producing an antibody disclosed herein comprises culturing a host transformed with a hybrid vector, such as E. coli or mammalian cells. The vector comprises, in an appropriate reading framework, one or more expression cassettes carrying a promoter operably linked to a first DNA sequence encoding a single peptide linked to a second DNA sequence encoding an antibody protein. Then, the antibody protein is collected and separated. Optionally, the expression cassette carries a promoter operably linked to a polycistronic, such as a bicistronic DNA sequence, encoding an antibody protein each operably individually linked to a single peptide in an appropriate reading framework.

The amplification of hybridoma cells or mammalian cells in vitro is carried out in an appropriate culture medium, which contains mammalian serum (e.g., fetal bovine serum) or trace elements and growth-sustaining supplements (e.g., normal mouse peritoneal effusion cells). , Spleen cells, bone marrow macrophages, feeder cells such as 2-aminoethanol, insulin, transferrin, low-density lipoprotein, oleic acid, etc.). Medium). Amplification of host cells, either bacterial cells or yeast cells, is similarly carried out in suitable culture media known in the art. For example, suitable culture media for bacteria are LE, NZCYM, NZYM, NZM, Terrific Broth, SOB, SOC, 2 x YT, or M9 minimal media. A suitable culture medium for yeast is YPD, YEPD, minimal medium, or Complete Minimal Dropout Medium.

In vitro production provides a relatively pure antibody preparation and allows scale-up to provide large quantities of the desired antibody. Techniques for culturing bacterial cells, yeast, plant, or mammalian cells are known in the art, and cultures in homogeneous suspensions (e.g., in airlift reactors or continuously stirred reactors), culture of perpetuated or captured cells (e.g., hollow fibers, Microcapsules, agarose microbeads or ceramic cartridges) and the like.

Large amounts of desired antibodies can be obtained by amplifying mammalian cells in vivo. To this end, hybridoma cells producing the desired antibody are injected into a histocompatibility mammal to induce the growth of an antibody-producing tumor. Optionally, prior to injection, animals are detonated with a hydrocarbon, preferably a mineral oil such as pristane (tetramethyl-pentadecane). After 1-3 weeks, the antibody is isolated from the body fluids of these mammals. For example, hybridoma cells obtained by fusion of appropriate myeloma cells with antibody-producing splenocytes derived from Balb/c mice, or transfected cells derived from the hybridoma cell line Sp2/0 producing the desired antibody. Optionally, Balb/c mice pre-treated with Pristine are injected intraperitoneally. After 1-2 weeks, ascites are collected from these animals.

Such techniques include, for example, Kohler and Milstein, (1975) Nature 256:495-497; U.S. Patent No. 4,376, 110; Harlow and Lane, Antibodies: a Laboratory Manual, (1988) Cold Spring Harbor. Techniques for the production of recombinant antibody molecules are described in the above references and, for example, W097/08320; U. S. Patent No. 5,427,908; U.S. Patent No. 5,508,717; Smith, 1985, Science, Vol. 225, pp 1315-1317; Parmley and Smith 1988, Gene 73, pp 305-318; De La Cruz et al, 1988, Journal of Biological Chemistry, 263 pp 4318-4322; U.S. Patent No. 5,403,484; U.S. Patent No. 5223409; W088/06630; WO92/15679; U.S. Patent No. 5780279; U.S. Patent 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. Academy of Sciences USA 97(2)(2000): 722-727. These references are purely for reference.

The cell culture supernatant is selected for the desired antibody by immunofluorescence staining of CLL cells, immunoblotting, enzymatic immunization, such as sandwich test or dart-test, or radioimmunoassay.

In order to isolate the antibody, the immunoglobulin in the culture supernatant or ascites is concentrated by, for example, precipitation using ammonium sulfate, dialysis against hygroscopic substances such as polyethylene glycol, filtration through a screening membrane, or the like. If necessary, antibodies can be obtained from conventional chromatographic methods, such as gel filtration, ion-exchange chromatography, chromatography on DEAE-cellulose and/or (immuno-) affinity chromatography, such as from the CLL cell lines disclosed herein. Purification by affinity chromatography using one or more derived surface polypeptides or proteins A or G.

In another embodiment, a process for making a bacterial cell line secreting antibodies against a bacterial cell line characterized by immunizing a suitable mammal, such as a rabbit, with a pooled CLL patient sample is provided. Phage display libraries produced from immunized rabbits construct and pan the desired antibodies according to methods known in the art (eg, methods disclosed in various references).

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

In another embodiment, a process for making a hybridoma cell line secreting a monoclonal antibody against a CLL cell line described herein is provided. In this process, a suitable mammal, e.g., a Balb/c mouse, has one or more polypeptides or antigenic fragments thereof derived from cells described herein, the cell line itself, or antigenicity carrying the purified polypeptides described herein. Is immunized with a carrier. The immunized mammalian antibody-producing cells are either grown briefly in culture or fused with cells of an appropriate myeloma cell line. Hybrid cells obtained in this fusion are cloned, and cell clones secreting the desired antibody are selected. 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-Ag 14, and the obtained hybrid cells are selected for secretion of the desired antibody, and positive cells Is cloned.

^{Injecting 10 6} to 10 ⁷ cells of the cell line according to the present invention subcutaneously and/or intraperitoneally for several months, for example, 2 to 4 months, for example 4 to 6 times, to immunize Balb/c mice. The manufacturing process of hybridoma cell lines characterized as being preferred. Splenocytes from the immunized mice are harvested 2 to 4 days after the final injection and fused with cells of the myeloma cell line PAI in the presence of a fusion promoter, preferably polyethylene glycol. Suitably, the myeloma cells are fused with a 3- to 20-fold excess of splenocytes derived from immunized mice in a solution containing approximately 30% to 50% polyethylene glycol of 4000 molecular weight. After fusion, the cells are expanded in an appropriate culture medium described herein and supplemented with a selection medium, e.g., HAT medium, at regular intervals so that conventional myeloma cells do not grow larger than the desired hybridoma cells.

In another embodiment, a recombinant DNA comprising an insert coding for a heavy chain variable domain and/or a light chain variable domain of an antibody directed against the cell lines described herein is generated. In the present specification, “DNA” encompasses a coding single-stranded DNA, a double-stranded DNA composed of the coding DNA and its complementary DNA, or their complementary (single-stranded) DNA itself.

Furthermore, the DNA encoding the heavy chain variable domain and/or the light chain variable domain of an antibody directed against the cell lines described herein is an enzyme carrying the actual DNA sequence encoding the heavy chain variable domain and/or the light chain variable domain, or a variant thereof. It may be chemically or chemically synthesized DNA. The actual DNA variant is DNA encoding the heavy chain variable domain and/or the light chain variable domain of the above-described antibody in which one or more amino acids have been deleted or substituted with one or more other amino acids. Suitably, the modification is located outside the CDRs of the heavy and/or light chain variable domains of the antibody in humanization and expression optimization. In addition, variant DNA encompasses silent variants in which one or more nucleotides have been replaced with other nucleotides with new codons encoding the same amino acid. In addition, the variant sequence encompasses the degenerate sequence. Degenerate sequences are degenerate from the potency of the genetic code in that an unlimited number of nucleotides are substituted for other nucleotides without changing the amino acid sequence originally encoded. Such degenerate sequences achieve optimal expression of heavy chain murine variable domains and/or light chain murine variable domains due to the different restriction sites and/or frequencies of specific codons favored by certain hosts, in particular E. coli. It is effective to do.

Variants encompass DNA variants obtained by in vitro mutagenesis of actual DNA according to methods known in the art.

For the combination of complete tetrameric immunoglobulin molecules and expression of chimeric antibodies, the recombinant DNA insert encoding the heavy and light chain variable domains is fused with the light and heavy chain constant domains, e.g., in an appropriate host cell after integration into a hybrid vector. Is transfected.

