US 20110059852A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2011/0059852 A1 Karsunky (43) Pub. Date: Mar. 10, 2011
(54) COMPOSITIONS AND METHODS FOR Related U.S. Application Data TREATINGHAEMATOLOGICAL PROLIFERATIVE DSORDERS OF MEYLOD (60) Provisional application No. 61/039,701, filed on Mar. ORIGIN 26, 2008. Publication Classification (75) Inventor: Holger Karsunky, Redwood City, (51) Int. Cl. CA (US) C40B 30/00 (2006.01) GOIN 33/566 (2006.01) CI2O I/68 (2006.01) (73) Assignee: Celerant Therapeutics, Inc., San GOIN 33/50 (2006.01) Carlos, CA (US) (52) U.S. Cl...... 506/7; 436/501; 435/7.1:435/6; 436/94 (21) Appl. No.: 12/934,120 (57) ABSTRACT The disclosure relates to methods and compositions effective (22) PCT Fled: Mar. 26, 2009 in the diagnosis, prognosis and treatment of human hemato poietic cancers. In particular, the disclosure provides tumor (86) PCT NO.: PCT/USO9/38459 associated genes that encode for cytokine receptors that are differentially expressed in hematopoietic tumor cells of S371 (c)(1), myeloid origin compared with other cells, e.g., normal stem (2), (4) Date: Nov. 5, 2010 cells. Patent Application Publication Mar. 10, 2011 Sheet 1 of 6 US 2011/0059852 A1
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88O8) i 79CO 79CO OSS Patent Application Publication Mar. 10, 2011 Sheet 3 of 6 US 2011/0059852 A1 O??aujo99)(ZOz|=ueeW TWWpOOIE XeWJO % gol 0 000€ 000|| Patent Application Publication US 2011/0059852 A1 g01»01 Patent Application Publication Mar. 10, 2011 Sheet 5 of 6 US 2011/0059852 A1 e O d 3 3 (C) w S Xe/NJO % s 8 s 8 8 W-OSS Patent Application Publication Mar. 10, 2011 Sheet 6 of 6 US 2011/0059852 A1 08 09 OZ XeWJO % g01 »01 US 2011/0059852 A1 Mar. 10, 2011 COMPOSITIONS AND METHODS FOR Subtypes based on morphological features and cytochemical TREATINGHAEMATOLOGICAL staining properties, and although the self-renewal character PROLIFERATIVE DSORDERS OF MEYLOD istic in most types of AML is attributable to leukemic cells ORIGIN having cell marker phenotypes consistent with HSCs (Bon net, D. and Dick, J. E., Nat. Med. 3(7):730–737 (1997)), the chromosomal abnormality associated with the AML M3 sub 0001. This application claims the benefit of priority under type is observed in cell populations with a cell marker phe 35 U.S.C. S 119 of U.S. application Ser. No. 61/039,701 (filed notype characteristic of more differentiated cells of the Mar. 26, 2008), which is incorporated herein by reference in myeloid lineage (CD34. CD38) whereas the HSC popula its entirety. tion in M3 does not carry the translocation (Turhan, A. G. et TECHNICAL FIELD al., Blood 79:2154-2161 (1995)). 0006 Gain of self-renewing characteristic in the commit 0002 The present disclosure relates generally to agents ted progenitor cell population is also suggested in chronic capable of specifically targeting cancer stem cell markers and myeloid leukemia (CML, also called chronic myelogenous methods of using the agents, particularly in diagnostic and leukemia, or chronic granulocytic leukemia), a disease com therapeutic treatments. In particular, the present disclosure monly associated with the Philadelphia chromosome, which provides cytokine receptors as novel cancer stem cell targets is a balanced translocation between chromosomes 9 and 22. that are expressed extracellularly and that are targeted by t(9:22). The translocation produces a fusion between the bcr antibodies and other agents disclosed herein. and c-abl genes and results in expression of a chimeric protein BCR-ABL with increased tyrosine kinase activity. Although BACKGROUND the HSC population in CML typically contains the chromo 0003. The cells of the hematopoietic system arise from somal abnormality, the BCR-ABL fusion protein is mainly multipotent progenitors, the hematopoietic stem cells expressed in the committed cells of myelomonocytic lineage (HSCs), which progress through a series of developmental rather than the HSCs, indicating that committed cells in the programs to ultimately form the terminally differentiated myeloid lineage may be the source of the leukemic cells cells of the myeloid or lymphoid lineage. It is believed that in rather than the HSCs. Additional evidence for the committed the initial stages of hematopoiesis, HSCs commit to two myeloid cells as being the Source of the leukemic clones in distinguishable oligopotent but developmentally restricted CML comes from studies of controlled expression of BCR progenitor cell types, the common lymphoid progenitors ABL in transgenic animals. Use of promoters active specifi (CLPs) and the common myeloid progenitor (CMPs). T lym cally in myeloid progenitor cells to force expression of BCR phocytes, B lymphocytes, natural killer (NK) cells, and lym ABL in committed cells but not in HSCs produces disease phoid dendritic cells develop from corresponding progenitor characteristic of CML in these transgenic animal models cells derived from the CLPs whereas erythroid cells, mega (Jaiswal. S. et al., Proc. Natl. Acad. Sci. USA 100:10002 karyocytes, granulocytes, macrophages, and myeloid den 10007 (2003)). dritic cells develop from their corresponding progenitor cells 0007 Although myeloproliferative disorders, such as derived from CMPs. Cell populations at each stage of differ AML and CML are typically associated with cytogenetic entiation are distinguishable from other cell populations in abnormalities, the cytogenetic defect may not be solely the hematopoietic pathway based on programmed expression responsible for the proliferative trait. In some instances, the of a unique set of cell markers. chromosomal abnormality is observed in normal cells, which 0004 Although HSCs are capable of self renewal—cell Suggests that accumulation of additional mutations in either division that results in at least one of the daughter cells having the HSCs or committed myeloid cells is required for full the same characteristics as the parent cell—the progenitor manifestation of the disease state. Even in CML, the disorder cells committed to the lymphoid or myeloid lineages lose displays a multiphasic course, beginning from a chronic their potential to self-renew. That is, mitotic cell division of phase, which after 3-5 years and up to 10 years, leads to an the committed progenitors leads to differentiated progeny accelerated or blastic phase similar to AML. The time period rather than generation of a cell with the same proliferative and required to transition from the chronic phase (less than 5% differentiation capacity as the parent cell. This loss of self blasts or promyelocytes) toa the blastic phase (>30% blasts in renewal potential is seen in the ability of committed progeni the peripheral blood or bone marrow) may reflect the time tors cells to maintain hematopoiesis only for a limited time needed to accumulate the mutations responsible for conver period (i.e., short term reconstitution) following transplanta sion of the chronic phase to the more aggressive blastic phase. tion of the progenitor cells into an immunocompromised For the most part, however, the leukemic cells appear to retain animal, as compared to an HSC, which can completely regen the cell marker phenotypes detectable in normal progenitor erate and maintain hematopoiesis during the life of the host cells. animal (i.e., long term reconstitution). 0008 Treatments for proliferative disorders normally rely 0005. It has been observed, however, that in certain dis on the sensitivity of proliferating cells to cytotoxic or cyto ease states of the hematopoietic system, dysregulation of static chemotherapeutic agents. For instance, buSulfan, a cellular regulatory pathways may lead to progenitor cells that bifunctional alkylating agent, and hydroxyurea, an inhibitor acquire the ability to self-renew. For instance, acute myeloid of ribonucleoside diphosphate, affect DNA synthesis and sta leukemia (AML, also called acute myelogenous leukemia) is bility, resulting intoxicity to dividing cells. Other therapeutic a myeloproliferative disorder marked, in part, by infiltration agents of similar activity include cytosine arabinoside (cyt of bone marrow by abnormal hematopoietic cells. Indeed, the arabine) and daunorubicin. However, the effects of these stem cell nature of cancer was first shown in AML (Lapidotet agents are non-discriminatory and as a result they have seri al., 1994 Nature 17:645-8). AML is categorized into different ous side effects due to toxicity to normal dividing cells. US 2011/0059852 A1 Mar. 10, 2011 0009. Another treatment used in patients with haemato hematopoietic cancers of myeloid origin. As described logical malignancies is bone marrow transplant (BMT), herein, the following cytokine receptor markers have been where the recipient's hematopoietic cells are eliminated with found to be associated with hematopoietic tumor cells radiation and/or chemotherapy (e.g., cyclophosphamide), (HTCs) of myeloid origin: colony Stimulating factor 1 recep and the hematopoietic system reconstituted by transplant of healthy hematopoietic stem cells. Typically, the transplant tor (CSF1R): interleukin 13 receptor, alpha 1 (IL13RA1): uses HLA matched allogeneic bone marrow cells from a interleukin 1 receptor accessory protein (IL1RAP); inter family member (HLA-identical) or a serologically matched feron-C. receptor 1 (IFNAR1); interleukin-5 receptor alpha altruistic donor (MUD). Approximately, <50% of recipients (IL5RA); insulin receptor (INSR); interleukin 1 receptor-like find a donor, with exactly matching histocompatibility. Trans 1 (IL1RL1); leukocyte receptor tyrosine (LTK); and tumor plant with less well matched donors marketed increases the associated calcium signal transducer 1 (TACSTD1). transplant related morbidity and mortality. This therapeutic 0015. In preferred embodiments, the disclosed cytokine approach has limited application because of its dependence receptor markers are differentially expressed in HTCs of on the availability of suitable donors and because the treat ments show better outcome for patients in the chronic or early myeloid origin compared to normal HSCs. In some embodi phase of the disease as compared to acute or late stages. ments, the disclosed cytokine receptor markers are differen 0010 Antibody therapy for cancer involves the use of tially expressed by at least about 2 fold. In other embodi antibodies, orantibody fragments, againstan antigento target ments, the cytokine receptor markers are differentially antigen-expressing tumor cells. Because antibody therapy expressed by at least about 3 fold. In other embodiments, the targets cells expressing a particular antigen, there is a possi cytokine receptor markers are differentially expressed by at bility of cross-reactivity with normal cells and can lead to least about 5 fold, etc. The present disclosure provides agents detrimental results. Substantial efforts have been directed to specifically directed to these markers that find use in thera finding tumor-specific antigens. Tumor-specific antigens are peutic and diagnostic applications. found almost exclusively on tumors or are expressed at a greater level in tumor cells than the corresponding normal 0016. The following cytokine receptor markers are over cells. Thus, tumor-specific antigens provide targets for anti expressed on the Surface of myelogenous HTCs: colony body targeting of cancer, or other disease-related, cells stimulating factor 1 receptor (CSF1R): interleukin 13 recep expressing the antigen, as well as providing markers for diag tor, alpha 1 (IL13RA1); interleukin 1 receptor accessory pro nosis, for example, by identifying increased levels of expres tein (IL1RAP); interferon-C. receptor 1 (IFNAR1); interleu Sion. In immunotherapy approaches, antibodies specific to kin-5 receptor alpha (IL5RA); insulin receptor (INSR); Such tumor-specific antigens can be conjugated to cytotoxic interleukin 1 receptor-like 1 (IL1RL1); leukocyte receptor compounds or can be used alone in immunotherapy. tyrosine (LTK); and tumor associated calcium signal trans 0.011 Immunotherapy as a treatment option against ducer 1 (TACSTD1). Agents specifically directed to these hematpoietic cancers, such as AML, is limited by the lack of markers can specifically bind HTCs of myeloid origin by tumor-associated antigens that are tumor-specific and that are virtue of binding to the Surface-expressed marker. shared among diverse patients. It is desirable to find other 0017 Compositions that specifically target the disclosed therapeutic agents that take advantage of the developmental myelogenous HTC markers (e.g., the cytokine receptors dis origins of the leukemic cells by exploiting the common char closed herein), interfering with the expression thereof or acteristics between leukemic cells and normal cell popula binding to the expressed products, are provided herein, as tions in the myeloid lineage. This approach would provide treatments that can Supplement traditional therapies for well as methods of using the same in the diagnosis, prognosis myeloid leukemias, or that can be used as an alternative and treatment of haematological proliferative disorders char treatment to directly target the stem cell fractions of leukemic acterized by Such markers. The compositions include anti cells. This approach also provides additional diagnostic and bodies that specifically bind one or more of the extracellu prognostic strategies, as well as strategies for monitoring the larly-expressed antigens associated with myelogenous HTCs efficacy of a therapeutic regimen. that can inhibit their proliferation and/or mediate their 0012 Generally, therapeutic treatment is more effective destruction. The invention further provides immortal cell when tailored to a specific type of hematopoietic cancer. lines that produce one or more such antibodies. Predicting and determining efficacy of a treatment regime 0018. In one aspect, the invention provides antibodies that over time is also valuable in terms of clinical management. It specifically bind to one or more of colony stimulating factor is thus desirable to find tumor-specific markers that can be 1 receptor (CSF1R): interleukin 13 receptor, alpha 1 used in more efficient and accurate diagnosis and prognosis of (IL 13RA1); interleukin 1 receptor accessory protein myeloiod leukemic disorders, such as AML. (IL1RAP); interferon-C. receptor 1 (IFNAR1); interleukin-5 0013 Cytokine receptors belong to families of receptor receptor alpha (IL5RA); insulin receptor (INSR); interleukin proteins, which are divided into two subsets on the basis of the 1 receptor-like 1 (IL1RL1); leukocyte receptor tyrosine presence or absence of particular sequence motifs. The two (LTK); and tumor associated calcium signal transducer 1 subsets are the hematopoietin-receptor family (also referred (TACSTD1) associated with HTCs of myeloid origin. In to as the class I cytokine receptor family) and the class II Some embodiments, the antibody is a monoclonal antibody, cytokine receptor Superfamily (many of which are receptors for example, an antibody which is produced from a hybri for interferons or interferon-like cytokines). In the hemato doma cell line. In preferred embodiments, the monoclonal poietin-receptor family, the C. chain often defines ligand antibody specifically binds to hematopoietic tumor cells of specificity of the receptor and the B or Y chain initiates intra myeloid origin including, without limitation, chronic cellular signaling. myeloid leukemia (CML) blasts, acute myeloid leukemia (AML) blasts, as well as to cells from the KG-1a, Pfeiffer, 3. SUMMARY MOLT-3, GA-10, Ramos, and Jurkat cell lines. In another 0014. The present invention provides methods and com embodiment, the monoclonal antibody specifically binds to positions effective in the diagnosis and treatment of human AML blasts. US 2011/0059852 A1 Mar. 10, 2011 0019. In preferred embodiments, the invention provides disclosed cytokine receptors. In another embodiment, the antibodies that specifically bind to one or more of the dis present invention provides methods of mediating the destruc closed cytokine receptors associated with myelogenous tion of HTCs of myeloid origin by contacting the HTCs with HTCs and thereby inhibit their proliferation and/or mediate a composition comprising an antibody or other agent directed their destruction, but do not mediate destruction of normal to one or more of the disclosed cytokine receptors. In one hematopoietic stems cells. In a preferred embodiment, the embodiment, the antibody is a monoclonal antibody that spe antibody is a monoclonal antibody. In some embodiments, cifically binds an epitope on a disclosed cytokine receptor or the antibody is an IgG isotype or a humanized antibody. In a portion thereof. In another embodiment, the composition one embodiment, the humanized antibody is from a trans comprises an antibody complex. genic animal that includes a human immunoglobulin gene. 0027. In another embodiment, a method of depleting 0020. In another embodiment, the invention provides an HTCs over-expressing one or more of the disclosed cytokine antibody complex having at least one antibody that specifi receptors in a subject in need thereof is provided in which the cally binds to one or more of the disclosed cytokine receptors Subject is administered a composition comprising an anti associated with HTCs of myeloid origin. In a preferred body or antibody complex as described herein. In yet another embodiment, the antibody complex comprises a multimer embodiment, the present invention provides a method of comprising a monoclonal antibody that binds to one of the treating a patient with a myelogenous haematological prolif disclosed cytokine receptors erative disorder characterized by over-expression in HTCs of 0021. In alternative embodiments, the antibodies of the one or more of the disclosed cytokine receptors where the present invention include detectable moieties, radioactive patient is administered a composition that includes an anti compounds (e.g. radioisotopes or radionuclides), or bioactive body or antibody complex or other agent as described herein. compounds (e.g. drugs or Small molecules). In some embodi 0028. In some embodiments, the methods of the present ments, the bioactive compound is a cytotoxic agent. invention are Suitable for treating a haematological prolifera 0022. In another embodiment, the invention provides tive disorder of myeloid orgin, including myoproliferative small molecules, which bind, preferably specifically, to one disorders such as, for example, chronic myeloid leukemia or more of the cytokine receptor polypeptides disclosed (CML) and/or acute myeloid leukemia (AML). herein. In preferred embodiments, the small molecule is a 0029. In another aspect, the present invention provides Small organic molecule, including Small organic molecules diagnostic methods for hematological proliferative disorders known in the art as being an agonist or antagonist of a of myeloid origin, where the level of an expression product polypeptide corresponding to a cytokine receptor disclosed corresponding to one or more of the disclosed cytokine recep herein. Small molecules known to bind polypeptides corre tors is detected. In one embodiment, the expression product is sponding to other cytokine receptors disclosed herein can a transcription product, such as RNA. Methods of detecting also find use in the Subject therapeutic, prognostic and/or the level of RNA include utilizing a specific hybridization diagnostic applications. probe or an array of such probes. In another embodiment, the 0023 Optionally, the small molecule is conjugated to a expression product is a translation product such as one or growth inhibitory agent or cytotoxic agent Such as a toxin, more of the cytokine receptor antigens corresponding to one including, for example, a maytansinoid or calicheamicin, an or more of the cytokine receptors disclosed herein. Methods antibiotic, a radioactive isotope, a nucleolytic enzyme, or the of detecting the level of an antigen include utilizing antibod like. The small molecules that find use in the therapeutic ies of the instant disclosure. The RNA or antigen level can be methods of the instant invention preferably induce death of a compared to control levels, e.g., levels obtained from Samples cell to which they bind. For diagnostic purposes, the small of normal HSCs. molecules can be detectably labeled and/or attached to a solid 0030. In another embodiment, the present invention pro Support. vides prognostic methods for predicting the efficacy of treat 0024. The subject agents and antibodies directed to the ing a haematological proliferative disorder of myeloid origin, disclosed cytokine receptors have significant therapeutic and where the level of an expression product corresponding to one diagnostic utilities and in additional aspects pharmaceutical or more of the disclosed cytokine receptors is detected and compositions, methods and kits are provided employing the wherein the expression product level is correlated with a Subject agents and antibodies for use in diagnosing and treat treatment outcome. In one embodiment, the expression prod ing haematological proliferative disorders characterized by uct is RNA and lower expression levels correlate with more the presence of one or more of the disclosed cytokine recep favorable outcomes. tors such as, e.g., acute myelogenous leukemia, acute 0031. In still another embodiment the present invention myelomonocytic leukemia, chronic myelogenous leukemia provides methods for monitoring the efficacy of treating a and acute myeloid leukemia. haematological proliferative disorder of myeloid origin, 0025. In one aspect, the present disclosure provides meth where the level of an expression product corresponding to one ods of using antibodies to target one or more of the disclosed or more of the disclosed cytokine receptors is detected at cytokine receptors. In the present teachings, the antibodies various time points; and where a change in the level is corre provide a basis for immunotherapeutic approaches in treating lated with treatment outcome. In one embodiment, the disorders involving hematopoietic tumor cells (HTCs) of expression product is RNA and decreasing levels indicate a myeloid origin, for example, myeloproliferative disorders positive response to treatment. such as chronic myeloid leukemia (CML) and acute myeloid 0032. In some embodiments, the methods of the present leukemia (AML). invention are Suitable for diagnosis, prognosis and monitor 0026. In one embodiment, the present invention provides ing of a haematological proliferative disorder of myeloid methods of inhibiting the proliferation of HTCs of myeloid origin, such as myoproliferative disorders. The present inven orign by contacting the HTCs with a composition comprising tion provides methods of diagnosis, prognosis and monitor an antibody or other agent directed to one or more of the ing of chronic myeloid leukemia (CML) and/or acute US 2011/0059852 A1 Mar. 10, 2011 myeloid leukemia (AML). In a particularly preferred embodi cells of the hematopoietic system, HSCs are typically defined ment, the haematological proliferative disorder is AML and by the presence of a characteristic set of cell markers. the level of RNA or antigen is detected using a test sample 0040 “Marker phenotyping refers to identification of comprising AML, HTCs, which is compared to control levels markers or antigens on cells for determining its phenotype obtained from a control sample of normal HSCs. (e.g., differentiation state and/or cell type). This may be done by immunophenotyping, which uses antibodies that recog 4. BRIEF DESCRIPTION OF THE DRAWINGS nize antigens present on a cell. The antibodies may be mono 0033 FIG. 1 shows the results of double sorting Lin clonal or polyclonal, but are generally chosen to have mini CD34CD90CD45RACD38 and LinCD34CD90 mal crossreactivity with other cell markers. It is to be understood that certain cell differentiation or cell surface CD45RACD38' cells from 3 samples taken from 3 indi markers are unique to the animal species from which the cells vidual mobilized peripheral blood (MPB) donors that are not are derived, while other cell markers will be common afflicted with AML. between species. These markers defining equivalent cell 0034 FIG. 2 shows the results of of double sorting Lin types between species are given the same marker identifica CD34+CD90+CD45RA-CD38- and Lin-CD34+CD90+ tion even though there are species differences in structure CD45RA-CD38+ cells from 3 samples taken from peripheral (e.g., amino acid sequence). Cell markers include cell Sur blood of 3 individual patients diagnosed with AML. faces molecules, also referred to in certain situations as cell 0035 FIG. 3 shows representative results of incubating differentiation (CD) markers, and gene expression markers. peripheral blood cells with an antibody specific for IL1RAP. The gene expression markers are those sets of expressed The samples were taken from an individual diagnosed with genes indicative of the cell type or differentiation state. In AML or an individual mobilized peripheral blood (MPB) part, the gene expression profile will reflect the cell surface donors not afflicted with AML. markers, although they may include non-cell Surface mol ecules. 