MXPA96004315A - Antibodies agonists against the flk2 / flt3y receiver uses of mis - Google Patents

Abstract

The present invention describes agonist antibodies, which bind to the extracellular domain of the f1K2 / f1t3 receptor, and thereby activate the domain of the intracellular kinase thereof. The labeled antibodies are useful as diagnostic agents for the detection of the presence of the f1k2 / f1t3 receptor in primitive hematopoietic cells, for example. The antibodies are capable of causing primitive hematopoietic cells to proliferate and / or differentiate, and thereby increase the repopulation of mature blood cell lines in a mammal that has undergone chemotherapy or radiotherapy, or bone marrow transplantation. The antibodies are also useful for the treatment of mammals that have suffered a decrease in blood cells, as a consequence of disease or hemorrhage, for example

Description

AGONIST ANTIBODIES AGAINST THE FLK2 / FLT3 RECEIVER AND USES THEREOF FIELD OF THE INVENTION This application relates to the agonist antibodies against the flk2 / flt3 receptor and the uses thereof. In particular, the invention relates to the use of antibodies to increase the proliferation and / or differentiation of primitive hematopoietic cells.

BACKGROUND OF THE INVENTION A. HEMATOPOYESIS The process of blood cell formation by which red and white blood cells are replaced through the division of cells located in the bone marrow is called hematopoiesis. For a review of hematopoiesis see Dexter and Spooncer (Ann.Rev.Cell Biol., 3_: 423-441 (1987)). There are many different types of blood cells which belong to different cell lines. Along each line, there are cells in REF: 23129 different stages of maturation. Mature blood cells are specialized for different functions. For example, erythrocytes are involved in the transport of 0? and CO ?; T and B lymphocytes are involved in immune responses mediated by cells and antibodies, respectively; platelets are required for blood coagulation; and the granulocytes and macrophages act as general scavengers and accessory cells. Granulocytes can also be divided into basophils, eosinophils, neutrophils and mast cells. Each of the various types of blood cells arises from cells of the pluripotential or totipotential line, which are capable of undergoing self-renewal or give rise to progenitor cells or Colony Forming Units (CFU) that produce a more limited array of cell types . As the progenitor cells progressively lose their ability to self-renew, they become increasingly restricted lines. It has been shown that progenitor cells can develop into ultipotential cells (termed "CFC-Mix" by Dexter and Spponcer, supra). Some of the CFC-Mix cells may undergo renovation, while others lead to restricted online progenitors that develop sooner or later in mature myeloid cells (eg, neutrophils, megakaryocytes, macrophages, basophils and erythroid cells). Thus, pluripotent progenitor cells are capable of giving rise to the lymphoid cell lines PreB and PreT, which differ in mature B and T lymphocytes, respectively. The progenitors are defined by their progeny, for example, the progenitor cells that form granulocyte / macrophage colonies (GM-CFU) differ from neutrophils or macrophages; the primitive erythroid burst formation units (BFU-E) are differentiated into erythroid colony forming units (CFU-E), which give rise to mature erythrocytes. Similarly, the Meg-CFU, Eos-CFU and Bas-CFU progenitors are able to differentiate into megakaryocytes, eosinophils and basophils, respectively. The number of pluripotent progenitor cells in the bone marrow is extremely low and has been estimated to be in the order of about one per 10,000 to one per 100,000 cells (Boggs et al., J. Clin. Inv., 70_: 242 (1982) and Harrison). and collaborators, PNAS, 85; 822 (1988)). Consequently, the characterization of progenitor cells has been difficult. Therefore, various protocols have been developed to enrich pluripotent progenitor cells. See, for example, Matthews et al., Cell, 6 * 5: 1143-1152 (1991); WO 02157; Orlic et al., Blood, 82 (3): 762-770 (1993); and Visser et al., Stem Cells, 11 (2): 49-55 (1993). It has been shown that various line-specific factors control the cell growth, differentiation and functioning of hematopoietic cells. These factors or cytokines include interleukins (eg, IL-3), granulocyte-macrophage colony stimulation factor (GM-CSF), macrophage colony stimulation factors (M-CSF), granulocyte colony stimulation factor (M-CSF), erythropoietin (Epo), lymphotoxin, steel factor (SLF), tumor necrosis factor (TNF) and gamma interferon . These growth factors have a broad spectrum of activity, from generalized to specific line roles in hematopoiesis, or a combination of both. For example, IL-3 appears to act on multipotential progenitor cells, as well as progenitors restricted to the granulocyte / macrophage, eosinophil, megakaryocyte, erythroid, or mast cell lines. On the other hand, Epo generally acts on clearly mature erythroid progenitor cells.

B. TYROSINE-KINASES Many situations involved in hematopoietic development stimulate receptor tyrosine kinases (pTKs). For example, c-kit pTK and its cognate ligand (KL) have been shown to play a role in hematopoiesis. Tyrosine kinases catalyze protein phosphorylation using tyrosine as a substrate for phosphorylation. Members of the tyrosine kinase family can be recognized by the presence of several amino acid regions conserved in the tyrosine kinase catalytic domain (Hanks et al., Science, 241: 42-52 (1988)). The tyrosine kinases of receptor proteins share a similar architecture, with an intracellular catalytic portion, a hydrophobic transmembrane domain and an extracellular ligand binding domain. The extracellular domains (ECDs), which are responsible for ligand binding and the transmission of biological signals, have shown that they are composed of a number of different structural portions. The intracellular domain comprises a catalytic protein tyrosine kinase. Ligand binding to the extracellular portion is believed to promote dimerization of pTK, which results in transphosphorylation and activation of the intracellular tyrosine kinase domain (see Schlessinger et al., Neuron, 9 ^: 383-391 (1992 )).

C. RECEIVER FLK2 / FLT3 A murine gene encoding a pTK has been identified, which is expressed in cell populations enriched for progenitor cells, and uncommitted primitive progenitor cells, and is termed "fetal liver kinase-2" or "flk-2" by Matthews. and collaborators in Cell, _6_5: 1143-52 (1991). Rosnet et al. Independently identified partial cDNA sequences for the same gene, which they call "flt3", from murine and human tissues (Genomics, 9: 380-385 (1991)). The full-length sequence flt3 has been published by Rosnet et al. In Oncogene, 6 ^: 1641-1650 (1991). The sequence for human flk2 is described in World Patent WO 93/10136. Kuczynski et al. Refer to a gene called "STK-1" which is said to be the human homolog of murine flk2 / flt3 (Blood, 82 (10): PA486 (1993)). Matthews et al isolated the flk2 cDNA from atopoietic h-e tissue enriched with progenitor cells. In order to enrich the progenitor cells, the murine fetal liver cells were fractionated using a monoclonal antibody to AA4.1 and an antibody cocktail produced against specific differentiation antigens, collectively referred to as "Lin". It was found that flk2 is expressed in the AA4"AA4 + Lin10 and AA4 + Linor cells, but not in AA4 cells", the population AA4 + Lin ~ '~ contained all the pluripotent progenitor cells in the long term.The population AA4"Linr had few cells of the pluripotential line but it contained ultipotential progenitors. The population AA4"was devoid of primitive clonogenic cells, but contained fewer primitive progenitors such as CFU-E. The expression of the flk2 populations in AA4" Sea "and AA4 + Sea * Lin" °, which are considered as populations of progenitor cells highly enriched, was further demonstrated. We studied the additional expression of flk2 in thymus of 14 days (stage in which the thymocyte population is highly enriched in primitive precursors). Flk2 mRNA was overexpressed in the most immature T lymphocyte population (CD4 ~ 8 ~ Thy-llc7IL-2R "). In total, the results of the experiments described in Matthews et al. Indicate that flk2 is expressed in hematopoietic cells more primitive pluripotent / progenitor cells The expression of poly (A) * RNA in fetal and adult tissues was also investigated by Matthews et al .. The expression of flk2 mRNA was observed in fetal brain and liver, as well as in brain tissues and of adult bone marrow Rosnet and colleagues observed in a similar way that the flt3 gene is expressed in placenta in various adult tissues including the gonads and the brain, as well as in hematopoietic cells (Oncogene, 6: 1641-1650 (1991)). The flt3 transcript identified by Rosnet et al. Was 3.7 kb in length, except in the testes, where two shorter transcripts were identified. Small et al. (USA PNAS, 91: 459-463 (1994)) have shown antisense oligonucleotides directed against the human homologue of the flk2 / flt3 gene inhibit colony formation in long-term bone marrow cultures, the results of which also indicate that flk2 / flt3 can transduce growth signals in hematopoietic progenitor cells. World Patent WO 94/01576 refers to a soluble form of the flk2 / flt3 receptor, designated flk-2 s, encoded by a 1.9 kb DNA fragment. Dosil et al. Prepared a chimeric receptor consisting of the ligand extracellular domain of the human fms pTK and the transmembrane and tyrosine kinase domains of murine flk2 / flt3. The chimeric receptor was shown to confer transformed properties to NIH 3T3 cells and sustained the long-term proliferation of the Ba / F3 cell line (a line of murine hematopoietic cells dependent on IL-3, which generates B lymphocytes in vi) in the absence of IL-3 (Mol, Cell, Biol., 13 (10): 6572-6585 (1993)). It was shown that flk2 / flt3 interacts with the p85 subunit of the 3 '- Pi kinase and induced tyrosine phosphorylation of the PLC, GAP and Shc proteins. Proteins 3'-kinase of Pl, PLC ?. GAP and Shc are intracellular substrate proteins that are known to be associated with pTKs. The flk2 / flt3 receptor is structurally related to the pTKs subclass III, such as the platelet-derived growth factor-a and β-receptors (PDGF-R), the receptor (c-fms) of the colony-stimulating factor (CSF-1) , also known as macrophage colony stimulation factor), M-CSF) and the Steel factor receptor (also known as mast cell growth factor, totipotent cell factor or ligand kit) (c-kit). These receptors form a subfamily of pTKs which have five immunoglobulin-like segments in their ECDs and the intracellular catalytic domains thereof are interrupted by a variable length "interkinase" hydrophilic sequence. The genes of this subclass of pTK appear to have greater growth and / or differentiation functions in various cells, particularly in the hematopoietic system and placental development (see Rosnet et al. In Genomics, supra). Signaling through the c-fms receptor regulates the survival, growth and differentiation of monocytes. The Steel factor (SLF) which interacts with c-kit stimulates the proliferation of cells in the myeloid and lymphoid lines, and is a potent synergistic factor in combination with other cytokines (Lyman et al., Oncogene, 3: 815-822 [ 1993]). The pTK of flk2 / flt3 is mentioned by several other authors. See, for example, Orlic et al., Upra; Birg et al., Blood, 80 (10): 2584-2593 [1992]; and Visser et al., supra. Lyman et al. Refer to the molecular cloning of the transmembrane ligand for flk2 / flt3 which is shown to activate the flk2 / flt3 receptor (Cell, 7_5: 1157-1167 [1993]). It was found that the protein is similar in size and structure to the cytokines, M-CSF and SLF. It was shown that the ligand flk2 / flt3 increases the incorporation of thymidine in the early precursors of hematopoietic cells. In their first publication, Lyman and colleagues refer to the production of polyclonal rabbit antibodies against the interkinease domain or the C-terminus of flk2 / flt3, which immunoprecipitated a band greater than 143 kDa and a more diffuse band of 158 kDa. A C-terminal peptide of the flt3 sequence containing the final 22 amino acids thereof was used to generate the antisera. See Lyman et al., Qncogene, 8: 815-822 [1993]. Maroc et al. Also refer to the production of polyclonal antibodies against the kinase-terminal domain of flk2 / flt3 for use in the study of the biochemical characteristics of this protein (see Oncogene, 8: 909-918 [1993]). Polyclonal rabbit immune serum was directed against a fusion of the interkinease domain of flk2 / flt3 with TrpE. However, agonist antibodies that are capable of activating the f receptor are capable of activating the flk2 / flt3 receptor have not been described to date.

D. THERAPEUTIC IMPLICATIONS Chemotherapies or radiotherapies cause dramatic reductions in blood cell populations in patients with cancer. At least 500,000 cancer patients undergo chemotherapy and radiation therapy in the United States and Europe each year, and another 200,000 in Japan. Bone marrow transplant therapy of value in aplastic anemia, primary immunodeficiency and acute leukemia (after total body irradiation) is becoming increasingly widely practiced by the medical community. At least 15,000 Americans have bone marrow transplants every year. Other diseases can cause a reduction in the complete or selected blood cell lines. Examples of these conditions include anemia (including macrocytic and aplastic anemia); thrombocytopenia; hypoplasia; immune thrombocytopenic purpura (autoimmune) (ITP), and ITP induced by HIV. A pharmaceutical product that is capable of increasing the reconstitution of blood cell populations in these patients could clearly be of therapeutic benefit. Accordingly, an object of the present invention is to provide agonist antibodies against the flk2 / flt3 receptor. The labeled antibodies can be used to detect the flk2 / flt3 receptor in biological samples. A further objective of this invention is to provide a method for increasing the proliferation or differentiation of primitive hematopoietic cells, thereby improving the repopulation of mature blood cell lines. This is desirable where a mammal has suffered a decrease in hematopoietic or mature blood cells as a consequence of disease, radiation or chemotherapy. This method is also useful for the generation of mature blood cell lines from ex vivo hematopoietic cells. These and other objectives will be apparent to someone of ordinary skill in the art, when considering the specification as a whole.

BRIEF DESCRIPTION OF THE INVENTION These objectives are achieved, in one aspect, by the provision of agonist antibodies against flk2 / flt3. In still another aspect, the present invention is based on the observation that such agonist antibodies against flk2 / flt3 are capable of increasing the proliferation and differentiation of primitive hematopoietic cells. Accordingly, the present invention relates to a method for improving the proliferation or differentiation of primitive hematopoietic cells, comprising contacting the primitive hematopoietic cells with an effective amount of an agonist antibody against the flk2 / flt3 receptor. In a preferred embodiment, the agonist antibody is a monoclonal antibody directed against an epitope in the extracellular domain of flk2 / flt3. In a further aspect, the present invention relates to a method for improving the repopulation of blood cell lines in a? mammal, comprising administering to the mammal a therapeutically effective amount of an agonist antibody against the flk2 / flt3 receptor.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 describes the results of the thymidine incorporation assay using the transfected flk2 / flt3 receptor in the cell line of IL-3, 32D-dependent cells. In the assay, the cells were deprived of IL-3 overnight and then stimulated for 24 hours and the incorporation of thymidine was determined. The transfected BAF-3 cells (BAF-3T) and progenitor BAF-3 cells were stimulated with (a) medium alone, (b) prein rabbit serum,. (c) 579A polyclonal antibody or (d) agonist monoclonal antibody IC2-310.

Figures 2A-F show the fractionation of progenitor cell populations of fetal liver and bone marrow. Figures 2A-D show the fractionation of fetal liver AA4 + cells of 14 days of gestation. AA4 cells were enriched by immune production and subsequently stained using Sca-1, CD34, flk2 and c-kit antibodies.AA4 cells were stained for flk-2 and Sca-1 (Figure 2A); c-kit and CD34 (Figure 2B); flk-2 and CD34 (Figure 2C) and flk-2 and c-kit (Figure 2D). Figures 2E and 2F describe the fractionation of bone marrow progenitor cells Lin10 with antibodies flk-2, CD34 and Sca-1. The bone marrow progenitor cells LínlQ were isolated by indirect expansion with magnetic spheres. The Lin cocktail was comprised of RA3-GB2, GR-1, MAC-1, CD4, CD8, Ter-119 and CD5. The bone marrow cells Lin: ° were stained for flk-2 and CD34 (Figure 2E); and Sca-1 and flk-2 (Figure 2F), (shown as a plot of dashed lines due to the very small population of Linl0Sca + flk-2 + cells in the bone marrow.) These experiments were repeated a minimum of four times and the staining profiles were identical.

