MXPA98002464A - Dendrit cellular stimulating factor - Google Patents

Abstract

The fit3 ligand can be used to generate large numbers of dendritic cells from hematopoietic progenitor and stem cells. The fit3 ligand can be used to increase the immune response in vivo, and expand the dendritic cells ex vivo. Said dendritic cells can then be used to present tumor antigens, viral or other antigens to natural T cells, they can be useful as auxiliary vacu

Description

FACTOR ESTI DENPRITICA CELL MULTA FIELD OF THE INVENTION The present invention relates to a dendritic cell stimulating factor, to methods for improving the immune response in vivo, to methods for expanding ex vivo dendritic cells, and to purified dendritic cell preparations, and to dendritic cell populations useful in the manipulation of mediated responses mediated by T cell and mediated by B cell.

BACKGROUND OF THE INVENTION The goal of vaccination is to provide effective immunity by establishing adequate levels of antibody and an injected population of cells that can rapidly expand under renewed contact with the antigen. The first contact with the antigen during vaccination should not be harmful to the recipient and usually consists of a pathogenically deficient antigen. A frequent difficulty with active immunization protocols is that the vaccine antigen does not possess sufficient immunogenicity to promote a strong immune response, and, therefore, a sufficient level of protection against an attack by the antigen itself. . In addition, certain antigens can produce only a weak response mediated by cell. antibody. For many antigens, both a strong humoral response and a strong cell-mediated response are desirable. For decades, researchers have experimented with various compounds to increase the immunogenicity of vaccines. The immunopotentiators, also known as adjuvants, of vaccines are compositions of matter that facilitate a strong immune response to a vaccine. In addition, the relatively weak immunogenicity of certain recombinant antigens has required that auxiliaries be more potent. The vaccine auxiliaries have different modes of action, affecting the immune response both quantitatively and qualitatively. These modes of action may be mobilizing the T cells, acting as stores and altering the circulation of lymphocytes, so that these cells remain anchored in draining lymph nodes. They also serve to focus the antigen on the immunization site, thus allowing T cells and specific B cells in the antigen to interact more efficiently with the antigen presenting cells. They can also stimulate the proliferation and differentiation of T cells and have effects on B cells, such as improving the production of different isotypes Ig. In addition, adjuvants can stimulate and affect the behavior of antigen presenting cells, particularly macrophages, making them more effective in presenting the antigen to T cells and B cells. Dendritic cells are a population of rare cells and heterogeneous with distinctive morphology and a wide tissue distribution. A discussion of the dendritic cell system and its role in immunogenicity is provided by Steinman, R. M. Annu. Rev. Immunol. , 9_: 271-296 (1991), incorporated herein by reference. Dendritic cells exhibit an unusual cell surface and can be characterized by the presence of cell surface markers CD1 a +, CD4 +, CD14", CD86 +, CD 1 1 c +, DEC-205 +, CD14 + or HLA-DR + The dendritic cells have a high capacity to sensitize T cells restricted by M HC and provide an effective path to present antigens to T cells in situ, both own antigens during the development of the T cell and foreign antigens during immunity. , there is a great interest in using ex vivo dendritic cells as adjuvants of tumor vaccine or infectious disease, see, for example, Romani et al., J. Exp. Med., 180: 83 (1994) .The use of dendritic cells. as immunostimulatory agents has been limited due to the low frequency of dendritic cells in the peripheral blood, the limited access capacity to lymphoid organs and the terminal state of differentiation of the cells. The dendritic cells originate from progenitors of CD34 + bone marrow, and the proliferation and maturation of dendritic cells can be improved through the cytokines GM-CSF (sargramostim, Leukine®, I mmunex Corporation, Seattle, Washington), TNF- a, a c-team ligand (also known as stem cell factor (SCF), or cell growth factor barley (MGF)) and interleukin-4. Therefore, an agent that stimulates the generation of large numbers of functionally mature dendritic cells, in vivo or in vitro, could be of great importance.

