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1. WO2004060280 - PROCEDES ET COMPOSITIONS POUR INHIBER LA CROISSANCE DE CELLULES MALIGNES HEMATOPOIETIQUES

Note: Texte fondé sur des processus automatiques de reconnaissance optique de caractères. Seule la version PDF a une valeur juridique

[ EN ]

METHODS AND COMPOSITIONS FOR INHIBITING THE
GROWTH OF HEMATOPOIETIC MALIGNANT CELLS

[0001] This application is a continuation-in-part of U.S. Application No.

09/456,886, filed December 8, 1999, which is a divisional of U.S. Application No. 08/991,862, filed December 16, 1997, now U.S. Patent No. 6,309,826, which is a continuation-in-part of U.S. Patent application No. 08/863,079, filed May 23, 1997, now abandoned.

FIELD OF THE INVENTION

[0002] This invention relates to cell biology, physiology and medicine, and concerns an 88kDa glycoprotein growth factor ( "GP88 " or "PCDGF") and compositions and methods which affect the expression and biological activity of GP88. These compositions and methods are useful for diagnosis and treatment of diseases including cancer.

REFERENCES
[0003] Several publications are referenced herein.

BACKGROUND OF THE INVENTION
[0004] The proliferation and differentiation of cells in multicellular organisms is subject to a highly regulated process. A distinguishing feature of cancer cells is the absence of control over this process; proliferation and differentiation become deregulated resulting in uncontrolled growth. Significant research efforts have been directed toward better understanding this difference between normal and tumor cells. One area of research focus is growth factors and, more specifically, autocrine growth stimulation.

[0005] Growth factors are polypeptides which carry messages to cells concerning growth, differentiation, migration and gene expression. Typically, growth factors are produced in one cell and act on another cell to stimulate proliferation. However, certain malignant cells, in culture, demonstrate a greater or absolute reliance on an autocrine growth mechanism. Malignant cells which observe this autocrine behavior circumvent the regulation of growth factor production by other cells and are therefore unregulated in their growth.

[0006] Study of autocrine growth control advances understanding of cell growth mechanisms and leads to important advances in the diagnosis and treatment of cancer. Toward this end, a number of growth factors have been studied, including insulin-like growth factors ( "IGF-I" and "IGF-II" ), gastrin- releasing peptide ( "GRP" ), transforming growth factors alpha and beta ( "TGF-a" and "TGF-b " ), and epidermal growth factor ( " EGF " ) .

[0007] The present invention is directed to a recently discovered growth factor. This growth factor was first discovered in the culture medium of a highly tumorigenic "PC cell line," an insulin-independent variant isolated from the teratoma derived adipogenic cell line 1246. This growth factor is referred to herein as "GP88. " GP88 has been purified and structurally characterized. Amino acid sequencing of GP88 indicates that GP88 has amino acid sequence similarities with the mouse granulin/epithelin precursor.

[0008] Granulins/epithelins ( "grn/epi" ) are 6kDa polypeptides and belong to a novel family of double cysteine rich polypeptides. U.S. Patent No. 5,416,192 (Shoyab et al.) is directed to 6 kDa epithelins, particularly epithelin 1 and epithelin 2. According to Shoyab, both epithelins are encoded by a common 63.5 kDa precursor, which is processed into smaller forms as soon as it is synthesized, so that the only natural products found in biological samples are the 6 kDa forms. Shoyab et al. teaches that the epithelin precursor is biologically inactive.

[0009] Contrary to the teachings of Shoyab et al., the inventor's laboratory has demonstrated that the precursor is not processed as soon as it is syntiiesized. Studies, conducted in part by this inventor, have demonstrated that the precursor (i.e., GP88) is in fact secreted as an 88kDa glycoprotein with an N-linked carbohydrate moiety of 20kDa. Analysis of the N-terminal sequence of GP88 indicates that GP88 starts at amino acid 17 of the grn/epi precursor, demonstrating that the first 17 amino acids from the protein sequence deduced from the precursor cDNA correspond to a signal peptide compatible with targeting for membrane localization or for secretion.

[0010] Also in contrast to the teachings of Shoyab et al., the inventor demonstrated that GP88 is biologically active and has growth promoting activity, particularly as an autocrine growth factor for the producer cells.

[0011] Hematopoietic malignancies are malignant blood diseases including various lymphomas and leukemias. Leukemias of B-cell lineage include, but are not limited to, acute lymphocytic leukemia, B cell lymphoma, and multiple myeloma. Multiple myeloma ("MM") is a clonal B-cell neoplasm and the second most prevalent blood cancer, representing 1% of all cancers and 2% of all cancer deaths. B-cells (or B -lymphocytes) are precursor cells that differentiate into plasma cells after exposure to particular antigens. Plasma cells produce immunoglobulins and have a limited life span. However, uncontrolled growth of plasma cells in a clonal lineage of B cells may lead to accumulation of plasma cells producing monoclonal immunoglobulins or immunoglobulin fragments (e.g., M protein). MM is characterized by bone degradation and fractures, anemia, increased risk of infection, and decreased production of platelets in addition to other symptoms. The incidence of MM, currently about 14,000 new cases per year, has been steadily increasing in the United States for several decades (Niesvizky, R., D. Siegel, and J. Michaeli. 1993, Blood Reviews 7, no. 1:24). There has been little improvement in the treatment of human MM over the past 25 years and there is no cure for the disease (Hawley, R.G., and L.C. Berger, 1998, Leukemia & Lymphoma 29, no. 5-6:465). The few available therapies for treatment of MM have severe side effects and are of limited efficacy. For nearly 3 decades, the standard treatment for human MM has been glucocorticoid and/or chemotherapy with melphalan and prednisone alone or combinations of alkylating agents such as glucocorticoids and anthracyclines (Oken, M. M., 1997, Cancer Investigation 15, no. 1:57). However, almost all patients with MM who initially respond to glucocorticoid therapy relapse, with a median survival of two to three years following diagnosis (Alexanian, R., and M. Dimopoulos, 1994, New England Journal of Medicine 330, no. 7:484). During the progression of MM to more aggressive forms of the disease, MM cells become insensitive to the killing effect of glucocorticoids leaving only the use of
chemotherapeutic agents to control the disease.

[0012] What is needed are new compositions and methods for treatment and diagnosis of MM, and particularly compositions and methods that inhibit the proliferation and survival of multiple myeloma cells.

SUMMARY OF INVENTION
[0013] The inventor has now unexpectedly discovered that a glycoprotein

(GP88), which is expressed in a tightly regulated fashion in normal cells, is overexpressed and unregulated in highly tumorigenic cells derived from the normal cells, that GP88 acts as a stringently required growth stimulator and survival factor for the tumorigenic cells and that inhibition of GP88 expression or action in the tumorigenic cells results in an inhibition of the tumorigenic properties of the overproducing cells.

[0014] The inventor has further discovered diat GP88 is overexpressed in hematopoietic malignant cells such as leukemia cells of B-cell lineage (e.g., acute lymphocytic leukemia, B cell lymphoma, and multiple myeloma). GP88 stimulates the tumorigenic properties of hematopoietic malignant cells while inhibition of GP88 expression and biological activity greatly reduces the tumorigenic properties of hematopoietic malignant cells. An embodiment of the invention provides methods of inhibiting the growth or viability of hematopoietic malignant cells . In one embodiment of the invention, a GP88 antagonist inhibits multiple myeloma cell growth. In another embodiment of the invention, a composition for inhibiting the growth or viability of hematopoietic malignant cells comprising a GP88 antagonist (e.g., an anti-GP88 antibody, or anti-GP88 nucleic acid) is provided. In yet another embodiment, a method of diagnosing B-cell leukemia is provided comprising detecting GP88 (e.g., GP88 protein, or nucleic acids encoding GP88) in a tissue sample containing B cells (e.g., tissue suspected of containing myeloma cells including, but not limited to blood, bone marrow, lymph, liver, and spleen) and diagnosing multiple myeloma by determining whether GP88 protein is present in the tissue sample. The presence of GP88 in B cells indicates multiple myeloma.

Alternatively, detecting GP88 in B-cells indicates the presence of leukemia cells of B-cell lineage. Thus, the presence of GP88 serves as a prognostic marker for B-cell leukemia.

[0015] The invention also provides methods for determining whether a patient is responding or responsive to glucocorticoid therapy by comparing the level of GP88 in a tissue sample containing B-cells at a first time with the level of GP88 in a tissue sample containing B-cells at a second time. Increased levels of GP88 in tissue samples over time indicate a patient is not responding or responsive to glucocorticoid therapy.

[0016] This invention provides GP88 antagonizing compositions capable of inhibiting the expression or activity of GP88, methods for treating diseases associated with a defect in GP88 quantity or activity such as but not limited to cancer in a mammal in tissues including, for example, blood, cerebrospinal fluid, serum, plasma, urine, nipple aspirate, liver, kidney, breast, bone, bone marrow, testes, brain, ovary, skin, and lung, methods for determining the susceptibility of a subject to diseases associated with a defect in GP88 expression or action, methods for measuring susceptibility to GP88 antagonizing therapy, and methods, reagents, and kits for the in vitro and in vivo detection of GP88 and tumorigenic activity in cells.

[0017] Additional objects and advantages of the invention will be set forth in part in the description that follows, and in part will be obvious from the description, or may be learned by the practice of the invention.

[0018] To achieve the objects and in accordance with the purpose of the invention, as embodied and properly described herein, the present invention provides compositions for diagnosis and treatment of diseases such as but not limited to multiple myeloma in which cells exhibit an altered expression of GP88 or altered response to GP88.

[0019] Use ofthe term "altered expression" herein means increased expression or overexpression of GP88 by a statistically significant amount as compared to corresponding normal cells or surrounding peripheral cells. The term "altered expression" also means expression which became unregulated or
constitutive without being necessarily elevated. Use of the terms increased or altered "response" to GP88 means a condition wherein increase in any of the biological functions (e.g., growth, diflferentiation, viral infectivity) conferred by GP88 results in the same or equivalent condition as altered expression of GP88.

[0020] Use of the term "GP88 " herein means epithelin/granulin precursor in cell extracts and extracellular fluids, and is intended to include not only GP88 according to the amino acid sequences included in figures 8 or 9, which are of mouse and human origins, but also GP88 of other species. "GP88" does not include epithelin 1 or epithelin 2 peptides as described in U.S. Patent No. 5,416,192 (Shoyab et al.). In addition, the term also includes functional derivatives thereof having additional components such as a carbohydrate moiety including a
glycoprotein or other modified structures.

[0021] Also intended by the term GP88 is any polypeptide fragment having at least 10 amino acids present in the above mentioned sequences.
Sequences of this length are useful as antigens and for making immunogenic conjugates with carriers for the production of antibodies specific for various epitopes of the entire protein. Such polypeptides are useful in screening such antibodies and in the methods directed to detection of GP88 in biological fluids. It is well known in the art that peptides are useful in generation of antibodies to larger proteins (P. Georgii-Hemming, H.J. Wiklund, O. Ljunggren, and K. Nilsson, 1996, Blood 88, no. 6:2250). In one embodiment of this invention, it is shown that peptides from 12-19 amino-acids in length have been successfully used to develop antibodies that recognize full length GP88.

[0022] The polypeptide of this invention may exist covalently or non-covalently bound to another molecule. For example, it may be fused to one or more other polypeptides via one or more peptide bonds such as glutathione transferase, poly-histidine, or myc tag.

[0023] The polypeptide is sufficiendy large to comprise an antigenetically distinct determinant or epitope which can be used as an immunogen to reproduce or test antibodies against GP88 or a functional derivative thereof.

[0024] One embodiment includes the polypeptide substantially free of other mammalian peptides. GP88 of the present invention can be biochemically or immunochemically purified from cells, tissues or a biological fluid. Alternatively, the polypeptide can be produced by recombinant means in a prokaryotic or eukaryotic expression system and host cells.