Figures 3A-D describe the dual-parameter fluorescence histograms of populations enriched with AA4 + SCA * flk-2"(Figure 3A) and Ú (Figure 3B) after cell selection, Figures 3C and 3D illustrate fluorescence histograms corresponding red obtained after acridine orange for populations enriched with AA4 + SCAT flk-2"(Figure 3C) and for AA4 + Sca + flk-2 + (Figure 3D). The cursor illustrated the fluorescence intensity used to discriminate G (lower fluorescence intensity) from the cells subjected to the cycle. The percentages of cells in each phase of the cell cycle are provided at the insertion.

Figure 4 describes the effect of IC2-310 on the formation of methyl cellulose colonies. The hematopoietic cells were seeded in methylcellulose after being co-cultured in the fetal liver stromal line 7-4 for 7 days in the presence or absence of the agonist antibody IC2-310. Methylcellulose cultures were established under conditions that form myeloid or lymphoid colonies. The colonies were then stored for CFC after 10 days in culture. The assays were performed in triplicate and repeated in three separate experiments.

DETAILED DESCRIPTION OF THE PREFERRED MODALITIES I. DEFINITIONS In general, the following words or phrases have the indicated definition when used in the description, examples and claims: The term "flk2 / flt3" when used herein refers to a polypeptide molecule comprising the native amino acid sequence, full-length, encoded by the gene variably known as flk2, klt3 and STK-1, from any species, including murine and human polypeptides having the amino acid sequences of SEQ ID NO: 2 and SEQ ID NO: 4, respectively, or variants of the amino acid sequence of such polypeptides. In general, the DNA encoding such variants is capable of hybridizing under strict conditions to the native flk2 / flt3 DNA sequence. This definition specifically covers the soluble forms of flk2 / flt3, from natural sources (see, for example, World Patent WO 94/01576), synthetically produced in vi tro or obtained by genetic manipulation including recombinant DNA technology methods, as well as various combinations of chains of such polypeptides. The amino acid sequence variants preferably share at least about 65% sequential homology, and more preferably at least about 75% sequential homology with any domain of a native amino acid sequence of flk2 / flt3. The definition covers especially variously glycosylated and non-glycosylated forms of flk2 / flt3. The flk2 / flt3 receptors of non-human or non-murine mammalian species (eg, bovine, equine, porcine, etc.) can, for example, be obtained by hybridization of cross-species, using probes obtained from the murine DNA sequence or human (SEQ ID NOS: 1 and 3, respectively) as a hybridization probe for isolating cDNA from mammalian cDNA libraries. Strict conditions are those that (1) employ low ionic strength and high temperature for washing, for example, 0.015 M sodium chloride / sodium citrate 0.0015 M / 0.1% NaDodS04 at 50 ° C; (2) employ during denaturation a denaturing agent such as formamide, for example, 59% formamide (vol / vol) with 0.1% bovine serum albumin / 0.1% Ficoll / 0.1% polyvinylpyrrolidone / sodium phosphate buffer 50 mM at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42 ° C; or (3) employ 50% formamide, 5 x SSC (0.75 M sodium chloride, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 x Denhardt's solution, Sperm salmon DNA sonicated (50 μg / ml), 0.1% SDS and 10% dextran sulfate at 42 ° C, washed at 42 ° C in 0.2 x SSC and 0.1% SDS. The term "extracellular domain" or "ECD" when used herein in relation to the flk2 / flt3 receptor refers to any polypeptide sequence that shares a ligand-binding function of the extracellular domain of the flk2 / flt3 receptor. The ligand binding function of the extracellular domain refers to the ability of the polypeptide to bind to at least one ligand flk2 / flt3 (e.g., the ligand described by Lyman et al., in Cell, supra). Consequently, it is not necessary to include the complete extracellular domain since smaller segments are commonly found, as suitable for binding to the ligand. The truncated extracellular domain is generally soluble. This term encompasses polypeptide sequences in which the hydrophobic transmembrane sequence (and, optionally, the C-terminal and / or N-terminal amino acids of the transmembrane domain) of the mature pTK has been deleted. Thus, the polypeptide containing the soluble extracellular domain can comprise the extracellular domain and the cytoplasmic domain of the flk2 / flt3 receptor. Alternatively, in the preferred embodiment, the polypeptide comprises only the extracellular domain of flk2 / flt3. In general, the ECD will comprise at least amino acid residues 1 to 542 of SEQ ID NOS: 2 or 4. "Antibodies (Abs)" are proteins that show binding specificity for a specific antigen. The native antibodies are usually heterotetrameric glycoproteins of approximately 150,000 daltons, composed of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by a covalent disulfide bond, while the number of disulfide bonds varies between the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced, intrachain disulfide bridges. Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains. Each light chain has a variable domain at one end (VL) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the variable domain of the light chain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the variable domains of light and heavy chains (Clothia et al., J. Mol. Biol., 186: 651-663 [1985]; Novotny and Haber, Proc. Nati. Acad. Sci. USA, 82: 4592-4596 [1985]). The term "antibody" is used in the broadest sense and specifically covers simple monoclonal antibodies (including agonist and antagonist antibodies), antibody compositions with polyepitopic specificity, as well as antibody fragments (eg, Fab, F (ab ') 2, and Fv), as long as they show the desired biological activity. The term "variable" refers to the fact that if these portions of the variable domains differ widely in sequence between antibodies, and they are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not uniformly distributed across the variable domains of the antibodies. This is concentrated in three segments called complementarity determination regions (CDRs) or hypervariable regions in the variable domains of the light chain and the heavy chain. The most highly conserved portions of the variable domains are called the framework or structure (FR). The variable domains of the native heavy and light chains each comprise four FR regions, which mostly adopt a β-sheet configuration, connected by three CDRs, which forms loops which are connected, and in some cases forming part of the structure of blade ß. The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen binding site of the antibodies (see Kabat, EA et al., Sequences of Proteins Immunological Interest National Institute of Health, Bethesda, MD [1987]). The constant domains are not directly involved in the binding of an antibody to an antigen, but show various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity. The digestion of the antibodies with papain produces two identical antigen binding fragments, called Fab fragments, each with a single antigen binding site, and a residual "Fe" fragment, whose name reflects its ability to rapidly crystallize. Does pepsin treatment produce an F (ab ') fragment? which has two antigen binding sites and is still capable of cross-linking to the antigen. "Fv" is the minimum antibody fragment which contains a complete antigen recognition and binding site. This region consists of a dimer of a variable domain of a heavy chain and a light chain in tight, non-covalent association. It is in this configuration that the three CDRs of each variable domain interact to define an antigen binding site on the surface of the VK-VL dimer. Collectively, the six CDRs confer specificity of binding to the antigen, to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind the antigen, albeit at a lower affinity than the entire binding site. The Fab fragment also contains the constant domain of the light chain and the first constant domain (CHi) of the heavy chain. The Fab 'fragments differ from the Fab fragments by the addition of a few residues at the carboxyl terminus of the CH.sub.i domain. of the heavy chain, including one or more cysteines from the hinge region of the antibody. Fab'-SH is the designation herein for Fab 'in which the cysteine residue or residues of the constant domains carry a free thiol group. The F (ab ') 2 antibody fragments were originally produced as pairs of Fab' fragments which have hinge cysteines therebetween. Other chemical couplings of the antibody fragments are also known. The light chains of the antibodies (immunoglobulin) of any vertebrate species can be assigned to one of two clearly distinct types, called kappa (K) and lambda (?) based on the amino acid sequences of their constant domains. Depending on the amino acid sequence of the constant domain of their heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these can further be divided into subclasses (isotypes), for example, IgG-1, IgG-2, IgG-3 and IgG4; IgA-1 and IgA-2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called a, delta, epsilon,?, And μ, respectively. Subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known. The term "monoclonal antibody" as used herein, refers to an antibody obtained from a population of substantially homogeneous antibodies, for example, the individual antibodies comprising the population are identical except for possible mutations of natural origin that may be present in smaller quantities. Monoclonal antibodies are highly specific, being directed against a single antigenic site. In addition, in contrast to conventional (polyclonal) antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous because they are synthesized by the hybridoma culture, not contaminated by other immunoglobulins. The "monoclonal" modifier indicates the character of the antibody, which is obtained from a substantially homogeneous population of antibodies, and which is not constructed to require production of the antibody by any particular method. For example, the monoclonal antibodies to be used according to the present invention can be made by the hybridoma method first described by Kohier and Milstein, Nature, 256: 495 [1975], or they can be made by recombinant DNA methods [see , for example, US Patent No. 4,816,567 (Cabilly et al.)].

Monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and / or light chain is identical with or homologous to the corresponding sequences in antibodies derived from a particular species, or which belong to a particular class or subclass of antibodies, while the rest of the chain or chains is identical with or homologous to the corresponding sequences in the antibodies derived from another species, or belonging to another class or subclass of antibodies, as well as to the fragments of such antibodies, as long as they show the desired biological activity (US Patent No. 4,816,567 (Cabilly et al., Morrison et al., Proc. Nati, Acad. Sci. USA, 81: 6851-6855 [1984]). The "humanized" forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab ', F (ab') 2 or other antigen-binding subsequences. of antibodies) which contain the minimal sequence derived from non-human immunoglobulin. For the most part, the humanized antibodies are human immunoglobulins (container antibody) in which the residues of a region of complementary determination (CDR) of the container, are replaced by residues of a CDR of a non-human species (donor antibody). , such as mouse, rat or rabbit that have the desired specificity, affinity and capacity. In some cases, structural Fv residues of human immunoglobulin are replaced by corresponding non-human residues. In addition, the humanized antibody may comprise residues that are found neither in the recipient's antibody nor in the imported CDR or in the structural sequences. These modifications are made to further refine and optimize the functioning of the antibodies. In general, the humanized antibody will substantially comprise all or at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin, and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody will also optimally comprise at least a portion of a constant region (Fe) of immunoglobulin, typically that of a human immunoglobulin. For additional details see: Jones et al., Nature, 321: 522-525 [1986]; Reichmann et al., Nature, 332: 323-329 [1988]; and Presta, Curr. Op. Struct. Biol., 2: 593-596 [1992]). By "agonist antibody" is meant an antibody that is capable of binding to and activating flk2 / flt3. For example, the agonist can bind to the extracellular domain of flk2 / flt3 and thereby cause dimerization of this receptor, resulting in transphosphorylation and activation of the intracellular catalytic kinase domain thereof. Accordingly, this may result in the stimulation of growth and / or differentiation of cells expressing the receptor. As described herein, these cells will generally comprise primitive / hematopoietic progenitor cell lines, and thus agonist antibodies will cause the primitive hematopoietic cells to differentiate and / or proliferate, which will lead in general to a repopulation of the lines of mature blood cells. The agonist antibodies herein are preferably against epitopes from the extracellular domain of flk2 / flt3. The term "agonist antibody" covers the anti-fIk2 agonist / flt3 monoclonal antibodies and the anti-fIk2 / flt3 agonist antibody compositions with polyepitopic specificity. In the preferred embodiment of the invention, the antibodies are monoclonal antibodies. In the most preferred embodiment, the monoclonal antibodies have the same biological characteristics as the monoclonal antibody produced by the hybridoma cell line deposited under ATCC Accession No. HB 11,557 of the American Collection of Species Collection (American Type Culture Collection). By "biological characteristics" are meant the in vi tro and / or in vi vi activities of the monoclonal antibody, for example, the ability to activate the kinase domain of flk2 / flt3, the ability to stimulate cell growth and / or differentiation of the primitive hematopoietic cells, and the binding characteristics of the antibody, etc. Accordingly, the antibody is preferably linked substantially to the same epitope as the anti-fIk2 / flt3 monoclonal antibody described herein. More preferably, the antibody will also have substantially the same or higher binding affinity for the antigen of the anti-flk2 / flt3 monoclonal antibody described herein. To determine whether a monoclonal antibody has the same specificity as the specifically described anti-fIk2 / flt3 antibody (for example, the antibody having the deposit of ATCC No. HB 11,557) one can, for example, use a competitive binding assay of ELISA. An "isolated" polypeptide (eg, antibody) means the polypeptide that has been identified and separated, and / or recovered from a component of its natural environment. The contaminating components of their natural environment are materials that could interfere with any diagnostic or therapeutic use for the polypeptide, and may include enzymes, hormones, and other protein or non-protein solutes. In preferred embodiments, for example, a polypeptide product comprising a monoclonal antibody of the present invention, will be purified from a cell culture or other synthetic environment (1) to more than 95% by weight protein, as determined by the Lowry method, and more preferably greater than 99% by weight; (2) to a degree sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence, by use of a gas or liquid phase sequencer (such as the sequencer commercially available from Applied Biosystems, Model 470, 477 , or 473), or (3) homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or, preferably, silver tincture. The term "therapeutically effective amount" is used to refer to an amount of any given molecule, sufficient for the prevention or treatment of a specific physiological condition or symptom. The therapeutically effective amount of the agonist antibody to be administered, will be governed by considerations such as the disorder to be treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the agent's distribution site, the method of administration, the administration scheme, and other factors known to medical practitioners, and is the minimum amount necessary to repopulate mature blood cell lines in patients who have undergone chemotherapy or radiation therapy or therapy for bone marrow transplantation, or any other conditions or diseases mentioned herein . "Primitive hematopoietic cells" means the most primitive or more impartial blood cells of the hematopoietic system. The blood cells may comprise the cells of the totipotential line and / or cells that are slightly involved in a particular blood cell line (eg, multipotential cells). The term "mammal" refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports or pet animals, such as dogs, horses, cats, cows, etc. Preferably, the mammal in the present is the human.

The term "cytokine" is a generic term for proteins released by a cell population that acts on another cell more as intercellular mediators. Examples of such cytokines are lymphokines, monokines and traditional polypeptide hormones. Included among the cytokines are growth hormone, insulin-like growth factors, human growth hormone, N-methionyl-human growth hormone, bovine growth hormone, parathyroid hormone, thyroxine, insulin, proinsulin, relaxin, prorrelaxin, glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH), hematopoietic growth factor, liver growth factor, fibroblast growth factor, prolactin, placental lactogen, tumor necrosis factor alpha and beta, inhibitory substance muleriana, peptide associated with mouse gonadotropin, inhibin, activin, vascular endothelial growth factor, integrin, thrombopoietin, nerve growth factors such as NGF-β, platelet growth factor, Transforming growth factors (TGFs) such as TGF-a and TGF-β, factor Y and growth 11 insulin-like, erythropoietin (EPO), osteoinductive factors, interferons such as interferon-alpha, -beta and -gamma, colony stimulation factors (CSFs) such as macrophage CSF (M-CSF), granulocyte-acrophage CSF (GM-CSF), and granulocyte CSF (G-CSF), interleukins (lis) such as IL-1, IL-la, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12 and other polypeptide factors including LIF, SCF, and the ligand kit. As used herein, it is understood that the above terms include proteins from natural sources and from recombinant cell culture. Similarly, the terms are intended to include biologically active equivalents; for example, that they differ the amino acid sequence by one or more amino acids or in the type or degree of glycosylation.