In addition, a human constant domain, e.g., γ1, γ2, γ3 or γ4, preferably γ1 or γ4, and having an insert that encodes the heavy chain murine variable domain of an antibody directed against the cell lines described herein. Present the DNA. In addition, a recombinant DNA having an insert fused to a human constant domain κ or λ, preferably κ and encoding the light chain murine variable domain of an antibody directed against the cell lines described herein is presented.

Another embodiment is a peptide that encodes a recombinant polypeptide in which the heavy chain variable domain and the light chain variable domain are linked by a spacer group, and optionally a signal sequence that facilitates processing of the antibody in a host cell and/or a peptide that facilitates purification of the antibody, Recombinant DNA comprising DNA encoding a cleavage site, a peptide spacer and/or an effector molecule. DNA encoding an effector molecule is DNA encoding an effector molecule effective for diagnostic or therapeutic applications. In particular, effector molecules, which are enzymes capable of catalyzing the activation of toxins or enzymes, such as prodrugs, are preferred. DNA encoding such an effector molecule carries a DNA sequence encoding a naturally occurring enzyme or toxin, or a variant thereof, and can be made by methods known in the art.

The antibodies and antibody fragments described herein are effective for diagnosis and treatment. Therefore, it provides a therapeutic or diagnostic composition comprising the antibody disclosed herein.

In the case of a diagnostic composition, the antibody is provided with a means for detecting such an antibody, for example an enzymatic means, a fluorescent means, a radioactive means or other means. The antibody and the detection means are provided simultaneously, individually or sequentially in the diagnostic kit.

Although the above details relate to antibodies, in some embodiments, polypeptides derived from such antibodies can be used in the present invention.

CLL Cell line uses

CLL cell lines are beneficial in many ways in that they provide an important tool for the development of diagnostic and therapeutic agents for CLL, cancer, and other disease states characterized by upregulated levels of OX-2/CD200, such as melanoma. Do.

The cell lines according to the present invention are useful for in vitro studies of the etiology, disease occurrence, and ecology of CLL and other disease states characterized by upregulated levels of OX-2/CD200. This assists in the identification of suitable agents that are effective in the treatment of these diseases.

The cell line for in vitro in vivo diagnosis of CLL, cancer, and other disease states (eg, melanoma) characterized by upregulated levels of OX-2/CD200; Other methods can also be used to produce polypeptides and/or monoclonal antibodies for selection and/or characterization of antibodies produced, for example, by panning antibody libraries with antigens derived from primary cells and/or CLL patients. .

Cell lines can be used as such, or antigens can be derived from them. Suitably, these antigens are cell-surface antigens specific for CLL. They can be isolated directly from cell lines according to the invention. Alternatively, cDNA expression libraries made from the cell lines described herein can be used to express CLL-specific antigens useful for selection and characterization of anti-CLL antibodies and identification of novel CLL-specific antigens.

Treatment of CLL using monoclonal antibody therapy has already been proposed in the art. Recently, Hainsworth (Oncologist 5(5)(2000) 376-334) described current therapeutics derived from monoclonal antibodies. In particular, lymphocytic leukemia is considered a promising candidate for this treatment modality due to the presence of multiple lymphocyte-specific antigens in lymphocyte tumors.

^{Existing antibody therapeutics (eg, Rituximab™} against CD20-antigen expressed on the surface of B-lymphocytes) have been successfully used for certain lymphoid diseases. However, in the case of CLL, the CD20 antigen is expressed at a lowered level on the surface of B-lymphocytes (Almasri et al., Am. J. Hematoi., 40(4)(1992) 259-263).

Therefore, the CLL cell line described herein enables the development of novel anti-CCL antibodies and polypeptides having specificity for one or more antigenic determinants of the CCL cell line of the present invention, and CLL, cancer, OX-2/CD200 It is used in the treatment and diagnosis of other disease states characterized by upregulated levels of.

The antibody or polypeptide binds to a receptor with which OX-2/CD200 normally interacts, thereby preventing or inhibiting the binding of OX-2/CD200 to the receptor. As another alternative, the antibody can prevent or inhibit normal or increased expression of OX-2/CD200 by binding to an antigen that modulates the expression of OX-2/CD200. Because the presence of OX-2/CD200 is associated with a reduced immune response, it interferes with the metabolic pathways of OX-2/CD200, allowing the patient's immune system to more effectively defend against disease conditions such as cancer or CLL. It is desirable.

In a particularly preferred 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 over-expressing OX-2/CD200 significantly reduce the production of Th1 cytokines, OX-2/CD200 upregulates anti-CD200 antibodies or polypeptides that bind to OX-2/CD200. The Th1 cytokine profile is restored when administered to an individual. Thus, these polypeptides and/or antibodies are effective therapeutic agents for the treatment of CLL and other cancers or diseases over-expressing OX-2/CD200.

Thus, in another embodiment, the methods of the present invention comprise testing for the presence of OX-2/CD200 in the subject and administering a polypeptide that binds to OX-2/CD200. In a particularly preferred embodiment, a CLL patient is examined for over-expression of OX-2/CD200, and an antibody that binds to OX-2/CD200 is administered to the patient. As detailed below, one such antibody is scFv-9 (Figure 9B) that binds to OX-2/CD200.

In order to enable those skilled in the art to more effectively perform the compositions and methods disclosed herein, the following examples are presented for purposes of illustration.