COMPENDIUM OF THE INVENTION Ligand flt3 ("ligand" flt3 or "flt3-L") is known to affect hematopoietic and progenitor stem cells. Surprisingly it was found that the ligand flt3 can also potentially stimulate the generation of downstream or intermediate cells, such as myeloid precursor cells, monocytic cells, macrophages, B cells, and dendritic cells of progenitor cells and the stem of CD34 + bone marrow. The present invention relates to a method for mobilizing dendritic cells in vivo, expanding dendritic cells ex vivo and to purified preparations of dendritic cells. The preparation of dendritic cells according to the invention could potentially find use as vaccine adjuvants. Also included within the embodiments of the invention is a method for preparing specific T cells in the antigen using dendritic cells mobilized with the ligand flt3. The invention provides the use of an effective amount of the ligand flt3 to increase or mobilize the numbers of intermediate cells in vivo, for example, in the peripheral blood, tissues or organs of the patient. Since the invention relates to the generation of large numbers of said downstream and intermediate cells (e.g., myeloid cells, monocytic cells and macrophages) of CD34 + cells using the ligand flt3, the focus is particularly on the dendritic cells. By increasing the amount of dendritic cells in a patient, said cells by themselves can be used to present the antigen to the T cells. For example, the antigen can be one that already exists in the patient, such as a tumor antigen, or a bacterial or viral antigen. Therefore, ligand f 113 can be used to increase the numbers of dendritic cells in vivo to enhance a patient's immune response against existing antigens. Alternatively, ligand flt3 can be administered before, concurrent with or subsequent to the administration of an antigen to a patient for immunization purposes. Thus, as a vaccine adjuvant, the ligand flt3 can generate large amounts of dendritic cells and other intermediate cells, in vivo, to more effectively present the antigen. The total response is a stronger and improved immune response and a more effective immunization for the antigen. The invention also provides a method for generating large amounts of ex vivo dendritic cells. After collection of CD34 + hematopoietic stem and progenitor cells from the patient, the ligand flt3 can be used to expand these cells in vitro (also known as ex vivo expansion) and to activate said CD34 + cells to differentiate dendritic cells from the lymphoid or myeloid line. The resulting collection of dendritic cells can be administered to a patient to provide a stronger and improved immune response to an antigen. Alternatively, the resulting dendritic cells find use as a vaccine adjuvant and can be administered prior to, concurrent with or subsequent to the administration of the antigen. The invention also provides a method for generating large quantities of dendritic cells that present the antigen, ex vivo. After the collection of hematopoietic progenitor and CD34 + stem cells from the patient, the ligand flt3 can be used to expand said cells in vitro and to activate said CD34 + cells to differentiate to dendritic cells. The resulting collection of dendritic cells is then exposed to an antigen and allowed to process and present the antigen in vitro (this procedure is sometimes referred to in the art as "antigen pulsation"). An alternative method for preparing dendritic cells that present antigen is to transfect the dendritic cells with a gene encoding a specific polypeptide in the antigen. Once the dendritic cells express the antigen, the dendritic cells that present the antigen can be administered to a patient. The invention also provides the ex vivo preparation of specific T cells in the antigen. Following the procedures described above to prepare large numbers of dendritic cells that present antigen, ex vivo, the dendritic cells presenting the antigen can be used to generate antigen-specific T cells from natural T cells that have been collected from a patient. After the antigen has been properly presented to the generated T cells, the T cells specific in the antigen can be administered to the patient. The invention also provides a method for increasing an immune response in a patient having an infectious disease, wherein the method comprises the step of administering an amount of ligand flt3 sufficient to increase the number of dendritic cells in the patient. The invention also provides a method for increasing an immune response in a patient having a cancerous or neoplastic disease, wherein the method comprises the step of administering a sufficient amount of the ligand flt3 to increase the number of dendritic cells in the patient. Said method provides means to improve the specific immune response in the patient's tumor. A method for improving a patient's autoimmune tolerance, wherein the method comprises the step of administering a sufficient amount of the ligand flt3 to increase the number of dendritic cells of the patient. In addition, methods are included to promote the survival of grafts and transplanted organs and tissues. The methods of the invention also include the use of a effective amount of a cytokine in sequential or concurrent combination with the ligand flt3. Said cytokines include, but are not limited to, interleukin ("lys") I L-3 and 11-4, a colony stimulation factor ("CSF") selected from the group consisting of the colony stimulating factor of the granulocyte macrophage. ("GM-CSF") or GM-CSF / I L3 fusions, or other cytokines such as TN Fa or a c-team ligand. The invention further includes a dendritic cell expansion medium comprising cell growth media, autologous serum, and flt3 ligand alone or in combination with a cytokine of the group listed above.

DETAILED DESCRIPTION OF THE INVENTION The invention is directed to the use of ligand flt3 to generate large numbers of intermediate cell types of progenitor cells and hematopoietic stem cells CD34 +. Such intermediate cell types include myeloid cells, monocytic cells, macrophages, B cells and dendritic cells. The large numbers of these intermediate cell types are not found naturally in vivo and can be generated by administering the ligand flt3. Said improvement in the total cell number can increase the immune response to an antigen in the host. Another embodiment of the invention is the isolation and use of said intermediate cell types as antigen presenting cells or their use as vaccine auxiliaries. The invention, since particularly focused on the modality with respect to dendritic cells, is also applicable to myeloid, monocytic and macrophage cell types. As used herein, the term "ligand flt3" refers to a genus of polypeptides that are described in the U.A. No. 5, 554,512, EP 0627487 A2 and in WO 94/28391, all incorporated herein by reference. A human flt3 ligand cDNA was deposited at American Type Culture Collection, Rockville, Maryland, USA (ATCC) on August 6, 1993, and assigned accession number ATCC 69382. The deposit was made under the terms of the Treaty of Budapest The ligand flt3 can be made according to the methods described in the documents cited above.