[0025] "Substantially free of other mammalian polypeptides" reflects the fact that the polypeptide can be synthesized in a prokaryotic or a non-mammalian or mammalian eukaryotic organism, if desired. Alternatively, methods are well known for the synthesis of polypeptides of desired sequences by chemical synthesis on solid phase supports and their subsequent separation from the support. Alternatively, the protein can be purified from tissues or fluids of mammals where it naturally occurs so that it is at least 90% pure (on a weight basis) or even 99% pure, if desired, of other mammalian polypeptides, and is d erefore substantially free from them. This can be achieved by subjecting the tissue extracts or fluids to standard protein purification such as on immunoabsorbants bearing antibodies reactive against the protein. One embodiment of the present invention describes purification methods for die purification of naturally occurring GP88 and of recombinant GP88 expressed in baculovirus infected insect cells. Alternatively, purification from such tissues or fluids can be acliieved by a combination of standard methods such as but not limited to the ones described in (Oken, M.M., 1997, Cancer Investigation 15, no. 1:57).

[0026] As an alternative to a native purified or recombinant glycoprotein or polypeptide, "GP88 " is intended to also include functional derivatives. By functional derivative is meant a "fragment," "variant," "analog," or "chemical derivative" of the protein or glycoprotein as defined below. A functional derivative retains at least a portion of the function of the full length GP88 which permits its utility in accordance with the present invention.

[0027] A "fragment" of GP88 refers to any subset of the molecule that is a shorter peptide that retains the tumorigenic properties of the full-length GP88 protein. This corresponds for example but is not limited to regions such as K19T and S14R for mouse GP88, and E19V and A14R (equivalent to murine K19T and S14R, respectively) for human GP88.

[0028] A "variant" of GP88 refers to a molecule substantially similar to either the entire peptide or a fragment thereof. Variant peptides may be prepared by direct chemical synthesis of the variant peptide using methods known in die art.

[0029] Alternatively, amino acid sequence variants ofthe peptide can be prepared by modifying the DNA which encodes the synthesized protein or peptide. Such variants include, for example, deletions, insertions, or substitutions of residues within the amino-acid sequence of GP88. Any combination of deletion, insertion, and substitution may also be made to arrive at the final construct, provided the final construct possesses the desired activity. The mutation that will be made in the DNA encoding the variant peptide must not alter the reading frame and preferably will not create complementary regions tiiat could produce secondary mRNA structures. At the genetic level these variants are prepared by site directed mutagenesis (G. G. Freund, D.T. Kulas, and R.A. Mooney, 1993, Journal of Immunology 151, no. 4:1811) of nucleotides in die DNA encoding the peptide molecule thereby producing DNA encoding the variant, and thereafter expressing the DNA in recombinant cell culture. The variant typically exhibits the same qualitative biological activity as the nonvariant peptide.

[0030] An "analog" of GP88 protein refers to a non-natural molecule substantially similar to either the entire molecule or a fragment thereof.

[0031] A "chemical derivative" contains additional chemical moieties not normally a part of the peptide or protein. Covalent modifications of the peptide are also included within the scope of this invention. Such modifications may be introduced into die molecule by reacting targeted amino-acid residues of he peptide with an organic derivatizing agent that is capable of reacting with selected side chains or terminal amino-acid residues. Most commonly derivatized residues are cysteinyl, histidyl, lysinyl, arginyl, tyrosyl, glutaminyl, asparaginyl and amino terminal residues. Hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl and threonyl residues, methylation of he alpha-amino groups of lysine, histidine, and histidine side chains, acetylation of the N-terminal amine and amidation of the C-terminal carboxylic groups. Such derivatized moieties may improve the solubility, absorption, biological half life and die like. The moieties may also eliminate or attenuate any undesirable side effect of the protein and the like. In addition, derivatization witii bifunctional agents is useful for cross-linking the peptide to water insoluble support matrices or to other macromolecular
carriers. Commonly used cross-linking agents include glutaraldehyde,
N-hydroxysuccinimide esters, homobifunctional imidoesters,
l,l-bis(-diazoloacetyl)-2-phenylethane, and bifunctional maleimides. Derivatizing agents such as methyl-3-[9p-azidophenyl)]dithiopropioimidate yield
photoactivatable intermediates that are capable of forming crosslinks in the presence of light. Alternatively, reactive water-insoluble matrices such as cyanogen bromide activated carbohydrates and the reactive substrates described in U.S. Patents
3,969,287 and 3,691,016 may be employed for protein immobilization.

[0032] Use of the term GP88 "antagonizing agents" herein means any composition that inhibits or blocks GP88 expression, production or secretion, or any composition that inhibits or blocks the biological activity of GP88. This can be achieved by any mode of action such as but not limited to the following:

[0033] (A) GP88 antagonizing agents include any reagent or molecule inhibiting GP88 expression or production including but not limited to: [0034] ( 1 ) antisense GP88 DNA or RNA molecules that inhibit GP88 expression by inhibiting GP88 translation;

[0035] (2) reagents (hormones, growth factors, small molecules) that inhibit GP88 mRNA and/or protein expression at the transcriptional, translational or post-transaltional levels;

[0036] (3) factors, reagents or hormones that inhibit GP88 secretion;

[0037] (B) GP88 antagonizing agents also include any reagent or molecule that will inliibit GP88 action or biological activity such as but not limited to:

[0038] (1) neutralizing antibodies to GP88 that bind the protein and prevent it from exerting its biological activity;

[0039] (2) antibodies to the GP88 receptor that prevent GP88 from binding to its receptor and from exerting its biological activity;

[0040] (3) competitive inhibitors of GP88 binding to its receptors (e.g., proteins, ribozymes, aptamers, small molecules);

[0041] (4) inhibitors of GP88 signaling pathways (e.g., proteins, ribozymes, aptamers, small molecules).

[0042] Specific examples presented herein provide a description of preferred embodiments, particularly the use of neutralizing antibodies to inhibit GP88 biological action and the growth of multiple myeloma cells; the use of antisense GP88 cDNA and antisense GP88 oligonucleotides to inhibit GP88 expression leading to inhibition of the tumorigenic properties of PC cells;
characterization of GP88 receptors on cell surfaces of several cell lines including the mammary epithelial cell line C57MG, the 1246 and PC cell fines and the mink lung epithelial cell line CCL64.

[0043] In one embodiment of the invention, the GP88 antagonizing agents are antisense oligonucleotides to GP88. The antisense oligonucleotides preferably inhibit GP88 expression by inhibiting translation of the GP88 protein. In another embodiment, the antagonizing agent is RNAi (RNA interference nucleic acids). RNAi are double-stranded RNA molecules that are homologous to the target gene (e.g., GP88) and interfere with the target gene's activity.

[0044] Alternatively, such a composition may comprise reagents, factors or hormones that inhibit GP88 expression by regulating GP88 gene transcriptional activity. Such a composition may comprise reagents, factors or hormones that inhibit GP88 post- translational modification and its secretion. Such a composition may comprise reagents that act as GP88 antagonists that block GP88 activity by competing with GP88 for binding to GP88 cell surface receptors. Alternatively, such a composition may comprise factors or reagents that inhibit the signaling pathway transduced by GP88 once binding to its receptors on diseased cells.

[0045] The composition may also comprise reagents that block GP88 action such as an antibody specific to GP88 that neutralizes its biological activity, or an antibody to the GP88 receptor that blocks its activity.

[0046] The antibodies of the invention (neutralizing and others) are preferably used as a treatment for multiple myeloma or other diseases in cells which exhibit an increased expression of GP88. By the term "neutralizing" it shall be understood that the antibody has the ability to inhibit or block the normal biological activity of GP88, including GP88's ability to stimulate cell proliferation, increase cell survival, or to induce tumor growth in experimental animals and in humans. An effective amount of anti-GP88 antibody is administered to an animal, including humans, by various routes. In an alternative embodiment, the anti-GP88 antibody is used as a diagnostic to detect cells which exhibit an altered (increased) expression of GP88 as occurring in diseases such as but not limited to cancers (e.g., multiple myeloma), and to identify diseased cells whose growth is dependent on GP88 and which will respond to GP88 antagonizing therapy. In yet another embodiment, the anti-GP88 antibody is used to deliver compounds such as cytotoxic factors or antisense oligonucleotides to cells expressing or responsive to GP88. The cytotoxic factors may be attached, linked, or associated with the anti-GP88 antibody.

[0047] The antisense oligonucleotides of the invention are also used as a treatment for cancer in cells which exhibit an increased expression of GP88, such as hematopoietic malignant cells (e.g., B-cell leukemia cells). An effective amount of the antisense oligonucleotide is administered to an animal, including humans, by various routes.

[0048] The present invention also provides a method for determining the susceptibility to diseases associated with a defect in GP88 expression or action which comprises obtaining a sample of biological fluid or tissue and measuring the amount of GP88 in die fluid or tissue or measuring the susceptibility of the cells to respond to GP88. In the case of cancer (e.g., hematopoietic malignancy), the amount of GP88 being proportional to the susceptibility to the cancer.

[0049] The present invention also provides a method for measuring the degree of severity of cancer (e.g., hematopoietic malignancy) which comprises obtaining a sample of biological fluid or tissue and measuring the amount of GP88 in the fluid or tissue sample, the amount of GP88 being proportional to the degree or severity of the cancer. In one embodiment of the invention, the tissue sample is derived from bone, bone marrow, or serum. In another embodiment of the invention, d e presence of GP88 in B cells is detected.

[0050] The present invention also provides a method for measuring susceptibility to GP88 antagonizing therapy which comprises obtaining a sample of the diseased tissue (biopsy) or a tissue suspected of being diseased, maintaining the cells derived from the sample in culture, treating the cells derived from the culture with anti-GP88 neutralizing antibody and determining if the neutralizing antibody inhibits the cell growth. The ability of the antibody to inhibit cell growth is indicative riiat the cells are dependent on GP88 to proliferate and is predictive that GP88 antagonizing therapy will be efficacious. In addition, d e invention provides methods for determining whether a patient is responding or responsive to glucocorticoid therapy by comparing the level of GP88 in a tissue sample taken at a first time with a tissue sample taken at a second time. Increased levels of GP88 in tissue samples containing B-cells indicates the patient is not responding or is not responsive to glucocorticoid therapy.

[0051] The present invention also provides a method for determining the susceptibility to cancer associated with an abnormality in GP88 receptor level or activity which comprises obtaining a sample of tissue and measuring the amount of GP88 receptor protein or mRNA in the tissue or measuring the kinase activity of the receptor in the tissue (GP88 binding to its receptor induces phosphorylation of cellular proteins including the receptor for GP88).

[0052] The present invention also provides a method for targeting GP88 antagonizing reagents to the diseased site by conjugating them to an anti-GP88 antibody or an anti-GP88 receptor antibody.

[0053] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments of d e invention and, together with the description, serve to explain the principles of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS
[0054] FIG. 1A compares the level of expression of GP88 protein in the

1246, 1246-3A and PC cell lines. Cells were cultured in DME-F12 medium supplemented with 2% fetal bovine serum (FBS). Immunoprecipitation and Western blot analysis with anti-K19T antibody measured GP88 expression levels.

[0055] FIG. IB compares the level of GP88 mRNA expression in the

1246, 1246-3A and PC cell lines. mRNA for RPL32 is used as an internal control for equal amounts of RNA loading.

[0056] FIG. 1C compares the expression of GP88 mRNA in 1246 cells

(left panel) and in PC cells (right panel) in serum-free and serum containing medium. The results show d at GP88 expression in 1246 cells is inhibited by the addition of fetal bovine serum whereas such inhibition is not observed in the highly tumorigenic PC cells.

[0057] FIG. 2 illustrates the effect of treatment of the highly tumorigenic

PC cells with increasing concentrations of anti-GP88 neutralizing antibody.

[0058] FIG. 3 shows C3H mice injected subcutaneously with 10° antisense GP88 transfected PC cells (bottom) and with empty vector transfected control PC cells (top).

[0059] FIG. 4 shows in vivo GP88 expression levels in C3H mice tumor tissues and in surrounding normal tissues.

[0060] FIG. 5 shows GP88 mRNA expression levels in estrogen receptor positive and estrogen receptor negative human mammary carcinoma cell lines.

[0061] FIG. 6 shows the effect of increasing concentrations of GP88 on the growth of the mouse mammary epithelial cell line C57.