II. ANTIBODY PRODUCTION (a) Polyclonal antibodies Polyclonal antibodies to flk2 / flt3 are generally produced in animals by multiple subcutaneous (se) or intraperitoneal (ip) injections of flk2 / flt3 and an adjuvant. It may be useful to conjugate flk2 / flt3 or a fragment containing the target amino acid sequence (e.g. ECD of flk2 / flt3) to a protein that is immunogenic in the species to be immunized, for example, keyhole limpet hemocyanin in the form of a lock hole, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor, using a bifunctional or derivatizing agent, for example the maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteine residues), N-hydrosuccinimide ( through Residues of Usin), glutaraldehyde, succinic anhydride, S0C12, or R1N = C = NR, where R and R1 are different alkyl groups. The animals are immunized against the immunogenic conjugates or derivatives by combining 1 mg or 1 μg of conjugate (for rabbits or mice, respectively) with 3 volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites. One month later the animals are challenged with 1/5 to 1/10 of the original amount of the conjugate in complete Freund's adjuvant by subcutaneous injection in multiple sites. 7 to 14 days later the animals are bled and the serum is assayed for the antibody titer for flk2 / flt3. The animals are challenged until the maximum title is reached. Preferably, the animal is challenged with a conjugate of the same flk2 / flt3 to a different protein and / or to the same protein, through a different cross-linking reagent. The conjugates can also be made in recombinant cell culture as fusion proteins. Also, aggregation agents such as alumina are used to increase the immune response. (b) Monoclonal antibodies Monoclonal antibodies are obtained from a population of substantially homogeneous antibodies, for example, the individual antibodies comprising the population are identical except for possible naturally occurring mutations, which may be present in minor amounts. Thus, the "monoclonal" modifier indicates the character of the antibody, which is not a mixture of discrete antibodies. For example, the flk2 / flt3 monoclonal antibodies of the invention can be made using the hybridoma method first described by Kohier and Milstein, Nature, 256: 495 [1975], or they can be made by recombinant DNA methods (US Patent No. 4,816,567 (Cabilly et al.)). In the hybridoma method, a mouse or other appropriate host animal, such as hamster, is immunized as described hereinabove to produce lymphocytes that produce or are capable of producing antibodies, which will bind specifically to the protein used for immunization . Alternatively, lymphocytes can be immunized in vi tro. The lymphocytes are then fused with myeloma cells using the appropriate fusion agent, such as polyethylene glycol, to form a hybridoma cell [Goding, Monoclonal Antibodies: Principles and Practice, pp. 625-974]. 59-103 (Academic Press, 1986)]. Hybridoma cells prepared in this way are seeded and grown in a suitable culture medium which preferably contains one or more substances that inhibit the growth or survival of the unfused, progenitor myeloma cells. For example, if the progenitor myeloma cells lack the enzyme hypoxanthine-guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas will typically include hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of cells deficient in HGPRT. Preferred myeloma cells are those that efficiently fuse, support stable high-level expression of the antibody by selected antibody-producing cells, and are sensitive to a medium such as the HAT medium. Among these, preferred myeloma cell lines are murine myeloma lines, such as those derived from mouse tumors MOPC-21 and MPC-11, available from the Salk Institute Cell Distribution Center, San Diego, California USA, and SP cells -2 available from the North American Species Crop Collection, Rockville, Maryland, USA. Human myeloma and mouse-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133: 3001 [1984], Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63, Marcel Dekker, Inc., New York, 1987). The culture medium in which the hybridoma cells develop is tested for the production of monoclonal antibodies directed against flk2 / flt3. Preferably, the binding specificity of the monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation, or by an in vi tro binding assay, such as in radioimmunoassay (RIA) or the enzyme-linked immunosorbent assay (ELISA). The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal. Biochem. , 107: 220 [1980]. After the hybridoma cells which produce antibodies of the desired specificity, affinity and / or activity are identified, the clones can be subcloned by limiting dilution methods and developed by standard methods (Goding, supra). Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle Medium or RPMI-1640 medium. In addition, hybridoma cells can be developed in vi ve as ascites tumors in an animal. The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, of ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography. The DNA encoding the monoclonal antibodies of the invention is easily isolated and sequenced using conventional methods (for example, by the use of oligonucleotide probes that are capable of binding specifically to the genes encoding the heavy and light chains of murine antibodies) . The hybridoma cells of the invention serve as a preferred source of such DNA. Once isolated, DNA can be placed within expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not produce another way the immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. DNA can also be modified, for example, by substituting the coding sequence for the human heavy and light chain constant domains, instead of the homologous murine sequences (Cabilly et al., Supra).; Morrison et al., Proc. Nati Acad. Sci., 81: 6851 [1984]), or by covalently joining to the sequence encoding the immunoglobulin, all or part of the coding sequence for a non-immunoglobulin polypeptide. Typically, such non-immunoglobulin polypeptides are substituted by the constant domains of an antibody of the invention, or these are substituted by the variable domains of an antigen combining site, of an antibody of the invention, to create a chimeric bivalent antibody comprising an antigen combining site that has specificity for the flk2 / flt3 receptor, and another antigen combining site that has specificity for a different antigen. Hybrid chimeric antibodies can also be prepared in vi tro using known methods in the chemistry of synthetic proteins, including those involving crosslinking agents. For example, immunotoxins can be constructed using a disulfide exchange reaction, or by the formation of a thioether bond. Examples of reagents suitable for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate. For diagnostic applications, the antibodies of the invention will typically be labeled with a detectable portion. The detectable portion can be any that is capable of producing, either directly or indirectly, a detectable signal. For example, the detectable portion can be a radioisotope such as 3H, 1C, 32P, 35S or 125I, a fluorescent or chemiluminescent compound, such as fluorescein isothiocyanate, rhodamine, or luciferin; radioactive isotopic labels, such as, for example, "25I, 2P, 1C or 3H, or an enzyme, such as alkaline phosphatase, beta-galactosidase or horseradish peroxidase.

Any method known in the art for separately conjugating the antibody to the detectable portion can be employed, including those methods described by Hunter et al., Nature, 144: 945 [1962]; David et al., Biochemistry, 1_3: 1014 [1974]; Pain et al., J. Immunol. Meth., 4_0: 219 [1981]; and Nygren, J. Histochem. and Cytochem., 3_0: 407 [1982]. The antibodies of the present invention can be employed in any known assay method, such as competitive binding assays, direct or indirect sandwich assays, and immunoprecipitation assays. Zola, Monoclonal Antibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc., 1987). Competitive binding assays rely on the ability of a labeled standard (which may be a flk2 / flt3 receptor or an immunologically reactive portion thereof) to compete with the analyte in the test sample (flk2 / flt3) to bind to a limited amount of the antibody. The amount of flk2 / flt3 in the test sample is inversely proportional to the amount of standard that is bound to the antibodies. To facilitate the determination of the amount of standard to be bound, antibodies are generally insolubilized before or after competition, so that the standard and the analyte that are bound to the antibodies can be conveniently separated from the standard and of the analyte that remain unbound. Sandwich assays involve the use of two antibodies, each capable of binding to a different immunogenic portion, or epitope, of the protein to be detected. In a sandwich assay, the analyte in the test sample is linked by a first antibody that is immobilized on a solid support, and thereafter it binds to a second antibody to the analyte, thereby forming an insoluble, three-part complex . David & Greene, US Patent No. 4,376,110. The second antibody can itself be labeled with a detectable portion (direct sandwich assays) or can be measured using an anti-immunoglobulin antibody that is labeled with a detectable portion (indirect sandwich assay). For example, one type of sandwich assay is an ELISA assay, in which case the detectable portion is an enzyme. (c) humanized and human antibodies Methods for the humanization of non-human antibodies are well known in the art. In general, a humanized antibody has one or more amino acid residues introduced therein from a non-human source. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from a "import" variable domain. Humanization can be essentially performed following the method of Winter et al. (Jones et al., Nature, 321: 522-525 [1986]; Riechmann et al., Nature, 332: 323-327 [1988]; Verhoeyen et al., Science, 239: 1534-1536 [1988], by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody.As a consequence, such "humanized" antibodies are chimeric antibodies (Cabilly et al., Supra), where In practice, humanized antibodies are typically substituted by human antibodies in which some CDR residues and possibly some FR residues are substituted by a non-human species. Residues of analogous sites in rodent antibodies The choice of human variable domains, both light and heavy, to be used in the Preparation of humanized antibodies is very important in order to reduce antigenicity. According to the so-called "best fit" method, the variable domain sequence of a rodent antibody is selected against the entire library of known sequences of the human variable domain. The human sequence that is closest to that of the rodent is then accepted as the human structure (FR) for the humanized antibody (Sims et al., J ^ Immunol., 151: 2296 [1993]; Clothia and Lesk, J. Mol. Biol., 196: 901 [1987]). Another method uses a particular structure derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same structure can be used for several different humanized antibodies (Cárter et al, Proc Nati Acad Sci USA, 8: 9: 4285 [1992], Presta et al., J. Immunol., 151: 2623 [1993]). It is also important that the antibodies are humanized with the retention of high affinity for the antigen and other favorable biological properties. To achieve this goal, according to a preferred method, the humanized antibodies are prepared by a process of analysis of the progenitor sequences and various conceptual humanized products, using three-dimensional models of the humanized progenitor sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs that illustrate and visually show the probable three-dimensional conformational structures of the selected candidate immunoglobulin sequences are available. The inspection of these visual representations allows the analysis of the possible role of the residues in the functioning of the candidate immunoglobulin sequence, for example, the analysis of the residues that influence the ability of the candidate immunoglobulin to bind to its antigen. In this way, the FR residues can be selected and combined from the consensus and import sequence, so that the desired characteristic of the antibody is achieved, such as the increased affinity for the antigen or target antigens. In general, CDR residues are directly and more substantially involved in the influence of antigen binding. For further details see World Patent WO 94/04679 published March 3, 1994. Alternatively, it is now possible to produce transgenic animals (e.g., mice) that are capable, after immunization, of producing a full repertoire of human antibodies in absence of endogenous immunoglobulin production. For example, the homozygous deletion or omission of the gene from the binding region (JH) of the heavy chain of the antibody in chimeric and germline mutant mice has been reported to result in complete inhibition of the production of the endogenous antibody. The transfer of the human germline immunoglobulin gene array in such a germline mutant mouse will result in the production of human antibodies after challenge with the antigen. See, for example, Jakovits et al., Proc. Nati Acad. Sci. USA, 90: 2551-255 [1983]; Jakovits et al., Nature, 362: 255-258 [1993]; Bruggermann et al., Year in Immuno. , 7_: 33 [1993], Human antibodies can also be produced in phage libraries (Hoogenboom and Winter, J. Mol. Biol., 227: 381 [1991], Marks et al., J. Mol. Biol., 222: 581 [1991]). The techniques of Cote et al. And Boerner et al. Are also available for the preparation of human monoclonal antibodies (Cote et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p.77 [1985] and Boerner et al., J. Im uno1., 147: 86:95 [1991]). (d) Selection for agonist antibodies In order to select antibodies that are agonists for the flk2 / flt3 receptor, the tyrosine phosphorylation assay of Holmes et al.

(Science, 256: 1205-1210 [1992]) is available. This test is described in detail in Example IC herein. Alternatively, a thymidine incorporation assay can be performed using the transfected flk2 / flt3 receptor within a cell line dependent on IL-3 (see Example IC in the present).

III. THERAPEUTIC USES FOR AGONIST ANTIBODIES ANTI-flk2 / flt3 The agonist antibodies of the present invention can be used to increase the repopulation of mature blood cell lines, in cells that have undergone chemotherapy or radiotherapy, or therapy for bone marrow transplantation. In general, the antibodies will act via an enhancement of the proliferation and / or differentiation of primitive hematopoietic cells. Antibodies can, for example, increase the proliferation and differentiation of lymphoid myeloid lines. Agonist antibodies are similarly useful for the treatment of diseases caused by a decrease in blood cells. Examples of these diseases include: anemia (including macrocytic and aplastic anemia); thrombocytopenia; hypoplasia; immune thrombocytopenic purpura (autoimmune) (ITP); and ITP induced by HIV. Also, agonist antibodies are useful for the treatment of patients who have suffered from hemorrhage. The antibodies described herein can be administered to a human patient, in a pharmaceutically acceptable dosage form, suitable for intravenous, subcutaneous or intramuscular administration. Such dosage forms encompass pharmaceutically acceptable carriers that are inherently non-toxic and non-therapeutic. Examples of such carriers include ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids. , water, salts, or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium acid phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, pilivinylpyrrolidone, cellulose-based substances, and polyethylene glycol. The molecules will typically be formulated in such vehicles at a concentration of about 0.1 mg / ml to 100 mg / ml. The pharmaceutical compositions can be prepared and formulated in dosage forms by methods known in the art; for example, see Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania, 15th Edition 1975. From about 1 to 500 μg / kg, preferably from about 1 to 100 μg / kg, more preferably from about 10 to 100 μg / kg, and still more preferably from 10 to 50 μg / kg. kg of antibody, is an initial candidate dose, suitable for administration to the patient, either by, for example, one or more separate administrations or by continuous infusion. For repeated administrations over several days or longer, depending on the condition, the treatment is repeated until a desired suppression of the symptoms of the disease occurs, or until the desired improvement in the patient's condition is achieved. The dose can be administered at intervals ranging from once a week to once every six months. The determination of the optimal dose and the optimal route and frequency of administration is well within the knowledge of those skilled in the art. Similarly, doses for other molecules within the scope of the present invention can be determined without undue experimentation. The treatment according to the present invention can be combined with other therapies to improve repopulation of hematopoietic blood cell lines. For example, the antibodies can be co-administered with other cytokines or hematopoietic growth factors that are capable of increasing the proliferation and / or differentiation of hematopoietic cells (e.g., Epo, interleukins; IL-1, IL-3, IL-6 , IL-11, GM-CSF, G-CSF, M-CSF, SLF, LIF, TNF, lymphotoxin, the ligand flk2 / flt3, ligand kit, IGF-1 and? -interferon, etc.