The term "I L-3" refers to a genus of interleukin-3 polypeptide, as described in the patent of E. U.A. No. 5, 108, 910, incorporated herein by reference. Such polypeptides include analogs having amino acid sequences that are substantially similar to the natural human interleukin-3 amino acid sequences described, for example, in EP Nos. 275,598 and 282, 185, each is incorporated herein by reference. The term "I L-3" also includes analogs and alleles of I L-3 molecules that exhibit at least some biological activity in common with natural human I L-3. Illustrative analogs of I L-3 are described in EP publication No. 282, 185. Other forms of I L-3 include I L-3 [Pro8Asp1 sAsp70] human, I L-3 [Ser8 Asp15Asp70] human and I L -3 [Ser8] human. A DNA sequence that encodes the human IL-3 protein suitable for use in the invention is publicly available from the American Type Culture Collection (ATCC) under accession number ATCC 67747. The nomenclature used herein with respect to amino acid sequences in brackets designates which amino acids differ from the natural human form. For example, human IL-3 [Ser8Asp15Asp70] refers to a human I L-3 protein in which amino acid 8 has been changed to a serine residue. Amino acid 15 has been changed to a residue of aspartic acid and amino acid 70 has been changed to a residue of aspartic acid. The term "I L-4" refers to a polypeptide as described by Mosley et al. , Cell 59: 335 (1989), Idzerda et al. , J. Exp. Med. 171: 861 (1990) and Galizzi et al. , Intl. Immunol. 2: 669 (1990, each of which is incorporated herein by reference.) I L-4 polypeptide includes analogs having an amino acid sequence that is substantially similar to the natural human IL-4 amino acid sequences described by Mosley et al. al., Idzerda et al., and Galizzi et al., and which are biologically active since they are capable of binding to an I L-4 receptor, transducing a biological signal initiated by binding the I-L-4 receptor, or by reacting cross-linked with anti-I L-4 antibodies The term "I L-4" also includes analogs of natural human L-4 molecules sufficient to retain the biological activity of natural human L-4. herein, "GM-CSF" refers to a genus of proteins as described in the patents of E.U.A.

Nos. 5,108,910 and 5,229,496, each of which is incorporated herein by reference. Such proteins include analogs having an amino acid sequence that is substantially similar to the natural human GM-CSF amino acid sequences (eg, as is publicly available ATCC 53157 or ATCC 39900), and which are biologically active since they are capable of binding to a GM-CSF receptor, transducing a biological signal initiated by binding the GM-CSF receptor, or cross-reacting with anti-GM-CSF antibodies. Amino acid sequences are described, for example, by Anderson et al., Proc. Nati Acad. Sci., USA 82_: 6250 (1985). The commercially available GM-CSF (sargramostim, Leukine®) is obtained from Immunex, Corp., Seattle, WA). The term "GM-CSF" also includes analogs of the natural human GM-CSF molecules described in the U.S. Patents. Nos. 5,108,910 and 5,229,496 sufficient to retain the biological activity of natural human GM-CSF. Illustrative analogs of GM-CSF include, for example, those described in EP Publication No. 212914 and WO 89/3881, each of which is incorporated herein by reference. Other GM-CSF analogs can also be used to construct fusion proteins with IL-3. A DNA sequence encoding a particularly preferred GM-CSF protein having potential glycosylation sites removed is publicly available from ATCC under the designation numbers ATCC 67231. The term "GM-CSF / IL-3 fusion protein" means a fusion of C-terminal to N-terminal of GM-CSF and IL-3. The fusion proteins are known and described in the patents of E.U.A. Nos. 5,199,942, 5,108,910 and 5,073,627, each of which is incorporated herein by reference. A preferred fusion protein is PIXY321, as described in the patent of E.U.A. No. 5,199,942. The term "c-kit ligand" also known as the Cell Growth Factor (FGM), or Stem Cell Factor (SCF), refers to a polypeptide described in EP 423,980, which is incorporated herein by reference, and which claims priority of the US patent application Series No. 589,701, filed on October 1, 1990. Said c-kit ligand polypeptide includes analogs having an amino acid sequence that is substantially similar to the ligand amino acid sequences of natural human c-kit., described in EP 423,980, and which are biologically active as they are capable of binding to a c-kit receptor, transducing a biological signal initiated by joining the c-kit receptor, or cross-reacting with anti-cDNA ligand antibodies. team-c. The term "c-kit ligand" also includes analogs of natural human c-team ligand molecules sufficient to retain the biological activity of the natural human c-team ligand. The term "auxiliary" refers to a substance that enhances, enhances or potentiates the immune response of the host to a vaccine antigen. The procedure for the "ex vivo expansion" of cells of the Stem and hematopoietic progenitors are described in the U.A. No. 5, 199,942, incorporated herein by reference. In summary, the term means a method comprising: (1) collecting stem cells and CD34 + hematopoietic progenitors from a patient from peripheral blood harvest or from bone marrow explants; and (2) expanding said cells ex vivo. In addition to the cell growth factors described in the patent 5, 199,942, other factors such as ligand flt3, I L-1, I L-3, ligand of equipment c can be used. The term "immunogenicity" means the relative effectiveness of an immunogen or antigen to induce an immune response. The term "substantially similar" means a variant amino acid sequence preferably that is at least 80% identical to a natural amino acid sequence, most preferably at least 90% identical. The percentage of identical aspect can be determined, for example, by comparing the sequence information using the GAP computer program, version 6.0, described by Devereux et al. (Nucí, Acids Res. 12: 387, 1984) and available from the University of Wisconsin Genetics Computer Group (UWGCG). The GAP program uses the alignment method of Needleman and Wunsch (J. Mol. Biol. 48: 443, 1970), as reviewed by Smith and Waterman (Adv. Appl. Math 2: 482, 1981). The preferred default parameters for the GAP program include: (1) a unary comparison matrix (containing a value of 1 for identities and 0 for non-identities) for nucleotides, and the matrix of heavy comparison of Gribskov and Burgess, Nucí. Acids Res. 14: 6745, 1986, as described by Schwartz and Dayhoff, eds. , Atlas of protein Sequence and Structure, National Biomedical Research Foundation, p. 353-358, 1979; (2) a penalty of 3.0 for each hole and an additional penalty of 0.10 for a symbol in each hole; and (3) no penalty for end gaps. The variants may comprise conservatively substituted sequences, meaning that a given amino acid residue is replaced by a residue having similar physiochemical characteristics. Examples of conservative substitutions include the substitution of one aliphatic residue for another, such as Me, Val, Leu, or Ala for another, or substitutions of one polar residue for another, such as between Lys and Arg; Glu and Asp; or Gln and Asn. Other conservative substitutions, for example, substitutions of complete regions having similar hydrophobicity characteristics, are well known. Variants of natural existence are also encompassed by the invention. Examples of such variants are proteins resulting from cases of binding of alternating mRNA or the proteolytic cleavage of the natural protein, wherein the natural biological property is retained. As used herein, "vaccine" means an organism or material that contains an antigen in a harmless manner. The vaccine is designed to trigger an immunoprotective response. The vaccine can be recombinant or non-recombinant. When a non-immune host is inoculated, the vaccine will cause active immunity to the organism or material, but it will not cause disease. The vaccines can take the form of, for example, a toxoid, which is defined as a toxin that has been detoxified, but still retains its main determinants; or an annihilated organism, such as a typhoid, cholera or polio; or attenuated organism, which are forms of living pathogens, but not virulent, or it can be an antigen encoded by said organism, or it can be a living tumor cell or an antigen present in a tumor cell. A variety of cell selection techniques are known to identify and separate stem cells or CD34 + hematopoietic progenitors from a population of cells. The methods and materials for identifying and selecting such cell types are known. For example, monoclonal antibodies can be used to bind to a marker protein or surface antigen protein found in stem cells or progenitors. Said markers or cell surface antigens for hematopoietic stem cells include CD34 and Thy-1. In a method, the antibodies are fixed to a surface, for example, glass beads, and contacted with a mixture of cells suspected of containing stem cells. This allows the antibodies to bind and secure the stem cells to the glass beads. Alternatively, the antibodies can be incubated with the cell mixture and the resulting combination contacted with a surface having an affinity for the antibody-cell complex. Unwanted cells and cellular matter are removed by providing a relatively pure population of stem cells. Stem cells or progenitors that have the CD34 marker, make up only about 1% to 3% of the mononuclear cells in the bone marrow. The number of stem cells or progenitors CD34 + in the peripheral blood is about 10- to 100 times less than in the bone marrow. With respect to particular aspects of the invention, the choice of stem cell selection means will depend on the desired phenotype of the cell to be isolated. Hematopoietic stem cells are selectable by virtue of their physical characteristics, such as expressing the flt3 receptor bound to the membrane, or having the following cell markers: CD34 or Thy-1. Monoclonal antibodies that recognize any of these antigens have been described in the U.A. No. 4,714,680 (anti-My-10), incorporated herein by reference, anti-CD34 is commercially available from Becton Dickinson, Franklin Lakes, BJ), and anti-Thy-1 monoclonal antibodies can be readily generated using the methods described by Dalchau et al. , J. Exp. Med. 149: 576 (1979), incorporated herein by reference. A flt3 receptor binding protein can also be used. Such as anti-flt3 monoclonal antibodies or the flt3 ligand. The cell-binding protein is contacted with the collected cell mixture and the combination is allowed to incubate for a sufficient period to allow binding of the desired cell to the cell-binding protein.