1 schematically illustrates a typical step involved in cell surface panning of an antibody library by magnetic-activated cell sorting (MACS).
2 is a graph showing the results of whole cell ELISA demonstrating the binding of selected scFv clones to primary B-CLL cells and the absence of binding to normal human PBMCs. In the figure, 2°+3° represents a negative control well stained with mouse anti-HA and detection anti-mouse antibody alone. In the figure, the RSC-S library represents a soluble antibody prepared from the original rabbit scFv library. In the figure, R3/RSC-S Pool represents a soluble antibody prepared from the entire pool of scFv antibodies after panning 3 times. The anti-CD5 antibody was used as a positive control to verify that an equal number of B-CLL and PBMC cells were plated in each well.
3 is a graph showing the results of whole cell ELISA comparing the binding of the selected scFv antibody to primary B-CLL cells and normal human B cells. The anti-CD19 antibody was used as a positive control to verify that an equal number of B-CLL and normal B cells were plated in each well. The other contrast is the same as described in the legend of FIG. 2.
Figures 4A and 4B show the results of whole cell ELISA used to determine whether scFv clones bind to patient-specific (ie, idiotype) or blood type-specific (ie, HLA) antigens. Each clone was tested for binding to PBMCs isolated from 3 different B-CLL patients. Clones bound to one patient sample are considered patient or blood type-specific.
5A and 5B show the results of 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-I is an erythropoietic cell line derived from erythroleukemia.
6A, 6B, 6C show the results of whole cell ELISA comparing the binding of scFv clones to primary B-CLL cells and cell lines derived from CLL-AAT and B-CLL patients. TF-I cells are included as negative controls.
7 shows the binding specificity of the scFv antibody according to the present invention in tricolor flow cytometry. In normal peripheral blood mononuclear cells, the antigen recognized by scFv-9 is expressed at moderate levels in B lymphocytes and weakly expressed in a subpopulation of T lymphocytes. PBMCs from normal donors were analyzed by tricolor flow cytometry using anti-CD5-FITC, anti-CD19-PerCP, and scFv-9/anti-HA-biotin/streptavidin-PE.
8A, 8B, and 8C show the expression levels of antigens recognized by the scFv antibody according to the present invention. Antigens recognized by scFv-3 and scFv-9 were over-expressed in primary CLL tumors from which the CLL-AAT cell line was derived. Primary PBMCs derived from CLL patients or PBMCs derived from normal donors used to establish CLL-AAT cell lines were stained with scFv antibody and analyzed by flow cytometry. scFv-3 and scFv-9 stain CLL cells much brighter than normal PBMCs as measured by mean fluorescence intensity.
9A, 9B, 9C provide a summary of the CDR sequences and binding specificities of the selected scFv antibodies. As shown in Figure 9B, the clone number in the left column is cited by scFv number 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-9), 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), D2.2b (scFv-16).
Figure 10 is the primary CLL cells described in Figures 8a-8c vs. This is a summary table of the results of flow cytometry comparing the expression levels of scFv antigens in normal PBMCs.
FIG. 11 is a table summarizing the results of flow cytometry comparing the expression level of scFv-9 antigen as a percentage of CD38+ cells in peripheral blood mononuclear cells isolated from 10 CLL patients.
12 shows the identification of the scFv antigen by immunoprecipitation and mass spectrometry. CLL-AAT cells were labeled with a 0.5 mg/ml sulfo-NHS-LC-Biotin (Pierce) solution in PBS pH 8.0 for 30'. After washing extensively with PBS to remove unreacted biotin, the cells were destroyed by nitrogen cavitation, and the microsomal fraction was separated by differential centrifugation. The microsome fraction was resuspended in NP40 lysis buffer and treated extensively with normal rabbit serum and protein A sepharose to ensure stability in advance. Antigen was immunoprecipitated with HA-labeled scFv antibody bound to murine anti-HA agarose beads (Roche). After immunoprecipitation, antigens were separated by SDS-PAGE and detected by Western blot or Coomassie G-250 staining using streptavidin-alkaline phosphatase (AP). An antibody that does not bind to CLL-AAT cells, scFv-7, was used as a negative control. Antigen bands were excised from Coomassie-stained gels and confirmed by mass spectrometry (MS). MALDI-MS was performed at the Proteomics Core Facility of The Scripps Research Institute (La Jolla, CA). μLC/MS/MS was performed at the Harvard Microchemistry Facility (Cambridge, MA).
13 shows that the three scFv antibodies specifically bind to 293-EBNA cells transiently transfected with human OX-2/CD200 cDNA clones. OX-2/CD200 cDNA was cloned from CLL cells by RT-PCR and inserted into 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, the cells were analyzed for binding to the scFv antibody by flow cytometry.
14 shows that the presence of OX-2/CD200 transfected cells induces down-regulation of Th1 cytokines such as IL-2 and IFN-γ. Addition of anti-OX 2/CD200 antibody at 30 μg/ml completely restored the Th1 response.
Figure 15 shows that the presence of CCL cells in hybrid lymphocyte reactants induces down-regulation of the Th1 response to IL-2.
Figure 16 shows that the presence of CCL cells in hybrid lymphocyte reactants induces down-regulation of the Th1 response to IFN-γ.
Figures 17a and b show the mean +/- SD of tumor volume for all NOD/SCID mice groups injected subcutaneously ^{with 4x10 6 RAJI cells in the presence and absence of human PBL.}
Figure 18 shows the results of statistical analysis performed using two parameter tests (Student's t-test and Welch's test) and one non-parametric test (Wilcox test).

Example 1

Cell line CLL - AAT Separation of

Establishment of the cell line

Peripheral blood was obtained from patients diagnosed with CLL. The total number of WBCs was 1.6x10 ⁸ /ml. Mononuclear cells were separated by Histopaque-1077 density gradient centrifugation (Sigma Diagnostics, St. Louis, MO). Cells were washed twice with IMDM (Iscove's Modified Dulbecco's Medium) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and resuspended in 5 ml of cold IMDM/10% FBS. Viable cells were counted by staining with trypan blue. Cells were mixed with an equal volume of 85% FBS/15% DMSO and frozen in liquid nitrogen in 1 ml portions for storage.

In immunophenotyping, ^{>90% of the CD45 +} lymphocyte population was found to express IgD, kappa light chain, CD5, CD19, and CD23. In addition, the population expressed IgM and CD20 at low levels. Approximately 50% of cells expressed CD38 at high levels. These cells were negative for the lambda light chain, CD10, and CD138.

Portions of cells were thawed and washed, 20% heat-inactivated FBS, 2 mM L-glutamine, 100 unit/ml penicillin, 100 μg/ml streptomycin, 50 μM 2-mercaptoethanol, 5 ng/ml recombinant human Resuspended at a density of ^{10 7} /ml in IMDM supplemented with IL-4 (R & D Systems, Minneapolis, MN). The cells were cultured in a humidified 5% CO ₂ atmosphere at 37°C. The medium was partially replaced at 4 day intervals until stable growth was observed. After 5 weeks, the total number of cells in the culture medium doubled at approximately 4 day intervals. The cell line was named CLL-AAT.

Characterization of the cell line

In the immunophenotype confirmation of the cell line by flow cytometry, high expression of IgM, kappa light chain, CD23, CD38, and CD138, intermediate expression of CD19 and CD20, and weak expression of IgD and CD5 were confirmed. The cell line was negative for lambda light chain, CD4, CD8, and CD10.

Immunophenotype confirmation of the cell line was also performed by whole cell ELISA using a panel of rabbit scFv antibodies selected for specific binding to primary B-CLL cells. All these CLL-specific scFv antibodies also recognized the CLL-AAT cell line. In contrast, most scFvs have two cell lines derived from B cell lymphoma: Ramos, Burkitt's lymphoma cell line; RL, did not bind to non-Hodgkin's lymphoma cell line.

Example 2

Screening of scFv antibodies against B- CLL -specific cell surface antigens using antibody phage display and cell surface panning

Immunization and scFv Antibody library Construction

Peripheral blood mononuclear cells (PBMC) were isolated from blood collected from CLL patients in Scripps Clinic (La Jolla, CA). Two rabbits were immunized with ^{2x10 7} PBMCs collected from 10 different CLL donors. Three immunizations, two subcutaneous infusions and a subsequent one intravenous infusion were performed at 3-week intervals. Serum titer was investigated 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 harvested from these animals. Total RNA was isolated from these tissues using Tri-Reagent (Molecular Research Center, Inc). The single-chain Fv (scFv) antibody phage display library was constructed as described in the existing literature (Barbas et al., (2001) Phage Display: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York). . For cell surface panning, phagemid particles from the re-amplified library were precipitated with polyethylene glycol (PEG) and resuspended in a phosphate buffer (PBS) containing 1% bovine serum albumin (BSA) and overnight in PBS. Dialysis was performed.

Cell surface In panning Antibody selection by

These libraries were enriched for CLL cell surface-specific antibodies by positive-negative selection using a magnetic-activated cell sorter (MACS), as revealed by Siegel et al. (1997, J. Immunol. Methods 206:73-85). Made it. Briefly, pagemid particles from the scFv antibody library were pre-incubated in MPBS (PBS, 2% fat-free milk powder in pH 7.4, 0.02% sodium azide) at 25° C. for 1 hour to block non-specific binding sites. Approximately 10 ⁷ primary CLL cells were labeled with mouse anti-CD5 IgG and mouse anti-CD19 IgG conjugated to paramagnetic microbeads (Miltenyi Biotec, Sunnyvale, CA). Unbound microbeads were removed by washing. Labeled CLL cells (“target cells”) were mixed with excess “antigen-negative absorber cells”, pelleted and resuspended in 50 μl (10 ¹⁰ -10 ¹¹ cfu) of phage particles. Absorber cells are responsible for absorbing phages that non-specifically adhere to the cell surface and specific for "common" antigens present on both target and absorber cells. The absorber cells used were TF-1 cells (human red leukemia cell line), or normal human B cells isolated from peripheral blood by immunomagnetic negative selection (StemSep system, Stem Cell Technologies, Vancouver, Canada). The ratio of absorber cells to target cells was approximately 10 times by volume. After 30 minutes incubation at 25° C., the cell/phage mixture was transferred to a ^{MiniMACS MS + separation column.} The column was washed twice with 0.5 ml of MPBS and once with 0.5 ml of PBS to remove unbound phage and absorber cells. Target cells were eluted from the column in 1 ml of PBS and pelleted in a microcentrifuge at a maximum rate of 15 seconds. The captured phage particles were eluted by resuspending the target cells in 200 μl of acid elution buffer (0.1 N HCl, adjusted to pH 2.2 with glycine, + 1 μg/ml BSA). After 10 minutes incubation at 25° C., the buffer was neutralized with 12 μl of 2M Tris base, pH 10.5, and the eluted phage was amplified in E. coli for subsequent panning. In each round of panning, the input and output grip titers were determined. The input titer is the total number of re-amplified phage particles added to the target cell/absorber cell mixture, and the output titer is the total number of captured phage eluted from the target cell. The enrichment factor (E) is calculated using the _{formula E=(R n} output/R _n input)/(R ₁ output/R _{1 input).} In most cases, ^{a concentration factor of 10 2} -10 ³ times was achieved in the 3rd or 4th round.

