MXPA94003806A

MXPA94003806A - Ligands for receivers f

Info

Publication number: MXPA94003806A
Application number: MXPA/A/1994/003806A
Authority: MX
Inventors: D Lyman Stewart; Patricia Beckmann M
Original assignee: Immunex Corporation
Priority date: 1993-05-24
Filing date: 1994-05-23
Publication date: 1999-06-01

Abstract

The present invention relates to ligands for flt3 receptors, capable of transducing signals of self-renewal to regulate the growth, proliferation or differentiation of progenitor cells and stem cells. The present invention is directed to flt3-L as an isolated protein, to DNA encoding flt-3-L, to host cells transfected with cDNA encoding flt-3-L, to compositions including flt-3-L , to methods to improve the transfer of genes to a mammal that uses flt-3-L, and to methods to improve transplants, which use flt-3-L. The flt-3-L finds use in the treatment of patients with anemia, AIDS and various cancer

Description

LIG? NDQS FOR FLT3 RECEIVERS INVENTION-RES; STEWAR? 0, LYMAN and H. PATRICIA BICKMftftN- citizens Mortßait-ericano », with address mn 312 N» 4St Street, Seaiti®, Washing on 98U5, E- and, A »; and 5454 Raga »Lans, Pouisbo- Washington 983"? o, E, lí. A- CA "5í? Í, BIEHTK 'mW-MEX CORPORATION, a» oei®düad Mortßasteri ana, or g **? * I? Aí? A and sjci-stßn * low ia »? - and« -s de la latado ds? e-elawar »with f > ratcíio ß-n 51 University -treet-Seattle-, Washington OSIOI, K. U- A- SUMMARY OF THE INVENTION Ligands are described for flt3 receptors. cf-? aces of transducing signals of self-renewal to regulate the growth, proliferation or differentiation of progenitor cells and stem cells- The present invention is directed to flt3-L as an isolated protein - to DNA encoding flt-3-L, a host cells transfected with cDNAs encoding flt-3-L, to compositions including flt-3-L, to methods for improving gene transfer to a mammal using flt-3-L, and to methods for improving transplants, using the flt-3-L. The flt-3-L finds use in the treatment of patients with anemia, AIDS and various cancers. > ? * * * # The present is a continuation in part of the Application of the United States of North America 08 / 209,502, filed on March 7, 1994, which is a continuation in part of the Application of the United States of North America 08/162, 407 , filed on December 3, 1993, which is a continuation in part of the Application of the United States of America 08 / 111,758, filed on August 25, 1993, which is a continuation in part of the Request from the States United States of America 08 / 106,463, filed on August 12, 1993, which in turn is a continuation in part of the United States Request for North America. ca 08 / 008,394, filed on May 24, 1993, abandoned.

FIELD OF THE INVENTION The present invention relates to mammalian flt-3 ligands, to nucleic acids encoding those ligands, to processes for the production of recombinant flt-3 ligands, to pharmaceutical compositions containing those ligands, and to their use in various therapies.

BACKGROUND OF THE INVENTION Blood cells originate from hematopoietic rod cells, which become compromised to differ along certain lineages, ie, erythroid, megakaryocytic, granulocytic, monocytic, and lymphocytic. . Cytokines that stimulate the proliferation and maturation of cell precursors are called colony stimulation factors ("CSFs"). Many CSFs are produced by T-lymphocytes, including interleukin-3 ("IL-3"), granulocyte-monocyte CSF (GM-CFS), granulocyte CSF (G-CSF), and monocyte CSF (M-CSF). These CSFs affect both mature cells and cane cells. So far, no factors have been discovered that are capable of predominantly affecting cane cells. Tyrosine kinase receptors ("TKRs") are the growth factor receptors that regulate the proliferation and differentiation of a number of cells (Yarden, Y. &Ullrich, A. Annu, Rev. Biochem., 51, 443 -478, 1938, and Cadena, DL &Gilí, GN FASEB J., ñ, 2332-2337, 1992). Certain TKRs work inside the hematopoietic system. For example, by signaling through the type 1 colony stimulation factor ("CSF-1"), the cfms receptor regulates the survival, growth and differentiation of monocytes (Stanley et al., J. Cell Bioche., 21, 151- 159, 1983). The steel factor ("SF", also known as mast cell growth factor, rod cell factor or cassette ligand), acting through the c-kit, stimulates the proliferation of cells in both the myeloid and lymphoid compartments . The flt3 (Rosnet et al. Oncogene, Q., 1641-1650, 1991) and the fIk-2 (Matthews et al., Cell, 65. 1143-1152, 1991) are variant forms of a TKR that is related to the receptors c -fms and c-kit. The flk-2 gene product is expressed in hematopoietic and progenitor cells, whereas the product of the flt3 gene has a more general distribution in the tissue. The receptor proteins of flt3 and flk-2 are similar in amino acid sequence and vary in two amino acid residues in the extracellular domain and differ in an amino acid segment 31 located near the C-terminus (Lyman et al., Oncogene, fi, 815- 822, 1993). It has been found that the ligand flt-3 ("flt3-L") regulates the growth and differentiation of progenitor and rod cells and is likely to have clinical utility in the treatment of hematopoietic disorders, in particular, aplastic anemia and myelodysplastic syndromes. . Additionally, flt3-L will be useful in syngeneic bone marrow transplants or derivatives of themselves in patients who are undergoing cytoreductive therapies, as well as cell expansion. The flt3-L will also be useful in gene therapy and rod cell mobilization systems. Cancer is treated with cytoreductive therapies that involve the administration of ionizing radiation of chemical toxins that rapidly kill the dividing cells. Side effects typically result from cytotoxic effects on normal cells and may limit the use of cytoreductive therapies. A common side effect is myelosuppression, or damage to the cells of the bone marrow that cause white and red blood cells and platelets. As a result of myelosuppression, patients develop cytopenia, or deficits in the blood cell, which increases the risk of infection and bleeding disorders. Cytopenias increase pathology, mortality, and lead to underdosage in cancer treatment. Many clinical researchers have manipulated cytoreductive therapy dosing regimens and schedules to increase the dosage for cancer therapy, while limiting the damage to the bone marrow- An attempt involves bone marrow or peripheral blood cell transplants in which the bone marrow or circulating hematopoietic stem or progenitor cells are removed, before cytoreductive therapy and then reinfused after therapy, to restore hematopoietic function. U.S. Patent No. 5,199,942, incorporated herein by reference, discloses a method for using the fusion proteins GM-CSF, IL-3, SF, GM-CSF / IL-3, erythropoietin ("EPO ") and combinations thereof, in transplantation regimes derived from itself. High-dose chemotherapy is therapeutically beneficial because it can produce an increased frequency of objective response in patients with metastatic cancers, particularly breast cancer, when compared to standard dose therapy. This may result in an extended disease-free remission for some patients with still poor prognosis. However, high-dose chemotherapy is toxic and many resulting clinical complications are related to infections, bleeding disorders and other effects associated with prolonged periods of myelosuppression. The myelodysplastic syndromes are cane cell disorders characterized by impaired cell maturation, progressive pancitofenia, and functional abnormalities of mature cells. These have also been characterized by varying degrees of cytophenia, erythropoiesis and myelopoiesis ineffective with bone marrow cells that are normal or increased in number and that have a peculiar morphology. Bennett et al. (Br. J. Hae atol.; ül: 189-199) divided these disorders into five subtypes: anemia with resistance to treatment, anemia with resistance to treatment with ringed sideroblasts, anemia with resistance to treatment with excess blasts, anemia with resistance to treatment with excess blasts in transformation, and chronic myelomonocytic leukemia. Although a significant percentage of these patients developed acute leukemia, the majority died from infections or hemorrhagic complications. The treatment of these syndromes with retinoids, vitamin D, and quinoa was not successful. The majority of patients who suffer from these syndromes are elderly and are not suitable candidates for bone marrow transplantation or aggressive angiocemic chemotherapy. Eplastic anemia is another disease entity that is characterized by bone marrow failure and severe pancitofenia. Unlike the myelodysplastic syndrome, in this disorder the bone marrow is acellular or hypocellular. Current treatments include bone marrow transplantation from a histocompatible donor or immunosuppressive treatment with antithymocyte globulin (ATG). Similar to myelodysplastic syndrome, the majority of patients with this syndrome are elderly and unsuitable for bone marrow transplantation or for aggressive antileukemic chemotherapy. Mortality in these patients is extraordinarily high due to infections or hemorrhagic complications. Anemia is common in patients with acquired immunodeficiency syndrome (AIDS). Usually anemia is more severe in patients receiving zidovudine therapy. Many important retroviral, anti-infective, and antineoplastic agents suppress erythropoiesis. It has been shown that recombinant EPO normalizes the patient's hematocrit and hemoglobin levels, however, usually very high doses are required. A growth factor that stimulates the proliferation of erythroid lineage along with or in combination with EPO or other growth factors can be used to treat these patients and reduce the required number of transfusions. A growth factor that can also increase the number of T cells would find a particular use in the treatment of patients with AIDS. = ÜM? RIfl-I? THE INVENTION The present invention relates to biologically active ligand flt3 (flt3-L) as an isolated or homogeneous protein. In addition, the present invention is directed to isolated DNAs encoding flt3-L and expression vectors that include cDNAs encoding flt3-L. Within the scope of the present invention are host cells that have been transfected or transformed with expression vectors that include a cDNA encoding flt3-L, and processes to produce flt3-L by culturing host cells under conditions that lead to the expression of flt3-L. The flt3-L can be used to prepare pharmaceutical compositions that are to be used in allogeneic, syngeneic or self-derived transplantation methods. The pharmaceutical compositions may include flt3-L alone or in combination with other growth factors, such as interleukins, colony stimulation factors, protein tyrosine kinases and cytokines. The present invention includes methods for using the flt3-L compositions in gene therapy and in the treatment of patients suffering from myelodysplastic syndrome, aplastic anemia, HIV infection (AIDS) and cancers, such as breast cancer, lymphoma, cancer of small cell lung, multiple myeloma, neuroblastoma, acute leukemia, testicular tumors, and ovarian cancer. The present invention also relates to antibodies, and in particular to monoclonal antibodies, which are immunoreactive with flt3-L. Also included in the present invention are fusion proteins that include a portion of flt3-L and the constant domain of an immunoglobulin protein. The present invention is also directed to the use of flt3-L in progenitor cell transplantation or procedures. of stick in peripheral blood. Typically, progenitor cells or peripheral blood cane cells are removed from a patient prior to myeloablative cytoreductive therapy, and then re-administered to the patient together with, or after cytoreductive therapy, to counteract the effects myelosuppressive of this therapy. The present invention allows the use of an effective amount of flt3-L in at least one of the following ways: (i) flt3-L is administered to the patient prior to the collection of the progenitor or cane cells, to increase or mobilize the number of those circulating cells; (ii) after the collection of the progenitor or rod cells of the patient, f3t.3-L is used to expand these cells x vivo-, and (iii) the flt3-L is administered to the patient after transplantation of the cells. progenitor or cane cells collected, to facilitate the grafting thereof. The method of transplantation of the present invention may further include the use of an effective amount of a cytokine in sequential or concurrent combination with flt3-L. These cytokines include, but are not limited to, interleukins ("IL") I-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL- 9, IL-10, IL-11, IL-12, IL-13, IL-14 or IL-15, a CSF selected from the group consisting of the fusions of G-CSF, GM-CSF, M-CSF , or GM-CSF / IL-3, or other growth factors such as CSF-1, SF, EPO, leukemia inhibitory factor ("LIF") or fibroblast growth factor ("FGF"). The flt3-L is also useful in the same way for syngeneic or allogeneic transplants. The present invention also includes a progenitor or rod cell expansion medium, including cell growth medium, serum derived from itself, and flt3-L alone or in combination with a cytokine from the group listed above. The present invention also includes the use of flt, 3-L to expand progenitor or rod cells collected from umbilical cord blood. The expansion can be performed with the flt3-L alone or in sequential or concurrent combination with a cytokine of the group listed above. The present invention also includes the use of flt3-L in gene therapy. The flt3-L allows the proliferation and culture of the first hematopoietic stem or progenitor cells that are to be transfected with exogenous DNA for use in gene therapy. Alternatively, a cDNA encoding flt3-L can be transfected into the cells in order to finally send its gene product to the target cell or tissue. In addition, the present invention includes the use of flt3-L to stimulate the production of erythroid cells in vivo for the treatment of anemia. This use includes the administration of flt3-L to the patient in need of that erythroid cell stimulus in conjunction with, or after cytoreductive therapy. The treatment may include the co-administration of another growth factor selected from the cytokines of the group listed above. Preferred cytokines for use in this treatment include EPO, IL-3, G-CSF and GM-CSF. This treatment is especially useful for patients with AIDS, and preferably for patients with AIDS who are receiving AZT therapy. Since flt3-L stimulates the production of rod cells, other non-hematopoietic rod cells that are carriers of flt3 receptors can be affected by the flt3-L of the present invention. Flt3-L is useful in IV fertilization procedures and can be used in vivo in the treatment of infertility conditions. In the intestine, the ligand flt3 is useful in the treatment of intestinal damage as a result of irradiation or chemotherapy. Flt3-L can also be used to treat patients infected with the human immunodeficiency virus (HIV). Such treatment would encompass the administration of flt3-L to stimulate in vivo production, as well as the ex vivo expansion of T cells and erythroid cells. This treatment can avoid T cell deficiency, in particular of CD4 positive T cells, and can raise the patient's immune response against the virus, thus improving the quality of life of the patient. The flt.3-L can be used to stimulate the rod cells that lead to the development of hair follicles, thus promoting hair growth. In addition, flt3-L can be linked to a solid phase matrix and used to purify by affinity or separate cells expressing flt3 or its cell surfaces. The present invention encompasses the separation of cells having the flt3 receptor on the surface thereof from a mixture of cells in solution, and includes contacting the cells in the mixture with a contact surface having a molecule which links to the flt3 in it, and the separation of the contact surface and the solution. Once separated, cells ex vivo can be expanded using flt3-L and administered to the patient.