5. DETAILED DESCRIPTION OF 0041 Lineage markers are cell Surface antigens that can be EMBODIMENTS used for immunophenotyping cells of a particular develop 5.1 Definitions mental lineage. For example, a set of Lin antigens compris ing CD2, CD3, CD4, CD5, CD8, NK1.1, B220, TER-119, 0036. For the following descriptions, the technical and Gr-1 can be used to identify mature murine blood cells. Cells scientific terms used herein will have the meanings com that do not express these marker antigens, or express them at monly understood by one of ordinary skill in the art, unless very low levels, are said to be lineage marker negative (Lin). specifically defined otherwise. Accordingly, the following The monoclonal antibody cocktails directed against these terms are intended to have the following meanings: lineage markers can be used to remove cells expressing these 0037. The terms “cancer stem cell (CSC) markers” or antigens from Source tissues (for example, bone marrow, “cancer stem cell (CSC) targets' as well as “hematopoietic umbilical cord blood, mobilized peripheral blood, fetal liver, tumor cell (HTC) markers' or “hematopoietic tumor cell and the like). This negative selection procedure yields a popu (HTC) targets' refer to genes and their expression products, lation of cells that is enriched for primitive hematopoietic such as mRNA and polypeptides, that have been found to be stem cells or very early progenitor cells or precursor cells that associated with HTCs by virture, for example, of increased do (not yet) express these markers (see, for example: KTLS expression and/or biological activity. For example, in the case cells). These cells are called lineage negative cells, abbrevi of the CSF1R marker disclosed herein, CSF1R mRNA tran ated Lin cells. Several Subpopulations of lineage negative scribed from the CSF1R gene is found at higher levels in cells have been identified that are enriched for hematopoietic samples comprising AML HTCs as compared with Samples stem cells. They include LinCD34" cells (Krause et al. comprising normal HSCs. An HTC associated with a given 1994), Lin Sca'"c-Kit'Thy1" cells (Fleming et al., 1993) marker is referred to herein as a “marker-HTC.” and human CD34"CD38 cell populations. 0038 “HTCs of myeloid origin” particularly refers to can 0042 Agent” refers to any molecule specifically directed cer stem cells derived from cells of the myeloid (nonlym to one or more of the disclosed HTC markers and that can act phoid) lineages, including monocytes, macrophages, neutro to inhibit, hinder and/or suppress a biological activity of the phils, basophils, eosinophils, erythrocytes, megakaryocytes/ HTC marker in a haematological proliferative disorder and/to platelets, dendritic cells and the like. HTCs of myeloid origin mediate destruction of the HTCs. Agents include any mol will be those found in myeloid leukemias, such as AML and ecule that specifically interacts with an HTC marker gene CML, where it is believed that progenitor cells commited to and/or expression product, including for example, antibodies myeloid lineages regain self-renewing characteristics. Vari that specifically bind to an antigen corresponding to a HTC ous forms of leukemia, for example, appear to have their marker to inhibit HTC proliferation and/or mediate their origins in a small population of HSCS or committed myeloid destruction; antisense molecules that interfere with the progenitor cells in which the cells acquire a combination of expression of an HTC marker; or molecules that interfere mutations that give rise to the malignant phenotype. The with a biological activity mediated by the HTC marker, such terms “HTCs of myeloid origin” and “myeloid HTCs” or as by sterically inhibiting interaction between an HTC marker “myelogenous HTCs are used herein interchangeably. and its ligand to interfere with activation of a cancer stem cell 0039) “Hematopoietic stem cell” or “HSC generally signal transduction pathway. The molecule may be one refers to clonogenic, self renewing pluripotent cells, capable known in the art, e.g., Small molecule agonists or antagonists of ultimately differentiating into all cell types of the hemato directed towards one or more of the HTC markers disclosed poietic system, including B cells T cells, NK cells, lymphoid herein. An antibody that specifically binds to an antigen cor dendritic cells, myeloid dendritic cells, granulocytes, mac responding to an HTC marker disclosed herein is referred to rophages, megakaryocytes, and erythroid cells. As with other as a “marker specific antibody.” US 2011/0059852 A1 Mar. 10, 2011 0043. A “small molecule”, “small molecule compound', immunoglobulin that retains the ability to bind antigen. These or 'small organic molecule' refers to a molecule having a include, as examples, F(ab), a monovalent fragment of V. C. molecular weight usually less than about 2000 daltons, alter and VC antibody domains; and F(ab')2 fragment, a bivalent natively less than about 1500, about 750, about 500, about fragment comprising two Fab fragments linked by a disulfide 250 or about 200 daltons in size, wherein such molecules are bridge at the hinge region. The term antibody also refers to known in the art to be capable of binding (preferably specifi recombinant single chain Fv fragments (ScPV) and bispecific cally binding) to a polypeptide corresponding to an cytokine molecules such as, e.g., diabodies, triabodies, and tetrabodies receptor disclosed herein. (see, e.g., U.S. Pat. No. 5,844,094). 0044) With regard to the binding of a small molecule com 004.6 Antibodies may be produced and used in many pound to a target molecule, the term “specifically interacts forms, including antibody complexes. As used herein, the with or “specifically binds” or is “directed to or towards' a term “antibody complex” or “antibody complexes” is used to particular product means binding that is measurably different mean a complex of one or more antibodies with another from a non-specific interaction. Specific binding can be mea antibody or with an antibody fragment or fragments, or a Sured, for example, by methods known in the art, e.g., using complex of two or more antibody fragments. Antibody com competition assays with a control molecule that is similar to plexes include multimeric forms of antibodies directed to the the target, for example, an excess of non-labeled target. A disclosed cytokine receptors such as homoconjugates and Small molecule compound that specifically binds a target can heteroconjugates as well as other cross-linked antibodies as have a Kd for the target of at least about 10M, alternatively describd herein. at least about 10 M, alternatively at least about 10 M, 0047 “Antigen' is to be construed broadly and refers to alternatively at least about 107M, alternatively at least about any molecule, composition, or particle that can bind specifi 10 M, alternatively at least about 10 M, alternatively at cally to an antibody. An antigen has one or more epitopes that least about 10' M, alternatively at least about 10' M, interact with the antibody, although it does not necessarily alternatively at least about 10' M, or greater. In one embodi induce production of that antibody. ment, the term “specific binding refers to binding where a 0048. The terms “cross-linked”, “cross-linking' and Small molecule compound binds to its particular target with grammatical equivalents thereof, refer to the attachment of out Substantially binding to any other polypeptide or macro two or more antibodies to form antibody complexes, and may molecule. also be referred to as multimerization. Cross-linking or mul 0045 Antibody refers to a composition comprising a timerization includes the attachment of two or more of the protein that binds specifically to a corresponding antigen and same antibodies (e.g. homodimerization), as well as the has a common, general structure of immunoglobulins. The attachment of two or more different antibodies (e.g. het term antibody specifically covers polyclonal antibodies, erodimerization). Those of skill in the art will also recognize monoclonal antibodies, dimers, multimers, multispecific that cross-linking or multimerization is also referred to as antibodies (e.g., bispecific antibodies), and antibody frag forming antibody homoconjugates and antibody heterocon ments, so long as they exhibit the desired biological activity. jugates. Such conjugates may involve the attachment of two Antibodies may be murine, human, humanized, chimeric, or or more monoclonal antibodies of the same clonal origin derived from other species. Typically, an antibody will com (homoconjugates) or the attachment of two or more antibod prise at least two heavy chains and two light chains intercon ies of different clonal origin (also referred to as heteroconju nected by disulfide bonds, which when combined form a gates or bispecific). Antibodies may be crosslinked by non binding domain that interacts with an antigen. Each heavy covalent or covalent attachment. Numerous techniques chain is comprised of a heavy chain variable region (V) and suitable for cross-linking will be appreciated by those of skill a heavy chain constant region (C). The heavy chain constant in the art. Non-covalent attachment may be achieved through region is comprised of three domains, C1, C2 and Cs, and the use of a secondary antibody that is specific to the primary may be of the mu, delta, gamma, alpha or epsilon isotype. antibody species. For example, a goat anti-mouse (GAM) Similarly, the light chain is comprised of a light chain variable secondary antibody may be used to cross-link amouse mono region (V) and a light chain constant region (C). The light clonal antibody. Covalent attachment may be achieved chain constant region is comprised of one domain, C., which through the use of chemical cross-linkers. may be of the kappa or lambda isotype. The VandV, regions 0049 “Epitope” refers to a determinant capable of specific can be further subdivided into regions of hypervariability, binding to an antibody. Epitopes are chemical features gen termed complementarity determining regions (CDR), inter erally present on Surfaces of molecules and accessible to spersed with regions that are more conserved, termed frame interaction with an antibody. Typical chemical features are work regions (FR). Each V, and V, is composed of three amino acids and Sugar moieties, having three-dimensional CDRs and four FRs, arranged from amino-terminus to car structural characteristics as well as chemical properties boxy-terminus in the following order: FR1, CDR1, FR2, including charge, hydrophilicity, and lipophilicity. Confor CDR2, FR3, CDR3, FR4. The variable regions of the heavy mational epitopes are distinguished from non-conforma and light chains contain a binding domain that interacts with tional epitopes by loss of reactivity with an antibody follow an antigen. The constant regions of the antibodies may medi ing a change in the spatial elements of the molecule without ate the binding of the immunoglobulin to host tissues or any change in the underlying chemical structure. factors, including various cells of the immune system (e.g., 0050 “Humanized antibody' refers to an immunoglobu effector cells) and the first component (Cld) of the classical lin molecule containing a minimal sequence derived from complement system. The heavy chain constant region medi non-human immunoglobulin. Humanized antibodies include ates binding of the immunoglobulin to host tissue or host human immunoglobulins (recipient antibody) in which resi factors, particularly through cellular receptors such as the Fc dues from a complementary determining region (CDR) of the receptors (e.g., FcyRI, FcyRII, FcyRIII, etc.). As used herein, recipient are replaced by residues from a CDR of a non antibody also includes an antigen binding portion of an human species (donor antibody) Such as mouse, rat or rabbit US 2011/0059852 A1 Mar. 10, 2011 having the desired specificity, affinity and capacity. In some typically 10-1000 times or more over background. “Specifi instances, Fv framework residues of the human immunoglo cally immunoreactive' or “antibody that specifically binds' bulin are replaced by corresponding non-human residues. also refers to an antibody that is capable of binding to an Humanized antibodies may also comprise residues which are antigen with sufficient affinity such that the antibody is useful found neither in the recipient antibody nor in the imported in targeting a cell having the antigenbound to its surface or in CDR or framework sequences. In general, a humanized anti targeting the soluble antigen itself. In Such embodiments, the body will comprise Substantially all of at least one, and typi extent of non-specific binding is the amount of binding at or cally two, variable domains, in which all or substantially all of below background and will typically be less than about 10%, the CDR regions correspond to those of a non-human immu preferably less than about 5%, and more preferably less than noglobulin and all or substantially all of the framework (FR) about 1% as determined by fluorescence activated cell sorting regions are those of a human immunoglobulin consensus (FACS) analysis or radioimmunoprecipitation (RIA), for sequence. A humanized antibody will also encompass immu example. noglobulins comprising at least a portion of an immunoglo 0058 “Recombinant antibody” refers to all antibodies bulin constant region (Fc), generally that of a human immu prepared and expressed, created or isolated by recombinant noglobulin (Jones et al., Nature 321:522-525 (1986); techniques. These include antibodies obtained from an ani Reichmann et al, Nature 332:323-329 (1988)). mal that is transgenic for the immunoglobulin locus, antibod 0051) “Immunogen refers to a substance, compound, or ies expressed from a recombinant expression vector, or anti composition which stimulates the production of an immune bodies created, prepared, and expressed by splicing of any response. immunoglobulin gene sequence to other nucleic acid 0052. The term “immunoglobulin locus’ refers to a Sequences. genetic element or set of linked genetic elements that com 0059. The term “associated with as in, for example, prise information that can be used by a B cell or B cell cytokine receptor markers being “associated with hemato precursor to express an immunoglobulin peptide. This pep poietic tumor cells (HTCs), refers to the case where the HTC tide can be a heavy chain peptide, a light chain peptide, or the marker genes (e.g., the cytokine receptor genes) are fusion of a heavy and a light chain peptide. In the case of an expressed at differential levels in HTCs as opposed to other unrearranged locus, the genetic elements are assembled by a mammalian cells, e.g., normal HSCs. That is, a transcription B cell precursor to form the gene encoding an immunoglo product, such as RNA, and/or a translation product, such as a bulin peptide. In the case of a rearranged locus, a gene encod polypeptide, corresponding to the cytokine receptor gene has ing an immunoglobulin peptide is contained within the locus. been found at differential levels in one or more samples 0053 “Isotype' refers to an antibody class defined by its comprising HTCs compared with one or more samples com heavy chain constant region. Heavy chains are generally clas prising other mammalian cells, e.g., normal HSCs. sified as gamma, mu, alpha, delta, epsilon and designated as 0060 “Expression' or “expressing as used herein refers IgG, IgM, IgA, Ig|D, and IgE. Variations within each isotype to both transcriptional and translational processes directed by are categorized into Subtypes, for example Subtypes of IgG a gene. That is, the terms refer to the process of converting are divided into IgG, IgG, IgG and IgG, while IgA is genetic information encoded in a nucleic acid sequence divided into IgA and IgA. The IgY isotype is specific to (gene) into RNA (e.g. mRNA rRNA, tRNA, snRNA, etc) birds. through transcription of the gene; and/or convereting genetic 0054 “Monoclonal antibody' or “monoclonal antibody information into protein through translation of mRNA. Simi composition” refers to a preparation of antibody molecules of larly, “expression product as used herein refers to a trancrip single molecular composition. A monoclonal antibody com tion or translation product of a gene, and includes, e.g., RNA position displays a single binding specificity and affinity for a (mRNA, tRNA, rRNA, snRNA, etc.) as well as polypeptides particular epitope. (intracellular, extracellular or Surface expressed proteins). 0055. The term “human monoclonal antibody” refers to 0061 “Differential expression'. “differentially antibodies displaying a single binding specificity which have expressed”, “differential levels, and the like, as used herein variable and/or constant regions (if present) derived from refers to a difference in the level of an expression product human germline immunoglobulin sequences. In one embodi corresponding to a marker in HTCs in comparison to other ment, the human monoclonal antibodies are produced by a mammalian cells, e.g., normal HSCs in particular. The differ hybridoma which includes a B cell obtained from a transgenic ence can be expressed as an expression ratio or signal ratio, non-human animal, e.g., a transgenic mouse, having a obtained from the quotient of the level of expression of an genome comprising a human heavy chain transgene and a expression product in HTCs over the level of expression of light chain transgene fused to an immortalized cell. the same expression product in HSCs. “Differential expres 0056 “Single chain Fv' or “scFv” refers to an antibody sion' generally refers to a difference in expression levels of at comprising the V and V, regions of an antibody, wherein least about 2 fold, at least about 3 fold, at least about 5 fold, at these domains are present in a single polypeptide chain. Gen least about 7 fold, at least about 10 fold, or at least about 15 erally, a scFv further comprises a polypeptide linker between fold. In particularly preferred embodiments, the difference in the V and V, domains which enables the schv to form the expression levels is at least about 20 fold, at least about 30 desired structure for antigen binding. fold, at least about 40 fold, or at least about 50 fold. In still 0057 “Specifically immunoreactive” or antibody that more preferred embodiments, the difference in expression “specifically binds’ refers to a binding reaction of the anti levels is at least about 70 fold, at least about 100 fold, at least body that is determinative of the presence of the antigen in a about 200 fold, or as much as nearly 300 fold, 400 fold or 500 heterogeneous population of antigens. The antibody may be fold. described as being “directed to’ or “directed against the 0062. The term “extracellularly-expressed’ refers to the particular antigen. Under a designated immunoassay condi case where expression products are found outside of a cell, tion, the antibody binds to the antigen at least two times, and whether existing entirely outside of the plasma membrane of US 2011/0059852 A1 Mar. 10, 2011 a cell (as in the case of secreted, Soluble products); or existing proximal downstream targets, resulting in a number of physi partly outside of a cell as in the case of Some membrane- (or ological effects, including cytoskeletal remodeling, gene Surface-) expressed products. Membrane-bound proteins transcription and protein translation. CSF1R activation is may be integral or peripheral, so long as at least a portion of known to promote the survival, proliferation and differentia the protein is accessible to antibodies outside the cell. The tion of mononuclear phagocytes, as well as the spreading and term membrane-bound includes membrane-expressed prod motility of macrophages. In osteoclasts, CSF1R synergizes ucts as well as receptor bound products that become associ with RANKL to regulate the differentiation of mononuclear ated with surface membranes by virtue of binding to a mem phagocytes to osteoclasts. brane-bound receptor. 