An alternative means for selecting stable stem cells is to induce cell death in the most committed lineage division cell types, using antimetabolites such as 5-fluorouracil (5-FU) or an alkylating agent such as 4-fluorouracil. -hydroxy cyclophosphamide (4-HC). Unstable cells are stimulated to proliferate and differentiate through the addition of growth factors that have little or no effect on the stem cells, causing stem cells to proliferate and differentiate, and making them more vulnerable to cytotoxic effects of 5-FU or 4-HC. See Berardi et al. , Science, 26_: 104 (1995), which is incorporated herein by reference. Isolation of stem cells or hematopoietic progenitors can be performed using, for example, affinity chromatography, magnetic beads coated with antibody, or antibodies fixed to a solid matrix, such as glass beads, flasks, etc. Antibodies that recognize a stem cell marker or progenitor can be fused or conjugated to other chemical moieties, such as biotin, which can be removed with a portion of avidin or streptavidin secured to a solid support; Fluorochromes useful in fluorescence activated cell sorting (FACS), or the like. Preferably, the isolation is achieved through an affinity column. The immunoaffinity columns can have any shape, but usually comprise a packed bed reactor. The bed packed in these bioreactors is preferably made from a porous material having a substantially uniform coating of a substrate. The porous material, which provides a high ratio of surface area to volume, allows the cell mixture to flow over a large contact area, while not impeding the flow of cells out of the bed. Typical substrates include avidin and streptavidin, while other conventional substrates can be used. The substrate must, either by its own properties, or by the addition of a chemical moiety, exhibit high affinity for a portion found in the cell binding protein, such as a monoclonal antibody. The monoclonal antibodies recognize a cell surface antigen on the cells that will be separated, and typically are also modified to present a portion of biotin. It is well known that biotin has a high affinity for avidin, and the affinity of these substances in this way removably secures the monoclonal antibody to the surface of the packed bed. Such columns are well known in the art, see Berenson, et al. , J. Cell Biochem. , 10D: 239 (1986). The column was washed with a PBS solution to remove unbound material. The target cells can be released from the beads using conventional methods. Immunoaffinity columns of the type described above utilizing biotinylated anti-CD34 monoclonal antibodies secured to a packed, avidin-coated bed are described, for example, in PCT Publication No. WO 93/08268. A variation of this method uses binding proteins from cell, such as monoclonal antibodies or ligand flt3 as described above, removably secured to a fixed surface in the isolation means. The bound cell binding protein is then contacted with the collected cell mixture and incubated for a sufficient period to allow isolation of the desired cells. Alternatively, monoclonal antibodies that recognize cell surface antigens may be labeled with a fluorescent tag, eg, chromophore or fluorophore, and separated through cell sorting according to the presence or absence of the amount of labeled product. The collected CD34 + cells are then exposed to their ligand flt3 alone or the ligand flt3 in concurrent or sequential combination with one or more of the following cytokines: GM-CSF, TNF-a, I L-3, IL-4, team ligand co fusion proteins GM-CSF / I L-3. The CD34 + cells are then allowed to differentiate and bind to cells of dendritic lineage. The dendritic cells are collected and can be either (a) administered to a patient in order to increase the immune system and the immune responses mediated by T cell or mediated by B cell to antigen, (b) exposed to an antigen before the administration of the dendritic cells to a patient, (c) transfected with a gene encoding a specific polypeptide in the antigen, or (d) exposed to an antigen and then allowed to process and present the antigen, ex vivo, to T cells collected from the patient after administration of the specific T cells in the antigen to the patient. More specifically, the invention provides for the use of an effective amount of a flt3 ligand to increase or mobilize dendritic cells in vivo, for example, in the peripheral blood of the patient or other tissues and organs, such as the spleen. By increasing the amount of the patient's dendritic cells, said cells themselves can be used to present the antigen to the T cells. For example, the antigen can be one that already exists within the patient, such as a tumor antigen, or a bacterial or viral antigen. Therefore, the ligand flt3 can be used to promote the immune response of the patient mediated by lymphocytes (for example, mediated by T cell or B cell) or mediated by myeloid, to the antigens already present thus potentially presenting an antigen presentation more effective to the T cells of the patient. Alternatively, ligand flt3 can be administered before, concurrent with or subsequent to the administration of an antigen to a patient for immunization purposes. In this way, as a vaccine adjuvant, the ligand flt3 can generate large amounts of dendritic cells in vivo to more effectively present the antigen. The total response is a stronger and improved immune response and a more effective immunization to the antigen. The systemic administration of the ligand flt3 is not only effective as a vaccine adjuvant, but also, as already discussed, is effective in increasing an immune response against previously existing antigens. For example, the inventors have shown that administration of the flt3 ligand to mice with tumor results in at least a significant reduction in the rate of tumor growth, and may result in rejection of the tumor in a large proportion of the tumors. mice. The data are presented in more detail in Example 3. Therefore, the ligand flt3 is an important cytokine in the generation of an immune response, in vivo, against the antigen. Due to its ability to generate dendritic cells, the ligand flt3 also finds use to promote the survival of transplanted tissues and organs. When allogeneic organs or other tissue are transplanted into a host, they can transfer stem cells, immature dendritic cells and mature dendritic cells from the donor. These cells are called transient cells, and said cells can be grafted into the host's hematopoietic system. In addition, stem cells, immature dendritic cells, and mature dendritic cells of the host can be grafted to the donor organ or tissue. It is then possible to establish a tolerance between the graft and the host, since the immature dendritic cells of the host and of the donor tissue interact with "other side" T cells. Such an interaction may include the removal of T cells that recognize the major histocompatibility complex (MHC) that the dendritic cells express. In this way, donor cells are "classified", so they fail to recognize and react against the host (ie, no graft against host disease) and the T cells are sorted, so that they fail to recognize and react against the graft (ie, no graft rejection). Thus, mutual tolerance can be achieved, and acceptance of the graft is improved. Administration of the ligand flt3 to the host or donor before transplantation could generate increased numbers of dendritic cells in said host p donor, and allow increased tolerance and graft survival. For the growth and cultivation of dendritic cells, a variety of growth and culture media can be used, and the composition of said media can easily be determined by one skilled in the art. Suitable growth media with solutions that contain nutrients or metabolic additives, and include those that are reduced in serum or serum-based. Representative examples of growth media are RPMI, TC 199, Dulbecco's medium modified with Iscoves (Iscove et al., FJ Exp. Med., 147: 923 (1978)), DMEM, Fisher's, alpha medium, NCTC, F -10, L-15 by Leibovitz, MEM and McCoy. Particular examples of nutrients that will be readily apparent to those skilled in the art include, serum albumin, transferin, lipids, cholesterol, a reducing agent such as 2-mercaptoethanol or monothioglycerol, pyruvate, butyrate and a glucocorticoid, such as 2- hydrocortisone hemisuccinate. More particularly, standard means include a source of energy, vitamins and other organic cell support compounds, such a pH regulator as HEPES, Tris, which act to stabilize the pH of the media, various inorganic salts. Particular reference is made to PCT publication No. WO 95/00632, wherein a variety of serum free cell growth media are described, said description is incorporated herein by reference. For any of the ex vivo methods of the invention, peripheral blood progenitor cells (PBPC) and peripheral blood stem cells (PBSC) are harvested using apheresis procedure known in the art. See, for example, Bishop et al. , Blood, vol. 83, No. 2, p. 610-616 (1994). Briefly, the PBPC and PBSC are collected using conventional devices, for example, a Haemonetics apheresis device Model V50 (Haemonetics, Braintree, MA). Harvests of 4 hours were performed, typically no more than five times a week until, for example, approximately 6.5 x 108 mononuclear cells (M NC) / kg of the patient were collected. The cells were suspended in standard media and then centrifuged to remove red blood cells and neutrophils. Cells located on the abutting surface between the two phases (also known in the art as a yellow coating) are removed and resuspended in H BSS. Suspended cells are predominantly mononuclear and a substantial portion of the cell mixture are stem cells first. The resulting stem cell suspension is then contacted with biotinylated anti-CD34 monoclonal antibodies or other binding means of cell. The contact period is maintained for a sufficient time to allow substantial interaction between the anti-CD34 monoclonal antibodies and the CD34 antigens on the surface of the stem cell. Typically, times of at least one hour are sufficient. The cell suspension is then contacted with the isolation means provided in the equipment. The isolation means may comprise a column packed with beads coated with avidin. Such columns are well known in the art, see Berenson et al. , J. Cell Biochem. , 10D: 239 (1986). The column is washed in a PBS solution to remove unbound material. Target stem cells can be released from the beads and the anti-CD34 monoclonal antibody using conventional methods. Stem cells obtained in this way can be frozen in a controlled regime freezer (for example, Cryo-Med, Mt. Clemens, MI), can then be stored in the vapor phase of liquid nitrogen. 10% dimethyl sulfoxide can be used as a cryoprotectant. After all the donor collections have been made, the stem cells thawed and emptied. The aliquots containing the stem cells, the culture medium, such as McCoy's 5A medium, 0.3% agar, and at least one of the expansion factors: recombinant human GM-CSF, recombinant human flt3 ligand, and molecules of fusion of recombinant human GM-CSF / IL-3 (PIXY321) at concentrations of approximately 200 U / ml, were cultured and expanded at 37 ° C in 5% CO2 in air completely moistened for 14 days. Optionally, IL-1 or human IL-4 can be added. The most preferred combination of expansion factors comprises the ligand flt3 plus either IL-3 or a fusion protein GM-CSF / IL-3. For in vivo administration in humans, the ligand flt3 can be formulated according to known methods that are used to prepare pharmaceutically useful compositions. The ligand flt3 can be combined in admixture, either as a single active material or with other known materials, with pharmaceutically acceptable diluents (e.g., Tris-HCl, acetate, phosphate), preservatives (e.g., Thimerosal, benzyl alcohol, parabens ), emulsifiers, solubilizers, auxiliaries and / or vehicles. Suitable carriers and their formulations are described in Remington's Pharmaceutical Sciences, 16th edition, 1980, Mack Publishing Co. In addition, such compositions may contain the ligand flt3 in complex with polyethylene glycol (PEG), metal ions, or be incorporated into polymeric compounds such such as polyacetic acid, polyglycolic acid, hydrogels, etc. , or be incorporated into liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, phantom erythrocytes or sphenoblasts. Said compositions will influence the physical state, the solubility, the stability, the release rate in vivo, and the clear rate in vivo of the ligand flt3. Ligand flt3 can be administered topically, parenterally or by inhalation. The term "parenteral" includes injections subcutaneous, intravenous, intramuscular, intracisternal injection. Or infusion techniques. These compositions will typically contain an effective amount of the ligand flt3, alone or in combination with an effective amount of any other active material. Said desired doses and drug concentrations contained in the compositions may vary depending on many factors, including the intended use, the body and age of the patient, and the route of administration. Preliminary doses can be determined according to test animals, and the dose classification for administration to humans can be done according to practices accepted by the art. Keeping this description in mind, typical doses of ligand flt3 can vary from about 10 μg per square meter to about 1000 μg per square meter. A preferred dose scale is of 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 invention.