A cDNA encoding a murine flt3 has been isolated and described in the SEQUENCE IDENTIFICATION NUMBER: 1. A cDNA encoding a human flt3 has also been isolated and is written in the SEQUENCE IDENTIFICATION NUMBER: 5. This discovery of the cDNAs encoding murine and human flt3-L, allows the construction of expression vectors including cDNAs encoding flt3-L; host cells transfected or transformed with the expression vectors; murine and human flt3-L, biologically active as homogeneous proteins; and immunorr-eactive antibodies with murine and human flt3-L. Flt3-L is useful in the augmentation, stimulation, proliferation or growth of cells expressing the flt.3 receptor, including non-hernatopoietic cells. Since the receptor flt3 is found in the brain, placenta, and tissues of nervous and hematopoietic origin, and finds distribution in the testis, ovaries, lymph node, spleen, thymus and fetal liver, it is possible to treatment of a variety of conditions associated with tissue damage thereof. Although they are not limited to these, the particular uses of flt3-L are described below. As used herein, the term "flt3-L" refers to a genus of polypeptides that binds and composes independently with the flt3 receptor found in progenitor and rod cells. The term "flt-3" encompasses the proteins having the amino acid sequence of 1 to 231 of the SEQUENCE IDENTIFICATION NUMBER: 2, or the amino acid sequence 1 to 235 of the SEQUENCE IDENTIFICATION NUMBER: 6, as well as to those proteins that have a higher degree of similarity or a high degree of identity with the amino acid sequence 1 to 231 of the SEQUENCE IDENTIFICATION NUMBER: 2, or amino acid sequence 1 to 235 of the SEQUENCE IDENTIFICATION NUMBER: 6, and whose proteins are biologically active and bind to the flt3 receptor. In addition, the term refers to biologically active gene products of the SEQUENCE IDENTIFICATION NUMBER: 1 or the SEQUENCE IDENTIFICATION NUMBER: 5. The term "flt3-L" also encompasses membrane-bound proteins (which include an intracellular region, a membrane region, and an extracellular region), and soluble or truncated proteins that mainly include the extracellular portion of the protein, retain the biological activity and are capable of being secreted. Specific examples of these soluble proteins are those which include amino acid sequence 28-163 of the SEQUENCE IDENTIFICATION NUMBER: 2, and amino acids 28-160 of the SEQUENCE IDENTIFICATION NUMBER: 6. The term "biologically active" as refers to flt3-L, means that flt3-L is able to bind to flt3. Alternatively, "biologically active" means that flt3-L is capable of transducing a stimulating signal to the cell through the flt3 bound to the membrane. "Isolated" means that flt3-L is free from association with other proteins or polypeptides, for example, as a recombinant host cell culture purification product or as a purified extract. A "variant of flt.3-L" as referred to herein, means a polypeptide substantially homologous to native flt3-L, but having an amino acid sequence different from that of native flt3-L (human, murine or other species). of mammals) due to one or more deletions, insertions or substitutions. Preferably, the variant amino acid sequence is at least 80 percent identical to xvae. amino acid sequence of native flt3-L, most preferably at least 90 percent identical. The identity of the percentage can be determined, for example, by comparing the sequence information using the GAP computer program, version 6.0, described by Devereux et al. (Nucí Acíds Bes.12: 387, 1984) and available with the University of Wisconsin Genetics Computer Group (UWGCC). The GAP program uses the Needleman & alignment method; Wunsch (J. Mol, Biol. 48: 443, 1970), as reviewed by Smith & Waterman (Adv. Appl. Math. 2: 482, 1981). Preferred error parameters for the GAP program include: (1) a unit comparison matrix (containing a value of 1 for identities and 0 for non-identities) for nucleotides, and the comparison matrix valued by Gribskov & Burgess, Nucí. Acids Bes. 14: 6745, 1986, as described by Schwartz & Dayhoff, editors, Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, pages 353-358, 1979; (2) a penalty of 3.0 for each separation and an additional penalty of 0.10 for each symbol in each separation; and (3) without punishment for extreme separations. The variants may include conservatively substituted sequences, this means that a given amino acid residue is replaced by a residue having similar physiochemical characteristics. Examples of substitutions c-oncervativ r. include the substitution of one aliphatic residue for another, such as Lie, Val, Leu, or Ala for each other, or the substitutions of one polar residue for another, such as between Lys and Arg; Glu and Asp; or Gln and Asn. Other of these conservative substitutions are well known, for example, substitutions of entire regions that have similar hydrophobicity characteristics. The present invention also encompasses flt3-L variants that occur naturally. Examples of these variants are proteins that result from alternating junction events of the mRNA or from cleavage of the flt3-L protein, where the binding property of flt3-L is retained. The alternating junction of mRNA can produce a truncated but biologically active flt3-L protein, such as, for example, a soluble form that occurs naturally in the protein. Variations that can be attributed to proteolysis include, for example, differences in the term N-flC- on expression in different types of host cells, due to the proteolytic removal of one or more terminal amino acids of the flt3-L protein. (generally from 1 to 5 terminal amino acids). The term "self-derived transplantation" is described in U.S. Patent No. 5,199,942, which is incorporated herein by reference. Briefly, the term means a method for conducting the progenitor cell or hematopoietic stem cell transplant derived from itself, which includes: (1) collecting progenitor cells or hematopoietic rod cells from a patient prior to cytoreductive therapy; (2) expanding the progenitor cells or hematopoietic rod cells ex vivo with flt3-L to provide a cell preparation that includes increased numbers of progenitor cells or hematopoietic rod cells; and (3) administering the cell preparation to the patient in conjunction with, or after, cytoreductive therapy. The progenitor and rod cells can be obtained from peripheral blood collection or from bone marrow explantations. Optionally, one or more cytokines, selected from the group listed above, can be combined with flt3-L to aid in the proliferation of particular types of hematopoietic cells or to affect the cellular function of the resulting proliferated hematopoietic cell population. Of the foregoing, SF, IL-1, IL-3, EPO, G-CSF, GM-CSF and GM-CSF / 1L-3 fusions are preferred, and the most preferred fusions are G-CSF, GM-CSF and GM-CSF / 1L-3. The term "allogeneic transplantation" means a method in which the bone marrow or progenitor cells or stem cells of the peripheral blood are removed from a mammal and administered to a different mammal of the same species. The term "syngeneic transplantation" means the transplantation of bone marrow among genetically identical mammals.

The method of transplantation of the present invention, described above, optionally includes a preliminary and live procedure that includes administration of flt3-L alone or in sequential or concurrent combination with a recruitment growth factor to a patient, to recruit hematopoietic cells within the peripheral blood before collection. The appropriate recruitment factors were listed above, and the preferred recruitment factors are flt3-L, SF, IL-1, and IL-3. The method of the present invention, described hereinbelow, optionally includes a subsequent, live procedure that includes administration of flt3-L alone or in sequential or concurrent combination with a graft growth factor to a patient, after transplantation of the cell preparation, to facilitate grafting and increase the proliferation of hematopoietic stem or progenitor cells grafted from the cell preparation. The appropriate graft factors were listed above, and the preferred graft factors are the GM-CSF, G-CSF, IL-3, IL-1, EPO and GM-CSF / IL-3 fusions. The present invention also includes a propanitor cell or rod cell expansion medium, which includes a cell growth medium, serum derived from itself, and flt3-L alone or in combination with a cytokine growth factor from the above list . Preferred growth factors are the SF, GM-CSF, IL-3, IL-1, G-CSF, EPO, and GM-CSF / 1L-3 fusions. In particular, flt-3 can be used to stimulate the proliferation of hernatopoietic and non-hematopoietic rod cells. This stimulation is beneficial when specific damage to the tissue has occurred. As such, flt3-L may be useful in the treatment of neurological damage and may be a growth factor for nerve cells. It is likely that flt3-L is useful in in vitro fertilization procedures and can also be used in vivo in the treatment of infertility conditions. The flt3-L could be useful in the treatment of intestinal damage that results from irradiation or chemotherapy .. Since the flt3 receptor is distributed in the cane cells reaching the development of hair follicles, the flt3-L could equally be useful to promote hair growth Since f3t3-L has been shown to stimulate the proliferation of T cells as well as erythrocytes (see Examples, below), flt3-L finds use in the treatment of patients infected with the human immunodeficiency virus (HIV). ). This treatment would encompass the administration of flt3-L to stimulate in vivo production, as well as the ex vivo expansion of T cells. In addition, flt3-L can prevent deficiency of CD4 + T cells. This treatment can raise or maintain the immune response of a patient against the virus, improving or maintaining, through this, the quality of life of a patient. In addition, this in vivo treatment would stimulate the cells of the erythroid lineage, thereby improving the hematocritic and hemoglobin levels of a patient. The flt3-L can be administered in its placement, alone or in sequential or concurrent combination with the selected cytokines of the group listed above. Flt3-L is useful in gene therapy because of its specificity for progenitor and rod cells. Gene therapy involves the administration of exogenous cells transfected with DNA to hosts that are allowed to graft. See, for example, Boggs, International J. Cell Cloning, fi: 80-96 (1990); Kohn et al., Cancer Invest. , 7 (2): 179-192 (1989); Lehn, Bone Marrow Transpl. ,: 287-293 (1990); and Verma, Scientific American, pages 68-84 (1990). Using gene therapy methods known in the art, a method for transferring a gene to a mammal includes the steps of (a) culturing early hematopoietic cells in a medium that includes flt3-L alone or in sequential or concurrent combination with a cytokine selected from the group listed above; (b) transfecting the cultured cells of step (a) with the exogenous gene; and (c) administering the transfected cells to the mammal. Within this method is the novel method of transfecting the progenitor or rod cells with a gene, which includes the steps of: (a) and (b) above. Furthermore, using the same methods or similar methods, the cDNA encoding flt3-L can be transfected into those sending cells, to send the gene product of flt3-L to target target. Example 1 describes the construction of a novel flt3-L fusion protein, which was used in the classification of flt3-L. Other antibody Fc regions can be substituted for the human IgGl Fc region described in Example 1. Other suitable Fc regions are those that can bind with high affinity to protein A or protein G, and include the Fc region of IgGl or fragments of the human or murine Fc IgGl region, for example, fragments that include at least the region of the hinge, so that interchain disulfide linkers will be formed. The flt3: Fc fusion protein offers the advantage that it can be easily purified. In addition, disulfide linkers are formed between the Fc regions of two separate fusion protein chains, creating dimers. The dlt receptor flt.3: Fc was chosen for the potential advantage of higher affinity binding to flt3-L, in view of the possibility that the ligand sought is multimeric. As described above, one aspect of the present invention is the soluble flt3-L polypeptides. The soluble flt3-L polypeptides include all or a portion of the extracellular domain of a native f "! T3-L, but lacks the transmembrane region that would cause retention of the polypeptide on a cell membrane. they conveniently include the native signal peptide (or a heterologous), when initially synthesized, to promote secretion, but the signal peptide is split over the secretion of flt3-L from the cell.The soluble flt3-L polypeptides encompassed The present invention retains the ability to bind to the receptor flt 3. In fact, the soluble flt3-L may also include part of the transmembrane region or part of the cytoplasmic domain or other sequences, provided that the protein of the latter can be secreted. soluble flt3-L soluble flt3-L can be identified (and distinguished from its insoluble, membrane-bound counterparts) by separating intact cells that express the desired protein of the culture medium, for example, by centrifugation, and assaying in the medium (supernatant) to see the presence of the desired protein. The presence of flt3-L in the medium indicates that the protein was secreted from the cells and, thus, is a soluble form of the desired protein. The soluble forms of flt3-L have many advantages over the native flt3-L binding protein. Purification of the proteins from the recombinant host cells is feasible, since the soluble proteins were secreted from the cells. In addition, soluble proteins are generally more suitable for intravenous administration. Examples of soluble flt3-L polypeptides include those that include a substantial portion of the extracellular domain of a native flt3-L protein. These mammalian soluble flt3-L proteins include amino acids 28 through 188 of the SEQUENCE IDENTIFICATION NUMBER: 2 or amino acids 28 through 182 of the SEQUENCE IDENTIFICATION NUMBER: 6. In addition, in the present invention they are including truncated soluble flt3-L proteins that include less than the entire extracellular domain. These truncated soluble proteins are represented by the sequence of amino acids 28-163 of the SEQUENCE IDENTIFICATION NUMBER: 2, and the amino acids of 28-160 of the SEQUENCE IDENTIFICATION NUMBER: 6. When initially expressed inside a host cell, the soluble flt.3-L may additionally include one of the heterologous signal peptides described below, which is functional within the host cells employed. Alternatively, the protein may include the native signal peptide, such that the mammalian flt3-L includes amino acids 1 through 188 of the SEQUENCE IDENTIFICATION NUMBER: 2 or amino acids 1 through 182 of the NUMBER OF IDENTIFICATION OF SEQUENCE: 6. In one embodiment of the present invention, flt3-L was expressed as a fusion protein that includes (from the terms N- to C-) the signal peptide of yeast factor a, a FLAGR peptide described below and in U.S. Patent No. 5,011,912, and soluble flt3-L consisting of amino acids 28 through 188 of the SEQUENCE IDENTIFICATION NUMBER: 2. Eeta recombinant fusion protein is expressed in, and secreted from the yeast cells. The FLAGR peptide facilitates the purification of the protein, and can subsequently be split from soluble flt3-L using bovine mucin enterokinase. The present invention encompasses isolated sequences of DNA encoding soluble flt3-L proteins. L? E1 truncated flt3-L, including soluble polypeptides, can be prepared by any of many conventional techniques. A desired DNA sequence can be synthesized chemically, using techniques known per se .. DNA fragments can also be produced by restriction endonuclease digestion of a full-length cloned DNA sequence, and isolated by agarose gel electrophoresis . Linkers containing restriction endonuclease cleavage site (s) can be used to insert the desired DNA fragment into an expression vector, or the fragment can be digested at the cleavage sites naturally present therein. The well-known polymerase chain reaction method can also be employed to amplify a DNA sequence encoding a desired protein fragment. As a further alternative, known mutagenesis techniques can be employed to insert a stop codon at a desired point, e.g., immediately downstream of the codon for the last amino acid of the extracellular domain. In another approach, the enzymatic treatment can be employed. (for example, using the exonuclease -F-sJ 31), to delete terminal nucleotides from a DNA fragment, to obtain a fragment having a particular desired term. Among the commercially available linkers are those that can be ligated to the blunt ends produced by the digestion of Bal 31, and which contain restriction endonuclease cleavage site (s). Alternatively, oligonucleotides that reconstruct the N- or C- terminus of the DNA fragment at a desired point can be synthesized and ligated to the DNA fragment. The synthesized oligonucleotide can contain a restriction endonuclease cleavage site upstream of the desired coding sequence and position an initiation codon (ATG) at the N- terminus of the coding sequence. As stated above, the present invention provides isolated or homogeneous flt3-L polypeptides, both recombinant or non-recombinant. Variants and derivatives of native flt3-L proteins that retain the desired biological activity (e.g., the ability to bind flt3) can be obtained by mutations of the nucleotide sequences encoding the native flt3-L polypeptides. Alterations of the native amino acid sequence can be achieved by any of many conventional methods. Mutations can be introduced at a particular site by synthesizing oligonucleotides containing a mutant sequence, flanked by restriction sites that allow ligation to fragments of the native sequence. After the ligation, the resulting reconstructed sequence encodes an analog having the desired insertion, substitution, or deletion of amino acids. Alternatively, site-specific oligonucleotide-directed mutagenesis methods can be employed to provide an altered gene wherein the predetermined codons can be altered by substitution, deletion or insertion. The exemplary methods for making the alterations stated above are described by Walder et al. (Gene 42: 133, 1986); Bauer and collaborators. { Gene 37:73, 1985); Craik (BioTechníques, January 1985, 12-19); Smith et al. (Genetic Engineering: Principles and Methods, Plenum Press, 1981); Kunkel (Proc. Natl. Acad. Sci. USA 82: 488, 1985); Kunkel et al. (Methods in Enzyme 1. 154: 367, 1987); and U.S. Patent Nos. 4,518,584 and 4,737,462, all of which are incorporated by reference. The flt3-L can be modified to create flt3-L derivatives by the formation of covalent or aggregative conjugates with other chemical moieties, such as glycosyl groups, lipids, phosphate, acetyl groups and the like. Covalent derivatives of flt3-L can be prepared by linking the chemical moieties to the functional groups in the amino acid side chains of flt3-L or in the N- term or the C- terminus of a flt3-L polypeptide or the extracellular domain of it. Other flt3-L derivatives within the scope of the present invention include covalent or aggregative conjugates of flt3-L or its fragments, with other proteins or polypeptides, such as by synthesis in recombinant culture as N- or C-terminal fusions. For example, the conjugate may include a signal or leader polypeptide sequence (e.g., the a-factor leader of Saccharomyces) in the N- terminus of a flt3-L polypeptide. The signal peptide or leader directs contranslationally or post-translationally the transfer of the conjugate from its synthesis site to a site inside or outside the membrane of the cell or cell wall. Flt3-L polypeptide fusions may include "peptides added to facilitate the purification and identification of flt3-L." These peptides include, for example, poly-His or the antigenic identification peptides described in the US Pat. No. 5,011,912 and Hopp et al., Bi o / Technol ogy fi: 1204, 1988. The present invention also includes flt3-L polypeptides with or without associated native-pattern glycosylation, flt3-L expressed in yeast expression systems. or mammalian (eg, COS-7 cells) may be similar to, or significantly different from, a native flt3-L polypeptide in molecular weight and glycosylation pattern, depending on the choice of the expression system. of flt3-L in bacterial expression systems, such as _g coli, provides non-glycosylated molecules The present invention encompasses equivalent DNA constructs ctes that encode various additions or substitutions of residues or amino acid sequences, or deletions of terminal or internal residues or sequences that are not needed for biological activity or for binding. For example, glycosylation sites N in the extracellular domain of flt3-L can be modified to prevent glycosylation, allowing the expression of a reduced carbohydrate analogue in mammalian and yeast expression systems. N-glycosylation sites in eukaryotic polypeptides are characterized by a triplet of amino acids Asn-X-Y, wherein X is any amino acid, except Pro and Y is Ser or Thr. The murine and human flt3-L proteins each include two of these triplets, at amino acids 127-129 and 152-154 of the SEQUENCE IDENTIFICATION NUMBER: 2, and at amino acids 126-128 and 150-152 of the SEQUENCE IDENTIFICATION NUMBER: 6, respectively. Substitutions, additions or deletions appropriate to the nucleotide sequence encoding these triplets will result in the prevention of binding of the carbohydrate residues in the side chain Asn. The alteration of a single nucleotide, chosen to replace the Aen with a different amino acid, for example, is sufficient to inactivate a glycosylation site N.