0068. The present invention provides anti-CSF1R anti 0063 “Subject' or “patient are used interchangeably and bodies, preferably monoclonal antibodies, that can specifi refer to, except where indicated, mammals such as humans cally bind to CSF1R antigen, e.g., CSF1R polypeptide and non-human primates, as well as rabbits, rats, mice, goats, exposed on the surface of HTCs of myeloid origin. As dis pigs, and other mammalian species. cussed in more detail below, the anti-CSF1R antibodies pref 0064 “Haematological proliferative disorder” or erably bind such myelogenous HTCs, thereby inhibiting their “hematopoietic proliferative disorder” refers to a condition proliferation and/or mediating their destruction. One of skill characterized by the clonal proliferation of one or more in the art will further recognize that antibodies known in the hematopoietic cells of the myeloid and/or lymphoid lineage. art can find use in the methods disclosed in the instant inven “Hematological proliferative disorer of the myeloid lineage' tion. For example, one or more anti-CSF1R antibodies com refers to conditions characterized by the clonal proliferation mercially available from Abcam, Abgent, Abnova, Antigenix primarily of one or more hematopoietic cells of the myeloid Amaerica, eBioscience, GeneTex, Lab Vision, Lifespan Bio lineage, rather than the lymphoid lineage. It is to be noted that sciences, Millipore, Novus Biologicals, R&D Systems, herein the term "of myeloid origin' is used interchangeably Sigma-Aldrich and Thermo Scientific Pierce may also find with the adjectives “myeloid” or “myelogenous.” Myelog use with respect to diagnostic and/or therapeutic applications enous hematopoietic proliferative disorders include, e.g., the taught herein. general classes of (a) dysmyelopoietic disease, (b) acute 0069 IL13RA1 occurs as 4 different splice variants and is myeloproliferative leukemia and (c) chronic myeloprolifera normally found on T and B cells, as well as on endothelial tive disease. Each general class is further categorized into cells. It is also referred to as CD213a1, CD213A1, NR4, different Subtypes, as is known in the art. IL13RA, IL-13R, IL-13RA and IL-13R-alpha-1. The IL13RA1 polypeptide is a type I transmembrane protein, 5.2 Hybridomas and Monoclonal Antibodies which interacts with IL4R to form IL13 receptor. The IL13 0065. The teachings of the present disclosure provide receptor in turn binds IL 13, and can partially replace the hybridoma cell lines and monoclonal antibodies that specifi common gamma chain in an IL-2 recptor complex. cally bind to one or more of the following cytokine receptor 0070 The present invention provides anti-IL13RA anti products: colony stimulating factor 1 receptor (CSF1R): bodies, preferably monoclonal antibodies, that can specifi interleukin 13 receptor, alpha 1 (IL13RA1); interleukin 1 cally bind to IL13RA antigen, e.g., IL13RA polypeptide receptor accessory protein (IL1RAP); interferon-C. receptor 1 exposed on the surface of HTCs of myeloid origin. As dis (IFNAR1); interleukin-5 receptor alpha (IL5RA); insulin cussed in more detail below, the anti-IL13RA antibodies pref receptor (INSR); interleukin 1 receptor-like 1 (IL1RL1):leu erably bind such myelogenous HTCs, thereby inhibiting their kocyte receptor tyrosine (LTK); and tumor associated cal proliferation and/or mediating their destruction. One of skill cium signal transducer 1 (TACSTD1) associated with in the art will further recognize that antibodies known in the hematopoietic tumor cells (HTCs) of myeloid origin. art can find use in the methods disclosed in the instant inven 0066. The following cytokine receptor markers are over tion. For example, one or more anti-IL13RA antibodies com expressed on the surface of HTCs of myeloid origin: colony mercially available from Abcam, Abnova, ABR-Affinity stimulating factor 1 receptor (CSF1R): interleukin 13 recep BioReagents, Atlas Antibodies, Cell Sciences, Diaclone, tor, alpha 1 (IL13RA1); interleukin 1 receptor accessory pro GeneTex, Lifespan Biosciences, Novus Biologicals, R&D tein (IL1RAP); interferon-C. receptor 1 (IFNAR1); interleu Systems and Strategic Diagnostics may also find use with kin-5 receptor alpha (IL5RA); insulin receptor (INSR); respect to diagnostic and/or therapeutic applications taught interleukin 1 receptor-like 1 (IL1RL1); leukocyte receptor herein. tyrosine (LTK); and tumor associated calcium signal trans (0071 IL1 RAP, also known as IL1 R3, IL-1 RAcP. ducer 1 (TACSTD1). The invention provides anti-CSF1R FLJ37788, occurs as 11 alternative splice forms, and is nor antibodies; anti-IL13RA1 antibodies; anti-IL1RAP antibod mally expressed on macrophages/monocytes, B cells, plate ies; anti-IFNAR1 antibodies; anti-IL5RA antibodies; anti lets, thymocytes, T cells and dendritic cells. IL1RAP is a type INSRantibodies; anti-IL1RL1 antibodies; anti-LTKantibod I transmembrane protein, but also occurs as a secreted ver ies; and anti-TACSTD1 antibodies, where the antibodies are Sion. The protein is known to generally mediate IL-1 depen preferably monoclonal antibodies. dent activation of NF-kB. 0067 CSF1R is a disulfide-linked homodimer, also 0072 The present invention provides anti-IL1RAP anti referred to in the art as CD115, 1436, CSF1R, 164770, bodies, preferably monoclonal antibodies, that can specifi P07333, C-FMS, CSFR, FIM2 and FMS, and formerly cally bind to IL1RAP antigen, e.g., IL1RAP polypeptide referred to as McDonough feline sarcoma viral (v-fms) onco exposed on the surface of HTCs of myeloid origin. As dis gene homolog. CSF1R is normally expressed on myeloid cussed in more detail below, the anti-IL1RAP antibodies cells, in particular progenitors and monocytes, and osteo preferably bind such myelogenous HTCs, thereby inhibiting clasts as a membrane bound polypeptide. Upon binding to its their proliferation and/or mediating their destruction. One of ligand CSF-1, the receptor undergoes tyrosine autophospho skill in the art will further recognize that antibodies known in rylation and Subsequently, phosphorylates other membrane the art can find use in the methods disclosed in the instant US 2011/0059852 A1 Mar. 10, 2011 invention. For example, one or more anti-IL1RAP antibodies available from R&D Diagnostics and LifeSpanierce may also commercially available from Abcam, Abnova Corporation, find use with respect to diagnostic and/or therapeutic appli GeneTex, Lifespan Biosciences and Novus Biologicals may cations taught herein. also find use with respect to diagnostic and/or therapeutic 0079 IL1RL1, also known as T1, ST2, DER4, ST2L, applications taught herein. ST2V, FIT-1, and MGC32623, is a member of the interleukin 1 receptor family. It may be induced by proinflammatory 0073. IFNAR1 is a type I membrane protein that forms one stimuli, and may be involved in the function of helper T cells. of the two chains of a receptor for interferons alpha and beta. 0080. The present invention provides anti-IL1RL1 anti Binding and activation of the receptor stimulates Janus pro bodies, preferably monoclonal antibodies, that can specifi tein kinases, which in turn phosphorylate several proteins, cally bind to IL1RL1 antigen, e.g., IL1RL1 polypeptide including STAT1 and STAT2. It may also function as an exposed on the surface of HTCs of myeloid origin. As dis antiviral factor. IFNAR1 is also known as AVP, IFRC, cussed in more detail below, the anti-IL5RA antibodies pref IFNBR, IFN-alpha-REC, IFNBR. erably bind such myelogenous HTCs, thereby inhibiting their 0074 The present invention provides anti-IFNAR1 anti proliferation and/or mediating their destruction. One of skill bodies, preferably monoclonal antibodies, that can specifi in the art will further recognize that antibodies known in the cally bind to IFNAR1 antigen, e.g., IFNAR1 polypeptide art can find use in the methods disclosed in the instant inven exposed on the surface of HTCs of myeloid origin. As dis tion. For example, one or more anti-IL1R1 antibodies com cussed in more detail below, the anti-IFNAR1 antibodies mercially available from Protein Tech may also find use with preferably bind such myelogenous HTCs, thereby inhibiting respect to diagnostic and/or therapeutic applications taught their proliferation and/or mediating their destruction. One of herein. skill in the art will further recognize that antibodies known in I0081 LTK is a member of the ros/insulin receptor family the art can find use in the methods disclosed in the instant of tyrosine kinases. Multiple transcript variants encoding dif invention. For example, one or more anti-IFNAR1 antibodies ferent isoforms have been found for this gene. LTK is also commercially available from Abcam, Novus Biologicals, and known as TYK1. R&D Systems may also find use with respect to diagnostic I0082. The present invention provides anti-LTK antibod and/or therapeutic applications taught herein. ies, preferably monoclonal antibodies, that can specifically 0075. IL5RA is an interleukin 5 specific subunit of a het bind to LTK antigen, e.g., LTK polypeptide exposed on the erodimeric cytokine receptor. The IL5 heterodimeric receptor surface of HTCs of myeloid origin. As discussed in more comprises a ligand specific alpha Subunit and a signal trans detail below, the anti-LTK antibodies preferably bind such ducing beta subunit shared by the receptors for interleukin 3 myelogenous HTCs, thereby inhibiting their proliferation (IL3) and colony stimulating factor 2 (CSF2/GMCSF). The and/or mediating their destruction. One of skill in the art will beta Subunit is activated by ligand binding, and is required for further recognize that antibodies known in the art can find use biological activity of IL5. IL5RA may interact with the syn in the methods disclosed in the instant invention. For decan binding protein (Syntenin), which is required for IL5 example, one or more anti-LTK antibodies commercially mediated activation of the transcription factor SOX4. Six available from Abcam, Abgent, Abnova, Lifespan Bio alternatively spliced variants encoding three distinct isoforms Sciences, Novus Biologicals, and Santa Cruz, Biologicals may have been reported. IL5RA is also known as CD125, also find use with respect to diagnostic and/or therapeutic CDw125, HSIL5R3, and MGC26560. applications taught herein. 0076. The present invention provides anti-IL5RA anti I0083) TACSTD1 is expressed on most normal epithelial bodies, preferably monoclonal antibodies, that can specifi cells and gastrointestinal carcinomas. It may function as a cally bind to IL5RA antigen, e.g., IL5RA polypeptide homotypic calcium-independent cell adhesion molecule and exposed on the surface of HTCs of myeloid origin. As dis is used as a target for immunotherapy of human carcinomas. cussed in more detail below, the anti-IL5RA antibodies pref TACSTD1 is also known as EGP, ESA, KSA, M4S1, MK1, erably bind such myelogenous HTCs, thereby inhibiting their EGP2, EGP34, EGP40, KS1/4, MIC18, TROP1, CO-17A, proliferation and/or mediating their destruction. One of skill EpCAM, hEGP2, CO17-1A, GA733-2, and TACST-1. in the art will further recognize that antibodies known in the I0084. The present invention provides anti-TACSTD1 anti art can find use in the methods disclosed in the instant inven bodies, preferably monoclonal antibodies, that can specifi tion. For example, one or more anti-IL5RA antibodies com cally bind to TACSTD1 antigen, e.g., TACSTD1 polypeptide mercially available from Abcam, Thermo Scientific Pierce, exposed on the surface of HTCs of myeloid origin. As dis GeneTex, Proteintech, and Atlas antibodies may also find use cussed in more detail below, the anti-TACSTD1 antibodies with respect to diagnostic and/or therapeutic applications preferably bind such myelogenous HTCs, thereby inhibiting taught herein. their proliferation and/or mediating their destruction. One of 0077. INSR, also known as HHF5 and CD220, stimulates skill in the art will further recognize that antibodies known in glucose uptake. the art can find use in the methods disclosed in the instant 0078. The present invention provides anti-INSR antibod invention. For example, one or more anti-TACSTD1 antibod ies, preferably monoclonal antibodies, that can specifically ies commercially available from Lifespan BioSciences may bind to INSR antigen, e.g., INSR polypeptide exposed on the also find use with respect to diagnostic and/or therapeutic surface of HTCs of myeloid origin. As discussed in more applications taught herein. detail below, the anti-INSR antibodies preferably bind such I0085 Monoclonal antibodies of the instant disclosure spe myelogenous HTCs, thereby inhibiting their proliferation cifically bind myelogenous HTCs by virtue of specific binding and/or mediating their destruction. One of skill in the art will to its target antigen. In preferred embodiments, the mono further recognize that antibodies known in the art can find use clonal antibody (or a derivative thereof) is specifically immu in the methods disclosed in the instant invention. For noreactive with cells of myeloid origin the hematopoietic example, one or more anti-INSR antibodies commercially system, such as granulocyte/macrophage progenitors (GMP). US 2011/0059852 A1 Mar. 10, 2011 KG-1a, K-562, Jurkat, CML blasts, and AML blasts. For sisting of the V and C domains; (iv) a Fv fragment con example, in some such embodiments, specific binding to sisting of the V, and V. domains of a single arm of an HTCs, by virtue of a cytokine receptor disclosed herein, antibody, (v) a dAb fragment (Ward et al., (1989) Nature mediates destruction of hematopoietic tumor cells of myeloid 341:544-546), which consists of a V. domain; and (vi) an origin. isolated complementarity determining region (CDR), and I0086 Antibodies can be produced readily by one skilled in (vii) bispecific single chain Fv dimers (PCT/US92/09965). the art. The general methodology for making monoclonal Furthermore, although the two domains of the Fv fragment, antibodies by hybridomas is now well known to the art. See, V, and V, are coded for by separate genes, they can be e.g., M. Schreier et al., Hybridoma Techniques (Cold Spring joined, using recombinant methods, by a synthetic linker that Harbor Laboratory); Hammerling et al., Monoclonal Anti enables them to be made as a single protein chain in which the bodies and T-Cell Hybridomas (Elsevier Biomedical Press). V, and V regions pair to form monovalent molecules As described above, the present disclosure provides methods (known as single chain Fv (scFV); see e.g., Bird et al. (1988) of producing the monoclonal antibodies or derivatives Science 242:423-426; and Huston et al. (1988) Proc. Natl. thereof. In some embodiments, these methods comprise cul Acad. Sci. USA85:5879-5883). Such single chain antibodies tivating a hybridoma cell under Suitable conditions, wherein are also intended to be encompassed within the term “anti the antibody is produced and obtaining the antibody and/or gen-binding portion of an antibody. These antibody frag derivative thereof from the cell and/or from the cell culture ments are obtained using conventional techniques known to medium. A specific example of making the monoclonal anti those with skill in the art, and the fragments are screened for bodies of the instant invention is also provided in the utility in the same manner as are intact antibodies. The anti Examples below. body fragments may be modified. For example, the molecules 0087. The antibodies can be purified by methods known to may be stabilized by the incorporation of disulphide bridges the skilled artisan. Purification methods include, among oth linking the VH and VL domains (Reiter et al., 1996, Nature ers, selective precipitation, liquid chromatography, HPLC, Biotech. 14:1239-1245). electrophoresis, chromatofocusing, and various affinity tech I0089. The present disclosure further provides fragments niques. Selective precipitation may use ammonium Sulfate, of the antibodies disclosed herein. Immunoglobulin mol ethanol (Cohn precipitation), polyethylene glycol, or others ecules can be cleaved into fragments. The antigen binding available in the art. Liquid chromatography mediums, region of the molecule can be divided into either F(ab') or include, among others, ion exchange medium DEAE, polyas Fab fragments. The F(ab')2 fragment is divalent and is useful partate), hydroxylapatite, size exclusion (e.g., those based on when the Fc region is either undesirable or not a required crosslinked agarose, acrylamide, dextran, etc.), hydrophobic feature. The Fab fragment is univalent and is useful when an matrixes (e.g., Blue Sepharose). Affinity techniques typically antibody has a very high avidity for its antigen. Eliminating rely on proteins that interact with the immunoglobulin Fc the Fc region from the antibody decreases non-specific bind domain. Protein Afrom Staphylococcus aureas can be used to ing between the Fc region and Fc receptor bearing cells. To purify antibodies that are based on human y1, y2, or y4 heavy generate Fab or F(ab) fragments, the antibodies are digested chains (Lindmarket al., J. Immunol. Meth. 62:1-13 (1983)). with an enzyme. Proteases that cleave at the hinge region of Protein G from C and G streptococci is useful for all mouse an immunoglobulin molecule preserve the disulfide bond(s) isotypes and for human Y3 (Guss et al., EMBO.J. 5:1567.1575 linking the F(ab') domain such that they remain together fol (1986)). Protein L, a Peptostreptococcus magnus cell-wall lowing cleavage. A suitable protease for this purpose is pep protein that binds immunoglobulins (Ig) through k light sin. For producing F(ab) fragments, proteases are chosen chain interactions (BD Bioscience/ClonTech. Palo Alto, Such that cleavage occurs above the hinge region containing Calif.), is useful for affinity purification of Ig subclasses IgM, the disulfide bonds that join the heavy chains but which leaves IgA, Ig), IgG, IgE and IgY. Recombinant forms of these intact the disulfide bond linking the heavy and light chain. A proteins are also commercially available. If the antibody con Suitable protease for making F(ab) fragments is papain. The tains metal binding residues. Such as phage display antibodies fragments are purified by the methods described above, with constructed to contain histidine tags, metal affinity chroma the exception of affinity techniques requiring the intact Fc tography may be used. When sufficient amounts of specific region (e.g., Protein A affinity chromatography). cell populations are available, antigen affinity matrices may 0090 Antibody fragments can be produced by limited be made with the cells to provide an affinity method for proteolysis of antibodies and are called proteolytic antibody purifying the antibodies. fragments. These include, but are not limited to, the follow 0088. The present invention provides the antibodies ing: F(ab')2 fragments, Fab' fragments, Fab'-SH fragments, described herein, as well as corresponding antibody frag and Fab fragments. “F(ab'), fragments' are released from an ments and antigen-binding portions. The terms “antibody antibody by limited exposure of the antibody to a proteolytic fragment’ or “antigen-binding portion of an antibody (or enzyme, e.g., pepsin or ficin. A F(ab') fragment comprises simply “antibody portion') of the present invention, as used two “arms, each of which comprises a variable region that is herein, refers to one or more fragments of an antibody that directed to and specifically binds a common antigen. The two retain the ability to specifically bind to an antigen. It has been Fab' molecules are joined by interchain disulfide bonds in the shown that the antigen-binding function of an antibody can be hinge regions of the heavy chains; the Fab' molecules may be performed by fragments of a full-length antibody. Examples directed toward the same (bivalent) or different (bispecific) of binding fragments encompassed within the term “antibody epitopes. "Fab' fragments' contain a single anti-binding fragment’ or “antigen-binding portion of an antibody domain comprising a Fab and an additional portion of the include (i) a Fab fragment, a monovalent fragment consisting heavy chain through the hinge region. “Fab'-SH fragments’ of the V, V, C, and C domains; (ii) a F(ab')2 fragment, a are typically produced from F(ab')2 fragments, which are held bivalent fragment comprising two Fab fragments linked by a together by disulfide bond(s) between the H chains in an disulfide bridge at the hinge region; (iii) a Fd fragment con F(ab')2 fragment. Treatment with a mild reducing agent Such US 2011/0059852 A1 Mar. 10, 2011 as, by way of non-limiting example, beta-mercaptoethy region of the recipient are replaced by residues from a hyper lamine, breaks the disulfide bond(s), and two Fab' fragments variable region of a non-human species (donorantibody) Such are released from one F(ab') fragment. Fab'-SH fragments as mouse, rat, rabbit or nonhuman primate having the desired are monovalent and monospecific. "Fab fragments' (i.e., an specificity, affinity, and capacity. In some instances, frame antibody fragment that contains the antigen-binding domain work region (FR) residues of the human immunoglobulin are and comprises a light chain and part of a heavy chain bridged replaced by corresponding non-human residues. Further by a disulfide bond) are produced by papain digestion of more, humanized antibodies may comprise residues that are intact antibodies. A convenient method is to use papain not found in the recipient antibody or in the donor antibody. immobilized on a resin so that the enzyme can be easily These modifications are made to further refine antibody per removed and the digestion terminated. Fab fragments do not formance. In general, the humanized antibody will comprise have the disulfide bond(s) between the H chains present in a substantially all of at least one, and typically two, variable F(ab')2 fragment. domains, in which all or substantially all of the hypervariable 0091 “Single-chain antibodies' are one type of antibody loops correspond to those of a non-human immunoglobulin fragment. The term single chain antibody is often abbreviated and all or substantially all of the FRs are those of a human as “scFv’ or “slfv.” These antibody fragments are produced immunoglobulin sequence. The humanized antibody option using molecular genetics and recombinant DNA technology. ally also will comprise at least a portion of an immunoglobu A single-chain antibody consists of a polypeptide chain that lin constant region (Fc), typically that of a human immuno comprises both a V and a V, domains which interact to form globulin. an antigen-binding site. The V and V, domains are usually 0096 Generally, in a humanized antibody, the entire anti linked by a peptide of 10 to 25 amino acid residues. body, except the CDRs, is encoded by a polynucleotide of 0092. The term “single-chain antibody' further includes human origin or is identical to such an antibody except within but is not limited to a disulfide-linked Fv (dsEv) in which two its CDRs. The CDRs, some or all of which are encoded by single-chain antibodies (each of which may be directed to a nucleic acids originating in a non-human organism, are different epitope) linked together by a disulfide bond; a bispe grafted into the beta-sheet framework of a human antibody cific slfv in which two discrete scFvs of different specificity is variable region to create an antibody, the specificity of which connected with a peptide linker; a diabody (a dimerized sR v is determined by the engrafted CDRs. The creation of such formed when the V, domain of a first sRv assembles with the antibodies is described in, e.g., WO 92/11018, Jones, 1986, V, domain of a second sPv and the V, domain of the first sEv Nature 321:522-525, Verhoeyen et al., 1988, Science 239: assembles with the V domain of the second sPv; the two 1534-1536. Humanized antibodies can also be generated antigen-binding regions of the diabody may be directed using mice with a genetically engineered immune system. towards the same or different epitopes); and a triabody (a Roque et al., 2004, Biotechnol. Prog. 20:639-654. trimerized sRV, formed in a manner similar to a diabody, but 0097. The present disclosure further provides humanized in which three antigen-binding domains are created in a and non-humanized antibodies. Humanized forms of non single complex; the three antigen binding domains may be human (e.g., mouse) antibodies are chimeric antibodies that directed towards the same or different epitopes). contain minimal sequence derived from non-human immu 0093. “Complementary determining region peptides” or noglobulin. Generally, humanized antibodies are human “CDR peptides are another form of an antibody fragment. A immunoglobulins (recipient antibody) in which residues CDR peptide (also known as “minimal recognition unit') is a from a hypervariable region of the recipient are replaced by peptide corresponding to a single complementarity-deter residues from a hyperVariable region of a non-human species mining region (CDR), and can be prepared by constructing (donor antibody) Such as mouse, rat, rabbit or nonhuman genes encoding the CDR of an antibody of interest. Such primate having the desired specificity, affinity, and capacity. genes are prepared, for example, by using the polymerase In some instances, framework region (FR) residues of the chain reaction to synthesize the variable region from RNA of human immunoglobulin are replaced by corresponding non antibody-producing cells. See, for example, Larrick et al., human residues. Furthermore, humanized antibodies may Methods: A Companion to Methods in Enzymology 2:106, comprise residues that are not found in the recipient antibody 1991. or in the donor antibody. These modifications are made to 0094. In "cysteine-modified antibodies,” a cysteine amino further refine antibody performance. In general, the human acid is inserted or substituted on the surface of antibody by ized antibody will comprise substantially all of at least one, genetic manipulation and used to conjugate the antibody to and typically two, variable domains, in which all or Substan another molecule via, e.g., a disulfide bridge. Cysteine Sub tially all of the hypervariable loops correspond to those of a stitutions or insertions for antibodies have been described non-human immunoglobulin and all or Substantially all of the (see U.S. Pat. No. 5.219,996). Methods for introducing Cys FRS are those of a human immunoglobulin sequence. The residues into the constant region of the IgG antibodies for use humanized antibody optionally also will comprise at least a in site-specific conjugation of antibodies are described by portion of an immunoglobulin constant region (Fc), typically Stimmel et al. (J. Biol. Chem 275:330445-30450, 2000). that of a human immunoglobulin. 