EXAMPLE 1 Generation of Dendritic Cells This Example describes a method for using a flt3 ligand to generate large numbers of ex vivo dendritic cells. The cells having the CD34 + phenotype are isolated as described above, for example, by first generating a yellow cell envelope using a procedure described supra. The cells of the yellow coat are then incubated with a specific monoclonal antibody CD34. The CD34 + cells, which are selected, are then cultured in an improved McCoy medium with 20 ng / ml each of GM-CSF, IL-4, TNF-α or 100 ng / ml ligand flt3 p ligand team c. The culture was continued for approximately two weeks at 37 ° C in 10% CO2 in moist air. The cells were then sorted through cytometry for the expression of CD1 a + and H LA-DR +. The combination of GM-CSF, I L-4 and TN F-a, resulted in an increase of six to seven times the number of cells obtained after two weeks of culture. The combination of ligand flt3 and the c-team ligand resulted in an additive increase of 12-13 fold in absolute numbers of cells. This was correlated with an 18-fold expansion with either the ligand flt3 or with the ligand of team c, or a 34-fold expansion with the combination of ligand flt3 and the team ligand c. The phenotypic analysis of the cells showed that between 60-70% of the cells were H LA-DR +, CD86 +, with 40-50% of the cells expressing CD1a in all factor combinations examined. The addition of ligand flt3 increased the absolute number of CD1 a + cells by 5 times. The c-team ligand increased those cells by 6.7 times, and the combination of the ligand flt3 and the c-team ligand by 1 1-fold. The analysis The functional status of the resulting cells in MLR revealed that the presence of ligand flt3 or ligand of team c did not affect the stimulating capacity of the resulting dendritic cells, while increasing the numbers obtained.