After panning Analysis of the concentrated antibody pool

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

1. In order to produce the whole pool in the form of HA-labeled soluble antibody , 2 ml of E. coli non-inhibitor strain (eg, TOP1OF') is 1 μl (10 ⁹ -10 ¹⁰ cfu) of phage. Infected with mid particles. The first library that was not panned was used 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 while stirring at 250 rpm. 8 ml of SB medium containing 50 µl/ml carbenicillin was added, and the culture solution was grown to an OD 600 of -0.8. IPTG was added to a final concentration of 1 mM to induce scFv expression from the Lac promoter, followed by stirring at 37° C. for 4 hours. The culture was centrifuged at 3000xg for 15'. The supernatant containing the soluble antibody was filtered and stored at -20°C in 1 ml portions.

2. Target cells vs. The binding of the scFv antibody pool to adsorbent cells was determined by flow cytometry using high affinity Rat Anti-HA (clone 3F10, Roche Molecular Biochemicals) as a secondary antibody and PE-conjugated Donkey Anti-Rat as a tertiary antibody. .

3. Target cells vs. The binding of the antibody pool to adsorbent cells was also determined by whole cell ELISA as described below.

After panning Individual scFv Selection of clones

In order to select individual scFv clones after panning, TOP10F' cells were infected with phage pool as described above, plated on LB plates containing carbenicillin and tetracycline, and incubated overnight at 37°C. Individual colonies were inoculated into deep 96-well plates containing 0.6-1.0 ml SB-carbenicillin medium per well. The culture was grown for 6-8 hours at 37°C in a HiGro stirred incubator (GeneMachines, San Carlos, CA) at 520 rpm. At this point, a 90 μl aliquot from each well was transferred to a deep 96-well plate containing 10 μl DMSO. These replicate plates were stored at -80°C. IPTG was added to the first plate at a final concentration of 1 mM and stirred for 3 hours. These plates were centrifuged at 3000xg for 15 minutes. The supernatant containing the soluble scFv antibody was transferred to another deep 96-well plate and stored at -20°C.

A sensitive whole-cell ELISA to select for HA-labeled scFv antibodies was developed:

1. The ELISA plate was coated with concanavalin A (0.1 M NaHCO ₃ , 10 mg/ml at pH 8.6, 0.1 mM CaCl ₂ ).

2. After washing the plate with PBS, 0.5-1×10 ⁵ target cells or adsorbent cells contained in 50 μl of PBS were added to each well, and centrifuged at 250×g for 10 minutes.

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

4. After washing with PBS, the non-specific binding sites are blocked with PBS containing 4% non-fat dry milk for 3 hours at room temperature.

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

6. The bound antibody is detected using Mouse Anti-HA secondary antibody (clone 12CA5) and alkaline phosphatase (AP)-conjugated Anti-Mouse IgG tertiary antibody. Using AMDEX AP-conjugated Sheep Anti-Mouse IgG (Amersham Pharmacia Biotech) as a tertiary antibody, approximately 10-fold amplification of the signal is achieved. AMDEX antibodies are conjugated to a plurality of AP molecules through the dextran backbone. Color is developed with alkaline phosphatase substrate PNPP and measured at 405 nm using a microplate reader.

Primary screening of scFv clones was performed by primary CLL cells vs. Performed by ELISA on normal human PBMCs. Clones positive for CLL cells and negative for normal PBMC were reselected by ELISA from normal human B cells, human B cell lines, TF-1 cells, and CLL-AAT cell lines. In addition, these clones were re-screened by ELISA in CLL cells isolated from 3 different patients to remove clones recognizing patient-specific or blood type-specific antigens. Results from a representative ELISA are shown in Figures 2-6 and summarized in Figures 9a-c.

The total number of unique scFv antibodies obtained was determined by DNA fingerprinting and sequencing. The scFv DNA insert was amplified from the plasmid by PCR and digested with the restriction enzyme BstNI. The resulting fragment was separated on a 4% agarose gel and stained with ethidium bromide. Clones with different restriction fragment patterns will have different amino acid sequences. Clones with the same pattern have similar or identical sequences. Clones with unique BstNI fingerprints were further analyzed by DNA sequencing. Twenty-five different sequences were found, which were grouped into 16 antibody groups as closely related complementarity determining regions (Figs. 9a-c).

For flow cytometry by scFv Characterization of antibodies

The binding specificity of several scFv antibodies was analyzed by tricolor flow cytometry (FIG. 7). PBMCs isolated from normal donors were stained with FITC-conjugated anti-CD5 and PerCP-conjugated anti-CD19. The scFv antibody staining was performed using biotin-conjugated anti-HA and PE-conjugated streptavidin as secondary antibodies. 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 different cell populations: a ^{subset of CD19 +} B lymphocytes and CD5 ⁺ T lymphocytes (FIG. 7 ). Further characterization of the T cell subtype ^{identified a subpopulation of CD4 +} CD8 ^- T _H cells (data not shown).

To determine whether the antigen recognized by the scFv antibody is over-expressed in primary CLL cells, PBMCs obtained 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 the positive cell population, CLL cells vs. It was possible to determine the relative expression level of the antigen in normal cells. The scFv-2 antibody stained CLL cells at a lower intensity than normal PBMCs, whereas both scFv-3 and scFv-6 stained CLL cells brighter than normal PBMCs. The fourth antibody, scFv-9, stained two of the five CLL samples more strongly than normal PBMCs, but slightly brighter staining for the other three CLL samples (FIG. 8 and Table 2). This suggests that antigens for scFv-3 and scFv-6 are approximately 2-fold over-expressed in a significant number of CLL tumors, while scFv-9 is 3-6-fold over-expressed in subtypes of CLL tumors.

CLL patients can be divided into two nearly equal groups: poor prognosis (mean survival of 8 years) and good prognosis (mean survival of 26 years). Several poor prognostic indicators have been identified in CLL, the most notable being the presence of the VH gene in the absence of somatic mutations and the presence of a high proportion of CD38 ⁺ B cells. Since scFv-9 recognizes antigens over-expressed only in subtypes of CLL patients, we tried to determine whether ^{scFv-9 antigen over-expression correlates with the percentage of CD38 + cells in blood samples obtained from 10 CLL patients (Fig.} 11). Its results suggest that scFv-9 antigen over-expression and CD38 ⁺ cell ratio are completely independent of each other.