[0062] FIG. 7 shows the growth properties and tumorigenic ability of PC cells transfected with a cytomegalovirus promoter controlled expression vector containing GP88 in antisense orientation and PC cells transfected with an empty vector.

[0063] FIG. 8 shows the nucleotide and deduced amino-acid sequence of mouse GP88. Peptide regions used as antigens to raise anti-GP88 antibodies K19T and S14R are underlined. The region cloned in the antisense orientation in the pCMV4 mammalian expression vector is indicated between brackets.

[0064] FIG. 9A shows the nucleotide sequence of human GP88 cDNA.

Indicated between brackets is the region cloned in the antisense orientation into the pcDNA3 mammalian expression system; and

[0065] FIG. 9B shows the deduced amino-acid sequence of human GP88.

The E19V region used as antigen to develop anti-human GP88 neutralizing antibody is underlined. It also indicates the region A14R equivalent to the mouse S14R region.

[0066] FIG. 10 shows the amino-acid sequence of mouse GP88 arranged to show the 7 and one-half repeats defined as granulins g, f, B, A, C, D and e (right side). This representation shows that the region K19T and S14R used to raise GP88 antibodies for developing anti-GP88 neutralizing antibodies is found between two epithlin/granulin repeats in what is considered a variant region. Indicated on the right hand side is the granulin classification of the repeats according to Bateman et al (6). Granulin B and granulin A are also defined as epithelin 2 and epithelin 1 respectively according to Plowman et al., 1992 (R. Alexanian, and M. Dimopoulos. 1994, New England Journal of Medicine 330, no. 7:484).

[0067] FIG. 11 shows a schematic representation of pCMV4 and a GP88 cDNA clone indicating die restriction sites used to clone GP88 antisense cDNA into the expression vector.

[0068] FIG. 12 shows the cross-linking of 125I-rGP88 to GP88 cell surface receptors on CCL-64 cells. The cross-linking reaction was carried out wid disuccinimidyl suberate (DSS). Reaction products were analyzed by SDS-PAGE on a 7% polyacrylamide gel.

[0069] FIG. 13 shows the cross-linking of 125I-rGP88 to GP88 cell surface receptors on 3T3 fibroblasts, PC cells and C57MG mammary epithelial cells. The results show that these various cell lines display GP88 cell surface receptors of similar molecular weight as the ones on CCL64 cells (FIG. 12).

[0070] FIG. 14 shows GP88 expression levels in non-tumorigenic MCF

10A and in malignant (MCF 7, MDA-468) human mammary epithelial cells.

[0071] FIG. 15 shows that GP88 expression is inhibited by antisense

GP88 cDNA transfection in human breast carcinoma MDA-468 cells.

[0072] FIG. 16 shows GP88 protein expression in various human hematological cell lines. GP88 is expressed in human multiple myeloma cell lines ARP-1 and RPMI 8226, human B cell lines Raji and Daudi, human macrophage cell line KOPM28, but not in human T cell lines Jurkat and KOPT-K1.

[0073] FIG. 17 shows that GP88 mRNA is expressed in human multiple myeloma cell lines ARP-1 and RPMI 8226.

[0074] FIGS. 18A and 18B show die effect of GP88 protein on the growth of RPMI 8226 cells. FIG. 18A shows that GP88 increases the live cell density of serum starved RPMI 8226 cells while FIG. 18B shows that GP88 increases d e percent viability of serum starved RPMI 8226.

[0075] FIGS. 19A and 19B show the effect of GP88 on the growth of

ARP-1 cells. FIG. 18A shows that GP88 increases the live cell density of serum starved ARP-1 cells while FIG. 18B shows that GP88 increases the percent viability of serum starved ARP-1 cells.

[0076] FIG. 20 shows the effect of anti-GP88 neutralizing antibody on the growth of RPMI 8226 cells. Treatment of RPMI 8226 cells with anti-GP88 antibody inhibited cell growth by 50% compared to cells that did not receive GP88 antibody (control AB) or cells treated with a combination of GP88 and anti-GP88 antibody.

[0077] FIGS. 21A and 21B show the effect of GP88 and PD98059 (a

MEK inhibitor) on cell growth and survival. GP88 increased bod live cell density (FIG. 21 A) and percent survival (FIG. 21B) in ARP-1 cells. These results show that GP88 activates the MAPK pathway in ARP-1 cells and that MAPK stimulates GP88-induced cell growth.

[0078] FIG. 22 shows that phosphorylation of Erkl and Erk2 d rough the

MAPK pathway is blocked by MEK inhibitor PD98059 in ARP-1 cells. These results show that GP88 activates the MAPK pathway in ARP-1 cells and that MAPK stimulates GP88-induced cell growth.

[0079] FIG. 23 shows that GP88 stimulated phosphorylation of Akl in

ARP-1 cells is blocked by PI3 K inhibitor LY294002. The results show d at GP88 activates the PI3 kinase signal pad way in ARP-1 cells.

[0080] FIG. 24 shows that GP88 does not induce phosphorylation of

STAT3. The results show that GP88 does not activate the JAK/STAT3 signal pathway in human multiple myeloma cells.

[0081] FIGS. 25A, 25B, and 25C show the results of triple-stained bone marrow smears from multiple myeloma patients. The bone marrow smears were stained for the presence of GP88 and for markers of he kappa and lambda light chains. The bone marrow smears were stained with DAPI (FIG. 25A), anti-human kappa/lambda chain antibody (FIG. 25B), and anti-GP88 antibody (25C).

[0082] FIG. 26 shows that the effect of dexamethasone on the expression of GP88 mRNA in multiple myeloma (ARP-1) cells. Dexamethasone significantiy inhibits d e expression of GP88 mRNA in ARP-1 cells.

[0083] FIGS. 27A and 27B shows the effects of GP88 on die cell growth

(27A) and viability (27B) of dexamethasone-treated ARP-1 cells. Dexamethasone decreases d e cell growth and viability of ARP-1 cells. GP88 partially reverses the negative effects of dexamethasone on the cell growth and viability of ARP-1 cells.

[0084] FIG. 28 shows the effect of GP88 on PARP cleavage in
dexamediasone -treated ARP-1 cells. GP88 significantiy reduces PARP cleavage at 24 and 48 hours following treatment with dexamethasone.

[0085] FIG. 29 shows GP88 protein expression in ARP-1 cells transfected with GP88 nucleic acid (lane 2) and an empty vector that does not contain GP88 nucleic acid (lane 1). GP88 is overexpressed in ARP-1 cells transfected with GP88 nucleic acid.

[0086] FIGS. 30A and 30B shows the effect of dexamethasone on the cell growth (30A) and viability (30B) of ARP-1 cells transfected with GP88 nucleic acid and ARP-1 cells transfected with an empty vector. The decrease in cell growth and viability of ARP-1 cells treated with dexamethasone is significantiy reduced in cells transfected with GP88 nucleic acid.

[0087] FIG. 31 shows the effect of dexamethasone on PARP cleavage in

ARP-1 cells overexpressing GP88. ARP-1 cells overexpressing GP88 have significantiy reduced levels of PARP cleavage.

DETAILED DESCRIPTION OF THE INVENTION
[0088] Reference will now be made in detail to the presendy preferred embodiments ofthe invention, which, together with the following examples, serve to explain the principles of the invention.

Biological Activity of GP88
[0089] The invention relates to GP88 and antitumor compositions useful for treating and diagnosing diseases linked to altered (increased) expression of GP88 (e.g., multiple myeloma). Alternatively this invention is used for treating and diagnosing diseases linked to increased responsiveness to GP88. Using a murine model system consisting of three cell lines, the inventor has shown that cells which overexpress GP88 form tumors. The parent cell line, 1246, is a C3H mouse adipogenic cell line which profiferates and differentiates into adipocytes in a defined medium under stringent regulation by insulin. The 1246 cells cannot form tumors in a syngeneic animal (C3H mouse) even when injected at a high cell density. An insulin independent cell line, 1246-3A, was isolated from 1246 cells maintained in insulin-free medium. The 1246-3A cells lost the ability to differentiate and form tumors when 106 are injected subcutaneously in syngeneic mice. A highly tumorigenic cell line, PC, was developed from 1246-3A cells by an in vitro-in vivo shuttle technique. The PC cells formed tumors when 104 cells were injected into syngeneic mice.

[0090] GP88 is overexpressed in the insulin-independent tumorigenic cell lines relative to the parent non-tumorigenic insulin-dependent cell line. Moreover, the degree of overexpression of GP88 positively correlates with the degree of tumorigenicity of these cells, demonstrating for the first time that GP88 is important in tumorigenesis (FIG. 1). With reference to FIG. 1, since GP88 is synthesized by cells but also secreted in culture medium, the level of GP88 was determined in cell lysates and in culture medium (CM). All cells were cultivated in DME/F12 nutrient medium supplemented with 2% fetal bovine serum. When cells reached confluency, culture medium (CM) was collected and cell lysates were prepared by incubation in buffer containing detergent followed by a 10,000 x g centrifugation. Cell lysate and conditioned medium were normalized by cell number. Samples from cell lysate and conditioned medium were analyzed by Western blot analysis using an anti-GP88 antibody, as explained below.

[0091] The development of a neutralizing antibody confirmed GP88's key role in tumorigenesis. When an anti-GP88 antibody directed to the K19T region of mouse GP88 was added to the culture medium, the growth of highly tumorigenic PC cells was inhibited in a dose dependent fashion (FIG. 2). With reference to FIG. 2, PC cells were cultivated in 96 well plates at a density 2 x 104 cells/well in
DME/F12 medium supplemented with human fibronectin (2 μg/ml) and human transferrin (10 μg/ml). Increasing concentrations of anti-GP88 IgG fraction were added to the wells after die cells were attached. Control cells were treated with equivalent concentrations of non-immune IgG. Two days later, 0.25 mCi of 3H-thymidine was added per well for 6 hrs. Cells were then harvested to count 3H-thymidine incorporated into DNA as a measure for cell proliferation.

[0092] Moreover, when the expression of GP88 was specifically inhibited by antisense GP88 cDNA in PC cells, the production of GP88 was reduced and these PC cells could no longer form tumors in syngeneic C3H mouse. In addition, these PC cells regained responsiveness to insulin. With reference to FIG. 3 and Tables 1 and 2, C3H female mice were injected subcutaneously with 106 antisense GP88 transfected PC cells (as explained below) or 10 ' empty vector transfected PC cells. Mice were monitored daily for tumor appearance. Photographs were taken 45 days after injection of die cells. The results show that mice injected widi antisense GP88 PC cells do not develop tumors, in contrast to the mice injected with empty vector transfected PC cells used as control.

Table 1. COMPARISON OF TUMORIGENIC PROPERTIES OF GP88 ANTISENSE TRANSFECTED CELLS, CONTROL TRANSFECTED
CELLS AND PC CELLS

CELLS AVERAGE NUMBER OF AVERAGE INJECTED DAY OF MICE WITH TUMOR TUMOR TUMORS WEIGHT (g)
DETECTION

PC 15±3.0 5/5 9.0±3.2

P14 15±3.7 5/5 7.8±2.7

ASGP88 0/5

PC: Control non- transfected cells
P-14: Empty vector control transfected PC cells
ASGP88: PC cells transfected with expression vector containing GP88
antisense cDNA
Tumors were excised and weighed at 45 days. -- indicates no tumor formation.

Table 2. COMPARISON OF PROPERTIES OF 1246, PC CELLS AND GP88 ANTISENSE CELLS

insulin GP88 antisense

independence transfection

1246 cells PC cells Antisense GP 88 cells

insulin responsive for insulin-independent for recovery of insulin
growth and growth differentiation responsiveness for growth differentiation deficient (differentiation?)
autocrine production of
insulin-related factor

cell surface insulin cell surface insulin cell surface insulin receptor receptor expression receptor expression very expression elevated
high low

GP88 expression low GP88 expression GP88 expression inhibited
constitutively high by antisense
GP88 expression No inhibition by serum
inhibited by serum
GP88 expression GP88 expression recovery of insulin
regulated by insulin constitutive regulation for endogenous
GP88 expression
non-tumoπgenic highly tumorigenic non-tumorigenic

[0093] Comparison of the expression of GP88 indicates that in vivo GP88 levels in tumors is dramatically higher than in normal tissues (FIG. 4). C3H mice were injected with 106 PC cells. Tumor bearing mice were euthanized. Tumors, fat pads and connective tissue were collected. Cell lysates were prepared by incubation in buffer containing detergent as described above for FIG. 1. Protein concentration of tissue extracts was determined, and equivalent amounts of proteins for each sample were analyzed by SDS-PAGE followed by Western blot analysis using anti-GP88 antibody to measure the content of GP88 in tissue extracts. The results showed that the level of GP88 in tumor extracts is at least 10-fold higher than in surrounding connective and fat tissues.