EXAMPLE 2 Use of Flt3-L in Dendritic Cell Expansion This Example describes a method for using the ligand flt3 for the expansion of dendritic cells. Prior to cell collection, it may be desirable to mobilize or increase the numbers of PBPC and PBSC in circulation. Mobilization can improve the collection of PBPC and PBSC, and can be achieved through intravenous administration of the ligand flt3 or sargramostim (Leukine®, Immunex Corporation, Seattle, Washington) to patients prior to harvesting said cells. Other factors such as CSF-1, GM-CSF, c-team ligand, 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 of they can also be administered in sequence, or in concurrent combination with the ligand flt3. PBPC and PBSC mobilized and unmobilized were collected using apheresis procedures known in the art. See, for example, Bishop et al., Blood, vol. 83, No. 2, p. 610-616 (1994). Briefly, PBPC and PBSC were collected using conventional devices, by example, a Haemonetics apheresis device Model V50 (Haemonetics, Braintree, MA). Harvests of 4 hours were typically made no more than five times a week until approximately 6.5 x 108 mononuclear cells (MNC) / kg of the patient were collected. The aliquots of PBPC and PBSC collected were analyzed for the content of the granulocyte-macrophage colony formation unit (CFU-GM) by diluting approximately 1: 6 with a balanced salt solution of Hank without calcium or magnesium (H BSS) and placing as a layer on the lymphocyte separation medium (Organon Teknika, Durham, North Carolina). After centrifugation, M NC was collected on the adjoining surface, washed and resuspended in H BSS. One milliliter aliquots containing approximately 300,000 MNC, modified McCoy 5A medium, 0.3% agar, 200 U / ml recombinant human GM-CSF, 200 μg / ml recombinant human L-3, and 200μ / ml of recombinant human G-CSF, were cultured at 37 ° C in 5% CO2 in fully moistened air for 14 days. Optionally, the ligand flt3 or the fusion molecules GM-CSF / I L-3 (PIXY 321) can be added to the cultures. These cultures are stained with Wright's stain, and the CFU-GM colonies were classified using a dissection microscope (Ward et al., Exp. Hematol., 16: 358 (1988).) Alternatively, colonies of CFU-GM can be isolated. be analyzed using the flow cytometry method CD34 / CD33 from Siena et al., Blood, Vol. 77, No. 2, pp. 400-409 (1991), or any other method known in the art.