Known methods for inactivating N-glycosylation sites include those described in U.S. Patent No. 5,071,972 and "P" 276,846, incorporated herein by reference. In another example, sequences encoding Cys residues that are not essential for biological activity can be altered to cause the Cys residues to be suppressed or replaced with other amino acids, preventing the formation of incorrect intramolecular disulfide bridges upon renaturation. Other equivalents are prepared by modifying adjacent dibesic amino acid residues to enhance expression in the yeast systems in which KEX2 protease activity is present. EP 212,914 describes the use of site-specific mutagenesis to inactivate the KEX2 protease processing sites in a protein. The KEX2 protease processing sites are inactivated by deletion, addition or substitution residues to alter the Arg-Arg, Arg-Lys, and Lys-Arg pairs to eliminate the occurrence of these adjacent basic residues. Lys-Lys pairings are considerably less susceptible to cleavage of KEX2, and the conversion of Arg-Lys or Lys-Arg to Lys-Lys represents a conservative and preferred approach to inactivate K? X2 sites. The murine and human flt3-L contain two K? X2 protease processing sites at amino acids 216-217 and 217-218 of the SEQUENCE IDENTIFICATION NUMBER: 2, and at amino acids 211-212 and 213-213 of the SEQUENCE IDENTIFICATION NUMBER: 6, respectively. Nucleic acid sequences within the scope of the present invention include isolated DNA and RNA sequences that hybridize to the native flt3-L nucleotide sequences described herein, under conditions of moderate or severe rigor, and that encode the flt3- L biologically active. Conditions of moderate rigor, as defined by Sambrook and collaborators Molecular Cloning: A Laboratory Manual, second edition, Volume 1, pages 1101-104, Cold Spring Harbor Laboratory Press, (1989), include the use of a prewashing solution of 5 X SSC, 0.5 percent SDS, 1.0 mM EDTA (pH 8.0) and hybridization conditions of approximately 55 ° C, 5 X SSC, overnight. Conditions of severe rigor include elevated hybridization and washing temperatures. The skilled artisan will recognize that the temperature and salt concentration of wash solution can be adjusted as necessary in accordance with factors such as the length of the probe. Due to the known degeneracy of the genetic code where more than one codon can encode the same amino acid, a DNA sequence can vary from that shown in the SEQUENCE IDENTIFICATION NUMBER: 1, and the SEQUENCE IDENTIFICATION NUMBER: 5 and still encode a flt3-L protein having the amino acid sequence of the SEQUENCE IDENTIFICATION NUMBER: 2, and the SEQUENCE IDENTIFICATION NUMBER: 6, respectively. These variant DNA sequences can be the result of silent mutations (for example, occurring during PCR amplification), or they can be the product of the deliberate mutagenesis of a native sequence. The present invention provides equivalent isolated DNA sequences, encoding biologically active flt3-L, selected from: (a) DNA derived from the coding region of a native mammalian flt3-L gene; (b) cDNA that includes the nucleotide sequence presented in the SEQUENCE IDENTIFICATION NUMBER: 1, or the SEQUENCE IDENTIFICATION NUMBER: 5; (c) DNA capable of hybridization to a DNA of (a) under moderate stringency conditions and encoding biologically active flt.3-L; and (d) DNA that is degenerate as a result of the genetic code to a DNA defined in (a), (b) or (o) and that encodes biologically active flt3-L. The present invention encompasses the flt3-L proteins encoded by these equivalent DNA sequences. The DNAs that are equivalent to the DNA sequence of the SEQUENCE IDENTIFICATION NUMBER: 1, or the SEQUENCE IDENTIFICATION NUMBER: 5, will hybridize under conditions of moderate rigor to the native DNA sequence encoding the polypeptides that include amino acid sequences of 28-163 of the SEQUENCE IDENTIFICATION NUMBER: 2, or 28-160 of the SEQUENCE IDENTIFICATION NUMBER: 6. Examples of the flt3-L proteins encoded by these DNAs include, but are not limited to, flt3-L fragments (soluble or membrane-bound) and flt3-L proteins. including inactivated N-glycosylation site (s), inactivated KEX2 protease (s) processing site (s), or conservative amino acid substitutions, as described above. Flt3-L proteins encoded by DNA derived from other mammalian species are also encompassed, where the DNA will hybridize to the cDNA of the SEQUENCE IDENTIFICATION NUMBER: 1, or the SEQUENCE IDENTIFICATION NUMBER: 5. The variants possessing the The skill required to bind the receptor flt3 can be identified by any suitable assay. The biological activity of flt3-L can be determined, for example, by competition or binding to the ligand binding domain of the flt3 receptor (i.e., competitive binding assays). One type of a competitive binding assay for a flt3-L polypeptide uses a soluble radiolabeled human flt3-L and intact cells expressing cell surface flt3 receptors. Instead of intact cells, one can substitute soluble flt3 receptors (such as a flt3: Fc fusion protein) bound to a solid phase through the interaction of a Protein A, Protein G or an antibody to the portions of the flt3 or Fc of the molecule, with the Fc region of the fusion protein. Another type of competitive binding assay utilizes soluble radiolabeled flt3 receptors such as a flt3: Fc fusion protein, and intact cells expressing flt3-L. Alternatively, soluble flt3-L can be linked to a solid phase to positively select cells expressing flt3. Competitive link tests can be carried out following conventional methodology. For example, the radiolabeled flt3-L can be used to compete with a putative flt3-L homolog to test the binding activity against the surface link flt3 receptors. Quantitative results can be obtained by competitive autoradiographic plate binding assays, or Scatchard points can be used to generate quantitative results. Alternatively, flt3-binding proteins, such as flt3-L and anti-flt3 antibodies, can be linked to a solid phase such as a column chromatography matrix or a similar substrate suitable for identifying, separating or purifying expressing cells. the receiver flt3 on its surface. The binding of the proteins that bind flt3 to a solid phase contact surface can be achieved by any means, for example, by constructing a flt3-L: Fc fusion protein and binding it to the solid phase through the Interaction of Protein A or Protein G. Various other means for attaching proteins to a solid phase are known in the art and are suitable for use in the present invention. For example, magnetic microspheres can be coated with proteins that bind to flt3 and keep them in the incubation vessel through a magnetic field. Suspensions of cell mixtures containing hematopoietic stem or progenitor cells are contacted with the solid phase having proteins that bind to the flt3 therein. Cells that have the flt3 receptor on their surface bind to the protein that binds to fixed flt3 and then cells that are not bound are washed. This method of affinity binding is useful for the purification, classification or separation of those cells that express the flt3 of the solution. In the art, release methods that select cells positively from the solid phase and encompass, for example, the use of enzymes. Preferably, these enzymes are non-toxic and non-harmful to cells and are preferably directed to the cleavage of the binding pattern of the cell surface. In the case of the interactions of flt3: flt3-L, the enzyme will unfold preferentially to the receptor flt3, thereby releasing the cell suspension resulting from the "foreign" flt3-L material. The population of the purified cell can then be expanded ex vivo, before transplantation to a patient in an amount sufficient to reconstitute the hernatopoietic and immune system of the patient. Alternatively, mixtures of cells suspected of containing flt3? "Cells can first be incubated with a protein that binds to the bistantin-3 flt-3, incubation periods typically being at least one hour in duration to ensure bond-sufficient to flt3, then the resulting mixture is passed through a column packed with avidin-coated beads, where the high affinity of biotin for avidin provides the cell link to the beads. use of beads coated with avidin, see Berenson et al, J, Cell. Biochem., 10D: 239 (1986) Washing of unbound material and release of bound cells is performed using conventional methods. previously, the appropriate proteins that bind to flt3 are flt3-L, anti-flt3 antibodies, and other proteins that are capable of binding by high affinity to flt3. The one that links to the flt3 is the one of flt3-L. As described above, the flt3-L of the present invention can be used to separate cells expressing flt3 receptors. In an alternative method, the flt3-L or an extracellular domain or fragment thereof can be conjugated to a detectable portion such as 3.251 to detect cells expressing flt3. Radiolabelling with Izej can be done by any of many standard methodologies that yield a functional 125I-Flet-L molecule, labeled for high specific activity. Or, an iodinated or bio-stannolate antibody can be used against the flt3 region or the Fc region of the molecule. Another detectable portion such as an enzyme that can catalyze a colorimetric or fluorometric reaction, biotin or avidin can be used. The cells that are to be tested for the expression of the flt3 receptor can be contacted with the labeled flt3-L. After incubation, unlabeled labeling flt3-L is removed and the linkage is measured using the detectable portion. The binding characteristics of flt3-L (including variants) can also be determined using soluble, conjugated flt3 receptors (e.g., 1? 5I-flt3: Fc) in competition assays similar to those described above. However, in this case intact cells expressing flt3 receptors, or soluble flt3 receptors, bound to a solid substrate, are used to measure the extent to which a sample containing putative flt3-L competes for binding to a flt3 soluble conjugate to flt3-L. Other assay media for flt3-L include the use of anti-flt3-L antibodies, cell lines that proliferate in response to flt3-L, or recombinant cell lines that express the flt3 receptor and proliferate in the presence of flt3-L . For example, the BAF / B03 cell line lacks flt3 and is IL-3 dependent. (See Hatakeyama et al., Cell, £ 2: 837-845 (1989)). BAF / B03 cells transfected with an expression vector that includes the flt3 receptor gene, proliferate in response to either IL-3 or flt3-L. An example of a suitable expression vector for transfection of flt3 is plasmid pCAV / OT, see Mosley et al., Cell, 59: 335-348 (1989). The flt3-L polypeptides can exist as oligomers, such as covalently bound or non-covalently linked dimers or trimers. The oligomers can be linked by disulfide linkers formed between cysteine residues in different flt3 polypeptides. In an embodiment of the present invention, a flt3-L dimer is created by flt3-L fusion to the Fc region of an antibody (e.g., IgGl) in a manner that does not interfere with the binding of flt3-L to the binding domain of the ligand flt3. The Fc polypeptide is preferably fused to the C-terminus of a soluble flt3-L (which includes only the extracellular domain). The general preparation of fusion proteins including heterologous polypeptides fused to various portions of polypeptides derived from the antibody (including the Fc domain) has been described, for example, Ashkenazi et al. (PNAS USA 88: 10535, 1991) and Byrn et al. (Nature 344: 677, 1990), incorporated herein by reference. A fusion of gene encoding flt3-L: Fc fusion protein is inserted into an appropriate expression vector. The flt3-L: Fc fusion proteins are allowed to assemble very much like the antibody molecules, over which the interchain disulfide bonds are formed between the Fc polypeptides, yielding the divalent flt3-L. If the fusion proteins are made with both heavy and light chains of an antibody, it is possible to form an oligomer of flt3-L with as many as four extracellular regions of flt3-L. Alternatively, one can bind two domains of soluble flt3 ~ L with a peptide bond. Recombinant expression vectors containing DNA encoding flt3-L can be prepared using well-known methods. Expression vectors include a flt3-L DNA sequence operably linked to suitable transcriptional or translational regulatory nucleotide sequences, such as those derived from a mammalian, microbial, viral, or insect gene. Examples of regulatory sequences include promoters, operators, or transcriptional enhancers, a ribosomal binding site of mRNA, and appropriate sequences that control the initiation and termination of transcription and translation. The nucleotide sequences are "operably linked" when the regulatory sequence is functionally related to the DNA sequence of flt3-L. In this manner, a promoter nucleotide sequence is operably linked to a DNA sequence of flt3-L if the promoter nucleotide sequence controls the transcription of the DNA sequence of flt3-L. Within the expression vector, the replication ability can additionally be incorporated into the desired host cells, usually conferred by a replication origin, and a selection gene by which the transformers are identified.