0095. The present disclosure further provides humanized (0098. It can be desirable to modify the antibodies of the and non-humanized antibodies. Humanized forms of non invention with respect to effector function, so as to enhance, human (e.g., mouse) antibodies are chimeric antibodies that e.g., the effectiveness of the antibody in treating cancer. For contain minimal sequence derived from non-human immu example, cysteine residue(u)can be introduced into the Fc noglobulin. Generally, humanized antibodies are non-human region, thereby allowing interchain disulfide bond formation antibodies that have had the variable-domain framework in this region. The homodimeric antibody thus generated can regions Swapped for sequences found in human antibodies. have improved internalization capability and/or increased The humanized antibodies may be human immunoglobulins complement-mediated cell killing and antibody-dependent (recipient antibody) in which residues from a hypervariable cellular cytotoxicity (ADCC). See Caronet al., J. Exp Med., US 2011/0059852 A1 Mar. 10, 2011 176:1191-1195 (1992) and Shopes, J. Immunol. 148:2918 bodies may be prepared, for example, as described in U.S. 2922 (1992). Homodimeric antibodies with enhanced anti Pat. No. 6,248.516, incorporated herein by reference in its tumor activity can also be prepared using heterobifunctional entirety. In some embodiments, the present invention pro cross-linkers as described in Wolff et al. Cancer Research, vides domain antibodies derived from an antibody that spe 53:2560-2565 (1993). Alternatively, an antibody can be engi cifically binds to an antigen corresponding to one of the neered that has dual Fc regions and can thereby have cytokine receptors disclosed herein. enhanced complement lysis and ADCC capabilities. See 0103) In another aspect, the present invention includes Stevenson et al., Anti-Cancer Drug Design, 3:219-230 multi-specific antibodies. Multi-specific antibodies include (1989). bispecific, trispecific, etc. antibodies. Bispecific antibodies 0099. In preferred embodiments, the antibodies described can be produced via recombinant means, for example by herein specifically bind to an antigen corresponding to one or using leucine Zipper moieties (i.e., from the Fos and Jun more of the disclosed cytokine receptors present on the cell proteins, which preferentially form heterodimers; Kostelny et surface of hematopoietic tumor cells (HTCs) that arose from al., 1992, J. Immnol. 148: 1547) or other lock and key inter progenitor cell populations in the myeloid lineage of the active domain structures as described in U.S. Pat. No. 5,582, hematopoietic system. Differentiation in the myeloid lineage 996. Additional useful techniques include those described in leads to formation of terminally differentiated cells that U.S. Pat. No. 5,959,083; and U.S. Pat. No. 5,807,706. In one include, among others, megakaryocytes, erythroid cells, mac embodiment, the present invention provides multi-specific rophages, basophils, eosinophils, neutrophils and myeloid antibodies that include an antibody that specifically binds an dendritic cells. These cells originate from hematopoietic stem antigen corresponding to a cytokine receptor disclosed cells (HSC), which differentiate through a series of progeni herein. In another embodiment, the multispecific antibody is tor cell populations displaying progressively restricted differ bispecific. entiation potential. The HSCs and the progenitor cell popu 0104 Bispecific antibodies are also sometimes referred to lations are identifiable from each other based on, among other as “diabodies.” These are antibodies that bind to two (or distinguishing characteristics, their capacity to differentiate more) different antigens. Also known in the art are triabodies into specific cell Subsets and the presence of a particular set of (a trimerized sRV, formed in a manner similar to a diabody, but cellular markers that is specific to the cell population. In some in which three antigen-binding domains are created in a embodiments, the monoclonal antibodies of the present dis single complex; the three antigen binding domains may be closure are directed to progenitor cells of the myeloid lineage directed towards the same or different epitopes) or a tetrabod that are marker-HTCs. In some embodiments, the mono ies (four antigen-binding domains created in a single complex clonal antibodies in the present disclosure are directed to where the four antigen binding domains may be directed committed myeloid progenitor cells that are marker+HTCs towards the same or different epitopes), and the like. Dia tria- and tetrabodies can be manufactured in a variety of ways 5.2.1 Modified Antibodies known in the art (Holliger and Winter, 1993, Current Opinion 0100. In another aspect, the present invention provides Biotechnol. 4:446-449), e.g., prepared chemically or from modified antibodies that are derived from an antibody that hybrid hybridomas. In addition, such antibodies and frag specifically binds an antigen corresponding to a cytokine ments thereof may be constructed by gene fusion (Tomlinson receptor disclosed herein. Modified antibodies also include et. al., 2000, Methods Enzymol. 326:461–479; WO94/13804: recombinant antibodies as described herein. Holliger et al., 1993, Proc. Natl. Acad. Sci. U.S.A. 90:6444 0101) Numerous types of modified or recombinant anti 6448, each of which is incorporated herein by reference in bodies will be appreciated by those of skill in the art. Suitable their entirety). In one embodiment, the diabody, triabody, or types of modified or recombinant antibodies include without tetrabody is derived from an antibody that specifically binds limitation, engineered murine monoclonal antibodies (e.g. an antigen corresponding to a cytokine receptor disclosed murine monoclonal antibodies, chimeric monoclonal anti herein. In a preferred embodiment, the diabody, triabody, or bodies, humanized monoclonal antibodies), domain antibod tetrabody is derived from two or more monoclonal antibodies ies (e.g. Fab., Fv, V. SclV, and dsFv fragments), multivalent that specifically bind to different antigens, each antigen cor or multispecific antibodies (e.g. diabodies, minibodies, min responding to different cytokine receptors disclosed herein. iantibodies, (sclfV), tribodies, and tetrabodies), and anti 0105. In another embodiment, the present invention pro body conjugates as described herein. vides minibodies, which are minimized antibody-like pro 0102. In one aspect, the present invention includes domain teins that include a scFV joined to a CH3 domain, that are antibodies. “Domain antibodies' are functional binding derived from an antibody that specifically binds an antigen domains of antibodies, corresponding to the variable regions corresponding to a cytokine receptor disclosed herein. Mini of either the heavy (VH) or light (VL) chains of human bodies can be made as described in the art (Hu et al., 1996, antibodies. Domain antibodies may have a molecular weight Cancer Res. 56:3055-3061). of approximately 13 kDa, or less than one-tenth the size of a 0106. In another embodiment, the present invention pro full antibody. They are well expressed in a variety of hosts vides binding domain-immunglobulin fusion proteins, where including bacterial, yeast, and mammalian cell systems. In the binding domain specifically binds an antigen correspond addition, domain antibodies are highly stable and retain activ ing to a HTC marker disclosed herein. The fusion protein may ity even after being Subjected to harsh conditions, such as include a marker specific binding domain polypeptide fused freeze-drying or heat denaturation. See, for example, U.S. to an immunoglobulin hinge region polypeptide, which is Pat. No. 6,291,158; 6,582,915; 6,593,081; 6,172,197; US fused to an immunoglobulin heavy chain CH2 constant Serial No. 2004/0110941; European Patent 0368684; U.S. region polypeptide fused to an immunoglobulin heavy chain Pat. No. 6,696,245, WOO4/058821, WOO4/003019 and CH3 constant region polypeptide. Under the present inven WO03/002609. In one embodiment, the domain antibody of tion, marker specific antibody fusion proteins can be made by the present invention is a single domain. Single domain anti methods appreciated by those of skill in the art (See published US 2011/0059852 A1 Mar. 10, 2011 U.S. Patent Application Nos. 20050238646, 20050202534, can result in a multimerized antibody with potent signalling 20050202028, 2005020023, 2005020212, 200501866216, activity. This has been particularly noted in the field of anti 20050180970, and 20050175614, each of which is incorpo tumor agents. For example, it has been reported that the rated herein by reference in their entirety). IgG-IgG homodimerization of anti-CD19, anti-CD20, anti 0107. In another embodiment, the present invention pro CD21, anti-CD22, and anti-Her-2 monoclonal antibodies vides a heavy-chain protein (V) derived from a marker confers potent anti-tumor ability to such homodimers specific antibody. Naturally-occuring heavy chain antibodies (Ghetie, M. et al. (1997) Proc. Natl. Acad. Sci., USA, Vol. 94, (e.g. camelidae antibodies having no light chains) have been pp-7509-7514 incorporated herein by reference in its utilitized to develop antibody-derived therapeutic proteins entirety). In addition, the homodimerization of monoclonal that typically retain the structure and functional properties of antibodies known to have anti-tumor activity, such as Ritux naturally-occuring heavy-chain antibodies. They are known imab R, can lead to an increase in effectiveness as an anti in the art as Nanobodies(R). Under the present invention, heavy tumor agent (Ghetie, M. (2001) Blood, Vo. 97:5: 1392-1398 chain proteins (V) derived from a marker specific heavy incorporated herein by reference in its entirety). chain antibody may be made by methods appreciated by those 0111. In some embodiments, the antibody complexes pro of skill in the art (See published U.S. Patent Application Nos. vided herein include multimeric forms of antibodies directed 20060246477, 20060211088, 20060149041, 200601 15470, to one or more of the antigens corresponding to one or more and 20050214857, each of which is incorporated herein by of the cytokine receptors disclosed herein. For example, anti reference in their entirety). bodies complexes of the invention may take the form of 0108. In one aspect, the present invention provides a modi antibody dimers, trimers, or higher-order multimers of mono fied antibody that is a human antibody. In one embodiment, meric immunoglobulin molecules. Crosslinking of antibod the marker specific antibodies described herein are fully ies can be done through various methods know in the art. For human antibodies. “Fully human antibody or “complete example, crosslinking of antibodies may be accomplished human antibody refers to a human antibody having only the through natural aggregation of antibodies, through chemical gene sequence of an antibody derived from a human chromo or recombinant linking techniques or other methods known in some. The anti-human marker specific complete human anti the art. For example, purified antibody preparations can spon body can be obtained by a method using a human antibody taneously form protein aggregates containing antibody producing mouse having a human chromosome fragment homodimers, and other higher-order antibody multimers. In containing the genes for a heavy chain and light chain of a one embodiment, the present invention provides homodimer human antibody see Tomizuka, K. et al., Nature Genetics, ized antibodies that specifically bind to an antigen corre 16, p. 133-143, 1997; Kuroiwa, Y. et al., Nuc. Acids Res., 26, sponding to a cytokine receptor disclosed herein. p. 3447-3448, 1998: Yoshida, H. et al., Animal Cell Technol 0112 Antibodies can be cross-linked or dimerized ogy: Basic and Applied Aspects Vol. 10, p.69-73 (Kitagawa, through linkage techniques known in the art (see Ghetie et al. Y., Matuda, T. and Iijima, S. eds.), Kluwer Academic Pub (1997) supra: Ghetie etal. (2001) supra). Non-covalent meth lishers, 1999; Tomizuka, K. et al., Proc. Natl. Acad. Sci. USA, ods of attachment may be utilized. In a specific embodiment, 97, 722-727, 2000 or obtained by a method for obtaining a crosslinking of antibodies can be achieved through the use of human antibody derived from a phage display selected from a secondary crosslinker antibody. The crosslinker antibody a human antibody library (see Wormstone, I. M. et al., Inves can be derived from a different animal compared to the anti tigative Ophthalmology & Visual Science. 43 (7), p.2301-8, body of interest. For example, a goat anti-mouse antibody 2002; Carmen, S. et al., Briefings in Functional Genomics (Fab specific) may be added to a mouse monoclonal antibody and Proteomics, 1 (2), p.189-203, 2002; Siriwardena, D. et to form a heterodimer. This bivalent crosslinker antibody al., Ophthalmology, 109(3), p.427-431, 2002). recognizes the Fab or Fc region of the two antibodies of 0109. In one aspect, the present invention provides a interest forming a homodimer. marker specific antibody that is an antibody analog, Some 0113. In one embodiment of the present invention, an anti times referred to as “synthetic antibodies. For example, a body that specifically binds to an antigen corresponding to a variety of recent work utilizes either alternative protein scaf cytokine receptor disclosed herein is cross-linked using a goat folds or artificial scaffolds with grafted CDRs. Such scaffolds anti-mouse antibody (GAM). In another embodiment, the include, but are not limited to, mutations introduced to stabi GAM crosslinker recognizes the Fab or Fc region of two lize the three-dimensional structure of the binding protein as antibodies, each of which specifically binds the same or two well as wholly synthetic scaffolds consisting for example of different antigens corresponding to the same or different biocompatible polymers. See, for example, Korndorfer et al., cytokine receptors disclosed herein. 2003, Proteins. Structure, Function, and Bioinformatics, Volume 53, Issue 1: 121-129. Roque et al., 2004, Biotechnol. 0114 Methods for covalent or chemical attachment of Prog. 20:639-654. In addition, peptide antibody mimetics antibodies may also be utilized. Chemical crosslinkers can be (“PAMs) can be used, as well as work based on antibody homo or heterobifunctional and will covalently bind with two mimetics utilizing fibronection components as a scaffold. antibodies forming a homodimer. Cross-linking agents are well known in the art; for example, homo-or hetero-bifunc tional linkers as are well known (see the 2006 Pierce Chemi 5.2.2 Cross-Linked Antibodies cal Company Crosslinking Reagents Technical Handbook; 0110. In one aspect, the present invention provides cross Hermanson, G. T., Bioconjugate Techniques, Academic linked antibodies that include two or more antibodies Press, San Diego, Calif. (1996); Aslam M. and Dent A. H., described herein attached to each other to form antibody Bioconjugation: protein coupling techniques for the biomedi complexes. Cross-linked antibodies are also referred to as cal sciences, Houndsmills, England: Macmillan Publishers antibody multimers, homoconjugates, and heteroconjugates. (1999); Pierce: Applications Handbook & Catalog, Perbio It has been observed in the art that the multimerization of an Science, Ermbodegem, Belgium (2003-2004); Haughland, antibody previously observed to have no signalling activity R. P., Handbook of Fluorescent Probes and Research Chemi US 2011/0059852 A1 Mar. 10, 2011 cals Eugene,9" Ed., Molecular Probes, OR (2003); and U.S. tion, transcription, translation, Secretion, membrane turnover, Pat. No. 5,747,641; all references incorporated herein by cell adhesion, signal transduction, cell death, and the like. A reference) Those of skill in the art will appreciate the suit bioactive compound includes pharmaceutical compounds. ability of various functional groups on the amino acid(s) of an 0119. In one aspect, the antibodies are conjugated to or antibody for modification, including cross-linking. Suitable modified to carry a detectable compound. Conjugating anti examples of chemical crosslinkers used for antibody bodies to detectable enzymes, fluorochromes, or ligands pro crosslinking include, but not limited to, SMCC succinimidyl vides a signal for visualization or quantitation of the target 4-(maleimidomethyl)cyclohexane-1-carboxylate, SATA antigen. Antibodies may be labeled with various enzymes to N-Succinimidyl S-acethylthio-acetate, hemi-succinate provide highly specific probes that both visualize the target esters of N-hydroxysuccinimide; sulfo-N-hydroxy-succin and amplify the signal by acting on a Substrate to produce a imide; hydroxybenzotriazole, and p-nitrophenol; dicyclo colored or chemiluminescent product. Horseradish peroxi hexylcarbodiimide (DCC), 1-(3-dimethylaminopropyl)-3- dase, alkaline phosphatase, glucose oxidase, and B-galactosi ethylcarbodiimide (ECD), and 1-(3-dimethylaminopropyl)- dase are the commonly used enzymes for this purpose. Fluo 3-ethylcarbodiimide methiodide (EDCI) (see, e.g., U.S. Pat. rochromes, Such as fluorescein isothiocyanate, No. 4,526,714, the disclosure of which is fully incorporated tetramethylrhodamine isothiocyanate (TRITC), phycoeryth by reference herein). Other linking reagents include glu rin, and Cy5, provide a colored reagent for visualization and tathione, 3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4 detection. Suitable fluorescent compounds are described in (3H)-one (DEPBT), onium salt-based coupling reagents, Haughland, R. P. Handbook of Fluorescent Probes and polyoxyethylene-based heterobifunctional cross-linking Research Chemicals Eugene, 9th Ed., Molecular Probes, OR reagents, and other reagents (Haitao, et al., Organ Lett 1:91 (2003). 94 (1999); Albericio et al., J Organic Chemistry 63:9678 0120 In another aspect, the conjugated compounds are 9683 (1998); Arpicco et al., Bioconjugate Chem. 8:327-337 chelating ligands, or macrocyclic organic chelating com (1997); Frischet al., Bioconjugate Chem. 7:180-186 (1996): pounds, particularly metal chelating compounds used to Deguchi et al., Bioconjugate Chem. 10:32-37 (1998); Beyer image intracellular ion concentrations or used as contrast et al., J. Med. Chem.41:2701-2708 (1998); Drouillat et al., J. agents for medical imaging purposes. Chelating ligands are Pharm. Sci. 87:25-30 (1998); Trimble et al., Bioconjugate ligands that can bind with more than one donor atom to the Chem. 8:416–423 (1997)). same central metal ion. Chelators or their complexes have 0115 Exemplary protocols for the formation of antibody found applications as MRI contrast agents, radiopharmaceu homodimers is given in U.S. Patent Publication tical applications, and luminescent probes. Conjugates of 2006.0062786, and Ghetie et al., (1997) supra, which are chelating compounds useful for assessing intracellular ion hereby incorporated by reference in their entirety. In a pre concentrations may be Voltage sensitive dyes and non-voltage ferred embodiment, the chemical cross-linker used is an sensitive dyes. Exemplary dye molecules for measuring intra SMCC or SATA crosslinker. cellular ion levels include, by way of example and not limi 0116. In addition, the antibody-antibody conjugates of tation, Quin-2: Fluo-3; Fura-Red; Calcium Green; Calcium this invention can be covalently bound to each other by tech Orange 550 580; Calcium Crimson; Rhod-2 550 575: SPQ; niques known in the art Such as the use of the heterobifunc SPA; MQAE: Fura-2; Mag-Fura-2: Mag-Funs-5; Di-4- tional cross-linking reagents, GMBS (maleimidobutryloxy ANEPPS: Di-8-ANEPPS; BCECF; SNAFL-1; SBFI; and succinimide), and SPDP (N-succinimidyl 3-(2-pyridyldithio) SBFI. propionate) see, e.g., Hardy, "Purification And Coupling Of I0121. In another embodiment, the ligands are chelating Fluorescent Proteins For Use In Flow Cytometry”. Handbook ligands that bind paramagnetic, Superparamagnetic or ferro Of Experimental Immunology, Volume 1, Immunochemistry, magnetic metals. These are useful as contrastagents for medi Weir et al. (eds.), pp. 31.4-31.124th Ed., (1986), and Ledbet cal imaging and for delivery of radioactive metals to selected ter et al. U.S. Pat. No. 6,010,902, each of which is incorpo cells. Metal chelating ligands, include, by way of example rated herein by reference in their entirety. and not limitation, diethylenetriaminepenta acetic acid 0117. In addition, antibodies may be linked via a thioether (DTPA); diethylenetriaminepenta acetic acid bis(methyla cross-link as described in U.S. Patent Publication mide); macrocyclic tetraamine 1,4,7,10-tetraazacyclodode 20060216284, U.S. Pat. No. 6,368,596, which is incorporated cane-N,N',N',N"-tetraacetic acid (DOTA); and porphyrins herein by reference. As will be appreciated by those skilled in (see, e.g., The Chemistry of Contrast Agents in Medical Mag the art, antibodies can be crosslinked at the Fab region. In netic Resonance Imaging, Merbach A. E. and Toth E., Ed., some embodiments, it is desirable that the chemical Wiley Interscience (2001)). Paramagnetic metal ions, which crosslinker not interact with the antigen-binding region of the are detectable in their chelated form by magnetic resonance antibody as this may affect antibody function. imaging, include, for example, iron(III), gadolinium(III), manganese (II and III), chromium(III), copper(II), dyspro 5.2.3 Conjugated Antibodies sium(III), terbium(III), holmium (III), erbium (III), and 0118. The antibodies disclosed herein can be conjugated europium(III). Paramagnetic metalions particularly useful as to inorganic or organic compounds, including, by way of magnetic resonance imaging contrast agents comprise iron example and not limitation, other proteins, nucleic acids, (III) and gadolinium(III) metal complexes. Other paramag carbohydrates, steroids, and lipids (see for example Green, et netic, Superparamagnetic or ferromagnetic are well known to al., Cancer Treatment Reviews, 26:269-286 (2000). The com those skilled in the art. pound may be bioactive. Bioactive refers to a compound 0.122. In another embodiment, the metal-chelate com having a physiological effect on the cell as compared to a cell prises a radioactive metal. Radioactive metals may be used not exposed to the compound. A physiological effect is a for diagnosis or as therapy based on delivery of small doses of change in a biological process, including, by way of example radiation to a specific site in the body. Targeted metallorad and not limitation, DNA replication and repair, recombina iopharmaceuticals are constructed by attaching the radioac US 2011/0059852 A1 Mar. 10, 2011 tive metalion to a metal chelating ligand. Such as those used mor activity of these agents resides in their ability to create for magnetic imaging, and delivering the chelate-complex to strand breaks in the cellular DNA. Conjugates to antibodies cells. An exemplary radioactive metal chelate complex is have been used to deliver these molecules into those tumor DTPA (see, e.g., U.S. Pat. No. 6,010,679). cells expressing antigens recognized by the antibody and 0123. In a further aspect, the conjugated compounds are shown to have potent antitumor activity with reduced peptide tags used for purposes of detection, particularly unwanted toxicity as compared to the unconjugated com through the use of antibodies directed against the peptide. pounds (Hinman, L. M. et al., Cancer Res. 53:3336-3342 Various tag polypeptides and their respective antibodies are (1993)). Conjugating the enediyne compounds to the compo well known in the art. Examples include poly-histidine (poly sitions described herein provides another method of targeting his) or poly-histidine-glycine (poly-his-gly) tags; the flu HA HTCs of myeloid origin. tag polypeptide and its antibody 12CA5 (Field et al., Mol. 0127 Radioactive compounds are useful as signals (e.g., Cell. Biol. 8:2159-2165 (1988)); the c-myc tag and the 8F9, tracers) or used to provide a therapeutic effect by their deliv 3C7,6E10, G4, B7 and 9E10 antibodies thereto (Evan et al., ery to a cell targeted (e.g., in the form of radiopharmaceutical Mol. Cell. Biol. 5:3610-3616 (1985)); and the Herpes Sim preparations) and may be attached to the antibodies by meth plex virus glycoprotein D (gD) tag and its antibody (Paborsky ods described below. Useful radioactive nuclides