Crops containing CFU-GM were frozen in a controlled-regime freezer (eg, CryoMed, Mt. Clemens, MI), then stored in the vapor phase of liquid nitrogen. As a cryoprotectant, 10% dimethylsulfoxide can be used. After all patient collections were made, cultures containing CFU-GM were thawed and emptied. The collection of thawed cells was contacted with the ligand flt3 either alone, sequentially or in combination concurrently with other cytokines listed above. Said exposure to the ligand flt3 will activate the CFU-GM to the dendritic cell lineage. The dendritic cells were reinfused intravenously to the patient.

EXAMPLE 3 Use of Flt3-L to Increase Immune Responses Anti-tumor This Example describes a method for using flt3-L to increase anti-tumor immune responses in vivo. Female C57BL / 10J (B10) mice (The Jackson Laboratory, Bar Harbor, ME) were injected with 5 x 10 5 fibrosarcoma B 10 tumor cells. Viable 10.5 via intradermal injection in a ventral position in a total volume of 50 μl. Lines B 10.2 and B 10.5 of fibrosarcoma are of B 10 origin and have been previously described, see Lynch et al. , Euro. J. Immunol., 21: 1403 (1991) incorporated herein by reference. Line B 10.2 of fibrosarcoma was induced through of subcutaneous implantation of a paraffin pellet containing 5 mg of methylcholanthrene, and line B10.5 was induced by chronic exposure to ultraviolet radiation. The tumor cell lines were maintained in vitro in MEM modified with a containing 5% FBS, 2 nM L-glutamine, 50 U / ml penicillin and 50 μg / ml streptomycin. Recombinant human flt3-L (10 μg / injection) was administered on a daily basis for a period of 19 days (unless otherwise noted) by subcutaneous injection in a total volume of 100 μl. Similarly, control mice were injected with a similar volume of pH buffer containing 100 ng of MSA. The growth rates were determined by plotting the size of the tumor against the time after the tumor attack. Tumor size was calculated as the product of two perpendicular diameters, measured through gauges, and expressed as the average tumor size only of those mice carrying a tumor within a particular treatment group. The number of mice bearing tumors compared to the number attacked for each treatment group at the end of an experiment are shown in the data below. From Table I, the data is a compilation of six different experiments, in which mice carrying tumors were treated either with ligand flt3 or with MSA. Complete regression of the tumor was observed in 19 of 50 mice treated with the ligand flt3, compared with 1 to 30 mice treated with MSA (p <0.0001 using the Fishers Exact Test). The observation that the rate of tumor growth in mice treated with the ligand flt3 (mean tumor size in mice bearing tumors to a 5-week post-tumor attack was 60 +/- 8 mm2) was significantly reduced, compared with mice treated with MSA (mean tumor size at a 5-week post-tumor attack was 185 +/- 17 mm2) it was also confirmed (p.0001 by Variation Analysis).