In addition, sequences encoding the appropriate signal peptides that are not naturally associated with flt3-L can be incorporated into the expression vectors. For example, a DNA sequence for a signal peptide (secretory leader) can be fused in frame to the flt3-L sequence, so that flt3-L is initially translated as a fusion protein that includes the signal peptide . A signal peptide that is functional in the intended host cells enhances the extracellular secretion of the flt3-L polypeptide. The signal peptide can be split from the flt3-L polypeptide on the secretion of flt3-L from the cell. Host cells suitable for the expression of flt3-L polypeptides include higher prokaryotic, yeast or eukaryotic cells. Suitable cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cell hosts are described, for example, in Pouwels et al. Cloning Vectors: A Laboratory Manual, Elsevier, New York, (1985). Cell-free translation systems can also be employed to produce the flt3-L polypeptides using RNAs derived from DNA constructs described herein. Prokaryotes include gram-negative or gram-positive organisms, for example E. coli or Bacilli. Prokaryotic host cells suitable for transformation include, for example, E. coli, Bacillus subtilis, Salmonella typhimurium, and various other species within the genus Pseudomonas, Streptomyces, and Staphylococcus. In a prokaryotic host cell, such as E. coli, a flt3-L polypeptide can include an N-terminal methionine residue to facilitate expression of the recombinant polypeptide in the prokaryotic host cell. The N-terminal Met can be split from the expressed recombinant flt3-L polypeptide. Expression vectors for use in prokaryotic host cells generally include one or more selectable phenotypic marker genes. A selectable phenotypic marker gene is, for example, a gene that encodes a protein that confers antibiotic resistance or that provides an autotrophic requirement. Examples of expression vectors useful for prokaryotic host cells include those derived from commercially available plasmids such as the cloning vector PBR322 (ATCC 37017). PBR322 contains genes for resistance to ampicillin and tetracycline and thus provides simple elements to identify transformed cells. To construct an expression vector using pBR322, an appropriate promoter and a DNA sequence of flt3-L are inserted into the vector pBR322. Other commercially available vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and pG? Ml (Promega Biotec, Madison, Wl, USA). Promoter sequences that are commonly used for prokaryotic host cell expression vectors include β-lactamase (penicillinase), lactose promoter system (Chang et al, Nature 275: 615, 1978, and Goedde et al., Nature 281: 544, 1979), tryptophan (trp) promoter system (Goeddel et al., Nucí Acids Ees .5: 4057, 1980; and EP-A-36776) and tac promoter (Maniatis, Molecular Cloning: A Labora tory Manual, Cold Spring Harbor Laboratory, page 412, 1982). A useful system of prokaryotic host cell expression employs a PL lamd phase promoter and a cL857ts thermolebile repressor sequence. Plasmid vectors available from the American Type Culture Collection incorporating derivatives of the lamda EL promoter, include plaube ida pHUB2 (resident in the MB9 strain of E. coli (ATCC 37092)) and pPLc28 (resident in the RR1 of E: coli ( ATCC 53082)). The flt3-L polypeptides can alternatively be expressed in yeast host cells, preferably of the genus Saccharomyces (e.g., S. cerevisiae). Other types of yeast can also be used, such as Plchls, K. lactis or Kluyveromyces. The ferment vectors will generally contain a replication sequence origin from a 2μ yeast plasmid, an autonomous replication sequence (ARS), a promoter region, sequences for polyadenylation, sequences for transcription termination, and a marker gene selectable Suitable promoter sequences for yeast vectors include, among others, promoters for metallothionein, 3-phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem. 255: 2073, 1980) or other glycolytic enzymes (Hess et al., J. Adv. Enzyme Reg. 7: 149, 1968; and Holland et al., Biochem. 17: 4900, 1978), such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hesokinase, pyruvate decarboxylase, phosphoructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate rnutase, pyruvate kinase, triosephosphate isomerase, isomerase of phosphoglucose, and glucokinase. Other vectors and promoters suitable for use in the expression of yeast are further described in Hitzeman, EPA-73,657 or Fleer et al., Gene, XQZ: 285-195 (1991); and van den Berg et al., Bio / Technology, fi: 135-139 (1990). Another alternative is the glucose-repressible ADH2 promoter described by Russell et al. (J. Biol. Chem. 25.3: 2674, 1982) and Beier et al. (Na ture 300: 724. 1982). Short vectors that can be duplicated in both yeast and E. coli, can be constructed by inserting DNA sequences from pBR322 for selection and replication in E. Coli (Amp1 gene and origin of the replica) within the yeast vectors described above. The leader sequence of the yeast a factor can be used for the direct secretion of the flt3-L polypeptide. The leader sequence of the a factor is generally inserted between the promoter sequence and the sequence of the structural gene. See, for example, Ku jan et al., Cel l fi0_: 933, 1982; Bitter et al., Proc. Na tl. Acad. Sci. USA, fil: 5330, 1984; U.S. Patent No. 4,546,082; and EP 324,274. Those skilled in the art are aware of other suitable leader sequences to facilitate the secretion of recombinant polypeptides from yeast hosts. A leader sequence can be modified near its 3 'end to contain one or more restriction sites. This will facilitate the fusion of the leader sequence to the structural gene. Those skilled in the art know the yeast transformation protocols. One of those protocols is described by Hinnen et al., Proc. Natl. Acad. Sel. USA Ifi: 1929, 1978. The protocol of Hinnen et al. Selects Trp- + transformers in a selective medium, where the selective medium consists of 0.67 percent yeast nitrogen base, 0.5 percent casamino acids, 2% glucose, 10 μg / milliliter of adenine and 20 μg / milliliter of uracil.The yeast host cells transformed by vectors containing the ADH2 promoter sequence can grow to induce expression in a "rich" medium. of a rich medium is one consisting of 1 percent yeast extract, 2 percent peptone, and 1 percent glucose, supplemented with 80 μg / milliliter of adenine and 80 μg / milliliter of uracil. ADH2 occurs when the glucose in the medium is depleted Mammalian or insect host cell culture systems can also be used to express recombinant flt3-L polypeptides Luckow &Summers, Bi o / Technology 6: 47 (1988), reviewed baculovirus systems for the production of heterologous proteins in insect cells. The established cell lines of mammalian origin can also be employed. Examples of suitable mammalian host cell lines include the COS-7 line of monkey kidney cells (ATCC CRL 1651) (Gl? Zman et al., Cell 2fi: 175, 1981), L cells, C127 cells, 3T3 cells. (ATCC CRL 163), Chinese hamster ovary cells (CHO), HeLa cells, and BHK cell lines (ATCC CRL 10), and the CV-1 / EBNA cell line, derived from the CVl cell line of African green monkey kidney (ATCC CCL 10), as described by McMahan et al. (EMBO J. 10: 2821, 1991). The transcriptional and translational control sequences for the expression vectors of mammalian host cells can be excised from viral genomes. The promoter sequences and the commonly used enhancer sequences are derived from the Polyoma virus, Adenovirus 2, Simian Virus 40 (SV 40), and human cytomegalovirus. DNA sequences derived from the SV40 viral genome can be used, eg, SV40 origin sites, early and late promoter, enhancer, splice, and polyadenylation, to provide other genetic elements for the expression of a structural gene sequence in a mammalian host cell. The early and latter viral promoters are particularly useful, because both are easily obtained from a viral genome as a fragment that may also contain replication viral origin (Fiers et al., Nature 273: 113. 1978). Smaller or larger SV40 fragments may also be used, provided that the approximately 250 bp sequence extending from the Hind III site to the Bgl I site located at the viral replication site is included. of the SV40. Exemplary expression vectors for use in mammalian host cells can be constructed as described by Okayama and Berg (Mol.Cell. Biol. Fi: 280, 1983). A useful system for stable, high-level expression of mammalian cDNAs in murine C127 murine epithelial cells can be constructed substantially as described by Cosman et al. (Mol.Immunol.23: 935. 1986). A useful high expression vector, PMLSV N1 / N4, described by Cosman et al., Nature 312: 768, has been deposited. 1984, as ATCC 39890. In EP-A-0367566, and US Patent Application Serial Number 07 / 701,415, filed May 16, 1991, incorporated herein by reference, is incorporated herein by reference. describe additional useful mammalian expression vectors. The vectors can be derived from retroviruses. In place of the native signal sequence, a heterologous signal sequence can be added, such as the signal sequence for IL-7 described in U.S. Patent No. 4,965,195; the signal sequence for the IL-2 receptor, described in Cosman et al., Nature. 312: 768 (1984); the IL-4 signal peptide described in EP 367,566; the IL-1 receptor type I signal peptide described in U.S. Patent No. 4,968,607; and the signal peptide of the IL-1 type II receptor described in EP 460,846. The flt3-L as an isolated or homogeneous protein, according to the present invention, can be produced by recombinant expression systems as described above or can be purified from naturally occurring cells. The flt3-L can be purified to a substantial homogeneity, as indicated by a single protein band on analysis by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). A process for producing flt3-L includes culturing a host cell transformed with an expression vector that includes a DNA sequence encoding flt3-L under conditions sufficient to promote expression of flt.3-L. The flt3-L is then recovered from the culture medium or cell extracts, depending on the expression system used. As those skilled in the art know, the methods for purifying a recombinant protein will vary according to factors such as the type of host cells employed and whether the recombinant protein is secreted or not within the culture medium. For example, when expression systems that secrete the recombinant protein are employed, the culture medium can first be concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. After the concentration step, the concentrate can be applied to a purification matrix such as a gel filtration medium. Alternatively, an anion exchange resin, for example, a matrix or substrate having pendant diethylaminoethyl groups (DEAE) may be employed. The matrices can be acrylamide, agarose, dextran, cellulose or other types commonly employed in protein purification. Alternatively, a cation exchange step can be employed. Suitable cation exchangers include various insoluble matrices including sulfopropyl or carboxymethyl groups. Sulfopropyl groups are preferred. Finally, one or more steps of high performance reverse phase liquid chromatography (RP-HPLC) can be employed, employing hydrophobic RP-HPLC medium (for example, gelatinous silica having pendant methyl groups or other aliphatic groups), to further purify the flt3-L. Some or all of the above purification steps, in various combinations, are well known and can be used to provide a substantially homogeneous recombinant protein. It is possible to use an affinity column that includes the ligand binding domain of flt3 receptors to affinity purify the expressed flt3-L polypeptides. The flt3-L polypeptides can be removed from an affinity column using conventional techniques, for example, in a high salt levigation buffer and then dialyzed into a lower salt buffer for use or by changing the pH or other components depending on the affinity matrix used. Alternatively, the affinity column can include an antibody that binds to flt3-L. Example 6 describes a method for using the flt3-L of the present invention to generate monoclonal antibodies directed against flt3-L- The recombinant protein produced in the bacterial culture is usually isolated by initial disruption of the host cells, centrifugation, extraction to from cell pills if it is an insoluble polypeptide, or from the supernatant fluid if it is a soluble polypeptide, followed by one or more steps of concentration, desalination, ion exchange, affinity purification or size exclusion chromatography. Finally, RP-HPLC can be used for the final purification process. Microbial cells can be broken by any convenient method, including freeze-thaw cyclization, sonication, mechanical disruption, or the cell dissolving agent. Transformed yeast host cells are preferably used to express flt3-L as a secreted polypeptide in order to simplify purification. The recombinant polypeptide secreted from a yeast host cell can be purified by methods analogous to those described by Urdal et al. (J. Chromatog, 22fi: 171, 1984). Ural et al. Describe two steps of reverse phase HPLC for the purification of human recombinant IL-2 on a preparative HPLC column. The counter-sense and sense oligonucleotides that include a single-stranded nucleic acid sequence (either RNA or DNA), capable of binding a target flt3-L mRNA sequence (forming a double) or to the flt3- sequence L in the double stranded DNA helix (forming a triple helix) can be made in accordance with the present invention. The sense and sense oligonucleotides, according to the present invention, include a fragment of the cDNA coding region of flt3-L. This fragment generally includes at least about 14 nucleotides, preferably from about 14 to about 30 nucleotides. The ability to create an antisense or sense oligonucleotide, based on the cDNA sequence for a given protein, is described in, for example, Stein &; Cohen, Cancer Res. 48: 2659, 1988 and van der Krol et al., BioTechnigues 6: 958, 1988.

The binding of the counter-sense and sense oligonucleotides to the target nucleic acid sequences results in the formation of complexes that block translation (RNA) or transcription (DNA) by one of many means, including increased degradation of the duplos, the premature termination of transcription or translation, or by other means. In this manner, anti-sense oligonucleotides can be used to block the expression of flt3-L proteins. The antisense and sense oligonucleotides also include oligonucleotides that have modified the sugar-phosphodiester backbones (or other sugar linkers, such as those described in W091 / 06629) and wherein these sugar linkers are resistant to endogenous nucleases. . These oligonucleotides with resistant sugar linkers are stable in vivo (ie, capable of resisting enzymatic degradation) but retain the sequence specificity to be capable of binding to the target nucleotide sequences. Other examples of sense or anti-sense oligonucleotides include those oligonucleotides that are covalently bound to organic portions, such as those described in WO 91/10448, and other portions that increase the affinity of the oligonucleotide for a target nucleic acid sequence. , such as poly- (L-lysine). Still further, intercalating agents, such as ellipticine, and alkylating agents or metal complexes can be attached to the antisense or sense oligonucleotides to modify the binding specificities of the anti-sense or sense oligonucleotide for the target nucleotide sequence. The antisense or sense oligonucleotides can be introduced into a cell containing the target nucleic acid sequence by any method of gene transfer, including, for example, transfection of DNA mediated by CaPO-a, electroporation, or by one of such gene transfer vectors or the Epstein-Barr virus, antisense and sense oligonucleotides are preferably introduced into a cell containing the target nucleic acid sequence, by inserting the antisense or sense oligonucleotide into a suitable retroviral vector, then contacting the cell with the retroviral vector containing the inserted sequence, either in vivo or ex vivo. Suitable retroviral vectors include, but are not limited to, the murine retrovirus M-MuLV, N2 (a retrovirus derived from M-MuLV), or the double copy vectors designated DCT5A, DCT5B and DCT5C (see PCT Application US 90/02656). Sense or anti-sense oligonucleotides can also be introduced into a cell containing the target nucleotide sequence, by the formation of a conjugate with a ligand binding molecule, as described in WO 91/04753. Suitable ligand-binding molecules include, but are not limited to, cell surface receptors, growth factors, other cytokines, or other ligands that bind to cell surface receptors. Preferably, the conjugation of the ligand-binding molecule does not substantially interfere with the ability of the molecule that binds a ligand to bind to its corresponding molecule or receptor, or blocks the entry of the oligonucleotide in sense or counter-sense or its conjugated version inside the cell. Alternatively, an antisense or sense oligonucleotide may be introduced from a cell containing the target nucleic acid sequence, by forming an oligonucleotide-lipid complex, as described in WO 90/10448. The oligonucleotide-lipid complex in sense or counter-sense of preference is dissociated inside the cell by an endogenous lipase. The flt3-L polypeptides of the present invention can be formulated according to known methods that are used to prepare pharmaceutically useful compositions. The flt3-L can be combined in colloidal aggregate, either as the sole active material or with other known active materials, with pharmaceutically suitable diluents (e.g., Tris-HCl, acetate, phosphate), preservatives (e.g., Thimerosal, benzyl alcohol, parabenoe), emulsifiers, solubilizers, auxiliaries and (or vehicles) Suitable vehicles and their formulations are described in Remington's Pharmaceutical Sciences, Idava, ed., 1980, Mack Publishing Co. In addition, these compositions may contain flt3-L in complex with polyethylene glycol (PEG), metal, or incorporated into polymeric compounds such as polyacetic acid, polyglycolic acid, hydrogels, etc., or incorporated into liposomes, microemulsions, micelles, unilamellar or muyilaminal vesicles, erythrocyte traces or spheroplasts.These compositions will influence the physical state, the solubility , stability, the range of live release, and the in vivo evacuation range of flt.3-L. Lt.3-L can also be conjugated to antibody against tissue-specific receptors, ligands or antigenes, or to ligands to ligands of specific tissue receptors. Where the flt3 receptor is found in neoplastic cells, flt3-L can be conjugated to a toxin wherein flt3-L is used to deliver the toxin to the specific site, or it can be used to sensitize the neoplastic cells to antineoplastic agents administered Subsequently. FIT3-L can be administered locally, parenterally, or by inhalation. The term "parenteral" includes subcutaneous, intravenous, intramuscular, intracranial, or infusion techniques. These compositions will typically contain an effective amount of flt3-L alone or in combination with an effective amount of any other active material. These desired doses and concentrations of drug contained in the compositions may vary depending on many factors, including the intended use, the weight and age of the patient, and the route of administration. Preliminary doses can be determined according to animal tests, and the classification of doses for human administration can be made according to accepted practices in the art. With the foregoing description in mind, typical doses of flt3-L may vary from about 10 μg per square meter to about 1000 μg per square meter. A preferred range of doses is in the order of about 100 μg per square meter to about 300 μg per square meter. In addition to the foregoing, the following examples are provided to illustrate the particular embodiments and not to limit the scope of the present invention.

EXAMPLE 1 Preparation of the Flt3: Fc Receptor Fusion Protein This example describes the cloning of murine flt3 cDNA, and the construction of an expression vector encoding a soluble murine fusion protein of flt3: Fc receptor for use in the detection of cDNA clones that encode flt3-L. Cloning with polymerase chain reaction (PCR) of the flt3 cDNA from a murine T cell was carried out using the oligonucleotide primers and methods described by Lyman et al., Oncogene, fi: 815-822, (1993), incorporated herein by reference. The cDNA sequence and the encoded amino acid sequence for mouse flt3 receptor are presented by Rosnet et al., Oncogene-, fi: 1641-1650, (1991), incorporated herein by reference. The mouse flt3 protein has an extracellular domain of amino acids 542, a transmembrane domain of amino acids 21, and a cytoplasmic domain of amino acids 437. Before fusing the murine flt3 cDNA to the N terminus of the cDNA encoding the Fc portion of a human IgG1 molecule, the amplified mouse flt3 cDNA fragment was inserted into an Asp718-NotI site of pCAV / NOT, described in PCT application WO 90/05183. The DNA encoding a single chain polypeptide including the Fc region of a human IgGl antibody was cloned into the Spel site of the pBLUESCRIPT SKK vector, which is commercially available from Stratagene Cloning Systems, La Jolla, California. This plasmid vector can be duplicated in E. coli and contains a polylinker segment that includes 21 unique restriction sites. A unique Bg / Ll site was introduced near the 5 'end of the inserted Fc encoding the sequence, so that the Bg /? Site. I encompasses the codons for amino acids three and four of the Fc polypeptide. The encoded Fc polypeptide extends from the region of the hinge to the native C terminus, ie, it is an Fc region of antibody of essentially complete length. The fragments of the Fc regions can also be used, for example, those that are truncated at the end of the C terminal. The fragments preferably contain multiple residues of cysteine (at least the cysteine residues in the hinge reaction), for allowing interchain disulfide bonds to form between the Fc portions of the polypeptide of two separate flt3: Fc fusion proteins, forming dimers as set forth above. A cleavage site of the restriction endonuclease Asp718 upstream of the coding region of flt3 was introduced. An Asp 718-VstI fragment was isolated from mouse flt3 cDNA (which included the entire extracellular domain, the transmembrane region, and a small portion of the cytoplasmic domain). The partial cDNA of Asp718-AFoeI flt3 described above was cloned into the pBLUESCRIPT SKR vector containing the Fc cDNA, such that the cDNA of flt3 was positioned upstream of the Fc cDNA. The single-strand DNA derived from the resulting gene fusion was rnutagenized by the method described by Kunkel (Proc. Natl. Acad. Sci. USA fi2: 488, 1985) and Kunkel et al. (Methods in Enzymol. 367, 1987) in order to perfectly fuse the entire extracellular domain of flt3 to the Fc sequence. The mageaged cDNA was sequenced to confirm that the appropriate nucleotides had been removed (ie, the transmembrane region and the DNA of the partial cytoplasmic domain were deleted) and that the sequences of flt3 and Fc were in the same frame reading. The fusion cDNA was then excised and inserted into a mammalian expression of the vector designated sfHAV-EO 409 that was cut with Sall-Notl, and the blunt Sali and Asp718 termini. The sfHAV-EO vector (also known as pDC406) is described by McMahan et al. (EMBO J., 10; Number 1Q: 2821-2832 (1991)). Flt3: Fc fusion proteins are preferably synthesized in mammalian recombinant cell culture. The expression vector containing the flt3: Fc fusion was transfected into CV-1 cells (ATCC CCL 70) and COS-7 cells (ATCC CRL 1651), both derived from monkey kidney. The expression level of flt3: Fc was relatively low in both CV-1 and COS-7 cells. In this manner, expression was attempted in 293 cells (transformed primary human embryonic kidney cells ATCC CRL 1573). The 293 cells transfected with the vector sfHAV-? 0 / flt3: Fc were cultured in roll bottles to allow transient expression of the fusion protein, which was secreted into the culture medium by the signal peptide of flt3. The fusion protein was purified on protein A Sepharose columns, levigated, and used to classify the cells by the ability to bind flt3: Fc, as described in Examples 2 and 3.