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

To identify antigens for these antibodies, antigens were immunoprecipitated from lysates prepared from microsomal fractions of cell-surface biotinylated CLL-AAT cells using scFv (FIG. 12). Immunoprecipitated antigens are purified by SDS-PAGE and matrix assisted laser desorption ionization mass spectrometry (MALDI-MS) or microcapillary reverse phase HPLC nano-electrospray direct thermal mass spectrometry (microcapillary reverse). It was confirmed by -phase HPLC nano-electrospray tandem mass spectrometry (μLC/MS/MS) (data not shown). ScFv-2 immunoprecipitated with 110 kd antigen from both RL and CLL-ALT cells (FIG. 12 ). The antigen was identified as a B cell-specific marker CD19 by MALDI-MS. Both ScFv-3 and scFv-6 were immunoprecipitated with 45 kd antigen from CLL-AAT cells. The antigen was identified by MALDI-MS as CD23, a known marker for CLL and activated B cells. ScFv-9 immunoprecipitated with 50 kd antigen from CLL-AAT cells (Fig. 12). The antigen was identified as OX-2/CD200, a known marker for ^{B cells, activated CD4 +} T cells, and thymocytes in μLC/MS/MS. OX-2/CD200 is also expressed in some non-lymphoid cells, such as neurological and endothelial cells.

Example 3

The ability of cells to over-express OX-2/CD200 to convert cytokine responses from Th1 responses (IL-2, IFN-γ) to Th2 responses (IL-4, IL-10) is monocytes obtained from one donor- Assessed in hybrid lymphocyte reactions using derived macrophages/dendritic cells and blood-derived T cells obtained from other donors. As a source of OX-2/CD200-expressing cells, OX-2/CD200 transfected EBNA cells or CLL patient samples as described below were used.

293- EBNA Cellular Transfection

293-EBNA cells (Invitrogen) were inoculated at ^{2.5×10 6 cells per 100 mm dish.} After 24 hours, these cells were transiently transfected using Polyfect reagent (QIAGEN) according to the manufacturer's instructions. Cells were co-transfected with 7.2 μg of OX-2/CD200 cDNA loaded on vector pCEP4 (Invitrogen) and 0.8 μg of pAdVAntage vector (Promega). As a negative control, cells were co-transfected with empty pCEP4 vector + pAdVAntage. 48 hours after transfection, approximately 90% of the cells expressed OX-2/CD200 on the surface in flow cytometry using the scFv-9 antibody.

From blood monocytes Dendrites Maturation of cells/macrophages

The buffy coat was obtained from San Diego Blood Bank, and primary blood lymphocytes (PBL) were isolated using Ficoll. Cells were adhered for 1 hour in EMEM (Eagles Minimal Essential Medium) containing 2% human serum, and then washed vigorously with PBS. After 3 days, the cells were cultured for 5 days in the presence of GM-CSF, IL-4, IFN-γ or M-CSF with or without the addition of lipopolysaccharide (LPS). The matured cells were harvested and irradiated at 2000 RAD using a γ-irradiator (Shepherd Mark I Model 30 irradiator (Cs ^{137 )).}

Hybrid lymphocyte reactants

Hybrid lymphocyte reactants were placed in a 24-well plate using 500,000 dendritic cells/macrophages and 1×10 ^{6 responder cells.} Responder cells were T cell-enriched lymphocytes purified from peripheral blood using Ficoll. T cells were cultured for 1 hour in a tissue culture flask, and a non-adherent cell fraction was collected and concentrated. 500,000 OX-2/CD200 transfected EDNA cells or CLL cells were macrophages/phase in the presence and absence of 30 μg/ml anti-CD200 antibody (scFv-9 converted to complete IgG) 2-4 hours before lymphocyte addition. It was added to the dendritic cells. The supernatant was collected after 48 and 68 hours and analyzed for the presence of cytokines.

scFv -9 perfection IgG Transition to

The light chain and heavy chain V genes of scFv-9 were amplified by overlapping PCR using primers that link the variable regions of each gene with 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 with specific primers, and the human lambda light chain constant region gene and the IgG1 heavy chain constant region CH1 gene were individually amplified with the following specific primers:

R9VL-F1 QP: 5'GGC CTC TAG ACA GCC TGT GCT GAC TCA GTC GCC CTC 3'(SEQ ID NO 103);

R9VL/hCL2-rev: 5'CGA GGG GGC AGC CTT GGG CTG ACC TGT GAC GGT CAG CTG GGT C 3'(SEQ ID NO 104);

R9VL/hCL2-F: 5'GAC CCA GCT GAC CGT CAC AGG TCA GCC CAA GGC TGC CCC CTC G 3'(SEQ ID NO 105);

R9VH-F1: 5'TCT AAT CTC GAG CAG CAG CAG CTG ATG GAG TCC G 3'(SEQ ID NO 106);

R9VH/hCG-rev: 5'GAC CGA TGG GCC CTT GGT GGA GGC TGA GGA GAC GGT GAC CAG GGT GC 3'(SEQ ID NO 107);

R9VH/hCG-F: 5'GCA CCC TGG TCA CCG TCT CCT CAG CCT CCA CCA AGG GCC CAT CGG TC 3'(SEQ ID NO 108);

hCL2-rev: 5'CCA CTG TCA GAG CTC CCG GGT AGA ACT C 3'(SEQ ID NO 109);

hCG-rev: 5'GTC ACC GGT TCG GGG AAG TAG TC 3'(SEQ ID NO 110).

The amplification product was purified and repeated PCR was performed.

The final product was digested with Xba I/Sac I (light chain) and Xho I/Pin AI (heavy chain) and cloned into a human Fab expression vector, PAX243hGL. DNA clones were analyzed for PCR errors by DNA sequencing. The hCMV IE promoter gene was inserted into the Not I/Xho I site (in front of the heavy chain). The vector was digested with Xba I/Pin AI/EcoR I/Nhe I, and the 3472 bp fragment carrying the light chain + hCMV IE promoter and heavy chain gene was transferred to the IgG1 expression vector at the Xba I/Pin AI site.

Cytokine analysis

The effect of scFv-9 converted to complete IgG on the cytokine profile in hybrid lymphocyte reactions was examined.

Cytokines found in the tissue culture supernatant, such as IL-2, IFN-γ, IL-4, IL-10, and IL-6 were quantified using ELISA. Corresponding capture and detection antibody pairs for each cytokine were purchased from R+D Systems (Minneapolis, MN), and standard curves for each cytokine were created using recombinant human cytokines. Anti-cytokine capture antibody was coated on the plate in PBS at the optimum concentration. After incubation overnight, the plates were washed and blocked with PBS containing 1% BSA and 5% sucrose for 1 hour. After washing three times with PBS containing 0.05% Tween, the supernatant was diluted 2-fold or 10-fold in PBS containing 1% BSA and added. The captured cytokines were detected with an appropriate biotinylated anti-cytokine antibody, and then alkaline phosphatase conjugated streptavidin and SigmaS substrate were added. 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 down-regulation of Th1 cytokines, such as IL-2 and IFN-γ. The addition of anti-CD200 antibody at 30 μg/ml completely restored the Th1 response, suggesting that the antibody blocks the interaction between OX-2/CD200 and its receptor.

As shown in Figures 15 and 16, the presence of CLL cells in the hybrid lymphocyte reaction resulted in down-regulation of the Th1 response (Figure 15 shows the results for IL-2; Figure 16 shows the results for IFN-γ. Shows the results). This has been applied to cells over-expressing OX-2/CD200 (IB, EM, HS, BH) as well as CLL cells that do not over-express OX-2/CD200 (JR, JG, GB). The expression level for is shown in Figure 11). However, the anti-CD200 antibody restored the Th1 response only in cells over-expressing OX-2/CD200, which in patients over-expressing OX-2/CD200, OX-2/CD200 and its receptors in macrophages. Elimination of the liver interaction suggests that the Th1 response is sufficiently restored. Other mechanisms were thought to be involved in the down-regulation of the Th1 response in patients who did not over-express OX-2/CD200.