IV. NON-THERAPEUTIC USES FOR ANTIBODIES ANTI-flk2 / flt3 Anti-fIk2 / flt3 antibodies are useful in diagnostic assays for flk2 / flt3, for example, for the detection of their expression in specific cells, tissues or serum. The antibodies are labeled with flk2 / flt3 and / or are immobilized on an insoluble matrix. In one embodiment of a receptor binding assay, an antibody composition is immobilized on an insoluble matrix, the test sample is contacted with the immobilized antibody composition to adsorb the flk2 / flt3, and then the family members. immobilized are contacted with a plurality of antibodies specific for each member, each antibody being individually identifiable as specific for a predetermined member of the family, or by unique labels such as discrete fluorophores or the like. By determining the presence and / or quantity of each unique marker, the relative proportion and quantity of each member of the family can be determined. The antibodies are also useful for the affinity purification of flk2 / flt3 from culture of recombinant cells or from natural sources. General affinity purification techniques are well known in the art, and any of these can be used for this purpose. Suitable diagnostic assays for anti-fIk2 / flt3 antibodies are well known per se. For example, competitive immunoassay, sandwich and spherical inhibition techniques are useful. Competitive methods employ a phase separation step as an integral part of the method, while spherical inhibition tests are conducted in a simple reaction mixture. Fundamentally, the same procedures are used for the flk2 / flt3 assay and for substances that bind to flk2 / flt3, although certain methods will be favored depending on the molecular weight of the substance being tested. Therefore, the substance to be tested is referred to herein as an analyte, regardless of its status otherwise as an antigen or antibody, and the proteins that bind to the analyte are referred to as binding partners, whether they are antibodies, cell surface receptors or antigens. The analytical methods for flk2 / flt3 or its antibodies all use one or more of the following reagents: labeled analyte analog, immobilized analyte analog, labeled binding partner, immobilized binding partner and spherical conjugates. The labeled reagents are also known as "trackers". The marker used (and this is also useful for labeling the flk2 / flt3 nucleic acid for use as a probe) is any detectable functional group that does not interfere with the binding of the analyte and its binding partner. Numerous markers are known for use in immunoassay, examples including portions that can be detected directly, such as fluorochrome markers, aminiscent and radioactive chemoi, as well as portions, such as enzymes, which must be reacted or derivatized to be detected. Examples of such labels include the radioisotopes J "P, 14C, 125I, ~ H and 131I, fluorophores such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luciferases, for example, firefly and bacterial luciferase (U.S. Patent No. 4,737,456), luciferin, 2,3-dihydrophthalazinodiones, malate dehydrogenase, urease, peroxidase such as horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, saccharide oxidases , for example, glucose oxidase, galactose oxidase, and glucose 6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase, coupled with an enzyme that employs hydrogen peroxide to oxidize a dye precursor such as HRP, lactoperoxidase, or microperoxidase, biotin-avidin, spin markers, bacteriophage markers, stable lnres radicals, and the like. Those of ordinary skill in the art will know of other suitable markers that may be employed in accordance with the present invention. The binding of these markers to flk2 / flt3, antibodies or fragments thereof, can be achieved using standard techniques commonly known to those of ordinary skill in the art. For example, coupling agents such as dialdehydes, carbodiimides, dimaleimides, bis-imidates, bis-diazotized benzidine and the like can be used to label or label the polypeptide with the fluorescent, chemiluminescent and enzymatic labels described above. See, for example, US Patent No. 3,940,475 (-fluorimetry) and 3,645,090 (enzymes); Hunter et al., Nature, 144: 945 [1962]; David et al., Biochemistry, 1_3: 1014-1021 [1974] Pain et al., J. Immunol. Methods, 40: 219-230 [1981]; Nygren, J. Histochem. and Cystochem., 30: 407-412 [1982]; O'Sullivan et al., Methods in Enzymology, de., J.J. Langone and H. Van Vunakis, Vol. 73 (Academic Press, New York, New York, 1981), pp. 147-166; Kennedy et al., Clin. Chim. Acta, 7_0_: 1-31 [1976]; and Schurs et al., Clin. Chim. Acta, 8 ^: 1-40 [1977], The coupling techniques mentioned in the last reference are the glutaraldehyde method, the periodate method, the dimaleimide method and the m-maleimidobenzyl-N-hydroxysuccinimide ester method . In the practice of the present invention, enzyme labels are a preferred embodiment.

No single enzyme is ideal for use as a marker in any conceivable assay. Rather, one must determine which enzyme is suitable for a particular assay system. The important criteria for the choice of enzymes are the number of productivity of the pure enzyme (the number of substrate molecules converted to product per enzyme site per unit of time), the purity of the enzyme preparation, the sensitivity of the detection of. its product, the ease and speed of detection of the enzymatic reaction, the absence of interference factors or enzyme-like activity in the test fluid, stability of the enzyme and its conjugate, availability and cost of the enzyme and its conjugate, and similar. Included among the enzymes used as preferred labels in the assays of the present invention are alkaline phosphatase, HRP, beta-galactosidase, urease, glucose oxidase, glucoamylase, malate dehydrogenase, and glucose-6-phosphate dehydrogenase. Urease is among the most preferred enzyme labels, particularly because of the chromogenic pH indicators that make its activity easily visible to the naked eye. The immobilization of reagents is required for certain test methods. Immobilization involves the separation of the binding partner from any analyte that remains free in solution. This is conventionally achieved either by the insolubilization of the binding partner or the analogous analyte before the test procedure, or by adsorption to a water-insoluble matrix or surface (Bennich et al., US Patent No. 3,720,760), by covalent coupling ( for example, using cross-linking with glutaraldehyde), or by insolubilization of the partner or the analogue subsequently, for example, by immunoprecipitation. Other test methods, known as competitive or sandwich assays, are well established and widely used in the commercial diagnostic industry. Competitive assays rely on the ability of a tracer analog to compete with the analyte in the test sample for a limited number of binding sites on a common binding partner. The link partner is generally insolubilized before or after the competition, and then the tracer and the analyte linked to the link partner are separated from the unlinked tracer and analyte. This separation is achieved by decantation (where the binding partner was preinsolubilized) or by centrifugation (where the binding partner was precipitated after the competitive reaction.The amount of the test sample analyte is inversely proportional to the amount of linked tracer, as measured by the amount of the labeling substance, the dose-response curves with known amounts of analyte are prepared and compared with the test results to quantitatively determine the amount of analyte present in the test sample. ELISA when enzymes are used as detectable markers Another competitive test species, called a "homogeneous" assay, does not require phase separation, here, a conjugate of an enzyme is prepared with the analyte, and is used such that when the Anti-analyte binds to the analyte, the presence of the anti-analyte modifies the activity of the enzyme. case, flk2 / flt3 or its immunologically active fragments are conjugated with a bifunctional organic bridge to an enzyme, such as peroxidase. The conjugates are selected for use with anti-fIk2 / flt3, so that the binding of anti-fIk2 / flt3 inhibits or makes the activity of the marker enzyme more potent. This method per se is widely practiced under the name of EMIT. Spherical conjugates are used in spherical impediment methods for homogeneous testing. These conjugates are synthesized by the covalent linkage of a low molecular weight hapten to a small analyte, so that the antibody to the hapten is substantially unable to bind to the conjugate at the same time as the anti-analyte. Under this test procedure, the analyte present in the test sample will bind to the anti-analyte, whereby the anti-hapten is allowed to bind to the conjugate, resulting in a change in the character of the conjugated hapten, for example , a change in fluorescence when the hapten is a fluorophore. Sandwich assays are particularly useful for the determination of flk2 / flt3 or antibodies against flk2 / flt3. In sequential sandwich assays a mobilized binding partner is used to adsorb the analyte from the test sample, the test sample is removed by washing, the bound analyte is used to adsorb the labeled binding partner, and the bound material is then separated from the residual tracker. The amount of linked tracer is directly proportional to the analyte of the test sample. In "simultaneous" sandwich assays the test sample is not separated before the addition of the labeled binding partner. A sequential sandwich assay using an anti-fIk2 / flt3 monoclonal antibody as an antibody and a polyclonal anti-fIk2 / flt3 antibody as the other, is useful when testing samples for flk2 / flt3 activity. The agonist antibodies bind to and activate flk2 / flt3, and are therefore useful diagnostic tools for further characterization of the biological activity of this receptor in vi tro and / or in vi vo. Agonist antibodies are also useful for diagnostic purposes. Similarly, the antibodies can be contacted with primitive hematopoietic cells and thereby lead to the proliferation and differentiation of mature blood cell lines. This is useful where mature blood cells are required for scientific or therapeutic purposes, or where differentiation of cells is under investigation. The above are merely exemplary diagnostic tests for antibodies to flk2 / flt3. Other methods now or later developed for the determination of these analytes are included within the scope of the present, including the bioassays described above. All citations throughout the specification and the references cited therein, are expressly incorporated by reference herein.

EXAMPLE 1 PRODUCTION OF AGONIST ANTIBODIES AGAINST THE RECEIVER flk2 / flt3 A. Cloning of the murine flk2 / fl t3 receptor The murine flk2 / flt3 receptor was cloned by RT-PCR from the RNA isolated from fetal livers of medium pregnancy mice. Six groups of overlapping primers were designed for the murine flt-3 sequence described by Rosnet et al. (Qncogene, _6: 1641-1650 [1991]). These primers were designed to amplify three segments of the genetic nucleotides 1-1307, 1308-1992, 1993-3096. The PCR products were then subcloned into pRK5.A. The sequence of the full-length flk2 / flt3 gene was identical to the sequence of the murine flt-3 gene published by Rosnet et al., Supra. An IgGi Fe fusion gene from the extracellular domain (ECD) of flk2 / flt3 was constructed, and the fusion protein was produced as previously described (Bennett et al., J. Biol. Chem., 2_66: 23060-23067 [ 1991]). The fusion protein was purified using protein A sepharose and the purified fusion protein was used for the generation of agonist antibodies against the extracellular domain of the flk2 / flt3 receptor.

B. Production of political bodies and monoclonal is Polyclonal antibodies were generated in New Zealand white rabbits against the flk2 / flt3 fusion protein. 4 μg of the fusion protein in 100 μl of PBS was emulsified with 100 μl of Freund's adjuvant (complete adjuvant for primary injection and incomplete adjuvant for all challenges.) For primary immunization and the first challenge, the protein was injected directly Within the popliteal lymph nodes (Sigel et al., Methods Enzymol., 93: 3-12 [1983]) .For the subsequent challenges, the protein was injected into subcutaneous and intramuscular sites.Injected 1.3 μg protein / kg of weight body every 3 weeks with bleeds taken 1 and 2 weeks after each challenge Baf-3 cells were transfected with the full-length flk2 / flt3 gene, and used to determine the specificity of the antibodies produced for the flk2 / flt3 fusion -IgG.Significant peak displacements were observed in the expression clones of flk2 / flt3 compared to the pre-immune serum or to the transfectant controls alone of the vector. 579A Anti-flk2 / flt3 monoclonal antibodies were produced by hyperimmunization of BALB / c mice intraperitoneally with the extracellular domain fusion protein of flk2 / flt3 (ECD) -Fe of human Igd in RIBI adjuvant (RIBI ImmunoChem Research, Hamilton, MT ) and fusion of Syrian hamster splenocytes with the mouse myeloma cell line P3X63 Ag8 Ul (Sanchez-Madrid et al., Immunol. J., 130: 309 [1983]). Antibodies were modified from ascites fluid using protein A-Sepharose / Repligen Corp., Cambridge, MA) and established affinity chromatography methods (Goding, J. Immunol.Methods, 2_0: 241-253 [1978].) The specificity of flk2 / flt3 was evaluated by flow cytometric analysis of BAF-3 cells transfected with the full-length receptor.The untransfected progenitor line it was used as a control.The 579A and polyclonal rabbit sera and the monoclonal antibody IC2-514 were also shown to immunoprecipitate the flk2 / flt3 receptor from transfected cell lines, labeled with S35-methionine.The anti-fIk2 / flt3 hybridomas murine, hamster, IC2-514 and IC2-310, were selected from the selection.The hybridoma IC2-310 was deposited with the ATCC on March 4, 1994 under accession number ATCC HB 11,557.The polyclonal and monoclonal agonist antibodies were selected by using the techniques described below.

C. Trials for an agonist bodies The agonist activity of 579A polyclonal antibodies and monoclonal antibody IC2-310 was determined using two assay systems: (a) a phosphotyrosine assay using the full-length murine flk2 / flt3 receptor, expressed on the cell line dependent on IL-3, BAF-3, and (b) a thymidine incorporation assay using the full-length receptor expressed on the IL-3-dependent cell line. BAF-3. BAF-3 cells were electroporesed with the full length flk2 / flt3 receptor as previously described (Colosi et al., J. Biol. Chem., 268: 12617-12623 [1992]). Tyrosine phosphorylation experiments were performed using the transfected flk2 / flt3 receptor within IL-3 dependent cells, as previously described (Holmes et al., Science, 256: 1205-1210 [1992]). Briefly, transfected Baf-3 (BAF-3T) or untransfected BAF-3 (BAF-3) cells were deprived of IL-3 and incubated with the suspected agonist antibody (polyclonal antiserum hybridoma supernatant, diluted a 1:20) or irrelevant control antibody for 30 minutes, used in IP lysis buffer (1% NP-40, 1 mM EDTA, 200 mM sodium chloride, 50 mM Tris-HCl, pH 8.0, 2 mM PMSF, Na3V04 2.5 M) and immunoprecipitated with 579A polyclonal antiserum. The used immunoprecipitates were separated on a 4-12% SDS-PAGE gradient gel, transferred to nitrocellulose and stained by Western using the antiphosphotyrosine antibody 4G10. Immunoprecipitation of immunoprecipitated material using anti-phosphotyrosine antibodies demonstrated the phosphorylation of the flk2 / flt3 receptor in response to IC2-310 and 579A, without phosphorylation of the 160 kD band observed for BAF-3 cells, untreated cells, or cells treated with an irrelevant control antibody. The molecular weight of the flk2 / flt3 receptor probably reflects extensive glycosylation of the receptor, as predicted by the sequential analysis (Matthews et al., Supra).

Similar molecular weight values have been obtained for the full-length receptor in other studies (Lyman et al., Upra and Maroc et al., 1993, supra). For the thymidine incorporation assay, 32D cells were deprived of IL-3 for 24 hours and then stimulated with antibody overnight, followed by an 8-hour pulse of one μCi of [5H] -thymidine. Thymidine incorporation was then determined using a cell harvester. 579A and IC2-310 gave significant stimulation of thymidine uptake in the transfected 32D cells. The specificity of these responses was demonstrated by the lack of response to irrelevant antibodies and hamster IgG (Figure 1). After the discovery of the agonist activity of IC2-310, the hybridoma supernatant was purified by protein A affinity. The purified antibody retained the activity at concentrations between 10-40 μg / ml. All subsequent in vitro trials routinely used 49 μg / ml. The polyclonal antibody (579A) and the monoclonal antibody IC2-310 were demonstrated in each of the above assays, as capable of activating the flk2 / flt3 receptor.

EXAMPLE 2 REPUBLICATION OF THE HEMATOPOYETIC SYSTEM This example established whether the flk2 / flt3 receptor was expressed or not on cells of the hematopoietic line, capable of long-term grafting of lethally irradiated hosts.

A. Ai sl es on the population of cell phones of the topographic line Populations of the hematopoietic line were isolated from AA4 + cells derived from fetal liver of medium gestation, as previously described (Jordán et al., Cell, 61: 953-963

[1990]) . AA4 cells were fractionated into Sea and Sea subpopulations, using the ly6 A / E conjugate phycoerythrin (Pharmingen). The CD34 + or CD34"populations of AA4 were derived using an affinity purified rabbit anti-mouse CD34 (Baumhueter). et al., Science, 262: 436-438 [1993]). The purified monoclonal antibody IC2-514 discussed in Example 1 was conjugated to biotin or phycoerythrin for FACS analysis. The anti-mouse-biotin c-kit conjugate was purchased from Pharmingen and all secondary and cocktail LIN antibodies were purchased from Caltag. The bone marrow hematopoietic line cells were obtained from C.57B / 6 Ly 5.1 or 5.2 mice, 8 to 12 weeks of age. The mononuclear cell fraction was isolated by density gradient (Aecudenz, Aecurate Biochemicals) and stained with the cocktail antibodies LIN as previously described (Jordán et al., Supra). Cells stained with LIN were removed via diminution of magnetic sphere (Dynal, Inc., Ploemacher et al., Blood, 7_4: 2755-2763 [1989] and Lin1 population stained with the appropriate antibodies.The stained cells were selected by exclusion with propidium iodide (1 μg / ml) and separated on an elite flow cytometer (Coulter Electronics, Haileah, Florida).