include, by et al., Protein Engineering 3:547-553 (1990)). Other tag way of example and not limitation, H, C, P, S, Cr, polypeptides include the Flag-peptide (Hopp et al., BioTech 57Co 5°Fe, 7Ga, 82Rb, 89Sr. 99Tc, 11 In, 123, 125I, 129I. 13 II, nology 6: 1204-1210 (1988)); the KT3 epitope peptide (Mar and 'Re. tin et al., Science 255:192-194 (1992)); tubulin epitope pep I0128. The conjugation of compounds to antibodies is well tide (Skinneret al., J. Biol. Chem. 266:15163-15166 (1991)); known to the skilled artisan, and typically takes advantage of and the T7 gene 10 protein peptide tag (Lutz-Freyermuth et functional groups present on or introduced onto the antibod al., Proc. Natl. Acad. Sci. USA 87:6393-6397 (1990)). ies and compound. Functional groups include, among others, 0124. In another embodiment, the conjugated compounds hydroxyl, amino, thio, imino, and carboxy moieties. Reaction may comprise toxins that cause cell death, or impair cell between functional groups may be aided by coupling reagents survival when introduced into a cell. A suitable toxin is and crosslinking agents. Crosslinking agents and linkers and campylobacter toxin CDT (Lara-Tejero, M., Science 290: corresponding methods for conjugation are described in Her 354-57 (2000)). Expression of the CdtB subunit, which has manson, G. T., Bioconjugate Techniques, Academic Press, homology to nucleases, causes cell cycle arrestand ultimately San Diego, Calif. (1996); Aslam M. and Dent AH., Biocon cell death. Another exemplary toxin is diptheria toxin (and jugation: protein coupling techniques for the biomedical sci similar Pseudomonas exotoxin), which functions by ADP ences, Houndsmills, England: Macmillan Publishers (1999); ribosylating ef-2 (elongation factor 2) molecule in the celland Pierce: Applications Handbook & Catalog, Perbio Science, preventing translation. Entry of the diptheria toxin A subunit Ermbodegem, Belgium (2003-2004); Haughland, R. P. induces cell death in cells containing the toxin fragment. Handbook of Fluorescent Probes and Research Chemicals Other useful toxins include cholera toxin and pertussis toxin Eugene,9' Ed., Molecular Probes, OR (2003); and U.S. Pat. (catalytic subunit-AADPribosylates the G protein regulating No. 5,747,641; all references incorporated herein by refer adenylate cyclase), pierisin from cabbage butterflys, an ence. Exemplary coupling or linking reagents include, by way inducers of apoptosis in mammalian cells (Watanabe, M., of example and not limitation, hemi-succinate esters of N-hy Proc. Natl. Acad. Sci. USA 96:10608-13 (1999)), ribosome droxysuccinimide; Sulfo-N-hydroxy-Succinimide; hydroxy inactivating toxins (e.g., ricin A chain, Gluck, A. et al., J. Mol. benzotriazole, and p-nitrophenol; dicyclohexylcarbodiimide Biol. 226:411-24 (1992)); and nigrin (Munoz, R. et al., Can (DCC), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide cer Lett. 167: 163-69 (2001)). (ECD), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiim 0.125 Bioactive compounds suitable for delivery by the ide methiodide (EDCI) (see, e.g., U.S. Pat. No. 4,526,714) the compositions herein, include, among others, chemotherapeu disclosure of which is fully incorporated by reference herein. tic compounds, including by way of example and not limita Other linking reagents include glutathione, 3-(diethoxyphos tion, vinblastin, bleomycin, taxol, cis-platin, adriamycin, and phoryloxy)-1,2,3-benzotriazin-4(3H)-one (DEPBT), onium mitomycin. Exemplary chemotherapeutic agents suitable for salt-based coupling reagents, polyoxyethylene-based hetero the present purposes are compounds acting on DNA synthesis bifunctional cross-linking reagents, and other reagents that and stability. For example, anti-neoplastic agents of the facilitate the coupling of antibodies to organic drugs and anthracyclin class of compounds act by causing strand breaks peptides and other ligands (Haitao, et al., Organ Lett 1:91-94 in the DNA and are used as standard therapy against cancer. (1999); Albericio et al., J Organic Chemistry 63:9678-9683 Exemplary anti-neoplastic agents of this class are daunoru (1998); Arpicco et al., Bioconjugate Chem. 8:327-337 bicin and doxorubicin. Coupling of these compounds to pro (1997); Frisch et al., Bioconjugate Chem. 7:180-186 (1996): teins, including antibodies, are described in Langer, M. et al., Deguchi et al., Bioconjugate Chem. 10:32-37 (1998); Beyer J. Med. Chem. 44(9): 1341-1348 (2001) and King, H. D. et al., et al., J. Med. Chem.41:2701-2708 (1998); Drouillat et al., J. Bioconjug. Chem. 10:279-288 (1999)). By attaching or link Pharm. Sci. 87:25-30 (1998); Trimble et al., Bioconjugate ing the antineoplastic agents to the antibodies, the compounds Chem. 8:416–423 (1997)). are delivered to HTCs of myeloid origin with a high degree of 0129. Techniques for conjugating therapeutic compounds specificity and promote killing of the targeted cells. to antibodies are also described in Arnon et al., “Monoclonal 0126. Other classes of antitumor agents are the enediyne Antibodies for Immunotargeting of Drugs in Cancers family of antibiotics, representative members of which Therapy,” in Monoclonal Antibodies and Cancer Therapy, include calicheamicins, neocarzinostatin, esperamincins, Reisfeld et al., ed., pp. 243-256, Alan R. Liss, Inc. (1985); dynemicins, kedarcidin, and maduropeptin (see, e.g., Smith, Thorpe, et al. “The Preparation and Cytotoxic Properties of A. L. and Nicolaou, K. C., J. Med. Chem. 39:2103-2117 Antibody Toxin Conjugates. Immunol. Rev. 62:119-58 (1996)). Similar to doxorubicin and daunorubicin, the antitu (1982); and Pietersz, G. A., “The linkage of cytotoxic drugs to US 2011/0059852 A1 Mar. 10, 2011 15 monoclonal antibodies for the treatment of cancer. Biocon backbones (or other Sugarlinkages, such as those described in jugate Chemistry 1(2):89-95 (1990), all references incorpo WO 91/06629) and wherein such sugar linkages are resistant rated herein by reference. to endogenous nucleases. Such oligonucleotides with resis tant Sugar linkages are stable in vivo (i.e., capable of resisting 6. ANTISENSE MOLECULES AND SMALL enzymatic degradation) but retain sequence specificity to be MOLECULE COMPOUNDS able to bind to target nucleotide sequences. Oligonucleotides 0130. Another aspect of the present invention relates to having modified backbones include those that retain a phos antisense and sense molecules comprising a singe-Stranded phorus atom in the backbone and those that do not have a nucleic acid sequence (either RNA or DNA) that can bind to phosphorus atom in the backbone. In other embodiments, mRNA (sense) or DNA (antisense) target sequences corre both the Sugar and the internucleoside linkage, i.e., the back sponding to a cytokine receptor disclosed herein. Antisense or bone, of the nucleotide units are replaced with different sense oligonucleotides, according to the present invention, groups. A further modification includes Locked Nucleic comprise a fragment of the coding region of DNA of the gene Acids (LNAs) in which the 2'-hdroxyl group is linked to the 3' corresponding to a cytokine receptor. Such a fragment gen or 4' carbon atom of the Sugar ring to form a bycyclic Sugar erally comprises at least about 14 nucleotides, preferably moiety. The sense or antisense oligonucleotide molecules from about 14 to about 30 nucleotides. Antisense or sense may also include nucleobase (base) modifications or Substi RNA or DNA molecules are generally at least about 5 nucle tutions. otides in length, alternatively at least about 15, at least about 0.134. The antisense and sense compounds used in accor 30, at least about 50, at least about 100, at least about 150, at dance with this invention may be conveniently and routinely least about 200, at least about 300, at least about 400, at least made through the well-known technique of Solid phase Syn about 500, at least about 700, at least about 800, or at least thesis. Equipment for Such synthesis is sold by several ven about 1000 nucleotides in length. The ability to derive an dors including, for example, Applied BioSystems (Foster antisense or a sense oligonucleotide, based upon a cDNA City, Calif.). Any other means for such synthesis known in the sequence encoding a given protein is described in, for art may additionally or alternatively be employed. It is well example, Stein and Cohen (Cancer Res. 48:2659, 1988) and known to use similar techniques to prepare modified oligo van der Krolet al. (BioTechniques 6:958, 1988). nucleotides. The compounds of the invention may also be 0131 Binding of antisense or sense oligonucleotides to admixed, encapsulated, conjugated or otherwise associated target nucleic acid sequences results in the formation of with other molecules, molecule structures or mixtures of duplexes that block transcription or translation of the target compounds, as for example, liposomes, receptor targeted sequence by one of several means, including enhanced deg molecules, oral, rectal, topical or other formulations, for radation of the duplexes, premature termination of transcrip assisting in uptake, distribution and/or absorption. tion or translation, or by other means. Such methods are 0.135 Antisense or sense oligonucleotides may be intro encompassed by the present invention. The antisense oligo duced into a cell containing the target nucleic acid sequence nucleotides thus may be used to block the expression of by any gene transfer method, including, for example, CaFO proteins corresponding to a cytokine receptor, wherein those mediated DNA transfection, electroporation, or by using marker proteins may play a role in the induction and/or per gene transfer vectors such as Epstein-Barr virus. In a pre sistance of myeloid leukemias in mammals. ferred procedure, an antisense or sense oligonucleotide is 0132 Preferred intragenic sites for antisense binding inserted into a suitable retroviral vector. A cell containing the include the region incorporating the translation initiation/ target nucleic acid sequence is contacted with the recombi start codon (5'-AUG/5'-ATG) or termination/stop codon (5'- nant retroviral vector, either in vivo or ex vivo. Suitable ret UAA, 5'-UAG and 5-UGA/5'-TAA, 5'-TAG and 5'-TGA) of roviral vectors include, but are not limited to, those derived the open reading frame (ORF) of the gene. These regions refer from the murine retrovirus M-Mul V. N2 (a retrovirus derived to a portion of the mRNA or gene that encompasses from from M-MuDV), or the double copy vectors designated about 25 to about 50 contiguous nucleotides in either direc DCT5A, DCT5B and DCT5C (see WO90/13641). tion (i.e., 5' or 3') from a translation initiation or termination 0.136 Sense or antisense oligonucleotides also may be codon. Other preferred regions for antisense binding include: introduced into a cell containing the target nucleotide introns; exons; intron-exonjunctions; the open reading frame sequence by formation of a conjugate with a ligand binding (ORF) or “coding region.” which is the region between the molecule, as described in WO 91/04753. Suitable ligand translation initiation codon and the translation termination binding molecules include, but are not limited to, cell Surface codon; the 5' cap of an mRNA which comprises an N7-me receptors, growth factors, other cytokines, or other ligands thylated guanosine residuejoined to the 5'-most residue of the that bind to cell surface receptors. Preferably, conjugation of mRNA via a 5'-5' triphosphate linkage and includes 5' cap the ligand binding molecule does not Substantially interfere structure itself as well as the first 50 nucleotides adjacent to with the ability of the ligand binding molecule to bind to its the cap; the 5' untranslated region (5' UTR), the portion of an corresponding molecule or receptor, or block entry of the mRNA in the 5' direction from the translation initiation sense or antisense oligonucleotide or its conjugated version codon, and thus including nucleotides between the 5' cap site into the cell. Alternatively, a sense oran antisense oligonucle and the translation initiation codon of an mRNA or corre otide may be introduced into a cell containing the target sponding nucleotides on the gene; and the 3' untranslated nucleic acid sequence by formation of an oligonucleotide region (3'UTR), the portion of an mRNA in the 3' direction lipid complex, as described in WO 90/10448. The sense or from the translation termination codon, and thus including antisense oligonucleotide-lipid complex is preferably disso nucleotides between the translation termination codon and 3' ciated within the cell by an endogenous lipase. end of an mRNA or corresponding nucleotides on the gene. 0.137 In another embodiment, the invention provides 0.133 Antisense or sense oligonucleotides further com small molecules, which bind, preferably specifically, to one prise oligonucleotides having modified Sugar-phosphodiester or more of the cytokine receptor polypeptides disclosed US 2011/0059852 A1 Mar. 10, 2011 herein. In preferred embodiments, the small molecule is a 0144. As used herein, “pharmaceutically acceptable car Small organic molecule, including Small organic molecules rier comprises any standard pharmaceutically accepted car known in the art as being an agonist or antagonist of a riers known to those of ordinary skill in the art in formulating polypeptide corresponding to a cytokine receptor disclosed pharmaceutical compositions. Thus, the antibodies, by them herein. selves, such as being present as pharmaceutically acceptable 0.138. In some embodiments, the small molecule is conju salts, or as conjugates, may be prepared as formulations in gated to a growth inhibitory agent or cytotoxic agent Such as pharmaceutically acceptable diluents; for example, Saline, a toxin. Such toxins include, for example, a maytansinoid or phosphate buffer saline (PBS), aqueous ethanol, or solutions calicheamicin, an antibiotic, a radioactive isotope, a nucle of glucose, mannitol, dextran, propylene glycol, oils (e.g., olytic enzyme, or the like. The small molecules that find use Vegetable oils, animal oils, synthetic oils, etc.), microcrystal in the therapeutic methods of the instant invention preferably line cellulose, carboxymethyl cellulose, hydroxylpropyl induce death of a cell to which they bind. As detailed above, binding to HTCs of myeloid origin may occur by virtue of methyl cellulose, magnesium Stearate, calcium phosphate, binding to a surface-expressed marker disclosded herein; or gelatin, polysorbate 80 or the like, or as solid formulations in by virtue of binding to the surface-expressed marker, which appropriate excipients. itself is bound to its recepetor on myelogenous HTCs. 0145 The pharmaceutical compositions will often further comprise one or more buffers (e.g., neutral buffered saline or 7. PHARMACEUTICAL COMPOSITIONS phosphate buffered saline), carbohydrates (e.g., glucose, mannose, Sucrose or dextrans), mannitol, proteins, polypep 0.139. In the preparation of pharmaceutical compositions tides oramino acids Such as glycine, antioxidants (e.g., ascor comprising the antibodies and/or antisense molecules and/or bic acid, sodium metabisulfite, butylated hydroxytoluene, Small molecule agonists orantagonists described in the teach butylated hydroxyanisole, etc.), bacteriostats, chelating ings herein, a variety of vehicles and excipients and routes of agents such as EDTA or glutathione, adjuvants (e.g., alu administration may be used, as will be apparent to the skilled minium hydroxide), solutes that render the formulation iso artisan. Representative formulation technology is taught in, tonic, hypotonic or weakly hypertonic with the blood of a interalia, Remington: The Science and Practice of Pharmacy, recipient, Suspending agents, thickening agents and/or pre 19th Ed., Mack Publishing Co., Easton, Pa. (1995) and Hand servatives. Alternatively, compositions of the present inven book of Pharmaceutical Excipients, 3rd Ed. Kibbe, A. H. ed., tion may be formulated as a lyophilizate. Washington D.C., American Pharmaceutical Association 0146 While any suitable carrier known to those of ordi (2000); hereby incorporated by reference in their entirety. nary skill in the art may be employed in the compositions of 0140. The pharmaceutical compositions will generally this invention, the type of carrier will typically vary depend comprise a pharmaceutically acceptable carrier and a phar ing on the mode of administration. Antibody compositions macologically effective amount of the antibodies, or mixture may be formulated for any appropriate manner of adminis of antibodies, or suitable salts thereof. Use of a mixture of tration, including for example, oral, nasal, mucosal, intrave monoclonal antibodies specific to a progenitor cell popula nous, intraperitoneal, intradermal, Subcutaneous, and intra tion as a therapeutic has a number of advantages. Abnormally muscular administration. proliferating cells have a tendency to mutate, and thus may 0147 For parenteral administration, the compositions can lose the antigen recognized by a single type of monoclonal be administered as injectable dosages of a solution or Suspen antibody. Moreover, antigen density of a single target antigen sion of the Substance in a physiologically acceptable diluent in the targeted cell could below such that there is insufficient with a pharmaceutical carrier that can be a sterile liquid Such triggering of the signals necessary for destruction of the cell as sterile pyrogen free water, oils, Saline, glycerol, polyeth by the immune system. The present disclosure addresses ylene glycol or ethanol. Additionally, auxiliary Substances, these issues by providing multiple cytokine receptors associ Such as wetting or emulsifying agents, Surfactants, pH buff ated with HTCs of myeloid origin that can be targeted by a ering Substances and the like can be present in compositions. mixture of different monoclonal antibodies. Other components of pharmaceutical compositions are those 0141 For known Small molecule agonists or antagonists, of petroleum, animal, vegetable, or synthetic origin, for pharmaceutical compositions can similarly be prepared based example, non-aqueous solutions of peanut oil, soybean oil, on known characteristics of the molecules. corn oil, cottonseed oil, ethyl oleate, and isopropyl myristate. 0142. The pharmaceutical composition may be formu Antibodies can be administered in the form of a depot injec lated as powders, granules, solutions, Suspensions, aerosols, tion or implant preparation which can be formulated in Such solids, pills, tablets, capsules, gels, topical cremes, supposi a manner as to permit a Sustained release of the active ingre tories, transdermal patches, and other formulations known in dient. An exemplary composition comprises antibody at 5 the art. mg/ml, formulated in aqueous buffer consisting of 50 mM 0143 For the purposes described herein, pharmaceuti L-histidine, 150 mM NaCl, adjusted to pH 6.0 with HC1. cally acceptable salts of the antibodies is intended to include 0.148. Typically, the compositions are prepared as any art recognized pharmaceutically acceptable salts includ injectables, either as liquid solutions or Suspensions; Solid or ing organic and inorganic acids and/or bases. Examples of powder forms suitable for reconstitution with suitable salts include Sodium, potassium, lithium, ammonium, cal vehicles, including by way example and not limitation, sterile cium, as well as primary, secondary, and tertiary amines, pyrogen free water, Saline, buffered solutions, dextrose solu esters of lower hydrocarbons, such as methyl, ethyl, and tion, etc., prior to injection. The preparation also can be propyl. Other salts include organic acids, such as acetic acid, emulsified or encapsulated in liposomes or micro particles propionic acid, pyruvic acid, maleic acid, Succinic acid, tar Such as polylactide, polyglycolide, or copolymers, as further taric acid, citric acid, benzoic acid, cinnamic acid, salicylic discussed below (see, e.g., Langer, Science 249:1527 (1990) acid, etc. and Hanes, Advanced Drug Delivery Rev. 28:97-119 (1997)). US 2011/0059852 A1 Mar. 10, 2011 0149 Additionally, the compositions may also be intro 0154) A variety of materials are useful for making micro duced or encapsulated into the lumen of liposomes for deliv particles. Non-biodegradable microcapsules and micropar ery and for extending their life time ex vivo or in vivo. As ticles include, but not limited to, those made of polysulfones, known in the art, liposomes can be categorized into various poly(acrylonitrile-co-vinyl chloride), ethylene-vinyl acetate, types: multilamellar (MLV), stable plurilamellar (SPLV), hydroxyethylmethacrylate-methyl-methacrylate copoly small unilamellar (SUV) or large unilamellar (LUV) vesicles. mers. These are useful for implantation purposes where the Liposomes can be prepared from various lipid compounds, encapsulated composition diffuses out from the capsules. In which may be synthetic or naturally occurring, including another aspect, the microcapsules and microparticles are phosphatidyl ethers and esters, such as phosphotidylserine, phosphotidylcholine, phosphatidyl ethanolamine, phosphati based on biodegradable polymers, preferably those that dis dylinositol, dimyristoylphosphatidylcholine; Steroids such as play low toxicity and are well tolerated by the immune sys cholesterol; cerebrosides; sphingomyelin; glycerolipids; and tem. These include protein based microcapsulates and micro other lipids (see, e.g., U.S. Pat. No. 5,833,948). particles made from fibrin, casein, serum albumin, collagen, 0150 Cationic lipids are also suitable for forming lipo gelatin, lecithin, chitosan, alginate or poly-amino acids Such Somes. Generally, the cationic lipids have a net positive as poly-lysine. Biodegradable synthetic polymers for encap charge and have a lipophilic portion, such as a sterol oran acyl Sulating may comprise polymers such as polylactide (PLA), or diacyl side chain. Preferably, the head group is positively polyglycolide (PGA), poly(lactide-co-glycolide) (PLGA), charged. Typical cationic lipids include 1,2-dioleyloxy-3-(tri poly(caprolactone), polydioxanone trimethylene carbonate, methylamino)propane; N-1-(2,3-ditetradecycloxy)propyl polyhybroxyalkonates (e.g., poly(3-hydroxybutyrate)), poly N,N-dimethyl-N-N-hydroxyethylammonium bromide; (y-ethyl glutamate), poly(DTH iminocarbony (bisphenol A N-1-(2,3-dioleyloxy)propyl-N,N-dimethyl-N-hydroxy iminocarbonate), poly (ortho ester), and polycyanoacrylate. ethylammonium bromide: N-1-(2,3-dioleyloxy)propyl-N, Various methods for making microparticles containing the N,N-trimethylammonium chloride: 3-N-(N',N'-dimethy Subject compositions are well known in the art, including laminoethane)carbamoylcholesterol; and dimethyldioctade solvent removal process (see, e.g., U.S. Pat. No. 4.389.330); cylammonium. emulsification and evaporation (May singer, D. et al., Exp. 0151. Liposomes also include vesicles derivatized with a Neuro. 141: 47-56 (1996); Jeffrey, H. et al., Pharm. Res. 10: hydrophilic polymer, as provided in U.S. Pat. Nos. 5,013.556 362-68 (1993)), spray drying, and extrusion methods. and 5,395,619, hereby incorporated by reference, (see also, 0.155. Another type of carrier is nanoparticles, which are Kono, K. et al., J. Controlled Release 68: 225-35 (2000); generally suitable for intravenous administrations. Submi Zalipsky, S. et al., Bioconjug. Chem. 6: 705-708 (1995)) to cron and nanoparticles are generally made from amphiphilic extend the circulation lifetime in vivo. Hydrophilic polymers diblock, triblock, or multiblock copolymers as is known in the for coating or derivation of the liposomes include polyethyl art. Polymers useful informing nanoparticles include, but are ene glycol, polyvinylpyrrolidone, polyvinylmethyl ether, limited to, poly(lactic acid) (PLA; see Zambaux et al., J. polyaspartamide, hydroxymethyl cellulose, hydroxyethyl Control Release 60: 179-188 (1999)), poly(lactide-co-gly cellulose, and the like. collide), blends of poly(lactide-co-glycolide) and polycarpro 0152 Liposomes are prepared by ways well known in the lactone, diblock polymer poly(1-leucine-block-l-glutamate), art (see, e.g., Szoka, F. et al., Ann. Rev. BiophyS. Bioeng. 