Anti-tumor rejection CD200 Animal models to examine the effectiveness of

A model was established in which RAJI lymphoma tumor growth was prevented by co-infusion of PBL. NOD / SCID mice were injected subcutaneously 1x10 ^6, 5X10 ⁶ or 4X10 ⁶ 10X10 ⁶ cells RAJI cells in load zone of the human PBL obtained from different donors to. Tumor length, width and body weight were measured three times a week. The mean +/-SD of tumor volume for all groups is shown in Figures 17A and B. Statistical analysis was performed using two parametric tests (Student t-test and Welch's test) and one non-parametric test (Wilcox test). The results of statistical analysis are shown in FIG. 18. The rejection response depends on the specific donor and total number of PBL cells. 1X10 ⁶ PBL was insufficient to prevent tumor growth. From day 22 to day 43, ^{donor 3 at 5x10 6} or 1x10 ⁷ at 5x10 6 or 1x10 7 starting from donor 2 and 36 at ^{5X10 6} PBL significantly reduced tumor growth. Donor 4 was very close to significance after 48 days.

To examine the effect of anti-CD200, RAJI cells were stably transfected with CD200. Animals were treated as described above. In the presence of CD200-transfected cells, tumors grew even in the presence of human PBL. Anti-CD200 antibody was administered to evaluate tumor rejection in this model.

In addition, a liquid tumor model was established. RAJI cells were injected intraperitoneally in NOD/SCID mice. These cells spread to the bone marrow, spleen, lymph nodes, and other organs, causing paralysis. Co-infusion of human PBL prevented or delayed tumor growth. Tumor growth was monitored in mice by assessing signs of movement impairment and paralysis. When these signs were observed, the mice were sacrificed and the total number of tumor cells was assessed by FACS analysis and PCR in various organs including bone marrow, spleen, lymph nodes, and blood.

Similar to the subcutaneous model, CD200 transfected cells were injected intraperitoneally. They grew even in the presence of human PBL. Anti-CD200 treatment resulted in tumor rejection or slowed tumor growth.

references

The following references are incorporated herein by reference in their entirety. Any portion of the content described in the reference that conflicts with the content described herein takes precedence over the present application.

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Various modifications to the embodiments disclosed herein are possible. As one of skill in the art would appreciate, certain sequences described herein may be modified somewhat without negatively affecting the function of the polypeptide, antibody or antibody fragment used for OX-2/CD200 binding. For example, substitutions of single or multiple amino acids in an antibody sequence that do not destroy the function of the antibody or antibody fragment can be performed frequently. Thus, polypeptides or antibodies that retain at least 70% homology to the specific antibodies described herein are within the scope of the present invention. In a preferred embodiment, antibodies that retain at least 80% homology to the specific antibodies described herein are contemplated. In another preferred embodiment, antibodies that retain at least 90% homology to the specific antibodies described herein are contemplated. For this reason, the above details are only examples of preferred embodiments and do not limit the present invention. Other modifications that do not depart from the spirit and scope of the present invention also belong to the present invention.