[0094] In normal cells (1246 cells, fibroblasts), the expression of GP88 is regulated, in particular by insulin, and inhibited by fetal bovine serum. In
tumorigenic cells, a loss of regulation of normal growth leads to the increased expression of GP88 and the acquisition of GP88 dependence for growth. Therefore, inhibition of GP88 expression and/or action is an effective approach to suppression of tumorigenesis. Detection of an elevated GP88 expression in biopsies provides diagnostic analysis of tumors that are responsive to GP88 inhibition therapy.

[0095] GP88 is also a tumor-inducing factor in human cancers. As seen in the 1246-3A cell line, a loss of responsiveness to insulin (or to IGF-I) and a concurrent increase in malignancy has been well documented in several human cancers including but not limited to breast cancers. Specifically, breast carcinoma is accompanied by the acquisition of an insulin/IGF-I autocrine loop, which is also the starting point of the development of tumorigenic properties in the mouse model system discussed above. Furthermore, GP88 expression is elevated in human breast carcinomas. More specifically, wid reference to FIG. 5, human GP88 was highly expressed in estrogen receptor positive and also in estrogen receptor negative insulin/IGF-I independent highly malignant cells. Also, GP88 is a potent growth factor for mammary epithelial cells (FIG. 6). The data in FIG. 5 was obtained by cultivating MCF7, MDA-MB-453 and MDA-MB-468 cells in DME/F12 medium supplemented with 10% fetal bovine serum (FBS). RNA was extracted from each cell line by the RNAzol method and poly-A+ RNA prepared. GP88 mRNA expression was examined by Northern blot analysis with 3 μg of poly- A+ RNA for each cell line using a 2P-labeled GP88 cDNA probe.

[0096] For Northern blot analysis of GP88 mRNA expression in rodent cells or tissues (mouse and rats), we used a mouse GP88 cDNA probe 311 bp in length starting at nucleotide 551 to 862 (corresponding to amino-acid sequence 160 to 270). RNA can be extracted by a variety of methods (Sambrook , Molecular Biology manual: 35) well known to people of ordinary skill in the art. The method of choice was to extract RNA using RNAzol (Cinnabiotech) or Trizol (Gibco-BRL) solutions which consists of a single step extraction by guanidinium isothiocyanate and phenol -chloroform.

[0097] For Northern blot analysis of GP88 mRNA expression in human cell lines, a 672 bp human GP88 cDNA probe was developed corresponding to nucleotide 1002 to 1674 (corresponding to amino-acid sequence 334-558) of human GP88. See example 8 for a detailed and specific description of the Northern blot analysis method used in the preferred embodiments.

[0098] With respect to FIG. 6, C57MG cells were cultivated in the presence of increasing concentrations of GP88 purified from PC cells conditioned medium (top panel), and recombinant GP88 expressed in insect cells (bottom panel), to demonstrate the growth stimulating effect of increasing concentrations of GP88 on the growth ofthe mouse mammary epithelial cell line C57MG.

[0099] A correlation between FIG-1 autocrine production and increased malignancy has also been well established for glioblastomas, teratocarcinomas and breast carcinomas. In these cancers, GP88 expression is also elevated in human tumors when compared to non-tumorigenic human fibroblasts and other human cell lines. GP88 promotes the growth of mammary carcinoma cells.

Anti-GP88 Antibodies
[00100] The invention provides compositions for treating and diagnosing diseases linked to increased expression of GP88. This also will apply to treatment and diagnosis of diseases linked to increased responsiveness to GP88. The compositions of this invention include anti-GP88 antibodies which neutralize the biological activity of GP88.

[00101] The present invention is also directed to an antibody specific for an epitope of GP88 and the use of such antibody to detect the presence or measure the quantity or concentration of GP88 molecule, a functional derivative thereof or a homologue from different animal species in a cell, a cell or tissue extract, culture medium or biological fluid. Moreover, anti-GP88 antibody can be used to target cytotoxic molecules to a specific site.

[00102] For use as antigen for development of antibodies, the GP88 protein naturally produced or expressed in recombinant form or functional derivative thereof, preferably having at least 9 amino-acids, is obtained and used to immunize an animal for production of polyclonal or monoclonal antibody. An antibody is said to be capable of binding a molecule if it is capable of reacting with the molecule to thereby bind the molecule to the antibody. The specific reaction is meant to indicate that the antigen will react in a highly selective manner with its
corresponding antibody and not with the multitude of other antibodies which may be evoked by other antigens.

[00103] The term antibody herein includes but is not limited to human and non-human polyclonal antibodies, human and non-human monoclonal antibodies (mAbs), chimeric antibodies, anti-idiotypic antibodies (anti-IdAb) and humanized antibodies. Polyclonal antibodies are heterogeneous populations of antibody molecules derived either from sera of animals immunized with an antigen or from chicken eggs. Monoclonal antibodies ( "mAbs" ) are substantially homogeneous populations of antibodies to specific antigens. mAbs may be obtained by methods known to those skilled in the art (U.S. Patent No. 4,376,110). Such antibodies may be of any immunological class including IgG, IgM, IgE, IgA, IgD and any subclass thereof. The hybridoma producing human and non-human antibodies to GP88 may be cultivated in vitro or in vivo. For production of a large amount of mAbs, in vivo is the presently preferred method of production. Briefly, cells from the individual hybridomas are injected intraperitoneally into pristane primed Balb/c mice or Nude mice to produce ascites fluid containing high concentrations of the desired mAbs. mAbs may be purified from such ascites fluids or from culture supernatants using standard chromatography methods well known to those of skill in the art.

[00104] Human monoclonal Ab to human GP88 can be prepared by immunizing transgenic mice expressing human immunoglobufin genes. Hybridoma produced by using lymphocytes from these transgenic animals will produce human immunoglobufin instead of mouse immunoglobufin.

[00105] Since most monoclonal antibodies are derived from murine source and other non-human sources, their clinical efficiency may be limited due to the immunogenicity of rodent mAbs administered to humans, weak recruitment of effector function and rapid clearance from serum. To circumvent these problems, the antigen- binding properties of murine antibodies can be conferred to human antibodies through a process called humanization. A humanized antibody contains the amino-acid sequences for the 6 complementarity-determining regions (CDRs) of the parent murine mAb which are grafted onto a human antibody framework. The low content of non-human sequences in humanized antibodies (around 5%) has proven effective in both reducing the immunogenicity and prolonging the serum half life in humans. Methods such as the ones using monovalent phage display and combinatorial library strategy for humanization of monoclonal antibodies are now widely applied to the humanization of a variety of antibodies and are known to people skilled in the art. These humanized antibodies and human antibodies developed with transgenic animals as described above are of great therapeutic use for several diseases including but not limited to cancer.

[00106] Hybridoma supernatants and sera are screened for the presence of antibody specific for GP88 by any number of immunoassays including dot blots and standard immunoassays (EIA or ELISA) which are well known in the art. Once a supernatant has been identified as having an antibody of interest, it may be further screened by Western blotting to identify the size of the antigen to which the antibody binds. One of ordinary skill in the art will know how to prepare and screen such hybridomas without undue experimentation in order to obtain a desired polyclonal or mAb.

[00107] Chimeric antibodies have different portions derived from different animal species. For example, a chimeric antibody might have a variable region from a murine mAb and a human immunoglobufin constant region. Chimeric antibodies and methods for their production are also known to those skilled in the art.

[00108] Accordingly, mAbs generated against GP88 may be used to induce human and non-human anti-IdAbs in suitable animals. Spleen cells from such immunized mice are used to produce hybridomas secreting human or non-human anti-Id mAbs. Further, the anti-Id mAbs can be coupled to a carrier such as Keyhole Limpet Hemocyanin (KLH) or bovine serum albumin (BSA) and used to immunize additional mice. Sera from these mice will contain human or non-human anti-anti-IdAb that have the binding properties ofthe original mAb specific for a GP88 polypeptide epitope. The anti-Id mAbs thus have their own idiotypic epitopes or idiotypes structurally similar to the epitope being evaluated.

[00109] The term antibody is also meant to include both intact molecules as well as fragments thereof such as, for example, Fab and F(ab')2, which are capable of binding to the antigen. Fab and F(ab')2 fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation and may have less non-specific tissue binding than an intact antibody. Such fragments are typically produced by proteolytic cleavage, using enzymes such as papain (to generate Fab fragments) and pepsin (to generate F(ab')2 fragments). It will be appreciated that Fab and F(ab')2 and other fragments of the antibodies useful in the present invention may be used for the detection or quantitation of GP88, and for treatment of pathological states related to GP88 expression, according to the methods disclosed herein for intact antibody molecules.

[00110] According to the present invention, antibodies that neutralize

GP88 activity in vitro can be used to neutralize GP88 activity in vivo to treat diseases associated with increased GP88 expression or increased responsiveness to GP88, such as but not limited to multiple myeloma. A subject, preferably a human subject, suffering from multiple myeloma or other disease associated with increased GP88 expression is treated with an antibody to GP88. Such treatment may be performed in conjunction with other anti-cancer or anti-viral therapy. A typical regimen comprises administration of an effective amount of the antibody specific for GP88 administered over a period of one or several weeks and including between about one and six months. The antibody of the present invention may be administered by any means that achieves its intended purpose. For example, administration may be by various routes including but not limited to subcutaneous, intravenous, intradermal, intramuscular, intraperitoneal and oral. Parenteral administration can be by bolus injection or by gradual perfusion over time.
Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions and emulsions, which may contain auxiliary agents or excipients known in the art. Pharmaceutical compositions such as tablets and capsules can also be prepared according to routine methods. It is understood that the dosage of will be dependent upon the age, sex and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment and die nature of the effect desired. The ranges of effective doses provided below are not intended to limit the invention and merely represent preferred dose ranges. However the most preferred dosage will be tailored to die individual subject as is understood and determinable by one skilled in the art. The total dose required for each treatment may be administered by multiple doses or in a single dose. Effective amounts of antibody are from about O.Olμg to about 100 mg/kg body weight and preferably from about 10 μg to about 50 mg/kg. Antibody may be administered alone or in conjunction wid other therapeutics directed to the same disease.

[00111 ] According to the present invention and concerning the
neutralizing antibody, GP88 neutralizing antibodies can be used in all therapeutic cases where it is necessary to inhibit GP88 biological activity, even though there may not necessarily be a change in GP88 expression, including cases where there is an overexpression of GP88 cell surface receptors and this in turn results in an increased biological activity, or where there is an alteration in GP88 signaling pathways or receptors leading to the fact that the signaling pathways are always "turned on. " In one embodiment, d e GP88 neutralizing antibodies are used to inhibit the growth of multiple myeloma cells. Neutralizing antibodies to growth factor and to growth factor receptors have been successfully used to inhibit the growth of cells whose proliferation is dependent on this growth factor. This has been the case for IGF-I receptor in human breast carcinoma cells and bombesin for lung cancer. The antibody to GP88 can also be used to deliver compounds such as, but not limited to, cytotoxic reagents such as toxins, oncotoxins, mitotoxins and immunotoxins, or antisense oligonucleotides, in order to specifically target them to cells expressing or responsive to GP88.

[00112] One region that allows antigen to develop a neutralizing antibody to GP88 is the 19 amino-acid region defined as K19T in the mouse GP88, and E19V in the human GP88 which is not located within the epithelin/granulin 6 kDa repeats but between these repeats, specifically between granulin A (epithelin 1) and granulin C in what is considered a variant region (see Figure 10). Without wishing to be bound by theory, it is believed that die region important for the biological activity of GP88 lies outside of the epithelin repeats.