9: 467-508 (1980)). One typical method is the lipid film hydra diblock and triblock poly(lactic acid) (PLA) and poly(ethyl tion technique in which lipid components are mixed in an ene oxide) (PEO) (De Jaeghere, F. et al., Pharm. Dev. Tech organic solvent followed by evaporation of the solvent to nol. 5: 473-83 (2000)), acrylates, arylamides, polystyrene, generate a lipid film. Hydration of the film in aqueous buffer and the like. As described for microparticles, nanoparticles Solution, preferably containing the Subject antibodies, results may be non-biodegradable or biodegradable. Nanoparticles in an emulsion, which is Sonicated or extruded to reduce the may be also be made from poly(alkylcyanoacrylate), for size and polydispersity. Other methods include reverse-phase example poly(butylcyanoacrylate), in which the therapeutic evaporation (see, e.g., Pidgeon, C. et al., Biochemistry 26: composition is absorbed onto the nanoparticles and coated 17-29 (1987); Duzgunes, N. et al., Biochim. Biophys. Acta. with surfactants (e.g., polysorbate 80). Methods for making 732: 289-99 (1983)), freezing and thawing of phospholipid nanoparticles are similar to those for making microparticles mixtures, and ether infusion. and include, among others, emulsion polymerization in con 0153. In another embodiment, the carriers are in the form tinuous aqueous phase, emulsification-evaporation, Solvent of microparticles, microcapsules, micropheres and nanopar displacement, and emulsification-diffusion techniques (see, ticles, which may be biodegradable or non-biodegradable e.g., Kreuter, J. Nano-particle Preparation and Applications, (see, e.g., “Microencapsulates: Methods and Industrial Appli in Microcapsules and Nanoparticles in Medicine and Phar cations.” in Drugs and Pharmaceutical Sciences, Benita, S. macy, pg. 125-148, (M. Donbrow, ed.) CRC Press, Boca ed, Vol 73, Marcel Dekker Inc., New York (1996); incorpo Rotan, Fla. (1991); incorporated herein by reference). rated herein by reference). As used herein, microparticles, 0156 The pharmaceutical compositions described herein microspheres, microcapsules and nanoparticles mean a par may be presented in unit-dose or multi-dose containers. Such ticle, which is typically a solid, containing the Substance to be as sealed ampoules or vials. Such containers are typically delivered. The substance is within the core of the particle or sealed in such a way to preserve the sterility and stability of attached to the particle's polymer network. Generally, the the formulation until use. In general, formulations may be difference between microparticles (or microcapsules or stored as Suspensions, Solutions or emulsions in oily or aque microspheres) and nanoparticles is one of size. As used ous vehicles, as indicated above. Alternatively, a pharmaceu herein, microparticles have a particle size range of about 1 to tical composition may be stored in a freeze-dried condition about >1000 microns. Nanoparticles have a particle size requiring only the addition of a sterile liquid carrier immedi range of about 10 to about 1000 nm. ately prior to use. US 2011/0059852 A1 Mar. 10, 2011 18 8. USE OF ANTIBODIES AND OTHER AGENTS groups of conditions: dysmyelopoietic disorder, acute myelo proliferative leukemia, and chronic myeloproliferative disor 8.1 Therapeutic Use of Antibodies and Small Mol der. ecules 016.1 Myelodysplastic Syndromes (MDS) include a group of closely-related blood formation disorders, in which 0157 Methods of immunotargeting cancer cells using the bone marrow shows qualitative and quantitative changes antibodies or antibody fragments are well known in the art. Suggestive of a preleukemic process, but having a chronic U.S. Pat. No. 6,306,393 describes the use of anti-CD22 anti course that does not necessarily terminate as acute leukemia. bodies in the immunotherapy of B-cell malignancies, and A variety of terms, including preleukemia, refractory anemia, U.S. Pat. No. 6,329.503 describes immunotargeting of cells refractory dysmyelopoietic anemia, Smoldering or Subacute that express serpentine transmembrane antigens. Antibodies leukemia, dysmyelopoietic syndrome (DMPS), and myelod described herein (including humanized or human mono ysplasia, have all been used to describe MDS. These condi clonal antibodies or fragments or other modifications thereof, tions are all characterized by a cellular marrow with impaired optionally conjugated to cytotoxic agents) can be introduced maturation (dysmyelopoiesis) and a reduction in the number of blood cells. DMPS is characterized by presence of mega into a patient such that the antibody binds to cancer cells or blastoids, megarkaryocyte dysplasia, and an increase in num their secreted expression products and mediates the destruc ber of abnormal blast cells, reflective of enhanced granulo tion of the cells and the tumor and/or inhibits the growth of the cyte maturation process. Patients with DMPS show cells or the tumor. Without intending to limit the disclosure, chromosomal abonormalities similar to those found in acute mechanisms by which Such antibodies can exerta therapeutic myeloid leukemia and progress to acute myeloid leukemia in effect may include complement-mediated cytolysis, anti a certain fraction of afflicted patients (Kardon, N. et al., body-dependent cellular cytotoxicity (ADCC), modulating Cancer 50(12):2834-2838 (1982)). the physiologic function of the tumor antigen, inhibiting 0162 Acute myeloproliferative leukemia (AML), also binding or signal transduction pathways, modulating tumor known as acute nonlymphocytic leukemia, acute myelocytic cell differentiation or proliferation, altering tumor angiogen leukemia, acute myeloblastic leukemia, and acute granulo esis factor profiles, modulating the secretion of immune cytic leukemia, is characterized by the presence of abnormal stimulating or tumor Suppressing cytokines and growth fac hematopoietic progenitor cells that have been blocked at an tors, modulating cellular adhesion, and/or by inducing apop undifferentiated or partially differentiated stage of matura tosis. The antibodies can also be conjugated to toxic or other tion, and thus are unable to differentiate into myeloid, eryth therapeutic agents, such as radioligands or cytosolic toxins, roid, and/or megakaryocytic cell lines. The abnormal cells discussed in detail above, and may also be used therapeuti block differentiation of normal progenitor cells in the bone cally to deliver the toxic ortherapeutic agent directly to tumor marrow, resulting in thrombocytopenia, anaemia, and granu cells. locytopenia. Diagnosis of AML is made when at least 30% of 0158. In preferred embodiments, the compositions have nucleated cells in the bone marrow are blasts. Acute myeloid applications to the treatment of conditions or diseases involv leukemia is further divided into subtypes M1 to M7 based on ing myeloid cells of the hematopoietic system. The present morphology of the proliferating cells and cytochemical stain disclosure further provides methods of using the antibodies to ing properties. target myeloid leukemic stem cells. For example, the disclo 0163 Chronic myeloproliferative disorders are a collec Sure provides methods of using the antibodies to treat disor tion of conditions characterized by increased number of ders involving cells of the myeloid lineages. Various diseases mature and immature granulocytes, erythrocytes, and plate have origins in the committed progenitor cell populations, or lets. Chronic myeloproliferative disorders can transition to involve progenitor cells by differentiation of diseased cells other forms within this group, with a tendency to terminate in through the myeloid pathway. acute myeloid leukemia. Specific diseases within this group 0159. By “treatment herein is meant therapeutic or pro include polycythemia Vera, chronic myeloid leukemia, agno phylactic treatment, or a Suppressive measure for the disease, genic myeloid leukemia, essential thrombocythemia, and disorder or undesirable condition. Treatment encompasses chronic neutrophilic leukemia. administration of the Subject antibodies in an appropriate 0164. The therapeutic preparations can use non-modified form prior to the onset of disease symptoms and/or after marker specific antibodies or antibodies conjugated with a clinical manifestations, or othermanifestations, of the disease therapeutic compound, such as a toxin or cytotoxic molecule, to reduce disease severity, halt disease progression, or elimi depending on the functionality of the antibody. Generally, nate the disease. Prevention of the disease includes prolong when non-modified antibodies are used, they will typically ing or delaying the onset of symptoms of the disorder or have a functional Fc region. By “functional Fc region' herein disease, preferably in a subject with increased susceptibility is meant a minimal sequence for effecting the biological to the disease. function of Fc. Such as binding to Fc receptors, particularly 0160 The antibodies described herein are particularly FcyR(e.g., FcyRI, FcyRII, and FcyRIII). Without being bound applicable to the treatment of myeloproliferative disorders, by theory, it is believed that the Fc region may affect the also referred to generally as hematopoietic malignancies, effectiveness of anti-tumor monoclonal antibodies by binding which are proliferative disorders involving cells of the to Fc receptors immune effector cells and modulating cell myeloid lineage. The term malignancy refers to growth and mediated cytotoxicity, endocytosis, phagocytosis, release of proliferation of one or more clones of abnormal cells. Leu inflammatory cytokines, complement mediated cytotoxicity, kemiatypically describes a condition in which abnormal cells and antigen presentation. In this regard, polyclonal antibod are present in the bone marrow and peripheral blood. Myelo ies, or mixtures of monoclonals, will be advantageous proliferative disorders, also called myeloid leukemias or because they will bind to different epitopes (of a single anti myelogenous leukemias, are categorized into three general gen or of different antigens corresponding to different cytok US 2011/0059852 A1 Mar. 10, 2011 ine receptors) and thus have a higher density of Fc on the cell 142praseodymium, 143praseodymium, 145praseody Surface as compared to when a single monoclonal antibody is mium, 149promethium, 150 europium, 165 dysprosium, used. Of course, to enhance their effectiveness in depleting 111 indium, 131 iodine, 125iodine, 123iodine, 88 lyt targeted cells, or where non-modified antibodies are not trium and 90 yttrium. Suitable radioactive radionuclides therapeutically effective, antibodies conjugated to toxins or include without limitation, Cu, 'Y, 'I, I, Re, Re, cytotoxic agents may be used. Thus, not only are the antibod '''At, ''Bi. These and other uses of the antibodies will be ies useful as therapeutic molecules themselves, they also find apparent to those of ordinary skill in the art in light of the utility in targeted delivery of therapeutic molecules to disclosures provided herein. myeloid cells. 0169. Known Small molecule agonists or antagonists can 0.165 Alternatively, where the antibodies exhibit a direct also find use in methods of treating of one or more of the effect on antigen and/or cell function, enhancement of the Fc myelogenous hematological proliferative disorders described receptor functionality may be less significant. For example, above. Generally, the methods comprise administration of a this may be the case where binding of the marker specific therapeutically effective amount of the small molecule (or antibody Sterically inhibits interaction between antigen and mixture of small molecules). Similarly as detailed above with its corresponding ligand. respect to immunotherapies, the therapeutic composition can 0166 The antibody compositions may be used either use the non-modified Small molecule or, optionally, the Small alone or in combination with other therapeutic agents to molecule is conjugated to a toxin or cytotoxic molecule or increase efficacy of traditional treatments for myeloid leuke growth inhibitory agent. For example, the Small molecule mias, or to target abnormal cells not targeted by the antibod may be conjugated to a maytansinoid or calicheamicin, an ies. Combining the antibody therapy method with a chemo antibiotic, a radioactive isotope, a nucleolytic enzyme, or the therapeutic, radiation or Surgical regimen may be preferred in like. patients that have not received chemotherapeutic treatment, 0170 Binding of the small molecule to a HTC of myeloid whereas treatment with the antibody therapy may be indi origin preferably induce cell death. Cell death may be medi cated for patients who have received one or more chemothera ated by the conjugated cytotoxic molecule or by the physi pies. Additionally, antibody therapy can also enable the use of ological response induced by the binding of the Small mol reduced dosages of concomitant chemotherapy, particularly ecule itself. in patients that do not tolerate the toxicity of the chemothera peutic agent very well. Furthermore, antibody treatment of 8.1.1. Administration and Dosages cancer patients with tumors resistant to chemotherapeutic (0171 The amount of the compositions needed for achiev agents might induce sensitivity and responsiveness to these ing a therapeutic effect will be determined empirically in agents in combination. accordance with conventional procedures for the particular 0167. In one aspect, the antibodies are used adjunctively purpose. Generally, for administering the compositions ex with therapeutic cytotoxic agents, including, by way of Vivo or in vivo for therapeutic purposes, the compositions are example and not limitation, buSulfan, thioguanine, idarubi given at a pharmacologically effective dose. By “pharmaco cin, cytosine arabinoside, 6-mercaptopurine, doxorubicin, logically effective amount’ or “pharmacologically effective daunorubicin, etoposide, and hydroxyurea. Other agents use dose' is meant an amount Sufficient to produce the desired ful as adjuncts to antibody therapy are compounds directed physiological effect or amount capable of achieving the specifically to an abnormal cellular molecule found in the desired result, particularly for treating or retreating the disor disease state. These agents can be disease specific. For der or disease condition, including reducing or eliminating example, for treating chronic myeloid leukemia arising from one or more symptoms or manifestations of the disorder or BCR-ABL activity, one class of useful compounds are inhibi disease. As an illustration, administration of antibodies to a tors of ablkinase activity, such as Imatinib, an inhibitor of patient suffering from a myeloproliferative disorder provides bcr-abl kinase, and antisense oligonucleotides against bcr a therapeutic benefit not only when the underlying disease is (e.g., Oblimersen). Other agents include, among others, inter eradicated or ameliorated, but also when the there is a feron-alpha, humanized anti-CD52, deacetylase inhibitor decrease in the severity or duration of the symptoms associ FR901228 (depsipeptide), and the like. ated with the disease. Therapeutic benefit also includes halt 0.168. In another aspect, isotopes are attached to the anti ing or slowing the progression of the underlying disease or bodies and/or fragments for therapeutic purposes. By “iso disorder, regardless of whether improvement is realized. tope' is meant atoms with the same number of protons and 0172. The amount administered to the subject will vary hence of the same element but with different numbers of depending upon the agent being administered, the purpose of neutrons (e.g., H vs. Hor D). The term "isotope' includes the administration, Such as prophylaxis or therapy, the state of “stable isotopes’, e.g. non-radioactive isotopes, as well as the Subject, the manner of administration, the number of “radioactive isotopes, e.g. those that decay over time, and administrations, the intervals between administrations, and radioactive radionuclides. In one embodiment, the antibodies the like. These can be determined empirically by those skilled and/or fragments are labeled with a radioisotope, which are in the art and may be adjusted based on the extent of the useful in radioimmunotherapy. Suitable radioisotopes therapeutic response. Factors to consider in determining an include without limitation an alpha-emitter, a beta-emitter, appropriate dose include, but are not limited to, size and and an Auger electron-emitter (Behrt, T. et al., (2000). Eur: J. weight of the Subject, the age and sex of the Subject, the Nuclear Med. Vol. 27 (7):753-765: Vallabhajosula, S. et al., J. severity of the symptoms, the stage of the disease, method of Nucl. Med. (2005) April; 46(4): 634-41). Such radioisotopes delivery of the agents, half-life of the agents, efficacy of the include without limitation 65Zinc, 140 neodymium, 177 agents, and what other therapy regimes have been or will be lutetium, 179 lutetium, 176mlutetium, 67 gallium, 159 administered. Stage of the disease to consider includes gallium, 161terbium, 153samarium, 169erbium, 175 whether the disease is acute or chronic, relapsing or remitting ytterbium, 161 holmium, 166holmium, 167thulium, phase, as well as the progressiveness of the disease. Deter US 2011/0059852 A1 Mar. 10, 2011 20 mining the dosages and times of administration for a thera done in a variety of ways, including, but not limited to, con peutically effective amount are well within the skill of the tinuously, Subcutaneously, intravenously, orally, topically, ordinary person in the art, in light of the disclosures provided transdermal, intraperitoneal, intramuscularly, and intravesi herein. cally. For example, microparticle, microsphere, and microen 0173 For any compositions of the present disclosure, the capsulate formulations are useful for oral, intramuscular, or therapeutically effective dose is readily determined by meth Subcutaneous administrations. Liposomes and nanoparticles ods well known in the art. For example, an initial effective are additionally Suitable for intravenous administrations. dose can be estimated from cell culture or other in vitro Administration of the pharmaceutical compositions may be assays. For example, Sliwkowsky, M.X. et al., Semin. Oncol. through a single route or concurrently by several routes. For 26(suppl. 12) 60-70 (1999) describes in vitro measurements instance, intraperitoneal administration can be accompanied of antibody dependent cellular cytoxicity. A dose can then be by intravenous injections. Preferably the therapeutic doses formulated in animal models to generate a circulating con are administered intravenously, intraperitonealy, intramuscu centration or tissue concentration, including that of the ICso larly, or Subcutaneously. In some embodiments, the Small as determined by the cell culture assays. A Suitable animal molecule compositions can be administered orally model for leukemia is described in detail in the Examples 0.178 The compositions may be administered once or sev below. eral times. In some embodiments, the compositions may be 0.174. In addition, the toxicity and therapeutic efficacy are administered once per day, a few or several times per day, or generally determined by cell culture assays and/or experi even multiple times per day, depending upon, among other mental animals, typically by determining a LDs (lethal dose things, the indication being treated and the judgement of the to 50% of the test population) and EDs (therapeutically prescribing physician. effectiveness in 50% of the test population). The dose ratio of 0179 Administration of the compositions may also be toxicity and therapeutic effectiveness is the therapeutic index. achieved through Sustained release or long-term delivery Preferred are compositions, individually or in combination, methods, which are well known to those skilled in the art. By exhibiting high therapeutic indices. Determination of the “sustained release or “long term release' as used herein is effective amount is well within the skill of those in the art, meant that the delivery system administers a pharmaceuti particularly given the detailed disclosure provided herein. cally therapeutic amount of Subject agents for more than a Guidance is also found in standard reference works, for day, preferably more thana week, and most preferable at least example Fingl and Woodbury, General Principles In: The about 30 days to about 60 days, or longer. Long term release Pharmaceutical Basis of Therapeutics pp. 1-46 (1975), and systems may comprise implantable Solids or gels containing the references cited therein. the antibodies, such as biodegradable polymers described 0175 To achieve an initial tolerizing dose, consideration above (Brown, D. M. et al., Anticancer Drugs 7: 507-513 is given to the possibility that the antibodies may be immu (1996)); pumps, including peristaltic pumps and fluorocar nogenic in humans and in non-human primates. The immune bonpropellant pumps; osmotic and mini-osmotic pumps; and response may be biologically significant and may impair the the like. therapeutic efficacy of the antibody even if the antibody is 0180. The method of the invention contemplates the partly or chiefly comprised of human immunoglobulin administration of marker specific monoclonal antibodies, as sequences such as, for example, in the case of a chimeric or well as combinations of different mAbs. As discussed above, humanized antibody. Within certain embodiments, an initial two or more monoclonal antibodies may provide an improved high dose of antibody is administered such that a degree of effect compared to a single antibody. For example, a combi immunological tolerance to the therapeutic antibody is estab nation of a monoclonal antibody with another monoclonal lished. The tolerizing dose is sufficient to prevent or reduce antibody that binds a different antigen, e.g., an antigen cor the induction of an antibody response to repeat administration responding to a different cytokine receptor, may provide an of the marker specific antibody. Preferred ranges for the tol improved effect compared to a single antibody. Such mab erizing dose are between 10 mg/kg body weight to 50 mg/kg "cocktails' may have certain advantages in as much as they body weight, inclusive. More preferred ranges for the toler contain mAbs, which exploit different effector mechanisms izing dose are between 20 and 40 mg/kg, inclusive. Still more or combine directly cytotoxic mAbs with mAbs that rely on preferred ranges for the tolerizing dose are between 20 and 25 immune effector functionality. Specific mAbs in combination mg/kg, inclusive. may exhibit synergistic therapeutic effects. Some methods of 0176 Within these therapeutic regimens, the therapeuti the invention also contemplate adminisration of one or more cally effective dose of antibodies is preferably administered known Small molecule agonists or antagonists, alone or in in the range of 0.1 to 10 mg/kg body weight, inclusive. More combination with one or more mabs described herein. The preferred second therapeutically effective doses are in the Small molecule in combination with specific mAbs may range of 0.2 to 5 mg/kg body weight, inclusive. Still more exhibit synergistic therapeutic effects. preferred therapeutically effective doses are in the range of 0.5 to 2 mg/kg, inclusive. Within alternative embodiments, 8.2 Diagnostic Use of Antibodies and Other Agents the Subsequent therapeutic dose or doses may be in the same 0181. The present invention further provides methods to or different formulation as the tolerizing dose and/or may be identify the presence of an antigen using the compositions of administered by the same or different route as the tolerizing the present invention, optionally conjugated or otherwise dose. associated with a suitable label. Such methods comprise incu 0177. For the purposes of this invention, the methods of bating a test sample with one or more of the marker specific administration are chosen depending on the condition being antibodies of the present invention and assaying for antibod treated, the form of the subjectantibodies or other agents, and ies that bind to components within the test sample. Conditions the pharmaceutical composition. Administration of the anti for incubating the antibody with a test sample may vary. body compositions or Small molecule compositions can be Incubation conditions depend on the format employed, the US 2011/0059852 A1 Mar. 10, 2011 detection methods employed, and the antibody used in the Additionally, variants of genes and gene expression products assay. One skilled in the art will recognize that any one of the including, for example, spliced variants and polymorphic commonly available immunological assay formats can alleles, can be detected. readily be adapted to employ antibodies of the present inven 0186 Methods of detecting the level of an RNA transcript tion (see Chard, T. An Introduction to Radioimmunoassay include, for example, utilizing a specific hybridization probe and Related Techniques, Elsevier Science Publishers, or an array of such probes. In a preferred embodiment, the Amsterdam, The Netherlands (1986); Bullock, G. R. et al., probe comprises a polynucleotide sequence that can hybrid Techniques in Immunocytochemistry, Academic Press, ize to all or a portion of the transcribed RNA sequence. Orlando, Fla. Vol. 1 (1982), Vol. 2 (1983), Vol. 3 (1985); 0187. The stringency conditions allowing hybridization Tijssen, P., Practice and Theory of immunoassays: Labora can be high to moderate to low. As used herein, conditions of tory Techniques in Biochemistry and Molecular Biology, moderate stringency refer to those known to the ordinarily Elsevier Science Publishers, Amsterdam, The Netherlands skilled artisian, e.g., as defined by Sambrook et al. Molecular (1985). Cloning: A Laboratory Manual, 2 ed. Vol. 1, pp. 1.101-104. 