B. Competitive repopulation The competitive repopulation technique used in the following experiment allows the comparison of the progenitor cell content of each of the derived populations. Competitive repopulation determines the proliferative capacity of two populations of donor hematopoietic cells (Harrison et al, Hematol, E., 21_: 206-219 [1993]). In this case, one million bone marrow cells were used as the competitor, and the content of progenitor cells of the population of potential progenitor cells, for example, AA4 + Sca + flx-2 + was measured relative to the population of the competitor.

Equivalents of progenitor cells (SCE) per 10,000 cells were determined using competitive repopulation analysis of genetically-labeled fetal liver or bone marrow in allelic C57BL / 6 mice at the Ly 5 locus, designated Ly 5.1 and Ly 5.2. The expression of Ly 5.1 in Ly 5.2 mice was determined at selected intervals after grafting. Progenitor or totipotent cell populations were isolated from young adult mice C57B1 / 6 Ly 5.1. Male mice, adults, young C57B / 6 Ly 5.2 were obtained from NCI and used as containers. A minimum of five animals was used per experimental group. Irradiation of the whole body (1100 cGy, 190 cGy / min) was administered as a single dose from a source of C137. In general, one million bone marrow cells of mice 5.2 were used as competitors, and the progenitor cell content of 1 x 104 cells and 5.1 of the population of potential progenitor cells, for example, AA4 * Sca + flk2 ~ measured in relation to the competing population. The cells were administered via tail vein injection and peripheral blood samples (50-100 μl) were obtained by retro-orbital sinus 4 weeks, 12 weeks and 6 months after reconstitution. The percentage of donor cells I and 5.1 was determined by staining with monoclonal antibody I and 5.2 conjugated to biotin (A20.1.7). To confirm repopulation by donor cells l and lll on all lines, the peripheral blood cells and the mononuclear fraction of bone marrow were stained with the following antibodies: B220 (lines of β cells, CD4 / 8 (T cell lines) , Gr-l / Mac-1 (myeloid lines) The number of progenitor or totipotent cells in each sample (equivalent of progenitor cells, SCE) was determined as described by Kiefer et al., (Blood, 78 (10): 2577 -2582 [1991]) The content of bone marrow progenitor cells was estimated to one progenitor cell per 105 total bone marrow cells (Harrison et al., Supra) .Therefore, 1 x 10 ° bone marrow cells used as a competitor, they contained 10 progenitor cells.This means that the contribution of the progenitor cell population I and 5.1 that is tested, is therefore defined by the equation: x T- (10 + x) = average fraction of repopulation% of l and 5.1 x = SCE The results are shown in Table 1. All lines were reconstituted by 5.1 donor cells. The results of the isolations of AA4 * kit * flk-2 * "represent a time point of 8 weeks.

TABLE 1 CELLULAR POPULATION 5 WEEKS WEEKS WEEKS SCE% 5.% 5.1 AA-i + S-a + f. - 72 ± 3 33 ± 4 30 ± 2 45 AA- Fl -2! ")? 9 ± 5 41 92 ± 2 57 AA4" D34 Fl? - 41 ± 12 66 ± 10 68 ± 9 11"34 f .. se ± ie "± 8 84 ± 4 rit" F; -.-_ * ol ± 6 16 '1 + 5 AA- Flk-2 () ,, ± 7 46 ± 10 AA4 *' i '75 ± 4 30 84 ± 5 0 ± 3 AA4 * -334 * * 't * 42 51 ± 14 10 49 ± 24 AA CDJ4 i' 2 ± 2 1 2 ± 6 4 ± 3 LI iQ: D34 Flk- 51 0 44 ± 25 53 + 22 11 Sca * F k- 50 40 ± 6 25 ± 1 Sc3 Flk- 53 ± 10 1 6 ^ + 12 20 In the fetal liver, the antibody AA4.1 (AA4) delineates a population of 1% of the cells in which all the activity of the totipotent progenitor cells resides (Jordán et al., Supra). The population AA4 * can be further enriched using antibodies to the antigen Sca-1 (ly6A / E). Population AA4 * Sea * contains all the activity of the totipotent progenitor cells (Matthews et al., Supra). Experiments using the CD34 polyclonal antibody demonstrated that 60% of the AA4 + population are CD34 positive (Figure 1). In addition, repopulation studies showed that all the activity of the progenitor cells lies in the AA4 + CD34 * fraction, and that the AA4 * CD34 population was not repopulated (Table 1) .It is also interesting to note that all the cells AA4 * CD34 * are positive for the expression of c-kit (Figure 2) and all AA4 * Sea * cells are also positive to CD34.The positive fraction to c-kit of population AA4 * was also shown to contain all The activity of the progenitor cells Progenitor or totipotent cell populations AA4 + Sea *, AA4 * CD34 *, and AA4 * kit * were used to investigate the expression of progenitor or totipotent cells of the flk2 / flt3 receptor (Figure 2). Repopulation studies demonstrated that the flk2 / flt3 receptor can be expressed on totipotent cells, but that the positive and negative totipotential cell fractions at flk2 / flt3 give rise to long-term reconstitution (Table 1).

In experiments (Table 1). In experiments using bone marrow as the source of the totipotential cells, the mononuclear fraction was secreted in a Lin10 population by separation by immunomagnetic spheres using the Lin cocktail of antibodies for mature hematopoietic cell types (Ploemacher et al., Blood, 74: 2755- 2763 [1989]). The LinL mononuclear cells: were fractionated into the totipotent cell fractions Linio CD34 * and Lin: ° Sea * (Figure 2). According to the fetal liver populations, these populations of totipotent cells of bone marrow gave positive subpopulations at flk2 / flt3 or negative at flk2 / flt3. These two subpopulations gave rise to long-term repopulation (Table 1). Previous experiments demonstrate the high content of totipotential cells from the AA4 * CD34r kit * and AA4 * Sea * flk-2 populations. "Furthermore, these indicate a general trend suggesting that the populations of flk2 / flt3 negative cells have a higher content of totipotential cells, when compared to the positive population at flk2 / flt3, relevant (Table 1) It should be noted that in the marrow there are very few LinJJ Sea * flk-2"cells, being the largest population of Lin10 Sea * positive to flk2 / flt3 (Figure 2). Fractionation of bone marrow mononuclear cells Lin10 in flk-2 * and flk-2"cells showed that LinlG flk-2 * cells were more potent in the short-term repopulation of the irradiated host (3.47 versus 1.25 equivalent totipotential cells or progenitors per 10,000 cells.) Of course, the repopulation from LinlQ flk-2 * cells was minimal after 12 weeks, suggesting that the Linio flk-2 * population is comprised of more committed parents than its flk-2 counterpart. * These experiments of reconstitution of lethally irradiated mice, clearly demonstrates that the flk2 / flt3 receptor tyrosine kinase is expressed on populations of totipotential cells, but very clearly, not all totipotential cells express flk2 / flt3. This finding was confirmed in all the different populations isolated from totipotent cells of the liver and bone marrow. Using competitive repopulation, it was demonstrated that fractions of totipotent cells positive to flk-2 have a significantly lower repopulation capacity than their relative negative counterparts flk-2. The most widely used marker for the study of human hematopoietic cells is cell surface expression of CD34. Several functional assays have shown that the CD34 positive subpopulation of human bone marrow contains virtually all primitive hematopoietic cells (Andrews et al., Med. JE., 169: 1721 [1989]; Berenson et al., Invest. JC. , 8_1: 951-955 [1988]; Civin et al., Immunol. ., 133: 157-165 [1984]; and Sutherland et al., Blood, "7_4: 1563-1569 [1989].) The polyclonal antibody specific for murine CD34 clearly demonstrated that according to the human homologue, the activity of the totipotential cell in murine hematopoiesis is confined to the CD34 fraction * Therefore, the murine hematopoietic totipotential cell phenotype from fetal liver is AA4 * LiniJ Sea * CD34 * kit * flk-2 + ". From the bone marrow, the totipotential cell phenotype is Lin ~ ° Sea * kit * CD34 * flk-2 * 7".

• JEMPLO 3 ANALYSIS OF THE EXPRESSION CELL CYCLE OF flk2 / flt3 Cell cycle analysis of hematopoietic totipotential cell populations has previously indicated that totipotent cell populations are heterogeneous in relation to cell cycle status (Fleming et al., Biol. JC., 1_22: 897-902 [1993] and Suda and collaborators, Physiol., JC., 117: 308 [1983]). In addition, the augmented repopulation has been attributed to those totipotential cells in the G,; / G-L phase, in comparison to the S / G2 / M subgroup of active proliferation (Fleming et al., Supra). If the expression of flk2 / flt3 on totipotential cells represents a population of totipotential cells potentially more compromised, showing decreased repopulation capacity, this can be reflected in the state of the cell cycle. Furthermore, it has been suggested that RNA expression of flk2 / flt3 is correlated with the cycle of totipotential cells (Orlic et al., Supra and Visser et al., Supra). Therefore, the cell cycle status of the population of totipotent cells of fetal liver (AA4 * Sea * was determined with respect to the expression of flk2 / flt3 (Figure 3).) An acridine orange staining technique was used, Two-step, to differentiate the cells in G0 phase of the Gi cells. When coupled with conventional cell cycle analysis of the DNA content histogram, this method allows the simultaneous analysis of the compartments of the cell cycle phase G0, Gx, S and G; M from any cell population Acidine orange staining of two steps was performed as previously detailed (Darzynkiewicz and Kapuscinski, Flow Cytometry and Sorting, 2nd edition, Wiley-Liss, 291-314; and Baumhueter et al., Science, 262: 436-438 [1993]. In summary, cells that had been selected after dual-parameter immunofluorescence staining were centrifuged (400 x 5 min) and resuspended in culture medium of RPMI 1640 cells with 10% fetal bovine serum at a final concentration of lOVml. To 0.3 ml of this cell suspension, a solution consisting of 0.45 ml of 0.1% Triton X-100 in 0.15 N sodium chloride and 0.08 N hydrochloric acid was added, and the mixture was incubated for 45 minutes on ice. To this solution, 1.8 ml of a solution consisting of 12 μm acridine orange (Polysciences, Inc.) was added in 0.2 M Na2HP0, 0.1 M citric acid, 10"3 M Na-EDTA and 0.15 M sodium chloride, and the sample was immediately analyzed by flow cytometry Red fluorescence (RNA) and green fluorescence (DNA content) were collected simultaneously by the addition of a 560 nm long-pass dichroic filter, coupled with a 525 + band pass filter / - 15 nm (green fluorescence) and a 630nm bandpass filter (red fluorescence) The G0 subpopulation was defined based on the red fluorescence of the peripheral blood mononuclear cells (PBMC) stained in parallel to the previously selected samples. A cursor was placed in the position corresponding to the intensity of the red resuscitation of 97% of PBMC, with the cells having the highest RNA contents above this position classified as populations in c iclo (for example, Gi, G2 S, G2M), and those at or below the cursor classified as Gr,. The enumeration of cell proportions in the cycle was performed by conventional cell cycle analysis using the algorithm of Dean and Jett, 1974; available in the computer software collection (software) Multicycle (Phoenix Flow Systems, San Diego, CA). It was shown that the main difference between the 2 subpopulations (AA4 * Sea * flk-2"and AA4 * Be" flk-2") was the increasing percentage of cells residing in G- in the population AA4 * Sea * flk -2"(Figure 3). The percentage of cells in S / G2 / M clearly indicated that these populations of totipotent cells of fetal liver contain many actively proliferating cells. Cell cycle analysis of the totipotential cell populations AA4 * kit * showed a much lower percentage of cells in G0, when compared with the population AA4 * Sea * flk-2. "However, little difference was found between the populations AA4 * kit * flk-2 * (Gc -12%, Gi - 39%, G2 / M - 8 %) and AA4 * kit * flk-2"(G0 - 7%, d-47%, S-41%, G: / M-5%). These data indicate that the flk2 / flt3 receptor is expressed by a subgroup of hematopoietic totipotential cells destined to differentiate to more committed progenitor cells. This hypothesis gains support from studies that have shown decreased radioprotective capacity in totipotent cells subjected to the cell cycle (Fleming et al., Biol. JC, 122: 897-902 [1993]), and the expression of mRNA from flk2 / flt3 in fractions of totipotent cells that are believed to be actively cycled (Orlic et al., Blood, £ 2: 762-770 [1993]; and Visser et al., Cells S., l_l_: 49-55 [1993]).

EXAMPLE 4 HEMATQPOYETIC TESTS OF AGONISTIC MONOCLONAL ANTIBODY FOR flk2 / flt3 A. If Dexter Cul tive Test System To assist in the evaluation of the biological function of the agonist antibody IC2-310, a Dexter culture assay system was developed using the immortalized stromal cell lines of the fetal liver. The fetal liver stromal cells were isolated by infection of primary cultures of fetal stroma with the recombinant retrovirus, as previously described (Larsson et al, Immunol. D., 1: 279-293 [1991]). Viral stocks of the recombinant retroviruses pZipSvtsA58 were prepared from previously characterized viruses that produce f2 cell lines (Cepko et al., Cell, 37: 1053-1062 [1984]; and Sharp et al., Biol. MC., 9: 1672-1681 [1989]). One of the resulting stromal cell lines designated 7-4 was used in these experiments. The following stromal cell / totipotential cell coculture assay was performed. The populations of hematopoietic totipotential cells were seeded at 10 4 cells / ml on the stromal line of fetal liver 7-4 in DMEM / F12 medium supplemented with 10% FCS. Cocultures were incubated at 37 ° C for 7 days. The content of totipotent cells was determined by competitive repopulation analysis before and after 7 days of cocultivation. The results obtained from each in vitro test system were confirmed in a minimum of three independent experiments. Growth factors were used at the following concentrations: KL-50 ng / ml; IL-3 - 1 ng / ml (Genzyme); GM-CSF - 0.2 ng / ml (R & D Systems); PDGF B / B - 2 ng / ml; and bFGF 2.5 ng / ml (Boehringer Mannheim, USA, Indianapolis, IN). The control wells were medium alone or medium impregnated with hamster Ig-G. After 7 days of co-culture, the resulting cell populations could only support the short-term repopulation of the irradiated host, as evidenced by the contribution of co-cultured donor cells at 4 weeks after grafting. However, after this early time point no additional contribution was observed from the cocultivated cells. The cocultivation from stroma 7-4 gave rise to the dramatic expansion in the number of cells. See Table 2 below.

TABLE 2 CELLULAR POPULATION CONTROL 310 \ '_ V_ * - 31 C AA4 + Kit * Flk-2 * 33 ± 14 52 ± 2 21 O ± l? 276 ± 12 AA ^ Kit * F1 .-- 2 *! ) 32 ± 3 28 ± 1 71 ± 12 84 ± 5 A * SCA * 8 ± 0.71 31 ± 2.1 120 ± 14 18 O ± l 3 AA4 * SCA () 6 ± J.71 6 ± 0.84 13 ± 0.3o 12 ± 0.35 AA4 * -D34 * F? F * 12 ± 2.6 22 ± 2.3 95 ± 6.1 1 _ 9 ± 7 JLAR POPULATION: 0 TR0L 310 KL - 3 15 ± 5 52 + 11 ^ ± 38 Line analysis of the resulting cell populations was performed using flow cytometric analysis and Wright Geisha staining of cytosine material. These analyzes demonstrated the presence of immature, myeloid and lymphoid progenitor cells (see Table 3 below).