[00113] The antibodies or fragments of antibodies useful in the present invention may also be used to quantitatively or qualitatively detect the presence of cells which express the GP88 protein. This can be accomplished by
immunofluorescence techniques employing a fluorescendy labeled antibody (see below) with fluorescent microscopic, flow cytometric, or fluorometric detection. The reaction of antibodies and polypeptides of the present invention may be detected by immunoassay methods well known in the art.

[00114] The antibodies of the present invention may be employed histologically as in light microscopy, immunofluorescence or immunoelectron microscopy, for in situ detection of the GP88 protein in tissues samples or biopsies. In situ detection may be accomplished by removing a histological specimen from a patient and applying the appropriately labeled antibody of the present invention. The antibody (or fragment) is preferably provided by applying or overlaying the labeled antibody (or fragment) to the biological sample. Through the use of such a procedure, it is possible to determine not only the presence of the GP88 protein but also its distribution in the examined tissue. Using the present invention, those of ordinary skill in the art will readily perceive that any wide variety of histological methods (such as staining procedures) can be modified in order to achieve such in situ detection.

[00115] Assays for GP88 typically comprise incubating a biological sample such as a biological fluid, a tissue extract, freshly harvested or cultured cells or their culture medium in the presence of a detectably labeled antibody capable of identifying the GP88 protein and detecting the antibody by any of a number of techniques well known in die art.

[00116] The biological sample may be treated with a solid phase support or carrier such as nitrocellulose or other solid support capable of immobilizing cells or ceU particles or soluble proteins. The support may then be washed followed by treatment with the detectably labeled anti-GP88 antibody. This is foUowed by wash of the support to remove unbound antibody. The amount of bound label on said support may then be detected by conventional means. By solid phase support is intended any support capable of binding antigen or antibodies such as but not limited to glass, polystyrene polypropylene, nylon, modified cellulose, or
polyacrylamide.

[00117] The binding activity ofa given lot of antibody to the GP88 protein may be determined according to weU known methods. Those skilled in the art will be able to determine operative and optimal assay conditions for each determination by employing routine experimentation.

[00118] Detection of the GP88 protein or functional derivative thereof and of a specific antibody for the protein may be accomplished by a variety of
immunoassays well known in the art such as enzyme linked immunoassays (EIA) or radioimmunoassays (RIA). Such assays are well known in the art and one of skill wiU readUy know how to carry out such assays using the anti-GP88 antibodies and GP88 protein of the present invention.

[00119] Such immunoassays are useful to detect and quantitate GP88 protein in serum or odier biological fluid as well as in tissues, cells, cell extracts, or biopsies. In a preferred embodiment, the concentration of GP88 is measured in a tissue specimen as a means for diagnosing cancer or other disease associated with increased expression of GP88.

[00120] The presence of certain types of cancers (e.g., multiple myeloma) and the degree of malignancy are said to be "proportional" to an increase in the level of the GP88 protein. The term "proportional" as used herein is not intended to be limited to a linear or constant relationship between the level of protein and the malignant properties of the cancer. The term "proportional" as used herein, is intended to indicate that an increased level of GP88 protein is related to appearance, recurrence or display of malignant properties of a cancer or other disease associated with increased expression of GP88 at ranges of concentration of the protein that can be readily determined by one skilled in the art.

[00121] Another embodiment of the invention relates to evaluating the efficacy of anti-cancer or anti-viral drug or agent by measuring the ability of the drug or agent to inhibit the expression or production of GP88. The antibodies of the present invention are useful in a method for evaluating anti-cancer or anti-viral drugs in that they can be employed to determine the amount of the GP88 protein in one of the above-mentioned immunoassays. Alternatively, the amount ofthe GP88 protein produced is measured by bioassay (cell proliferation assay ) as described herein. The bioassay and immunoassay can be used in combination for a more precise assessment.

[00122] An additional embodiment is directed to an assay for diagnosing cancers or other diseases associated with an increase in GP88 expression based on measuring in a tissue or biological fluid the amount of mRNA sequences present that encode GP88 or a functional derivative thereof, preferably using an RNA-DNA hybridization assay. The presence of certain cancers and the degree of malignancy is proportional to the amount of such mRNA present. For such assays the source of mRNA will be biopsies and surrounding tissues. The preferred technique for measuring the amount of mRNA is a hybridization assay using DNA of
complementarity base sequence.

[00123] Another related embodiment is directed to an assay for diagnosing cancers or other diseases associated with an increase in GP88 responsiveness based on measuring on a tissue biopsy whether treatment with anti-GP88 neutralizing antibody will inhibit its growth or other biological activity.

[00124] Another related embodiment is a method for measuring the efficacy of anti-cancer or anti-viral drug or agent which comprises the steps of measuring the agent's effect on inhibiting the expression of mRNA for GP88.
Sirmlarly such method can be used to identify or evaluate the efficacy of GP88 antagonizing agents by measuring the ability of said agent to inhibit the production of GP88 mRNA.

[00125] Nucleic acid detection assays, especially hybridization assays, can be based on any characteristic of the nucleic acid molecule such as its size, sequence, or susceptibility to digestion by restriction endonucleases. The sensitivity of such assays can be increased by altering d e manner in which detection is reported or signaled to the observer. A wide variety of labels have been extensively developed and used by those of ordinary skill in the art, including enzymatic, radioisotopic, fluorescent, chemical labels and modified bases.

[00126] One method for overcoming the sensitivity limitation of a nucleic acid for detection is to selectively amplify the nucleic acid prior to performing the assay. This method has been referred as the "polymerase chain reaction" or PCR (U.S. Pat. Nos. 4,683,202 and 4,582,788). The PCR reaction provides a method for selectively increasing the concentration of a particular nucleic acid sequence even when that sequence has not been previously purified and is present only in a single copy in a particular sample.

GP88 Antisense Components

[00127] This invention also provides GP88 antisense components. The constitutive expression of antisense RNA in ceUs has been shown to inhibit the expression of more than 20 genes and the list continues to grow. Possible mechanisms for antisense effects are the blockage of translation or prevention of splicing, both of which have been observed in vitro. Interference with splicing allows the use of intron sequences which should be less conserved and therefore result in greater specificity, inhibiting expression of a gene product of one species but not its homologue in another species. Alternatively, nucleic acid sequences which inhibit or interfere with gene expression (e.g., RNAi, ribozymes, aptamers) can be used to inhibit or interfere with the activity of RNA or DNA encoding GP88.

[00128] The term antisense component corresponds to an RNA sequence as well as a DNA sequence coding therefor, which is sufficiently complementary to a particular mRNA molecule, for which the antisense RNA is specific, to cause molecular hybridization between the antisense RNA and die mRNA such that translation of the mRNA is inhibited. Such hybridization can occur under in vivo conditions. The action of the antisense RNA results in specific inhibition of gene expression in the cells.

[00129] According to the present invention, transfection of B-cell leukemia cells with DNA antisense to the GP88 cDNA inhibits endogenous GP88 expression and inhibits tumorigenicity of the antisense cDNA transfected ceUs. This antisense DNA must have sufficient complementarity, about 18-30 nucleotides in length, to the GP88 gene so that the antisense RNA can hybridize to the GP88 gene (or mRNA) and inhibit GP88 gene expression regardless of whether the action is at the level of splicing, transcription, or translation. The degree of inhibition is readily discernible to one skiUed in the art without undue experimentation given the teachings herein and preferably is sufficient to inhibit the growth of cells whose proliferation is dependent on the expression of GP88. One of ordinary skill in the art will recognize that the antisense RNA approach is but a number of known mechanisms which can be employed to block specific gene expression.

[00130] The antisense components of the present invention may be hybridizable to any of several portions of the target GP88 cDNA, including the coding sequence, 3' or 5' untranslated regions, or other intronic sequences, or to GP88 mRNA. As is readily discernible by one of ordinary skill in the art, the minimal amount of homology required by the present invention is that sufficient to result in hybridization to the GP88 DNA or mRNA and in inhibition of
transcription of the DNA, or translation or function of the mRNA, preferably without affecting the function of other mRNA molecules and the expression of other unrelated genes.

[00131] Antisense RNA is delivered to a cell by transformation or transfection via a vector, including retroviral vectors and plasmids, into which has been placed DNA encoding the antisense RNA with the appropriate regulatory sequences including a promoter to result in expression of the antisense RNA in a host ceU. Stable transfection of various antisense expression vectors containing GP88 cDNA fragments in the antisense orientation have been performed. One can also deliver antisense components to cells using a retroviral vector. Delivery can also be achieved by liposomes.

[00132] For purpose of antisense technology for in vivo therapy, the currently preferred method is to use antisense oligonucleotides, instead of performing stable transfection of an antisense cDNA fragment constructed into an expression vector. Antisense oligonucleotides having a size of 15-30 bases in length and with sequences hybridizable to any of several portions of the target GP88 cDNA, including the coding sequence, 3' or 5' untranslated regions, or other intronic sequences, or to GP88 mRNA, are preferred. Sequences for the antisense oligonucleotides to GP88 are preferably selected as being the ones that have the most potent antisense effects. Factors that govern a target site for the antisense oligonucleotide sequence are related to the length of the oligonucleotide, binding affinity, and accessibility ofthe target sequence. Sequences may be screened in vitro for potency of their antisense activity by measuring inhibition of GP88 protein translation and GP88 related phenotype, e.g., inhibition of cell proliferation in cells in culture. In general it is known that most regions of the RNA (51 and 3' untranslated regions, AUG initiation, coding, splice junctions and introns) can be targeted using antisense oligonucleotides.

[00133] The preferred GP88 antisense oligonucleotides are those oligonucleotides which are stable, have a high resilience to nucleases (enzymes that could potentially degrade oligonucleotides), possess suitable pharmacokinetics to allow them to traffic to disease tissue at non-toxic doses, and have the ability to cross through plasma membranes.

[00134] Phosphorothioate antisense oligonucleotides may be used.
Modifications of the phosphodiester linkage as well as of the heterocycle or the sugar may provide an increase in efficiency. With respect to modification of the
phosphodiester linkage, phophorothioate may be used. An N3'-P5'
phosphoramidate linkage has been described as stabilizing oligonucleotides to nucleases and increasing the binding to RNA. Peptide nucleic acid (PNA) linkage is a complete replacement of the ribose and phosphodiester backbone and is stable to nucleases, increases the binding affinity to RNA, and does not allow cleavage by RNAse H. Its basic structure is also amenable to modifications that may allow its optimization as an antisense component. With respect to modifications of the heterocycle, certain heterocycle modifications have proven to augment antisense effects without interfering with RNAse H activity. An example of such modification is C-5 thiazole modification. FinaUy, modification of the sugar may also be considered. 2'-O-propyl and 2'-methoxyethoxy ribose modifications stabilize oligonucleotides to nucleases in ceU culture and in vivo. Cell culture and in vivo tumor experiments using these types of oligonucleotides targeted to c-raf-1 resulted in enhanced potency.

[00135] The delivery route will be the one that provides the best antisense effect as measured according to the criteria described above. In vitro cell culture assays and in vivo tumor growth assays using antisense oligonucleotides have shown that delivery mediated by cationic liposomes, by retroviral vectors and direct delivery are efficient. Another possible delivery mode is targeting using antibody to cell surface markers for the tumor cells. Antibody to GP88 or to its receptor may serve this purpose.