EXAMPLE 2, Classification of Cells by Flt3: Fc Link Approximately 100 different primary cells and cell lines falling within the following general categories were tested: murine fetal brain primary cells, murine fetal liver cell lines , rat fetal brain cell lines, carcinoma cell lines (fibroblastoids) of human lung, human and murine lymphoid and myeloid cell lines, and for flt3: Fc binding. The cell lines were incubated with flt3: Fc, followed by a bi-tinned anti-human Fc antibody, followed by streptavidin-phycoerythrin (Becton Dickinson). The bio-stannolate antibody was purchased with Jackson Immunoresearch Laboratories. Streptavidin bound to the binder molecule bound to the anti-human Fc antibody, which in turn was linked to the Fc portion of the flt.3: Fc fusion protein. Phycoerythrin is a fluorescent phycobiliprotein that serves as a detectable label. The level of the fluorescence signal was measured for each cell type using a FACScan® flow cytometer (Becton Dickinson). The cell types that were considered positive for the bond of flt3: Fc were identified.

Isolation and Cloning of the murine T-cell cDNA library flt3 L cDNA library A murine T-cell cDNA library was chosen from the P7B-0.3A4 cell line as a possible source of flt3-L cDNA. P7B-0.3A4 is a murine T-cell clone that is Th l ^ - * -, CD4 ~, CD8-, TCRab ±, 0.044 - * -. This was originally cloned at a cell density of 0.33 cells / well in the pre-presence of r-HuIL-7 and immobilized anti-CD3 MAb, and grew in continuous culture for more than 1 year by passing once a week in medium containing 15 ng / ml of r-HuIL-7. The originating cell line was derived from SJL / J mouse lymph node cells immunized with 50 mmoles of PLP139-151 peptide and 100 μg of Mycobacterium tuberculosis H37Ra in Freund's Incomplete Auxiliary. PLP is the proteolipid protein component of the medullary sheath of the central nervous system. The peptide composed of amino acids 139-151 had previously been shown as the encephalogenic peptide in experimental autoimmune encephalomyelitis (EAE), a murine model for multiple sclerosis in SJL / J mouse. / Touhy, V.K., Z. Lu, R.A. Laursen and M.B. Leee; 1989. Identification of an encephalothogenic determinant of proteolipid protein of rhielin for mouse SJL. J. Immuno 1. 142: 1523). After initial culture in the presence of the antigen, the maternal cell line, designated PLP7, had been in continuous culture with rHuIL-7 (and without antigen) for more than 6 months before cloning. P7B-0.3A4 proliferated only in reepheeta at very high concentrations of PLP139-1S1 peptide in the presence of irradiated syngeneic splenocytes and is neither enecephalogenic nor alresponsive. This clone proliferated in response to immobilized anti-CD3 MAb, 11-2, and IL-7, but not to IL-4. The bond of flt3: Fc was observed in urine T cells and in human T cells, and therefore a murine T cell line (0.3A4) was chosen because of its ease of growth. A cDNA library of 0.3A4 of rnurin was prepared in sfHAV-EO as described in McMahan et al (EMBO J., 1, Number 10, 2821-2832, 1991). The sfHAV-EO is a vector of expression that also duplicates in E. coli. The sfHAV-EO contains replication origins derived from the SV40 Epstein-Barr virus and pBR322 and is a derivative of HAV-EO described by Dower et al., J. Immunol. 142: 4314 (1989). The sfHAV-EO differs from the HAV-EO by the deletion of the Intron present in the tripartite leader sequence 2 of the adenovirus in HAV-EO. Briefly, the murine T cell cDNA was cloned into the SalI site of sfHAV-EO by an adapter method similar to that described by Haymerle et al. (Nucí Acids Res. 4: 8615, 1986), using the following adapter pair. oligonucleotide: 5 'TCGACTGGAACGAGACGACCTGCT 3' NO. ID. SEO: 3 3 'GACCTTGCTCTGCTGGAGGA 5' NO. ID. DE SEC.:4 Double-layered cDNA was prepared, finished blunt, prepared at random, from 0.3A4 poly (A) + RNA essentially as described by Gubler and Hoffman, Gene, 25 .: 263-269 (1983), using a Pharmacia DNA kit. Previous adapters were added to the cDNA as described by Haymerle et al. The betjo molecular weight material was removed by passage over Sephacryl S-1000 at 65 ° C, and the cDNA was ligated into sfHAV-E0410, which had been previously cut with salt and ligated into the same oligonucleotide pair. This vector was designated as sfHAV-E0410. DNA was electroporated (Dower et al., Nucleic Acids Res., Lfi: 6127-6145, (1983) in E. coli DH10B, and after one hour grew at 37 ° C, the transformed cells were frozen in one milliliter aliquots. in the SOC medium (Hanahan et al., J. Mol. Biol. lfifi: 557-580, (1983)) containing 20% glycerol, an aliquot was titrated to determine the number of ampicillin-resistant colonies. The resulting A4 had 1.84 million clones.The E. coli cells of strain DH10B tranefected with the cDNA library in sfHAV-E0410 were plated to provide approximately 1600 colonies per plate.The colonies were scraped from each plate, combined , and plasmid DNA was prepared from each combination, then the combined DNA, representing approximately 1600 colonies, was used to transfect a subconfluent layer of CV-1 / EBNA-1 cells using DEAE-dextran followed by chlorine treatment, similar to described by Luthman et al., Nucí. Acids Res. 11: 1295, (1983) and McCutchan et al., J. Na tl. Cancez * Inst. 41: 351, (1986). The CV-1 / EBNA cell line (ATCC CRL10478) constitutively expressed the nuclear antigen EBV 1 driven from the CMV immediate early enhancer / promoter. CV-1 / EBNA was derived from the green monkey kidney cell line CV-1 (ATCC CCL 70), as described by McMahan et al. (EMBO J. 10_: 2821, 1991). In order to transfect the CV-1 / EBNA cells with the cDNA library, the cells were maintained in complete medium (Dulbecco's modified Eagle's medium (DMEM) containing calf fetal serum at 10 percent (volume / volume), 50 U / milliliter of penicillin, 50 U / milliliter of streptomycin, 2mM L-glutamine) and plated at a density of approximately 2 x 10 B cells / well in single-well framed slides (Lab-Tek). The slides were pretreated with 1 milliliter of human fibronectin (10 μg / milliliters in PBS) for 30 minutes followed by 1 wash with PBS. The medium was removed from the adherent layer of cells, and replaced with 1.5 milliliters of complete medium containing 66.6 μM chlorine sulfate. Then two tenths of DNA solution (2 μg of DNA, 0.5 milligram / milliliter of DEAE-dextran in complete medium containing chlorocin) were added to the cells, and incubated for 5 hours. After incubation, the medium was removed and the cells were shocked by the addition of complete medium containing 10 percent DMSO for 2.5 to 20 minutes followed by replacement of the solution with fresh complete medium. The cells were cultured for 2 to 3 days to allow transient expression of the inserted sequences.

The transfected monolayers of CV-1 / EBNA cells were tested to see expression of flt3-L by slide autoradiography essentially as described by Gearing et al. (EMBO J. fi: 3667, 1989). The transfected CV-1 / EBNA cells (adhered to framed slides) were washed once with non-fat dry milk binding medium (BM-NFDM) (RPMI 1640 medium containing 25 milligrams / milliliter of bovine serum albumin (BSA). ), 2 milligrams / milliliter of eidium azide, 20 M of HEPES, pH 7.2, and 50 milligrams / milliliters of dry milk without fat). The cells were incubated with flt3: Fc in BM-NFDM (1 μg / milliliters) for 1 hour at room temperature. After incubation, the monolayers of cells were washed on the slides framed three times with BM-NFDM to remove the unbound fusion protein flt3: Fc and then incubated with 40 ng / milliliters of mouse anti-human Fc antibody 125I (described below ) (a 1:50 dilution) for 1 hour at room temperature. The cells were washed three times with BM-NFDM, followed by 2 washes with buffered saline (PBS) to remove the anti-human Fc antibody ISBJ and unbound mouse. The cells were fixed by incubation for 30 minutes at room temperature in 2.5 percent glutaraldehyde in PBS, pH 7.3, washed twice in PBS and air dried. The framed slides containing the cells were exposed in a Phophorimager (Molecular Dynamics) overnight, then soaked in Kodak's GTNB-2 photographic emulsion (6x dilution in water) and exposed to darkness for 3-5 days at 4oC in a light-proof box. The slides were then developed for approximately 4 minutes in Kodak D19 developer (40 grams / 500 milliliters of water), rinsed in water and fixed in Agfa G433C fixative. The slides were examined individually with a microscope at a magnification of 25-40X and positive cells expressing flt3-L were identified by the presence of autoradiographic silver beads against a background light. The mouse antihuman Fc antibody was obtained with Jackson Laboratories. This antibody showed a minimal binding to the Fc proteins linked to the FCT receptor. The antibody was labeled using the Chloramine T method. Briefly, a Sephadex G-25 column was prepared in accordance with the manufacturer's instructions. The column was pretreated with 10 column volumes of PBS containing 1 percent bovine serum albumin to reduce non-specific adsorption of the antibody to the column and resin. The unbound bovine serum albumin was then washed from the column with 5 volumes of PBS lacking bovine serum albumin. In a microfuge tube, 10 μg of antibody (dissolved in 10 μl of PBS) was added to 50 μl of 50 mM sodium phosphate buffer (pH 7.2), 2.0 mCi of vehicle-free Na12SI was added and the solution was mixed well . Then 15 μl of a freshly prepared solution of chloramine T (2 milligrams / 1 milliliter in 0.1M sodium phosphate buffer (pH 7.2)) was added and the mixture was incubated for 30 minutes at room temperature, and then immediately applied. mix to the Sephadex G-25 column. The radiolabelled antibody was then levitated from the column, through the collection of 100-150 μl of levigant fractions. Bovine serum albumin was added to the levigated fractions containing the radiolabeled antibody at a final concentration of 1 percent. Radioiodination yielded specific activities in the range of 5-10 x 10? E cpm / nmol of protein. Using the slide autoradiography approach, the approximately 1,840,000 cDNA in combinations of approximately 1,600 haeta cDNAs were selected that the assay of a transfected combination showed multiple clearly positive cells for the flt3: Fc binding. After this combination was divided into combinations of 500 and again selected by, slide autoradiography and a positive combination was identified. This combination was divided into combinations of 100 and again selected. Individual colonies of this combination of 100 were selected until a clone (clone # 6C) was identified that directed the synthesis of a surface protein with detectable linker activity of flt3: Fc. This clone was isolated, and its 0.88 kb cDNA insert was made sequential. The nucleotide and encoded amino acid sequences of the coding region of the cDNA of murine ligand flt3 of clone # 6C were presented in SEQUENCE IDENTIFICATION NUMBERS: 1 and 2. The cDNA insert is 0.88 kb in length. The reading frame opened inside this sequence can encode a protein of 231 amino acids. In this manner, the DNA and the amino acid sequences encoded for the open reading frame of 231 amino acids were presented in the SEQUENCE IDENTIFICATION NUMBERS: 1 and 2. In the SEQUENCE IDENTIFICATION NUMBER protein: 2 ee a protein of transmembrane of type I, with an N-terminal signal peptide (amino acids 1 to 27), an extracellular domain (amino acid 28 to 188), a tranenembrane domain (amino acids 189-211) and a cytoplasmic domain (amino acids 212-231) . The predicted molecular weight of the native protein after cleavage of the signal sequence is 23,164 daltons. The mature protein had an estimated pl of 9,372. There are -56 bp of unencoded sequence 5 'and 126 bp of unencoded sequence 3' flanking the coding region, including the added cDNA adapters. The cloning procedure described above also produced a murine flt ligand of clone # 5H, which is identical to clone # 6C starting at nucleotide 49 and continuing through nucleotide 545 (corresponding to amino acid 163) of NUMBER OF SEQUENCE IDENTIFICATION: 1. Clone # 5H differs completely from that point on, and represents an alternative splice construction. The vector sfHAV-E0410 containing the cDNA of flt3-L in the DH10B cells of E. coli was deposited with the American Type Culture Collection, Rockville, MD, USA (ATCC) on April 20, 1993 and the number was assigned to it. of accession ATCC 69286. The deposit was made under the terms of the Budapest Treaty.

EJKMP Q 4 A cDNA encoding human flt3-L was cloned from a randomly loaded cDNA library of T cell 22 of human clone lamda gtlO as described by Sims et al., PNAS, fifi: 8946-8950 (1989). The library was selected with a Foot I fragment of 413 bp that corresponded to the extracellular domain of murine flt3-L (nucleotides 103-516 of the SEQUENCE IDENTIFICATION NUMBER: 1). The fragment was randomly loaded, hybridized overnight to the library filters at 55 ° C in oligomeric prehybridization buffer. The fragment was then washed at 55 ° C at 2 x SSC / 0.1 percent SDS for one hour, followed by 1 x SSC / 0.1 percent SDS for one hour and then by 0.5 x SSC / 0.1 percent SDS for one hour. The DNA was extracted from the positive fage plates, and the inserts were amplified by PCR using oligonucleotides specific for the fage arms. The DNA was then sequenced, and the sequence for clone # 9 is shown in the SEQUENCE IDENTIFICATION NUMBER: 5. Additional human flt3-L cDNA was isolated from the same randomly loaded cDNA library as described above. by selecting the library with a fragment of the extracellular domain of the murine clone # 5H cDNA that includes a cDNA sequence that essentially corresponds to nucleotides 128-541 of the SEQUENCE IDENTIFICATION NUMBER:! Sequencing of the 988 bp cDNA from clone # 9 revealed an open reading frame of 705 bp surrounded by 29 bp of an uncoded 5 'and 250 bp sequence of an uncoded 3' sequence. The 3 'uncoded region did not contain a poly A end. There were no frame stop codons upstream of the initiator methionine. The open reading frame encodes a type I transmembrane protein of 235 amino acids as shown by amino acids 1-235 of the SEQUENCE IDENTIFICATION NUMBER: 6. The protein has an N-terminal signal peptide of 26 or 27 amino acids alternatively . There is a slightly higher probability that the N-terminal signal peptide is followed by an extracellular domain of 156 or 155 amino acids (for signal peptides of 26 and 27 amino acids, respectively); a transmembrane domain of 23 amino acids and a cytoplasmic domain of 30 amino acids. Human flt3-L shares all 72 percent amino acid identity and 78 percent amino acid similarity with murine flt3-L. The pBLUECRIPT SK (-) vector containing the human flt3-L cDNA from clone # 9 was deposited with the American Type Culture Collection, Rockville, MD, USA (ATCC) on August 6, 1993 and assigned the number of Access ATCC 69382. The deposit was made under the terms of the Budapest Treaty.