0182. The sample to be assessed can be any sample that Cold Spring Harbor Laboratory Press, (1989). Morerate strin contains an expression product (e.g., RNA transcript or extra gency conditions include use of a prewashing solution for cellular protein). A “test sample generally refers to a sample nitrocellulose filters 5xSSC, 0.5% SDS, 1.0 mM EDTA (pH obtained or derived from a patient afflicted with, or suspected 8.0), hybridization conditions of 50% formamide, 6xSSC at of being afflicted with, a hematological proliferative disorder 42°C. (or other similar hybridization solution, such as Stark's of myeloid origin. The test samples of the present invention solution, in 50% formamide at 42°C.), and washing condi include cells, protein or membrane extracts of cells, or bio tions of about 60° C., 0.5xSSC, 0.1% SDS. High stringency logical fluids. Samples can comprise brain, blood, serum, conditions usually involve, for example, hybridization con plasma, lymphatic fluid, bone marrow, plasma, lymph, urine, ditions as above, with washing at 68° C., 0.2xSSC, 0.1% tissue, mucus, sputum, saliva or other cell samples. The test SDS. The skilled artisan will recognize that the temperature sample used will vary based on the assay format, nature of the and wash solution salt concentration can be adjusted as nec detection method and the tissues, cells or extracts used and essary according to factors such as the length of the probe. the sample to be assayed. Methods for preparing protein The hybridization probe can be of any length and usually extracts or membrane extracts of cells are well known in the consists of at least about 5 nucleotides, at least about 10, at art and can be readily adapted in order to obtain a sample least about 15, at least about 20, or at least about 30 nucle which is compatible with the system utilized. otides. Longer lengths are suitable to lower stringency con 0183 In preferred embodiments, the test sample is ditions. obtained from peripheral blood, particularly from mobilized 0188 The probe may include natural (i.e. A, G, U.C. or T) peripheral blood (MPB). The sample initially obtained from a or modified bases (7-deazaguanosine, inosine, etc.). In addi Subject is generally enriched for stem cells. Hematopoietic tion, the bases in probes may be joined by a linkage other than stem cells can be identified by the presence or absence of a phosphodiester bond, so long as the bond does not interfere certain Surface markers, as described above, including, e.g., with hybridization. Thus, probes may be peptide nucleic CD34"; CD38; as well as Lin and/or CD90". acids in which the constituent bases are joined by peptide 0184 The present invention provides diagnostic methods bonds rather than phosphodiester linkages. for hematological proliferative disorders of myeloid origin, 0189 In some embodiments, more than one hybridization where the level of an expression product of one or more of the probe is used, e.g., two, three, four, five, 10 or more probes, up disclosed cytokine receptors is detected. “Detected,” and its to, including and beyond as many probes as cytokine recep grammatical variations, refers to assessing, measuring, read tors disclosed herein. In some embodiments, different probes ing, or otherwise determining a value for the level or amount are directed to different RNA products, e.g., RNA products of expression product corresponding to one of more of the corresponding to two or more different cytokine receptors cytokine receptors disclosed herein. An expression product disclosed herein. For example, probes for detecting the "corresponds to a designated cytokine receptor when it is expression level of two, three, four, five, or more of the derived therefrom via transcription and/or translation of the cytokine receptor disclosed herein may be used. gene encoding the designated cytokine receptor. Expression 0190. In preferred embodiments, the probes are immobi levels refer to the amount of expression of the expression lized, e.g., on an array, in different known locations. An array product, as described herein. A value for an expression level refers to a solid support with a surface to which a plurality of is also referred to as a “signal.” The values for expression different nucleic acid sequences (probes) are attached. The levels can be absolute or relative values, e.g., values provided array can be prepared either synthetically or biosynthetically, in comparison to control levels. The values for expression and can assume a variety of formats, e.g., libraries of com levels can be raw values, or values that are optionally res pounds tethered to resin beads, silica chips, or other solid caled, filtered and/or normalized. The approach used will Supports, as well as libraries of nucleic acids prepared by depend, for example, on the nature of the expression product spotting nucleic acids onto a substrate. The Solid Support can (e.g., RNA or polypeptide) as well as specific characteristics be any material or group of materials having a rigid or semi of the product, and the intended use for the data. rigid surface or Surfaces. Generally, at least one Surface of the 0185. For example, in one embodiment, the expression Solid Support will be substantially flat, although in some case product is a transcription product, such as RNA. RNA the array will include wells, raised regions, pins, etched includes, e.g. mRNA rRNA, tRNA, snRNA, and the like, trenches, or the like. Although a planar array Surface is pre inclulding any nucleic acid molecule that is transcribed from ferred, the array may be fabricated on a surface of virtually a gene. The nucleic acid molecule levels measured can be any shape or even a multiplicity of Surfaces. Solid Support(s) derived directly from the gene or, alternatively, from a corre can also take the form of beads, resins, fibers such as fiber sponding regulatory gene or regulatory sequence element. optics, gels, microspheres, or other geometric configurations. US 2011/0059852 A1 Mar. 10, 2011 22 See U.S. Pat. Nos. 5,770,358, 5,789,162, 5,708,153, 6,040, detected and measured. Methods of detecting the level of 193 and 5,800,992, which are hereby incorporated in their proteins preferably involve utilizing antibodies of the instant entirety for all purposes. disclosure, as discussed above. In some embodiments, Small 0191 Such arrays are generally termed “microarrays', or molecules known to bind to a polypeptide corresponding to a colloquially “chips, and have been described in the art, for disclosed cytokine receptor can similarly be detectably example, U.S. Pat. Nos. 5,143,854, 5,445,934, 5,744,305, labeled and/or attached to a solid support. 5,677, 195, 6,040,193, 5,424,186 and Fodor et al., Science, 0196. In some embodiments, more than one antibody is 251:767-777 (1991), each of which is incorporated by refer used, e.g., two or more mabs. In some embodiments, differ ence in its entirety for all purposes. These arrays may gener ent mAbs are directed to a proteinaceous product expressed ally be produced using mechanical synthesis methods or light from two or more different cytokine receptors disclosed directed synthesis methods, which can incorporate a combi herein. For example, antibodies for detecting the expression nation of photolithographic methods and solid phase synthe level of two, three, four, five, or more of the different cytokine sis methods. Techniques for the synthesis of these arrays receptors disclosed herein may be used. In some embodi using mechanical synthesis methods are described in, e.g., ments, both RNA and protein levels are detected in a given U.S. Pat. No. 5,384.261, incorporated herein by reference in sample or multiple samples from an individual. its entirety for all purposes. Commercially available probes 0197) The RNA or antigen level can be compared to con and arrays can be used, including, for example, Affymetrix trol levels, e.g., levels obtained using normal HSCs. A control human 0133 Plus 2.0 Array. sample comprising one or more normal HSCs can be 0.192 In a preferred embodiment, the expression product obtained, e.g., from an individual not afflicted with a hema is mRNA and the mRNA levels are obtained by contacting the tological proliferative disorder, e.g., not afflicted with a hema sample with a suitable microarray, and determining the extent tological proliferative disorder of myeloid origin. Preferably, of hybridization of the nucleic acid in the sample to the probes the control sample is obtained in a manner similar to that used on the microarray. to obtain the test sample, e.g., being obtained from the same 0193 For example, mRNA levels can be obtained from a organs, tissues or fluids (as detailed above with respect to test GeneChipTM probe array or Microarray (Affymetrix, Inc.) samples); and sorted to enrich for corresponding cell types. (U.S. Pat. Nos. 5,631,734, 5,874,219, 5,861,242, 5,858,659, Similarly, the level of RNA or antigen preferably is assesed in 5,856,174, 5,843,655, 5,837,832, 5,834,758, 5,770,722, the control sample in a manner similar to that used to obtain 5,770,456, 5,733,729, 5,556,752, all of which are incorpo the test values. Methods are known in the art for permitting rated herein by reference in their entirety), and the expression direct comparison of test and control levels, and a specific levels can be calculated with software (e.g., Affymetrix example of Such comparison is detailed below in Example 1. GENECHIPTM software). Briefly, nucleic acids (e.g., mRNA) In some embodiments, control levels are provided by previ from a sample which has been Subjected to particular strin ously-obtained data, Such as from control samples that have gency conditions hybridize to the probes on the chip. The been detected in prior assays; from published data; and/or nucleic acid to be analyzed (e.g., mRNA corresponding to an from accessible databases. HTC marker) is isolated, amplified and labeled with a detect 0198 Diagnosis is based on a correlation between the able label, (e.g., “P or fluorescent label) prior to hybridiza level of a given expression product in a test sample compared tion to the arrays. Once hybridization occurs, the arrays are to that in a control sample. For example, as detailed in inserted into a scanner which can detect patterns of hybrid Example 1 below, mRNA levels of the cytokine receptors ization. The hybridization data are collected as light emitted disclosed herein are higher in AML HTC samples as com from the labeled groups, which are now bound to the probe pared to normal HSC samples. Preferably, the difference in array. The probes that perfectly match the mRNA correspond expression levels is at least about 2 fold, at least about 3 fold, ing to an HTC marker produce a stronger signal than those at least about 5 fold, at least about 7 fold, at least about 10 fold that have mismatches. Since the sequence and position of or at least about 15 fold. In particularly preferred embodi each probe on the array are known, by complementarity, the ments, the difference in expression levels is at least about 20 identity of the nucleic acid applied to the probe is determined. fold, at least about 30 fold, at least about 40 fold or at least Quantitation from the hybridization of labeled mRNA/DNA about 50 fold. In still more preferred embodiments, the dif microarray can be performed by Scanning the microarrays to ference in expression levels is at least about 70 fold, at least measure the amount of hybridization at each position on the about 100 fold, at least about 200, fold or as much as nearly microarray. This can be performed with, for example, an 300 fold, 400 fold or 500 fold. For example, IL13RA1 mRNA Affymetrix scanner (Affymetrix, Santa Clara, Calif.). levels in AML, HTCs is over 35 times that in normal HSCS Microarrays are only one method of detecting RNA levels. (see Table 1). Accordinlgy, the information provided by the Other methods known in the art or developed in the future can present disclosure, alone or in conjunction with other test be used with the present invention. results, aids in Sample classification and diagnosis of hema 0194 In another embodiment, the expression product is a tological proliferative disorders of myeloid origin, such as translation product, e.g. one or more cytokine receptors dis AML. closed herein. Cytokine receptor proteins include any 0199. In some embodiments, more than one test sample polypeptide or derivative thereof, including, e.g., peptides, and/or more than one control sample are obtained and/or glycoproteins, lipoproteins and nucleic acid-protein com detected. For example, as illustrated in Example 1 below, all plexes. of 3 normal samples showed low levels of expression of each 0.195 Techniques for protein detection and quantitation cytokine receptor, while at least 2 out of 3 AML samples are known in the art. For example, antibodies specific for the showed high expression levels. Alternatively, AML samples protein or polypeptide can be obtained using methods which over-expressed the cytokine receptor by at least about 5 fold are routine in the art, and the specific binding of Such anti compared to normal HSCs. Diagnosis may take into account bodies to protein or polypeptide expression products can be the difference in expression levels between more than one test US 2011/0059852 A1 Mar. 10, 2011 sample and/or more than one control sample. In some treatment regime; times at intervals during a treatment embodiments, repeat assays are performed using a given regime; and times after the conclusion of treatment regime. sample. The time intervals can include several hours; a day: 2, 3, or 4 0200. In some embodiments, expression of more than one days; a week; 2, 3, 4, 5 or 6 weeks; a month, 2, 3, 4, 5, or 6 cytokine receptor can be detected. The different cytokine months, a year, or several years post-initiation of a treatment receptors can be detected simultaneously. For example, in regime. In some embodiments, the treatment is one disclosed some embodiments, two, three, four, five, or more of the herein, e.g., one or more of the therapeutic uses of the anti different cytokine receptors disclosed herein are detected. bodies described herein. In some embodiments, the treatment The detection of numerous genes can provide a more accurate is a treatment otherwise known or used or to be used in theart; evaluation of the sample. The correlation between expression ora combination of such treatments with one or more of those product levels and a given disease, such as AML, can be disclosed herein. determined using a variety of methods. Methods of classify ing samples are described, for example, in U.S. patent appli 0206. The disclosure above regarding, for example, test cation Ser. No. 09/544,627, filed Apr. 6, 2000 by Golub et al., and control samples; stem cell sorting; use of one or more the teachings of which are incorporated herein by reference in cytokine receptors; detection of RNA and/or protein levels: their entirety. correlation of data and so forth, as provided in the case of 0201 The present invention also provides prognostic diagnostic methods, also applies to the monitoring methods methods for predicting the efficacy of treating a haematologi disclosed herein. cal proliferative disorder of myeloid origin, where the level of 0207. In a preferred embodiment, the expression product an expression product of one or more of the disclosed cytok is RNA and RNA levels in test samples are compared to ine receptors is detected and wherein the expression product control levels. In a paticularly preferred embodiment, the level is correlated with a treatment outcome. “Treatment out expression product is mRNA and the mRNA levels are come as used herein refers to the efficacy of a treatment with obtained by contacting the sample with a suitable microarray, respect to a disease, that is, the response of the disease to a and determining the extent of hybridization of the nucleic particular treatment. The levels of expression products can be acid in the sample to the probes on the microarray. used to assess the likelihood that a given disorder will respond 0208 Monitoring the efficacy of a treatment is useful, e.g., well to a particular treatment, or to determine which of a in facilitating clinical management of the disease, e.g., where number of treatment options is more preferable. In some decisions must be made as to whether to continue a treatment embodiments, the treatment is one disclosed herein, e.g., one course or advance to other treatment options. Whether a or more of the therapeutic uses of the antibodies described myeloid leukemia is responding positively to a treatment can herein. In some embodiments, the treatment is a treatment be determined based on changes in the level of a given expres otherwise known or used or to be used in the art; or a combi sion product (or products) in test samples taken at various nation of such treatments with one or more of those disclosed time points, e.g., before and after administration of a particu herein. lar treatment. 0202 The disclosure above regarding, for example, test 0209. A shift in expression product levels from a level and control samples; stem cell sorting; use of one or more correlated with HTCs of myeloid origin towards a level cor cytokine receptors; detection of RNA and/or antigen levels: related with normal HSCs is evidence of an effective thera correlation of data and so forth, as provided in the case of peutic regime. In some embodiments, a reduction in the diagnostic methods, also applies to the prognostic methods expression product level corresponding to one or more of the disclosed herein. cytokine receptors disclosed herein indicates a positive 0203. In a preferred embodiment, the expression product response to treatment. The reduction indicates a trend towards is RNA and RNA levels in test samples are compared to levels seen in normal HSC samples. For example, as detailed control levels. In a paticularly preferred embodiment, the in Example 1 below, the expression product level in AML expression product is mRNA and the mRNA levels are HTCs is higher than that in normal HSCs for each of the obtained by contacting the sample with a suitable microarray, cytokine receptors disclosed herein. A reduction in expres and determining the extent of hybridization of the nucleic sion level in one or more of the disclosed cytokine receptors, acid in the sample to the probes on the microarray. e.g., in test samples obtained after the commencement of 0204. In particularly preferred embodiments, the expres treatment, would indicate a positive response. sion product is mRNA and lower mRNA levels correlate with 0210. In preferred embodiments, the expression level for more favorable treatment outcomes. For example, a TRGV9 one or more HTC markers is reduced at least about 5 fold, at expression level that is about 10 times higher than control least about 7 fold, at least about 10 fold or at least about 15 levels indicates a more favorable outcome than where the fold, during the course of treatment. In particularly preferred TRGV9 expression level is about 30 times higher. In some embodiments, the expression level for one or more HTC embodiments, the expression levels of more than one cytok markers is reduced at least about 20 fold, at least about 30 ine receptor is be detected; and lower expression levels of fold, at least about 40 fold or at least about 50 fold, during the multiple markers is further indication of a more favorable course of treatment. In still more preferred embodiments, the treatment OutCOme. expression level for one or more HTC markers is reduced at 0205 The present invention also provides methods for least about 70 fold, at least about 100 fold, at least about 200 monitoring the efficacy of treating a haematological prolif fold, or as much as nearly about 300 fold, about 400 fold or erative disorder of myeloid origin, where the level of an about 500 fold, during the course of treatment. For example, expression product of one or more of the disclosed cytokine IL5RA mRNA levels in AML, HTCs is almost 750 times that receptors is detected at various time points and correlated in normal HSCs (see Table 1). A reduction to 200, 100, 50, 20, with treatment outcome. The various time points can include, 10, or just 2 times the IL5RA mRNA levels in normal HSCs for example, time of diagnosis, times prior to commencing a is evidence of an effective therapeutic regime. US 2011/0059852 A1 Mar. 10, 2011 24 0211. In some embodiments, the methods of the present 0215. In some embodiments, the kit is a diagnostic kit for invention are suitable for diagnosis prognosis and/or moni use in detecting test samples. The kit can include a control toring of a haematological proliferative disorder of myeloid antibody that does not react with the antigen to be assayed, origin, such as myoproliferative disorders. The present inven along with a marker specific antibody or antigen-binding tion also provides methods of diagnosis, prognosis and moni fragment thereof which specifically binds to an antigen cor toring of a myoproliferative disorder that is chronic myeloid responding to a cytokine receptor disclosed herein. Further, leukemia (CML) and/or acute myeloid leukemia (AML). In a Such a kit can include a means for detecting the binding of particularly preferred embodiment, the myelogenous haema said antibody to the antigen (for example, the antibody may tological proliferative disorder is AML and the level of RNA be conjugated to a fluorescent compound such as fluorescein or antigen is detected using a test sample comprising AML or rhodamine which can be detected by flow cytometry). HTCs, compared to control levels obtained from a control 0216. In preferred embodiments, the diagnostic kit sample of normal HSCs. includes a substantially isolated antibody that specifically binds an antigen corresponding to an HTC marker (e.g., a 9. KITS cytokine receptor disclosed herein), as well as means for detecting antigen-antibody binding. In some embodiments, 0212. In another aspect of the present invention, kits are the antibody is attached to a solid Support. In some embodi provided which contain one or more of the necessary reagents ments, the antibody is a monoclonal