American Type Culture Collection PTA-3920 20011211

SEQUENCE LISTING <110> Alexion Pharmaceuticals, Inc. Bowdish, Katherine S. McWhirter, John Kretz-Rommel, Anke <120> POLYPEPTIDES AND ANTIBODIES DERIVED FROM CHRONIC LYMPHOCYTIC LEUKEMIA CELLS AND USES THEREOF <130> 60 CIP III (1087-60 CIP III) <140> 10/894,672 <141> 2004-07-20 <150> US 10/736,188 <151> 2003-12-15 <150> US 10/379,151 <151> 2003-03-04 <150> PCT/US01/47931 <151> 2001-12-10 <150> US 60/254,113 <151> 2000-12-08 <160> 110 <170> PatentIn version 3.2 <210> 1 <211> 14 <212> PRT <213> rabbit <400> 1 Thr Leu Ser Thr Gly Tyr Ser Val Gly Ser Tyr Val Ile Ala 1 5 10 <210> 2 <211> 11 <212> PRT <213> rabbit <400> 2 Gln Ala Ser Glu Ser Ile Arg Asn Tyr Leu Ala 1 5 10 <210> 3 <211> 11 <212> PRT <213> rabbit <400> 3 Gln Ala Ser Glu Ser Ile Ser Asn Trp Leu Ala 1 5 10 <210> 4 <211> 11 <212> PRT <213> rabbit <400> 4 Gln Ala Ser Glu Ser Ile Ser Asn Tyr Leu Ala 1 5 10 <210> 5 <211> 11 <212> PRT <213> rabbit <400> 5 Gln Ala Ser Gln Asn Ile Tyr Ser Asn Leu Ala 1 5 10 <210> 6 <211> 11 <212> PRT <213> rabbit <400> 6 Gln Ala Ser Gln Ser Val Asn Asn Leu Leu Ala 1 5 10 <210> 7 <211> 11 <212> PRT <213> rabbit <400> 7 Gln Ala Ser Glu Ser Ile Asn Asn Tyr Leu Ala 1 5 10 <210> 8 <211> 11 <212> PRT <213> rabbit <400> 8 Leu Ala Ser Glu Asn Val Tyr Ser Ala Val Ala 1 5 10 <210> 9 <211> 11 <212> PRT <213> rabbit <400> 9 Leu Ala Ser Glu Asn Val Tyr Gly Ala Val Ala 1 5 10 <210> 10 <211> 11 <212> PRT <213> rabbit <400> 10 Gln Ala Ser Gln Ser Ile Ser Asn Leu Leu Ala 1 5 10 <210> 11 <211> 11 <212> PRT <213> rabbit <400> 11 Leu Ala Ser Glu Asn Val Ala Ser Thr Val Ser 1 5 10 <210> 12 <211> 14 <212> PRT <213> rabbit <400> 12 Thr Leu Ser Thr Gly Tyr Ser Val Gly Glu Tyr Pro Val Val 1 5 10 <210> 13 <211> 14 <212> PRT <213> rabbit <400> 13 Thr Leu Arg Thr Gly Tyr Ser Val Gly Glu Tyr Pro Leu Val 1 5 10 <210> 14 <211> 11 <212> PRT <213> rabbit <400> 14 Leu Ala Ser Glu Asp Ile Tyr Ser Gly Leu Ser 1 5 10 <210> 15 <211> 11 <212> PRT <213> rabbit <400> 15 Gln Ala Ser Gln Ser Val Ser Asn Leu Leu Ala 1 5 10 <210> 16 <211> 11 <212> PRT <213> rabbit <400> 16 Gln Ala Ser Glu Asp Ile Glu Ser Tyr Leu Ala 1 5 10 <210> 17 <211> 12 <212> PRT <213> rabbit <400> 17 Gln Ser Ser Gln Ser Ile Ala Gly Ala Tyr Leu Ser 1 5 10 <210> 18 <211> 10 <212> PRT <213> rabbit <400> 18 His Ser Glu Glu Ala Lys His Gln Gly Ser 1 5 10 <210> 19 <211> 7 <212> PRT <213> rabbit <400> 19 Gly Ala Ser Asn Leu Glu Ser 1 5 <210> 20 <211> 7 <212> PRT <213> rabbit <400> 20 Arg Ala Ser Thr Leu Ala Ser 1 5 <210> 21 <211> 7 <212> PRT <213> rabbit <400> 21 Leu Ala Phe Thr Leu Ala Ser 1 5 <210> 22 <211> 7 <212> PRT <213> rabbit <400> 22 Gly Ala Ser Asp Leu Glu Ser 1 5 <210> 23 <211> 10 <212> PRT <213> rabbit <400> 23 His Thr Asp Asp Ile Lys His Gln Gly Ser 1 5 10 <210> 24 <211> 7 <212> PRT <213> rabbit <400> 24 Leu Ala Ser Lys Leu Ala Ser 1 5 <210> 25 <211> 13 <212> PRT <213> rabbit <400> 25 Ala Thr Ala His Gly Ser Gly Ser Ser Phe His Val Val 1 5 10 <210> 26 <211> 10 <212> PRT <213> rabbit <400> 26 Gln Ser Gly Asp Tyr Ser Ala Gly Leu Thr 1 5 10 <210> 27 <211> 10 <212> PRT <213> rabbit <400> 27 Gln Ser Gly Tyr Tyr Ser Ala Gly Leu Thr 1 5 10 <210> 28 <211> 10 <212> PRT <213> rabbit <400> 28 Gln Ser Gly Tyr Tyr Ser Ala Gly Val Thr 1 5 10 <210> 29 <211> 14 <212> PRT <213> rabbit <400> 29 Gln Gly Gly Asp Tyr Ser Ser Ser Ser Ser Tyr Gly Tyr Gly 1 5 10 <210> 30 <211> 10 <212> PRT <213> rabbit <400> 30 Gln Ser Gly Tyr Tyr Ser Pro Gly Val Thr 1 5 10 <210> 31 <211> 10 <212> PRT <213> rabbit <400> 31 Gln Ser Gly Tyr Tyr Ser Gly Gly Ala Thr 1 5 10 <210> 32 <211> 9 <212> PRT <213> rabbit <400> 32 Gln Gly Tyr Ser Ser Tyr Pro Pro Thr 1 5 <210> 33 <211> 8 <212> PRT <213> rabbit <400> 33 Gln Gly Tyr Ser Ser Tyr Pro Thr 1 5 <210> 34 <211> 12 <212> PRT <213> rabbit <400> 34 Ala Gly Tyr Lys Ser Ser Ser Thr Asp Gly Ile Ala 1 5 10 <210> 35 <211> 11 <212> PRT <213> rabbit <400> 35 Gln Ser Gly Tyr Tyr Ser Ala Gly His Leu Thr 1 5 10 <210> 36 <211> 12 <212> PRT <213> rabbit <400> 36 Leu Gly Gly Phe Gly Tyr Ser Thr Thr Gly Leu Thr 1 5 10 <210> 37 <211> 13 <212> PRT <213> rabbit <400> 37 Ala Ile Ala His Gly Thr Glu Ser Ser Phe His Val Val 1 5 10 <210> 38 <211> 13 <212> PRT <213> rabbit <400> 38 Ala Thr Gly His Gly Ser Gly Ser Ser Ala Gly Val Val 1 5 10 <210> 39 <211> 12 <212> PRT <213> rabbit <400> 39 Leu Gly Gly Tyr Pro Tyr Ser Ser Thr Gly Thr Ala 1 5 10 <210> 40 <211> 11 <212> PRT <213> rabbit <400> 40 Gln Ser Gly Trp Tyr Ser Ala Gly Ala Leu Thr 1 5 10 <210> 41 <211> 11 <212> PRT <213> rabbit <400> 41 Gln Ser Gly Tyr Tyr Arg Ala Gly Asp Leu Thr 1 5 10 <210> 42 <211> 11 <212> PRT <213> rabbit <400> 42 Gln Ser Gly Tyr Tyr Ser Ala Gly Ala Leu Thr 1 5 10 <210> 43 <211> 10 <212> PRT <213> rabbit <400> 43 Gln Ser Asn Ala Trp Ser Val Gly Met Thr 1 5 10 <210> 44 <211> 10 <212> PRT <213> rabbit <400> 44 Ala Ala Gln Tyr Ser Gly Asn Ile Tyr Thr 1 5 10 <210> 45 <211> 5 <212> PRT <213> rabbit <400> 45 Asn Tyr Ala Met Thr 1 5 <210> 46 <211> 5 <212> PRT <213> rabbit <400> 46 Ser Tyr Gly Leu Ser 1 5 <210> 47 <211> 5 <212> PRT <213> rabbit <400> 47 Thr Tyr Gly Val Ser 1 5 <210> 48 <211> 5 <212> PRT <213> rabbit <400> 48 Ser Asn Ala Met Gly 1 5 <210> 49 <211> 5 <212> PRT <213> rabbit <400> 49 Thr Asn Ala Met Gly 1 5 <210> 50 <211> 6 <212> PRT <213> rabbit <400> 50 Ser Ser Asp Trp Ile Cys 1 5 <210> 51 <211> 5 <212> PRT <213> rabbit <400> 51 Ser Asp Val Ile Ser 1 5 <210> 52 <211> 5 <212> PRT <213> rabbit <400> 52 Thr Tyr Ala Met Gly 1 5 <210> 53 <211> 5 <212> PRT <213> rabbit <400> 53 Ser Asn Ala Met Thr 1 5 <210> 54 <211> 5 <212> PRT <213> rabbit <400> 54 Asp Phe Ala Met Ser 1 5 <210> 55 <211> 5 <212> PRT <213> rabbit <400> 55 Ser Tyr Gly Met Asn 1 5 <210> 56 <211> 5 <212> PRT <213> rabbit <400> 56 Ser Asn Ala Met Ser 1 5 <210> 57 <211> 5 <212> PRT <213> rabbit <400> 57 Thr Asn Ala Ile Ser 1 5 <210> 58 <211> 5 <212> PRT <213> rabbit <400> 58 Ser Tyr Tyr Met Ser 1 5 <210> 59 <211> 5 <212> PRT <213> rabbit <400> 59 Ser Tyr Thr Met Ser 1 5 <210> 60 <211> 5 <212> PRT <213> rabbit <400> 60 Ser Asn Ala Ile Ser 1 5 <210> 61 <211> 5 <212> PRT <213> rabbit <400> 61 Thr Asn Ala Met Ser 1 5 <210> 62 <211> 6 <212> PRT <213> rabbit <400> 62 Ser Ser Tyr Trp Ile Cys 1 5 <210> 63 <211> 5 <212> PRT <213> rabbit <400> 63 Asn Tyr Gly Val Asn 1 5 <210> 64 <211> 15 <212> PRT <213> rabbit <400> 64 Ile Ile Ser Ser Asn Gly Gly Ala Asp Tyr Ala Ser Trp Ala Lys 1 5 10 15 <210> 65 <211> 16 <212> PRT <213> rabbit <400> 65 Tyr Phe Asp Pro Val Phe Gly Asn Ile Tyr Tyr Ala Thr Trp Val Asp 1 5 10 15 <210> 66 <211> 16 <212> PRT <213> rabbit <400> 66 Tyr Asn Asp Pro Ile Phe Gly Asn Thr Tyr Tyr Ala Thr Trp Val Asn 1 5 10 15 <210> 67 <211> 15 <212> PRT <213> rabbit <400> 67 Ile Ile Ser Ser Ser Gly Gly Thr Tyr Tyr Ala Ser Trp Ala Lys 1 5 10 15 <210> 68 <211> 15 <212> PRT <213> rabbit <400> 68 Ile Ile Ser Ser Ser Gly Ser Thr Tyr Tyr Ala Ser Trp Ala Lys 1 5 10 15 <210> 69 <211> 17 <212> PRT <213> rabbit <400> 69 Cys Ile Tyr Thr Gly Ser Ser Ser Ser Thr Trp Tyr Ala Ser Trp Ala 1 5 10 15 Lys <210> 70 <211> 16 <212> PRT <213> rabbit <400> 70 Tyr Ile Tyr Thr Gly Asp Gly Ser Thr Asp Tyr Ala Ser Trp Val Asn 1 5 10 15 <210> 71 <211> 15 <212> PRT <213> rabbit <400> 71 Ser Ile Tyr Ala Ser Arg Ser Pro Tyr Tyr Ala Ser Trp Ala Lys 1 5 10 15 <210> 72 <211> 15 <212> PRT <213> rabbit <400> 72 Thr Ile Ile Tyr Gly Asp Asn Thr Tyr Tyr Ala Ser Trp Ala Lys 1 5 10 15 <210> 73 <211> 17 <212> PRT <213> rabbit <400> 73 Val Val Tyr Ala Gly Thr Arg Gly Asp Thr Tyr Tyr Ala Asn Trp Ala 1 5 10 15 Lys <210> 74 <211> 16 <212> PRT <213> rabbit <400> 74 Tyr Ile Asp Pro Asp Tyr Gly Ser Thr Tyr Tyr Ala Ser Trp Val Asn 1 5 10 15 <210> 75 <211> 15 <212> PRT <213> rabbit <400> 75 Ile Thr Tyr Pro Ser Gly Asn Val Tyr Tyr Ala Ser Trp Ala Lys 1 5 10 15 <210> 76 <211> 15 <212> PRT <213> rabbit <400> 76 Tyr Ser Ser Tyr Gly Asn Asn Ala His Tyr Thr Asn Trp Ala Lys 1 5 10 15 <210> 77 <211> 15 <212> PRT <213> rabbit <400> 77 Ile Ile Ile Gly Ser Gly Thr Thr Tyr Tyr Ala Asn Trp Ala Lys 1 5 10 15 <210> 78 <211> 15 <212> PRT <213> rabbit <400> 78 Ile Ile Ser Ser Ser Gly Thr Ser Tyr Tyr Ala Thr Trp Ala Lys 1 5 10 15 <210> 79 <211> 15 <212> PRT <213> rabbit <400> 79 Ile Ile Ser Ser Ser Gly Ser Ala Tyr Tyr Ala Thr Trp Ala Lys 1 5 10 15 <210> 80 <211> 15 <212> PRT <213> rabbit <400> 80 Ile Ile Val Gly Ser Gly Thr Thr Tyr Tyr Ala Asp Trp Ala Lys 1 5 10 15 <210> 81 <211> 15 <212> PRT <213> rabbit <400> 81 Thr Ile Thr Tyr Gly Thr Asn Ala Tyr Tyr Ala Ser Trp Ala Lys 1 5 10 15 <210> 82 <211> 17 <212> PRT <213> rabbit <400> 82 Cys Ile Tyr Thr Gly Ser Asn Gly Ser Thr Tyr Tyr Ala Ser Trp Ala 1 5 10 15 Lys <210> 83 <211> 16 <212> PRT <213> rabbit <400> 83 Tyr Ile Asp Pro Val Phe Gly Ser Thr Tyr Tyr Ala Ser Trp Val Asn 1 5 10 15 <210> 84 <211> 17 <212> PRT <213> rabbit <400> 84 Asp Asp Glu Gly Tyr Asp Asp Tyr Gly Asp Tyr Met Gly Tyr Phe Thr 1 5 10 15 Leu <210> 85 <211> 14 <212> PRT <213> rabbit <400> 85 Asp Arg Ile Tyr Val Ser Ser Val Gly Tyr Ala Phe Asn Leu 1 5 10 <210> 86 <211> 14 <212> PRT <213> rabbit <220> <221> MISC_FEATURE <222> (11)..(14) <223> Xaa = is an unknown amino acid <400> 86 Asp Arg Ala Tyr Ala Ser Ser Ser Gly Tyr Xaa Xaa Xaa Xaa 1 5 10 <210> 87 <211> 13 <212> PRT <213> rabbit <400> 87 Asp Trp Ile Ala Ala Gly Lys Ser Tyr Gly Leu Asp Leu 1 5 10 <210> 88 <211> 9 <212> PRT <213> rabbit <400> 88 Arg Tyr Thr Gly Asp Asn Gly Asn Leu 1 5 <210> 89 <211> 13 <212> PRT <213> rabbit <400> 89 Asp Ala Ala Tyr Ala Gly Tyr Gly Trp Ile Phe Asn Leu 1 5 10 <210> 90 <211> 11 <212> PRT <213> rabbit <400> 90 Gly Asp Ala Gly Ser Ile Pro Tyr Phe Lys Leu 1 5 10 <210> 91 <211> 7 <212> PRT <213> rabbit <400> 91 Gly Asn Val Phe Ser Asp Leu 1 5 <210> 92 <211> 7 <212> PRT <213> rabbit <400> 92 Gly Leu Thr Tyr Tyr Pro Leu 1 5 <210> 93 <211> 12 <212> PRT <213> rabbit <400> 93 Gly Ala Tyr Ser Gly Tyr Pro Ser Tyr Phe Asn Leu 1 5 10 <210> 94 <211> 5 <212> PRT <213> rabbit <400> 94 Gly Phe Phe Asn Leu 1 5 <210> 95 <211> 7 <212> PRT <213> rabbit <400> 95 Gly Asn Ala Tyr Ser Asp Leu 1 5 <210> 96 <211> 22 <212> PRT <213> rabbit <400> 96 Asp Gln Pro Ile Ile Tyr Gly Ala Tyr Gly Asp Tyr Gly Leu Ala Thr 1 5 10 15 Gly Thr Arg Leu Asp Leu 20 <210> 97 <211> 22 <212> PRT <213> rabbit <400> 97 Asp Gln Pro Ile Ile Asp Ala Ala Tyr Gly Asp Tyr Gly Ile Ala Thr 1 5 10 15 Gly Thr Arg Leu Asp Leu 20 <210> 98 <211> 22 <212> PRT <213> rabbit <400> 98 Asp Gln Pro Ile Ile Thr Thr Asp Tyr Gly Gly Tyr Gly Ile Ala Thr 1 5 10 15 Gly Thr Arg Leu Asp Leu 20 <210> 99 <211> 19 <212> PRT <213> rabbit <400> 99 Asp Gln Pro Ile Thr Tyr Ala Gly Tyr Gly Tyr Ala Thr Gly Thr Arg 1 5 10 15 Leu Asp Leu <210> 100 <211> 7 <212> PRT <213> rabbit <400> 100 Gly Asn Thr Tyr Ser Asp Leu 1 5 <210> 101 <211> 13 <212> PRT <213> rabbit <400> 101 Ala Tyr Ile Tyr Tyr Gly Gly Tyr Gly Phe Phe Asp Leu 1 5 10 <210> 102 <211> 10 <212> PRT <213> rabbit <400> 102 Glu Ala Ser Phe Tyr Tyr Gly Met Asp Leu 1 5 10 <210> 103 <211> 36 <212> DNA <213> artificial sequence <220> <223> primer <400> 103 ggcctctaga cagcctgtgc tgactcagtc gccctc 36 <210> 104 <211> 43 <212> DNA <213> artificial sequence <220> <223> primer <400> 104 cgagggggca gccttgggct gacctgtgac ggtcagctgg gtc 43 <210> 105 <211> 43 <212> DNA <213> artificial sequence <220> <223> primer <400> 105 gacccagctg accgtcacag gtcagcccaa ggctgccccc tcg 43 <210> 106 <211> 34 <212> DNA <213> artificial sequence <220> <223> primer <400> 106 tctaatctcg agcagcagca gctgatggag tccg 34 <210> 107 <211> 47 <212> DNA <213> artificial sequence <220> <223> primer <400> 107 gaccgatggg cccttggtgg aggctgagga gacggtgacc agggtgc 47 <210> 108 <211> 47 <212> DNA <213> artificial sequence <220> <223> primer <400> 108 gcaccctggt caccgtctcc tcagcctcca ccaagggccc atcggtc 47 <210> 109 <211> 28 <212> DNA <213> artificial sequence <220> <223> primer <400> 109 ccactgtcag agctcccggg tagaagtc 28 <210> 110 <211> 23 <212> DNA <213> artificial sequence <220> <223> primer <400> 110 gtcaccggtt cggggaagta gtc 23

Claims (1)

Cell line "CLL-AAT" deposited under ATCC accession number PTA-3920

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