EXSHPLO 5 Expression of Flt3-L in Yeast For the expression of soluble flt3-L in yeast, the first synthetic oligonucleotide was used to amplify, by PCR (Mullís &Faloona, Meth. Enzymol., 155: 335-350, 1987) , the entire extracellular coding domain of flt3-L between the end of the signal peptide and the start of the transmembrane segment. The first 5 '(5' -AATTGGTACCTTTGGATAAAAGAGACTACAAGGACGACGATGACAAGACACC-TGACCTGACTGTTACTTCAGCCAC-3 ') of the SEQUENCE IDENTIFICATION NUMBER: 7 encoded a portion of the alpha factor leader and an antigenic octapeptide, the FLAG sequence was fused in frame with the predicted mature N term of the flt3-L. The 3 'oligonucleotide (5'-ATATGGATCCCTACTGCCTGGGCCGAGGC-TCTGGGAG-3') of the SEQUENCE IDENTIFICATION NUMBER: 8 created a stop codon after Gln-189, just in the putative transmembrane region. The DNA fragment generated by PCR was ligated into a yeast expression vector (for expression in K. lactis) which directs the secretion of the recombinant product into the yeast medium (Fleer et al., Gene, 10.7: 285-195 (1991), and van den Berg et al., Bio / Technology, fi: 135-139 (1990)). The FLAG: flt3-L fusion protein was purified from yeast broth by affinity chromatography as described previously (Hopp et al., Biotechnology, fi: 1204-1210, 1988).

EXAMPLE 6 Honoclonal Antibodies to Flt3-L This example illustrates a method for preparing monoclonal antibodies to flt3-L. The flt3-L was expressed in mammalian host cells such as COS-7 or CV-1 / EBNA-1 cells and purified using flt3: Fc affinity chromatography. The purified flt3-L, a fragment thereof or the extracellular domain, synthetic peptides or cells expressing flt3-L, can be used to generate monoclonal antibodies against flt3-L, using conventional techniques, for example, those techniques described in U.S. Patent No. 4,411,993. Briefly, mice were immunized with flt3-L as an immunogen emulsified in complete Freund's helper, and injected in amounts ranging from 10 to 100 μg, subcutaneously or intraperitoneally. Ten or twelve days afterwards, animals immunized with additional flt3-L emulsified in complete Freund's helper were boosted. The mice were periodically strengthened from now on on a weekly or biweekly immunization schedule. Serum samples were periodically taken by retro-orbital bleeding or removal of the tip of the tail to test flt3-L antibodies by spot spot assay, ELISA (Enzyme Linked Immunosorbent Assay) or inhibition of flt3 linkage. After detection of an appropriate antibody titer, the positive animals were given a final intravenous injection of flt3-L in saline. Three or four days later, the animals were sacrificed, the spleen cells were harvested, and the spleen cells were fused to a murine myeloma cell line, for example, NSl or preferably P3x63Ag8.653 (ATCC CRL 1580). . The fusions generated hybridoma cells, which were plated on multiple microtiter plates in a selective medium of HAT (hypoxanthene, aminopterin and thymidine), to inhibit the proliferation of unfused cells, myeloma hybrids, and hybrids of Spleen cells Hybridoma cells were selected by ELISA for reactivity against purified flt3-L, by adaptations of the techniques described by Engvall et al., Iimnunochem. , fi: 871, 1971 and in the patent of the United States of North America number 4,703,004. A preferred selection technique is the antibody capture technique described by Beckmann et al., (J. Immunol., 144: 4212, 1990). Hybridoma cells positive can be injected intraperitoneally into a syngeneic BALB / c mouse to produce ascites containing high concentrations of anti-flt3-L monoclonal antibodies. Alternatively, the hybridoma cells can grow in vi tro in bottles or roller bottles by various techniques. Monoclonal antibodies produced in mouse ascites can be purified by precipitation of ammonium sulfate, followed by gel exclusion chromatography. Alternatively, affinity chromatography based on the binding of the antibody to protein A or protein G can also be used, as also affinity chromatography based on binding to flt3-L can be used.

EXAMPLE 7 Use of Flt3-L Solo v in Combination with IL-7 or IL-3 This example shows the stimulation and proliferation of fetal liver cells AA4.1 - * -, by compositions containing flt3-L and IL-7; as well as the stimulation and proliferation of c-ki t positive cells (c-lriú +), by means of compositions containing flt3-L and IL-3. Cells expressing AA4.1 positive (AA4.1"4") were isolated from the livers of fetal mice of day 14 C57BL / 6 by placing in Optilux 100-millimeter plastic Petri dishes (Falcon No. 1001, Oxnard, CA). Plates were coated overnight at 4 ° C in PBS plus 1 percent fetal bovine serum (FBS) containing 10 μg / milliliter of AA4.1 antibody (McKearn et al., J. Immuno 1. 132: 332-339, 1984) and then extensively washed with PBS plus 1 percent FBS before use. A single cell suspension of liver cells was added at 10"7 cells / well in PBS plus 1 percent FBS and allowed to adhere to the plates for two hours at 4 [deg.] C. The plates were washed extensively, and then adhered cells were harvested by scraping for analysis or for further use in the hematopoiesis assays described below.FACS analysis using the AA4.1 antibody, demonstrated a population of AA4.1'i "cells from >95 percent. C-ki t + pluripotent cane cells were purified from adult mouse bone marrow (de Vries et al., J. Exp. Med., Llfi: 1503-1509, 1992; and Visser and de Vries, Methods in Cell Biol., 1993). , submitted). Low-density cells (<1078 grams / cubic centimeter) positive for lectin wheat germ agglutinin and negative for antigens recognized by monoclonal antibodies B220 and 15-1.4.1 (Viseer and collaborator, Meth. In Cell Biol., Fifi: 451-468, 1990), were able to divide into subpopulations of cells that expressed or not the c-ki t by using the factor of biostained steel. The c-ki t + fraction has been shown to contain pluripotent hematopoietic stem cells (de Vries et al., Science 235 .: 989-991, 1992; Visser and de Vries, Methods in Cell Biol., 1993, submitted; collaborators, 1993, submitted). Fetal liver cells AA4.1 - * - were cultured in recombinant IL-7 (U.S. Patent No. 4,965,195) at 100 ng / milliliter and recombinant flt3-L at 250 ng / milliliter. The flt3-L was used in three different ways in the experiments: (1) as present in CVL / EBNA cells transfected with Flt3-L, fixed; (2) as culture supernatants concentrated from these same CVl / EBNA cells transfected with Flt3-L; and (3) as a purified and isolated polypeptide preparation from yeast supernatant as described in Example 5.

Hematopoiesis assays The proliferation of c-ki t + stick cells, and AA4.1"* - cells from fetal liver, was tested in [3H] -thymidine incorporation assays as described essentially by Vriee et al., J. Exp. Med., 173_: 1205-1211, 1991. C-Jrii --- + purified cane cells were cultured at 37 ° C in a fully humidified atmosphere of 6.5 percent COs and 7 percent O2 in air for 96 hours. horae Recombinant murine IL-3 was used at a final concentration of 100 ng / milliliter Subsequently, the cells were pulsed with 2 μCi per well of [3 H] -thymidine (81 Ci / mmol, Amersham Corp., Arlington Heights, IL) and were incubated in IL-7, flt3-L and flt3-L + IL-7 for 48 hours, followed by a pulse of [3H] -thymidine from seie horae. Table 1 shows the results of the flt3- L and IL-7, and in Table II the results of flt3-L and IL-3 are shown.

TABLE I Effect of FIt3-L and IL-7 on Proliferation of Fetal Liver Cells AA4.1 + Factor -CO? Tc L flt3 ~ L IL-7 flt3-L + IL-7 Incorporation 100 1000 100 4200 of [3H ] -thymidine (CPM) The combination of flt3-L and IL-7 produced a response that was approximately four times greater than flt3-L alone and approximately 40 times greater than IL-7 alone.

TABLE II Effect of Flt3-L and IL-7 on Proliferation of Q Cells zMi ± Control Factor nt.3-L IL-3 flt3-L + IL-3 (Vector only) incorporation 100 1800 3000 9100 of [3H] - Thymidine (CPM) The culture supernatant of the CVl / EBNA cells transfected with the flt3-L cDNA stimulated the proliferation of the c-ki t + rod cells approximately 18 times larger than the culture supernatant of the CVl / EBNA cells transfected with the vector of expression alone. The addition of IL-3 to flt3-L containing supernatant, showed a synergistic effect, with approximately twice the observed degree of proliferation than would be expected if the effects were additive.

EXAMPLE 3 Construction of Flt3-L: Fc Fusion Protein This example describes a method for constructing a fusion protein that includes an extracellular region of flt3-L and the Fc domain of a human immunoglobulin. The methods are essentially the same as those described in Example 1 for the construction of a flt3: Fc fusion protein. Before fusing an flt3-L cDNA to the N-terminus of the cDNA encoding the Fc portion of a human IgG1 molecule, the cDNA fragment of flt3-L is inserted into an AspJ18-? Ao6I site of pCAV / NOT, described in PCT Application WO 90/05183. The DNA encoding a single chain polypeptide including the Fc region of a human IgGl antibody was cloned into the Spel site of the pBLUESCRIPT SKR vector, which is commercially available from Stratagene Cloning Systems, La Jolla, California. This plasmid vector can be duplicated in E. coli and contains a polylinker segment that includes 21 unique restriction sites. A unique Bg / 11 site is then introduced near the 5 'end of the inserted Fc encoding the sequence, so that the Bg / I l site spans the codons for amino acids three and four of the Fc polypeptide. The encoded Fc polypeptide extends from the hinge region of the N-terminus to the native C-terminus, ie, an antibody Fc region of substantially complete length. The fragments of the Fc regions can also be used, for example, those that are truncated at the end of the N-terminal. Preferably, the fragments contain multiple residues of cysteine (at least the cysteine residues in the hinge reaction) for allowing interchain disulfide bonds to be formed between the Fc portions of the polypeptide of two separate flt3: Fc fusion proteins, forming dimers. A partial cDNA of Asp718-Stul flt3 can be cloned into an AspJ18-SpeI site of the pBLUESCRIPT SKR vector containing the Fc cDNA, such that the cDNA of flt3 upstream of the Fc cDNA was positioned. The single-stranded DNA sequence derived from the resulting gene fusion, can be affected by the temperate directed mutagenesis described by Kunkel (Proc. Natl. Acad. Sci. USA fi2: 488, 1985) and Kunkel et al (Methods in Enzymol., 154: 367, 1987) in order to perfectly fuse all the extracellular domain of flt3 to the Fc sequence. The resulting DNA can then be sequenced to confirm that the appropriate nucleotides had been removed (ie, the transmembrane region and the partial cytoplasmic domain DNA were deleted) and that the sequences of flt3 and Fc were in the same reading frame. The fusion cDNA was then excised and inserted into the mammalian expression vector pCAV / NOT that was cut with Asp718-Notl. ). Flt3: Fc fusion proteins are preferably synthesized in mammalian recombinant cell culture. The expression vector containing the flt3: Fc fusion was then transfected into CV-1 cells (ATCC CCL 70) and COS-7 cells (ATCC CRL 1651). It is also possible to express 293 cells (primary human embryonic kidney cells transformadae ATCC CRL 1573). The 293 cells transfected with the vector pCAV / NOT / flt3-L: Fc were cultured in roller bottles to allow transient expression of the fusion protein, which was secreted into the culture medium by the flt3 signal peptide. The fusion protein can be purified on Sepharose columns of protein A.

EJ PLO 9 Generation of Transgenic Mice Overexpressing Fl 3-L This example describes a procedure that is used to generate transgenic mice that overexpress flt3-L. Transgenic mice expressing flt3-L were studied to determine the biological effects of overexpression. Mouse pronuclei (B16 / J) were microinjected with flt3-L DNA according to the method described by Gordon et al, Science 214: 1244-1246, (1981). In general, fertilized mouse ovules that had visible pronuclei were first placed in an injection chamber and held in place with a small pipette. An injection pipette was then used to inject the gene encoding flt3-L (clone # 6C) into the pronuclei of the ovule. Then, the injected ovules either (i) were transferred into the oviduct of a pseudopregnant female p.c. at day 0.5; (ii) were cultured in vi tro to the stage of two célulets (overnight) and transferred into the oviduct of a pseudo-pregnant female p.c. at day 0.5; or (iii) were cultured in vivo to the blastocyst stage and transferred into the uterus of a pseudopregnant female p.c. to day 2.5. Preferably, any of the first two options can be used since they avoid extended culture in vi tro, and preferably, approximately 20 to 30 microinjected ovules should be transferred, to avoid small baits. &TKMPI-0 10 Flt3-L Stimulates the Proliferation of Erioid Cells in the Spleen This example describes the effect of flt3-L on the production of erythroid cells in the spleen of transgenic mice. Transgenic mice were generated in accordance with the procedures of Example 10. The mice were sacrificed and each intact spleen was made into a single cell suspension. The suspended cells were rotated and then resuspended in 10 milliliters of medium containing PBS + 1 percent fetal bovine serum. Here the cell counts were performed using a hemocytometer. Each cell specimen was counted with Trypan Blue dye, to obtain a viable total cell count per millimeter of medium, according to the following formula: (REC + WBC) / milliliter, where RBC is the red blood cell count and WBC means the white blood cell count. Afterwards, each specimen was counted with Turk's tincture to obtain a total white blood cell count per milliliter of medium. The total red blood cell count per millimeter was calculated for each specimen by subtracting the total white blood cell count per millimeter minus the total viable cell count per millimeter. In the following Table III the results are shown.

TABLE III Erythroid Cell Proliferation in the Spleen of Mice Total Total Total Cell Viable Mouse White Cell Red Cell (million (million (million cells / ml) cells / ml) cells / ml) Control 1 29.7 27 2.7 Control 2 31 24.6 6.4 Transgenic 1 44.7 25.6 19.1 Transgenic 2 37.3 28.4 8.9 Of the data in Table III, the white blood cell counts per millimeter were approximately the same as those of the control mice. However, the red blood cell counts of the spleens of the doe transgenic mice were approximately two to three times higher than those observed in the control mice.

The flt3-L stimulates and ee increases in cells of the erythroid lineage, possibly through the stimulation of the erythroid progenitor cells, through the stimulation of the cells that produce erythropoietin, or by blocking a mechanism that inhibits erythropoiesis.

EXAMPLE 11 Flt3-L Stimulates the Proliferation of T Cells and Early B Cells? Spinal cord from 9 week transgenic mice generated in accordance with Example 10 was selected for the presence of several T and B cell phenotype markers, using antibodies that are immunoreactive with those markers. The following markers were investigated: marker B220, which is specific to the B cell lineage; the surface marker IgM (slgM), which is specific to mature B cells; the S7 marker (CD43), which is an early B cell marker; the Cane Cell Antigen 1 marker (SCA-1), which is a marker for activated T cells and B cells; CD4, which is a marker for T helper cells and some rod cells; and the Mac-1 marker, which is specific to macrophages, these were selected using well-known antibodies against these markers. The following Table IV shows the data obtained from the selection of bone marrow.

Two transgenic mice of the same litter were analyzed against a normal mouse from the same litter (control), and an unrelated normal mouse (control).