antibody. The detecting to carry out methods of the present invention. Specifically, means of the kit can include a second, labeled monoclonal Some embodiments provide a compartment kit having one or antibody. Alternatively, or in addition, the detecting means more containers, which comprise: (a) a first container com can include a labeled, competing antigen. prising one of the marker specific antibodies or complexes of 0217. In one diagnostic configuration, the test sample is the present invention, e.g., a first monoclonal antibody that reacted with a solid phase reagent having a surface-bound specifically binds a first antigen corresponding to one of the antigen, where the antigen corresponds to one (or more) of the cytokine receptors disclosed herein; and (b) one or more other cytokine receptors disclosed herein. After washing to remov containers comprising one or more of the following: wash ing unbound components, the reagent can be reacted with reagents, reagents capable of detecting presence of the anti reporter-labeled anti-human antibody to determine the body, and/or another marker specific antibody or complex of amount of anti-antigen antibody bound to the Solid Support. the present invention, e.g., a second monoclonal antibody that Such methods are well known and have been extensively specifically binds a second antigen corresponding to a differ described in the art. The reporter label can be an enzyme, for ent cytokine receptor disclosed herein. In some embodi example, which is detected by incubating the solid phase with ments, kits include additional containers, e.g., comprising a Suitable fluorometric, luminescent or calorimetric Substrate, third, fourth, fifth, etc., antibodies directed to antigens corre as is standard in the art. sponding to a third, fourth, fifth, etc., cytokine receptor dis 0218. The solid surface bearing bound antigens and/or closed herein. In some embodiments, additional containers antibodies, as described above, can be prepared by known include one or more known Small molecule agonists or techniques for attaching protein material to Solid Support antagonists that find use in the prognostic, diagnostic and/or material. Suitable solid support materials include, for therapeutic methods taught herein. example and without limitation, polymeric beads, dip Sticks, 0213. The kit typically contains containers which may be 96-well plate or filter material. In some embodiments, a small formed from a variety of materials such as glass or plastic, and molecule known to bind to a polypeptide corresponding to a can include, for example, bottles, vials, Syringes, and test disclosed cytokine receptor can be attached to a Solid Support, tubes. A compartment kit includes any kit in which reagents based on its chemical structure by methods known in the art. are contained in separate containers. Such containers include 0219. A text label typically accompanies the kit, and Small glass containers, plastic containers or strips of plastic or includes any writing or recorded material, which may be paper. Such containers allow efficient transfer of reagents electronic or computer readable form (e.g., disk, optical disc, from one compartment to another compartment, such that the or tape) providing instructions or other information for using samples and reagents are not cross-contaminated, and the one or more of the contents of the kit. In some embodiments, agents or solutions of each container can be added in a quan the label indicates that the contents are used for diagnosing or titative fashion from one compartment to another. One skilled treating the disorder of choice, such as a hematopoietic pro in the art will readily recognize that the disclosed antibodies liferatvie disorder of myeloid origin, according to one or of the present invention can be readily incorporated into one more of the methods described herein. In some embodiments, of the established kit formats, which are well known in the art. AML represents the disorder to be diagnosed and/or treated 0214) Provided herein are kits which include a composi using the contents of the kit. In some embodiments, CML tion described herein. In some embodiments the kit comprises represents the disorder to be diagnosed and/or treated using a hybridoma, complex, antibody and/or mixtures of antibod the contents of the kit. ies disclosed herein. In some embodiments, kits for therapeu tic applications are provided. Such as a kit housing a pharma 10. EXAMPLES ceutical formulation, e.g., one or more of the pharmaceutical compositions described herein. In some embodiments, the 10.1 Example 1 kits contain at least one additional reagent, including other antibodies, other monoclonal antibodies directed to HSCs, Identification of Cytokine Receptors Associated with other agents described herein, committed progenitor cells, HTCS polyclonal antibodies, or mixtures of the antibodies as 0220 HTC markers were identified by comparing RNA reagents for detecting myeloid cell types. Frozen or fixed transcript levels in normal HSCs and in AML CSCs for a forms of HSCs, CMPs, GMP and/or MEPs reactive with the variety of genes using micorarrays. Specifically, data was antibodies and reagents form additional contents of the kits. obtained for test samples comprising AML LinCD34' US 2011/0059852 A1 Mar. 10, 2011 CD38 cells, where three AML samples were taken from peripheral blood; LinCD34"CD38 and LinCD34"CD38" TABLE 2 cells were double sorted. The sorting strategy produced cells Normal CD38 CD38 AML which were also over 90% CD90. The sorting strategy pro HSC sample CSC sample duced purities greater than 98%. PoS. Average PoS. Average 0221) To allow for direct comparison, data was then samples signal samples signal Signal obtained from control samples comprising normal HSCs. A Gene detected intensity detected intensity ratio sample was taken from mobilized peripheral blood (MPB) of CSF1R O3 36 2.3 193 5 each of three individual donors and LinCD34CD90 IFNAR1 O3 23 3.3 112 5 CD45RACD38 and LinCD34CD90CD45RACD38 IL13RA1 3.3 179 3.3 606 3 cells were double sorted. The sorting strategy produced a IL1RAP 1.3 S4 3.3 803 15 ILSRA O3 7 2.3 1137 162 purity of over 99%. LILRA1 O3 13 2.3 44 3 0222 For both sorting strategies, the Lineage (Lin) cock INSR O3 33 3.3 192 6 IL1RL1 O3 50 3.3 136 7 tail included antibodies against CD2, CD3, CD11b, CD15, LTK O3 28 2.3 132 5 CD19, CD41, and CD235. Exemplary dot plots of control TACSTD1 3.3 2O1 2.3 2474 12 samples and AML samples are shown in FIGS. 1 and 2. respectively. 0227 Comparison of published data by Galet al. (“Gene 0223 Total RNA was then extracted, the RNA was reverse expression profiles of AML derived stem cells; similarity to transcribed and in vitro transcribed to ultimately yield fluo hematopoietic stem cells. Leukemia 2006, 20: 2147-2154, rochrome labeled cRNA probes from the transcripts. Tran incorporated herein by reference in its entirety) with control script levels were detected using Affymetrix whole Human samples described herein corroborates use of CSF1R, and Genome U133 Plus 2.0 Array. Briefly, the gene array was LTK as cancer stem cell targets (See U.S. Patent Application hybridized and read out. Signal intensities, probe set ID # and No. 61/039,701, incorporated herein by reference). absence/presencescore for each probe set was tabulated using 0228. As shown in FIG. 3., f expression of IL1RAP by MS Excel. AML CSCs was detectably by flow cytometric analysis using 0224. The signal values corresponding to RNA transcript the 3D8 antibody from Novus Biological (Littleton, Colo.). In levels in AML test samples and control samples (LinCD34' contrast, expression of IL1RAP by normal HSCs is undetect CD90"CD45RACD38 normal HSCs) were then directly able by flow cytometric analysis (FIG. 3). compared using MS Excel and Fisher's t-test. 0225 HTC marker genes were then selected based on the 10.2 Example 2 following criteria: (1) significance of signal intensity differ Production and In Vivo Efficacy of Monoclonal Anti ence between test and control samples cohorts p-0.05; (2) bodies genes not expressed in all 3 normal HSC samples (scored absent by Affymetrix software); (3) genes expressed in at 10.2.1. Preparation of Monoclonal Antibodies that least 2 out of 3 AML samples (scored present by Affymetrix Specifically Bindan Cytokine Receptor Associated software); (4) AMUHSC signal ratio 25 or 5) genes whose with HTCS expression was known to be extracellular and a cytokine receptor. 0229 Techniques for producing the monoclonal antibod ies are known in the art and are described, for instance, in 0226 Tables 1 and 2 below shows comparison for 10 Goding, Monoclonal Antibodies: Principles and Practice, pp. genes that were over-expressed in at least 2 out of the 3 of the 59-103 (Academic Press, 1986). Immunogens that may be CD38 or CD38", respectively, AMLCSCsamples compared employed include a purified polypeptide corresponding to to all three of the normal HSC samples. “Pos. samples’ refers colony stimulating factor 1 receptor (CSF1R): interleukin 13 to positive samples, scored as present by Affymetrix soft receptor, alpha 1 (IL13RA1); interleukin 1 receptor accessory ware (in the case of normal HSCs) or giving a signal intensity protein (IL1RAP); interferon-C. receptor 1 (IFNAR1); inter greater than 25 (in the case of AML CSCs). leukin-5 receptor (IL5R); insulin receptor (INSR); interleu kin 1 receptor-like 1 (IL1RL1):leukocyte receptor tyrosine TABLE 1. (LTK); or tumor associated calcium signal transducer 1 Normal CD38 CD38 AML (TACSTD1) as well as fusion proteins containing the same. HSC sample CSC sample Alternatively, cells expressing recombinant CSF1R: IL13RA1; IL1RAP: IFNAR1, IL5RA, INSR, IL1RL1, LTK PoS. Average PoS. Average or TACSTD1, on the cell surface, may be used. Selection of samples signal samples signal Signal the immunogen can be made by the skilled artisan without Gene detected intensity detected intensity ratio undue experimentation. CSF1R O3 26 2.3 89 3 IFNAR1 O3 30 2.3 59 2 0230 Mice, such as Balb/c, are immunized with the IL13RA1 O3 14 3.3 467 33 selected immunogen, emulsified in complete Freund's adju IL1RAP O3 25 3.3 411 16 vant and injected Subcutaneously or intraperitoneally in an ILSRA O3 8 2.3 6OOO 750 amount from 1-100 micrograms. Alternatively, the immuno INSR O3 49 3.3 132 3 IL1RL1 O3 18 2.3 342 19 gen is emulsified in MPL-TDM adjuvant (Ribi Immu LTK O3 39 2.3 126 3 nochemical Research, Hamilton, Mont.) and injected into the TACSTD1 3.3 189 2.3 1561 8 animal's hind foot pads. The immunized mice are then boosted 10 to 12 days later with additional immunogen emul sified in the selected adjuvant. Thereafter, for several weeks, US 2011/0059852 A1 Mar. 10, 2011 26 the mice may also be boosted with additional immunization transplantation. A Subset of the animals is sacrificed weekly injections. Serum samples may be periodically obtained from and bone marrow and spleen analyzed for human CD34' the mice by retro-orbital bleeding for testing in ELISA assays cells. to detect antibodies directed to the HTC marker polypeptide. 0236 Patient samples with engraftment potential are 0231. After a suitable antibody titer has been detected, the selected for use in antibody efficacy studies. For efficacy animals "positive' for antibodies can be injected with a final studies, CMUAML cells are transplanted and the test mono intravenous injection of the immunogen corresponding to an clonal antibody or control antibody will be injected on a HTC marker. Three to four days later, the mice are sacrificed schedule. Alternate schedules include once to 3 times per and the spleen cells harvested. The spleen cells are then fused week, 1-3 injections per week for 1-4 weeks, or 1-2 per week (using 35% polyethylene glycol) to a selected murine for 1-4 months. Injections can be intravenous by tail vein myeloma cell line such as P3X63AgU.1, available from injection, intraperitoneal, Subcutaneous, or intramuscular. ATCC, No. CRL 1597. The fusions generate hybridoma cells Following completion of the treatment schedule, animals are which can then be plated in 96 well tissue culture plates sacrificed and tissues collected for analysis. Peripheral blood, containing HAT (hypoxanthine, amininopterin, and thymi spleen and bone marrow can be evaluated by FACS analysis dine) medium to inhibit proliferation of non-fused cells, for the presence of human phenotypic CML cancer stem cells, myeloma hybrids, and spleen cell hybrids. CD34" marker" HTCs, detectable in the bone marrow and 0232. The hybridoma cells then can be screened in an spleen at the conclusion of the treatment. Philadelphia (Ph) ELISA for reactivity against the immunogen corresponding chromosome can be assayed by PCR to determine whether to the HTC marker. Determination of “positive’ hybridoma the cells are CML or normal. cells secreting the desired monoclonal antibodies directed 0237 As an example, eleven mice can be transplanted against the HTC marker polypeptide is within the skill in the with CML sample (MISIRB 31104.750), 5x10° cells/mouse. art. Mice can be conditioned with 250 rad TBI (X-ray source, 0233. The positive hybridoma cells can be injected intra Faxitron CP160), and anti-asialo GM1. The anti-asialo GM1 peritoneally into Syngeneic Balb/c mice to produce ascites is injected by intraperitoneal injection on days 0, 5 and 11. At containing the monoclonal antibodies directed to polypeptide 4 weeks post transplant half the mice in each group will begin corresponding to the HTC marker. Alternatively, the hybri receiving intraperitoneal or intravenous injections of a clone doma cells can be grown in tissue culture flasks or roller of a marker specific mAb, described herein, or control anti bottles. Purification of the monoclonal antibodies produced body, 250-1000 mg/dose, two times a week for 4 weeks. in the ascites can be accomplished using ammonium Sulfate Additionally, a group of 40 mice are also transplanted with precipitation, followed by gel exclusion chromatography, as CML (MISIRB 31104.750) as above. Antibody administra known in the art. Alternatively, affinity chromatography tion begins at the time of transplant. Mice are injected by based upon binding of antibody to protein A or protein G can intraperitoneal injection with 0.5-1 mg/dose of antibody, be employed, also as known in the art. twice a week for 8 weeks. Alternatively, CML cells isolated from previously engrafted mice will be serially transplanted. 10.2.2. In Vivo Efficacy of Monoclonal Antibodies Some of these secondary recipients will be treated with the Against Myeloid Leukemia marker specific mAb clone at time of transplantation by intra 0234. In vivo models of human cancer are useful to deter peritoneal injection with 0.5-1 mg/dose of antibody, twice a mine preclinical efficacy of candidate therapeutic agents. For week for 8 weeks. Following the above treatments with the monoclonal antibodies, studies in appropriate animal models marker specific mAb clone, the mice are analyzed by flow help evaluate target cell lysis and tumor eradication under cytometry for tumor burden, expression of CD34 and expres physiological conditions in vivo. Several groups have sion of the specific HTC marker gene. The number and fre described engraftment of CML chronic phase (CP), acceler quency of human cells in the bone marrow and spleen will be ated phase (AP), and/or blast phase (BP) and AML cells into determined for all mice surviving to the end of the study. SCID and NOD/SCID mice. In general, generation of chi Human cells (marker+) will be sorted by FACS from both meric animals showing engraftment of human CML cells is groups of mice for serial transplantation to determine if cells more consistent in NOD/SCID mice (See Dazzi, F et al. with functional cancer stem cell potential are present. Blood 92: 1390-1396 (1998); Wang, J. C. Y. etal, Blood 91: 0238 For secondary transplant of CML cells, CD34" 2406-2414 (1998); Dick, Jetal Blood 87: 1539-1548 (1996): marker" cells can be sorted from the bone marrow and spleen Bonnet, D et al. Blood 106: 4086-4092 (2005)). In vivo effi of several mice for transplantation. NOD/SCID analysis for cacy of monoclonal antibodies against CML and/or normal the secondary transplant can be performed 8 or 10 weeks post GMP and not HSC can be determined using the NOD/SCID transplant with the CD34 compartment of bone marrow. human CML model. 0239 Determination of whether the marker specific 0235 Xenograft animals can be generated as described by monoclonal antibody clone can eliminate or reduce tumor Dazzi et al. Briefly, NOD/SCID mice are bred in house or burden in mice transplanted with primary human AML blast purchased from a commercial Supplier (Jackson Laborato crisis cells. Twenty-five mice can be transplanted with AML ries) and housed under pathogen-free conditions. Prior to cells injected at 10x10 CD34"marker" cells/mouse. Cells are injection of cells, animals are irradiated (250 cGy, X-ray injected intravenously into the tail vein or the post lateral source). Cryopreserved cells from a CML or AML patient aspect of the orbital cavity. Mice are conditioned with 250 rad obtained from peripheral blood, mobilized peripheral blood, TBI (X-ray source, Faxitron CP160), and anti-asialo GM1. or bone marrow are analyzed by flow cytometry to determine The anti-asialo GM1 is injected IP on days 0, 5 and 11. the percentage of CD34" cells in the sample. Samples con Beginning 4 weeks post transplant, mice are randomized into taining 1 to 10x10 CD34" cells are injected IV into the 2 groups, therapy and control. The group receiving the thera conditioned mouse in a total Volume of 1 mL. Alternatively, peutic is injected I.P. with the marker specific mAb clone. CD34" cells can be sorted from the sample by FACS prior to Antibody will be administered by intraperitoneal injection 2 US 2011/0059852 A1 Mar. 10, 2011 27 times a week, for 4 weeks. Antibody concentration will be 1 exhaustive or to limit the invention to the precise forms dis mg per injection (a total of 2 mg/mouse/week). Volume will closed, and obviously many modifications and variations are vary depending on the antibody lot used. Mouse IgG will be possible in light of the above teaching. The embodiments used as the control article; it will be prepared in the same were chosen and described in order to best explain the prin diluent and injected at the same concentration and Volume as ciples of the invention and its practical application, to thereby the monoclonal antibody. Mice will be sacrificed 2-3 days enable others skilled in the art to best utilize the invention and following the last injection of marker specific antibody clone. various embodiments with various modifications as are Suited Bone marrow and spleen will be isolated and counted. Tissues to the particular use contemplated. It is intended that the will be analyzed by FACS for expression of CD34 and the scope of the invention be defined by the Claims appended specific HTC marker gene. The number and frequency of hereto and their equivalents. human cells in the bone marrow and spleen will be deter 0243 All patents, patent applications, publications, and mined for all mice surviving to the end of the study. Human references cited herein are expressly incorporated by refer cells will be sorted by FACS from both groups of mice for ence to the same extent as if each individual publication or serial transplantation to determine if cells with functional patent application was specifically and individually indicated cancer stem cell potential are present post-antibody treat to be incorporated by reference. 1-15. (canceled) ment. Alternatively, mice will begin antibody treatment at the 16. A method of diagnosing a myelogenous haematologi time of AML cell transplant. These mice will be injected with cal proliferative disorder in a patient, comprising 0.5mg of antibody 2 times a week for 8 weeks. Analysis will obtaining a test sample from said patient, and proceed as described above. detecting in said test sample a level of an expression prod 0240. In addition, the efficacy of the maker specific mAb uct corresponding to interleukin 1 receptor accessory clone can be tested for efficacy in leukemia in vivo models protein (IL1RAP), wherein the level of said expression using human cell lines expressing the corresponding antigen. product in said sample in comparison with a control is Immunocompromised animals will be inoculated with indicative of said myelogenous haematological prolif human leukemia cell lines recognized by the marker specific erative disorder. mAb clone. The efficacy of the clone will be tested using 17. The method of claim 16, wherein said expression prod multiple cell lines. The cell lines used should maintain a uct is mRNA. primitive phenotype in vivo with Sustained expression of the 18. The method of claim 16, wherein the levels of at least epitope recognized by the marker specific mAb clone. Such two different expression products are detected. cell lines will be identified and used as appropriate. Each cell 19. The method of claim 16, wherein said detecting uses a line will be characterized using different routes of adminis microarray. tration; intravenous, Subcutaneous or intraperitoneal injec 20. The method of claim 16, wherein the myelogenous tion. NOD/SCID mice will be tested for tumor engraftment at haematological proliferative disorder diagnosed is AML. the site of injection and Subsequent invasion of bone marrow 21. A method of monitoring the efficacy of treating a myel and spleen. Animals will be treated with the marker specific ogenous haematological proliferative disorder in a patient, mAb clone beginning at the time of tumor inoculation or comprising following tumor engraftment with injections starting 1-4 obtaining a test sample from said patient at two or more weeks post cell administration. Antibody will be adminis time points during said treatment, and tered by intraperitoneal injection 2 times a week, for 4 weeks. detecting in each of said test samples a level of an expres Mouse IgG will be used as the control article, injected at the sion product corresponding to: same concentration and Volume as the marker specific mAb wherein a reduction in the level of said expression product clone. Test cells will be administered at a single site. Animals over time indicates a positive response to treatment. are weighed weekly and observed for clinical signs of toxicity 22. The method of claim 21, wherein said expression prod and death for the duration of treatment. Animals are observed uct is mRNA. and palpated for the formation of nodules at the site of injec 23. The method of claim 21, wherein the levels of at least tion twice weekly. Detected nodules will be measured in two two different expression products are detected at two or more dimensions and findings recorded. The injection site is of said time points. exposed at the end of study and tumor removed and measured 24. The method of claim 21, wherein said detecting uses a and weighed. In addition, a sample of bone marrow, spleen microarray. and tumor mass are to be removed for phenotyping by FACS. 25. The method of claim 21, wherein the myelogenous These tissues will be disassociated and prepared for analysis haematological proliferative disorder monitored is AML. by flow cytometry. Tissues will be screened for expression of 26-28. (canceled) one or more of the human HTC markers and the marker 29. The method of claim 16, wherein the expression prod specific mAb clone. uct is protein. 0241. Using the above described in vivo models, it can be 30. The method of claim 29, wherein the detecting uses an shown that treatment with monoclonal antibody composi antibody. tions of the present invention is effective in reducing tumor 31. The method of claim 21, wherein the expression prod size, ameliorating one or more symptoms and/or prolonging uct is protein. Survival of mice in the therapy group. 32. The method of claim 31, wherein the detecting uses an 0242. The foregoing descriptions of specific embodiments antibody. of the present invention have been presented for purposes of illustration and description. They are not intended to be