Inhibiting The Growth of Hematopoietic Malignant Cells
[00136] Preferred embodiments ofthe invention are directed to methods and compositions for reducing, interfering with, and/or inhibiting the growth and proliferation of hematopoietic malignant ceUs. Hematopoietic cells are divided into three categories: erythroid, myeloid and lymphoid cells. The erythroid ceUs are red blood cells and their precursors. Myeloid cells include monocytes, granulocytes, basophils, eosinophUs and megakaryocytes. Myeloma is a type of cancer originating from myeloid ceUs (monocytes). Hematopoietic malignant ceUs include, but are not limited to leukemias (e.g., ALL (Acute lymphoblastic leukemia), AML (acute myelogenous leukemia), CML (chronic myelogenous leukemia), acute bilineage leukemia, acute undifferentiated leukemia, chronic lymphocytic leukemia, juvenile chronic myelogenous leukemia, prolymphocytic leukemia, MDS (myelodysplastic syndromes), acquired idiopathic sideroblastic anemia, acute myelofibrosis, chronic myelomonocytic leukemia, essential thrombocythemia, myelodysplastic disorders, myelofibrosis myeloid metaplasia, paroxysmal nocturnal hemoglobinuria,
polycythemia vera, refractory anemia, refractory anemia with excess blasts (RAEB), refractory anemia with excess blasts in transformation (RAEB-T)), and lymphomas (e.g., Hodgkin lymphoma, non-Hodgkin lymphoma, plasma cell dyscrasia, multiple myeloma, plasma cell leukemia, waldenstrom macroglobulinemia).

[00137] As described above, hematopoietic malignant ceUs express elevated levels of GP88. The present invention demonstrates that GP88 is the first growth factor shown to be a prognostic indicator of hematopoietic malignancies (e.g., B-cell leukemias such as multiple myeloma), and that GP88 antagonists reduce, inhibit, and/or interfere with the growth of hematopoietic malignant cells.

[00138] As shown in FIGS. 16 and 17, GP88 protein (FIG. 16) and mRNA (FIG. 17) is overexpressed in human multiple myeloma ceU lines ARP-1 and RPMI 8226 and human B cell lines Raji and Daudi and not expressed in human T-cell lines Jurkat and KOPTI-K1. In addition, GP88 stimulates growth and increases the percent survival of multiple myeloma ceUs. The live ceU density (i.e., growth) and viability of RPMI 8226 multiple myeloma cells increased in a dose dependant manner in response to increased amounts of GP88 (FIGS. 18A and 18B). As shown in FIG. 18A, the live cell density of RPMI 8226 cells increased by 3-fold in the presence of 200 ng/ml of GP88. Likewise, the percent survival of RPMI 8226 ceUs increased by 2-fold after 48 hours in the presence of 100 or 200 ng/ml of GP88. The growth and viability response of RPMI 8226 myeloma ceUs to GP88 is similar to that of myeloma cells to IL-6 (compare FIG. 18A col. 3 to col. 4 and FIG. 18B col. 3 to col. 4). SimUar results were obtained witii ARP-1 multiple myeloma cells. The live cell density of ARP-1 ceUs more than doubled in the presence of 200 ng/ml of GP88. (FIG. 19A). The percent survival of ARP-1 multiple myeloma ceUs doubled after 48 hours in the presence of 200 ng/ml of GP88. IL-6 also doubled both the live cell density and percent survival after 48 hours of ARP-1 cells. (FIGS. 19A and 19B). Reducing, inhibiting, or interfering with the growth stimulatory and survival effects of GP88 on myeloma cells reduces the growth and survival of multiple myeloma cells, providing a therapeutic benefit to multiple myeloma patients.

[00139] In one embodiment of the invention, a method of inhibiting, reducing, or interfering with the growth of hematopoietic malignant cells (e.g., B-cell leukemias ceUs such as myeloma ceUs) by contacting hematopoietic malignant cells witii a GP88 antagonist is provided. As described above, GP88 antagonists (e.g., anti-GP88 antibodies) inhibit the growth of myeloma cells. In another embodiment of the invention, the hematopoietic malignant ceUs are human myeloma cells.

[00140] GP88 antagonists (e.g., anti-GP88 antibodies or antibody fragments, and GP88 smaU molecules) bind to GP88 secreted from the ceU and inhibit and/or interfere with the biological activity of GP88. GP88 antagonists can, for example, bind to GP88 and prevent GP88 from binding to its receptor on the ceU surface. GP88 antagonists (e.g., anti-GP88 antisense polynucleotides) can also enter the ceU and inhibit or interfere with the expression of the GP88 protein. For example, anti-GP88 antisense polynucleotides can hybridize with mRNA encoding GP88 and block translation of the GP88 protein. Alternatively, the GP88 antagonist may be conjugated or linked to another molecule capable of interfering or inhibiting cell growth (e.g., toxins, antibodies, antibody fragments, and nucleic acids). GP88 antagonists also can interfere with the biological activity of GP88 by binding to a molecule other than GP88. For example, GP88 antagonists can bind to, inhibit, and/or interfere with the activity of the GP88 receptor and thus interfere with the binding of GP88 to its receptor.

[00141] The term "GP88 antagonist" refers to any composition that inhibits or blocks GP88 expression, production or secretion, or any composition that inhibits or blocks the biological activity of GP88 including, but not limited to, anti- GP88 antibodies, anti-GP88 antisense polynucleotides, anti-GP88 receptor antibodies, anti-GP88 smaU molecules. In one embodiment of the invention, the GP88 antagonist is an anti-GP88 antibody or antibody fragment. The term
"antibody fragment" refers to any section, portion, or part of an antibody that retains the antigen binding properties of the antibody. Anti-GP88 antibodies also include antibody fragments, humanized antibodies, humanized antibody fragments and can be made as described above.

[00142] The term "contacting" refers to delivering GP88 antagonist to hematopoietic malignant ceUs (e.g., leukemia cells of B-cell lineage) wherein the GP88 antagonist can interact with the cell either directly (e.g., binding to GP88 inside the ceU) or indirectly (e.g., binding to GP88 and preventing GP88 from direcdy contacting myeloma cells). A GP88 antagonist may be injected into the blood stream of a patient suffering from a hematopoietic malignancy to bind GP88 and prevent GP88 from stimulating hematopoietic malignant ceU growth. GP88 antagonist may also be microinjected into a cell by shooting pellets coated with GP88 antagonist inside the cell in order to prevent secretion of GP88.
Hematopoietic malignant cells may also be transfected with nucleic acid encoding a GP88 antagonist. Alternatively, patients can be treated with a GP88 small molecule antagonist to block GP88 activity. Contacting hematopoietic malignant cells with GP88 antagonist blocks the activity of GP88 and therefore inhibits, reduces, and/or interferes with the growth ofthe cells. GP88 antagonists such as anti-GP88 antibodies and anti-GP88 antisense nucleic acids can be made and administered by any suitable mechanism (e.g., injection, and aerosol) as described above.

[00143] Administration of GP88 antagonists to hematopoietic malignant ceUs significantiy reduces the growth of the ceUs. For example, anti- GP88 neutralizing antibody inhibits the growth of RPMI 8226 multiple myeloma cells by about 50% while treatment of the same ceUs with non-immuno rabbit IgG did not show any significant inhibition of ceU growth. (FIG. 20). Addition of exogenous GP88 reversed the inhibitory effect of the anti-GP88 neutralizing antibody. (FIG. 20). The reversal of GP88 antagonist induced growth inhibition by the addition of exogenous GP88 demonstrates that GP88 is a growth factor for myeloma cells. The growth of myeloma cells can be measured by several methods including, but not limited to, measuring the live cell density in vitro by staining cells with trypan blue, uptake of radioactive nucleotides, cell mass, BudR incorporation, ELISA, cell metabolism, spectroscopy, and direct measurement of the dimensions of a tumor mass.

[00144] Preferred embodiments of the invention are also directed to methods of diagnosing B-ceU leukemia by detecting GP88 in tissue samples containing B-cells (e.g., blood, bone marrow, lymph, spleen, liver). The presence of GP88 in tissue samples containing B-cells indicates B-ceU leukemia. GP88 protein or nucleic acid can be detected as described above. Also provided are methods of diagnosing B-cell leukemia by detecting the presence of GP88 in B-ceUs. The presence of GP88 in B-ceUs indicates B-cell leukemia.

[00145] In another embodiment of the invention, the presence of GP88 in bone marrow cells indicates the presence of multiple myeloma ceUs. The presence of immunoglobufin lambda or kappa light chains in bone marrow cells is a marker for neoplastic or potentiaUy neoplastic myeloma ceUs. Hitzman et al., Immunoperoxidase staining of bone marrow sections, Cancer 48(ll):2438-46 (1981). Immunostaining bone marrow sections for the presence of lambda or kappa immunoglobufin light chains allows for detection of myeloma cases that are difficult to diagnose such as nonsecretory myeloma. Id. As shown in Table 1, such myeloma cells that stain positive for kappa or lambda light chains also stain positive for GP88.

Table 1 Expression of Ig light chain and GP88 in bone marrow smears
From multiple myeloma patients
Patients Ig K chain Ig λ chain GP88
1 +
2 - 3 - +
4 + +
5a - 5b + +
6 - 7 - +
8 + +
9 - 10 + +
1 1 + +
12 +

GP88 is not detected in bone marrow ceUs from patients in remission or in ceUs that do not express kappa or lambda immunoglobufin light chains. Furthermore, patients in remission for multiple myeloma (e.g., Patient 5a) do not express GP88. Patient 5a relapsed and displayed the symptoms of multiple myeloma (Patient 5b). Patient 5b was positive for both the kappa light chain and GP88. Thus, GP88 serves as a biological marker for multiple myeloma. An example of a triple stain for the presence of a control (DAPI), kappa/lambda light chains, and GP88 in the same patient sample is shown in FIGS. 25A, 25B, and 25C respectively. Detecting the presence of GP88 in bone marrow cells is indicative of whether multiple myeloma ceUs are present. The presence of GP88 can be detected by GP88 antagonists (e.g., anti-GP88 antibodies, anti-GP88 nucleic acid) using a variety of methodologies including, but not limited to, immunostaining, immunofluorescence, in situ hybridization, western blot, northern blot, and southern blot.

The Presence of GP88 Indicates Whether A Patient Is Responding or
Responsive to Anti- Cancer Therapy
[00146] Anti-cancer agents such as glucocorticoids and glucocorticoid analogs (e.g., dexamethasone, prednisolone, methylprednisolone, hydrocortisone, betamethasone, prednisone, fludrocortisone, cortisone, corticosterone,
triamcinolone, and paramethasone) alone or in combination with chemotherapy (e.g., alkylating agents) are used to treat patients with hematopoietic malignancies (e.g., B-cell leukemia). However, certain patients may not be responsive to anticancer therapy. In addition, it is well known that patients that are initially responsive to anti-cancer therapy develop resistance and no longer respond to the drugs.

[00147] For example, prolonged systemic exposure to glucocorticoids may have severe adverse side effects such as: ( 1 ) endocrine and metabolic disturbances including, but not limited to, Cushing-like syndrome, hirsutism, menstrual irregularities, premature epiphyseal closure, secondary adrenocortical and pituitary unresponsiveness, decreased glucose tolerance, and negative nitrogen and calcium balance; (2) fluid and electrolyte disturbances such as sodium and fluid retention, hypertension, potassium loss, and hypokalaemic alkalosis; (3) musculo-skeletal effects (e.g., myopathy, abdominal distension, osteoporosis, aseptic necrosis of femoral and humeral heads); (4) gastro-intestinal effects including gastric and duodenal ulceration, perforation, and hemorrhage; (5) dermatological effects such as impaired wound healing, skin atrophy, striae, petechiae and ecchymoses, bruising, facial erythema, increased sweating, and acne; (6) central nervous system effects (e.g., psychic disturbances ranging from euphoria to frank psychotic manifestations, convulsions, pseudotumor cerebri (benign intracranial hypertension) with vomiting and papUloedema); (7) ophthalmic effects including glaucoma, increased intraocular pressure, posterior subcapsular cataracts; and (8) immunosuppressive effects such as increased susceptibility to infections, decreased responsiveness to vaccination and skin tests. Thus, unnecessary exposure to anti-cancer therapy (e.g., glucocorticoids, such as dexamethasone), should be limited to the extent possible to avoid causing complications and discomfort without significant positive benefits.