TABLE 3 ANALYSIS OF CYTOPSINE AA 4 + 3 3 *% of% of Blast% I q MNC Mieloi is L-.nfo? De = Med 3 ío 47 13 IC2-J10 10 30: < -_- '13 - L 35 IC2-31C / GMCSF 31 52 < 10 / KL / GMC3F 23 FACS ANALYSIS AA4% MAC-1 * Gr-1 * CD4 / CD "O111 z _ b5 64 11 63 Many cells maintained the expression of Sea, flk2 / flt3, c-kit and CD34.The multiple-line potential of this haematopoietic microenvironment was also The hematopoietic cells were harvested from coculture of totipotent cells / stromal cells after 7 days and stained for FACS analysis using the MAC-1 antibody (macrophages), GR-1 (granulocytes), ß220 ( ß cells), CD4 / 8 (T cell markers) These data are presented from a representative experiment The experiments were repeated a maximum of three times The results are shown in Table 3. This was confirmed by analysis of cytosine which identified a variety of hematopoietic cell types, including those of the myeloid and lymphoid series. The cytosine differentials from the cell populations of the line AA4 * CD34 * kit * after cocultivation with the stromatic cell line 7-4, for 7 days, were cocultivated in the presence of the indicated growth factors. Blasts are immature phenotype cells that contain precursors for many lines. Large mononuclear cells (lg MNC) are cells of intermediate size and differentiation that contain many precursor cells of the myeloid and lymphoid lines. The myeloid cells are cells of mature myeloid images. Lymphoid cells are cells that show characteristics of plasma or lymphocytic cells. See Table 3. Totipotential cells plated in the presence of agonist antibody IC2-310 gave rise to a proliferative event greater than that observed when they were plated on 7-4 alone. However, IC2.310 did not induce the proliferation of non-totipotential cell populations, such as AA4 * Sea. "In addition, IC2-310 had no effect on totipotential cell populations that do not express flk2 / flt3 (Table 2). The FACS of these cells again demonstrated the presence of several potential lines (Table 3) As with cells developed on 7-4 alone, cells stimulated with IC2-310 were only able to repopulate in the short term. the IC2-310 antibody was highly increased in combination with KL (Table 2) The cycopsin analysis of the cocultivated cells demonstrated a significant drop in the percentage of cells of the blasto type with a concomitant increase in the percentage of cells of the myeloid lines , including myeloblasts, myelocytes, promyelocytes or metamyelocytes (Table 3) Taken together, these data demonstrate the complete proliferation that results from the impedance of the flk2 / flt3 receptor. In addition, these illustrate that in the concept of cocultivation on the stromatic cell line 7-4, this proliferative event is accompanied by differentiation to more mature hematopoietic phenotypes. It is clear that the activation of the flk2 / flt3 receptor promotes the proliferation and differentiation of hematopoietic totipotential cells when these are co-cultured with stroma. This proliferation is more clearly evidenced by the increases in the number of cells and in the colony-forming cells, obtained after the activation of the totipotent cells with the agonist antibody IC2-310-, Conversely, the agonist antibody has • little effect on non-totipotential cell populations. The proliferation of the hematopoietic system by IC2-310 seems to be restricted to totipotent cell populations, and gives rise to an expanded population or more mature hematopoietic phenotypes.

B. Effect of monoclonal anti body IC2 - 310 on the formation of colons on methyl cel ulosa.

Hematopoietic colony assays were performed to determine the effects of the IC2-310 antibody on the potential for colony formation of primitive hematopoietic populations. Tests were performed on methylcellulose in the presence of conditioned WEHI media supplemented with KL in order to test the myeloid potential of the input cell, or alternatively, in the presence of IL-7 and KL to test the ß-lymphoid potential (McNiece et al. , Immunol. J., 146: 3785-3790 [1991]). Standard myeloid colony assays in methyl cellulose were performed using complete methyl cellulose medium (Ste Technologies, Inc., # M3430) with the addition of the 50 ng / ml kit ligand, KL (R & D Systems, Minneapolis, MN). The colonies were counted after 10 days in culture, only the colonies of more than 50 cells were evaluated. Lymphoid colonies were produced using methyl cellulose base (Stem Cell Technologies) with 50 ng / ml ligand kit and 50 ng / ml murine IL-7 (R &D Systems, Minneapolis, MN), see McNiece et al., Supra. The cytosine analysis of the resulting colonies was performed as previously described (Testa and Molineux, Hematopoiesis: Oxford IRL Press). The populations of hematopoietic totipotential cells plated on cells 7-4 and then removed after 7 days, were able to form myeloid cell-forming colonies (CFCs) and lymphoid CFCs. When the cocultivation on 7-4 was performed in the presence of the agonist antibody IC2-310, there was an approximately 5-fold increase in myeloid CFCs and a 12-fold increase in lymphoid CFCs (Figure 4). The cytosine data revealed that the myeloid colonies are mixed line, but these represented mainly the subgroup of granulocytes / macrophages. The analysis of the colonies produced in the presence of IL-7 and KL, demonstrated a B220f IgM phenotype. "Most of these cells were also stained for the S7 marker, which is considered to stain the cells of the fk line before the pre-β stage (Hardy et al., J, Ex. Med., 173: 1213-1225 [1991]) Once again, the proliferative effect of IC2-310 was restricted to the population of totipotential cells. effect on the AA4 * Sca "cell population, which does not contain totipotent cells (Figure 4). These results support the observations that the IC2-310 antibody is only capable of stimulating the proliferation of the most primitive hematopoietic cell populations.

Deposit of materials The following culture has been deposited with the North American Collection of Species Crops, 12301 Parklawn Drive, Rockville, MD, USA (ATCC): ATCC Hybridoma No. Date of Deposit anti-FLK2 / FLT3 ATCC HB 11,557 March 4, 1994 This deposit was made under the provisions of the Budapest Treaty on Recognition International Deposit of Microorganisms for the Purpose of Patent Procedure and the Regulations under it (Budapest Treaty). This ensures the maintenance of a viable crop for 30 years from the date of deposit. The agency will be made available by the ATCC under the terms of the Treaty of Budapest, and subject to an agreement between Genentech, Inc. and ATCC, which ensures the permanent and unrestricted availability of the progeny of the crop to the public, after the issuance of the relevant North American Patent, or after it remains open to the public of any US or foreign patent application, whichever comes first , and ensure the availability of the progeny to someone determined by the United States Patent and Trademark Commissioner, who is qualified for this, in accordance with 35 USC Section 122 and the rules of the Commissioner pursuant thereto (including CFR section 1.14). with particular reference to 886 OG 638). The assignee of the present application has agreed that if the cultivation in deposit must die or be lost or destroyed when cultivated under suitable conditions, this will be promptly replaced after notification, with a viable specimen of the same crop. The availability of the deposited strain is not considered as a license to practice the invention in contravention of the rights granted under the authority of any government, in accordance with its patent laws. It is considered that the above-described specification is sufficient to make it possible for someone of experience in the technique practice the invention. The present invention is not limited in scope by the deposited culture, since it is intended that the modality deposited as a simple illustration of one aspect of the invention and any culture that is functionally equivalent, is within the scope of this invention. The deposit of the material herein does not constitute an admission that the written description, contained herein, is inadequate to make it possible to practice any aspect of the invention, including the best modality thereof, nor is it to be considered as limiting of the scope of the claims to the specific illustration that this represents. Of course, various modifications of the invention, in addition to those shown and described herein, will be apparent to those of skill in the art from the foregoing description, and fall within the scope of the appended claims.