TABLE IV Effect of overexpression of flt3-L in Transgenic Mice Percentage of Positive Cells Marker Control No Transgé-Transgé-Relacion Control the same sole # 1 sole # 2 swim litter B220 30.64 27.17 45.84 48.78 slgM 3.54 2.41 1.94 1.14 S7 ( CD43) 54.43 45.44 46.11 50.59 SCA-1 10.92 11.74 19.45 27.37 CD4 6.94 8.72 12.21 14.05 Mac-1 36.80 27.15 21.39 18.63 The above data indicate that overexpression of flt3-L in mice leads to an increase in the number of B cells, as indicated by the increase of B220- * cells and SCA-1 cells. B220-4- by FACS indicated an increase in proB (HSA-, S7 ~ * -) cells.The increase in CD4"1- cells indicated an approximately two-fold increase in T cells and rod cells. The decrease in cells bearing the slgM marker indicated that flt3-L does not stimulate the proliferation of mature B cells. These data indicate that flt3-L increases cells with a rod cell, T cell or early B cell phenotype, and does not stimulate the proliferation of mature B cells or macrophages.

EXAMPLE 12 Analysis of the Thymus of Mice Overexpressing Flt, 3-L This example describes the thymus analysis of transgenic mice generated in accordance with the procedure of Example 10. Seie adult mice were sacrificed, each approximately three months of age. The thymus was removed from each mouse and a single cell suspension was made. The FACS analysis showed that a total change in the number of cells did not occur and that the mice showed no change in the proportions of thymocytes maturing using the markers: CD4 vs. CD8; CD3 vs. aßTCR (T cell receptor); and CD3 vs. rdTCR (T cell receptor). However, a change occurred in the proportions of certain cell types within the CD4- and CD8 ~ compartment (that is, the early cells with respect to development, which represents approximately 12 percent to 3 percent of total cells of the thymus). Specifically, CD4 ~ and CD8 ~ cells in the thymus were developed in three stages. Stage 1 represents the cells that had the negative markers ("IL-2R-") of the receptor Pgp-1"f", HSA "1- and IL-2. After stage 1, the thymic cells developed to stage 2, which consisted of cells that had markers Pgp-1"1-, HSA" 1"1- and IL-2R? - ^, and then to stage 3, characterized by cells that had the Pgp-1 markers "'-'''-, HSA" 1"* - and IL-2R- Thymic cells were reduced in stage 2 of the transgenic mice by approximately 50 percent, while the population of cells in the stage 3 was proportionally increased.This data suggests that flt3-L carries the thymic cells from stage 2 to stage 3 of development, indicating that flt3-L is active on early T cells.

Use of Flt3-L in Peripheral Cane Cell Transplant This example describes a method for using flt3-L in peripheric, self-derived (PSC) or peripheral blood progenitor cell (PBPC) cell transplantation. Typically, transplantation of PBPC and PSC is performed in patients whose bone marrow is unsuitable for collection due to, for example, bone marrow abnormality or malignant involvement.

Prior to the cell collection, it may be desirable to mobilize or increase the numbers of PBPC and circulating PSCs. Mobilization can improve the collection of PBPC and PSC, and can be achieved through intravenous administration of flt3-L to patients prior to collection of those cells. Other growth factors can also be administered such as CSF-1, GM-CSF, SF, G-CSF, EPO, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, GM-CSF / IL-3 fusion proteins, LIF, FGF and combinations thereof, in sequence or in concurrent combination with the flt3-L. PBPC and PSC mobilized or non-mobilized are collected, yielding apheresis procedures known in the art. See, for example, Bishop et al., Blood, volume 83, Number 2, pages 610-616 (1994). Briefly, PBPCs and PSCs are collected using conventional devices, for example, a Haemonetics Aspheresis dietary model V50 (Haemonetics, Braintree, MA). Collections of four hours are typically performed, no more than five times a week, until approximately 6.5 x 10e mononuclear cells (MNC) / kilogram of the patient are collected. Aliquots of PBPC and PSC collected by granulocyte-macrophage colony formation unit (CFU-GM) content are assayed by approximately 1: 6 dilution of Hank's balanced salt solution without calcium or magnesium (HBSS) and were placed on a lymphocyte separation medium (Organon Teknika, Durham, North Carolina). After centrifugation, MNC were collected in the interfacial zone, washed and resuspended in HBSS. One milliliter of aliquots containing approximately 300,000 MNC, modified medium 5A of McCoy, 0.3 percent agar, 200 U / milliliter of recombinant human GM-CSF, 200 U / milliliter of recombinant human IL-3, and 200 U / ml were cultured. milliliter of recombinant human G-CSF, at 37 ° C in 5% C0s in fully humidified air for 14 days. Optionally, fusion molecules (PIXY 321) flt3-L or GM-CSF / IL-3 can be added to the cultures. These cultures were stained with Wright's stain, and the CFU-GM colonies were counted using a dissecting microscope (Ward et al, Exp. Hematol., 16: 358 (1988).) Alternatively, CFU colonies can be tested. -GM using the CD34 / CD33 flow cytometry method of Siena et al., Blood, Volume 77, Number 2, pages 400-409 (1991), or any other method known in the art. GM in a controlled-range freezer (eg, Cryo-Med, Mt. Clemens, MI), then stored in the vapor phase of liquid nitrogen.You can use ten percent dimethylsulfoxide as a cryoprotectant. All patient collections were made, all cultures containing CFU-GM were thawed and pooled, The thawed cell collection either intravenously infused back into the patient, or ex vivo expanded before reinfusing. Live of the combined cells can be performed using the flt3-L as a growth factor either alone, sequentially or in concurrent combination with other cytokines listed above. The methods of this ex vivo expansion are well known in the art. The cells, either expanded or not expanded, are infused intravenously back into the patient. To facilitate grafting of the transplanted cells, flt3-L is administered simultaneously with, or subsequent to reinfusion. This administration of flt3-L is done alone, sequentially or in concurrent combination with other cytokines selected from the above list.

EXAMPLE 14 Purification of Progenitor Cells and Hematopoetic Caps Using Flt3-L This example describes a method for purifying progenitor cells and hematopoietic rod cells from a suspension containing a mixture of cells. Cellos were collected from the bone marrow or peripheral blood, using conventional procedures. The cells were suspended in standard medium and then centrifuged to remove the red and neutrophil blood cells. The cells located in the interface between the two phases are separated (also known in the art as the polished cover) and are resuspended. These cells are predominantly mononuclear and represent a substantial portion of the early progenitor and hematopoietic rod cells. Subsequently, the reagent cell suspension was incubated with bioethanol-laden flt3-L for a sufficient time to allow a substantial interaction of flt3: flt3-L. Typically, incubation times of at least one hour are sufficient. After incubation, the cell suspension was passed, under the force of gravity, through a column packed with avidin-coated beads. These columns are well known in the art, see Berenson et al., J. Cell Biochem. , 101 > : 239 (1986). The column was washed with a PBS solution to remove the unbound material. The target cells can be released from the globules and flt3-L, using conventional methods.

LIST OF SEQUENCE (1) GENERAL INFORMATION: (i) APPLICANT: Lyman, Stewart D. Beckmann, M. Patricia (ii) TITLE OF THE INVENTION: Ligands for Receptors flt3 (iii) SEQUENCE NUMBER: 8 (iv) ADDRESS FOR CORRESPONDENCE: (A) RECIPIENT: Stephen L. Malaska, Immunex Corporation (B) STREET: 51 University Street (C) CITY: Seattle (D) STATE: Washington (E) COUNTRY: United States of America (F) ) CP: 98101 (v) COMPUTER READING FORM: (A) TYPE OF DRIVE: Floppy disk (B) COMPUTER: Apple Macintosh (C) OPERATING SYSTEM: Macintosh 7.0.1 (D) SOFTWARE: Microsoft Word, Version # 5.1 (vi) CURRENT REQUEST DATA: (A) APPLICATION NUMBER: -to be assigned- (B) SUBMISSION DATE: March 7, 1994 (C) CLASSIFICATION: (vii) PREVIOUS APPLICATION DATA: (A) APPLICATION NUMBER: 08 / 162,407 (B) SUBMISSION DATE: December 3, 1993 (C) CLASSIFICATION: (vii) PREVIOUS APPLICATION DATA: (A) APPLICATION NUMBER: 08 / 111,728 (B) SUBMISSION DATE: August 25, 1993 (C) CLASSIFICATION: (vii) PREVIOUS APPLICATION DATA: (A) APPLICATION NUMBER: 08 / 106,463 (B) SUBMISSION DATE: August 12, 1993 (C) CLASSIFICATION: (vii) PREVIOUS APPLICATION DATA: (A) APPLICATION NUMBER: 08 / 068,394 (B) SUBMISSION DATE: May 24, 1993 (C) CLASSIFICATION: (viii) ATTORNEY / AGENT INFORMATION: (A) NAME: Malaska , Stephen L. (B) REGISTRATION NUMBER: 32,655 (C) REFERENCE / INTERNAL ATTORNEY NUMBER: 2813-D (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: (206) 587-0430 (B) TELEFAX: (206) 233-0644 (C) TELEX: 756822 (2) INFORMATION FOR THE IDENTIFICATION NUMBER OF SEQUENCE: 1: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 879 base pairs (B) TYPE: nucleic acid (C) CORDS: single (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA to mRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) CHARACTERISTICS: (A) NAME / KEY: miscellaneous characteristic (B) LOCATION: 1.25 (ix) CHARACTERISTICS: (A) NAME / KEY: miscellaneous characteristic (B) LOCATION: 855..879 (i) CHARACTERISTICS: (A) NAME / KEY: CDS (B) LOCATION: 57..752 (xi) SEQUENCE DESCRIPTION: IDENTIFICATION NUMBER OF SEQUENCE:!: GTCGACTGGA ACGAGACGAC CTGCTCTGTC ACAGGCATGA GGGGTCCCCG GCAGAG 56 ATG ACA GTG CTG GCG CCA GCC TGG AGC CCA AAT TCC TCC CTG TTG CTG 104 Met Thr Val Leu Ala Pro Wing Trp Ser Pro Asn Ser Ser Leu Leu Leu 1 5 10 15 CTG TTG CTG CTG CTG AGT CCT TGC CTG CGG GGG ACA CCT GAC TGT TAC 152 Leu Leu Leu Leu Leu Ser Pro Cys Leu Arg Gly Thr Pro Asp Cys Tyr 20 25 30 TTC AGC CAC AGT CCC ATC TCC TCC AAC TTC AAA GTG AAG TTT AGA GAG 200 Phe Ser His Ser Pro lie Be As As Phe Lys Val Lys Phe Arg Glu 35 40 45 TTG ACT GAC CAC CTG CTT AAA GAT TAC CCA GTC ACT GTG GCC GTC AAT 248 Leu Thr Asp His Leu Leu Lys Asp Tyr Pro Val Thr Val Wing Val Asn 50 55 60 CTT CAG GAC GAG AAG CAC TGC AAG GCC TTG TGG AGC CTC TTC CTA GCC 296 Leu Gln Asp Glu Lys His Cys Lys Ala Leu Trp Ser Leu Phe Leu Wing 65 70 75 80 CAG CGC TGG ATA GAG CAA CTG AAG ACT GTG GCA GGG TCT AAG ATG CAA 344 Gln Arg Trp He Glu Gln Leu Lys Thr Val Wing Gly Ser Lys Met Gln 85 90 95 ACG CTT GAG GAC GAC AAC ACC GAG ATA CAT TTT GTC ACC TCA TGT 392 Thr Leu Leu Glu Asp Val Asn Thr Glu He His Phe Val Thr Ser Cys 100 105 110 ACC TTC CAG CCC CTA CCA GAA TGT CTG CGA TTC GTC CAG ACC AAC ATC 440 Thr Phe Gln Pro Leu Pro Glu Cys Leu Arg Phe Val Gln Thr Asn He 115 120 125 TCC CAC CTG AAG GAC ACC TGC ACA CAG CTG CTT GCT CTG AAG CCC 488 Ser His Leu Leu Lys Asp Thr Cys Thr Gln Leu Leu Wing Leu Lys Pro 130 135 140 TGT ATC GGG AAG GCC TGC CAG AAT TTC TCT CGG TGC CTG GAG GTG CAG 536 Cys He Gly Lys Ala Cys Gln Asn Phe Ser Arg Cys Leu Glu Val Gln 145 150 155 160 TGC CAG CCG GAC TCC TCC ACC CTG CTG CCC CCA AGG AGT CCC ATA GCC 584 Cys Gln Pro Asp Ser Ser Thr Leu Leu Pro Pro Arg Ser Pro He Wing 165 170 175 CTA GAA GCC ACG GAG CTC CCA GAG CCT CGG CCC AGG CAG CTG TTG CTC 632 Leu Glu Ala Thr Glu Leu Pro Glu Pro Arg Pro Arg Gln Leu Leu Leu 180 185 190 CTG CTG CTG CTG CTG CTG CTC ACA CTG GTG CTG CTG GCA GCC GCC TGG 680 Leu Leu Leu Leu Leu Pro Leu Thu Leu Val Leu Leu Wing Wing Trp 195 200 205 GGC CTT CGC TGG CAA AGG GCA AGA AGG AGG GGG GAG CTC CAC CCT GGG 728 Gly Leu Arg Trp Gln Arg Wing Arg Arg Arg Gly Glu Leu His Pro Gly 210 215 220 GTG CCC CTC CCC TCC CAT CCC TAGGATTCGA GCCTTGTGCA TCGTTGACTC 779 Val Pro Leu Pro Ser His Pro 225 230 AGCCAGGGTC TTATCTCGGT TACACCTGTA ATCTCAGCCC TTGGGAGCCC AGAGCAGGAT 839 TGCTGAATGG TCTGGAGCAG GTCGTCTCGT TCCAGTCGAC 879 (2) INFORMATION FOR THE SEQUENCE IDENTIFICATION NUMBER: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 231 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) SEQUENCE DESCRIPTION: IDENTIFICATION NUMBER OF SEQUENCE: 2: Met Thr Val Leu Ala Pro Ala Trp Ser Pro Asn Be Ser Leu Leu Leu 1 5 10 15 Leu Leu Leu Leu Be Pro Cys Leu Arg Gly Thr Pro Asp Cys Tyr 20 25 30 Phe Ser His Ser Pro Be Ser As As Phe Lys Val Lys Phe Arg Glu 35 40 45 Leu Thr Asp His Leu Leu Lys Asp Tyr Pro Val Thr Val Wing Val Asn 50 55 60 Leu Gln Asp Glu Lys His Cys Lys Wing Leu Trp Ser Leu Phe Leu Wing 65 70 75 80 Gln Arg Trp He Glu Gln Leu Lys Thr Val Wing Gly Ser Lys Met Gln 85 90 95 Thr Leu Leu Glu Asp Val Asn Thr Glu He His Phe Val Thr Ser Cys 100 105 110 Thr Phe Gln Pro Leu Pro Glu Cys Leu Arg Phe Val Gln Thr Asn He 115 120 125 Ser His Leu Leu Lys Asp Thr Cys Thr Gln Leu Leu Wing Leu Lys Pro 130 135 140 Cys He Gly Lys Wing Cys Gln Asn Phe Ser Arg Cys Leu Glu Val Gln 145 150 155 160 Cys Gln Pro Asp Be Ser Thr Leu Leu Pro Pro Arg Ser Pro He Wing 165 170 175 Leu Glu Wing Thr Glu Leu Pro Glu Pro Arg Pro Arg Gln Leu Leu Leu 180 185 190 Leu Leu Leu Leu Leu Pro Leu Leu Val Leu Leu Ala Ala Ala Trp 195 200 205 Gly Leu Arg Trp Gln Arg Ala Arg Arg Arg Gly Glu Leu His Pro Gly 210 215 220 Val Pro Leu Pro Ser His Pro 225 230 (2) INFORMATION FOR THE SEQUENCE IDENTIFICATION NUMBER: 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) CORDS: simple (D) TOPOLOGY: linear (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQUENCE IDENTIFICATION NUMBER: 3: TCGACTGGAA CGAGACGACC TGCT (2) INFORMATION FOR THE SEQUENCE IDENTIFICATION NUMBER: 4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) CORDS: simple (D) TOPOLOGY: linear (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: IDENTIFICATION NUMBER OF SEQUENCE: 4: AGCAGGTCGT CTCGTTCCAG (2) INFORMATION FOR THE IDENTIFICATION NUMBER OF SEQUENCE: 5: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 988 base pairs (B) TYPE: nucleic acid (C) CORDS: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA to mRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) 'FEATURE: (A) NAME / KEY: CDS (B) LOCATION: 30..734 (xi) SEQUENCE DESCRIPTION: IDENTIFICATION NUMBER SEQUENCE: 5: CGGCCGGAAT TCCGGGGCCC CCGGCCGAA ATG ACA GTG CTG GCG CCA GCC TGG 53 Met Thr Val Leu Ala Pro Ala Trp 1 5 AGC CCA ACA ACC TAT CTC CTC CTG CTG CTG CTG CTG AGC TCG GGA CTC 101 Being Pro Thr Thr Tyr Leu Leu Leu Leu Leu Leu Being Ser Gly Leu 10 15 20 AGT GGG ACC CAG GAC TGC TCC TTC CAA CAC AGC CCC ATC TCC TCC GCC 149 Ser Gly Thr Gln Asp Cys Ser Phe Gln His Ser Pro He Ser Ser Asp 25 30 35 40 TTC GCT GTC AAA ATC CGT GAG CTG TCT GAC TAC CTG CTT CAA GAT TAC 197 Phe Wing Val Lys He Arg Glu Leu Ser Asp Tyr Leu Leu Gln Asp Tyr 45 50 55 CCA GTC ACC GTG GCC TCC AAC CTG CAG GAC GAG GAG CTC TGC GGG GGC 245 Pro Val Thr Val Wing As Asn Leu Gln Asp Glu Glu Leu Cys Gly Gly 60 65 70 CTC TGG CGG CTG GTC CTG GCA CAG CGC TGG ATG GAG CGG CTC AAG ACT 293 Leu Trp Arg Leu Val Leu Ala Gln Arg Trp Met Glu Arg Leu Lys Thr 75 80 85 GTC GCT GGG TCC AAG ATG CAA GGC TTG CTG GAG CGC GTG AAC ACG GAG 341 Val Wing Gly Ser Lys Met Gln Gly Leu Leu Glu Arg Val Asn Thr Glu 90 95 100 ATA CAC TTT GTC ACC AAA TGT GCC TTT CAG CCC CCC CCC AGC TGT CTT 389 He His Phe Val Thr Lys Cys Ala Phe Gln Pro Pro Pro Ser Cys Leu 105 110 115 120 CGC TTC GTC CAG ACC AAC ATC TCC CGC CTC CTG CAG GAG ACC TCC GAG 43 Arg Phe Val Gln Thr Asn He Be Arg Leu Leu Gln Glu Thr Ser Glu 125 130 135 CAG CTG GTG GCG CTG AAG CCC TGG ATC ACT CGC CAG AAC TTC TCC CGG 48 Gln Leu Val Wing Leu Lys Pro Trp He Thr Arg Gln Asn Phe Ser Arg 140 1 45 150 TGC CTG GAG CTG CAG TGT CAG CCC GAC TCC TCA ACC CTG CCA CCC CCA 53 Cys Leu Glu Leu Gln Cys Gln Pro Asp Ser Ser Thr Leu Pro Pro Pro 155 160 165 TGG AGT CCC CGG CCC CTG GAG GCC ACA GCC CCG ACA GCC CCG CAG CCC 58 Trp Ser Pro Arg Pro Leu Glu Wing Thr Wing Pro Thr Wing Pro Gln Pro 170 175 180 CCT CTG CTC CT CT CTG CTG CTG CCC GTG GGC CTG CTG CTG CTG GCC 62 Pro Leu Leu Leu Leu Leu Leu Leu Pro Val Gly Leu Leu Leu Leu Leu Wing 185 190 195 200 GCT GCC TGG TGC CTG CAC TGG CAG AGG ACG CGG CGG AGG ACA CCC CGC 67 Wing Wing Trp Cys Leu His Trp Gln Arg Thr Arg Arg Arg Thr Pro Arg 205 210 215 CCT GGG GAG CAG GTG CCC CCC GTC CCC AGT CCC CAG GAC CTG CTG CTT 72 Pro Gly Glu Gln Val Pro Pro Val Pro Ser Pro Gln Asp Leu Leu Leu 220 225 230 GTG GAG CAC TGACCTGGCC AAGGCCTCAT CCTGCGGAGC CTTAAACAAC 77 Val Glu His 235 GCAGTGAGAC AGACATCTAT CATCCCATTT TACAGGGGAG GATACTGAGG CACACAGAGG 83 GGAGTCACCA GCCAGAGGAT GTATAGCCTG GACACAGAGG AAGTTGGCTA GAGGCCGGTC 89 CCTTCCTTGG GCCCCTCTCA TTCCCTCCCC AGAATGGAGG CAACGCCAGA ATCCAGCACC 95 GGCCCCATTT ACCCAACTCT GAACAAAGCC CCCG 98 (2) INFORMATION FOR THE SEQUENCE IDENTIFICATION NUMBER: 6: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 235 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) SEQUENCE DESCRIPTION: IDENTIFICATION NUMBER SEQUENCE: 6: Met Thr Val Leu Ala Pro Ala Trp Ser Pro Thr Thr Tyr Leu Leu Leu 1 5 10 15 Leu Leu Leu Le Ser Ser Gly Leu Ser Gly Thr Gln Asp Cys Ser Phe 20 25 30 Gln His Ser Pro He Ser Be Asp Phe Wing Val Lys He Arg Glu Leu 35 40 45 Be Asp Tyr Leu Leu Gln Asp Tyr Pro Val Thr Val Wing As Asn Leu 50 55 60 Gln Asp Glu Glu Leu Cys Gly Gly Leu Trp Arg Leu Val Leu Wing Gln 65 70 75 80 Arg Trp Met Glu Arg Leu Lys Thr Val Wing Gly Ser Lys Met Gln Gly 85 90 95 Leu Leu Glu Arg Val Asn Thr Glu He His Phe Val Thr Lys Cys Ala 100 105 110 Phe Gln Pro Pro Pro Ser Cys Leu Arg Phe Val Gln Thr Asn He Ser 115 120 125 Arg Leu Leu Gln Glu Thr Ser Glu Gln Leu Val Wing Leu Lys Pro Trp 130 135 140 He Thr Arg Gln Asn Phe Ser Arg Cys Leu Glu Leu Gln Cys Gln Pro 145 150 155 160 Asp Ser Be Thr Leu Pro Pro Pro Be Pro Pro Arg P Lou Lou Glu Wing 165 170 175 Thr Wing Pro Thr Wing Pro Gln Pro Pro Leu Leu Leu Leu Leu Leu 180 185 190 Pro Val Gly Leu Leu Leu Wing Ala Wing Trp Cys Leu His Trp Gln 195 200 205 Arg Thr Arg Arg Arg Thr Pro Arg Pro Gly Glu Gln Val Pro Pro Val 210 215 220 Pro Ser Pro Gln Asp Leu Leu Leu Val Glu His 225 230 235 (2) INFORMATION FOR THE SEQUENCE IDENTIFICATION NUMBER: 7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 71 base pairs (B) TYPE: nucleic acid (C) CORDS: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA to mRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQUENCE IDENTIFICATION NUMBER: 7: AATTGGTACC TTTGGATAAA AGAGACTACA AGGACGACGA TGACAAGACA CCTGACTGTT 60 71 ACTTCAGCCA C (2) INFORMATION FOR THE IDENTIFICATION NUMBER OF SEQUENCE: 8: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 37 base pairs (B) TYPE: nucleic acid (C) CORDS: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA to mRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQUENCE IDENTIFICATION NUMBER: 8: ATATGGATCC CTACTGCCTG GGCCGAGGCT CTGGGAG NOVELTY THE INVENTION Having defined the above invention, it considers it a novelty and, therefore, the content of the following is claimed as property:

Claims

CLAIMS 1. An isolated flt3 ligand polypeptide (flt3-L).
2. A polypeptide, according to claim 1, characterized in that it is a murine flt3-L.
3. A polypeptide, according to claim 1, characterized in that it is a human flt3-L.
4. A polypeptide, according to claim 3, characterized in that it includes amino acids 1-235 of the SEQUENCE IDENTIFICATION NUMBER: 6.
5. A polypeptide, according to claim 1, characterized in that it is a soluble flt3-L.
6. A polypeptide, according to claim 5, characterized in that it includes amino acids 28-160 or 28-182 of the SEQUENCE IDENTIFICATION NUMBER: 6.
7. A polypeptide, in accordance with claim 3. , characterized in that it is encoded by the cDNA insert of the vector sfHAVE0410 in DH10B cells of E. coli having the accession number ATCC 69382.
8. An isolated DNA sequence encoding a flt3-L polypeptide.
9. An isolated DNA sequence, according to claim 8, which encodes a murine flt3-L polypeptide.
10. An isolated DNA sequence, according to claim 8, which encodes a human flt3-L polypeptide.
11. An isolated DNA sequence, according to claim 8, which encodes amino acid sequence 28-160 or 28-182 of the SEQUENCE IDENTIFICATION NUMBER: 6.
12. A DNA, in accordance with the claimed in claim 8, selected from the group consisting of: (a) cDNA derived from the coding region of a flt3-L gene; (b) cDNA sequences selected from the group consisting of the SEQUENCE IDENTIFICATION NUMBER:! and the SEQUENCE IDENTIFICATION NUMBER: 5; (c) DNA sequences that hybridize under conditions of moderate stringency to the cDNA of (a) or (b), and whose DNA sequences encode flt3-L; (d) DNA sequences which, due to the degeneracy of the genetic code, encode flt3-L polypeptides having the amino acid sequence of the polypeptides encoded by the DNA sequences of (a), (b) or (c) ).
13. An expression vector that includes a DNA sequence, in accordance with claim 8.
14. An expression vector that includes a DNA sequence, according to claim as claimed in claim 9.
15. A vector of expression that includes a DNA sequence, according to claim 10.
16. An expression vector that includes a DNA sequence, according to claim as claimed in claim 11.
17. An expression vector that includes a DNA sequence, according to claim 12.
18. A host cell transfected or transformed with the expression vector, according to claim 13.
19. A host cell transfected or transformed with the vector of expression, in accordance with that claimed in claim 14.
20. A homing cell tranefected or transformed with the expression vector, in accordance with the Claimed in claim 15.
21. A host cell transfected or transformed with the expression vector, according to claim claimed in claim 16.
22. A host cell transfected or transformed with the expression vector, in accordance with that claimed in claim 17.
23. A process for producing a flt3-L polypeptide, which includes culturing a host cell, according to claim 18, under conditions that promote expression, and recovering the polypeptide from the host medium. culture.
24. A process for producing a flt.3-L polypeptide, which includes culturing a host cell, according to claim 19, under conditions that promote expression, and recovering the polypeptide from the culture medium.
25. A process for producing a flt3-L polypeptide, which includes culturing a host cell, according to claim 20, under conditions that promote expulsion, and recovering the polypeptide from the culture medium.
26. A process for producing a flt3-L polypeptide, which includes culturing a host cell, according to claim 21, under conditions that promote expression, and recovering the polypeptide from the culture medium.
27. A process for producing a flt3-L polypeptide, which includes culturing a host cell, according to claim 22, under conditions that promote expression, and recovering the polypeptide from the culture medium.
28. An antibody that is immunoreactive with a flt3-L polypeptide.
29. An antibody, according to claim 28, characterized in that it is a monoclonal antibody.
30. A pharmaceutical composition that includes an effective amount of a flt3-L polypeptide, in accordance with claim 1, and a pharmaceutically acceptable carrier, excipient or diluent.
31. A pharmaceutical composition that includes an effective amount of a flt3-L polypeptide, in accordance with claim 3, and a pharmaceutically acceptable carrier, excipient or diluent.
32. A pharmaceutical composition that includes an effective amount of a flt3-L polypeptide, in accordance with claim 5, and a pharmaceutically acceptable carrier, excipient or diluent.
33. A method for conducting the transplantation derived from itself in a patient receiving cytoreductive therapy, which includes: (a) collecting progenitor cells or hematopoietic rod cells from a patient prior to cytoreductive therapy; and (b) administer the cells collected to the patient after cytoreductive therapy; wherein the method also includes at least one of the following steps: (i) administering an effective amount of flt3-L to the patient to increase the number of circulating progenitor cells or cane before collection cells; (ii) expanding the progenitor cells or ex vivo rod cells by contacting them with an effective amount of flt3-L; and (iii) administering an effective amount of flt3-L to the patient to facilitate grafting of the progenitor or cane cells transplanted into the patient.
34. A method, according to claim 33, wherein flt3-L is used in combination with a cytokine selected from the group consisting of CSF-1, GM-CSF, SF, G-CSF, EPO, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, fusion proteins GM-CSF / IL-3, LIF, FGF and sequential or concurrent combinations thereof.
35. A method, according to claim 34, wherein flt3-L is used in combination with a cytokine selected from the group consisting of GM-CSF, SF, G-CSF, EPO, IL- 3 and GM-CSF / IL-3 fusion proteins.
36. A method for transfecting an exogenous gene into an early hematopoietic cell, which includes the steps of: (a) culturing the early hematopoietic cells in medium that includes an effective amount of a flt3-L polypeptide; and (b) transfecting the cultivated cells of the patch (a) with the gene.
37. A method for transferring an exogenous gene to a mammal that includes the steps of: (a) culturing the early hematopoietic cells in medium that includes an effective amount of a flt3-L polypeptide; and (b) transfecting the cultured cells of step (a) with the gene; and (c) administering the transfected cells to the mammal. 39. A method for stimulating T-cell proliferation in a mammal, including administering to the mammal an effective amount of a flt3-L polypeptide, in accordance with claim 1. 40. A method for stimulating proliferation of cells of the erythroid lineage in the spleen of a mammal, including administration to the mammal of an effective amount of a flt3-L polypeptide, in accordance with claim 1. 41. A method, in accordance with claimed in claim 40, characterized in that it also includes the administration of an effective amount of EPO. 42. A method for treating a patient having symptoms of myelodysplastic syndrome, including administering to the patient an effective amount of a flt3-L polypeptide, in accordance with claim 1 and, optionally, an effective amount. of one or more growth factors selected from the group consisting of CSF-1, GM-CSF, SF, G-CSF, EPO, IL-1, IL-2, IL-3, IL-4, IL-5 , IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, fusion proteins GM-CSF / IL- 3, LIF, FGF. 43. A method for treating a patient who has symptoms of anemia, including - administering to the patient an effective amount of a flt3-L polypeptide, in accordance with claim 1 and, optionally, an effective amount of one or more growth factors selected from the group consisting of CSF-1, GM-CSF, SF, G-CSF, EPO, IL-1, IL-2, IL-3, I -4, IL-5, IL -6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, GM-CSF / IL-3 fusion proteins, LIF, FGF. 44. A method for treating a patient having symptoms of acquired immunodeficiency syndrome, including administering to the patient an effective amount of a flt3-L polypeptide, as claimed in claim 1 and, optionally, an amount effective of one or more growth factors selected from the group consisting of CSF-1, GM-CSF, SF, G-CSF, EPO, IL-1, IL-2, IL-3, IL-4, IL- 5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, GM-CSF / IL fusion proteins -3, LIF, FGF. 45. A method, according to claim 44, wherein the patient is receiving AZT therapy. 46. A transgenic non-human mammal, all of which cells of origin and somatic cells contain a DNA sequence, in accordance with that claimed in claim 8, introduced into that mammal, or an ancestor of that mammal, in an embryonic state. 47. A method for separating cells having the flt3 receptor on the surface thereof from a mixture of cells in suspension, which includes contacting the cells in the mixture with a contact surface having a protein that binds to the flt3 on it, and separate the contact surface and the suspension. 48. A method, according to claim 48, wherein the protein that binds to flt3 is flt3-L. Sa testinaónio lo cuai. sign to previous SR this City © Mé ico. » r. > a, the 23? i &s-d * l month? @ m and of 199. POE IMHÜMEX CORFOfi? TIOK APOOBRADO Ing. j & Jßr Sa elbow