[00148] Dexamethasone induces apoptosis of multiple myeloma cells. As shown in FIG. 26, dexamethasone also inhibits GP88 protein expression. GP88 protein expression was measured by Western blot analysis of conditioned media collected by ARP-1 cell cultures in the presence and absence of dexamethasone alone and in combination with IL-6 (FIG. 26). Dexamethasone significantiy inhibited die expression of GP88 protein. The addition of exogenous GP88 overcomes the apoptosis-inducing effects of dexamethasone (FIG. 27A and 27B). As shown in FIGS. 27A and 27B, GP88 significantly increased both cell growth (FIG. 27A) and cell viabUity (FIG. 27B) of ARP-1 cells treated with dexamethasone. FIG. 28 shows that GP88 significantly reduces the cleavage of an apoptosis marker PARP (Poly (ADP-ribose) polymerase) in dexametiiasone-treated ARP-1 cells.
Cleavage of PARP into two fragments is a marker of cell apoptosis. Thus, GP88 has an anti-apoptotic effect and can inhibit dexamethasone-induced killing of B-cell leukemia ceUs.

[00149] Increased levels of GP88 in MM ceUs are responsible for the transition of MM cells to a glucocorticoid resistant form. As shown in FIG. 29, cells transfected with GP88 (ARP-1/PCDGF) produced ten times more GP88 than untransfected cells or control ARP-1 cells that were transfected with empty vector (ARP-l/EV). (FIG. 29). MM cells transfected with GP88 show an increased growth rate and viability (resistance to the killing effect of dexamethasone) (FIGS. 30A and 30B). As shown in FIG. 30A, the ARP-1/PCDGF cells had a higher growth rate and were more resistant to the apoptotic effects of dexamethasone (columns 1 and 2) than the ARP-1/empty vector control cells (columns 3 and 4). Likewise, the ARP-1/PCDGF ceUs showed increased viabUity in response to the addition of dexamethasone (columns 1 and 2) that the ARP-1/empty vector ceUs. Thus, the presence of GP88 indicates that B-cells are or have become
dexamethasone-resistant.

[00150] Methods of determining whether a patient is responding or responsive to anti-cancer therapy by detecting the presence of GP88 in a tissue sample containing B-cells are also provided by the invention. The term
"responding" to anti-cancer therapy refers to patients who are receiving anti-cancer therapy. One embodiment of the invention wUl determine if such patients should continue to receive anti-cancer therapy. The term "responsive" to anti-cancer therapy refers to patients who are not yet receiving anti-cancer therapy. Another embodiment of the invention will determine if such patients should begin to receive anti-cancer therapy. Increased levels of GP88 in tissue samples (e.g., detectable increase in the level of GP88) containing B-ceUs over time indicate that the patient is not responding or responsive to anti-cancer therapy (e.g., glucocorticoids such as dexamethasone). Alternatively, increased levels of GP88 in B-cells compared to normal or peripheral tissues is sufficient to indicate that the patient is not responding or responsive to glucocorticoid therapy. GP88 protein and/or nucleic acids (e.g., DNA or RNA encoding GP88) can be detected as described above (e.g., using anti-GP88 antibodies, antisense nucleic acids). In another embodiment, the GP88 level in an individual patient's B cells or tissues containing B-cells can be periodically monitored. An increased level of GP88 in a patient's B-cells or in tissues containing B-cells over time indicates that the patient is not responding or responsive to anticancer therapy.

Recombinant GP88
[00151] The present invention is also directed to DNA expression systems for expressing a recombinant GP88 polypeptide or a functional derivative thereof substantially free of other mammalian DNA sequences. Such DNA may be double or single stranded. The DNA sequence should preferably have about 20 or more nucleotides to allow hybridization to another polynucleotide. In order to achieve higher specificity of hybridization, characterized by the absence of hybridization to sequences other than those encoding the GP88 protein or a homologue or functional derivative d ereof, a length of at least 50 nucleotides is preferred.

[00152] The present invention is also directed to the above DNA molecules, expressible vehicles or vectors as well as hosts transfected or transformed with the vehicles and capable of expressing the polypeptide. Such hosts may be prokaryotic, preferably bacteria, or eukaryotic, preferably yeast, mammalian or insect ceUs. A preferred vector system includes baculovirus expressed in insect cells. The DNA can be incorporated into host organisms by transformation, transduction, transfection, infection or related processes known in the art. In addition to DNA and mRNA sequences encoding the GP88 polypeptide, the invention also provides methods for expression of the nucleic acid sequence. Further, the genetic sequences and oligonucleotides allow identification and cloning of additional polypeptides having sequence homology to the polypeptide GP88 described here.

[00153] An expression vector is a vector which (due to the presence of appropriate transcriptional and/or translational control sequences) is capable of expressing a DNA (or cDNA) molecule which has been cloned into the vector and thereby produces a polypeptide or protein. Expression of the cloned sequence occurs when the expression vector is introduced into an appropriate host ceU. If a prokaryotic expression vector is employed, then the appropriate host cell would be any prokaryotic cell capable of expressing the cloned sequence. SimUarly, if an eukaryotic expression system is employed, then the appropriate host ceU would be any eukaryotic cell capable of expressing the cloned sequence. Baculovirus vector, for example, can be used to clone GP88 cDNA and subsequentiy express the cDNA in insect cells.

[00154] A DNA sequence encoding GP88 polypeptide or its functional derivatives may be recombined with vector DNA in accordance with conventional techniques including blunt-ended or staggered ended termini for ligation, restriction enzyme digestion to provide appropriate termini, filling in cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and ligation witii proper enzyme ligases. Techniques for such manipulations are discussed in (35).

[00155] A nucleic acid molecule is capable of expressing a polypeptide if it contains nucleotide sequences which contain transcriptional and translational regulatory information and such sequences are operably linked to nucleotide sequences which encode the polypeptide. An operable linkage is a linkage in which the regulatory DNA sequences and the DNA sequence sought to be expressed are connected in such a way as to permit gene expression. The precise nature of the regulatory regions needed for gene expression may vary from organism to organism but shall in general include a promoter region, which in prokaryotes contains both the promoter (which directs the initiation of RNA transcription) as weU as the DNA sequences which when transcribed into RNA wUl signal the initiation of protein synd esis. Such regions wUl normally include those 5' non-coding sequences involved with the initiation of transcription, translation such as the TATA box, capping sequence, CAAT sequence and the like.

[00156] If desired, the 3' non-coding region to the gene sequence encoding the protein may be obtained by described methods (screening appropriate cDNA library or PCR amplification). This region may be retained for the presence of transcriptional termination regulatory sequences such as termination and polyadenylation. Thus, by retaining the 3' region naturally contiguous to the DNA sequence coding for the protein, the transcriptional termination signals may be provided. Where the transcription termination signals are not provided or satisfactorily functional in the expression host cells, then a 3' region from another gene may be substituted.

[00157] Two DNA sequences such as a promoter region sequence and

GP88 encoding sequence are said to be operably linked if the nature of the linkage between the sequences does not result in d e introduction of a frame-shift mutation or interfere with the ability ofthe promoter sequence to direct transcription of the polypeptide gene sequence.

[00158] The promoter sequences may be prokaryotic, eukaryotic or viral.

Suitable promoters are inducible, repressible or constitutive. Examples of suitable prokaryotic promoters are reviewed by.

[00159] Eukaryotic promoters include but are not limited to the promoter for the mouse methaUothionein I gene, the TK promoter of Herpes Virus, the gene gal4 promoter, the SV40 early promoter, the mouse mammary tumor virus
(MMTV) promoter, and the cytomegalovirus (CMV) promoter. Strong promoters are preferred. Examples of such promoters are those which recognize the T3, SP6 and T7 polymerases, the PL promoter of bacteriophage lambda, the recA promoter, the promoter of the mouse methaUothionein I gene, the SV40 promoter and the CMV promoter.

[00160] It is to be understood that application of the teachings of the present invention to a specific problem or environment will be within the capabUity of one having ordinary skiU in the art in light of the teachings contained herein. The present invention is more fully illustrated by the following non-limiting examples.

EXAMPLE 1
Cell lines and reagents
[00161] Daudi, Raji, KOPM-28, ARP-1, RPMI 8226, Jurkat, KOPT-K1, and HL-60 were obtained from the American Type Culture CoUection (ATCC,

Manhassas, VA). RPMI 1640 medium, FBS, and Trizol was obtained from
Invitrogen life technologies (Carlsbad, CA). Alexa 456 conjugated goat anti mouse IgG F(ab')2 and Alexa 488 conjugated goat anti rabbit IgG F(ab')2 were obtained from Molecular Probes (Eugene, OR). IL-6 was obtained from Upstate
Biotechnology Inc. (Lack Placid, NY). PD98059, anti phosph-MAPK antibody, anti phosph-Akt antibody, anti Akt antibody, anti phosph-tyr-STAT3 were obtained from New England Biolabs (Beverly, MA). Anti STAT3 was obtained from BD Biosciences. Anti MAPK antibody was obtained from Santa Cruz Biotechnology (Santa Cruze, CA). LY194002 was obtained from Biomol (Plymouth Meeting, PA). Supersignal Western chemiluminescent substrate was obtained from Pierce (Rockford, IL). ImmobUon-P transfer membranes were obtained from Millipore (Bedford, MA). Monoclonal antibodies to anti human K or λ light chains were obtained from Dako (Carpinteria, CA). Protein A sepharose was obtained from Amersham Pharmacia Biotech (Piscataway, NJ). GP88 and anti-GP88 antibody were purified in our lab. AU other reagents were obtained from Sigma.

GP88 protein expression
[00162] Daudi, Raji, KOPM-28, ARP-1 , RPMI 8226, Jurkat, HL-60, and

KOPT-K1 were cultured at a density of 1X105 cells/ml in RPMI medium
supplemented witii 10% FBS. Until the cells reach a density of 1X10° cells/ml, the culture media equivalent to 1.5xl07 live cells were collected to measure GP88 protein expression. Immunoprecipitaion and Western Blot analysis were carried out as described previously (18) using 50ug/ml anti-GP88 F(ab') conjugated to HRP as the detecting antibody.

Northern blot analysis
[00163] RPMI 8226 and ARP-1 ceU were cultured in 10% FBS RPMI medium. RNA isolation was carried out using Trizol. Northern Blot analysis was carried out as described previously (18).
Cell growth and survival assay
[00164] RPMI 8226 or ARP-1 cells were cultured in 10% FBS RPMI.

Before the assay, cells were washed by serum free RPMI 1640 twice and cultured in serum free RPMI 1640 medium for 24 hours. GP88 or IL-6 was added to media at indicated concentration. Live cell density and viability were determined by trypan blue exclusion and ceU counting. Experiments were carried out in tripHcate sets with results expressed as mean ± SD.

Anti-GP88 Neutralizing Assay
[00165] RPMI 8226 cells were cultured in 10% FBS RPMI 1640, washed by RPMI 1640 twice, and cultured in RPMI 1640 media at 1X105 ceUs/ml.
Affinity purified anti-GP88, non-immuno rabbit IgG, or affinity purified anti-GP88 antibody with GP88 was added as appropriate. After 48 hours, live ceU density was checked by trypan blue staining and cell counting. Experiments were carried out in triplicate sets and the result was expressed as mean ± SD.

MAPK assay
[00166] ARP-1 cells were cultured in 10% FBS RPMI 1640 medium, washed by RPMI 1640 twice, and resuspended at 2.5X105 live cells/ml in RPMI 1640. After overnight starvation, ARP-1 ceUs were either treated with or without

30μM PD98059 for 60 min. GP88 was added to final concentration of 200ng/ml except wells for negative controls. Ten milliliters of ceU culture was used for each sample. After ten minutes of incubation, the ceUs were lysed by loading buffer. Cell lysates were separated on a 12.5% SDS-PAGE gel. The phosph-MAPK and total MAPK proteins were detected by anti-phoph-MAPK and anti MAPK antibodies respectively using Western blot analysis.

Akt assay
[00167] ARP-1 cells were cultured in 10% FBS RPMI 1640 medium, washed in RPMI 1640 twice, and resuspended at 2.5X105 live cells/ml in RPMI 1640. After overnight starvation, ARP-1 cells were either treated with or without 50μM LY194002 for ten minutes. GP88 was added to the experimental wells on a microtiter plate at a final concentration of 200ng/ml. GP88 was not added to control weUs. Ten millUiters of cell culture was used for each sample. After ten minutes of incubation, cells were lysed by loading buffer. Cell lysates were separated on a 12.5% SDS-PAGE gel. The phosph-Akt and total Akt proteins were detected by anti-phoph-Akt and anti Akt antibodies respectively using Western blot analysis.