LIST OF SEQUENCES (1) GENERAL INFORMATION: (i) APPLICANT: GENENTECH, INC. (ii) TITLE OF THE INVENTION: AGONIST ANTIBODIES AGAINST THE FLK2 / FLT3 RECEIVER AND USES THEREOF (iii) SEQUENCE NUMBER: 4 (iv) ADDRESS FOR CORRESPONDENCE: (A) RECIPIENT: Genentech, Inc. (B) STREET: 460 Point San Bruno Blvd (C) CITY: South San Francisco (D) STATE: California (E) COUNTRY: United States of America (F) POSTAL CODE: 94080 (v) COMPUTER LEGIBLE FORM: (A) TYPE OF MEDIA: 5.25-inch, 360-kb floppy disk (B) COMPUTER: IBM-compatible PC (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) SOFTWARE: skate (Genentech) (vi) DATA OF THE CURRENT APPLICATION: (A) APPLICATION NUMBER (B) DATE OF PRESENTATION (C) CLASSIFICATION: (vii) PRIORITY APPLICATION DATA (A) APPLICATION NUMBER (B) SUBMISSION DATE: (viii) ATTORNEY / AGENT INFORMATION: (A) NAME: Wendy M. Lee (B) REGISTRATION NUMBER: 00,000 (C) REFERENCE NUMBER / DOSSIER 879PCT (ix) TELECOMMUNICATION INFORMATION (A) TELEPHONE: 415 / 225-1994 (B) TELEFAX: 415 / 952-9881 (C) TELEX: 910 / 371-7168 (2) INFORMATION FOR SEQ ID NO: 1: (i) CHARACTERISTICS OF THE SEQUENCE: A) LENGTH: 3521 bases B) TYPE: nucleic acid C) TYPE OF CHAIN: simple D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 1: GCCACCTGCA GCCCGGGGCG CGCCGCTGGG ACCOCATCAC AOOCTOOOCC 50 GGCGGCCTGG CTACCGCGCG CTCCGGAGGC CATGCGGOCO TTOßCOCAOC 100 GCAGCGACCG GCGGCTGCTG CtGCTtGTTO G? TOTCAGT AATGATTCTT 150 GAGACCGTTA CAAACCAAOA CCTOCCTGTG ATCAAGTOTO TTTTAATCAG 200 TCATGAGAAC AATGGCTCAT CAGCGGGAAA GCCATCATCO TACCGAATOG 250 TGCGAGGATC CCCAGAAGAC CTCCAGTGT? CCCCGAGGCO CCAGAGTOAA 300 GGGACGGTAT ATGAAGCGGC CACCGTGGAG GTGGCCGAOT CTOOOTCCAT 350 CACCCTGCAA GTGCAGCTCG CCACCCCAGG GGACCTTTCC TOCCTCTOOO 400 TCTTTAAGCA CAGCTCCCTG GGCTGCCAGC CGCACTTTQA TTTACAAAAC 450 AGAGGAATCG TTTCCATGGC CATCTTGAAC GTGACAGAGA CCC? OOC? 03 500 AGAATACCTA CTCCATATTC AGAGCGAAGC CGCCAACTAC ACAOTACTOT 550 TCACAGTGAA TGTAAGAGAT ACACAGCTGT ATGTGCTAAG GAGACCTTAC 600 TTTAGGAAGA TGGAAAACCA GGATGCACTG CTCTGCATCT CCGAGOOTGT 650 TCCGGAGCCC ACTGTGGAGT GGGtGCTCTs CAOCTCCCAC AßßOAAAOCT 700 GTAAAGAAGA AGGCCCTGCT GTTGTCAGAA AOGAOGAAAA GCTACTTCAT 750 GAGTTGTTCG GAACAGACAT CAGATGCTGT OCTAGAAATG CACTOGOCCG 800 CGAATCGACC AAGCTOTTCA CCATAQATCT AAACCAGGCT CCTCAOAGCA 850 CACTGCCCCA GTTATTCCTG AAAGTGGGGG AACCCTTGTQ GATCAC-TOT 900 AAGGCCATCC ATGTGAACCA TGGATTCGGG CTCACCTGGG AGCTOGAAGA 950 CAAAGCCCTG GAGGAGGGCA GCTACTTTGA GATGAGTACC TACTCCACAA 1000 ACAGGACCAT GATTCOGATT CTCTTOGCCT TTGTGTCTTC CaTGOOAAOO 1050 AACGACACCG GATATTACAC CTGCTCTTCC TCAAAGCACC CCAGCCAßTC 1100 AscGttssto ACCATCCTAG AAAAAGGOTT TATAAACGCT ACCAOCTCOC 1150 AAGAAGAGTA TGAAATTGAC CCGTACGAAA AGTTCTGCTT CTCAOTCAOG 1200 TTTAAAGCGT ACCCACGAAT CCGATGCACG TOGATCTTCT CTCAAGCCTC 1250 AJTTCCTTGT GAACAGAGAG GCCTGGAGGA TßGGTACAGC ATATCTAAAT 1300 TTTGCGATCA TAAGAACAAG CCAGGAGAGT ACATATTCTA TGCAGAAAAT 1350 GATGACGCCC AGTTCACCAA AATGTTCACG CTGAATATAA GAAAGAAACC 1400 TCAAGTGCTA GCAAATGCCT CAGCCAGCCA OGCGTCCTGT TCCTCTOATO 1450 GCTACCCGCT ACCCTCTTOG ACCTGGAAGA AGTGTTCGGA CAAATCTCCC 1500 AATTGCACGG AGGAAATCCC AGAAGGAGTT TGGAATAAAA AGGCTAACAG 1550 AAAAGTGTTT GGCCAGTGGG TGTCGAGCAG TACTCTAAAT ATOAGTGAGß 1600 CCGGGAAAGG GCTTCTOGTC AAATGCTGTG CGTACAATTC TATGGGCACG 1650 TCTTGCGAAA CCATCTTTTT AAACTCACCA OGCCCCTTCC CTTTCATCCA 1700 AGACAACATC TCCTTCTATG CGACCATTGG GCTCTGTCTC CCCTTCATTQ 1750 TTGTTCTCAT TGTGTTGATC TGCCACAAAT ACAAAAAGCA ATTTAGOTAC 1800 GAGAGTCAGC TGCAGATGAT CCAGGTGACT GGCCCCCTGG ATAACGAOTA 1850 CTTCTACGTT GACTTCAGGG ACTATOAATA TGACCTTAAa TOOOAOTTCC January 00 CGAGAGAGAA CTTAGAGTTT GGGAAGGTCC TOGGGTCTGG CGCTTTCGGG 1950 AGGGTGATGA ACGCCACGGC CTATGGCATT AGTAAAACGG GAGTCTCAAT 2000 TCAGGTGGCG GTGAAGATGC TAAAAGAGAA AGCTGACAGC TGTGAAAAAG 2050 AAGCTCTCAT GTCGGAGCTC AAAATGATGA CCCACCTGGO ACACCATGAC 2100 AACATCGTGA ATCTGCTGGG GGCATGCACA CTOTCAGOOC CAGTOTACTT GATTTTTGAA 2150 TATTGTTGCT ATGGTGACCT CCTCAACTAC CTAAOAAOTA 2200 AAAGAGAGAA GTTTCACAGG ACATGGACAG AGATTTTTAA GGAACATAAT 2250 TTCAGTTTTT ACCCTACTTT CCAOGCACAT TCAAATTCCA OCATOCCTOO 2300 TTCACGAGAA GTTCAGTTAC ACCCGCCCTT GOATCAGCTC TCAOOOTTCA 2350 ATGGGAATTC AATTCATTCT GAAGATGAGA TTGAATATO? AAACCAGAAG 2400 AGGCTGGCAG AAGAAGAGGA GGAAGATTTO AACGTGCTGA CGTTTGAAGA 2450 CCTCCTTTGC TTTGCGTACC AAGTOGCCAA AGGCATGGAA TTCCTOOAOT 2500 TCAAGTCGTG TGTCCACAGA GACCTGGCAG CCAOGAATGT OTTOOTCACC 2550 CACGGGAAGG TGGTGAAGAT CTGTGACTTT GGACTOGCCC GAOACATCCT 2600 GAGCGACTCC AGCTACGTCG TCAGOGGCAA CGCACOGCTG CCGOTOAAGT 2650 GGATGGCACC CGAGAGCTTA TTTGAAGGGA TCTACACAAT CAAßAGTOAC 2700 GTCTGGTCCT ACGGCATCCT TCTCTGGGAG ATATTTTCAC TOGOTGTGAA 2750 CCCTTACCCT GGCATTCCTG TCGACGCTAA CTTCTATAAA CTOATTCAGA 2800 GTGGATTTAA AATGGAGCAG CCATTCTATG CCACAGAAGO GATATACTTT 2850 GTAATGCAAT CCTGCTGGGC TTTTGACTCA AGGAAGCGOC CATCCTTCCC 2900 CAACCTGACT TCATTTTTAG GATGTCAGCT OGCAGAGGCA OAAOAAOCGA 2950 TGTATCAGAA CATOGGTGGC AACGTCCCAG AACATCCATC CATCTACCAA 3000 AACAGGCGGC CCCTCAGCAG AGAGGCGGGC TCAGAGCCGC CATCOCCACA 3050 GGCCCAGGTG AAGATTCACA GAGAAAGAAG TTAGCGAGOA OOCCTTOGAC 3100 CCCGCCACCC TAGCAGGCTO TAGACCGCAO AGCCAAGATT AOCCTCOCCT 3150 CTGAGGAAGC GCCCTACAGG CCGTTGCTTC GCTGCACTTT TCTCTAGATO 3200 CTGTCTGCCA TTACTCCAAA GTGACTTCTA TAAAATCAAA CCTCTCCTCQ 3250 CACAGGCGGG AGAGCCAATA ATGAGACTTO TTGGTGAGCC COCCTACCCT 3300 GGGGGGGCCTT TCCAGGCCCC CCAGOCTTGA GOGGAAAGCC ATOTATCTGA 3350 AATATAGTAT ATTCTTGTAA ATACGTGAAA CAAACCAAAC CCOTGTTTTO 3400 CTAAGGGAAA GCTAAATATG ATTTTTAAAA ATCTATGTTT TAAAATACTA 3450 TGTAACTTTT TCATCTATTT AGTGATATAT TTTATOOATO GAAATAAACT 3500 TTCTACTGTA ßAAAAAAAA? A 352 (2) INFORMATION FOR SEQ ID NO: 2: (i) CHARACTERISTICS OF THE SEQUENCE: A) LENGTH: 1000 amino acids B) TYPE: amino acid D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2: Mat Arg Ala Leu Ala Gln Arg Sar Aap Arg Arg Lau Leu Lau Lau 1 5 10 15 Val val Leu Ser Val Mat lia Lau Glu Thr Val Thr Aan Gln Aap 20 25 30 Leu Pro Val He Lyß Cya Val Lau Ha Ser Hia Glu? An? An Gly 35 40 45 Be Being Wing Gly Lya Pro Sar Sar Tyr Arg Mat Val Arg Gly Ser 50 55 60 Pro Glu Aap Leu Gln Cya Thr Pro Arg Arg Oln Sar Glu Oly Thr 65 70 75 Val Tyr Glu Ala Ala Thr Val Olu Val Ala Olu Sar Gly Sar Zle 80 85 90 Thr Leu G n Val Gln Lau Wing Thr Pro Gly Aap Lau Sar Cya Lau 95 100 105 Trp Val Phe Lya Hia Ser Ser Leu Q and Cya Oln Pro His Pha? Sp 110 115 120 Leu Gln Asn Arg Gly? Le Val Ser Met Ala Ha Lau Asn Val Thr 125 130 13S Glu Thr Gln Wing Gly Glu Tyr Lau Lau Hia? La ßln Sar Glu Wing 140 145 150 Wing Asn Tyr Thr Val Leu Phß Thr Val Aßn Val Arg Aap Thr Gln 155 160 165 Leu Tyr Val Leu Arg Arg Pro Tyr Phe Arg Lya Mat Olu Asn Oln 170 175 180 Aap Ala Leu Leu Cya Ha Ser Olu Oly Val Pro Olu Pro Thr Val 185 190 195 Glu Trp Val Leu Cya Ser Ser Hia Arg Glu Sar Cyß Lys Olu Olu 200 205 210 Gly Pro Ala Val Val Arg Lya Glu ßlu Lya Val Lau Hia Glu Lau 215 220 225 Phe Gly Thr Aap Ha Arg Cya Cys Wing Arg Asn Wing Lau Gly? Rg 230 235 240 Glu Ser Thr Lya Leu Phe Thr He Asp Leu? Sn Oln Wing Pro Gln 245 250 255 Be Thr Leu Pro Gln Lau Phe Lau Lya Val Oly Olu Pro Lau Trp 260 265 270 He? Rg Cya Lya Ala Ha Hia Val Aan Hia Oly Pha Oly Lau Thr 275 280 285 Trp Glu Lau Olu Aap Lya Ala Lau Olu Olu Oly S * x Tyr Pha Glu 290 295 300 Mßt Ser Thr Tyr Ser Thr Aan Arg Thr Mat Ha? Rg Ha Lau Lau 305 310 315 Wing Phe Val Ser Ser Val G and Arg Asn? Sp Thr Oly Tyr Tyr Thr 320 325 330 Cya Ser Sar Ser Lya Kia Pro Sar Gln Ser Ala Lau Val Thr lia 335 340 345 Lau Glu Lya ßly Phß He Asn Wing Thr Ser Sar Oln Olu Olu Tyr 350 355 360 Glu He Aap Pro Tyr Glu Lya Pha Cya Phß Ser Val Arg Pha Lys 365 370 375 Ala Tyr Pro? Rg He? Rg Cya Thr Trp Ha Pha Smr Oln Ala Ser 380 385 390 Phe Pro Cys Glu Gln? Rg Gly Leu Glu? Sp ßly Tyr Sar Ha Ser 395 400 405 Lya Phe Cys? Sp His Lya? Sn Lyß Pro Gly Glu Tyr Ha Pha Tyr 410 415 420? The Glu? ßn? Sp? Sp? The Gln Pha Thr Lya Mat Pha Thr Lau? Sn 425 - 430 435 He? Rg Lys Lys Pro Gln Val Lau? La? Sn? La Sar Ala Sar ßln 440 445 450? The Ser Cya Ser Ser? Ap Gly Tyr Pro Leu Pro Sar Trp Thr Trp 455 460 465 Lys Lys Cys Ser .'vap Lyß Sar Pro? Sn Cys Thr Olu Olu He Pro 470 475 480 Glu Gly Val Trp? Sn Lyß Lyß? La? ßn? Rg Lyß Val Pha Gly ßln 485 490 495 Trp Val Being Ser Thr Leu? ßn Met Being Olu? The Oly Lyß ßly 500 505 510 Leu Leu Val Lya Cya Cya? Tyr? ßn Sar Mat Oly Thr Sar Cy? 515 520 525 Glu Thr He Phe Leu? Sn Ser Pro Oly Pro Pha Pro Pha Ha Oln 530 535 540? ßp Asn He Ser Phe Tyr? The Thr Ha Gly Lau Cy? Lau Pro Pha 545 550 555 He Val Val Leu He Val Leu He Cya Hiß Lyß Tyr Lyß Lyß Gln 560 565 570 Phe Arg Tyr Glu Be Gln Leu Gln Met He Oln Val Thr Oly Pro 575 580 585 Leu Asp Asn Glu Tyr Phe Tyr Val? Sp Pha Arg? ßp Tyr Olu Tyr 590 595 600? Sp Leu Lys Trp Olu Phe Pro? Rg Olu? Sn Lau 'Olu Pha ßly Ly? 605 610 615 Val Lau Gly Ser Gly? The Pha ßly? Rg Val Mat Asn? The Thr Wing 620 625 630 Tyr Gly He Ser Lyß Thr ßly Val Sar ll * Oln Val? The Val Lyß 635 640 645 Met Leu Lyß Glu Lyß? La? Sp Sar Cyß Glu Lyß Glu? La Lau Mat 650 655 660 Sar Glu Leu Lyß Mat Mat Thr Kia Lau Gly Hiß Hiß? ßp Asn XI * 665 670 675 Val? Sn Lau Leu Gly? La Cya Thr Lau Sar Oly Pro Val Tyr Lau 680 685 690 He Phe Glu Tyr Cya Cya Tyr Oly? Sp Lau Lau? Sn Tyr Lau? Rg 695 700 705 Ser Lyß? Rg Glu Lyß Phß Hiß? Rg Thr Trp Thr Olu Zlß Pha Lyß 710 715 720 Glu Hiß? Sn Phß Ser Phe Tyr Pro Thr Ph? Oln? The Hia Sar? An 725 730 735 Ser Ser Met Pro Gly Ser? Rg? Lu Val Oln Lau Hia Pro Pro Leu 740 745 750? Sp Gln Leu Ser Gly Phe? An Oly ? ßn Ser Ha Hiß Sar Olu? sp 755 760 765 Glu llß Glu Tyr Glu? ßn Gln Lyß? Rg Leu? The Olu Olu Olu Glu 770 775 780 Glu Asp Leu? An Val Leu Thr Phe Glu? Ap Leu Leu Cy? Pha? La 785 790 795 Tyr Gln Val? Lya Gly Mat Olu Pha Lau? Lis Pha Ly? Sar Cy? 800 805 810 Val Hia? Rg? Ap Lau? La? La? Rg? ßn Val Lau Val Thr Kiß Gly 815 820 825 Lys Val Val Lyß He Cyß? ßp Pha Oly Leu? La? Rg? ßp ll * Lau 830 835 840 Sar Asp Being Ser Tyr Val Val? Rg ßly? ßn? La? Rg Lau Pro Val 845 850 855 Lya Trp Met the Pro ßlu Ser Leu Phß Glu ßly Ha Tyr Thr? La 860 865 870 Lys Ser Asp Val Trp Ser Tyr ßly He Leu Lau Trp Glu lß Phß 875 880 885 Ser Leu Gly Val Asn Pro Tyr Pro Gly? Le Pro Val? Ap? La? An 890 895 900 Phe Tyr Lys Leu? Le Gln Ser Gly Phe Lya Mat Olu Qln Pro Phe 905 910 915 Tyr? Thr Glu Gly Ha Tyr Pha Val M? T Gln Sar Cy? Trp? La 920 925 930 Phe? Ap Ser? Rg Lya? Rg Pro Sar Ph? Pro? ßn Lau Thr Sar Pha 935 940 945 Leu Gly Cyn Oln Leu? Glu? Olu Olu? Mat Tyr ßln Asn 950 955 960 Mat Gly Gly? ßn Val Pro Glu Hiß Pro Ser He Tyr Oln? Sn? Rg 965 970 975? Rg Pro Leu Ser? Rg Olu? The Oly Sar Olu Pro Pro Pro Gln 980 985 990? The Gln Val Lya? Le Hi? ? rg Olu? rg Ser 995 1000 (2) INFORMATION FOR SEQ ID NO: 3: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 3475 bases (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 3: CG? GGCGGC? TCCGAGGGCT GGGCCGOCßC CCTOOOOOAC CCCOOOCTCC 50 GGAGGCCATG CCGGCGTTGG CGCGCßACßC GGOCACCOTO CCOCTOCTCG 100 TTGTTTTTTC TGC ?? TGAT? TTTßOG? CT? TT? CAA? TC? Aβ? TCTGCCT 150 GTG? TCAAGT GTGTTTT ?? T C ?? TC? T ?? ß A? CA? TG? TT C? TCAOTOOO 200 GAAGTCATCA TCAT? TCCCA TGGTATC? ß? ? TCCCCOß ?? GACCTCGOGT 250 GTGCGTTG? G ACCCCAGAGC TCAOGOACAO T? TACOAAOC TOCCOCTOTO 300 GAAGTGGATG TATCTOCTTC C? TC? C? CT? C? ßT? TEST? TCOATOCCCC 350 AGGGA? C? TT TCCTGTCTCT GGGTCTTTA? GC? C? ßCTCC CTOAATTOCC 400 AGCC? C? TTT TG? TTT? C ?? C? G? GOA? TTOTTTCC? T OOTCATTTTO 450? AAATG? CAG AAACCCAAGC TGGA? T? C CT? CTTTTT? TTCAGAGTOA 500 AGCTACCAAT TACACAATAT TGTTTAC? ßT G? OT? T ?? ß? ?? TCCTOC 550 TTTACACATT A? GAAGACCT TACTTTAGAA AAATGGAAA? CC? ßßACOCC 600 CTGGTCTGCA T? TCTGAGAG CGTTCC? ßAβ CCß? TCGTßß ?? TßßGTßCT 650 TTGCGATTCA C? GGGGkG ??? GCTßT ?? ß? Aß ?? ? TCC? OCTOTTOTTA 700? AAAGGAGG? A ??? GT? CTCT CATGA? TT? T TTGGG? C? AOA CATAAOOTOC 750 TGTGCC GAA ATGAACTGGG CAGGOAATGC? CC? @OCTOT TCACAAT? ßA 800 TCT? AATCAA ACTCCTC? G? CCACATTOCC? CAATT? TTT CTTAAAOTAO 850 GGGAACCCTT ATGG? T ?? ßG TGC ??? GCT? TTCATOTOAA CCATOOATTC 900 GGGCTCACCT GGG ?? TT? G? ? AACAA? OCA CTCG? OG? ßO OCAACTACTT 950 TG? GATGAGT ACCT? TTC? A C ??? C? O ?? C T? TG? T? COO ATTCTOTGTO 1000 CTTTTGTATC ATC? GTGGC? ? G ??? CG? C? CCGGAT? CT? CACTTOTTCC 1050 TCTTCAAAGC ATCCC? GTCA? TC? ßCTTT? ßTT? CC? TC? TAO? ß? S? 1100? TTTATAAAT GCTACC? TT CAAOTGA? ß? TTATOAAATT ßACCAAT? Tß 1150 AAGAGTTTTG TTTTTCTGTC? GGTTT ??? G CCT? CCCACA? ATCAO? TOT 1200 ACGTGGACCT TCTCTCG? AA? TCATTTCCT TGTG? GCA ?? AOOOTCTTA 1250 TAACGGATAC AGC? T? TCC? ? GTTTTsC ?? TCATA? ßCAC CAOCCAOOAß 1300? ATATATATT CCATßC? ß ?? ? ATG? TO? Tß CCCAATTT? C CAAAATOTTC 1350 ACGCTGAAT? TA? ß ?? GG ?? ACCTCAAGTO CTCßCAß ?? O CATCGGCAAG 1400 TC? GGCGTCC TOTTTCTCGO ATGO? T? CCC? TT? CCATCT TOOACCTOOA 1450? GAAGTGTTC AG? C ?? OTCT CCC ?? CTßC? CAGAAGAOAT CACAOA? OßA 1500 GTCTGGAATA GA ?? GGCTAA CAG ???? ßTß TTTßOACAßT OOOTOTCßAO 1550 CAGTACTCTA ?? C? TG? GTG AAGCCATA ?? AGOOTTCCTO OTCAAOTOCT 1600 GTGC? T? CAA TTCCCTTGGC ACATCTTQT6 Aß? Cß? TCCT TTT? ? CTCT 1650 CCAGGCCCCT TCCCTTTCAT CCAAG? CAAC? TCTCATTCT? TOCA? CAAT 1700 TGGTGTTTGT CTCCTCTTCA TTGTCGTTTT A? CCCTGCTA? TTTGTCACA 1750 AGTACAAAAA GCAATTTAGG TATGAAAGCC AßCT? C? ß? T OOTACAOOT? 1800 ACCGGCTCCT CAG? T? ATGA GTACTTCTAC GTT?? TTTC? G? ß ?? T? Tß? 1850 ATATGATCTC AAATGGG? GT TTCC ?? G? O? ??? TTT? G? ß TTTOOO? AOO 1900 T? CTAGGATC? GGTGCTTTT GG ???? OTO? TO ?? COCAAC AßCTT? Tßß? 1950 ATTAGC ??? A CAOGAGTCTC ?? TCCAßOTT GCCGTCAAA? TOCTßA ?? ßA 2000 AA ?? GCAG? C AGCTCTGAAA G? G? O? CACT CATGTCA? AA CTCA? G? TO? 2050 TG? CCC? GCT ßOGA? OCCAC GAGAATATTO TOAACCTOCT OGOGGCGTGC 2100 ACACTGTCAG GACCA? TTT? CTTG? TTTTT Q? T? CTOTT? CT? TOOTOA 2150 TCTTCTC? AC T? TCT? GAA GTAAAAGAG? ? AAATTTCAC AßGACTTGGA 2200 C? GAGATTTT CAAGG? AC? C ?? TTTC? ßTT TTT? CCCC? C TTTCCA? TCA 2250 C? TCCAAATT CCAGCATGCC TGGTTCAAGA G? AGTTCAGA T? C? CCCGG? 2300 CTCGG? TCAA ATCTC? GGGC TTC? TGßG ?? TTC? TTTCAC TCTOAA?? TO 2350? ATTGAATA TGA ??? CC? • AAGßCTßß AAGAAGAGOA ßGACTTGAAT 2400 GTGCTT • CAT TTGA • ß • TCT TCTTTßCTTT ßCAT • TCAAO TTOCCA • OO 2450 • TGG • TTT CTGO • TTT? OTCOTOTOT TCAC? OA? C CTOOCCOCCA 2500 GG? ACGTGCT TGTC? CCCAC GGG ??? GTO? TGAAGATATG TG? CTTTGG? 2550 TTGGCTCG? G? T? TC? TG? ß T? TTCCAAC T? T? TTOTCA O? Os? CA? TOC 2600 CCGTCTGCCT OTA ??? TßG? TGGCCCCCß? ?? GCCTGTTT ßAAOOC? TCT 2650 ACACCATTAA OAGTG? TGTC TGGTC? T? T? ß? AT? TT? CT ßTO? AA? TC 2700 TTCTCACTTG GTGTGA? TCC TT? CCCT? SCC? TTCC? STT?? TOCTAACTT 2750 CTACAAACTG ATTCAAAATG ß? TTT ???? T ßß? TCAGCCA TTTT? TßCT? 2800 C? GAAGAAAT AT? C? TT? T? ? TGC ?, TCCT ß? TßOGCTTT T? CTCAAOO 2850 AAACGGCCAT CCTTCCCTAA TTTGACTTCG TTTTT? ßß? T GTC? GCTGGC 2900 ? GATGCAGAA GAAGCGATOT ATCAGA? TGT? G? TGOCCGT OTTTC? O? T 2950 GTCCTCACAC CTACCAAAAC AGGCG? CCTT TC? GC? G? G? O? TOO? TTTO 3000 GGGCTACTCT CTCCGCAGGC TC? GßTCßAA ß? TTCßT? O? OOAACAATTT 3050 AGTTTT? AGG ACTTC? TCCC TCCACCTATC CCT? ACAGOC TGTAOATT? C 3100 C? AAACA? G? TTAATTTCAT CACTAAAAGA? ATCTATTA TCA? CTOCTß 3150 CTTCACCAGA CTTTTCTCT? GAAGCCGTCT GCGTTTACTC TTOTTTTCA? 3200 ? GGGACTTTT GTA ??? TCAA ATCATCCTGT C? C ?? OOCAO ß? OßAOCTOA 3250 T? ATGAACTT T? TTOG? GCA TTG? TCTGCA TCCAAOOCCT TCTCAOOCCO 3300 ßCTTG? GTG? TTGTßT? CC Tß ?? ßT? CAß T? T? TTCTTO T ??? TACAT? 3350 AAACAAAAGC ATTTTOCTAA GGAG ?? OCT? ? T? TG? TTTT TT? AOTCT? T 3400 GTTTTAAAAT A? TATGTAAA TTTTTCA? CT ATTTA? T? T? T? TTT? T? 3450 ßGTGGG? ATA A ?? TTTCT? C TACAG 3475 INFORMATION FOR SEQ ID NO: 4: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 993 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 4: Met Pro Ala Leu Ala Arg Asp ßly ßly Gln Lau Pro Lau Lau Val 1 5 10 15 Val Phe Being Wing Met He Phe Oly Thr He Thr Asn Oln? ßp Lau 20 25 30 Pro Val He Lya Cyß Val Leu He? An Hiß Lyß? Sn? ßn? ßp Sar 35 40 45 Being Val Gly Lya Being Being Tyr Pro Mßt Val Sar Olu Sar Pro 50 55 60 Glu? Sp Leu Gly Cy?? La Lau? Rg Pro Gln Sar Sar Oly Thr Val 65 70 75 Tyr Glu Ala Ala? Val Glu Val? ßp Val Sar? La Sar Ha Thr 80 85 90 Leu Gln Val Leu Val Asp Ala Pro ßly Aan Ha Sar Cya Leu Trp 95 100 105 Val Phe Lys His Ser Ser Leu? ßn Cyn Gln Pro Hiß Pha? ßp Lau 110 115 120 Gln Asn Arg Gly Val Val Sar Mat Val? La Lau Ly? Mat Thr ßlu 125 130 135 Thr Gln Wing Gly Glu Tyr Lau Lau Pha He Gln Sar Olu? The Thr 140 145 150? Sn Tyr Thr He Leu Phe Thr Val Sar He? Rg? Sn Thr Lau Lau 155 160 165 Tyr Thr Leu? Rg? Rg Pro Tyr Pha? Rg Lya Mat Glu? ßn ßln? Sp 170 175 180? The Leu Val Cya 11 * Ser ßlu Sar Val Pro ßlu Pro ll * Val ßlu 185 190 195 Trp Val Leu Cya Pro? Val Val Lyß Lyß ßlu Olu Lyß Val Lau Hiß Olu Leu Phi 215 220 225 ßly Met? Ap Ha? Rg Cyß Cyß? La? Rg? Sn ßlu Lau Oly Arg Olu 230 235 240 Cys Thr? Rg Leu Phe Thr He? Sp Lau? Sn Oln Thr Pro Oln Thr 245 250 255 Thr Leu Pro ßln Lau Pha Leu Lyß Val ßly ßlu Pro Lau Trp? 260 265 270 Arg Cys Lys Ala Val His Val Aan His Gly Phe Gly Leu Thr Trp 275 28'0 285 Glu Leu Glu Asn Lys Ala Leu Glu Glu Gly Asn Tyr Phe Glu Met 290 295 300 Being Thr Tyr Being Thr Asn Arg Thr Met le Arg He Leu Phe Ala 305 310 315 Phe Val Ser Ser Val Ala Arg Asn Asp Thr Gly Tyr Tyr Thr Cyß 320 325 330 Being Ser Lya His Pro Being Gln Ser Ala Leu Val Thr le Val 335 340 345 Glu Lys Gly Phe He Aßn? The Thr? An Ser Ser ßlu? Ap Tyr Glu 350 355 360 He? Sp Gln Tyr Glu Glu Pha Cya Phe Ser Val? Rg Phe Lya? The 365 370 375 Tyr Pro Gln He? Rg Cy? Thr Trp Thr Phe Ser? Rg Lya Ser Ph? 380 385 390 Pro Cys Glu Gln Lys Gly Leu? Sp? An Gly Tyr Ser He Ser Lya 395 400 405 Phe Cys Asn Hiß Lys His Gln Pro Gly Glu Tyr 11 * Phe His Wing 410 415 420 Glu Asn Aßp Asp Ala Gln Phe Thr Lys Met Phe Thr Leu Aßn? Le 425 430 435 Arg Arg Lys Pro Gln Val Leu Ala Glu Wing Ser? La Ser Gln? 440 445 450 Ser Cys Phe Ser? S Gly Tyr Pro Leu Pro Ser Trp Thr Trp Lys 455 460 465 Lys Cys Ser? Sp Lys Ser Pro? Sn Cy? Thr Glu Glu He Thr Glu 470 475 480 Gly Val Trp? An? Rg Lya? La? ßn? Rg Lys Val Phe Gly ßln Trp 485 490 495 Val Ser Ser Ser Thr Leu? Sn Met Ser ßlu? La? La Lyß ßly Phe 500 505 510 Leu Val Lya Cya Cyß? The Tyr? ßn Ser Leu Gly Thr Ser Cya ßlu 515 520 525 Thr He Leu Leu? An Ser Pro ßly Pro Phe Pro Phe He ßln? ßp 530 535 540? Sn He Ser Phe Tyr? The Thr Ha ßly and Val Cya Lau Lau Ph? He 545 550 555 Val Val Leu Thr Leu Leu? Le Cyß Hiß Lyß Tyr Lyß Lyß ßln Pha 560 565 570? Rg Tyr Glu Ser Gln Leu Gln Met Val ßln Val Thr ßly Ser Ser 575 580 585 Asp Asn Glu Tyr Ph? Tyr Val? Ap Ph?? Rg Glu Tyr Glu Tyr? ßp 590 595 600 Leu Lyß Trp Glu Phe Pro? Rg Glu? ßn Leu Glu Phe Gly Lyß Val 605 610 615 Leu Gly Ser Gly? The Phe Gly Lyß Val Met Asn Ala Thr? The Tyr 620 '625 630 Gly He Ser Lys Thr Gly Val Ser He Gln Val? Val Lyß Met 635 640 645 Leu Lys Glu Lys? The? Sp Ser Ser Glu? Rg Glu? The Leu Met Ser 650 655 660 Glu Leu Lys Met Met Thr Gln Leu Gly Ser His Glu? Sn He Val 665 670 675 Asn: .eu Leu Gly Ala Cyß Thr Leu Ser Gly Pro He Tyr Leu He 680 685 690 Phe Glu Tyr Cyß Cys Tyr Gly? ßp Leu Leu? Sn Tyr Leu? Rg Ser 695 700 705 Lys? Rg 3-a Lys Phe His Arg Thr Trp Thr ßlu lß Phß Lya Glu 710 715 720 His Asn Phe Ser Phe Tyr Pro Thr Phe Gln Ser Hia Pro? Sn Ser 725 730 735 Ser Met Pro Gly Ser? Rg Glu Val Gln He His Pro? ßp Ser? Sp 740 745 750 Gln le Ser Gly Leu His Gly? Sn Ser Phe Hi? Ser Glu? Sp Glu 755 760 765 He Glu Tyr Glu? Sn Gln Lys? Rg Leu Glu Glu Glu ßlu? ßp Leu 770 775 780 Asn Val Leu Thr Phe Glu Asp Leu Leu Cys Phe Wing Tyr Gln Val 785 790 795 Ala Lys Gly Met Glu Phe leu Glu Phe Lys Ser Cya Val His Arg 800 805 810 Asp Leu Ala Ala Arg Asn Val Leu Val Thr His Gly Lys Val Val 815 820 825 Lys? Le Cys Aap Phe Gly Leu Ala? Rg? Sp l? Met Ser? Sp Ser 830 835 840 Asn Tyr Val Val Arg Gly Asn? La? Rg Leu Pro Val Lys Trp Met 845 850 855 Wing Pro Glu Ser Leu Phe Glu ßly He Tyr Thr He Lyß Sar Asp 860 865 870 Val Trp Ser Tyr ßly He Leu Leu Trp Glu He Phß Ser Leu Gly 875 880 885 Val Asn Pro Tyr Pro Gly? L? Pro Val? ßp Ala? Sn Ph? Tyr Lys 890 895 900 Leu He Gln? Sn Gly Phe Lys Met? S Oln Pro Phe Tyr? The Thr 905 910 915 Glu Glu He Tyr He He Mßt ßln Ser Cyß Trp? The Phß? ßp Ser 920 925 930? Rg Lys Arg Pro Ser Phe Pro? ßn Leu Thr Ser Phe Leu ßly Cyß 935 940 945 Gln Leu Ala Asp Ala ßlu Glu Ala Met Tyr Gln? Sn Val? Sp ßly 950 955 960 Pro Val Ser Glu Cyß Pro Hiß Thr Tyr ßln? ßn? Rg? Rg Pro Phe 96S 970 975 Ser? Rg Glu M? T? ßp Leu Gly Leu Leu Ser Pro ßln? The Oln Val 980 985 990 Glu? Ap Ser 993 It is noted that in relation to this date, the best method known to the applicant for - or to carry out the aforementioned invention is the clear result of the present description of the invention.