TABLE I Fibrosarcoma Compound Material +/- Flt3-L of Six Tumor Size Experiments (mm2) The size of the tumor was sharply delayed with the ligand flt3 compared to the control. Therefore, the data show that the ligand flt3 is an important cytokine in the increase of the immune response against foreign antigens, and in particular against cancer.

Claims (9)

CLAIMS 1. A method for increasing an immune response in a patient, comprising the step of administering a quantity of ligand flt3 to the patient, sufficient to generate an increase in the number of dendritic cells of the patient. 2. A method according to claim 1, further comprising the step of administering one or more molecules selected from the group consisting of GM-CSF, IL-4, TNF-a, IL-3, c-team ligand, and mergers of GM-CSF and IL-3. 3. A method for increasing an immune response in a patient having an infectious disease, comprising the step of administering the ligand flt3 in an amount sufficient to generate an increase in the number of dendritic cells of the patient. 4. A method according to claim 3, further comprising the step of administering one or more molecules selected from the group consisting of GM-CSF, IL-4, TNF-a, IL-3, c-team ligand, and mergers of GM-CSF and IL-3. 5. A method according to claim 3, wherein the infectious disease is HIV (acquired immunodeficiency virus). 6. A method for increasing an immune response in a patient having a cancerous or neoplastic disease, comprising the step of administering the ligand flt3 in an amount enough to generate an increase in the number of dendritic cells of the patient. 7. A method according to claim 6, further comprising the step of administering one or more of the molecules selected from the group consisting of GM-CSF, IL-4, TNF-a, IL-3, ligand equipment c, and mergers of GM-CSF and IL-3. 8. A dendritic cell preparation having at least two cell surface markers, selected from the group consisting of CD1a, HLA-DR and CD86, produced by contacting stem cells or hematopoietic progenitors with ligand flt3. 9 - A dendritic cell preparation according to claim 8, further produced by contacting stem cells or hematopoietic progenitors with a molecule selected from the group consisting of GM-CSF, IL-4, TNF-a, IL-3 , ligand of team c, and mergers of GM-CSF and IL-3. 10 - A dendritic cell population expressing the antigen produced by the method of: (a) contacting stem cells or hematopoietic progenitors with ligand flt3 in an amount sufficient to generate a dendritic cell population; (b) either (i) exposing the dendritic cells to a specific peptide in the antigen, or (ii) transfecting the dendritic cells with a gene encoding a peptide specific to the antigen; (c) allow dendritic cells to process and express the antigen; and (d) purifying the dendritic cells expressing the antigen.

1. A dendritic cell population according to claim 10, wherein step (a) of the method further comprises contacting stem cells or hematopoietic progenitors with a molecule selected from the group consisting of GM-CSF , I L-4, TN Fa, IL-3, c-team ligand, and GM-CSF and I L-3 fusions. 1

2. A method for activating stem cells or hematopoietic progenitors to a dendritic cell lineage, comprising contacting said stem cells or hematopoietic progenitors with ligand flt

3. 13. A method for preparing a dendritic cell population that is presented to the antigen, comprising the steps of: (a) contacting stem cells or hematopoietic progenitors with the ligand flt3 in an amount sufficient to generate a population of dendritic cell; (b) either (i) exposing the dendritic cells to a specific peptide to the antigen, or (ii) transfecting the dendritic cells with a gene encoding a peptide specific for the antigen; (c) allowing the dendritic cells to process and express the antigen; and (d) purifying the dendritic cells expressing the antigen. 14 - A method according to claim 13, wherein step (a) further comprises contacting the stem cells or hematopoietic progenitors with a molecule selected from the group consisting of GM-CSF, IL-4, TNF-α, IL-3, c-team ligand, and GM-CSF and IL-3 fusions. 15. A method for preparing antigen-specific T cells comprising the steps of: (a) contacting stem cells or hematopoietic progenitors with ligand flt3 in an amount sufficient to generate a dendritic cell population; (b) either (i) exposing the dendritic cells to a specific peptide to the antigen, or (ii) transfecting the dendritic cells with a gene encoding a peptide specific for the antigen; (c) allowing the dendritic cells to process and express the antigen; and (d) allowing the dendritic cells to present the antigen to the T cells. 16. A method for improving an immune response in a mammal to a vaccine antigen, comprising the steps of administering to said mammal an immunogenic amount of the antigen. vaccine antigen and an amount to increase the immunogenicity of ligand flt3 in concurrent or sequential combination with said vaccine antigen. 17 - A vaccine adjuvant comprising a molecule selected from the group consisting of the ligand of team c and the ligand flt3. 18.- A method to induce tolerance of graft tissue in a host, comprising administering the ligand flt3 to the host in an amount sufficient to increase the number of dendritic cells. 19.- A dendritic cell expansion medium comprising an effective amount of ligand flt3 and a cytokine selected from the group consisting of I L-3, I L-4, GM-CSF, TN F, ligand of equipment C and proteins of fusion GM-CSF / I L-3.