STAT3 assay
[00168] ARP-1 cells were cultured in 10% FBS RPMI 1640 medium, washed twice in RPMI 1640 medium, and resuspended at 2.5X105 live cells/ml in RPMI 1640. After starvation of the ceU culture overnight, ARP-1 ceUs were treated with 200ng/ml GP88 or lOng/ml IL-6 for 15 min. Cells were lysed by loading buffer and separated on a 7.5% SDS-PAGE. 3X10° ceUs were used for each sample.

The phosph-tyr-STAT3 and total STAT3 proteins were detected by anti-phoph-tyr-STAT3 and anti STAT3 antibodies respectively using Western blot analysis.
Immunocytochemistry studies
[00169] Bone marrow smears obtained from multiple myeloma patients at the University of Maryland Greenbaum Cancer Center were fixed for 15 minutes on ice with 2% paraformaldehyde in PBS, washed by PBS, and permeabilized with 0.2% Triton X100 for 15 minutes at room temperature. The slides were stained with 0.85 μg/ml rabbit anti-human GP88 antibody at room temperature for 1 hour, washed by PBS, and incubated witii secondary 2μg/ml Alexa 488-conjugated goat anti rabbit IgG F(ab')2 at room temperature for 1 hour. These slides were also stained with 0.25μg/ml monoclonal antibodies to anti human K or λ light chains at room temperature for 1 hour, washed by PBS, and followed by incubation with lμg/ml Alexa456 conjugated goat anti mouse IgG F(ab')2 at room temperature for 1 hour. FinaUy, samples were stained by 0.5 μg/ml DAPI at room temperature for 15 minutes. Stained bone marrow samples were observed witii Olympus BX40 fluorescence microscope equipped with 100W mercury lamp and appropriate filters.

GP88 expression in human hematological cell line
[00170] We examined GP88 expression in several human leukemic cell lines. Samples examined were standardized to the same cell number. FIG. 16 shows GP88 protein expression was high in human B cell lines (Raji and Daudi) and human MM cell lines (ARP-1 and RPMI 8226). In contrast, no GP88 was produced in human T cell lines (Jurkat and KOPT-K1 ) and promyelocytic leukemia (HL-60). A low level of GP88 was found in macrophage ceU line (KOPM-28). HL-60 is a promyelocytic ceU line that can be induced to differentiate terminally to granulocyte-like ceUs or monocyte/macrophage-fike cells upon exposure to different reagents (19). These results show that GP88 is preferentiaUy expressed by hematological malignancies of B cell lineage. The level of GP88 mRNA expression in the MM cell lines ARP-1 and RPMI 8226 is shown in FIG. 17.

GP88 function in two human MM cell lines: RPMI 8226 and ARP-1
[00171] The effect of exogenously added GP88 on the growth and survival of RPMI 8226 (FIG. 18) and ARP-1 (FIG. 19) was examined and compared to IL-6, a known paracrine growth stimulator of MM cell growth. As shown in FIG. 3 A, RPMI 8226 cells were starved in RPMI medium only for 24 hours, then GP88 or IL-6 was added to medium. After 24 hour treatment 50 ng/ml (5.7x10-7 M), 100 ng/ml (1.1x10-6 M), 200 ng/ml (2.3x10-6 M) GP88, and lOng/ml (4.5x10-7 M) IL-6 stimulated the growth of RPMI 8226 cells by 1.3, 1.5, 1.5, and 1.6-fold, respectively. After 48 hour treatment, 50, 100, 200 ng/ml GP88, and lOng/ml IL-6 stimulated the growth of RPMI 8226 cells by 1.7, 2.5, 2.6, and 2.8 fold, respectively. These data show that GP88 stimulates the growth of RPMI 8226 cells in a dose and time dependent fashion similarly to IL-6. In addition to stimulating the growth of human MM ceUs, exogenous GP88 also stimulated cell survival of RPMI 8226 simUarly to IL-6 (FIG. 18B).

[00172] Dex-sensitive ARP-1 cells exhibited similar growth and survival effects in response to GP88. As shown in FIG. 19A, ARP-1 cells were starved in serum-free medium for 24 hours, then GP88 or IL-6 was added to medium. At 24 hours of treatment, 200ng/ml GP88 and lOng/ml IL-6 stimulated the growth of ARP-1 cells by 1.3 and 1.4 fold, respectively. After 48 hours of treatment,
200ng/ml GP88 and lOng/ml IL-6 stimulated the growth of ARP-1 cells by 2.3 and 2.6 fold, respectively. Similarly exogenous GP88 also stimulated cell survival of ARP-1 (FIG. 19B).

Effect of anti-GP88 neutralizing antibody on the growth of RPMI 8226
cells
[00173] In order to check whether GP88 produced and secreted by MM ceUs was required for ceU growth, we examined the effect of anti-GP88 neutralizing antibody on the growth of RPMI 8226 ceUs. We have shown previously that this antibody was able to inhibit the proliferation of breast cancer ceUs overexpressing

GP88 (20). As shown in FIG. 20, treatment of RPMI 8226 cell with 200μg/ml affinity purified anti-GP88 antibody inliibited RPMI 8226 ceU growth by about 50% in serum free condition. However, treatment of RPMI 8226 cells with 200μg/ml non-immuno rabbit IgG did not significantiy inhibit RPMI 8226 ceU growth.
Addition of exogenous 200ng/ml GP88 prevented the inhibition effect of anti- GP88 antibody. These results show that GP88 stimulated MM ceU growth in an autocrine fashion.

Signaling pathway stimulated by GP88 in ARP-1 cells
[00174] We examined signal pathways involved in growth factor signal transduction to determine their role, if any, in the GP88 signal transduction pathway in MM cells. MAPK signal pathway plays a key role in proliferation process and MAPK activity is stimulated in response to many different growth factors (21, 22). PI3K signal pathway is primarily associated with survival and cell growth regulation (23, 24). FIG. 21 shows that stimulation of ARP-1 ceU growth and survival by 200ng/ml of GP88 was blocked by the MEK inhibitor PD98059 at 30μM. FIG.

22 shows 200ng/ml GP88 activated the phosphorylation of Erkl and Erk2 and this phosphorylation was also inhibited by 30μM PD98059. Together, these results show that GP88 activated MAPK signal patiiway in ARP-1 cells and that MAPK was responsible for stimulation of cell growth by GP88. To determine the role of the PI3K signal pathway, we assessed the phosphorylation of Akt. Akt contains an amino-terminal pleckstrin homology (PH) domain that binds phosphorylated lipids at the membrane in response to activation of PI3 kinase (25, 26). FIG. 23 shows that GP88 stimulates the phosphorylation of Akt in ARP-1 ceUs and tiiis
phosphorylation was inhibited by PI3 K inhibitor LY294002 at 50μM. These results showed that GP88 activated PI3 kinase signal pathway in ARP-1 cells.

[00175] MAPK and JAK/STAT pathways are two important signaling pathways in human MM cells induced by IL-6 (6). In order to check whether GP88 activates JAK/STAT pathways in human MM cells, the phosphorylation of STAT3 was assessed foUowing stimulation of MM ceUs by 200ng/ml GP88 or lOng/ml IL-6. As shown in FIG. 24, only IL-6, but not GP88, stimulates the phosphorylation of STAT3. These data suggest that GP88 does not activate the JAK/STAT3 pathway in human MM cells.

Immunocytochemistry studies of human patient bone marrow smears
[00176] We examined GP88 expression in 13 bone marrow biopsy samples from patients with multiple myeloma by immunocytochemistry staining of GP88 and human κ/λ light chains. The presence K or λ light chains in bone marrow ceUs is a marker of myeloma cells (27). Table 1 demonstrates that GP88 was
overexpressed in bone marrow smears of MM patients. Staining of the samples with anti-human K or λ light chains showed that the myeloma ceUs that stained positive for GP88 were positive for K or λ light chains indicating that the ceUs that overexpress GP88 in bone marrow smears of MM patients are the multiple myeloma cells. GP88 -positive cells were not observed in the bone marrow smears from patients in remission (patients 2, 5a, 6 and 9) where K or λ light chain-positive cells were not detected. It is important to note that when the relapse of MM disease occurred in patient 5 a, GP88 expression was detected in the bone marrow samples and co-localized with ceUs expressing K light chains (5b in Table 1). The κ/λ chain positive cells showed 100% GP88 positive staining by counting 100 κ/λ chain positive ceUs. A typical triple staining by DAPI, κ/λ chain, and GP88 is shown in FIG. 25. These data clearly indicated that GP88 expression is associated with myeloma ceUs from all MM patients examined and correlated weU with the presence of the disease.

The Effects of Dexamethasone (Dex) and IL-6 effect on GP88 protein
expression in ARP-1 cells.
[00177] ARP-1 cells were seeded in 10% CT-FBS RPMI 1640 in the presence of 10-7M Dex or lOng/ml IL-6 added alone or in combination. Control ceUs were cultivated with vehicle medium diat did not contain Dex or IL-6. After

48 hours, the ceU culture medium was changed to RPMI 1640 for 24 hours and the conditioned medium was collected. The GP88 secreted in the conditioned medium was measured by immunoprecipitation and western blot analysis (FIG. 26). Dex inhibited the expression of GP88 in Dex-treated ARP-1 cells.

The Effects of Exogenous Addition of GP88 and IL-6 on Dex-induced Cell
Death.
[00178] ARP-1 ceUs were cultured in media containing 10% CT-FBS RPMI

1640 medium in the presence of 10-7 M Dex, 200ng/ml GP88, or lOng/ml IL-6 added alone or in combination. After 48 hours, the five ceU density and cell viability were checked by trypan blue exclusion and counting with a hemocytometer. FIG.

27A shows the effect on live cell density and FIG. 27B shows the effect on cell viability. GP88 increased the growth and viability of Dex-treated ARP-1 cells.

The Effects of GP88 on PARP cleavage in ARP-1 cells
[00179] ARP-1 ceUs were seeded and treated with 10-7 M Dex, 200ng/ml

PCDGF, and lOng/ml IL-6 as described above. CeUs were collected at 24 hours and 48 hours. ARP-1 cells were lysed and lOOμg protein per lane were used to analyze PARP cleavage by SDS-PAGE and western blot analysis. GP88 inhibited the apoptotic effects of Dex on Dex-treated ARP-1 cells (FIG. 28).

Overexpression of GP88 in ARP-1 cells.
[00180] Dexamethasone-sensitive human MM cell line ARP-1 was transfected with expression vector pcDNA3 containing a CMV promoter, a neomycin resistant gene, and GP88 cDNA by electroporation. Transfected ceUs were selected in the presence of G418. GP88 expression in the cell culture media was detected by immunoprecipitation and western blot analysis. As shown in FIG. 29, ARP-1 cells transfected with GP88 (ARP-1/PCDGF) had elevated levels of GP88 compared to ceUs transfected witii an empty expression vector (ARP-l/EV).

The Effects of Dex on ARP-1 and GP88 Overexpressed ARP-1 cells.
[00181] ARP-1/PCDGF ceUs that overexpress GP88 and ARP-l/EV were cultured in 10% CT-FBS RPMI with or without 10-7 M Dex. CeU density (A) and viability (B) were measured after 24 hours. As shown in FIGS. 30A and 30B, Dex-induced reduction in cell growtii and viability was significantly reduced in cells with elevated levels of GP88 (columns 1 and 2) compared to ceUs that did not express GP88 (columns 3 and 4).

The Effects of GP88 Over Expression Dex-induced PARP cleavage.
[00182] Empty vector control and PCDGF overexpressing ARP-1 cells were treated with or without 10-7 M Dex. After 48 hours, the cells were lysed to measure the expression of intact and cleaved PARP (FIG. 31). Cells overexpressing GP88 (ARP-1/PCDGF) showed greatly reduced cleavage of PARP compared to cells that did not express GP88. Thus, GP88 inhibits the apoptotic effects of dexamethasone on ARP-1 ceUs.

REFERENCES
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