Having described the invention as antece-15 of, the content of the following is claimed as property:

Claims (17)

1. An agonist antibody, characterized in that it binds to the tyrosine kinase receptor flk2 / flt3 (flk2 / flt3).

2. The antibody according to claim 1, characterized in that it comprises a monoclonal antibody.

3. The antibody according to claim 1, characterized in that the antibody binds to the extracellular domain of flk2 / flt3 and activates the tyrosine kinase domain thereof.

4. The antibody according to claim 1, characterized in that the antibody increases the pro? I "T5er? Tion or differentiation of primitive hematopoietic cells.

5. The antibody according to claim 4, characterized in that the antibody increases the proliferation and differentiation of the myeloid and lymphoid blood cell lines.

6. The antibody according to claim 4, characterized in that the antibody causes a synergistic increase in the proliferation and differentiation of the primitive hematopoietic cells, in combination with a cytokine.

7. An isolated nucleic acid, characterized in that it encodes the antibody according to claim 1.

8. A pharmaceutical composition, characterized in that it comprises the antibody according to claim 1 in an effective amount for the activation of a tyrosine kinase domain of flk2 / flt3, and a pharmaceutically acceptable carrier.

9. The pharmaceutical composition according to claim 8, further characterized in that it comprises a cytokine capable of increasing the proliferation or differentiation of hematopoietic cells.

10. The composition according to claim 9, characterized in that the cytokine is selected from the group consisting of erythropoietin (EPO), an interleukin, granulocyte-macrophage colony stimulation factor (GM-CSF), granulocyte CSF (G-CSF) ), Macrophage CSF (M-CSF), Steel factor (SLF), tumor necrosis factor (TNF), lymphotoxin, flk2 ligand / flt3, ligand kit, insulin growth factor I (IGF-I) and? -interferon .

11. The composition according to claim 10, characterized in that the cytokine is the Steel factor (SLF).

12. The method for increasing the proliferation or differentiation of primitive hematopoietic cells, characterized in that it comprises contacting the hematopoietic cells with an effective amount of the agonist antibody according to claim 1.

13. The method according to claim 12, characterized in that the agonist antibody is a monoclonal antibody directed against an epitope in the extracellular domain of flk2 / flt3.

14. The method according to claim 12, characterized in that the agonist antibody causes the differentiation or proliferation of the myeloid and lymphoid blood cell lines.

15. The method according to claim 12, further characterized in that it comprises contacting the hematopoietic cells with the Steel factor (SLF).

16. A method for increasing the repopulation of mature blood cell lines in a mammal, characterized in that it comprises administering to a mammal a therapeutically effective amount of the agonist antibody according to claim 1.

17. The method according to claim 16, characterized in that the mammal has undergone chemotherapy, radiation therapy, or therapy for bone marrow transplantation, or has suffered a decrease in blood cell lines, as a consequence of having suffered haemorrhage or disease.